Changes in the hydroxyproline,hexosamine and sialic acid of the diabetic human and β, β′-iminodipropionitrile-treated rat retinal vascular systems

Summary The retinal vasculature has been isolated from nondiabetic and diabetic post mortem human eyes by controlled trypsin digestion. Chemical analysis demonstrated increases in the hydroxyproline and hexosamine contents in diabetes. There was no general increase in the sialic acid content. These results have been related to histological preparations of sectors of the same retinas. Administration of, -iminodipropionitrile to rats caused a retinopathy characterised by endothelial cell proliferation, increased PAS-positivity and microaneurysms. Chemical analysis of retinal vascular systems from, -iminodipropionitrile-treated rats revealed increases in hydroxyproline, hexosamine and sialic acid contents.

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Änderungen des Gehaltes an Hydroxyprolin, Hexosamin und N- azetyl- Neuraminsäure in den Netzhautgefäen von menschlichen Diabetikern und mit , Iminodiproprionitril behandelten Ratten

Zusammenfassung Die Netzhautgefäße wurden post-mortal aus diabetischen und nichtdiabetischen Augen mit Hilfe einer kontrollierten Trypsin-Behandlung gewonnen. Bei der chemischen Analyse fand sich ein Anstieg des Hydroxyprolin- und Hexosamingehaltes in den diabetischen Augen. Der N-azetyl-Neuraminsäuregehalt war im allgemeinen nicht erhöht. Diese Resultate wurden in Beziehung gebracht zu den histologischen Befunden, die an den einzelnen Sektoren der gleichen Retina erhoben wurden. Verabreichung von, -Iminodiproprionitril an Ratten bewirkte eine Retinopathie unter den Zeichen einer Wucherung der Endothelzellen, verstärkter PAS-Anfärbbarkeit und Mikroaneurysmen. Die chemische Analyse der Netzhautgefäße von mit, -Iminodiproprionitril behandelten Ratten zeigte einen erhöhten Gehalt an Hydroxyprolin, Hexosamin und N-azetyl-Neuraminsäure.

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Variations de l'hydroxyproline, de l'hexosamine et de l'acide sialique dans les systèmes vasculaires rétiniens de l'homme diabétique et du rat traité par le , -iminodipropionitrile

Résumé Le système vasculaire rétinien a été isolé, par digestion trypsique contrôlée, après la mort, à partir d'yeux humains de non-diabétiques et de diabétiques. L'analyse chimique a révélé l'augmentation du contenu en hydroxyproline et en hexosamine, dans le diabète. Il n'y avait pas d'augmentation générale du contenu en acide sialique. Ces résultats ont été rapprochés des préparations histologiques de portions des mêmes rétines. L'administration de, -iminodipropionitrile à des rats provoquait une rétinopathie caractérisée par une prolifération des cellules endothéliales, une PAS-positivité augmentée et des microanévrismes. L'analyse chimique des systèmes vasculaires rétiniens des rats traités par le,-iminodipropionitrile, a révélé l'augmentation du contenu en hydroxyproline, en hexosamine et en acide sialique.

     

Glycosaminoglycano-Hydrolasen im menschlichen Serum

Zusammenfassung Von 200 klinisch gesunden Probanden wurden — in 6 Altersgruppen — die Normalwerte der Serum-Glykosaminoglyzanohydrolasen: Hyaluronidase, -Glukuronidase und -Azetylglukosaminidase ermittelt. Dabei fand sich eine hohe Enzymaktivität der 3 lysosomalen Enzyme im Säuglingsalter und eine Abnahme der Enzymaktivität in der Altersgruppe: 1–15 Jahre. Während die Hyaluronidase zwischen 1 und 80 Jahren etwa gleiche Aktivitäten zeigte, fand sich bei der -Glukuronidase und der -Azetylglukosaminidase ein deutlicher Aktivitätsanstieg mit höherem Lebensalter, der jedoch nur bei der -Azetylglukosaminidase die Enzymaktivität in der ersten Altersgruppe erreichte.Für die -Azetylglukosaminidase wurden pH-Optimum, optimale Substratund Enzymkonzentration bestimmt.

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Summary In the serum of 20 healthy persons, devided into 6 different age-groups, enzyme activity of Hyaluronidase, -Glukuronidase and -Acetylglucosaminidase was determined. High enzymatic activities of the three lysosomal enzymes was found in the first year of life where upon the enzyme activity dropped between twenty and thirty years. Hyaluronidase activity remained relatively constant between twenty and eighty years. In contrast -Glukuronidase and -Azetylglukosaminidase activity increased with advancing age. While -Glukuronidase of aged persons nerver reached the level of the first year activity, the values for -Acetylglucosaminidase can be equivalent in very old and very young persons.In the specific case of -Acetylglucosaminidase pH-optimum, optimal substrate and enzyme-concentration was determined.

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1 m U=freigesetztes Phenolphthalein bzw. Phenol je 1 ml Serum und Min bei 37°C.

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Mir Unterstützung der Deutschen Forschungsgemeinschaft.     

