Regulation of N-acetylglucosaminyltransferase V activity. Kinetic comparisons of parental, Rous sarcoma virus-transformed BHK, and L-phytohemagglutinin-resistant BHK cells using synthetic substrates and an inhibitory substrate analog

Baby hamster kidney (BHK) cells transformed with Rous sarcoma virus, RS-BHK cells, demonstrate a 2.5-fold increase in the activity of N-acetylglucosaminyltransferase V (GlcNAc-T V, EC 2.4.1.155), and this increase in activity appears to be specific for this enzyme. By contrast, a lectin-resistant BHK cell line selected for its ability to grow in high levels of L-phytohemagglutinin, LP3.3, is characterized by a specific decrease in its GlcNAc-T V activity. To test if these alterations in the apparent Vmax of GlcNAc-T V are due to changes in the efficiency of populations of enzymes in RS-BHK and LP3.3 cells compared to the parental BHK cells, we have compared the kinetic properties of the enzymes from these three sources. The Km constants observed for both the sugar nucleotide donor (UDP-GlcNAc) and two synthetic trisaccharide acceptors were indistinguishable. The Vmax values toward three synthetic acceptors were also determined first for the BHK GlcNAc-T V, and they varied by over 5-fold. When these values were measured for the variant and transformed cell enzymes, however, similar 5-fold differences were still observed, although the absolute values for these acceptors were all higher or lower for the RS-BHK and LP3.3 enzymes, respectively. In addition, we have synthesized a deoxygenated analog of the specific GlcNAc-T V acceptor, beta GlcNAc(1,2) alpha Man(1,6) beta ManOR, where the reactive 6'-OH group has been removed, and the resulting trisaccharide was found to be a competitive inhibitor of the enzyme. The Ki for this inhibitor was near 70 microM for the GlcNAc-T V from all three sources. These kinetic comparisons demonstrate that the enzymes from the three cell types have kinetically indistinguishable active sites. These results suggest that the differences in the apparent Vmax values among the cell types are most likely due to alterations in the number of active molecules rather than in the modulation of either their catalytic activities or specificities.     

Defects in CD8+ regulatory T cells in the lamina propria of patients with inflammatory bowel disease

Mucosal tolerance is believed to be partly mediated by regulatory T cells. Intestinal epithelial cells (IECs) may play an important role in the generation of such regulatory cells, because they are able to process and present Ag to T cells. Furthermore, we have previously demonstrated that IECs are able to generate regulatory CD8(+) T cells in vitro. In the present study, we have analyzed lamina propria (LP) lymphocytes for the presence of such regulatory CD8(+) T cells in normal individuals as well as in patients with inflammatory bowel disease (IBD). The results of the present study show that LP CD8(+) T cells derived from normal controls possess regulatory activity, whereas both unfractionated LP lymphocytes and purified LP CD4(+) T cells do not. The LP CD8(+) T cells suppress Ig production by pokeweed mitogen-stimulated PBMCs by 31-80%, in a cell contact-dependent manner. No significant difference in suppression between CD28(+) and CD28(-)CD8(+) LP T cells was observed. In contrast to CD8(+) T cells from normal LP, CD8(+) T cells isolated from LP of IBD patients, did not suppress Ig production by pokeweed mitogen-stimulated PBMC (five of six ulcerative colitis specimens; six of six Crohn's disease specimens). Furthermore, we demonstrate that the frequency of TCR Vbeta5.1-positive CD8(+) T cells, which we previously have demonstrated to be regulatory and to be expanded by IECs in vitro, is decreased in IBD LP compared with normal LP. In conclusion, this study demonstrates that CD8(+) T cells with regulatory activity are present in the LP of normal healthy individuals, but not in patients with IBD, suggesting that these cells might play an active role in mucosal tolerance.     

