Nuclear polymorphism and nuclear size in precarcinomatous and carcinomatous lesions in rat colon and liver

The role of nuclear polymorphism and nuclear size in an analysis of the differences between colon and liver tumors in rats as well as in an analysis of hepatic dysplasia was studied. It was shown that colon tumors which developed following treatment of animals with a direct carcinogen alone or with a carcinogen followed by secondary bile acid were characterized by low incidence of nuclear polymorphism and by an increased nuclear size in epithelial cells. Metastatic liver tumors in rats with colon tumors were characterized by a high value for the coefficient of nuclear form polymorphism and by a significant decrease in nuclear size. Hepatic dysplasia which developed as a result of prolonged treatment with secondary bile acids was characterized by high rate of nuclear form polymorphism and by a significant increase in nuclear size. The obtained results suggest that nuclear polymorphism is dependent upon the type of tissue or organ involved in the cancerous transformation and that it may have significance as a diagnostic marker of precarcinomatous and carcinomatous lesions of digestive organs only when used in combination with other analyses.     

Conformation and proteolysis of glucagon and insulin in surfactant and lipid solutions

The effects of micelles of nonionic, zwitterionic, anionic and cationic surfactants and lipids on the conformation of glucagon and insulin have been investigated by circular dichroism and intrinsic protein fluorescence. The influence of these amphipathic compounds on the hydrolysis, monitored by HPLC, of glucagon and insulin by trypsin and chymotrypsin has also been studied. The alpha-helix content of glucagon was increased to a similar extent by all the micelles, irrespective of their charge and of whether they were synthetic surfactants or phospholipids. The amphipathic compounds always induced a blue-shift in the wavelength of maximum emission of fluorescence of glucagon of about 9 nm, whereas the fluorescence intensity was increased in some cases and decreased in others. The circular dichroism of insulin was also modified in some cases. Some amphipathic compounds protected glucagon against proteolysis by trypsin and chymotrypsin very markedly, whereas others did not protect at all or only slightly protected the hormone. Two hypotheses have been formulated to explain the different results. Hydrolysis of insulin was generally not influenced by surfactants and lipids.     

Differential regulation of gelatinase A and B and TIMP-1 and -2 by TNFalpha and HIV virions in astrocytes

Changes in the fine balance between matrix metalloproteinases and their tissue inhibitors, which drives extracellular matrix turnover, may be critical to central nervous system inflammation in HIV infection as well as in neurotoxicity. Although they do not produce virus when infected by HIV, astrocytes may be directly affected by the virion, because some viral proteins are known to transduce signaling in brain cells and are also sensitive to the major proinflammatory cytokine TNFalpha. We therefore studied the effects of HIV and TNFalpha on MMP-2, MMP-9 and their inhibitors, TIMP-1 and TIMP-2, in astrocytes, by zymography and ELISA, respectively, or by RT-PCR for both of them. HIV slightly increased the production of pro-MMP-2 and pro-MMP-9 by astrocytes, in a dose-dependent manner. TNFalpha strongly induced pro-MMP-9. TIMP-1 and TIMP-2 levels were affected only slightly, if at all, by HIV and TNFalpha. Thus, astrocyte/HIV contact may lead to extracellular matrix activation, which may be strongly amplified by the inflammatory response. Our data strongly suggest that, besides their physiological production of MMP-2, astrocytes would be a major source of MMP-9 in the inflamed brain.     

Glucose and lactate turnover and gluconeogenesis in chronic metabolic acidosis and alkalosis in normal and diabetic dogs

