Comparison of disk diffusion,the E test,and detection ofmecA for determination of methicillin resistance in coagulase-negative staphylococci

The aim of this study was to find a reliable, fast, and simple alternative to the methicillin disk method for determination of methicillin resistance in coagulase-negative staphylococci, since results of this method are often difficult to read due to growth within the zone of inhibition. The sensitivity of 319 strains of coagulase-negative Staphylococci to a 5 g methicillin disk on Mueller-Hinton agar using an incubation period of 48 h was compared with that of 1 (1 g and 5 g oxacillin disks on Mueller-Hinton agar with or without 2% NaCl, using an incubation period of 24 h. The detection ofmecA (MecAgen) by the polymerase chain reaction was used as a standard. Minimum inhibitory concentrations were determined by means of the E test. Of the 225mecA-positive strains, 190, 215, and 193 were resistant to 5 g methicillin, 1 g oxacillin and 5 g oxacillin disks on Mueller-Hinton agar, respectively, and 216, 218, and 223 were resistant on Mueller-Hinton agar with 2% NaCl. Of the 94mecA-negative strains, 89, 93, and 94 were susceptible to 5 g methicillin, 1 g oxacillin, and 5 g oxacillin disks on Mueller-Hinton agar, respectively, and 92, 93, and 94 were susceptible on Mueller-Hinton agar with 2% NaCl. Using breakpoints of 2 g/ml for oxacillin resistance and 8 g/ml for methicillin resistance, the E test yielded sensitivities of 99.6 and 99.1% and specificities of 97.9 and 98.9% after 48 h of incubation. The 5 g oxacillin disk was faster and easier to read than the methicillin disk and correlated better with detection ofmecA than the methicillin disk or the 1 g oxacillin disk.     

Facilitation by 5-hydroxytryptamine of ATP-activated current in rat pheochromocytoma cells

The effects of 5-hydroxytryptamine (5-HT) on an inward current activated by extracellular ATP were investigated in rat pheochromocytoma PC12 cells. Under whole-cell voltage-clamp conditions 5-HT (10 M) reversibly enhanced the amplitude of the current activated by 30 M ATP. The enhancement may not be due to an increase in the number of functional channels because the current activated by 300 M ATP was not remarkably augmented compared with the current activated by 30 M ATP. The current enhancement by 100 M 5-HT was less obvious than that by 10 M 5-HT. When the current kinetics were compared, activation of the ATP-evoked current was accelerated to the same extent by either 10 or 100 M 5-HT, whereas deactivation was largely more accelerated by 100 M 5-HT. Propranolol (10 M), a 5-HT₁ receptor antagonist, or LY53857 (10 M), a 5-HT₂ receptor antagonist, exerted an agonistic effect: the ATP-activated current was facilitated by these compounds. Metoclopramide (10 M), a 5-HT₃ receptor antagonist, neither facilitated the ATP-activated current, nor blocked the current facilitation by 5-HT. Guanosine 5-O-(2-thiodiphosphate) (GDP[S]) (2 mM), the non-hydrolysable analog of guanosine 5-triphosphate (GTP), or K-252a (2 M), a protein kinase inhibitor, did not affect the facilitation by 5-HT of the ATP-activated current when they were included in the intracellular solution. The ATP-activated current pre-facilitated by 10 M dopamine was not enhanced by 10 M 5-HT. Similarly, the pre-facilitation by 5-HT attenuated the current enhancement by dopamine. The results suggest that 5-HT facilitates the ATP-activated channels through receptors that are not readily classified into conventional subclasses of 5-HT receptors. The reciprocal masking between the current facilitation by 5-HT and that by dopamine, combined with their sensitivities to the compounds involved in the intracellular solution, indicates that the facilitation by 5-HT may share not all, but some, common cellular mechanism with that by dopamine.     

