Characterization of precursor and secreted forms of human angiotensinogen.

To define the basis of the heterogeneity of angiotensinogen, we have characterized the immunoreactivity of high molecular weight (HMW) and low molecular weight (LMW) plasma angiotensinogen, the angiotensinogen precursor synthesized by cell-free translation, and angiotensinogen secreted by human hepatoma (Hep G2) cells. Angiotensinogen precursor synthesized by rabbit reticulocyte lysate primed with RNA prepared from liver or Hep G2 cells was compared with angiotensinogen secreted by Hep G2 cells by using immunoprecipitation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). So as to assess the contribution of N-glycosylation of angiotensinogen, Hep G2 cells were incubated in the presence of tunicamycin. Glycosylation of secreted angiotensinogen was further characterized by using chromatography on concanavalin A-Sepharose, digestion with neuraminidase, and treatment with trifluoromethane sulfonic acid. In Sephadex G-200 column chromatography, HMW plasma angiotensinogen eluted just after the column void volume and was clearly separated from LMW angiotensinogen which eluted just before bovine serum albumin. Both HMW and LMW plasma angiotensinogen were shown to bind to monoclonal and polyclonal antibodies raised against pure LMW angiotensinogen. Only one angiotensinogen precursor (mol wt 50,000) was identified by cell-free translation which, after cleavage by renin, was reduced to mol wt 45,600. Angiotensinogen secreted by Hep G2 cells showed electrophoretic heterogeneity (mol wt 53,100-65,400). Tunicamycin-treated Hep G2 cells secreted five discrete forms of angiotensinogen, a predominant form of mol wt 46,200, with other forms (mol wt 46,800, 48,100, 49,200, and 49,600) representing 10% of secreted angiotensinogen. All five forms showed a similar reduction in molecular weight after cleavage by renin. The predominant 46,200-mol wt protein represented nonglycosylated angiotensinogen in that, after cleavage by renin, it had an electrophoretic mobility (mol wt 45,600) identical to the desangiotensin I-angiotensinogen resulting from renin cleavage of the angiotensinogen precursor. The other higher molecular weight forms of angiotensinogen secreted by tunicamycin-treated Hep G2 cells were shown to represent O-glycosylated angiotensinogen in that they were reduced to 46,200 mol wt by treatment with trifluoromethane sulfonic acid. Dexamethasone (10(-7) and 10(-6)M) stimulated angiotensinogen secretion by Hep G2 cells two- to fourfold, both in the absence and presence of tunicamycin. However, a small stimulatory effect of mestranol (10(-7) M) was evident only in the presence of tunicamycin. Neither dexamethasone nor mestranol influenced the electrophoretic pattern (SDS-PAGE) of angiotensinogen secreted by Hep G2 cells. However, when incubation media were chromatographed on Sephadex G-200 with subsequent immunoprecipitation of the column fractions, both dexamethasone and mestranol were shown to stimulate the secretion of HMW angiotensinogen (eluting just after the column void volume) which, on SDS-PAGE, migrated in a position identical to LMW angiotensinogen. From these studies, we conclude that all forms of human angiotensinogen are derived from a single precursor. The heterogeneity of secreted angiotensinogen represents differences in posttranslational processing of angiotensinogen. This processing includes both N- and O-glycosylation, and also the formation of HMW complexes (HMW angiotensinogen) through association either with other angiotensinogen molecules or with some other protein(s) whose secretion by hepatocytes is stimulated by glucocorticoids and estrogens.     