The turnover rate of glucose, the irreversible disposal rate of lactate, and the rate of gluconeogenesis from lactate were calculated by tracer methods in four normal and four alloxan-diabetic dogs under control conditions as well as in chronic, stable metabolic acidosis and alkalosis. Acidosis was produced by feeding dogs 0.8-1 g.kg-1.day-1NH4Cl over 1 week, alkalosis was produced by feeding dogs a chloride-free diet and injections of furosemide. Mean plasma pH in the three states were 7.28 +/- 0.013, 7.40 +/- 0.024, and 7.51 +/- 0.015 in normal dogs, and 7.22 +/- 0.025, 7.42 +/- 0.009, and 7.49 +/- 0.002 in the diabetic dogs. Respective mean plasma bicarbonate levels were 14.6 +/- 0.88, 22.0 +/- 0.80, and 32.4 +/- 1.88 mequiv. in normal dogs, and 12.3 +/- 1.30, 22.6 +/- 0.66, and 35.0 +/- 1.14 mequiv. in diabetic animals. In normal dogs shifts in acid-base balance had no effect on the level of plasma glucose or the turnover rate of glucose. In diabetic dogs plasma glucose level was significantly elevated by alkalosis. Plasma lactate was positively correlated with plasma pH (r = 0.69, p less than 0.01) and was in general higher in diabetic than in normal animals. The increment in concentration was due to a decreased clearance of lactate from the plasma. The irreversible disposal rate was not changed by the acid-base status. Whereas a larger fraction of lactate removed from the plasma appeared in glucose in diabetic animals, this fraction was not changed significantly by shifts in the acid-base status.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cutinase unfolding and stabilization by trehalose and mannosylglycerate

The unfolding of cutinase at pH 4.5 was induced by increasing the temperature and guanidine hydrochloride concentration in the presence of potassium chloride, trehalose, and mannosylglycerate potassium salt. Protein thermal unfolding approached a two-state process, since the unfolding transitions were coincident within experimental error when assessed by near-ultraviolet (UV) difference, tryptophyl, and 8-anilino-1-naphthalene sulfonic acid (ANS) fluorescence spectroscopy. Trehalose at 0.5 M increased the temperature at which 50% of cutinase is unfolded by 3 degrees C. Unfolding induced by guanidine hydrochloride is clearly a non-two-state process. The presence of a stable intermediate was detected because unfolding assessed by near-UV difference spectroscopy occurs earlier than unfolding assessed by tryptophyl fluorescence. The intermediate is molten globule in character: the ANS fluorescence is higher than in the presence of the folded or unfolded state, showing native-like secondary structure and losing many tertiary interactions of the folded state, i.e., those surrounding the tyrosyl microenvironment. The stabilization effect of trehalose and mannosylglycerate was quantified by fitting the unfolding transitions to a model proposed by Staniforth et al. (Biochemistry 1993;32:3842-3851). This model takes into consideration the increase in solvation energies of the amino acid side-chains as the denaturant concentration was increased and the fraction of amino acid side-chains that become exposed in the unfolded structure of cutinase. Trehalose and mannosylglycerate stabilize the folded state relative to the intermediate by 1.4-1.6 and 1.6 kcal/mol and the intermediate relative to the unfolded state by 1.0 and 1.5 kcal/mol, respectively.     

壳聚糖酶的克隆表达与壳寡糖的制备分析

目的:克隆壳聚糖酶基因于大肠杆菌中实现高表达,制备壳寡糖。方法:以枯草芽孢杆菌总DNA为模板扩增壳聚糖酶基因(CSN),克隆至载体pET23a(+)上,转化菌株BL21(DE3)。重组子经0.5 mmol/L IPTG诱导后,SDS-PAGE和质谱检测与鉴定重组酶。酶纯化后水解壳聚糖,薄层色谱分析其水解产物。结果:质谱证明壳聚糖酶(31.5kDa)成功表达,表达量占菌体总蛋白的45%左右。纯化后重组酶浓度为900 mg/L,纯度95%、回收率85%,酶活力为10 000 U/mg。壳聚糖降解产物为壳二糖至壳四糖。结论:原核表达载体pET23a(+)-CSN构建正确,壳聚糖酶表达量与活性高,适用于水解壳聚糖制备壳寡糖。     

Uptake of native and deglycosylated ricin A-chain immunotoxins by mouse liver parenchymal and non-parenchymal cells in vitro and in vivo