In vitro efficacy and fungicidal activity of voriconazole againstAspergillus andFusarium species

Twenty-nineAspergillus isolates and 25Fusarium isolates underwent in vitro antifungal susceptibility testing by a broth macrodilution procedure adapted from the National Committee for Clinical Laboratory Standards guidelines. The MIC50s of both voriconazole and amphotericin B were 0.5 g/ml and 1 g/ml against species ofAspergillus andFusarium, respectively, while the MIC90s of both agents were 1 and 2 g/ml. Voriconazole was more active in vitro than amphotericin B: the geometric mean MICs of voriconazole and amphotericin B againstAspergillus spp. were 0.36 g/ml and 0.64 g/ml, respectively. Voriconazole also demonstrated fungicidal activity againstAspergillus spp., with 86% (24/29) of isolates exhibiting minimum lethal concentrations of 4 g/ml.     

Topical anti-inflammatory properties of flutrimazole,a new imidazole antifungal agent

The topical anti-inflammatory properties of flutrimazole, a new imidazole antifungal, have been evaluated. Flutrimazole inhibited mouse ear oedema induced by arachidonic acid, tetradecanoylphorbolacetate and dithranol, with IC₅₀ values of 3.32, 0.55 and 2.42 mols/ear, respectively. Ketoconazole showed similar potency in arachidonic acid and dithranol models (IC₅₀=3.76 and 2.41 mols/ear) whereas it was less active against tetradecanoylphorbol acetate (IC₅₀=1.96 mols/ear). The standard anti-inflammatory sodium diclofenac was overall slightly more potent than antifungals (IC₅₀=2.23, 0.57 and 0.57 mols/ear against arachidonic acid, tetradecanoylphorbol acetate and dithranol, respectively). Both 2% flutrimazole and 2% ketoconazole creams, applied topically, inhibited carrageenan-induced rat paw oedema by about 40%. Under the same conditions, 1% flutrimazole and diclofenac creams inhibited by 26 and 54%, respectively. Flutrimazole may work through the inhibition of 5-lipoxygenase, as it inhibited LTB₄ production by human granulocytes with an IC₅₀ value of 11 M (IC₅₀ value for ketoconazole was 17 M), whereas ram seminal vesicle cyclooxygenase was only inhibited by 16% at a concentration of 25 M. Drugs such as flutrimazole, with dual anti-inflammatory/antifungal activity, may be advantageous in the treatment of topical fungal infections with an inflammatory component.accepted by I. Ahnfelt-Rønne     

Effects of inhibitors and ion substitutions on oscillations of cell membrane potential in cells expressing the RAS oncogene

Previous studies revealed that in NIH fibroblasts expressing the ras oncogene but not in other NIH fibroblasts, bradykinin leads to sustained, calcium dependent oscillations of cell membrane potential by repetitive activation of calcium-sensitive K⁺ channels. The present study has been performed to test for ion and inhibitor sensitivity of these oscillations. Both, Lys-bradykinin (kallidin) and bradykinin, but not any shorter peptide tested, maintained the oscillations. The oscillations are abolished in the presence of the K⁺ channel blocker barium (10 nmol/l). The amplitude but not the frequency of the oscillations is dependent on the extracellular potassium concentration. The oscillations are not dependent on the presence of extracellular sodium, bicarbonate or chloride. The oscillations are abolished in the absence of extracellular calcium and their frequency is significantly decreased at reduced extracellular calcium (to 0.2 mmol/l). The oscillations are not inhibited by acute administration of ouabain (0.1 mmol/l), by dimethylamiloride (100 mol/l), furosemide (1 mmol/l) and hydrochlorothiazide (100 mol/l), by cobalt (100 mol/l), zinc (100 mol/l), gadolinium (100 mol/l), verapamil (10 mol/l) and diltiazem (10 mol/l), but are abolished in the presence of 100 mol/l lanthanum, 1 mmol/l cadmium, 10 mol/l nifedipine, 25 mol/l SK & F 96365 and 200 mol/l TMB-8. Stimulation of calcium entry by 10 mol/l ionomycin is frequently followed by oscillations of cell membrane potential even in the absence of bradykinin. In conclusion, in cells expressing the ras oncogene bradykinin leads to sustained activation of calcium channels at the cell membrane, which cause oscillations of the cell membrane potential by triggering intracellular calcium release.     