Sensitive and specific enzymatic assay for the determination of precursor forms of prostate-specific antigen after an activation step

BACKGROUND: The proteome of the serine protease prostate-specific antigen (PSA) and its enzymatic properties have been clarified only recently. We have developed a specific and sensitive method for the measurement of active PSA and used it to measure proPSA in blood. METHODS: We used the synthetic peptide KGISSQY, which possesses a PSA-specific cleavage site, as substrate. To ascertain the specificity of the assay, we used an anti-PSA monoclonal antibody that captures known forms of PSA. An activation step enabled us to measure proPSA by converting it to mature, active PSA. RESULTS: The detection limit of the optimized assay was 0.5 microg/L. In blood samples from patients, the activation step substantially increased the concentration of active PSA, thus showing the presence of proPSA in the samples. ProPSA was 0-79% (median, 45%) of the amount of free PSA in 15 samples with total PSA concentrations of 5.3-423 microg/L. In samples obtained from three benign prostatic hyperplasia (BPH) patients after transurethal resection of the prostate, no significant increase in activity was detected after the activation step, thus showing that proPSA was not a portion of free PSA in plasma of BPH patients. CONCLUSIONS: Proforms of PSA are a considerable fraction of free PSA in the blood of patients with increased total PSA. The approach described can be used to study the diagnostic value of proPSA and active PSA in patients with BPH and prostate cancer.     

T-Lymphocyte precursors. I. Synergy between precursor and mature T lymphocytes in the response to concanavalin A

When mouse bone marrow cells are mixed with cortisol-resistant thymocytes and stimulated in vitro with concanavalin A, the mitogenic response observed is much greater than additive, that is, it is synergistic. Between 94 and 96% of responding cells could be identified as T cells (Thy-1 positive) and of these, 79-100% derived from the cortisol-resistant thymocyte population, not from the bone marrow. Purified macrophages could not replace bone marrow; and marrow depleted of mature T or B cells worked as well as normal marrow. Thus, T and B cells and macrophages were ruled out as the synergizing cell of bone marrow. Nude spleen contained 10 times as many precursors of T cells as did nude marrow and was 10 times better at synergy with cortisol-resistant thymocytes. This implication of the pre-T cell as synergizer was supported by the finding that the synergistic activity of marrow was lost on preincubation, but maintained if the preincubation medium contained thymosin or cyclic AMP. Thus, the ability to enhance the response of relatively mature T cells to Con A is a property of pre-T cells. It is anticipated that this property will allow more detailed studies of T-cell precursor development in mice, and possibly in man.     

Effect of fluoroquinolones on the in-vitro proliferation of myeloid precursor cells

The effects of three fluoroquinolones (ofloxacin, pefloxacin and rufloxacin) on the in-vitro proliferation of murine myeloid cells were studied. Their activity was compared with that of nalidixic acid and novobiocin. Therapeutic concentrations of quinolones do not affect the physiological course of myelopoiesis. Only very high concentrations of drug (greater than 70 mg/l) affect bone marrow cell growth producing a dose-dependent inhibition. Because quinolones are active on topoisomerase II from eukaryotic cells they can modulate mammalian cell growth.     

Leukocytes from four patients with complete or partial Leu-CAM deficiency contain the common beta-subunit precursor and beta-subunit messenger RNA.

Deficiency of a family of three leukocyte adhesion molecules (Leu-CAM) is associated with recurrent and life-threatening bacterial infections in man. Each of the three antigens, Mo1, LFA-1, and Leu M5 has a distinct alpha subunit noncovalently associated with a common beta subunit that appears to be required for the expression of these antigens on the cell surface. To investigate the molecular basis of Leu-CAM deficiency, we studied leukocytes from four unrelated patients suffering from complete or partial Leu-CAM deficiency using immunoprecipitation of metabolically labeled proteins, RNA extraction, and Northern blot analysis. We found that B cells from all four patients synthesized a normal sized beta subunit precursor that either failed to "mature" or matured only partially to the membrane expressed form. B cells from all four patients also had a single normal sized beta subunit mRNA of approximately 3.4 kb. Leu-CAM deficiency, in these unrelated patients, is not due to the absence of the beta chain gene or to aberrant splicing of its mRNA and are consistent with a defective beta subunit gene resulting in abnormal posttranslational processing of the synthesized molecule.     