Endotoxin-lipoprotein complex formation as a factor in atherogenesis: associations with hyperlipidemia and with lecithin:cholesterol acyltransferase activity

A potential role of endotoxin–lipoprotein (bacterial lipopolysaccharide–lipoprotein, LPS–LP) complex formation as a pathogenic factor for atherosclerosis has not been studied yet. The aim of this study was to test the hypothesis that in endotoxinemia in humans hyperlipidemia associated with atherosclerosis development can favor an excessive LPS–LP complex formation, and endotoxin presented in blood can inhibit lecithin:cholesterol acyltransferase (LCAT), one of the key enzymes of reverse cholesterol transport. Endotoxin-binding capacity of lipoproteins (LP) in patients with normolipidemia and hyperlipidemia types IIa and IV was estimated from label incorporation into different LP fractions isolated by means of sequential ultracentrifugation following serum preincubation with Salmonella minnesota R595 ¹²⁵I-labeled LPS. The effect of varied concentrations of S. minnesota R595 LPS on LCAT activity was evaluated from the overall esterifying activity of serum using [1,2-³H₂]cholesterol-labeled substrate. The elevation of low density LP (LDL) and very low density LP (VLDL) contents in blood serum in hyperlipidemia types IIa and IV, respectively, resulted in significant elevation of LPS binding to these fractions. LPS added to the blood serum leads to the dose-dependent decrease in LCAT activity. The revealed phenomena of elevated LPS binding to atherogenic LP fractions in hypercholesterolemia and endotoxin-induced LCAT inhibition suggest the pathogenic role of LPS–LP complexes in atherogenesis.     

Glutathione S-transferase and UDP-glucuronyltransferase activity in primary cultures of rainbow trout gill epithelial cells

The objective of this study was to characterize rainbow trout (Oncorhynchus mykiss) gill epithelial cells in primary culture by evaluating their ability to maintain glutathione and glucuronide conjugating enzymes. The activity and inducibility of the phase II enzymes was investigated as a function of culture time. Glutathione S-transferase (GST) and UDP-glucuronyltransferase (UDPGT) enzyme activities were measured in freshly isolated cells and in cells cultured for 7 and 12 days. GST activity, determined with 1-chloro-2,4-dinitrobenzene, decreased gradually to 72% after 7 days and to 38% after 12 days in culture compared with freshly isolated cells. There was no significant difference between UDPGT activities in freshly isolated cells compared with cells cultured up to 12 days although a transient decrease in activity was observed at day 7. In vitro induction of the enzymes was studied using beta-naphtoflavone (BNF) and 3-methylcholanthrene (3-MC) as inducers. GST activity increased 2-fold after exposure to BNF and 1.5-fold after 3-MC exposure for 48 h in 7 days old cultures. No induction was observed in 12 days old cultures. UDPGT activity was not induced either at day 7 or 12.     

Gastrointestinal immunology: cell types in the lamina propria--a morphological review.

Defense mechanisms--including immune responses--of the gastrointestinal (GI) system rely on a delicate balance of multidirectional interactions of different components of the GI mucosa. The majority of the cells involved in immune reactions are in the lamina propria (LP) and in the submucosa. Several biologically active substances (enzymes, neurotransmitters, humoral mediators) and their receptors have been reported to be present in LP cells. These cells are in close morphological connections with the surface epithelial cells and with the surrounding vessels and nerve fibers, suggesting a functional association with them. In this paper cell types of the LP will be reviewed from a morphological aspect. In summary, LP cells can be classified as basic structural elements (fibroblasts, fibrocytes, vascular endothelial and smooth muscle cells), blood cells (granulocytes, mast cells, macrophages, T and B lymphocytes, plasma cells), and occasional epithelial and endocrine cells of the surface epithelium. Nerve fibers and terminals, but not neuronal perikarya, can also be seen in the LP. The appearance and the proportion of LP cells strongly depend on the functional activity of the GI system at any given time. Their number and distribution might be significantly altered in certain pathological conditions (infections, inflammations, ulcerations, and other GI disorders). We hope, this review may help clinicians, pathologists, and researchers in the recognition of LP cell types, and in demonstrating their activation, migration and proliferation in different physiological and pathological conditions.     

Decrease in phosphofructokinase activity during blood preservation and the effect of intracellular ATP

The activities of 15 enzymes, including all the glycolytic enzymes, were observed during the preservation of human red cells at 4°C. After 8 weeks, phosphofructokinase (PFK) activity had decreased most. The decrease was prevented by the addition of adenine and inosine to the preservation medium, but not at all by inosine alone and only slightly by adenine alone. This decrease paralleled that of the decrease of intracellular ATP. PFK in the hemolysate was inactivated rapidly, but the inactivation was effectively prevented by ATP. The decrease in glycolytic activity during preservation was concluded to be a result of the loss of PFK activity, and this in turn was due to the decrease of ATP.     