Relations between axons and oligodendroglial cells during initial myelination. II. The individual axon

Summary Axo-glial relations in the ventral funiculus of the spinal cord (SC) and in the corpus callosum (CC) of the cat were examined by electron microscopy during initial myelination. In addition to random transverse and longitudinal sections from several stages, two series of sections were studied. As a first step in myelination the axons become ensheathed by one to three uncompacted glial lamellae (E-sheaths). E-sheaths present a length range from <5 m to 149 m (SC) or to 93 m (CC). E-sheaths are more frequent along SC-axons than CC-axons, and the mean E-sheath is 3.3-fold longer in the former compared to the latter. In both areas naked axon portions occur between successive E-sheaths, but these gaps are insufficient to allow elongation of all short E-sheaths into long ones. Sheaths composed of mixed compacted (M-sheaths) and uncompacted segments have a length range of 66–212 m in the SC and 66–171 m in the CC. In relation to the undifferentiated terminations of E-sheaths or mixed E/M-sheaths, undercoated axolemmal domains are always lacking. Fully compacted sheaths were not found in the series from the SC. In the CC, 141–212 m long compact sheaths were found, with tight axoglial junctions at their terminations. Axolemmal domains with a nodal undercoating occur in relation to some of these terminations. In both areas, individual developing axons present a chaotic mixture of naked, ensheathed and myelinated portions; bulges with clusters of vesiculotubular profiles are frequent along naked and ensheathed axonal portions, particularly in the SC. The axon diameter is clearly larger in myelinated than in naked portions of the same axon. On the basis of these results, we propose that the early glial sheaths of developing CNS axons actively elongate and undergo extensive remodelling before compaction. The maximal length of uncompacted E-sheaths, and the sheath length at which axoglial junctions and nodes of Ranvier form, are markedly different in the two areas.     

Activity of trimethoprim/ sulfamethoxazole plus polymyxin B against multiresistantStenotrophomonas maltophilia

The inhibitory activity of eight antibiotics and the inhibitory and bactericidal activities of combinations of trimethoprim/sulfamethoxazole (TMP/SMX) plus three fixed concentrations of polymyxin B (0.01 g/ml, 0.1 g/ml and 0.5 g/ml) against 30 multiresistant strains ofStenotrophomonas maltophilia were tested. Polymyxin B at 0.01 g/ml modified the inhibitory activity of TMP/SMX against only 40% of strains. At 0.1 g/ml and 0.5 g/ml, polymyxin B enhanced the inhibitory activity of TMP/SMX activity against all strains. Polymyxin B enhanced the bactericidal activity of TMP/SMX only at concentrations near the minimum inhibitory concentration of polymyxin B alone.     

Provisional quality control parameters and interpretive criteria for testing susceptibility ofStreptococcus pneumoniae andHaemophilus influenzae to quinupristin/dalfopristin (RP59500)

Studies were undertaken to select tentative criteria for susceptibility testing of quinupristin/dalfopristin againstStreptococcus pneumoniae andHaemophilus influenzae. Against 612 isolates ofStreptococcus pneumoniae, MICs of quinupristin/dalfopristin were 1.0 g/ml for all but one strain. With a tentative MIC breakpoint of either 1.0 g/ml or 2.0 g/ml for susceptible, a disk diffusion zone diameter breakpoint of 19 mm embraced all but two of the susceptible pneumococci; 16 mm included all strains. ForHaemophilus influenzae, MICs of quinupristin/dalfopristin clustered near the tentative breakpoints; 91.5% of the MICs were 2.0 to 8.0 g/ml. This precluded satisfactory performance of the disk diffusion test in discriminating between resistant and susceptible isolates unless MIC breakpoints are modified for this species: clinical experience will be needed before that can be justified. Based on data from a multilaboratory study, the following quality control limits are proposed forStreptococcus pneumoniae ATCC 49619 when testing quinupristin/dalfopristin: 0.25 to 1.0 g/ml for broth microdilution tests and 19 to 24 mm for disk diffusion tests. For tests ofHaemophilus influenzae ATCC 29247, MIC limits are 2.0 to 16 g/ml; disk tests were very reproducible but are not yet recommended.     