The therapeutic activity of ricin A-chain immunotoxins is undermined by their rapid clearance from the bloodstream of animals by the liver. This uptake has generally been attributed to recognition of the mannose-terminating oligosaccharides present on ricin A-chain by receptors present on the non-parenchymal (Kupffer and sinusoidal) cells of the liver. However, we demonstrate here that, in the mouse, the liver uptake of a ricin A-chain immunotoxin occurs in both parenchymal and non-parenchymal cells in equal amounts. This is in contrast to the situation in the rat, where uptake of the immunotoxin is predominantly by the non-parenchymal cells. Recognition of sugar residues on the A-chain portion of the immunotoxin plays an important role in the liver uptake by both cell types in both species. However it is not the only mechanism since, firstly, an immunotoxin containing ricin A-chain which had been effectively deglycosylated with metaperiodate and cyanoborohydride was still trapped to a significant extent by hepatic non-parenchymal cells after it was injected into mice. Secondly, deglycosylation, while eliminating uptake of the free A-chain by parenchymal and non-parenchymal cells in vitro, only reduced the uptake of an immunotoxin by either cell type by about half. Thirdly, the addition of excess D-mannose or L-fucose inhibited the uptake of free A-chain by mouse liver cell cultures by more than 80% but only inhibited the uptake of the native A-chain immunotoxin by about half and had little effect on the uptake of the deglycosylated ricin A-chain immunotoxin. Recognition of the antibody portion of the immunotoxin by liver cells seems improbable, since antibody alone or an antibody-bovine serum albumin conjugate were not taken up in appreciable amounts by the cultures. Possibly attachment of the A-chain to the antibody exposes sites on the A-chain that are recognised by liver cells in vitro and in vivo.     

Effect of Substrate Concentration and Organic and Inorganic Compounds on the Occurrence and Rate of Mineralization and Cometabolism

Isopropyl N-phenylcarbamate (IPC) at 400 pg and 1 μg/ml was mineralized in samples of sewage, but only the lower concentration was mineralized in lake water samples in a 50-day period. IPC at 1 μg/ml disappeared from lake water, but it was converted to organic products. Mineralization of IPC at 400 pg/ml in lake water was enhanced by additions of inorganic nutrients or a mixture of nonchlorinated water pollutants but not by yeast extract or mixtures containing aromatic compounds or excretions of primary producers. The mineralization of 200 pg of 2,4-dichlorophenoxyacetate per ml of lake water was not affected by additions of low levels of yeast extract or compounds excreted by primary producers but was enhanced by low concentrations of mixtures of water pollutants. It is suggested that some chemicals that are found to be converted only to organic products, presumably by cometabolism, in tests using the concentrations commonly employed in laboratory evaluations may be mineralized at the lower concentrations prevailing in natural waters.     

Lysosomal degradation of glycoproteins and glycosaminoglycans. Efflux and recycling of sulphate and N-acetylhexosamines.

Lysosomal degradation of the carbohydrate portion of glycoproteins and glycosaminoglycans produces monosaccharides and sulphate, which must efflux from the lysosomes before re-entering biosynthetic pathways. We examined the degradation of glycoproteins and glycosaminoglycans by lysosomes isolated from cultured human diploid fibroblasts. Cells were grown for 24 h in medium containing [3H]glucosamine and [35S]sulphate. When lysosomes are isolated from these cells, they contain label primarily in macromolecules (glycoproteins and glycosaminoglycans). Glycoprotein degradation by isolated lysosomes was followed by measuring the release of tritiated sugars from macromolecules and efflux of these sugars from the organelles. Glycosaminoglycan degradation was monitored by the release of both tritiated sugars and [35S]sulphate. During macromolecule degradation, the total amounts of free [35S]sulphate, N-acetyl[3H]glucosamine and N-acetyl[3H]galactosamine found outside the lysosome parallels the amounts of these products released by degradation. The total degradation of glycoproteins and glycosaminoglycans by intact cultured cells was also examined. The lysosomal contribution to degradation was assessed by measuring inhibition by the lysosomotropic amine NH4Cl. After 48 h incubation, inhibition by NH4Cl exceeded 55% of glycoprotein and 72% of glycosaminoglycan degradation. Recycling of [3H]hexosamines and [35S]sulphate by intact cells was estimated by measuring the appearance of 'newly synthesized' radioactively labelled macromolecules in the medium. Sulphate does not appear to be appreciably recycled. N-Acetylglucosamine and N-acetylgalactosamine, on the other hand, are reutilized to a significant extent.     

