Studies of the alkaline phosphatase in the sperm, testes and accessory sex glands of bulls

Studied were the activity and properties of alkaline phosphatase (AP) in the seminal plasma, spermatozoa washed with physiologic saline, testes, and accessory sexual glands of a bull. The AP activity was highest in the seminal plasma (2493.2 IU/l) and lowest in the Kupffer gland (257.3 IU/kg crude tissue, on an average). In washed spermatozoa it proved by 10 per cent lower than the activity in seminal plasma. At 56 degrees C as much as 60-80 per cent of the AP in the investigated materials was inactivated for 30 min. AP was shown to be inactivated strongly (up to 84-97 per cent) by urea (3.8 M). L-arginine (10(-2) M) and EDTA (10(-3) M) inactivated to an equal extent AP in all studied organs. L-phenylalanine inactivated AP weakly (13-15 per cent) in the testis and the epididymis, and more strongly (32-57 per cent) in the accessory glands and the plasma. Agar electrophoresis revealed three to four isoenzymes of AP in the seminal plasma, three isoenzymes in the testis and the epididymis, and two isoenzymes in the accessory sexual glands and in spermatozoa washed with physioogic saline.     

Serum total, heat and urea stable alkaline phosphatase activities in relation to ABO blood groups and secretor phenotypes

The relationship between serum alkaline phosphatase (AP) isoenzymes, ABO blood groups and secretor phenotypes was evaluated in 125 Nigerian voluntary blood donors. The serum total AP activity patterns were group O > Group B > Group AB > Group A, but only the differences between A/B, A/O and AB/O were significant. The total and activities of the different isoenzymes were lowest in group A, while highest values were found in group O. The different activities for group AB were intermediate between the levels for A and B. The activities of the different isoenzymes tended to be higher in secretors than in non-secretors. Our results may suggest that both the blood group and secretor genes are strong determinants of AP isoenzyme patterns in Nigeria.     

Hemagglutinating activity of extracellular alkaline metalloendopeptidases from Vibrio sp. NUF-BPP1

Alkaline metalloendopeptidase (metalloprotease) AP1 (48 kDa) from Vibrio sp. isolated from the intestine of a five-barred goatfish (Parupeneus trifasciatus) was reported in our previous paper to produce AP2 (36 kDa) by releasing a peptide fragment (molecular mass of about 12 kDa) from the C-terminal end of AP1 by autodigestion. AP1 strongly agglutinated fish (flounder, Paralichthys olivaceus) and rabbit erythrocytes, and weakly chicken erythrocytes. In contrast, AP2 had no significant hemagglutinating activity toward any erythrocytes tested, except for weak activity on flounder erythrocytes, suggesting that the C-terminal region of AP1 may be required for the strong hemagglutinating activity. The optimum temperature for the hemagglutinating activity of AP1 was found to be lower than that for the proteolytic activity. At acidic pHs (below pH 7.5), the hemagglutinating activity of AP1 decreased, and its pH profile resembled that of the proteolytic activity. The hemagglutinating activity of AP1 was not observed in the presence of o-phenanthroline or synthetic and proteinous substrates, but different kinds of saccharides and lipids had no effect. While the proteolytic activity of AP1 was not affected by CaCl2, the hemagglutinating activity of AP1 decreased with increases in CaCl2 concentrations. These results suggested that the hemagglutinating activity of these proteases (AP1 and AP2) was most likely caused by their proteolytic action on erythrocyte cell surfaces.     

Interferon and tumor necrosis factor production by peripheral blood leukocytes of patients with infectious mononucleosis

Blood samples from 29 patients with infectious mononucleosis (IM) in phases of acute disease and convalescence were obtained. Interferon alpha (IFN-alpha) and tumor necrosis factor alpha (TNF-alpha) activity was detected in sera of patients both in: acute and convalescence phase, however when IFN titers were higher in the acute than convalescence phase, TNF titers were the highest in convalescence. In the whole blood assay Newcastle disease virus (NDV), phytohemagglutinin (PHA) and concanavalin A (ConA) and lipopolysaccharide (LPS) were used as cytokine inducers. A significant decrease in IFN titer induced in vitro with NDV, PHA and ConA was observed in blood leukocytes of patients in the acute IM phase. In convalescence the ability of blood leukocyte of IM patients to produce IFN returned to normal, comparable with control. However, blood leukocytes of IM patients in the acute phase produced more TNF in response to LPS than in convalescence. The role of the observed overproduction of TNF in the course of IM similar to that in HIV infection should be elucidated.     