Intracellular precursor forms of plasma proteins: their functions and possible occurrence in plasma

An attempt is made to review the steps in the biosynthesis of plasma proteins by the liver and to point out intermediate precursor forms that might escape to the plasma. With the use of immunochemical techniques such as immunofixation electrophoresis it is possible to identify these precursors when they present an unusual bands on protein electrophoresis. The discovery of the role of vitamin K in the formation of the blood-clotting proteins, for example, followed the immunochemical detection of an abnormal form of inactive prothrombin in plasma. The chief types of precursors likely to occur in blood are forms that are incompletely glycosylated, phosphorylated, or sulfated, or which are single peptide chains of multichain proteins. Precursors containing basic propeptides such as proinsulin or proalbumin may also appear. By being alert to the possible appearance of intracellular forms in plasma, the clinical chemist may be able to relate their presence to the nature of the disease process responsible.     

兔耳瘢痕成熟过程中组织内氧分压的变化

制作兔耳瘢痕从形成到萎缩成熟的动物模型,采用经皮氧监测仪测定术后14,30,60,90d瘢痕和正常皮肤组织内氧分压,并作病理学检查。术后14d创面有肉芽生长,部分上皮化,鲜红色,组织内有大量的炎性细胞及新生血管,氧分压为(0.81±0.25)kPa;术后30d为明显高出皮面的瘢痕,鲜红色,组织内有大量的成纤维细胞及微血管,氧分压为(1.98±0.26)kPa;术后60d,瘢痕萎缩,颜色变淡,组织内成纤维细胞及微血管数目减少,氧分压为(7.86±0.60)kPa;术后90d,瘢痕变平,颜色变白,组织结构接近正常皮肤,氧分压为(13.70±1.16)kPa,术后不同时间点氧分压比较差异有显著性意义(P<0.01)。结果显示随着瘢痕的萎缩成熟,瘢痕组织内氧分压逐渐增加。提示组织内氧分压的提高可能有助于瘢痕萎缩成熟。     

Extracellular proteolysis in the adult murine brain.

Plasminogen activators are important mediators of extracellular metabolism. In the nervous system, plasminogen activators are thought to be involved in the remodeling events required for cell migration during development and regeneration. We have now explored the expression of the plasminogen activator/plasmin system in the adult murine central nervous system. Tissue-type plasminogen activator is synthesized by neurons of most brain regions, while prominent tissue-type plasminogen activator-catalyzed proteolysis is restricted to discrete areas, in particular within the hippocampus and hypothalamus. Our observations indicate that tissue-type plasminogen activator-catalyzed proteolysis in neural tissues is not limited to ontogeny, but may also contribute to adult central nervous system physiology, for instance by influencing neuronal plasticity and synaptic reorganization. The identification of an extracellular proteolytic system active in the adult central nervous system may also help gain insights into the pathogeny of neurodegenerative disorders associated with extracellular protein deposition.     

Studies on the multiple forms of gamma-glutamyltransferase.

In sera of 53 patients with highly raised gamma-glutamyltransferase (gamma-GT) (EC 2.3.2.2) activity, the gamma-GT was separated by column chromatography on Sephadex DEAE A-50 into two fractions. In 35 sera only one gamma-GT fraction was found at a conductivity of 2.0--2.5 mS and in 18 sera two gamma-GT fractions were found at a conductivity of 0.2 mS and again at 2.0--2.5 mS. There was no opvious correlation to the underlying disease. In 20 sera the additional parameters, protein, protein electrophoresis, free and esterified cholesterol, bilirubin, triglycerides, lipoprotein pattern and lipoprotein X were examined. We recognized, that the two forms of the gamma-GT are not true isoenzymes but that the separation of the gamma-GT into two fractions is caused by a different distribution of the cholesterol containing lipoprotei,s.     

