Posttranslational modifications of α-tubulin of Toxoplasma gondii

The posttranslational modifications of -tubulin of Toxoplasma gondii were characterized by antibodies and biochemical analysis of the carboxy-terminal peptide. -Tubulin is acetylated and glutamylated. Side chains with up to three glutamate residues are linked to Glu445 of T. gondii -tubulin. The data suggest that the site of glutamylation on -tubulin is conserved over a broad range of species.     

Differential effects of interleukin-1α and β on the arachidonic acid cascade in human synovial cells and chondrocytes in culture

The effects of interleukin-1 and were tested on the [³H]-arachidonic acid release and the prostaglandin synthesis by human cultured synovial cells and chondrocytes. Both forms of interleukin-1 stimulated the arachidonic acid release but interleukin-1 was more potent than IL-1. Human synovial cells and chondrocytes synthesized three types of prostaglandins upon stimulation with interleukin-1 or : prostaglandin E₂, F₂ and 6-keto-prostaglandin F₁. Regarding the synthesis of these prostaglandins, IL-1 was again more potent than IL-1. A comparison between interleukin-1-stimulated synovial cells and chondrocytes revealed neither significant quantitative nor qualitative differences in both the arachidonic acid release and the prostaglandin synthesis.     

Distribution of renal integrin receptors and their ligands in congenital nephrotic syndrome of the finnish type

The aim of this study was to examine the distribution of 1 and v integrins (Ints) and some of their ligands in the kidneys of patients with congenital nephrotic syndrome of the Finnish type (CNF) and in controls using indirect immunofluorescence with monoclonal antibodies. The mesangial reactivity of Int 1 and Int 1 subunits was more variable and an increased glomerular reactivity with Int 3 and Int-6 antibodies was found in CNF kidneys than in controls. Int 2 subunit was either completely missing from or found in significantly lesser amounts in CNF kidney glomeruli. The immunoreactivity for Int v was more variable, fainter and also more granular in CNF samples than in control kidneys. The glomerular reactivity for Int 5 was more diffuse and weaker, and in sclerotic Bowman's capsules more intense in CNF kidneys than in controls. Immunoreactivity for Int 6 was restricted and was comparable in extent in CNF and control kidneys. Of the extracellular matrix components studied, the expression of EDAFn, EDBFn, OncFn, Ln 2 chain, Ln 1 chain and tenascin was increased. This is also seen in several glomerular diseases with inflammation and sclerosis. Immunoreactivity for vitronectin was decreased. Several differences were found in the intensity or location of the immunostaining for the 1 and v Ints and their ligands in CNF kidneys compared with controls, which have not been found in any other proteinuric disease. Disturbed Int expression pattern in CNF may specifically reflect the disturbance of glomerular function caused by the primary defect in this disease.     

Simultaneous determination of histamine andNα-Methylhistamine in biological samples by an improved enzymatic single isotope assay

A modified highly sensitive and specific radioenzymatic assay for the simultaneous determination of histamine (HA) andN-methylhistamine (N-MH) in tissues and body fluids is described. In the presence of the enzyme histamine-N-methyltransferase and of the methyl donor [³H]-S-adenosylmethionine, the transmethylation process was about seven times more effective for HA than forN-MH. In the same conditions only very low amounts ofN,N-dimethylhistamine (N,N-DMH) were converted into its 1-methylated derivative. The high degree of specificity attained by this method is due to the rapid quantitative extraction of the biological fluids, to the partially purified enzyme preparation and to the thin-layer chromatography system used which allows an excellent separation of the³H-1-methyl products of HA,N-MH andN,N-DMH. This method is highly sensitive for the assay of HA andN-MH (detection limit 10 and 50 picograms, respectively), but due to lack in sensitivity, it cannot be extended to the measurement ofN,N-DMH. The HA content of 20–30 samples can be determined in duplicate by one person in a working day. The concentrations of HA measured by this method in different biological samples (human whole blood, plasma, urine, gastric juice and skin biopsies) were in good agreement with the values reported in the literature. The presence of minute amounts ofN-MH in the human gastric juice was established by rigorous checking.Part of this paper was presented at the Thirteenth Meeting of the European Histamine Research Society, in Florence, Italy, May 16–19, 1984.     

