Role of PGF2α in luteolysis based on inhibition of PGF2α synthesis in the mare

The effects of inhibition of PGF2α synthesis on luteolysis in mares and on the incidence of prolonged luteal activity were studied in controls and in a group treated with flunixin meglumine (FM), a PGF2α inhibitor (n = 6/group). The FM was given every 8 hours (1.0 mg/kg) on each of Days 14.0 to 16.7. Concentration (pg/mL) of PGF2α metabolite averaged over 8 hours of hourly blood sampling at the beginning of each day, was lower in the FM group than in the controls on Day 14 after ovulation (6.7 ± 1.3 vs. 13.8 ± 2.9, P < 0.05), Day 15 (15.0 ± 3.9 vs. 35.2 ± 10.4, P < 0.10), and Day 16 (21.9 ± 5.7 vs. 54.7 ± 11.4, P < 0.03). Concentration (ng/mL) of progesterone (P4) was greater in the FM group than in the controls on Day 14 (10.1 ± 0.9 vs. 7.7 ± 0.9, P < 0.08), Day 15 (9.2 ± 1.0 vs. 4.3 ± 1.0, P < 0.008), and Day 16 (5.6 ± 1.6 vs. 1.2 ± 0.4, P < 0.02). The interval from ovulation to the beginning of a decrease in P4 and to the end of luteolysis (P4 < 1 ng/mL) was each delayed (P < 0.03) by ∼1 day in the FM group. Intervals involving the luteal phase were long (statistical outliers, P < 0.05) in two mares in the FM group, indicating prolonged luteal activity. Results supported the hypotheses that (1) inhibition of PGF2α synthesis interferes with luteolysis in mares and (2) inhibition of PGF2α at the expected time of luteolysis may lead to prolonged luteal activity.     

Metabolites of PGF2α in Blood Plasma and Urine as Parameters of PGF2α Release in Cattle

The metabolism of PGF2α in cattle results initially in the formation of 15-keto-13,14-dihydro-PGF2α (15-ketodihydro-PGF2α) and later the 11-ketotetranor PGF metabolites. Both types of metabolites appear in the peripheral circulation and finally the 11-ketotetranor PGF metabolites are found in large quantities in the urine in a species-related pattern. Several approaches can be made to the quantitative analysis of PGF2α release during reproductive studies. First, assay of the 15-ketodihydro-PGF2α metabolite in the peripheral circulation; second, analysis of the longer-lived 11-ketotetranor PGF metabolites in the peripheral circulation; and finally analysis of the latter metabolites in the urine. The antibodies used in radioimmunoassays of both types of metabolites of PGF2α were found to be specific and the results agree well with those obtained earlier by mass spectrometric analysis. The assay of 11-ketotetranor PGF metabolites was used to study the excretion of urinary metabolites in the cow after i.v. infusion of PGF2α and also during the normal estrous cycle and early pregnancy. These studies suggest that 11-ketotetranor PGF metabolites in cow urine serve as a good parameter of PGF2α release, especially for long–term studies, but when a precise pattern of PGF2α release is required, measurement of 15-ketodihydro-PGF2α levels in frequently collected plasma samples is preferable.     

Heterogeneity of β2-Microglobulin in Human Urine

Purification and characterization of β₂-microglobulin from human urine was performed. The yield was 30.1%, and 150.4 mg of β₂-microglobulin was obtained. The final preparation of β₂-microglobulin obtained showed three bands on disc gel electrophoresis at pH 9.5, and all of them have immunological activity. However, these three bands migrated as a single band on disc gel electrophoresis at pH 4.3. It is concluded that the three bands observed on disc gel electrophoresis at pH 9.5 were charge isomers. The isoelectric points of isomers were determined by isotachophoresis and two of them were 5.4 and 5.9 respectively, while the other one was not determined.     

Involvement of γ-aminobutyric acid transporter 2 in the hepatic uptake of taurine in rats