Microwave catalyzed carbothermic reduction of zinc oxide and zinc ferrite: effect of microwave energy on the reaction activation energy

Recently, more attention has been paid to the use of microwave (MW) energy in accelerating chemical reactions. The effect of microwave energy on the reduction of zinc oxide and zinc ferrite was investigated. The results indicated that the temperatures required to initiate zinc oxide and zinc ferrite reduction under MW heating were 550 and 450 °C, respectively, while under conventional thermal (CT) heating, were 950 and 850 °C, respectively. Apparently, the MW reaction had a negative standard Gibbs free energy (ΔG) at a lower temperature (∼400 °C) when compared to the CT reaction. Additionally, the activation energy (E_a) substantially decreased from 223.7 and 221.1 kJ mol⁻¹ under CT heating to 64.8 and 32.9 kJ mol⁻¹ under MW heating for Zn oxide and zinc ferrite, respectively. The enhancement in zinc reduction under MW energy was due to the rapid and bulk heating phenomena of MWs as well as the interactions occurring between the electromagnetic MW pattern and the molecules of heated materials.

Catalytic effect of microwave energy on carbothermic reduction of zinc ferrite.     

Effect of allopurinol on Trypanosoma cruzi: metabolism and biological activity in intracellular and bloodstream forms.

Allopurinol (4-hydroxypyrazolo [3,4-d]pyrimidine) is an effective agent in vitro against Trypanosoma cruzi. The important forms of this parasite, with respect to the pathogenesis of Chagas' disease in man, are the bloodstream (trypomastigote) and the intracellular forms. Experiments with radiolabeled allopurinol and analysis of the metabolic products of this compound by high-performance liquid chromatography showed that both the bloodstream and the intracellular forms of T. cruzi metabolize allopurinol in the same manner as has been shown for the epimastigotes in vitro. The metabolic pathways for pyrazolopyrimidines in the pathogenic forms were demonstrated with organisms isolated from infected animals and a tissue culture system infected with T. cruzi. Treatment of infected tissue culture with allopurinol eradicated the infection. This investigation implies that allopurinol may be useful in chemotherapy of T. cruzi infections, a supposition which has been borne out in one animal study.     

CD40不同形式的活化对CD40突变的RPMI8226细胞增殖和表型的影响

目的 分析CD40抗体或配体不同形式刺激对RPMI8226细胞增殖及表面共刺激分子和黏附分子表达的影响,探讨RPMI8226细胞CD40基因突变在其生物学行为中的作用.方法 用RT-PCR方法和DNA序列测定检测RPMI8226细胞CD40基因突变体,分析RPMI8226细胞经CD40抗体或配体四种不同方式(CD40单抗、CD40单抗包板、rhsCD40L和CD40L转基因细胞)刺激后,其体外生长曲线、细胞表型及细胞周期的变化,并利用激光共聚焦显微镜对RPMI8226细胞表面CD40信号转导体的形成进行初步分析.结果 RPMI8226细胞表达CD40突变体(TCA→TTA,Ser→Leu),但是该点突变并未影响hmuCD40的抗原表位.三种激发CD40活化的分子(CD40单抗、rhsCD40L和CD40L转基因细胞)并不影响RPMI8226细胞的体外增殖,但是CD40单抗包板能明显抑制RPMI8226细胞的增殖[(2.5±0.6)×105 vs (7.8±1.2)×105,P＜0.05],并产生明显的G1期阻滞[(58.0±3.6)% vs (42.0±2.3)%,P＜0.05].RPMI8226细胞上黏附分子和共刺激分子的表达无明显变化.激光共聚焦显微镜分析结果显示,在CD40被激活后能形成CD40信号传导的复合体.结论 RPMI8226细胞高表达一种CD40基因突变体,该突变体能在被激活后形成相应的信号传导复合体.     

Relationship between rate and extent of G protein activation: comparison between full and partial opioid agonists.