Modulation of human polymorphonuclear neutrophil functions by α1acid glycoprotein

₁-Acid glycoprotein (₁-AGP), a naturally occurring human plasma protein and acute-phase reactant, was extracted by a two-step procedure from sera collected from four healthy men. Its activity was testedin vitro on human polymorphonuclear (PMN) functions (migration, aggregation, O ₂ ^– generation). ₁,-AGP was not chemoattractant but inhibited the PMN response to the chemoattractant formylmethionyl-leucyl-phenylalanine without affecting spontaneous migration (Boyden and agarose methods of assessment). At concentrations between 0.15 and 0.45 mg/ml, ₁AGP exerted an aggregating effect with a maximal effective concentration of 0.3 mg/ml. ₁-AGP inhibited superoxide generation by PMNs stimulated either by opsonized zymosan or phorbol myristate acetate. This inhibition varied according to the intensity of the stimulation. At low stimulus concentrations, a dose-dependent inhibition of membrane-associated PMN responsiveness to soluble or particulate stimuli was observed. These findings suggest that ₁-AGP may be able to prevent PMN activation in the course of inflammatory processesin vivo.     

Clinical and laboratory features of patients with an inherited deficiency of neutrophil membrane complement receptor type 3 (CR3) and the related membrane antigens LFA-1 and p150,95

Over the last 3 years a group of more than 20 patients has been described worldwide who have a similar history of recurrent bacterial infections and an inherited deficiency of three related leukocyte membrane surface antigens known as CR₃, LFA-1 (lymphocyte function-associated antigen type 1), and p150,95 (function unknown). These antigens share a common -chain structure linked noncovalently to one of three distinct -chain types. It is believed that the patients with this disease have a reduced or absent ability to synthesize the common subunit of the antigen family, resulting in absent or reduced expression of all three antigen family members on different leukocyte types. Neutrophils have a reduced phagocytic and respiratory burst response to bacteria and yeast as well as a reduced ability to adhere to various substrates and migrate into sites of infection.In vitro functional studies of normal neutrophils, monocytes, and lymphocytes treated with monoclonal antibodies to the individual and chains of these antigens suggest that most of the clinical features of the patients may be due to the neutrophil and monocyte deficiency of CR₃. Although natural killer-cell activity is diminished or absent, no immune deficiency of the patients' lymphocytes attributable to the absence of LFA-1 has been detected. Diagnosis of this disease has been facilitated by the commercial availability of monoclonal antibodies specific for the chains of CR₃ and p150,95.     

Serum interferon activity in inflammatory bowel disease

Serum interferon (IFN) of-class was studied in 64 consecutive patients, 26 with Crohn's disease, 38 with ulcerative colitis, and in 34 healthy volunteers. Detectable IFN- in 10 patients was associated with a moderate to severe activity of chronic inflammatory bowel disease (CIBD). However, 19 of 28 patients (68%) with activity in their disease did not have elevated IFN- levels. The three groups, ulcerative colitis, Crohn's disease, and healthy volunteers did not reveal any statistically significant difference in serum IFN-, as four of 34 healthy controls without intercurrent infections had elevated levels as well. Possible effects of, , and classes of IFN on endogenous arachidonic acid (AA) release and metabolism in human neutrophils was investigated in a substudy. IFN- caused a dose-dependent release of AA from phospholipids and metabolism of a modest fraction of leukotriene B₄ (LTB₄) and 5-hydroxyeicosatetraenoic acid (5-HETE) at doses reaching a maximum of 100 IU/ml. IFN of the and classes did not exert such effects. Addition of complement 5a to cells activated by IFN- caused induction of increased 5-li-poxygenase activity with unchanged release of AA. As only 16% of all CIBD patients had elevated IFN- levels as compared to 12% among the group of healthy volunteers, IFN- does not seem to be of importance for the perpetuation of the inflammatory reaction in ulcerative colitis or Crohn's disease, and other factors may therefore be responsible for activation of the inflammatory cells to production of LTB₄ and 5-HETE.     