Uptake and metabolism of radiolabelled GM1-ganglioside in skin fibroblasts from controls and patients with GM1-gangliosidosis

Summary The uptake and metabolism of [3-³H-sphingosine]GM1-ganglioside was measured in cultured skin fibroblasts from controls and patients with infantile, juvenile and adult GM1-gangliosidosis. When dissolved in medium with phosphatidylserine, GM1-ganglioside was efficiently taken up by cultured skin fibroblasts and transferred into lysosomes. A linear increase in GM1-ganglioside endocytosis was shown with phosphatidylserine concentrations of up to 40m/ml. A pulse-chase study revealed that [³H]GM1-ganglioside was metabolized to GM2-ganglioside, GM3-ganglioside, ceramide dihexoside, ceramide monohexoside, ceramide and sphingosine. Sphingosine was recycled to sphingomyelin. In a 20-h pulse study, cell lines from patients with GM1-gangliosidosis of infantile, juvenile and adult types hydrolysed 2–5%, 20–44% and 54–58% of the total endocytosed GM1-ganglioside respectively. These values were lower than in control cells (64.17 ± 5.43% (n=10)). The hydrolysis rates of exogenous [³H]GM1-ganglioside in cultured fibroblasts from patients with various types of GM1-gangliosidosis closely reflected the clinical severity.Abbreviations GM1 Gal13GalNAc14(NeuAc23)Gal14Galc-1-ceramide - GM2 GalNAc14(NeuAc23)Gal14Glc-1-ceramide - GM3 NeuAc23Gal14Glc-1-ceramide - CDH Gal14Glc-1-ceramide     

Beta lactamase resistance of newer cephalosporins and antimicrobial effectiveness against gram-negative bacilli

Summary Three newer cephalosporins (cefamandole, cefoxitin and cefazaflur) were investigated, in comparison with three older agents (cephalothin, cephaloridine and cefazolin) to determine their stability to -lactamases of gram-negative bacilli, and to correlate this with their antibacterial activity. Nine of the 17 bacterial strains employed produced broad-spectrum -lactamases; the remaining eight produced cephalosporinases. The cephalosporins were highly active against bacteria producing broad-spectrum -lactamases; they were less active against organisms producing cephalosporinases. All of the cephalosporinase-producing strains were resistant to cephalothin and cephaloridine. With the other cephalosporins the correlation between hydrolysis by cephalosporinases and resistance of the organisms was poor. Four of eight cephalosporinase-producing strains were resistant to cefoxitin, which was completely resistant to hydrolysis by the -lactamases. Cefozolin, cefamandole and cefazaflur inhibited several of these strains in spite of destruction by the -lactamase. Several cephalosporins need to be used in antimicrobial susceptibility testing of gram-negative bacilli.

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Beta-Lactamase-Resistenz der neuen Cephalosporine und die antimikrobielle Wirksamkeit gegen gramnegative Bazillen

Zusammenfassung Drei neue Cephalosporine (Cefamandol, Cefoxitin und Cefazaflur) wurden im Vergleich zur drei älteren Präparaten der Cephalosporinreihe (Cephalothin, Cephaloridin und Cefazolin) untersucht, um ihre -Lactamase-Stabilität gegenüber gram-negativen Bazillen abzugrenzen und das Ergebnis mit ihrer antibakteriellen Aktivität zu korrelieren. Neun der 17 verwendeten Stämme produzierten Breitspektrum--Lactamasen. Die restlichen acht produzierten Cephalosporinasen. Die Cephalosporine waren hoch aktiv gegen breitspektrum--lactamase-produzierende Bakterien; sie waren weniger aktiv gegen Keime, die Cephalosporinase bildeten. Alle cephalosporinase-produzierenden Keime waren cephalothin-und cephalosporidin-resistent; bei den anderen Cephalosporinen war die Korrelation zwischen der Hydrolyse durch Cephalosporinasen und der Bakterienresistenz gering. Vier der acht cephalosporinase-produzierenden Stämme erwiesen sich als cefoxitin-resistent. Cefoxitin war jedoch komplett resistent gegenüber der -Lactamasehydrolyse. Cefazolin, Cefamandol und Cefazaflur hemmten einige dieser Stämme trotz der Zerstörung durch die -Lactamasen. Es wird postuliert, daß verschiedene Cephalosporine für die Empfindlichkeitstestung gram-negativer Bazillen erforderlich sind.

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This work was supported by grants from the Lilly Research Laboratories and Smith Kline and French Laboratories.     