Regulation of interleukin-1β and tumor necrosis factor secretion by the human monocytic leukemia cell line,THP-1

Activated macrophages synthesize and release the potent polypeptides, interleukin-1 (IL-1) and tumor necrosis factor (TNF). In an effort to identify the cellular signals which control cytokine production by activated macrophages, we have developed anin vitro model employing the human THP-1 cell line. In the present study, THP-1 cells primed by 1.6 M phorbol 12-myristate-13-acetate (TPA) for 4 hr demonstrated a dose-and time-dependent release of IL-1 and TNF upon activation by 20 g/ml LPS. BSA/anti-BSA-coated latex beads were also a potent stimulus for IL-1 secretion. Moreover, the combination of a suboptimal concentration of LPS (200 ng/ml) plus interferon- (0.03–333 U/ml) greatly enhanced IL-1 production. Resting THP-1 monocytes not primed by TPA did not secrete IL-1 or TNF. These distinct patterns of cytokine production may be related to the developmental stages of macrophage activation.     

Papillary cystic tumours of the pancreas: An analysis by nuclear morphometry

Papillary cystic tumour (PCT) is a rare, low-grade malignant pancreatic neoplasm, in which the histological criteria for malignancy are still uncertain. We performed a histological examination of 3 metastasizing PCTs, while comparing them with 18 non-metastasizing PCTs, using a computed image analyser. The mean maximum nuclear diameter, the mean standard deviation (SD) of the nuclear diameter, the mean nuclear area and the nuclear-nonnuclear (N/NN) ratio obtained by the image analyser of the metastasizing PCTs (7.23 m, 2.21 m, 30.45 m², 36.41%) were all significantly larger than those of the non-metastasizing PCT (6.34 m, 1.59 m, 23.66 m², 23.74%;P<0.005,P< 0.005,P<0.005,P<0.001 respectively). However, there were no statistical differences in either the nuclear ellipsoidity or nuclear regularity. These results suggested that nuclear morphometry might be a useful parameter to define metastatic potential, in addition to histological variables such as venous invasion, nuclear grade and mitotic rate.     

Treatment of fish parasites

For chemotherapy of fish parasitized by monogeneans, a novel triazine derivative, 2-[3,5--dichloro-4-(4-methyl-sulfonylphenoxy)-phenyl]-1-methyl-hexahydro-1,2,4-triazine-3,5-dion (HOE 092 V), was tested in vivo against the gill- and skin-parasitizing speciesDactylogyrus extensus, D. vastator, andGyrodactylus arcuatus. Naturally infected fish were incubated in aerated, separate tanks at 22°C for 1,2,3, and 4 h in water containing 0, 1, 5, 10, or 15 g HOE 092 V/ml, whereasPseudodactylogyrus bini was tested in vitro at 10 g HOE 092 V/ml for 2.25 h. As seen by means of transmission electron microscopy, in vivo treatment againstD. extensus caused vacuolization and lysis of the parasite's tegument at a dose as low as 1 g/ml over a 3-h exposure period. Higher doses, such as 5 and 10 g/ml over the same exposure period, produced lesions in the circular and longitudinal musculature ofD. extensus and differing degrees of damage to the ciliary cells of protonephridia and immature vitelline cells. There was 100% mortality inD. vastator when incubation was done with 10 g HOE 092 V/ml for 4 h (G. arcuatus: 5 g/ml for 4 h; 10 g/ml for 1 h) and inP. bini after 2.25 h in vitro exposure. In all species tested, the anterior portion and the opisthaptor region were most sensitive to the drug action. This study shows that fish infected withGyrodactylus spp.,Dactylogyrus spp., and/orPseudodactylogyrus spp. can be treated successfully in a water bath containing 10 g HOE 092 V/ml.Abbreviations BL basal lamina - BM basal membrane - C caverna - CC canal cell - CI cilium - CIT cilium destroyed by treatment - CM circular musculature - CT connective tissue - DB dense body - EI electron-dense inclusion - EP electron-dense particles marking the boundary of holes - HC heterochromatin - I invagination of the tegument - IC immature vitelline cell - L lumen - LD lipid droplet - LM longitudinal musculature - LV lyzed immature vitelline cell - M mitochondrion - MC mature vitelline cell - MF myosin filament - MFI muscle fiber - MS membrane stack - N nucleus - NM nuclear membrane - OC outer membrane of cilium - OM outer membrane - P parenchyma - R ribosome - V vesicle - VA vacuole - VD vitellary droplet - VF vitelline follicle - YG Yolk globule - YL content of yolk globule     