Fetuin as a marker of cortical plate cells in the fetal cow neocortex: a comparison of the distribution of fetuin, alpha 2HS-glycoprotein, alpha-fetoprotein and albumin during early development

Summary Fetuin, ₂HS-glycoprotein (₂HS), -fetoprotein (AFP) and albumin have been shown to be present in some regions of the neocortex in two early stages of development of the cow brain using PAP immunocytochemistry. In the pre-cortical plate stage fibres of the primordial plexiform layer stained positively for fetuin. No staining was seen for albumin but plasma and cerebrospinal fluid (CSF) were positive for ₂HS and AFP. In the early cortical plate stage the strongest fetuin positive staining was seen in the earliest formed cells of the plate. ₂HS staining was much less intense but similar in distribution. The possible role of fetuin, or related glycoproteins, in cortical plate differentiation is discussed. Staining for AFP and for albumin was seen mainly in the ventricular zone and marginal zone fibres, and had a similar distribution and intensity for both proteins. Plasma and CSF stained for all four proteins. Tests showed some cross-reactivity between fetuin and anti-₂HS and, to a much lesser extent, between antisera to AFP and albumin and antigens denatured by fixation.     

Immunocytochemical characterization of porcine zona pellucida during follicular development

Sections of bovine ovaries fixed in Bouin's fluid or methanol-acetic acid and embedded in paraffin were incubated with chicken polyclonal antibodies to HPLC-purified zona glycoproteins ZP3 and ZP3. Oocytes of primordial follicles as well as of primary follicles showed weak labelling with anti-ZP3 and anti-ZP3. No immunostaining could be observed in the follicle cells. The ZP of primary follicles displayed distinct immunoreactivity for both ZP3 and ZP3. In secondary follicles, distinct labelling with anti-ZP3 and weak labelling with anti-ZP3 could be seen in the oocyte. The ZP showed immunoreactivity with antibodies to ZP3 and ZP3. Both antibodies labelled single follicle cells. In tertiary follicles, the oocytes were weakly labelled with anti-ZP3 and anti-ZP3. Some granulosa cells showed staining for ZP3 and ZP3. The ZP displayed strong immunoreactivity for ZP3 and ZP3. Cells of the corona radiata were strongly immunopositive for ZP3 and ZP3. Similar histotopography of immunoreactive cells could be seen in preovulatory follicles. The characteristic pattern observed for the distribution of ZP3 and ZP3 strongly suggests that in the porcine ovary both the oocyte and the follicle cells contribute to the synthesis of the ZP, perhaps in sequence.     

Mating-type suppression of the DNA-repair defect of the yeast rad6Δ mutation requires the activity of genes in the RAD52 epistasis group

The RAD6 gene of Saccharomyces cerevisiae is required for post-replication repair of UV-damaged DNA, UV mutagenesis, and sporulation. Here, we show that the radiation sensitivity of a MAT a rad6 strain can be suppressed by the MAT2 gene carried on a multicopy plasmid. The a1-2 suppression is specific to the RAD6 pathway, as mutations in genes required for nucleotide excision repair or for recombinational repair do not show such mating-type suppression. The a1-2 suppression of the rad6 mutation requires the activity of the RAD52 group of genes, suggesting that suppression occurs by channelling of post-replication gaps present in the rad6 mutant into the RAD52 recombinational repair pathway. The a1-2 repressor could mediate this suppression via an enhancement in the expression, or the activity, of recombination genes.     