Taurine is essential for the hepatic synthesis of bile salts and, although taurine is synthesized mainly in pericentral hepatocytes, taurine and taurine-conjugated bile acids are abundant in periportal hepatocytes. One possible explanation for this discrepancy is that the active supply of taurine to hepatocytes from the blood stream is a key regulatory factor. The purpose of the present study is to investigate and identify the transporter responsible for taurine uptake by periportal hepatocytes. An in vivo bolus injection of [(3)H]taurine into the rat portal vein demonstrated that 25% of the injected [(3)H]taurine was taken up by the liver on a single pass. The in vivo uptake was significantly inhibited by GABA, taurine, β-alanine, and nipecotic acid, a GABA transporter (GAT) inhibitor, each at a concentration of 10 mM. The characteristics of Na(+)- and Cl(-)-dependent [(3)H]taurine uptake by freshly isolated rat hepatocytes were consistent with those of GAT2 (solute carrier SLC6A13). Indeed, the K(m) value of the saturable uptake (594 μM) was close to that of mouse SLC6A13-mediated taurine transport. Although GABA, taurine, and β-alanine inhibited the [(3)H]taurine uptake by > 50%, each at a concentration of 10 mM, GABA caused a marked inhibition with an IC(50) value of 95 μM. The [(3)H]taurine uptake exhibited a significant reduction when the GAT2 gene was silenced. Immunohistochemical analysis showed that GAT2 was localized on the sinusoidal membrane of the hepatocytes predominantly in the periportal region. These results suggest that GAT2 is responsible for taurine transport from the circulating blood to hepatocytes predominantly in the periportal region.     

Study of the Efficacy of a Pronucleotide of 2-Chloro-2′-Deoxyadenosine in Deoxycytidine Kinase-Deficient Lymphoma Cells

2-Chloro-2 ′-deoxyadenosine (CdA, cladribine) is a nucleoside analogue (NA) used for the treatment of lymphoproliferative disorders. Phosphorylation of the drug to CdAMP by deoxycytidine kinase (dCK) and its subsequent conversion to CdATP is essential for its efficacy. DCK deficiency is a common mechanism of resistance to NA, which could be overcome by the pronucleotide approach. The latter consists of using the nucleoside monophosphate conjugated to a lipophilic group enabling CdAMP to enter the cells by passive diffusion. In this study, we show that cycloSaligenyl-2-chloro-2 ′-deoxyadenosine monophosphate (cycloSal-CdAMP) is 10-fold more potent that CdA in a dCK-deficient lymphoma cell line. These results suggest that the use of cycloSal-nucleotides could be a strategy to counteract resistance caused by dCK deficiency.     

Role of Zinc in Zinc-α2-Glycoprotein Metabolism in Obesity: a Review of Literature

Biological Trace Element Research - Excessive adipose tissue promotes the manifestation of endocrine disorders such as reduction of the secretion of zinc-α2-glycoprotein (ZAG), an adipokine...     

Critical Function of γH2A in S-Phase

Phosphorylation of histone H2AX by ATM and ATR establishes a chromatin recruitment platform for DNA damage response proteins. Phospho-H2AX (γH2AX) has been most intensively studied in the context of DNA double-strand breaks caused by exogenous clastogens, but recent studies suggest that DNA replication stress also triggers formation of γH2A (ortholog of γH2AX) in Schizosaccharomyces pombe. Here, a focused genetic screen in fission yeast reveals that γH2A is critical when there are defects in Replication Factor C (RFC), which loads proliferating cell nuclear antigen (PCNA) clamp onto duplex DNA. Surprisingly Chk1, Cds1/Chk2 and the Rad9-Hus1-Rad1 checkpoint clamp, which are crucial for surviving many genotoxins, are fully dispensable in RFC-defective cells. Immunoblot analysis confirms that Rad9-Hus1-Rad1 is not required for formation of γH2A by Rad3/ATR in S-phase. Defects in DNA polymerase epsilon, which binds PCNA in the replisome, also create an acute need for γH2A. These requirements for γH2A were traced to its role in docking with Brc1, which is a 6-BRCT-domain protein that is structurally related to budding yeast Rtt107 and mammalian PTIP. Brc1, which localizes at stalled replication forks by binding γH2A, prevents aberrant formation of Replication Protein A (RPA) foci in RFC-impaired cells, suggesting that Brc1-coated chromatin stabilizes replisomes when PCNA or DNA polymerase availability limits DNA synthesis.     