Effects of verapamil on excitation-contraction coupling in single crab muscle fibers

Summary The effects of verapamil and its optical isomers on the electrical and mechanical characteristics of single muscle fibers ofCallinectes danae were studied. Verapamil (10–20 g/ml) blocked the procaine-and TEA-induced spikes; the blockade was preceded by reduction in the rate of rise of the up-stroke and increase in the duration of the action potentials. Inhibition of Ba-spikes required higher concentrations of verapamil (>50 g/ml). These concentrations reduced the amplitude of the normally occurring graded electrogenic membrane responses and reduced the rate of development of the current-induced tensions. With lower concentrations (10–30 g/ml) verapamil enhanced the negative afterpotentials and the peak amplitude of the local contractions elicited by depolarizing current pulses, while the graded membrane responses were not markedly modified. Verapamil (1–100 g/ml) did not affect the resting membrane potential but increased the effective membrane resistance. Determination of the cable characteristics by DC pulses indicated that verapamil (1–10 g/ml) shortens the membrane length constant, increases the specific resistivity of the sarcoplasm and, in most cases, increases the membrane time constant. Verapamil (10 g/ml) induced tension in these crab fibers. The contractions were potentiated in Na-deficient media, by increase in [Ca]₀, and by membrane depolarization; Ca-free salines depressed, and procaine abolished these contractions. The results suggest that verapamil affects both Ca and K conductances and interferes with the Ca-sequestering mechanisms of these fibers. The (–)-isomer of verapamil was more effective than the (+)-isomer with respect to tension development, prolongation and subsequent blockade of procainespikes and enhancement of current-induced after-potentials and contractions.This work was supported in part with grants from CNP_q (TC-16.897) and from CEPG-UFRJ     

Der basale Noradrenalinspiegel im peripheren ven?sen Blut des Menschen

Zusammenfassung 1. Mit einer in den wesentlichen Punkten auf das Verfahren von Häggendal zurückführenden Technik wurden die Katecholamine, insbesondere der Noradrenalinspiegel, im venösen Armvenenblut bei kreislaufgesunden Probanden zwischen 20 und 68 Jahren unter Ruhebedingungen untersucht.2. Die Noradrenalin-Konzentration betrug im Mittel aus 95 Einzeluntersuchungen 0,282 g/l Plasma (Häggendal 0,300 g), die Adrenalin-Konzentration war 0,056 g/l (Häggendal 0). Während die Kreislaufgrößen (Mitteldruck und Herzfrequenz)nach 20 min Ruhe und 10 min nach dem Einsetzen der Kanüle schon praktisch basale Werte erreichten, ist dies beim venösen Noradrenalin-Spiegel erst ca. 20 min nach dem letzten vorausgegangenen Stress der Fall.3. Die Aufgliederung des Materials nach dem Alter der Probanden zeigt eine ansteigende Tendenz der Noradrenalinkonzentration mit zunehmenden Jahren.4. Die Aufgliederung von insgesamt 238 Proben aus 95 Versuchen nach dem Mitteldruck und der Herzfrequenz zeigt jeweils eine signifikante positive Korrelation sowohl zwischen Druck als auch Frequenz und NA-Gehalt des venösen Blutes.5. Der mittlere NA-Spiegel von 16 weiblichen Probanden lag mit 0,250 g etwas unter dem Gesamtdurchschnitt, sowohl nach der Alters- als auch nach der Druck-Aufgliederung; keine Signifikanz.

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Summary 1. Catecholamines, especially noradrenaline, are determined in the cubital vein blood of normal individuals aged between 20 and 68. A slightly modified Häggendal-procedure is used.2. The average concentration of noradrenaline is 0.282 g/l plasma (Häggendal 0.3 g), the concentration of adrenaline 0.056 g/l (Häggendal 0). Blood pressure and heart rate are back to normal 10 min after the insertion of the cannula, the noradrenaline level, however, only 20 min after the last antecedent stress.3. A classification of the results according to age shows increasing noradrenaline levels with increasing age.4. A classification according to average blood pressure and heart rate shows significantly positive correlations between pressure, heart rate, and noradrenaline content of the venous blood.5. The average noradrenaline concentration in 16 females is 0.25 g/l plasma as compared to 0.28 g total average. The difference is not regarded as significant.

