Inhibition of phagocytosis of polymorphonuclear leucocytes by adenosine and HoCl3 in vitro

Phagocytosis of polymorphonuclear leucocytes treated with NaF, HoCl3 and adenosine were studied. The highest concentration used was 25 mM of NaF, 25 mM of adenosine and 5 mM of HoCl3. It was ascertained that these substances, inhibitors of erythrocyte contractile protein, inhibit both phagocytosis and ability of polymorphonuclear leucocytes to change their shape. These unfavourable effects may be induced by the chemicals interfering with polymorphonuclear leucocytes contractile protein. NaF, HoCl3 and adenosine are also responsible for morphological changes in the cell nucleus.     

Regulation of 3-hydroxy-3-methylglutaryl-coenzyme A reductase activity in mouse uterine epithelial cells.

The regulation of 3-hydroxy-3-methylglutaryl-CoA reductase was studied in mouse uterine epithelium. The enzyme was rapidly inactivated during incubation with ATP/Mg2+ in vitro, and could be re-activated by incubation with partially purified rat liver phosphoprotein phosphatase. Enzyme activity was rapidly inhibited by mevalonate injection in vivo to approx. 30% of control. The percentage of total enzyme active in vivo was measured by inclusion of NaF in the isolation buffers. The percentage of enzyme active in vivo 18 h after stimulation by oestrogens remained at approx. 25% after inhibition of activity by mevalonate injection, cholesterol feeding or progesterone pretreatment. However, 9 h after oestrogen stimulation, cholesterol feeding inhibited enzyme activity to 57% of control, 94% of which was in the active form. We conclude that, although all components for a reversible phosphorylative regulation of 3-hydroxy-3-methylglutaryl-CoA reductase activity are present in uterine epithelial cells, a role in the rapid changes in epithelial enzyme activity has not been demonstrated.     

Differential effect of extracellular calcium on the Na(+)-K+ pump activity in intact polymorphonuclear leucocytes and erythrocytes

The effect of extracellular calcium on the Na(+)-K+ pump activity in human polymorphonuclear leucocytes and erythrocytes was studied and compared with the activity in mixed peritoneal leucocytes from rats. While there was maximal decrease in the pump activity (25-30%) of leucocytes from both rat and human by calcium 0.6 mM, a concentration of 0.1 mM caused a substantial decrease indicating a high sensitivity for extracellular calcium. In contrast, calcium had no effect on the pump activity in erythrocytes. The effect of calcium on the pump activity in leucocytes may be due to regulation of the influx of sodium across the plasma membrane, since in human leucocytes calcium had no effect on the pump activity if the cells were loaded with sodium.     

Substrate recognition of the membrane-associated sialidase NEU3 requires a hydrophobic aglycone

The human neuraminidases (NEU) consist of a family of four isoforms (NEU1-NEU4). Members of this enzyme family are proposed to have important roles in health and disease through regulation of the composition of cellular sialosides. The NEU3 isoform is a membrane-associated enzyme that cleaves glycolipid substrates. However, few reports have examined the substrate specificity of the enzyme for non-natural substrates. We report here a series of 11 synthetic trisaccharides that feature modifications of the aglycone or the Neu5Ac residue of an octyl β-sialyllactoside. The time course of substrate cleavage by NEU3 was monitored using an electrospray ionization mass spectrometry assay to obtain relative rates (k(rel)). We observed that NEU3 substrate activity was directly dependent upon the hydrophobicity of the aglycone but had no apparent requirement for features of the ceramide headgroup. We also observed that trisaccharides with incorporated azide groups in the Neu5Ac residue at either C9 or the N5-Ac position were substrates, and in the case of the N5-azidoacetyl derivative, the activity was superior to that of GM3. However, the incorporation of larger aryl groups was tolerated only at C9, but not at N5-Ac. We propose a two-site model for enzyme recognition, requiring interaction at both the Neu5Ac residue and the hydrophobic aglycone.     

Sex-linked hereditary thrombocytopenia with immunological defects.