Acute hepatic coma. Experimental study on the predictive value of clinical-chemical findings for the prognosis of acute hepatic coma

In order to determine the validity of clinical-chemical parameters for the prognosis of hepatic failure, 28 pigs were subjected to liver ischemia for 40--160 minutes duration. The following parameters were studied: GOT, GPT, gamma-GT, LAP, LDH, GlDH, AP and isoenzymes, total bilirubin, potassium, sodium and chloride. In a statistical comparison in the surviving animals, an unexplainable increase in GlDH activity was observed. In the other clinical-chemical parameters none was seen to be of use for the prognosis for either life or death in acute hepatic failure.     

Serum lactate-dehydrogenase isoenzymes in HIV positive children. A longitudinal study

The impairment of humoral immunity with rapid turn-over of cellular B clones in children with HIV infection is known as well as the conduct of LDH isoenzymes in B cell lymphoproliferative diseases like Burkitt's lymphoma. Therefore, serum lactate-dehydrogenase activity (LD, EC 1.1.1.27) and its isoenzymes have been evaluated twice (within 12 months) in 11 children with HIV infection with respect to a control group (30 subjects). Furthermore, the relationship between those and other clinical and immunologic parameters (total lymphocytes, CD4/CD8, immunoglobulins, classification according to the Atlanta CDC 1987) has been studied. HIV infected children have shown a significant decrease in LD1 rates, which was directly correlated to CD4/CD8 values. After the follow-up, this correlation became even more significant. Thus, these findings may suggest the usefulness of LDH isoenzymes evaluation as a marker of disease activity in children with HIV infection.     

Effects of lesions to amygdala, ventral subiculum, medial prefrontal cortex, and nucleus accumbens on the reaction to novelty: Implications for limbic—striatal interactions.

The effects of bilateral excitotoxic lesions of 3 major sources of afferents to the ventral striatum (nucleus accumbens) were compared on an open field test of food neophobia allowing the choice between familiar and novel food. Whereas lesions of the basolateral amygdala and ventral subiculum had qualitatively similar effects to reduce food neophobia (although not affecting the latency to eat), amygdala lesions increased and the ventral subiculum decreased locomotor activity. In contrast, damage to the ventromedial prelimbic prefrontal cortex only affected initial food choice and latency measures. By comparison, excitotoxic lesions of the nucleus accumbens itself and intra-accumbens infusion of the N-methyl-{d}-aspartate (NMDA) receptor antagonist AP5 increased activity and attenuated food neophobia. Results are discussed in terms of the role of limbic and prefrontal neuronal networks converging in the nucleus accumbens to control different aspects of the behavioral response to novelty. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Strategies to improve the nutritive value of rice bran in poultry diets. IV. Effects of addition of fish meal and a microbial phytase to duckling diets on bird performance and amino acid digestibility

1. Ducklings were given diets with vegetable protein (VP) and 0 or 600 g rice bran/kg; fish meal (60 g/kg) and a phytase (+, -) were added to the diets (VP + AP). An additional 40 g soyabean meal/kg was added to the diet with rice bran (VP ++). Amino acid digestibility and mineral retention were measured in the lower ileum of ducklings killed at 23 d of age. Acid insoluble ash was used as an inert marker. Trypsin and amylase activities were also measured and weights of the pancreas and small intestine recorded at slaughter. 2. Addition of soyabean meal (VP ++) to the diet with rice bran improved growth rate and food intake compared to the diet without (VP) and gave the same food intake and growth rate as the comparable basal diet (VP) without rice bran. Fish meal improved growth rate on the diets without rice bran and improved food intake on this diet (VP + AP). Rice bran depressed growth rate and food conversion ratio (FCR); protein source affected growth rate, food intake and FCR; phytase increased food intake only. There were several interactions. 3. Determined total amino acid composition of the diets appeared to meet the essential amino acid requirements of ducklings. Rice bran depressed the ileal digestibility of virtually all amino acids and phytase had no direct effect, although there were interactions. Fish meal addition to diets with rice bran improved the apparent digestibility of several essential amino acids as well as that of dry matter and crude protein. 4. Ileal retention of some minerals and tibia ash content were reduced by rice bran. Fish meal and phytase inclusion increased P retention and ash in tibia. 5. Higher intestinal trypsin activity and increased pancreas size were seen in ducklings on diets with rice bran compared to those without. Intestinal amylase activity was reduced in ducklings given rice bran, probably because of its low starch content. 6. The stimulating effect of fish meal on duckling performance was probably caused in part by the improvement in the digestibility of some amino acids. The addition of small amounts of minerals in fish meal may have increased mineral retention. Phytase gave benefits anticipated from our previous work, but also improved lysine and threonine digestibility in diets containing vegetable protein only.     