Acute ethanol consumption synergizes with trauma to increase monocyte tumor necrosis factor α production late postinjury

The hypothesis that acute ethanol uptake plus trauma can synergize to increase immunosuppression was tested. We found that, unlike non-alcohol-exposed patients, patients with acute alcohol use prior to trauma have a transient decrease in monocyte tumor necrosis factor (TNF) production during the very early postinjury (0–3 days) period. However, TNF production by these alcoholexposed patients' monocytes (MØ) became hyperelevated late postinjury (>9 days). Consequently, these massively elevated MØ TNF levels can contribute to posttrauma immunosuppression after acute alcohol use. We also demonstrate that normal monocyte activation with the superantigen,Staphylococcus enterotoxin B (SEB), results in a preferential induction of cellassociated MØ TNF production, described as characteristic of immunosuppressed trauma patients. Acutein vitro ethanol treatment down-regulated the elevated TNF production by trauma patients' MØ after either SEB, muramyl-dipeptide (MDP), interferon- plus MDP, or lipopolysaccharide (LPS) stimulation. Both SEB- and LPS-induced TNF mRNA induction was inhibited by acute alcohol treatment in normal MØ, indicating that ethanol can regulate cytokine gene expression. An additional immunosuppressive effect of acute ethanol's stimulation was suggested by its induction of elevated transforming growth factor production in trauma patients' activated MØ.     

NADPH oxidase priming and p47phox phosphorylation in neutrophils from synovial fluid of patients with rheumatoid arthritis and spondylarthropathy

Superoxide anion (O2°-)production by neutrophil NADPH oxidase participates in arthritic joint lesion formation. Proinflammatory cytokines such as tumor necrosis factor (TNF), interleukin 8 (IL-8) and granulocyte/macrophage-colony stimulating factor (GM-CSF) have a priming effect on neutrophil NADPH oxidase activity. NADPH oxidase activation is dependent on phosphorylation of p47phox, a cytosolic component of the enzyme. We studied O2°-production and p47phox phosphorylation in synovial fluid (SF) from patients with rheumatoid arthritis (RA) and spondylarthropathy (SpA) according to TNF, IL-8 and GM-CSF levels. O2°-production by neutrophils isolated from SF of all the arthritis patients (RA and SpA) was higher than that of circulating resting neutrophils and when stimulated with fMLP or PMA. In addition, p47phox was partially phosphorylated in SF neutrophils compared to circulating neutrophils. High levels of TNF and IL-8 (but not GM-CSF) are detected in patient's SF (compared to circulating blood levels). TNF levels were significantly higher in RA than in SpA SF. These results suggest that increased NADPH oxidase activity could be involved in arthritic joint inflammation through increased p47phox phosphorylation. This could be the result of the presence of high levels of priming agents such as TNF and IL-8 but not GM-CSF.     

Binding of α-actinin to F-actin or to tropomyosin F-actin is a function of both α-actinin concentration and gel structure

Summary We have studied by electron microscopy as well as by measurements of low shear viscosity, rigidity and binding, the effect of-actinin on the gel formed at 37° C with F-actin and with tropomyosin-decorated F-actin. Contrary to previous reports in the literature,-actinin at nanomolar concentrations is an efficient actin gelling protein, even at 37° C, provided that the concentration of actin (or of tropomyosin-decorated F-actin) is low (1.2–2.4 m). The binding of-actinin to F-actin, as a function of actin concentration, is anomalous. The amount of bound-actinin increases when actin concentration increases from 0 to 1.2 m but does not change significantly when actin concentration is further increased up to 48 m. A similar result is obtained with tropomyosin-decorated F-actin.These observations can be explained by an hypothesis that binding is a function of the-actinin — F-actin association constant as well as of the rigidity of the gel. When the concentration of actin increases, the rigidity of the gel also increases and more work is required to bring two actin filaments to the reaction distance with-actinin and, consequently, a larger-actinin concentration is required to attain the same ratio of bound-actinin to actin monomers in the filaments.     