14 cases of severe thrombocytopenia in one family are presented. Case histories, clinical examination, analyses of platelets, haemoglobin, reticulocytes, leucocytes, eosinophilocytes, differential counts of leucocytes, serum immunoglobulin IgA, IgM, IgG, IgE concentrations, complement fixing platelet antibodies, isohaemagglutinins, colour perception, determination of red cell and serum groups as well as HL-A types were obtained from a total of 59 members of the family. The in vitro blast transformation response of blood lymphocytes was studied in 6 patients and 45 relatives. The pattern of transmission of the disease was in full agreement with X-linked recessive inheritance. Investigation of the immune system revealed impaired responses to microbial antigens in the 6 patients so studied. All relatives examined had normal haematological status, whereas approximately half showed a subnormal response to one microbial extract. The low responders were evenly distributed within the family, and it was not possible to correlate low response and presumed carrier state.     

Leucocyte glucose-6-phosphate dehydrogenase (G-6-PD) activity in G-6-PD deficient Chinese

The activity of glucose-6-phosphate dehydrogenase (G-6-PD) in leucocytes was studied for erythrocyte G-6-PD deficiency using 49 hemizygous males, 16 heterozygous females, and 19 normal controls. The mean G-6-PD activity in leucocytes of the affected neonates (9.2 +/- 5.4 units) and the children (11.2 +/- 5.3 units) were significantly lower than those of normal newborns (22.9 +/- 5.1 units, P less than 0.01). Seventy percent of the effected newborns and 58% of the children with G-6-PD deficiency had the leucocyte enzyme activity of less than 13 IU/10(9)WBC. The leucocyte enzyme activity (14.6 +/- 8.6 units) of 16 heterozygous G-6-PD deficient mothers was also lower than that of normal controls (23.1 +/- 7.0 units). The present study thus concludes that, in G-6-PD deficient Chinese, the enzyme defect is demonstrable not only in erythrocytes but also in leucocytes.     

Isolation and characterization of cDNAs encoding wheat 3-hydroxy-3-methylglutaryl coenzyme A reductase.

The enzyme 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR, EC 1.1.1.34) is a key enzyme in the isoprenoid biosynthetic pathway. We have isolated partial cDNAs from wheat (Triticum aestivum) using the polymerase chain reaction. Comparison of deduced amino acid sequences of these cDNAs shows that they represent a small family of genes that share a high degree of sequence homology among themselves as well as among genes from other organisms including tomato, Arabidopsis, hamster, human, Drosophila, and yeast. Southern blot analysis reveals the presence of at least four genes. Our results concerning the tissue-specific expression as well as developmental regulation of these HMGR cDNAs highlight the important role of this enzyme in the growth and development of wheat.     

The effect of phagocytosis of Candida albicans blastospores on alkaline phosphatase levels in rat polymorphonuclear leucocytes

Summary The phagocytic function of young polymorphonuclear leucocytes with high levels of leucocyte alkaline phosphatase, which are present in the peripheral blood during an inflammatory response, was compared with that of normal polymorphonuclear leucocytes.Candida albicans blastospores were used as phagocytic targets. The phagocytic index of polymorphonuclear leucocytes with low levels of leucocyte alkaline phosphatase was higher than that of cells with high levels of enzyme.Monitoring of leucocyte alkaline phosphatase levels with increasing times of incubation of leucocytes with the blastospores showed a progressive decline in the level of the enzyme. Thus loss of the enzyme is linked to the phagocytic function, although the mechanism of this dynamic process is unclear.     