Amylin reduces food intake more potently than calcitonin gene-related peptide (CGRP) when injected into the lateral brain ventricle in rats

Amylin and the structurally and functionally related peptide calcitonin gene-related peptide (CGRP) have been shown to reduce food intake in rats. The aim of the present study was to compare the anorectic potency of both peptides over a wide dose range when administered into the lateral brain ventricle (ICV). Furthermore, we also tested the influence of a lesion in the area postrema/nucleus of the solitary tract (AP/NTS) region on the anorectic effects of amylin and CGRP after ICV administration because AP/NTS lesion has been shown to reduce the anorectic effects of both peptides when injected intraperitoneally (IP). Amylin [1-510 pmol/rat (0.004-2 microg/rat) ICV] and CGRP [1-131 pmol/rat (0.004-0.5 microg/rat) ICV] dose-dependently reduced food intake in food-deprived rats. At a dose of 26 pmol/rat (0.1 microg/rat), amylin almost completely suppressed food intake for 1 h after injection. Amylin [EC50 = 2 pmol/rat (0.007 microg/rat)] was markedly more potent than CGRP [57 pmol/rat (0.215 microg/rat)] with regard to its anorectic effect. A lesion in the AP/NTS region did not influence the anorectic effects of amylin and CGRP after administration into the lateral ventricle. It is concluded that amylin is more potent than CGRP in reducing food intake after administration into the lateral brain ventricle. Receptors in the forebrain may mediate the anorectic effects of both peptides when administered via this route.     

Purification of peroxidase isoenzymes from turnip roots

Simple reproducible procedures for purification of the main soluble (S) and ionically bound (IB) cationic peroxidase isoenzymes from turnip roots were established. The procedures included ammonium sulfate precipitation of the isoenzymes, chromatographic separation of the main isoenzymes using cellulose phosphate columns and purification to homogeneity by hydrophobic interaction chromatography on phenyl Sepharose columns. The specific activity of the phenyl Sepharose purified S and IB isoenzymes were 2760 and 896 units/mg protein with 140 and 4.8 fold increase over the crude extract and 38 and 13% recovery. The pH maxima and K(m) for phenol and H2O2 of purified S and IB were determined.     

Effect of intraocular lens design on neodymium:YAG laser capsulotomy rates

The activity of alkaline phosphatase (AP) was studied in adults of highly active (HA) and low active (LA) Drosophila melanogaster strains and their F1 hybrids, both under normal conditions and after a heat shock (38 degrees C). Under normal conditions, the HA strain expressed a higher AP activity compared to that in the wild-type strain Canton-S and dominated in respect to this character. The AP activity showed a sexual dimorphism, as it was higher in females of both strains. Heat shock (38 degrees C) induced no alterations in the AP activity of D. melanogaster.     

N-acetyl-beta-hexosaminidase activity and isoenzymes in human gastric adenocarcinoma

The enhancement of lysosomal beta-hexosaminidase degradative activity in different human cancer tissues is fairly well documented. Gastric tumors have attracted considerable attention on the basis of their social incidence and clinical recurrence. Here we report a comparative study of beta-hexosaminidase activity and of its isoenzymes beta-hexosaminidase A (HA) and beta-hexosaminidase B (HB) from gastric adenocarcinoma and normal mucosa. Tumor beta-hexosaminidase activity from crude extracts and chromatographically resolved HA and HB forms were analyzed as regards their physicochemical and enzymatic properties and were compared to similar samples obtained from control tissue. The existence of one active site in the beta-hexosaminidase enzyme responsible for both N-acetyl-beta-D-glucosaminidase and N-acetyl-beta-D- galactosaminidase activities was determined. Apart from their relative contributions to beta-hexosaminidase activities, two major differences appeared in tumor HA and HB forms with respect to the corresponding controls: (1) the presence of an atypical heat-stable HB isoenzyme in gastric adenocarcinoma, and (2) a significantly increased Vmax of the HA form acting on both p-nitrophenyl-N-acetyl-beta-D-glucosaminide and p-nitrophenyl-N-acetyl-beta-D-galactosaminide substrates. The results show that the beta-hexosaminidase HA and HB isoenzymes from gastric adenocarcinoma display different patterns of response from the same forms from other human tumors.     