     

2 μm plasmid copy number in different yeast strains and repartition of endogenous and 2 μm chimeric plasmids in transformed strains

By using the renaturation kinetics technique we tried to get informations about the maintenance of the 2 m plasmid in yeast cells. For this purpose we determined the 2 m plasmid copy number: in various yeast strains, in a special set of mutants, in cells treated with ethidium bromide and cycloheximide and in different yeast strains obtained by transformation with 2 m chimeric plasmids.According to the strain used the proportion of 2m DNA varied from 1.1% to 3.9%, which corresponds to 24 to 88 2 m molecules per haploid genome. The particular multiresistant mutant, where the frequent loss of oligomycine resistance is correlated with the loss of extractible covalently closed circular DNA, contained 39 2 m copies per haploid genome. In the partial revertant oligomycine sensitive all the 2 m DNA sequences were lost. (Less than 0.1 copy per haploid genome.)Ethidium bromide did not affect the 2 m copy number while cycloheximide induces an increase of 36%.When a strain containing 88 2 m DNA copies per haploid genome is transformed with 2 m chimeric plasmids there is no significative change in the total number of plasmid: 36 copies of endogenous and 44 of chimeric plasmid per haploid genome. When 2 m chimeric plasmids were introduced in our 2 m-less strain despite the stability of the transformants, there is only 8 copies per haploid genome.     

A paramyxovirus-like virus isolated from pemphigus-disease in man

Summary Electron optical studies of an infectious agent isolated from individuals suffering frompemphigus vulgaris, dermatitis herpetiformis Duhring andepidermolysis bullosa revealed a paramyxovirus-like virus. The pleomorphic but approximately spherical particles showed a mean diameter of 150 to 200 m, however, also extreme values of 80 and 500 m could be measured. Two main features, an outer envelope covered with clubshaped, 11 m long projections and an internal helical component consisting of elipsoidal subunits were readily observable. For the helical component an overall diameter of 18 m, an inner diameter of 4.5–6 m and a pitch height of 5–6 m was estimated.After centrifugation in CsCl-density-gradients peak-infectivity-titres of Freon-purified virus were found in fractions with a medium density of 1.285 g/ml.     

Inhibitory effects of E-5110 on interleukin-1 generation from human monocytes

Effects of E-5110, a novel non-steroidal antiinflammatory drug, on interleukin-1 (IL-1) generation from human monocytes were studiedin vitro. E-5110 reduced the amounts of extra- and intracellular IL-1 activity induced by lipopolysaccharide (LPS, 1 g/ml) in a dose-dependent manner (1–10M). E-5110 also inhibited the IL-1 generation induced by antigen-antibody complexes, opsonized zymosan and silica particles. It was suggested that the inhibition of IL-1 generation by E-5110 was independent of the inhibitory effects on arachidonate cyclooxygenase and/or lipoxygenase because indomethacin, piroxicam, BW755C and AA861 had no effects on IL-1 generation. Hydrocortisone (IC₅₀:0.084 M), aurothioglucose (11.5 M) and lobenzarit (75.0 M), which are clinically effective antirheumatic drugs, also inhibited IL-1 generation, like E-5110 (1.21 M). It is expected that E-5110 will be superior to classical non-steroidal antiinflammatory drugs in medical treatment of rheumatoid arthritis.     

Intrinsic cholinergic excitation in the rat neostriatum: Nicotinic and muscarinic receptors

Summary An in vitro slice technique was employed to study the receptors involved in intrinsic cholinergic excitation in the rat neostriatum. The locally evoked synaptic potentials were suppressed by antinicotinic agents, mecamylamine (10 M), d-tubocurarine (3 M) or hexamethonium (100 M), but not by the antimuscarinic agent atropine (100 M). If the slices were exposed to an acetylcholinesterase (AChE)-inhibitor (paraoxon 1–20 M, physostigmine 0.1–0.5 M), the synaptic potentials were potentiated. The amplitude of the orthodromic population spike increased, and it was further facilitated when the stimulus frequencies were raised from 1–3 Hz to 10–30 Hz. The frequency facilitation following exposure to an AChE-inhibitor was blocked by atropine (1–100 M). Intracellular recording indicated that a slow depolarizing potential caused the frequency potentiation of the orthodromic discharges. Apparently rat neostriatum is similar to cholinergic systems in sympathetic ganglia and spinal Renshaw cells, in that nicotinic receptors mediate fast excitation and muscarinic receptors mediate slow excitation.     