β2-Adrenergic regulation of prostaglandin D2 receptor in rabbit platelets

[³H]Prostaglandin D₂ binding to rabbit platelets was increased by about 150% in the presence of β-adrenoceptor agonist, isoproterenol. The isoproterenol-induced potentiation of the [³H]prostaglandin D₂ binding gave a bell-shaped dose-response relationship (maximum response at 3·10⁻⁸ M) in a stereospecific manner. Similar and moderate potentiation was obtained with terbutaline. On the other hand, β-adrenoceptor antagonists such as alprenolol, propranolol and butoxamine (β₂-specific) had no potentiating effect on [³H]prostaglandin D₂ binding; rather, they abolished the isoproterenol-induced increase of [³H]prostaglandin D₂ binding. The β₁-specific antagonist, metoprolol, did not have any effect. Rabbit platelets were found to possess one [³H]prostaglandin D₂ binding site (K_d = 6·10⁻⁷ M, B_max = 787 fmol/mg protein). In the presence of isoproterenol at 3·10⁻⁸ M, B_max was increased with unaltering K_d value. Isoproterenol did not increase [³H]prostaglandin E₁, [³H]prostaglandin E₂ and [³H]prostaglandin F_2α bindings to platelets. The potential effect of isoproterenol was mimicked by forskolin, theophylline, dibutyryl cyclic AMP, prostaglandin E₁ and prostaglandin I₂, but it was abolished by 2′, 5′-dideoxyadenosine, an inhibitor of adenylate cyclase, indicating that elevated level of cyclic AMP may be available for the induction of the increase of [³H]prostaglandin D₂ binding. Prostaglandin D₂-induced cyclic AMP synthesis and antiaggregation activity were also augmented in the presence of isoproterenol. These results suggest a β₂-adrenoceptor-mediated cyclic AMP-dependent mechanism for the regulation of prostaglandin D₂ receptor binding in rabbit platelets.     

The involvement of microRNAs in Type 2 diabetes

T2D (Type 2 diabetes mellitus) is a major health issue that has reached epidemic status worldwide. T2D is a progressive metabolic disorder characterized by reduced insulin sensitivity, insulin resistance and pancreatic β-cell dysfunction. Improper treatment of TD2 can lead to severe complications such as heart disease, stroke, kidney failure, blindness and nerve damage. The aetiology and molecular mechanisms of T2D are not fully understood, but compelling evidence points to a link between T2D, obesity, dyslipidaemia and insulin resistance. Although T2D seems to be strongly linked to environmental factors such as nutrition and lifestyle, studies have shown that genetic factors, such as polymorphisms associated with metabolic genes, imprinting, fetal programming and miRNA (microRNA) expression, could also contribute to the development of this disease. miRNAs are small 22-25-nt-long untranslated RNAs that negatively regulate the translation of mRNAs. miRNAs are involved in a large number of biological functions such as development, metabolism, immunity and diseases such as cancer, cardiovascular diseases and diabetes. The present review examines the various miRNAs that have been identified as being potentially involved in T2D, focusing on the insulin-sensitive organs: white adipose tissue, liver, skeletal muscle and the insulin-producing pancreatic β-cells.     

A novel anti-proliferative role of HMGA2?in induction of apoptosis through caspase 2?in primary human fibroblast cells

The HMGA2 (high-mobility group AT-hook) protein has previously been shown as an oncoprotein, whereas ectopic expression of HMGA2 is found to induce growth arrest in primary cells. The precise mechanisms underlying this phenomenon remain to be unravelled. In the present study, we determined that HMGA2 was able to induce apoptosis in WI38 primary human cells. We show that WI38 cells expressing high level of HMGA2 were arrested at G2/M phase and exhibited apoptotic nuclear phenotypes. Meanwhile, the cleaved caspase 3 (cysteine aspartic acid-specific protease 3) was detected 8 days after HMGA2 overexpression. Flow cytometric analysis confirmed that the ratio of cells undergoing apoptosis increased dramatically. Concurrently, other major apoptotic markers were also detected, including the up-regulation of p53, Bax and cleaved caspase 9, down-regulation of Bcl-2; as well as release of cytochrome c from the mitochondria. We further demonstrate that the shRNA (small-hairpin RNA)-mediated Apaf1 (apoptotic protease activating factor 1) silencing partially rescued the HMGA2-induced apoptosis, which was accompanied by the decrease of cleaved caspase-3 level and a decline of cell death ratio. Our results also reveal that γH2A was accumulated in nuclei during the HMGA2-induced apoptosis along with the up-regulation of cleaved caspase 2, suggesting that the HMGA2-induced apoptosis was dependent on the pathway of DNA damage. Overall, the present study unravelled a novel function of HMGA2 in induction of apoptosis in human primary cell lines, and provided clues for clarification of the mechanistic action of HMGA2 in addition to its function as an oncoprotein.     