Antioxidant status and lipid peroxidation in the blood of breast cancer patients of different ages

Oxidative stress is considered to be implicated in the pathophysiology of breast cancers. In this study we investigated the level of oxidative stress and antioxidant (AO) status in the blood of breast cancer patients of different ages. The level of lipid hydroperoxides (LP) was measured in blood plasma and the activities of copper, zinc superoxide dismutase (CuZnSOD), catalase (CAT), glutathione peroxidase (GPx), and glutathione reductase (GR) enzymes, as well as the level of total glutathione (GSH) and CuZnSOD protein were measured in blood cells of breast cancer patients and age-matched healthy subjects. Our results showed that breast carcinoma is related to increase of lipid peroxidation in plasma with concomitant decrease of AO defense capacity in blood cells, which becomes more pronounced during aging of the patients. Suppression of CuZnSOD activity related to breast cancer is most likely caused by decreased de novo synthesis of this enzyme. Similar patterns of suppression in CuZnSOD and CAT activities related to aging were recorded both in controls and patients. Age-related decrease in CuZnSOD activity seems not to be caused by altered protein levels of this enzyme. Suppression of AO enzymes associated with breast cancer and aging is most likely the cause of increased levels of reactive oxygen species (ROS). Our results indicate significant role of oxidative-induced injury in the breast carcinogenesis, particularly during the later stages of aging. Overall, our data support the importance of endogenous AOs in the etiology of breast cancer across all levels of predicted risk.     

Study of lysosomal enzymes in chick embryo fibroblasts infected with microorganisms of family Halprowiaceae (Chlamydiaceae)

It is shown that infection of chick embryo fibroblasts with agents of paratrachoma and meningopneumonia Halprowiaceae (Chlamydiaceae) causes a sharp decrease of the activities of lysosomal enzymes, e.g. acidic alpha-glucosidase, beta-glucuronidase, beta-galactosidase, alpha-mannosidase, acid phosphatase, etc. The activity of cytosol enzymes (neutral alpha-glucosidase, amylo-1,6-glucosidase) does not change, however. A decrease in the activities of lysosomal enzymes in infected fibroblasts occurs some time later after inoculation and is due to a release of lysosomal enzymes from the fibroblasts into the culture medium, without loss of cell integrity. No changes in the activity of lysosomal enzymes in fibroblasts and culture medium is observed in the case of inoculation of cells with a killed agents, as well as after contact of cells with a suspension of normal chick embryo yolk sacs. The release of lysosomal enzymes from halprowiae-infected chick embryo fibroblasts probably occurs by the exocytosis.     

PTH-dependent adenylyl cyclase activation in SaOS-2 cells: passage dependent effects on G protein interactions

Parathyroid hormone (PTH) sensitive adenylyl cyclase activity (ACA) in SaOS-2 cells varies as a function of cell passage. In early passage (EP) cells (< 6), ACA in response to PTH and forskolin (FOR) was relatively low and equivalent, whereas in late passage (LP) cells (> 22), PTH exceeded FOR dependent ACA. Potential biochemical mechanisms for this passage dependent change in ACA were considered. In EP, prolonged exposure to pertussis toxin (PT) markedly enhanced ACA activity in response to PTH, Isoproterenol and Gpp(NH)p, whereas ACA in response to FOR was decreased. In contrast, the identical treatment of LP with PT diminished all ACA in response to PTH, Gpp(NH)p, and FOR. The dose dependent effects of PT on subsequent [(32)P]ADP-ribosylation of its substrates, GTPase activity, as well as FOR-dependent ACA, were equivalent in EP and LP. The relative amounts of G(alpha)i and G(alpha)s proteins, as determined both by Western blot, PT and cholera toxin (CT) dependent [(32)P]ADP-ribosylation, were quantitatively similar in EP and LP. Western blot levels of G(alpha)s and G(alpha)i proteins were not influenced by prior exposure to PT. Both PT and CT dependent [(32)P]ADP-ribosylation were dose-dependently decreased following exposure to PT. However, the PT-dependent decline in CT-dependent [(32)P]ADP-ribosylation occurred with enhanced sensitivity in LP. The protein synthesis inhibitor cycloheximide partially reversed the PT associated decrease in FOR dependent ACA in EP. In contrast, cycloheximide completely reversed the PT associated decrease in FOR and as well as PTH dependent ACA in LP. G(alpha)s activity, revealed by cyc(-) reconstitution, was not altered either by cell passage or exposure to PT. The results suggest that the coupling between the components of the complex may be pivotally important in the differential responsiveness of early and late passage SaOS-2 cells to PTH.     