Down-regulated β-adrenoceptors in severely failing human ventricles: Uniform regional distribution,but no increased internalization

Summary In chronic heart failure cardiac -adrenoceptors are decreased. In this study we investigated whether a) in severely failing human ventricles -adrenoceptors are uniformly decreased or regional variations exist, and b) the -adrenoceptor decrease is caused by increased internalization or is a real loss in -adrenoceptors. For this purpose we assessed -adrenoceptor number and subtype distribution in a particulate fraction (mainly sarcolemmal plasma membranes) and a light vesicle fraction of right and left ventricular segments (obtained by cutting transversal, rings of 2 cm from the midventricular regions) of explanted hearts from 2 patients with end-stage congestive dilated cardiomyopathy (DCM) and one patient with end-stage ischemic cardiomyopathy (ICM). In all three hearts ventricular -adrenoceptor number was very low (7.5–10 and 21–26 fmol/mg protein in DCM, 15–22 fmol/mg protein in ICM compared to 68–74 fmol/mg protein in non-failing ventricles). -Adrenoceptors were uniformly decreased over the whole ventricular region and no considerable regional variations existed. The same held true for ₁- and ₂-adrenoceptors. In ICM decrease in -adrenoceptors was due to a concomitant reduction in ₁- and ₂-adrenoceptors, in DCM it was mainly caused by ₁-adrenoceptor down-regulation. In all ventricular segments investigated light vesicle -adrenoceptors amounted to about 5–7% of total ventricular -adrenoceptors and this was not significantly different from non-failing left ventricles. We conclude that a) in severely failing human ventricles -adrenoceptors are evenly down-regulated and no regional variations exist and b) the decrease in -adrenoceptors is not due to enhanced internalization but is a real loss of -adrenoceptors.Abbreviations DCM dillted cardiomyopathy - ICM ischemic cardiomyopathy - ICI 118,551 erythro-(±)-1-(7-methylindan-4-yloxy)-3-isopropylaminobutan-2-ol hydrochloride - CGP 12177 (±)-4-(3-tertiarybutylamino-2-hydroxypropoxy)-benzimidazole-2-on hydrochloride - ICYP (–) [¹²⁵I]-Iodocyanopindolol     

α- andβ-mannosidoses

Summary Clinical, pathological and biochemical findings in the mannosidoses are described. Family studies showed granulocyte-rich white cell fractions to be the tissue of choice for carrier detection in-mannosidosis. Metabolic labelling studies using [³H] mannose demonstrated accumulation of Man1-4GlcNAc in cultured skin fibroblasts from a patient with this condition. Alternative methods of egress from lysosomes were suggested for this compound by its secretion into culture medium and apparent reduction of storage with time in cultures.-mannosidase deficient goats are not thought to be a true animal model of the human condition, as although they showed a similar enzyme deficiency, the clinical presentation is much more severe and the major storage material (Man1-4GlcNAc1-4GlcNAc) is different.     

Perspectives of beta-lactamases inhibitors in therapy of infections caused by Escherichia coli or Klebsiella with plasmidic resistance to third generation cephalosporins

Summary New plasmidic -lactamases inactivating so far stable cephalosporins, aztreonam and cephamycins restrict the use of these antibiotics in therapy of infections, e.g., byEscherichia coli and Klebsiella. Thus, combinations of -lactamase inhibitors and -lactam antibiotics were investigatedin vitro with regard to their therapeutic perspectives. Minimal inhibitory concentrations and the kinetics of killing in a pharmacodynamic model were determined. Extended broad spectrum betalactamases (EBS--lactamases) representative both for the TEM- and SHV-type were included. None of the available fixed combinations of penicillins and -lactamase inhibitors appears useful for therapy of infections caused by producers of EBS--lactamases. In contrast, combinations of piperacillin and tazobactam or sulbactam plus cephalosporins (cefoperazone, cefotaxime, ceftazidime) or aztreonam are highly active (both by their MICs and bactericidal activity) against TEM-type EBS--lactamases, but less promising for the SHV-type EBS--lactamases, and plasmidic cephamycinase. Of the -lactams available, the monobactam carumonam and the carbapenems (imipenem, meropenem) remain safe in infections caused byE. coli and Klebsiella EBSBase producers.

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Therapeutische Perspektiven von -Laktamase-Inhibitoren bei Infektionen durch Escherichia coli oder Klebsiella mit plasmid-determinierter Resistenz gegenüber Drittgenerationscephalosporinen

Zusammenfassung Neue plasmid-kodierte -Laktamasen inaktivieren bisher stabile Cephalosporine, Aztreonam und Cephamycine. Sie schränken damit die therapeutische Sicherheit dieser Antibiotika bei Infektionen zum Beispiel durchEscherichia coli oder Klebsiellaarten ein. Deshalb wurden die therapeutischen Perspektiven von Kombinationen aus -Laktamase-Inhibitoren und -Laktam-Antibiotikain vitro analysiert. Die minimalen Hemmkonzentrationen (MHK) wurden gemessen. Zu einem pharmakokinetischen Modell wurde die Abtötungskinetik bestimmt. Einbezogen wurden Enzyme sowohl aus der TEM- als auch der SHV-Reihe. Keine der beiden zugelassenen festen Kombinationen aus Penicillinen und -Laktamase-Inhibitoren erwies sich als erfolgversprechend für die Therapie von Infektionen durch Stämme, welche -Laktamasen mit erweitertem Spektrum produzieren. Demgegenüber waren Kombinationen aus Piperacillin und Tazobactam oder Sulbactam mit Cephalosporinen (Cefoperazon, Cefotaxim, Ceftazidim) oder Aztreonam sowohl aufgrund ihrer MHK-Werte als auch ihrer bakteriziden Aktivität im pharmakodynamischen Modell hochaktiv gegenüber TEM--Laktamasen mit erweitertem Spektrum, jedoch weniger wirksam bei Stämmen mit neuen Enzymen der SHV-Reihe und plasmid-kodierter Cephamycinasen. Unter den derzeit vorhandenen -Laktam-Antibiotika kommt anhand ihrerIn-vitro-Aktivität dem Monobactam Carumonam sowie den Carbapenemen (Impipenem, Meropenem) auch ohne -Laktamase-Inhibitor die größte therapeutische Sicherheit bei Infektionen durchE. coli und Klebsiella-Stämme mit der Fähigkeit zur Synthese von -Laktamasen mit erweitertem Spektrum zu.