Neurogenesis and aging: FGF-2 and HB-EGF restore neurogenesis in hippocampus and subventricular zone of aged mice

Neurogenesis, which may contribute to the ability of the adult brain to function normally and adapt to disease, nevertheless declines with advancing age. Adult neurogenesis can be enhanced by administration of growth factors, but whether the aged brain remains responsive to these factors is unknown. We compared the effects of intracerebroventricular fibroblast growth factor (FGF)-2 and heparin-binding epidermal growth factor-like growth factor (HB-EGF) on neurogenesis in the hippocampal dentate subgranular zone (SGZ) and the subventricular zone (SVZ) of young adult (3-month) and aged (20-month) mice. Neurogenesis, measured by labelling with bromodeoxyuridine (BrdU) and by expression of doublecortin, was reduced by approximately 90% in SGZ and by approximately 50% in SVZ of aged mice. HB-EGF increased BrdU labelling in SGZ at 3 months by approximately 60% and at 20 months by approximately 450%, which increased the number of BrdU-labelled cells in SGZ of aged mice to approximately 25% of that in young adults. FGF-2 also stimulated BrdU labelling in SGZ, by approximately 25% at 3 months and by approximately 250% at 20 months, increasing the number of newborn neurones in older mice to approximately 20% of that in younger mice. In SVZ, HB-EGF and FGF-2 increased BrdU incorporation by approximately 140% at 3 months and approximately 170% at 20 months, so the number of BrdU-labelled cells was comparable in untreated 3-month-old and growth factor-treated 20-month-old mice. These results demonstrate that the aged brain retains the capacity to respond to exogenous growth factors with increased neurogenesis, which may have implications for the therapeutic potential of neurogenesis enhancement in age-associated neurological disorders.     

Antioxidant and anti-AGE therapeutics: evaluation and perspectives

Diabetic patients exhibit an oxidative stress status, that is an imbalance between reactive oxygen species and antioxidant defences, in favour of the first ones. This oxidative stress, together with formation of advanced glycation endproducts (AGEs), is involved in diabetic complications. It could thus be of great interest to propose antioxidant and/or anti-AGE therapeutics as complementary treatment in these patients. Antioxidants can be classical molecules such as vitamin E, lipoic acid or N-acetylcysteine. Thus, vitamin E supplementation can improve insulin efficiency and glycemic equilibrium, as shown by the decrease of glycaemia, glycated haemoglobin and fructosamine values. In addition, this kind of supplementation lowers plasma lipid peroxidation and oxidizability of low density lipoproteins, which is involved in the atherogenesis process. Moreover, it allows to fight against complications such as retinopathy. A second category is represented by molecules able to fight against the effects of glycation end-products (AGEs). They can act: either by preventing cellular action of AGEs; this is obtained with soluble receptors of advanced glycation endproducts (sRAGE); or by inhibiting AGE formation (scavenging of reactive carbonyl intermediates). Nucleophilic compounds such as pyridoxamine, tenilsetam, 2,3-diaminophenazone, OPB-9195 or aminoguanidine can act in this way. Aminoguanidine is able to limit the development of the main diabetes-associated complications in animals. A double-blind clinical assay has been conducted in type 2 diabetic patients in the United States and the Canada, in order to determine if aminoguanidine is able to slow down the progression of diabetes-induced nephropathy. We will discuss about another guanidic molecule, i.e. metformin, which is also able to scavenge AGEs, in the last part of this review. A third category of molecules is constituted by oral antidiabetic molecules exhibiting antioxidant properties. They are thiazolidinediones (troglitazone) and sulfonylureas (gliclazide). Troglitazone and gliclazide can thus decrease LDL oxidizability and monocyte adhesion to endothelial cells, which is an early step in the atherogenesis process and which is stimulated by oxidised LDLs. Finally, a prospective way is devoted to oral antidiabetic drugs exhibiting both antioxidant and anti-AGE properties. A very used antidiabetic drug of interest is metformin (dimethylbiguanide), since it can prevent diabetes complications not only by lowering glycaemia, but also by inhibiting AGE formation and by stimulating antioxidant defences. The latter therapeutic approach constitutes a future way in the diabetes area, in order both to obtain a better glycemic control and a least development of diabetic complications.     