Crosslinked enzyme aggregates in hierarchically-ordered mesoporous silica: a simple and effective method for enzyme stabilization

alpha-chymotrypsin (CT) and lipase (LP) were immobilized in hierarchically-ordered mesocellular mesoporous silica (HMMS) in a simple but effective way for the enzyme stabilization, which was achieved by the enzyme adsorption followed by glutaraldehyde (GA) crosslinking. This resulted in the formation of nanometer scale crosslinked enzyme aggregates (CLEAs) entrapped in the mesocellular pores of HMMS (37 nm), which did not leach out of HMMS through narrow mesoporous channels (13 nm). CLEA of alpha-chymotrypsin (CLEA-CT) in HMMS showed a high enzyme loading capacity and significantly increased enzyme stability. No activity decrease of CLEA-CT was observed for 2 weeks under even rigorously shaking condition, while adsorbed CT in HMMS and free CT showed a rapid inactivation due to the enzyme leaching and presumably autolysis, respectively. With the CLEA-CT in HMMS, however, there was no tryptic digestion observed suggesting that the CLEA-CT is not susceptible to autolysis. Moreover, CLEA of lipase (CLEA-LP) in HMMS retained 30% specific activity of free lipase with greatly enhanced stability. This work demonstrates that HMMS can be efficiently employed as host materials for enzyme immobilization leading to highly enhanced stability of the immobilized enzymes with high enzyme loading and activity.     

Involvement of nitric oxide in noradrenaline-induced changes in the activity of antioxidant enzymes and lipid peroxidation in rat brown adipose tissue and heart

The effect of exogenous noradrenaline (NA) (1.6 mg.kg(-1) i.p., 35 min prior sacrifice) on the activity of antioxidant enzymes (AOE) copper zinc superoxide dismutase (CuZnSOD), manganese superoxide dismutase (MnSOD) and catalase (CAT), as well as lipid peroxides (LP) concentration were studied in the rat interscapular brown adipose tissue (IBAT) and heart of saline (controls) and N(omega)-nitro-L-arginine methyl ester (L-NAME) treated rats (10 mg.kg(-1), i.p., during 3 days and 20 min before NA). NA differently affects both AOE activities and LP production in the IBAT and heart. Thus, NA inhibited the activity of all IBAT AOE and LP production while in the heart it markedly increased CAT activity only, but had no effect on any of SODs activities and LP concentration. L-NAME, a nitric oxide synthase blocker, completely abolished the NA-induced inhibition of the IBAT AOE and LP production, whereas in the heart it was without effect. In conclusion, these results indicate that both NA and L-NAME effects on AOE activity and LP production are tissue specific and also suggest that nitric oxide mediates the NA-induced inhibition of AOE activity and LP production in the IBAT only.     

HIOMT drives the photoperiodic changes in the amplitude of the melatonin peak of the Siberian hamster

In the pineal, melatonin (Mel) is synthesized from serotonin by arylalkylamine-N-acetyltransferase (AA-NAT) and hydroxyindole-O-methyltransferase (HIOMT). Although it is clear that AA-NAT drives the daily rhythm in Mel synthesis, the mechanisms involved in the photoperiodic changes of the amplitude of the Mel peak, as observed in the Siberian hamster, remain to be determined. We investigated the characteristics of AA-NAT and HIOMT in Siberian hamsters kept either under a short (SP) or a long photoperiod (LP). The amplitude of the nocturnal peak of Mel was about two times higher under SP than under LP, whereas AA-NAT activity was about two times smaller under SP. In contrast, a twofold increase of HIOMT activity was observed under SP compared with LP. No change in the affinity of the enzymes for their substrates was observed between the two photoperiods. Our data strongly suggest that the photoperiodic variations in the amplitude of the nocturnal peak of Mel are driven by HIOMT, thereby promoting an important physiological role for this enzyme in the seasonal regulation of Mel production.     