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Symposium 2nd Biennial Conference on Chemotherapy of Infectious Diseases and Malignancies, Montreux, March 5^th–8^th, 1989.

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Supported by Pfizer GmbH, Karlsruhe, FR Germany.     

The effect of ceftriaxone on the anaerobic bacterial flora and the bacterial enzymatic activity in the intestinal tract

Summary The normal flora of the intestinal tract, mainly consisting of anaerobic bacteria, protects the host against colonization by pathogenic microorganisms. Antimicrobial treatment with ceftriaxone may influence the colonic microflora and as a consequence, the protective effect. Ten healthy volunteers received 1 g of ceftriaxone intramuscularly for five days. This resulted in a significant decrease (p<0.05) of the mean cultural counts (± SEM) of total anaerobes from 10.67 (0.11) (prior to treatment) to 9.02 (0.45) and 8.97 (0.46) at days 3 and 5, respectively (during treatment). After treatment (days 10 and 15–19), the cultural counts of anaerobes returned to 10.17 (0.16) and 10.44 (0.18), respectively. Bacterial enzymes may serve as an indicator of protective microflora. - aspartylpeptidase and deoxycholate hydrolase activity was determined in faecal supernatants of the volunteers and compared with anaerobic culturing. Both enzymatic activities show a significant correlation with the total number of anaerobes present at day 3 of ceftriaxone treatment. At day 5 and 8 only -aspartylpeptidase showed significant correlations with cultural counts of total anaerobes,Bacteroides spp. or bifidobacteria. At day 15 to 19 (ten to 14 days after treatment) -aspartylpeptidase showed only a significant correlation with the number ofBacteroides spp. This indicates that changes in the indigenous bacterial flora during and shortly after treatment with ceftriaxone can be monitored by determination of -aspartylpeptidase. Recovery of the intestinal flora is difficult to assess in this manner.

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Einfluß von Ceftriaxon auf die anaerobe Flora und die bakterielle Enzymaktivität im Intestinaltrakt

Zusammenfassung Die normale Flora des Intestinaltraktes besteht vorweigend aus anaeroben Bakterien und schützt den Wirt gegen eine Kolonisation durch pathogene Mikroorganismen. Eine antimikrobielle Therapie mit Ceftriaxon kann die Mikroflora des Dickdarms beeinträchtigen und damit auch deren protektiven Effekt. Zehn gesunde Probanden erhielten fünf Tage lang 1 g Ceftriaxon intramuskulär appliziert. Dies führte zu einer signifikanten Abnahme der mittleren Koloniebildnerzahlen von 10,67 (SEM ± 0,11) vor Applikation auf 9,02 (± 0,45) nach drei und auf 8,97 (± 0,46) nach fünf Tagen (p<0,05). Nach zehn und 15 bis 19 Tagen im Anschluß an die Antibiotikagabe kehrten die Anaerobier-Koloniebildnerzahlen auf 10,17 (± 0,16) bzw. 10,44 (± 0,18) zurück. Bakterienenzyme können als Indikator für die protektive Mikroflora dienen. In Überständen von Stuhlproben der Probanden wurden -Aspartylpeptidase und Desoxycholat-Hydrolase bestimmt und mit den Anaerobier-Kulturen verglichen. Zwischen den Aktivitäten beider Enzyme und der am Tag 3 gemessenen Anaerobier-Gesamtzahl fand sich eine signifikante Korrelation. Am Tag 5 und Tag 8 zeigte nur die -Aspartylpeptidase eine signifikante Korrelation mit den Gesamt-Kolonie-bildnerzahlen der Anaerobier sowie mit den Zahlen vonBacteroides spp. oder Bifidobakterien. An den Tagen 15 bis 19 (zehn bis 14 Tage nach Antibiotikagabe) bestand nur zwischen der Zahl vonBacteroides spp. und -Aspartylpeptidase eine signifikante Korrelation. Nach Behandlung mit Ceftriaxon lassen sich folglich Veränderungen der bakteriellen Flora kurzfristig durch Bestimmung der -Aspartylpeptidase erfassen, weniger gut aber die Erholung der Darmflora.