Paraoxonases and cardiovascular diseases: pharmacological and nutritional influences

PURPOSE OF REVIEW: To summarize the new articles published in the last year on paraoxonases, including their expression in cardiovascular diseases, and regulation by pharmacological and nutritional means. RECENT FINDINGS: The elucidation of the crystal structure of the paraoxonase 1 (PON1) gene, obtained by directed evolution, shows that it consists of a six-bladed beta-propeller with a unique active site. PON1 is present in HDL but also in lipoprotein-deficient serum, in VLDL and in chylomicrons. PON1 protects lipids in lipoproteins, in macrophages and in erythrocytes from oxidation. Cellular PON2 and PON3 were also shown to reduce oxidative stress. Beyond its antioxidative properties, PON1 possesses additional antiatherogenic properties against macrophage foam cell formation: attenuation of cholesterol and oxidized lipids influx, inhibition of macrophage cholesterol biosynthesis and stimulation of macrophage cholesterol efflux. The PON1 gene is regulated by Sp1 and protein kinase C, whereas the PON2 gene in macrophages is regulated by nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. PON1 activity and mass are both reduced in cardiovascular diseases and the hypocholesterolemic drugs, statins, increase serum PON1 activity (by reducing oxidative stress, or by upregulating hepatic PON1 expression). Expression of cellular PON2, like PON1, was upregulated by statins. Nutritional antioxidants, such as polyphenols, increase PON1 mRNA expression and activity, by an aryl hydrocarbon receptor-dependent mechanism. SUMMARY: The elucidation of PON1 structure and its active center has enabled a better understanding of its mechanism of action, including its physio-pathological substrate(s). Some drugs and nutrients including dietary antioxidants and polyphenols considerably increase the activities of paraoxonases which, in turn, can reduce oxidative stress and atherosclerosis development.     

Comparative electron cytochemical and biochemical study of ATPase and beta-glycerophosphatase activity in thymocytes, leukocytes and erythrocytes]

A study was made of the effect of procedures (freezing-thawing prior to incubation, prefixation with formaldehyde and glutaraldehyde, incubation with DMSO) on the activity of ATPase and beta-glycerophosphatase in leucocytes and erythrocytes of man, and of the effect of these procedures and of homogenization on ATPase activity in the cells of the rat thymus. The homogenization of rat thymocytes decreases ATPase activity by 15%. A repeated freezing-thawing results in a 15% decrease of ATPase activity in the cells of the rat thymus. The homogenization of rat thymocytes decreases ATPase activity in rat thymocytes, in a 2% decrease in human leucocytes, and in a 21% increase in human erythrocytes. Beta-glycerophosphatase activity in leucocytes and in erythrocytes increases thereby by 89 and 38%. Incorporation of 5% DMSO into the medium increases ATPase activity in human leucocytes and erythrocytes by 17 and 16%, while thymocytes this activity drops by 27%. Beta-glycerophosphatase activity increases thereby in leucocytes by 26 and in erythrocytes by 11.5%, resp.     