Intracellular peroxidation processes in chronic alcohol intoxication

The data available in literature concerning the induction of lipid peroxidation (LP) with chronic alcohol administration are systematized. LP can be considered as one of the main processes leading to cellular membrane damage. The cytotoxic activity is attributed not only to the free radicals but also to the final products of the lipid hydroperoxide decomposition, such as malonic dialdehyde and 4-hydroxyalkenals. Data about antioxidative defence enzymes (glutathione peroxidase and transferase, catalase superoxide dismutase) and less investigated protein factors which inhibit LP are summarized; particular attention is paid to changes in their activity during chronic alcoholization. Molecular mechanisms underlying the LP stimulation in the liver tissue against a background of ethanol ingestion are analyzed. New data are presented on the role of peroxisomes in the development of alcohol cardiomyopathy.     

Lipoprotein metabolism in patients with diabetes of type II

Changes in contents of blood serum lipoproteins (LP) and activity of enzymes of their transformation were investigated in patients with diabetes mellitus of type II. It was found out that mechanisms of increasing of apoB-containing LP in these patients were on the one hand in enhancing of the processes of their new-formation in the liver. On the other hand, retardation of their arrival to peripheral tissues might be observed, that as a whole resulted in continuous LPLD circulation in blood. Above-mentioned processes were associated with a reciprocal decreasing of the amount of all the apoA-containing LP fractions examined. To a certain extent mechanisms of disturbance of LP metabolism in the circulation bed were stipulated by changes of function of the enzyme catalyzing their transformation.     

Comparative study of antioxidant enzymes in tissues surrounding implant in rabbits

There is a possible role of reactive oxygen species (SROS) in the complication of implants although there is presently little information. The aim of this study was to investigate the alterations in lipid peroxidation (LP) and antioxidant enzyme activities in tissues surrounding implants in rabbits. Thirty New Zealand albino male rabbits were used. They were randomly divided into five groups. The first group (I) was used as control. Groups II, III, IV and V were implanted with stainless steel, ceramic, titanium and polyethylene, respectively. One month after the administration of implant, the tissues surrounding the implant were carefully removed for antioxidant enzyme analysis. Glucose-6-phosphate dehydrogenase (G6PD), glutathione reductase (GR), superoxide dismutases (SOD), glutathione peroxidase (GPx), catalase (CAT) in tissues surrounding the implants in the groups II, III and IV were significantly (p<0.05-p<0.001) lower than in the control group although glutathione S-transferase (GST) activities and LP values were increased. CAT activity and LP level did not decrease in group V. In conclusion, these data demonstrate that there is an increase in lipid peroxidation in the tissues surrounding ceramic and titanium implants of animals whereas there is a decrease in antioxidant enzymes. Oxidative stress plays a very important role in the complications of ceramic and titanium implants. The polyethylene implant seems to be the best of the four implant materials tested.     

Photoperiodic regulation of body mass, food intake, hibernation, and reproduction in intact and castrated male European hamsters,Cricetus cricetus

A group of sexually active male European hamsters were raised either in short-photoperiod conditions (SP; LD 8:16) or in long-photoperiod conditions (LP; LD 16:8) from their capture at the end of the hibernation period. Another group of hamsters was castrated in April and gonadectomized animals were maintained in SP and cold (7 degrees C) or in a succession of SP and LP plus cold. Another group, castrated in May or in September and raised in LP conditions, was transferred in September to SP conditions and cold. 1. Normal hamsters raised in continuous SP or LP apparently did not show signs of rhythmic behavior, except possibly in gonadal activity. 2. Body weight increased continuously, plasma testosterone levels oscillated between 1.5 and 2.5 ng/ml, and animals raised in SP and in cold did not enter hibernation. 3. Similar results were also found in castrated animals kept in SP conditions and cold. 4. The sequence LP-SP induced a decrease in food intake and body weight and a decrease in plasma testosterone levels and triggered entry into hibernation in both intact and castrated animals. 5. After 6 months continuously in SP and with exposure to cold spontaneous recrudescence in food intake and body weight occurred and hibernation ended in both intact and castrated animals. 6. In normal animals a spontaneous increase in plasma testosterone levels was observed. 7. In both normal and gonadectomized animals the phase of refractoriness could be broken by exposure to LP conditions. 8. The critical photoperiod lies between 15 and 15.5 h. These results demonstrate that the European hamster is a photoperiodic species.(ABSTRACT TRUNCATED AT 250 WORDS)     