     

Die β-Laktamase-Inhibitoren und ihre klinische Bedeutung

Zusammenfassung Zu den verschiedenen Möglichkeiten der Überwindung einer -Laktamase-bedingten Resistenz von Mikroorganismen gehört der Einsatz von Enzyminhibitoren, die selbst keine nennenswerte eigene antimikrobielle Aktivität aufweisen, jedoch in Kombination mit einem Breitspektrumantibiotikum vom -Laktamtyp synergistisch wirken. Auf diese Weise gelangen -Laktam-resistente Bakterien erneut in das Wirkungsspektrum von Substanzen wie Penicillin G oder Ampicillin, die aufgrund steigender Resistenzentwicklung in den letzten Jahren ihre therapeutische Effizienz zu verlieren drohen. 6--Bromopenicillansäure und die sogenannten Olivansäuren weisen eine bemerkenswerte Hemmpotenz gegenüber verschiedenen -Laktamasen auf. Die mikrobiologischen und bisher vorliegenden pharmakokinetischen Daten eines Penicillansäuresulfons, das ebenfalls signifikante Hemmeigenschaften verschiedener klinisch relevanter -Laktamasen besitzt, werden diskutiert. Von Clavulansäure, einem Stoffwechselprodukt vonStreptomyces clavuligerus mit -Laktamstruktur konnte ebenfalls gezeigt werden, daß es ein progressiver Hemmstoff der -Laktamasen vom Richmond-Typ II-V ist. Neben den bisher vorliegendenIn-vitro-Untersuchungen werden auch Ergebnisse klinischer Studien mit der Kombination Clavulansäure und Amoxicillin erwähnt.

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Inhibitors of -lactamases and their clinical significance

Summary One of the various possibilities of overcoming bacterial resistance due to -lactamase production is with enzyme inhibitors. These have no remarkable intrinsic antimicrobial activity, but act synergistically in combination with a broad spectrum antibiotic of the -lactam type. Thus -lactam resistant bacteria are once again within the antibacterial spectrum of substances like penicillin G or ampicillin, which have been in danger of losing their therapeutical effectiveness in recent years due to an ever increasing development of resistance. 6--bromopenicillanic acid and the so-called olivanic acids exhibit remarkable inhibitory properties against several -lactamases. Microbiological and pharmacokinetic data published recently on a penicillanic acid sulfone, which also shows significant inhibitory properties against several clinically relevant -lactamases, are discussed in this paper. Clavulanic acid, a recently discovered product ofStreptomyces clavuligerus with a -lactam structure, acts as a progressive inhibitor of Richmond type II-V -lactamases. In addition to microbiological and enzyme-kineticin vitro data, results of clinical studies with the combination clavulanic acid and amoxicillin are summarized.

     

Macrophages enhance binding of beta-VLDL and cholesterol ester accumulation in cultured aortic smooth muscle cells

Summary The effect of macrophages on the uptake of -very low-density lipoprotein (-VLDL) by smooth muscle cells (SMC) expressing different morphological phenotypes was examined in culture. The SMC were grown alone and in co-culture with macrophages for four days, then incubated with different concentrations of¹²⁵I--VLDL for 3 h at 4°C or with 75 ug/ml -VLDL for 24h at 37°C. The binding of -VLDL to SMC at 4°C was enhanced in the presence of macrophages irrespective of the phenotype expressed by SMC. This occurred through modification of the lipoprotein, since binding of re-isolated macrophage-conditioned -VLDL to SMC was 12.5 times that of fresh -VLDL. This modified form of -VLDL competed with fresh -VLDL for binding to SMC. Binding was inhibited in the presence of probucol, suggesting that an oxidative mechanism may be involved.The presence of macrophages also enhanced the accumulation of -VLDL-derived cholesterol in SMC. While most of this is a consequence of the enhanced binding, macrophages may also act directly on SMC to increase cholesterol accumulation, since the activity of acid cholesterol ester hydrolase and neutral cholesterol ester hydrolase in SMC was reduced in the presence of macrophages.     

Zur cytostatischen Wirkung von N-Methyl-N-β-chloräthyl-hydrazin und seinem Benzaldehydhydrazon

Zusammenfassung In N-Methyl-N--chloräthylhydrazin bzw. seinem Benzaldehydhydrazon liegen zwei neue cancerotoxische Methylhydrazine vor. Sie übetreffen in vitro und in vivo bei intraperitonealer Applikation die Hemmwirkung von Procarbazin auf das Ehrlich-Carcinom der Maus und das Yoshida-Sarkom der Ratte in der Ascitesform. Die etwas geringere Wirksamkeit des Hydrazons beim soliden Tumor und tumorferner Applikation läßt auf eine noch nicht optimale Transportform des N-Methyl-N--chloräthylhydrazins schließen. Das Hydrazon besitzt eine geringere Toxicität als Procarbazin.