Demonstration and structural comparison of receptors for insulin-like growth factor-I and -II (IGF-I and -II) in brain and blood-brain barrier

Specific receptors for insulin-like growth factors (IGF) I and II on microvessel-free rat brain cell membranes (RBCM) and in the microvessels that constitute the blood-brain barrier (BBB) were identified and characterized by means of affinity cross-linking techniques and specific anti-receptor antibodies. Two different models of BBB were examined: isolated rat brain capillaries and cultured bovine brain microvessel endothelial cells. Cross-linking with 125-I-IGF-I, followed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), revealed an alpha subunit of apparent Mr = 138,000 in both BBB preparations, compared to 120,000 in RBCM. Cross-linking was inhibited by unlabeled IGF and insulin, but not by antibody directed against the IGF-II receptor. When 125-I-IGF-II was cross-linked, followed by SDS-PAGE under reducing conditions, a major band of apparent Mr = 250,000 was identified in RBCM and both BBB preparations. This band, which migrated with an approximately equivalent Mr in both brain and BBB membranes, was inhibited by unlabeled IGF and by antibody specific for the IGF-II receptor. Thus, both rat and bovine brain microvessels possess classical Type I and II IGF receptors. While the alpha subunit of the Type I receptor of brain is smaller than that of the BBB, the Type II receptor of brain and BBB appear to be structurally and immunologically identical.     

Aging and prostacyclin responses in aorta and platelets from WKY and SHR rats

In spontaneously hypertensive rat (SHR) aorta, prostacyclin is an endothelium-derived contracting factor contributing to the endothelial dysfunction. This study was designed to determine whether the impairment of the prostacyclin response is influenced by aging and whether such a dysfunction is observed in platelets. Isometric tension was measured in aortic rings, and aggregation was studied in platelet-rich plasma taken from 3-, 6-, and 15-mo-old Wistar-Kyoto rats (WKY) and SHR. In aorta from 3- and 6-mo-old WKY, prostacyclin and beraprost [prostacyclin receptor (IP) agonists] produced relaxations that were enhanced by Triplion (thromboxane-prostanoid receptor antagonist). In 15-mo-old WKY, the relaxations to beraprost were maintained, but not those to prostacyclin. In SHR aorta, prostacyclin or beraprost produced no or minor relaxations, which, in younger SHR, were enhanced by Triplion. In both strains, the relaxations were inhibited by CAY-10441 (IP receptor antagonist). The relaxations to forskolin and isoproterenol were reduced with aging. When compared with those of WKY, the relaxations to isoproterenol were reduced in 3- but not in 6- or 15-mo-old SHR, whereas those to forskolin were consistently diminished at any given age. Whatever the age, prostacyclin and beraprost produced CAY-10441-sensitive inhibitions of ADP-induced platelet aggregation. Both agonists were more potent in SHR than in WKY. Therefore, in platelets from WKY and SHR, the IP receptor-dependent antiaggregant response is functional and maintained during aging. In aorta from WKY those responses are reduced by aging and, in SHR, are already compromised at 3 mo. This dysfunction of the IP receptor is only partially explained by a general dysfunction of the adenylate cyclase pathway.     

Fungal Communities and Disease Symptoms on Stem Bases of Wheat and Barley and Effects of Seed Treatments Containing Fluquinconazole and Prochloraz

Three successive crops of winter wheat or barley were grown as second, third and fourth cereals. Communities of fungi on shoot bases, identified after isolation on agar media, were more diverse (determined by number of taxa identified) on wheat than on barley, and their diversity increased from year to year. Diversity was not affected by seed treatments containing fluquinconazole or prochloraz. Eyespot (caused by Tapesia spp.) and brown foot rot (caused by Fusarium spp. or Microdochium nivale ) increased from year to year. Eyespot, brown foot rot (after the first year) and sharp eyespot (which remained infrequent), assessed in summer (June), affected wheat more than barley. Eyespot severity was increased slightly on barley by treatments containing fluquinconazole, formulated with or without prochloraz, in the second year (third cereal), when it was also decreased slightly on wheat by fluquinconazole plus prochloraz, except in plots where the treatment had been applied for two successive years. The increases or decreases in eyespot in the second year were accompanied by, respectively, decreases or increases in the frequency of Idriella bolleyi where fluquinconazole was applied alone. Although the eyespot pathogen Tapesia yallundae (but not Tapesia acuformis ) is sensitive to fluquinconazole in vitro , seed treatment, applied principally to control take-all disease, is likely to have only a small effect against eyespot (or other stem-base diseases), and then only on wheat and when formulated with prochloraz.