Lipid peroxidation in the myocardium in experimental infarct

Changes in the content of lipid peroxidation (LP) products and activities of antioxidant enzymes--superoxide dismutase, glutathione peroxidase and catalase in myocardium of rats after experimental infarction as well as after pretreatment with antioxidant ionol, beta-adrenoblocker inderal and verapamil, an inhibitor of slow Ca2+-channels have been studied. In the left ventricles of the control animals decreased levels of LP-products (Schiff bases and lipid hydroperoxides) have been registered as compared with right ventricles, accompanied by increased activity of antioxidant enzymes in the left ventricles. In experimental infarction the level of LP products increases and activity of antioxidant enzymes decreases both in ischemic and nonischemic regions of the heart. In nonischemic zone these changes can be prevented by pretreatment with inderal and ionol but not with verapamil.     

Regulation of enzymes and isoenzymes of carbohydrate metabolism in the yeast Saccharomyces cerevisiae

An electrophoretic method has been devised to investigate the changes in the enzymes and isoenzymes of carbohydrate metabolism, upon adding glucose to derepressed yeast cells. (i) Of the glycolytic enzymes tested, enolase II, pyruvate kinase and pyruvate decarboxylase were markedly increased. This increase was accompanied by an overall increase in glycolytic activity and was prevented by cycloheximide, an inhibitor of protein synthesis. (ii) In contrast, respiratory activity decreased after adding glucose. This decrease was clearly shown to be the result of repression of respiratory enzymes. A rapid decrease within a few minutes of adding glucose, by analogy with the so-called ' Crabtree effect', was not observed in yeast. (iii) The gluconeogenic enzymes, fructose-1,6-bisphosphatase and malate dehydrogenase, which are inactivated after adding glucose, showed no significant changes in electrophoretic mobilities. Hence, there was no evidence of enzyme modifications, which were postulated as initiating degradation. However, it was possible to investigate cytoplasmic and mitochondrial malate dehydrogenase isoenzymes separately. Synthesis of the mitochondrial isoenzyme was repressed, whereas only cytoplasmic malate dehydrogenase was subject to glucose inactivation.     

Metabolism of androgens in the seminal vesicles and the different lobes of the prostate in young mature rats

Oxidation and reduction of 4-androstene-3,17-dione (androstenedione), 17 beta-hydroxy-4-androsten-3-one (testosterone), 17 beta-hydroxy-5 alpha-androstan-3-one (DHT), 5 alpha-androstan-3 alpha,17 beta-diol (3 alpha-A'diol) and 5 alpha-androstane-3 beta,17 beta-diol (3 beta-A'diol) were measured in homogenates from ventral (VP), dorsal (DP) and lateral prostate (LP), the coagulating gland (CG) and seminal vesicle (SV) of the intact sexually mature rat using NAD(H) or NADP(H) as cofactors. The specific activity of the various enzymes varied significantly between the different organs. 5 alpha-Reductase activity was highest in the DP and the CG, and undetectable in the LP. 17 beta-Hydroxysteroid oxidoreductase (17 beta-HSOR) activity was mainly confined to the LP. 3 alpha-Hydroxysteroid oxidoreductase (3 alpha-HSOR) activity was also highest in the LP. In the VP the highest 3 alpha-HSOR activity was recorded using NAD(H) as cofactor. In the other organs, similar or higher enzymatic activities were measured using NADP(H) as added cofactor. 3 beta-Hydroxysteroid oxidoreductase (3 beta-HSOR) activity was high in the LP and low or undetectable in the other tissues. Our results indicate that isoenzymes of 3 alpha-HSOR, 3 beta-HSOR and 17 beta-HSOR are present in the accessory sex organs of the rat.     

Effects of UV Radiation on Photolyase and Implications with Regards to Photoreactivation following Low- and Medium-Pressure UV Disinfection

Photolyase activity following exposure to low-pressure (LP) and medium-pressure (MP) UV lamps was evaluated. MP UV irradiation resulted in a greater reduction in photolyase activity than LP UV radiation. The results suggest that oxidation of the flavin adenine dinucleotide in photolyase may have caused the decrease in activity.