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On the cytosic action of N-methyl-N--chloraethyl-hydrazine and its benzaldehydhydrazone

Summary N-Methyl-N--chloroethyl-hydrazine and its benzaldehydhydrazone are two new cytostatic methylhydrazines. Administered intraperitoneally, they are more effective in inhibiting the ascites tumor growth (Ehrlich's carcinoma in mice and Yoshida sarcoma in rats) than procarbazine in vitro as well as in vivo. The intraperitoneal administration of hydrazone shows a minor effect on the solid tumor. This may be explained by a different pharmacocinetical behaviour. Hydrazone is less toxic than procarbazine.

     

Diagnosis of the mucopolysaccharidoses using cultured skin fibroblasts and amniotic fluid cells

Assay of-L-iduronidase, heparin sulphamidase,N-acetyl--D-glucosaminidase, arylsulphatase B,-L-fucosidase,-glucuronidase,-galactosidase and-D-mannosidase in cultured cells is described. Activities in deficient fibroblast strains are compared to control fibroblast strains. The first case of Sanfilippo B in the United Kingdom is reported. A comparison of enzyme activities in cultured fibroblasts and amniotic fluid cells is made.     

Functional and morphological effects of interleukin-1β on the perfused rat pancreas

Summary We recently reported a potentiating effect of recombinant human interleukin-1 on glucose-stimulated insulin release from the isolated perfused pancreas. With the aim of determining whether the stimulatory effect of recombinant interleukin-1 on the B cell in the intact gland was modulated by varying the concentration, time of exposure to recombinant interleukin-1 or B-cell activity, and to elucidate a possible mechanism of action, we measured in the perfused rat pancreas the release of insulin, glucagon and/or prostaglandin E₂ according to the following three different protocols: (1) perfusion with 20 ng/ml of recombinant interleukin-1 for 92 min at 5 and 20 mmol/1 D-glucose (2) perfusion with varying concentrations of recombinant interleukin-1 ranging from 0.1×10^–3 ng/ml to 100 ng/ml at 5 and 20 mmol/l D-glucose (3) perfusion with 20 ng/ml of recombinant interleukin-1 at 5,11 or 20 mmol/l D-glucose. Furthermore, in a separate set of experiments we examined the influence of the cytokine on the morphology of the endocrine pancreas. Interleukin-1 stimulated insulin secretion at 11 and 20 mmol/l D-glucose and potentiated first as well as second phase insulin release in a dose-dependent fashion, with decreasing effect at higher concentrations. Glucagon secretion was also stimulated by recombinant interleukin-1, irrespective of increasing glucose (5, 11, 20 mmol/l) and insulin concentrations. The potentiating effect of recombinant interleukin-1 on insulin secretion was evident even after discontinued perfusion with the cytokine, suggesting a priming effect on B-cell function. Furthermore, we did not observe any relation between the recombinant interleukin-1 mediated insulin and glucagon release and prostaglandin E₂. Electron microscopy of the pancreata perfused with recombinant interleukin-1 revealed significant B cell and to a lesser extent A-cell lysis as well as induction of cell protrusions (blebs) in B cells only, accompanied by peripheral degranulation and rearrangement of rough endoplasmatic reticulum. We suggest that in addition to a paracrine effect of locally produced interleukin-1 systemic interleukin-1 may have an endocrine effect on A- and B-cell function and viability. Interleukin-1 should be considered to be a physiological modulator of insulin and glucagon secretion e.g. during the acute phase response, but also as a pathogenetic factor in Type 1 (insulin-dependent) diabetes mellitus.     

Detection of β-ureidopropionase deficiency with HPLC–electrospray tandem mass spectrometry and confirmation of the defect at the enzyme level

The pyrimidine bases uracil and thymine are degraded via the consecutive action of three enzymes to -alanine and -aminoisobutyric acid, respectively. To date, a number of patients have been described with a deficiency of dihydropyrimidine dehydrogenase and dihydropyrimidinase, the first two enzymes of the pyrimidine degradation pathway. In this study, we demonstrate that the first patient presenting with N-carbamyl--amino aciduria, due to a deficiency of -ureidopropionase, was easily diagnosed at the metabolite level using HPLC–tandem mass spectrometry. Urinary analysis showed strongly elevated levels of N-carbamyl--alanine and N-carbamyl--aminoisobutyric acid, with normal or moderately increased levels of the pyrimidine bases and the dihydropyrimidines, respectively. The deficiency of -ureidopropionase was confirmed by measuring all three enzymes of the pyrimidine degradation pathway. No activity of -ureidopropionase could be detected in a liver biopsy of the patient, while a normal activity of dihydropyrimidine dehydrogenase and dihydropyrimidinase was present. Thus, HPLC–tandem mass specrometry proved to be a powerful tool for the initial diagnosis of patients with deficiency of -ureidopropionase.     