STAT5 signaling in expression of the α-subunit of interleukin-2 receptor in human blood lymphocytes

A comparative study of STAT3 and STAT5 activity (assessed by tyrosine phosphorylation level) and the expression of an α-subunit of the interleukin-2 receptor (examined by cytophotometric evaluation of CD25 cell number) during phytohemaglutinin (PHA)-induced proliferation of human blood lymphocytes (HBLs) has been carried out. It was found that the level of STAT3 phosphorylation was high both in resting and competent HBLs and remained unchanged in the presence of PHA or interleukin-2 (IL-2). In contrast to STAT3, phosphorylation of STAT5 was not seen either in resting or competent HBL. In the presence of PHA, STAT5 phosphorylation was observed no earlier than in 2–5 h; maximal phosphorylation was detected after 24 h. In competent HBLs, exogenous IL-2 induced high phosphorylation of STAT5 in 30 min that was retained for the next 24–48 h. Alterations in the level of tyrosine phosphorylation of STAT5 correlated with CD25 expression. WHI-P131, a JAK3 kinase inhibitor, prevents STAT5 activation, CD25 surface expression, and lymphocyte proliferation. It is concluded that JAK3/STAT5 signaling via an IL-2 receptor is necessary to support the long-term expression of a high-affinity αβγ_c-receptor of IL-2 and HBL optimal proliferation.     

Equilibration of Label in Malate during Dark Fixation of CO(2) in Kalanchoë fedtschenkoi

In vitro studies of dark ¹⁴CO₂ fixation with isolated cell aggregates of Kalanchoë fedtschenkoi showed that malate synthesized after 20 sec is predominantly (85 to 92%) labeled at carbon 4, while after 20 min only 65 to 69% of the radioactivity was located in this position. The intramolecular labeling pattern of malate could not be changed by supplementing the cells with carboxylation reaction substrates such as ribulose diphosphate or phosphoenolpyruvate. The kinetic decline of label at carbon 4 of malate occurs independently of CO₂ fixation, since 4-¹⁴C-labeled aspartate fed to the cells gave rise to malate labeled 62% at carbon 4 after 20 min. Furthermore, the cells were capable of converting fed malate to fumarate. It is concluded that synthesis of malate during dark CO₂ fixation is accomplished by a single carboxylation step via phosphoenolpyruvate carboxylase and labeling patterns observed in malate are a consequence of the action of fumarase.     

Identification of structural motifs in the E2 glycoprotein of Chikungunya involved in virus–host interaction

Chikungunya fever is one of the reemerging vector-borne diseases. It has become a major global health problem especially in the developing countries. There are no vaccines or specific antiviral drugs available to date. This study reports small molecule inhibitors of envelope glycoprotein 2 (E2 glycoprotein) which are predicted based on Chikungunya virus–host interactions. E2 glycoprotein of Chikungunya virus interacts at 216 residue of the host receptor protein which plays a vital role in initiating infection. Understanding the structural aspects of E2 glycoprotein is crucial to develop specific inhibitors to prevent the virus binding from host receptors. In silico method was adopted to predict the sequence motifs of envelope protein, as the method like yeast two hybrid system is laborious, time consuming, and costly. The E2 glycoprotein structure of the Indian isolate was modeled using two templates (2XFC and 3JOC) and then validated. The class III PDZ domain binding motif was found to be identified at 213–216 amino acids. The corresponding peptide structures which recognize the PDZ domain binding motif were identified by the literature search and were used for generating five point pharmacophore model (ADDDR) containing acceptor, donor and aromatic ring features. Databases such as Asinex, TosLab and Maybridge were searched for the matches for the predicted pharmacophore model. Two compounds were identified as lead molecules as their glide score is?>?5?kcal/mol. Since the pharmacophore model is developed based on Chikungunya virus–host interaction, it can be used for designing promising antiviral lead compounds for the treatment of Chikungunya fever.An animated Interactive 3D Complement (I3DC) is available in Proteopedia at http://proteopedia.org/w/Journal:JBSD:21     

Frequent loss of heterozygosity in the β2-microglobulin region of chromosome 15 in primary human tumors