Enzyme secretion by the isolated rabbit pancreas: Absence of a relation with the Na−K activated ATPase

Summary The isolated rabbit pancreas secretes protein and -amylase at a relatively constant rate during an eight-hour period. Ouabain (10^–5 and 10^–6 M) did not alter enzyme secretion, but inhibited flow (65% and 28% respectively). Acetazolamide (10^–3 M) had no effect on pancreatic enzyme secretion, while flow was inhibited by 25%. Na azide (10^–2 M) failed to affect protein and -amylase secretion. Flow was inhibited by approximately 88%. NaF (10^–2M) increased both protein and -amylase secretion, while flow remained virtually unchanged. Gassing of the bathing fluid with 95% N₂–5% CO₂ reduced protein and -amylase secretion by 57 and 64% respectively, while flow was decreased by 77%. Lowering of the sodium concentration in the bathing fluid by 85% decreased -amylase secretion by 46%. Flow was inhibited by 77%. Return to the standard solution caused initially an increase of -amylase secretion (86%), followed by a decrease. These results strongly suggest that the enzyme secretion of the pancreas is not coupled to the sodium pump, responsible for fluid and electrolyte secretion.     

Down-regulation of tumor necrosis factor α activity by acute ethanol treatment in human peripheral blood monocytes

As the most commonly used drug that can modulate both metabolic and immune pathways, ethanol is evaluated in this report as a regulator of tumor necrosis factor (TNF) production in human peripheral blood monocytes (M) in combination with a variety of stimuli. While acute ethanol treatment did not induce TNF in M, it was a potent down-regulator of M TNF production whether induced by the combination of interferon- plus muramyl dipeptide (MDP) (P<0.001), lipopolysaccharide (LPS) alone (P<0.01), or interferon- plus LPS. Down-regulation of M TNF by ethanol was dose dependent and statistically significant in the biologically relevant, 25–150 mM, ethanol concentration range. We also demonstrate that these ethanol concentrations did not affect M viability. TNF down-regulation by ethanol was most effective when ethanol was administered 4 hr prior to MDP stimulation; however, it was also effective—though to a lesser extent—if it was added at the time of MDP stimulation. Furthermore, ethanol also down-regulated TNF production of thein vivo preactivated M of trauma patients, which produce hyperelevated levels of TNF. We have previously shown that the majority of posttrauma elevated M TNF is produced by the M subpopulation expressing high-affinity type I Fc receptors (FcRI). When the FcRI cross-linking-stimulated M subpopulation was treated with acute ethanol, TNF production was suppressed again both inin vivo preactivated M of trauma patients and in M of normal controls. In experiments utilizing cyclooxygenase inhibitor, we also demonstrate that ethanol has a direct, prostaglandin E₂-independent, effect on M TNF production. These results demonstrate that acute ethanol exposure has the potential to down-regulate M production of TNF significantly regardless of the TNF-inducing stimulus. Decreased capacity of M to produce TNF might, therefore, contribute to the immunological and metabolic abnormalities described after ethanol uptake.     

Effects of acetazolamide on medullary extracellular pH and{\text{P}}_{{\text{CO}}_{\text{2}} } and on ventilation in peripherally chemodenervated catsand on ventilation in peripherally chemodenervated cats

Cyclic guanosine monophosphate (GMP) productions by alpha rat atrial natriuretic peptide 1–28 (-rANP), carbamylcholine or sodium nitroprusside were assessed in isolated glomeruli microdissected from collagenase-treated kidneys of 2- to 34-day-old and adult rats. In both young and adult animals, -rANP-stimulated cyclic GMP generation was proportional to the number of glomeruli and was enhanced in a dose-dependent and saturable fashion with increasing -rANP concentrations. The apparent activation constant values were 6.4 nM for 5-day-old and 9.7 nM for adult rats. Maximal doses of either -rANP or rANP 5–28 elicited similar responses in young and adult animals. Clear differences appeared between the developmental patterns of cyclic GMP productions stimulated by either -rANP, carbamylcholine or sodium nitroprusside. The response to -rANP was very large in the youngest rats tested, declined sharply during the suckling period and represented about 1.6 times the adult control level in 34-day-old rats. In contrast, the response to carbamylcholine was low after birth and rose progressively with age up to the adult level at the end of the weaning period, and the response to nitroprusside seemed to be independent of the animal's age.     