Opioid agonists acting at their receptors alter intracellular events by initiating activation of various types of Gi/Go proteins. This can be measured by the binding of the stable GTP analog [(35)S]guanosine-5'-O-(3-thio)triphosphate ([(35)S]GTPgammaS). In this study agonist efficacy is defined by the degree to which an opioid stimulates the binding of [(35)S]GTPgammaS. This allows for a definition of full and partial agonists; a full agonist causing a greater stimulation of [(35)S]GTPgammaS binding than a partial agonist. The hypothesis that the rate of agonist-stimulated [(35)S]GTPgammaS binding is dependent upon agonist efficacy was tested using membranes from C6 glioma cells expressing mu- or delta-opioid receptors. At maximal concentrations the rate of agonist-stimulated [(35)S]GTPgammaS binding followed the efficacy of mu-agonists in stimulating [(35)S]GTPgammaS binding, i.e., [D-Ala(2),N-Me-Phe(4),Gly(5)-ol]-enkephalin > morphine > meperidine > butorphanol > nalbuphine. At submaximal concentrations of mu- or delta-full agonists the [(35)S]GTPgammaS association rate was also reduced, such that the rate of [(35)S]GTPgammaS binding correlated with the extent of [(35)S]GTPgammaS bound, whether this binding was stimulated by a full agonist or a partial agonist. Agonists also stimulated [(35)S]GTPgammaS dissociation, showing that binding of this stable nucleotide was reversible. Comparison of the delta-agonists [D-Ser(2),Leu(5)]-enkephalin-Thr and (+/-)-4-((alpha-R*)-alpha-((2S*,5R*)-4-allyl-2,5-dimethyl-1-piperazinyl)-3-hydroxylbenzyl)-N,N-diethylbenzamide, a compound with slow dissociation kinetics, showed the measured rate of G protein activation was not influenced by the agonist switching between receptors. The results are consistent with the idea that the active state(s) of the receptor induced by full or partial agonists is the same, but the number of activated receptors determines the rate of G protein activation.     

Effect of insulin on mitochondrial oxidative phosphorylation and energy charge of the perfused guinea pig liver.

The effect of insulin was investigated in the isolated guinea pig liver perfused with Krebs-Ringer bicarbonate buffer containing red blood cells and albumin. In the mitochondria isolated from livers perfused with 10 units of insulin per hour, the phosphorylative activity with glutamate as a substrate increased to about 160 per cent of control 60 minutes after the beginning of perfusion (p less than 0.01). Such an enhanced phosphorylative activity was accompanied by increases in the respiratory control ratio, state 3 respiration, and P/O ratio. On the other hand, in the liver perfused with insulin, the levels of the energy charge and adenine nucleotide quotient increased to a significant degree as compared to the liver without insulin (p less than 0.01 and p less than 0.05, respectively). It is suggested that insulin plays an important role as a portal factor in regulating mitochondrial oxidative phosphorylation and the levels of the phosphorylated adenine nucleotides.     

溶血磷脂酸对小胶质细胞活化和吞噬功能的影响

目的:观察溶血磷脂酸(LPA)对小胶质细胞活化及其吞噬功能的影响。方法:小胶质细胞系BV2细胞复苏后传代培养,取对数生长期细胞分为对照组和LPA组,LPA干预30min后各组加入FITC标记的葡聚糖,在干预后1h、2h后收取细胞,用流式细胞仪测定各孔FITC-葡聚糖微粒吞噬率及荧光强度。结果:LPA干预1h时,吞噬率较对照组明显增加(P<0.05),2h时两组细胞吞噬率达到最高值。LPA干预后1h及2h时,吞噬荧光强度较对照组明显增加(P<0.01),而且吞噬率与荧光强度的乘积较对照组亦明显增加(P<0.01)。结论:LPA可提高BV2细胞活化及吞噬功能,提示LPA在小胶质细胞活化及吞噬过程中发挥了重要作用。