Action of cytochalasin E on polymorphonuclear leucocytes of guinea pig peritoneal exudates

Actions of cytochalasins on polymorphonuclear leucocytes of guinea pig peritoneal exudates have been studied. When the leucocytes were treated with cytochalasin E, complete disappearance of pseudopods was observed during a time, which would be related with the inhibition of particle ingestion. Intracellular granules migrated to the cell periphery in ectoplasma and were afterwards excluded from the cells. Later, formation of cytoplasmic protuberations with irregular shapes and variable diameters (“zeiosis”) and of large vacuoles were observed. Similar changes were also observed in monocytes. These morphological changes were accompanied with metabolic alterations which mimicked those observed during phagocytosis: appearance of cyanide-insensitive respiration, stimulation of hexose monophosphate oxidative pathway and release of superoxide anions and lysosomal enzymes into suspending medium. Other members of the cytochalasin family, cytochalasin C and D, similarly induced release of superoxide anions, whereas cytochalasin A and B had no such effect on leucocytes. Cytochalasin E seemed to have dual effects on leucocytes, namely, (1) inhibition of cytokinesis which is a common effect of the cytochalasin family; and (2) triggering of metabolic changes and degranulation which are characteristics of phagocytotic process.     

Dietary Modification of the Activation State of Intestinal 3-Hydroxy-3-methylglutaryl Coenzyme A (HMG-CoA) Reductase

To ascertain whether the phosphorylation-dephosphorylation reaction is actually involved in the in vivo regulation of intestinal 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, dietary modulation of the activation state of the enzyme was studied in isolated epithelial cells of rats. Substitution of a sucrose-enriched semipurified diet for the commercial non-purified diet caused a significant increase in jejunal activity with a concomitant decrease in ileal activity. Jejunal activity increased without influencing the activation state whereas at the early stage of dietary manipulation, there was a rapid decrease in apparent activity compared to total activity in the ileum, hence the reduction of the activation state. These observations favor the view that the phosphorylation (inactivation) reaction is responsible for the regulation of intestinal HMG-CoA reductase in vivo. In contrast, dietary fat-dependent stimulation of jejunal reductase activity was mainly attributable to an increase in enzyme protein rather than in the level of the activation. The results suggest a complex controlling feature of the cholesterol synthesis in the intestine.     

14-3-3 proteins--a role in the regulation of melatonin biosynthesis

14-3-3 proteins compose a large family of proteins that exist primarily as homo- and heterodimers within all eukaryotic cells. They are engaged in the regulation of numerous cellular processes, including melatonin biosynthesis. Melatonin, the hormone of darkness, is synthesized in a diurnal or circadian rhythm, with high levels at night. It has been demonstrated that cAMP levels and PKA activity in melatonin-synthesizing cells (pinealocytes and retinal photoreceptors) increase at night. PKA phosphorylates serotonin N-acetyltransferase (AANAT; the penultimate and key regulatory enzyme in melatonin biosynthesis pathway) at its N- (Thr31) and C-(Ser205)terminal region. Phosphorylated of AANAT bind to 14-3-3 proteins. The formation of pAANAT/14-3-3 complex stabilizes the enzyme and protects it against proteolytic destruction. Furthermore, this complex induces allosteric changes of the AANAT molecule resulting in an increase of the enzyme activity; this in turn enhances melatonin production by several fold. Exposure to light at night decreases intracellular cAMP level with concomitant dephosphorylation of pAANAT, its dissociation from 14-3-3 dimers, proteosomal proteolysis of free AANAT molecules, and finally turning off the melatonin production.     

Structural and biochemical characterization of the prenylated flavin mononucleotide-dependent indole-3-carboxylic acid decarboxylase

The ubiquitous UbiD family of reversible decarboxylases is implicated in a wide range of microbial processes and depends on the prenylated flavin mononucleotide cofactor for catalysis. However, only a handful of UbiD family members have been characterized in detail, and comparison between these has suggested considerable variability in enzyme dynamics and mechanism linked to substrate specificity. In this study, we provide structural and biochemical insights into the indole-3-carboxylic acid decarboxylase, representing an UbiD enzyme activity distinct from those previously studied. Structural insights from crystal structure determination combined with small-angle X-ray scattering measurements reveal that the enzyme likely undergoes an open-closed transition as a consequence of domain motion, an event that is likely coupled to catalysis. We also demonstrate that the indole-3-carboxylic acid decarboxylase can be coupled with carboxylic acid reductase to produce indole-3-carboxyaldehyde from indole + CO₂ under ambient conditions. These insights provide further evidence for a common mode of action in the widespread UbiD enzyme family.     