Genetic characterization of an α-specific gene responsible for sexual agglutinability in Saccharomyces cerevisiae: mapping and gene dose effect

Summary A recessive ag1 mutation leads to specific defect in sexual agglutinability specifically in cells of the yeast Saccharomyces cerevisiae. The cryptopleurine resistance gene cry^R 1, closely linked to the mating type locus, was used to select / strains which emerged from / strains by mitotic nonreciprocal recombination, to genetically analyse ag1, since ag1 is expressed only in mating type. The ag1 gene was found to be linked to the centromere tightly, to met3 at 4.4 cM, and to ilv3 at 12 cM on chromosome X. Sexual agglutinability of cells was shown to be dependent on the dose of the AG1 gene, using / isogenic strains carrying AG1/AG1, AG1/ag1 or ag1/ag1. The sst2-1 mutation did not suppress the ag1 mutation. Based on these results, function of the AG1 gene is discussed.Abbreviations cM centimorgan - FDS first division segregation - NPD nonparental ditype - PD parental ditype - SDS second division segregation - TT tetratype     

Gene forα2(I) collagen is on mouse chromosome 6 not 16

Collagen is the most abundant protein in the animal world and a principal component, of the extracellular matrix of tissues. Type I collagen is composed of two ₁ chains and one 2 chain. The human 2(I) locus (COL1A2)has been assigned to human chromosome 7q21.3–q22.1. Here, we report the mapping of its murine counterpart Colla-2to mouse chromosome 6 (MMU6) by Southern blotting using somatic cell hybrids. This result disagrees with the previously reported mapping of Colla-2to MMU16 by immunochemical techniques. Our results are supported by comparative mapping data showing conserved homology between regions of human chromosome 7 and mouse chromosome 6.     

Clearances ofCandida albicans-derived α- and β-linked mannose residues in sera from patients with candidiasis

Two monoclonal antibodies (MAbs) reacting with differentCandida albicans mannan oligomannosidic epitopes were used to detect antigens in human sera. The first MAb reacted with -linked mannose residues shared by mannoproteins, and the second displayed reactivity against -1,2-linked oligomannosides shared by phospholipomannan. Two latex agglutination tests performed after dissociation of serum immune complexes were 100% specific. In a retrospective analysis of 39 sera from 20 patients with candidiasis, each test detected seven patients; in combination, they detected ten. More than 60% of positive samples were positive with only one test. These results demonstrate that the clearance ofCandida albicans antigens from the blood differs according to the type of oligomannoside and glycoconjugate. Antigen detection kits with MAbs to differentCandida albicans mannan epitopes could provide a logical rationale to compensate for the transitory character of mannanemia detection during candidal infections.     

Expression ofβ1 integrins in non-neoplastic mammary epithelium,fibroadenoma and carcinoma of the breast

1 Integrins were examined immunohisto-chemically in normal and mastopathic mammary glands, 12 benign tumours and 90 carcinomas of the breast using monoclonal antibodies against1 and1 to6 subunits. When compared with epithelial cells of non-neoplastic mammary glands and of benign tumours, carcinoma cells showed considerable quantitative changes in the pattern of2,3 and6 subunit expression. In contrast, the distribution pattern of1,1,4 and5 antigens corresponded to the situation observed in non-neoplastic mammary gland epithelium in most instances. An abnormal expression of2 was found in 71.0% of the carcinomas ranging from a remarkably low number of2-positive tumour cells in 27.5% of the cases to a complete absence of the2 molecule in 43.5% of the carcinomas. Of the carcinomas 39.9% exhibited quantitative changes in3 expression with an abnormally low content of3-positive neoplastic cells in 15.4% and a complete absence of this molecule in 24.5% of the cases. Expression of6 was abnormal in 73.2% of the carcinomas, consisting in a greater number of6-negative tumour cells in 31.9% and in a complete absence of6 in 41.3% of the tumours. The abnormally low expression/absence of2 and3 subunits correlated with oestrogen receptor negativity (P<0.033 andP<0.04, respectively). In addition, abnormally low expression/absence of2 correlated with poor differentiation of the tumours (P< 0.014). The quantitative changes in the expression pattern of1-associated subunits in breast carcinomas may cause a disturbed cell-cell and/or cell-matrix interaction that increases the invasive and migratory property of the tumour cells.