The effects of parabiosis on serum and kidney glycosidase activities in spontaneously diabetic mice

Summary Spontaneously diabetic non-obese mice of the ICR strain were newly inbred in Shionogi laboratory, Japan. Animals became diabetic suddenly, more frequently and severely in females. Blood glucose levels were 452±73 mg/100 ml with serum insulin levels of < 1.0 U/ml in the fed state. Parabiosis with normal control ICR mice for 2 weeks decreased the blood glucose level to 260±51 mg/ 100ml (P<0.01) and resulted in serum insulin levels of 46.0±18.0 U/ml (P<0.01). Kidney homogenate -N-acetylglucosaminidase and -galactosidase activities were reduced in diabetic mice (42% and 44% decrease respectively) (P<0.025 and P<0.001), and restored almost to normal after 2 weeks of parabiosis. Renal -mannosidase activity was decreased 43% (P<0.001) in the diabetic mice but unaffected by parabiosis. Serum -N-acetylglucosaminidase, -galactosidase and -glucosidase activities were significantly increased in diabetic mice (179%; 233% and 58% increase respectively) (P<0.005, P<0.001 and P<0.001), and returned to normal with parabiosis.     

Rapid molecular characterization of mutations leading to unstable hemoglobin β-chain variants

Summary Characterization of unstable hemoglobins by protein analysis is often difficult. However, it is facilitated by DNA analysis, especially in the case of hyperunstable -chain variants, which produce a -thalassemia phenotype. We have applied an efficient strategy to the detection of such variants at the DNA level, based on computer-designed denaturing gradient gel electrophoresis (DGGE) of amplified DNA fragments. This approach makes it possible to detect any anomaly in the -globin gene. We describe the use of the DGGE method for rapid characterization of -chain variants and report a new missense mutation in the -globin gene third exon, 127 CAG-CGG/Gln-Arg, which is responsible for the synthesis of a highly unstable hemoglobin.     

Oligosaccharides accumulated in the bovineβ-mannosidosis kidney

Summary The phenotype of bovine-mannosidosis (-mannosidase deficiency), recently identified in Salers cattle, is similar to the caprine form of the disease (Abbittet al., 1991). This investigation was designed to characterize accumulated kidney oligosaccharides in bovine-mannosidosis. Oligosaccharides were extracted from the kidney of an affected Salers calf and purified by chromatographic techniques. The amount of accumulating oligosaccharides in 1 g of wet tissue was about 21µmol. Structures of derivatized oligosaccharides were characterized by high-performance liquid chromatography, mass spectrometry, methylation analysis and sequential exoglycosidase digestions. The major accumulating oligosaccharides were Man1-4GlcNAc and Man1-4GlcNAc1-4GlcNAc. Oligosaccharides accumulating in minor amounts were Man1-4GlcNAc1-4Man1-4GlcNAc, Man1-6Man1-4GlcNAc1-4GlcNAc and Man1-4GlcNAc1-4Man1-4GlcNAc1-4GlcNAc. As in caprine-mannosidosis, oligosaccharides with terminal-mannose residues and cleaved as well as uncleaved chitobiose linkages were identified in bovine-mannosidosis kidney. The accumulating oligosaccharides in tissue were thus identical in bovine and caprine-mannosidosis; however, the source of the novel oligosaccharides remains to be determined.     

Incidence of strains producing extended spectrum β-lactamases in Argentina

Summary The incidence of strains producing transferable -lactamases capable of hydrolyzing third generation cephalosporins or aminothiazole-oximino substituted monobactams in five Buenos Aires hospitals during a four month period was studied. These enzymes were categorized by 1) MIC 1 mg/l for third generation cephalosporins; 2) MIC < 0.06 mg/l for third generation cephalosporins combined with clavulanic acid or sulbactam; 3) sensitivity to imipenem or cephamycins (excluding permeability mutants); and 4) transferable resistance by conjugation. -lactamases hydrolyzing aminothiazole-oximino substituted monobactams were produced by 17.2% of Enterobacteriaceae from intensive care unit patients; 3.6% from inpatients of other units and 1.2% from outpatients. Producers were mainlyKlebsiella spp. (45/46) resistant to aminoglycosides (most of them AAC 3 — AAC 6 producers). Three strains had a an isoelectric point of 6.0, two of 6.5 and three of 7.7. TEM-1 -lactamase (isoelectric point 5.4) was detected in 6/8 strains. An inocolum effect was observed in 40/46 strains. AKlebsiella pneumoniae strain preserved since 1982 also produced a transferable -lactamase hydrolyzing aminothiazole-oximino substituted monobactams.

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Vorkommen von Bakterienstämmen mit erweitertem Breitspektrum -Laktamasen in Argentinien