Aspects of the purine nucleoside phosphorylase (PNP) deficient state produced in normal rats following oral administration of 8-amino-9-(2-thienylmethyl)guanine (PD 119,229), a novel inhibitor of PNP

8-Amino-9-(2-thienylmethyl)guanine (PD 119,229; 2,8-diamino-1,9-dihydro-9-(2-thienylmethyl)-6H-purine-6-one monohydrochloride) is a potent inhibitor of human purine nucleoside phosphorylase (PNP). The effects of orally administered PD 119,229 on the plasma concentration of the PNP substrates, inosine and guanosine, were determined using normal rats. In time course studies following administration of a single 3 mg/kg dose of PD 119,229, both inosine and guanosine were statistically significantly elevated as soon as one hr postdose. Plasma inosine elevation was maximal ten hr after dosing, reaching a mean of 2.13 M (14.2-fold vehicle). Guanosine was maximally elevated at three hr following a single 3 mg/kg oral dose, reaching a mean of 0.77 M (4.5-fold vehicle). In dose-response studies in which blood specimens were obtained one hr following oral administration of PD 119,229 at doses of 1.5 to 50 mg/kg, maximal mean inosine elevation (1.71 M or 57-fold vehicle) occurred at 15 mg/kg, with a plateauing or decline in inosine concentration noted at higher doses. The maximal mean plasma guanosine concentration was achieved at 50 mg/kg (mean of 0.2 M, or 6.7-fold vehicle). Substantially greater nucleoside elevation was not observed following multiple oral 15 mg/kg doses, nor when nucleoside levels were assessed at two to four hr following a large oral dose. However, inosine levels reaching 15 M, and guanosine concentrations approaching 2 M, were occasionally noted in individual rats. It is concluded that oral administration of PD 119,229 can simulate, in normal rats, one of the more readily detectable biochemical abnormalities of the PNP deficient state in humans.     

A light and electron microscopic study of the intraneuronal transport of horseradish peroxidase and wheat germ agglutinin-peroxidase conjugates in the rat visual system

Summary The ability of horseradish peroxidase (HRP) and the lectin wheat germ agglutinin (WGA) covalently conjugated with HRP to label retrogradely dorsal lateral geniculate nucleus (dLGN) neurons, subsequent to injections of either marker into rat striate cortex, was assessed by counting labelled neurons in the dLGN. Rats injected with either marker in concentrations ranging from 0.1 to 100g/l of HRP either free or coupled to WGA were perfused 24 h later and their brains incubated using the chromagen tetramethyl benzidine. At high concentrations (2–100g/1), comparable numbers of labelled neurons were observed in the dLGN but at low concentrations (0.1–1.0g/1), WGA-HRP labelled 2–5 times more dLGN neurons than did unconjugated HRP. The sugarN-acetylglucosamine, and free WGA added in excess to WGA-HRP, abolished the retrograde labelling of dLGN neurons.In additional rats, which received striate cortex injections of 100g/1 of either free HRP or HRP coupled to WGA, the injection site was studied with electron microscopy after survivals of 30 min to 24 h. Similar organelles in neuronal perikarya, dendrites and axons were labelled by both markers, with the exception that only rats injected with WGA-HRP had labelled GERL in some of their neurons in striate cortex.It was concluded from these studies that: (1) WGA-HRP is a more sensitive retrograde marker than free HRP at low concentrations in the rat visual system; (2) WGA-HRP binds specifically to moieties with terminalN-acetylglucosamine; and (3) WGA-HRP, but not free HRP, is localized to neuronal GERL of striate cortex subsequent to endocytosis.