Nucleotide excision repair 3' endonuclease XPG stimulates the activity of base excision repairenzyme thymine glycol DNA glycosylase

An ionizing radiation-induced DNA lesion, thymine glycol, is removed from DNA by a thymine glycol DNA glycosylase with an apurinic/apyrimidinic (AP) lyase activity encoded by the Escherichia coli endonuclease III ( nth ) gene and its homolog in humans. Cells from Cockayne syndrome patients with mutations in the XPG gene show approximately 2-fold reduced global repair of thymine glycol. Hence, I decided to investigate the molecular mechanism of the effect of XPG protein observed in vivo on thymine glycol removal by studying the interactions of XPG protein and human endonuclease III (HsNTH) protein in vitro and the effect of XPG protein on the activity of HsNTH protein on a substrate containing thymine glycol. The XPG protein stimulates the binding of HsNTH protein to its substrate and increases its glycosylase/AP lyase activity by a factor of approximately 2 through direct interaction between the two proteins. These results provide in vitro evidence for a second function of XPG protein in DNA repair and a mechanistic basis for its stimulatory activity on HsNTH protein.     

Transcription factor AP2 is required for expression of the rat transforming growth factor-alpha gene

DNase I footprinting of the rat TGF alpha promoter in the presence of crude cell nuclear extract revealed three sites of protein-DNA interaction (Fp-A, Fp-B, Fp-C) in the region from -222 to +73. Mutation of specific sites within the Fp-A and Fp-B regions reduced expression of a TGF alpha promoter-reporter gene (TGF alphaLUC) from 50-90% in transiently transfected CHO cells, indicating the importance of protein/DNA interactions at these sites. Since Fp-A contained a perfect AP2 consensus sequence (5'-GCCNNNGGC-3') as its center, we investigated the possibility that AP2 binding is important for TGF alpha promoter activity. A double-stranded oligonucleotide spanning Fp-A displayed a distinct mobility shift in the presence of nuclear extract that was inhibited by an excess of known functional AP2-binding sequence. Moreover, a similar mobility shift occurred in the presence of purified AP2 protein, and the further addition of AP2 antibody produced a supershifted complex. More refined DNase I footprinting of a smaller, oligonucleotide probe in the presence of purified AP2 protein revealed a protected region that included the putative AP2 binding site. Additionally, co-transfection of an AP2 expression vector increased TGF alphaLUC expression 25-fold in Drosophila Schneider cells. These various findings corroborate a role for AP2 in TGF alpha promoter activity. The Fp-B region contains a T5 motif that has been previously suggested to function as an atypical TATA box. An Fp-B oligonucleotide displayed a specific gel mobility shift in the presence of a TATA binding protein (TBP)-TFIIA complex, and the further addition of TBP antibody produced a supershift. These results confirm that protein binding within Fp-B is functionally important, and they also indicate that the T5 motif functions as a TBP binding site.     

Hexokinase isoenzymes in the rat placenta

The phosphorylation of glucose to glucose-6-phosphate, the first enzymatic step for glucose utilization is catalysed by a family of four hexokinase isoenzymes (HKI-IV) which display a tissue-specific distribution. The expression of HK isoenzymes was investigated in the rat placenta. High levels of HKI and HKII mRNA were found in the junctional and the labyrinthine zones. HKIII mRNA was present at low levels in the junctional zone and glucokinase (HKIV) mRNA was not detected, indicating that HKI and HKII are the two major placental HK isoenzymes. HKII activity was increased in placenta of insulinopenic diabetic rats. This regulation is likely to support the increase in glucose utilization and storage characteristics of the enlarged placentae of diabetic rats.     

Human CYP2D6 and metabolism of m-chlorophenylpiperazine

BACKGROUND: Metabolic drug-drug interactions can occur between drugs that are substrates or inhibitors of the same cytochrome P450 (CYP) isoenzymes, but can be prevented by knowing which isoenzymes are primarily responsible for a drug's metabolism. m-Chlorophenylpiperazine (mCPP) is a psychopharmacologically active metabolite of four different psychiatric drugs. The present experiments were designed to identify the CYP isoenzymes involved in the metabolism of mCPP to its main metabolite p-hydroxy-mCPP (OH-mCPP). METHODS: The rate of production of OH-mCPP from mCPP was correlated with isoform activities in a panel of human liver microsomes, was assessed using a panel of individual complementary DNA-expressed human CYP isoenzymes, and was investigated in the presence of a specific inhibitor of CYP2D6. RESULTS: OH-mCPP production correlated significantly with CYP2D6 activity in human liver microsomes. Furthermore, incubations with microsomes from cells expressing CYP2D6 resulted in OH-mCPP formation, whereas no mCPP was formed from incubations with microsomes from cells expressing other individual isoforms. Finally, when the specific CYP2D6 inhibitor quinidine was preincubated with either human liver microsomes or cells expressing human CYP2D6, there was a concentration-dependent decrease in the production of OH-mCPP. CONCLUSIONS: These results confirm that CYP2D6 is the isoform responsible for the p-hydroxylation of mCPP, and indicate that caution should be exercised in coprescribing inhibitors or substrates of CYP2D6 with drugs that have mCPP as a metabolite.     