Downregulation or total loss of HLA class I expression on tumor cells is known as a mechanism of cancer immune escape. Alterations of the HLA phenotype are frequently due to mutations affecting genes encoding the HLA class I heavy chains located on chromosome 6p21 or the β2-microglobulin (β2m) gene encoding the light chain of the HLA complex located on chromosome 15q21. Frequently irreversible total loss of HLA class I molecules is due to the coincidence of two molecular events, the mutation of one β2m gene and the loss of the second copy. The latter is detectable as loss of heterozygosity (LOH) of microsatellite markers in the β2m region on chromosome 15q21 (LOH-15q21). Thus, LOH-15q21 might be an important event in the processes of HLA class I downregulation and total loss. Here we studied the frequency of LOH-15q21 in tumor tissues of different entities. By determining the status of heterozygosity of two microsatellite markers we detected LOH-15q21 in 44% of bladder carcinomas (n = 69), in 35% of colon carcinomas (n = 95), in 16% of melanomas (n = 70) but only in 7% of renal cancers (n = 45). Moreover, we observed a frequent coincidence of LOH-15q21 and LOH-6p21 in colorectal carcinoma, bladder carcinoma and melanoma, but not for renal carcinoma. We believe that the high incidence of LOH-15q21 in some malignancies and especially the coincidence of LOH-15q21 and LOH-6p21 might have a strong impact on tumor immunogenicity and on the efficiency of cancer immunotherapy.     

Doses of prostaglandin F2α effective for induction of parturition in goats

Twelve mixed breed does were injected with different doses of prostaglandin F₂α (PGF₂α) or saline on day 144 of gestation. Four each received single intramuscular injections of 5.0 or 2.5 mg PGF_2α, or 1.0 ml saline (controls). Systemic progesterone (P₄) concentrations were determined daily from day 144 until the day of kidding. Does receiving 5.0 mg PGF₂α, 2.5 mg PGF₂α, or saline kidded within mean (± SD) hours and range (hours) of 35 ± 8.6 and 28–48, 43 ± 11.8 and 29–57, and 111 ± 79.1 and 41–200, respectively. Mean (± SD) concentrations of P₄ (ng/ml) on the day of injection and on day 1 postinjection were 5.2 ± 2.6 and 0.7 ± 0.9, 5.3 ± 2.2 and 1.1 ± 1.0, and 6.4 ± 3.9 and 4.1 ± 2.6 for does receiving 5.0 mg PGF₂α, 2.5 mg PGF₂α, or saline, respectively. It was concluded that 5.0 mg and 2.5 mg PGF₂α effectively shortened the interval from injection to parturition, but that this interval was not as predictable as that previously reported with 20 mg PGF₂α.     

Isolation of some precursors of 2-methylpyrrole in the Elson–Morgan reaction

1-C-(1-Acetylacetonyl)-2-deoxy-2-(1-methyl-3-oxobut-1-enyl)amino -d-galactitol is obtained from the condensation of 2-amino-2-deoxy-d-galactose with pentane-2,4-dione in anhydrous solvent. On treatment with hot alkali it gives 2-methylpyrrole with 37% yield. By acid hydrolysis under mild conditions the compound loses the N substituent and from the resulting unstable derivative 2-methylpyrrole is obtained (52% yield). It is concluded that derivatives of aminohexoses substituted at C-1 with a dioxopentyl chain are the precursors of 2-methylpyrrole in the Cessi & Serafini-Cessi (1963) modification of the Elson-Morgan reaction. As demonstrated previously, products of condensation of aminohexoses with pentane-2,4-dione at the amino group are not converted directly into 2-methylpyrrole, but this step provides protection of the amino group during condensation at C-1.     

The roles of DNA polymerases κ and ι in the error-free bypass of N2-carboxyalkyl-2'-deoxyguanosine lesions in mammalian cells

To counteract the deleterious effects of DNA damage, cells are equipped with specialized polymerases to bypass DNA lesions. Previous biochemical studies revealed that DinB family DNA polymerases, including Escherichia coli DNA polymerase IV and human DNA polymerase κ, efficiently incorporate the correct nucleotide opposite some N(2)-modified 2'-deoxyguanosine derivatives. Herein, we used shuttle vector technology and demonstrated that deficiency in Polk or Poli in mouse embryonic fibroblast (MEF) cells resulted in elevated frequencies of G→T and G→A mutations at N(2)-(1-carboxyethyl)-2'-deoxyguanosine (N(2)-CEdG) and N(2)-carboxymethyl-2'-deoxyguanosine (N(2)-CMdG) sites. Steady-state kinetic measurements revealed that human DNA polymerase ι preferentially inserts the correct nucleotide, dCMP, opposite N(2)-CEdG lesions. In contrast, no mutation was found after the N(2)-CEdG- and N(2)-CMdG-bearing plasmids were replicated in POLH-deficient human cells or Rev3-deficient MEF cells. Together, our results revealed that, in mammalian cells, both polymerases κ and ι are necessary for the error-free bypass of N(2)-CEdG and N(2)-CMdG. However, in the absence of polymerase κ or ι, other translesion synthesis polymerase(s) could incorporate nucleotide(s) opposite these lesions but would do so inaccurately.