Zusammenfassung Während einer Zeit von vier Monaten wurde an fünf Krankenhäusern in Buenos Aires nach dem Vorkommen von Stämmen mit Bildung übertragbarer -Laktamasen gesucht, die Cephalosporine der dritten Generation oder aminothiazol-oximino-substituierte Monobaktame hydrolysieren. Kriterien für die Selektion dieser Enzyme waren 1) MHK-Werte von 1 mg/l für Cephalosporine der dritten Generation; 2) MHK-Werte von < 0,06 mg/l für Drittgenerationscephalosporine in Kombination mit Clavulansäure oder Sulbactam; 3) Empfindlichkeit gegenüber Imipenem oder Cephamycinen; 4) Resistenz übertragbar. Zur Bildung von -Laktamasen mit hydrolytischer Aktivität gegen Aminothiazol-oximinosubstituierte Monobaktame waren 17,2% der Enterobacteriaceae-Isolate von Intensivpflegepatienten befähigt, 3,6% der Isolate von Patienten von anderen Stationen und 1,2% der Isolate von ambulanten Patienten. Die meisten der Stämme, die diese Enzyme bildeten (45/46), waren aminoglykosidresistenteKlebsiella spp. (Synthese von AAC 3-AAC 6). Der isoelektrische Punkt lag bei drei Enzymen bei 6,0, in zwei Fällen bei 6,5 und in drei Fällen bei 7,7. Sechs von acht Stämmen bildeten TEM-1 -Laktamase. Bei 40 der 46 Stämme wurde ein Inokulumeffekt festgestellt. Die Bildung übertragbarer -Laktamasen mit hydrolytischer Wirkung gegen Aminothiazolyl-oximino-substitutierte Monobaktame wurde in einem bereits seit 1982 gelagertenKlebsiella pneumoniae-Stamm nachgewiesen.

     

The antigenicity of human hemoglobins

Summary The correlation of the antigenicities among native hemoglobins and their subunit chains were investigated by the absorption of antisera and the combination of urea added immunoelectrophoresis with double diffusion. Alphachain showed identity with Hb-F but partial identity with -chain and Hb-A. Beta-chain showed identity with Hb-A but -chain and Hb-F showed partial identity with this chain. Gamma-chain showed identity only with Hb-F and its antigenicity was considered as being different from those of - or -chains.The lines of -, -and -chains were reconfirmed from the facts that the appearance of them depended always on the existence of anti-Hb-A or anti-Hb-F antibodies in the absorbed antisera and the minor component lines of

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Zusammenfassung Die Zusammenhänge der Antigenität zwischen nativen Hämoglobinen und deren Unterketten wurden mit der Absorption der Antiseren und der Kombination der Harnstoff-Immunelektrophorese und Doppeldiffusion untersucht. Die -Kette zeigte Identität mit Hb-F, aber nur partielle Identität mit der -Kette und Hb-A. Die -Kette war in ihrer Antigenität mit Hb-A identisch, die -Kette und Hb-F waren teilweise identisch mit der -Kette. Die -Kette zeigte die Identität mit Hb-F; es wird angenommen, daß ihre Antigenität verschieden von der -oder -Ketten ist.Für das Auftreten der Linien der -, - und -Ketten müssen Anti-Hb-A-oder Anti-Hb-F-Antikörper in den absorbierten Antiseren vorhanden sein, außerdem fusionieren die schwächeren Linien der Doppeldiffusion nicht mit irgendwelchen Linien der Unterketten. Auch gereinigte - oder -Ketten wurden zur Feststellung ihrer Linien benutzt.

     

Desensitization pattern of cardiac β-adrenoceptor subtypes by prolonged in vivo infusion of pindolol and celiprolol in rats

Summary The regulatory effects of pindolol and celiprolol on cardiac -adrenoceptor density were studied in vivo in order to assess the subtype selectivity of their partial agonistic activity (PAA). The substances were continuously administered to rats for 1 week by means of implanted osmotic minipumps. The density of -adrenoceptor subtypes were estimated from ICYP saturation binding experiments performed on cardiac ventricular plasma membranes in the presence of a highly selective antagonist (CGP 20172 A or ICI 118,551). Both antagonists were employed at concentrations as high as to block one subtype only without affecting the complementary subtype. For control purposes, rats were also treated with isoprenaline (0.4 mg/kg/h) and propranolol (0.15 mg/kg/h), or vehicle. Pindolol (0.036 mg/kg/h) and celiprolol (0.36 mg/kg/h) reduced the density of ventricular ₂-adrenoceptors by 46% and 23%, respectively, which — in the case of pindolol — was significant when compared to the non-treated controls. Both compounds, however, produced a small, but distinct increase in the number of ₁-adrenoceptors by approximately 26%. This finding is in contrast to the propranolol-induced upregulation of both ₁- and ₂-adrenoceptors by approximately 80%. Since supramaximal doses of each drug were administered, a significant smaller increase of ₁-adrenoceptors by pindolol and celiprolol —as compared to the increase produced by propranolol — can be interpreted as evidence for a PAA of pindolol and celiprolol on ₁-adrenoceptors as well. Isoprenaline as a full agonist caused a marked loss of of both -adrenoceptor subtypes. Although it exhibits equal affinity at both subtypes the decrease amounted to 80% of the ₂- but only to 54% of the ₁-adrenoceptors density. This indicates that the down-regulation of cardiac -adrenoceptors in general seems to be more pronounced at the ₂-than at the ₁-adrenoceptors population. We conclude that the subtype desensitization pattern of agents with intrinsic activity precludes the determination of subtype-selectivity, since ₁- and ₂-adrenoceptors appear to differ in their sensitivity presumably as a result of subtype specific baseline desensitization produced by endogenous catecholamines.