14-3-3蛋白在细胞周期调节中的作用

14-3-3是一个在真核细胞中广泛表达、功能复杂的蛋白家族,主要通过磷酸化依赖的方式与靶蛋白结合,从而发挥其调控作用。细胞周期的调节对维持基因组的稳定性至关重要。近年来的研究发现,14-3—3蛋白可以和越来越多的细胞周期调节蛋白相互作用,调节G2／M期和G1／S期转换,从而对细胞周期起调控作用。简要综述了14—3—3蛋白在细胞周期调节中的作用。     

Activation of guanylate cyclase and the regulatory role of cyclic 3',5'-guanosine monophosphate

The effect of dithiothreitol on the activity of soluble guanylate cyclase and on enzyme activation by sodium nitroprusside and free stable radical was studied. A higher degree of oxidation of guanylate cyclase from rat platelets in comparison with that of the enzyme from human platelets was found, which influences both the value of the enzyme activity and its regulation. It was shown that dithiothreitol enhanced the stimulating effect of nitroprusside but inhibited the activation of guanylate cyclase by free radical, which was suggestive of a difference in the mechanisms of the activating effect of these agents. A scheme of the biological role of cyclic 3',5'-guanosine monophosphate was proposed. On the basis of this scheme, different pathological states caused by disturbances in the functions of guanylate cyclase were identified and investigated.     

Phagocytosis and leucocyte enzymes in protein–calorie malnutrition

1. Enzymes pertinent to bactericidal activities of leucocytes were assayed in children suffering from protein-calorie malnutrition. 2. Leucocytes obtained from malnourished and control children contained similar activities for glucose 6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase. Granule-bound NADPH oxidase activity was low in leucocytes isolated from malnourished patients and failed to show the phagocytic stimulation which is normally seen in control leucocytes. Further, leucocytes obtained from malnourished patients did not release the acid phosphatase from lysosomes during phagocytosis, unlike those from controls. 3. Treatment of the malnourishment with a diet high in calories and protein resulted in significant increase in the activities of glucose 6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase and NADPH oxidase and in releasing the acid phosphatase from the lysosomes into the supernatant fraction during phagocytosis. 4. The significance of these enzyme changes are discussed in relation to the increased susceptibility of these patients to infection.     

白细胞酯酶同工酶对胃癌诊断价值的探讨

本文应用薄平板等电聚焦电泳法对15例未经手术治疗的胃癌患者进行了外周血白细胞酯酶同工酶的分离.结果:正常人外周血白细胞酯酶同工酶显示12条带,等电点(PI)为3.73～7.80.胃癌患者显示11条带,与正常相比第十二条带缺如,PI为3.80～7.90.同工酶各分带与正常相比,酶活性发生明显异常(P<0.01).提示;胃癌患者酯酶同工酶的改变可能是胃癌早期诊断的酶学指标.     

Evidence for polymorphonuclear-leucocyte-derived proteinases in arthritic cartilage.

1. An enzyme that degrades proteoglycan at neutral pH was extracted with 4 M-guanidine hydrochloride from the articular cartilage of rabbits with antigen-induced arthritis. 2. The enzyme had an apparent molecular weight on Ultrogel AcA 54 of about 8000 and was optimally active at pH 7.5 in Tris/HCl buffer containing 0.2 M-NaCl. The partially purified preparation was totally inhibited by 0.01 mM-N-acetyldialanylprolylvalylchloromethane, severely inhibited by 2 mM-phenylmethanesulphonyl fluoride and soya-bean trypsin inhibitor (200 microgram/ml) and slightly inhibited by 10 mM-EDTA. Marked inhibition was also obtained with a cytosolic fraction prepared from rabbit polymorphonuclear leucocytes. 3. All properties of the enzyme were virtually identical with those of an 'elastase-like' proteinase that was isolated from rabbit polymorphonuclear-leucocyte granules. 4. The results are consistent with the idea that cartilage proteoglycan degradation in acute joint inflammation is due at least partly to the diffusion into the cartilage of proteinases derived from synovial-fluid polymorphonuclear leucocytes.