Molecular cloning and expression of a cDNA encoding a novel isoenzyme of protein kinase C (nPKC). A new member of the nPKC family expressed in skeletal muscle, megakaryoblastic cells, and platelets

At least seven bacteriophage lambda clones encoding structurally related but unique polypeptides with PKC activity have been isolated from mammalian brain, epidermis, and lung cDNA libraries. The possibility that additional isoenzymes are expressed in human blood platelets or megakaryoblastoid human erythroleukemia cells was examined by polymerase chain reaction amplification of reverse transcribed RNA employing oligonucleotide primers corresponding to conserved peptide sequences. cDNAs encoding a novel PKC-related sequence, designated PKC-theta, and four (alpha, beta, delta, and eta) previously identified isoenzymes were isolated from reverse transcribed total RNA of human erythroleukemia cells and platelets. PKC-theta lacks a conserved region (C2) that is present in the calcium-dependent isoenzymes and therefore belongs to the group of novel, or nPKC, isoenzymes. Significantly increased [3H] phorbol 12,13-dibutyrate binding and cytoskeleton-associated calcium-independent PKC activity were found in COS cells expressing the transfected cDNA. Northern transfer analysis of mRNA from various human tissues revealed high level expression of PKC-theta in skeletal muscle, lung, and brain, and minimal expression in cardiac muscle, placenta, and liver. These findings extend the PKC family and suggest a novel approach to the study of diversity within this pathway of intracellular signal transduction.     

Purification and characterization of hepatic glutathione transferases from an insectivorous marsupial, the brown antechinus (Antechinus stuartii)

1. Five unique glutathione transferase isoenzymes were purified from the hepatic cytosol of an insectivorous marsupial, the brown antechinus. The purified GSTs were characterized by structural and catalytic properties including apparent molecular weight and isoelectric point, specificity towards model substrates, kinetic parameters, sensitivity to inhibitors and cross-reactivity with antisera raised against human GSTs. 2. An alpha class GST, Antechinus GST 1-1, predominated in the hepatic cytosol, representing 71% of the total GST purified. The substrate specificity of Antechinus GST 1-1 was similar to that of other alpha class GSTs, particularly with respect to its high activity with cumene hydroperoxide. The mu class was represented by three GST isoenzymes, Antechinus GST 3-3, GST 3-4 and GST 4-4. These isoenzymes represented 8, 2 and 10% of the total GST purified respectively. A single GST, Antechinus GST 22, belonged to the pi class of GSTs and represented 12% of the total GST purified. The hepatic GST isoenzyme ratio (by class) observed in the brown antechinus was more similar to that observed in the human than in rat. 3. A previous study investigating a herbivorous marsupial, the brushtail possum (Trichosurus vulpecula) also identified a predominant hepatic GST belonging to the alpha class and displaying peroxidase activity. The evolutionary conservation of a similar predominant GST isoenzyme in these marsupials suggests that they play an important role in the detoxication metabolism of these unique mammals.     

The role of colour and duplex Doppler ultrasound in the assessment of thyroid nodules

After chronic sympathectomy or sinoaortic denervation (SAD), arterial pressure (AP) becomes extremely unstable, especially because of movement-related depressor episodes. The simultaneous measurement of AP and regional blood flows in sympathectomized and SAD rats indicates that these depressor episodes are accompanied by strong regional vasodilations, possibly involving an autoregulatory component. The sympathetic nervous system, mainly through baroreflex modulation of its activity, overrides these responses and thereby, considerably limits the AP variability. In the conscious unrestrained rat, AP fluctuates in a narrow range (variation coefficients calculated over 1-hour beat-to-beat recordings are typically approximately 5%). This variability of AP involves sympathetically-mediated pressor episodes that are coupled to behavior and alerting environmental stimuli. Regarding the latter, studies in SAD rats point to an opposing interaction between centrally-induced sympathoexcitation and baroreflex activation. Another component of normal AP variability appears as an oscillation centered around 0.4 Hz. Spectral analysis of AP and regional hemodynamic variables indicates that this oscillation is secondary to rhythmic fluctuations in the vasomotor sympathetic tone that are synchronized by the arterial baroreceptor reflex. It is concluded that both stability and normal variability of AP critically depend on the baroreflex control of the sympathetic vascular tone.