Biomedical subjects

A Sadhu

Publications and source records attributed to A Sadhu.

8 recordsLinked to original sources

PubMed1998

Characterization of protein phosphatases in mouse oocytes.

Okadaic acid (OA) enhances the resumption of meiosis in mouse oocytes, indicating that serine/threonine protein phosphatase-1 (PP1) and/or PP2A is involved. However, specific identification of PP1 and/or PP2A in mouse oocytes has not been reported. Here we demonstrate that fully grown germinal vesicle-intact (GVI) mouse oocytes contain mRNA corresponding to two isotypes of PP1, PP1alpha and PP1gamma. In addition, the transcript for PP2A was also present. At the protein level only PP1alpha and PP2A were recognized in fully grown GVI oocytes by Western blot analysis. Neither of the PP1gamma spliced variant proteins, PP1gamma1 and PP1gamma2, was detectable. Immunohistochemical analysis of ovarian tissue from gonadotropin-stimulated adult mice resulted in subcellular localization of both PP1alpha and PP2A, but not PP1gamma, in oocytes from all stages of folliculogenesis. In primordial oocytes, PP1alpha and PP2A were present in the cytoplasm. In more advanced stages of oogenesis, PP1alpha, although still present in the cytoplasm, was highly concentrated in the nucleus, whereas PP2A was predominantly cytoplasmic with a distinct reduction in the nuclear area. Both PP1alpha and PP2A were immunodetectable in oocytes during the prepubertal period. Eleven-day-old mouse oocytes, considered OA-insensitive and germinal vesicle breakdown (GVB)-incompetent, displayed both PP1alpha and PP2A predominantly in the cytoplasm. By 15 days of age mouse oocytes, which are beginning to acquire OA sensitivity and GVB competence, showed a relocation of PP1alpha into the nucleoplasm while PP2A remained predominantly cytoplasmic. This is the first specific identification of PP1alpha and PP2A in mouse oocytes. The differential localization of PP1alpha and PP2A, in addition to the relocation of PP1alpha during the acquisition of meiotic competence, suggests that these PPs have distinct regulatory roles during the resumption of meiosis.

Animals↗

PubMed1998

Transient exposure of rhesus macaque oocytes to calyculin-A and okadaic acid stimulates germinal vesicle breakdown permitting subsequent development and fertilization.

Exposure of mammalian oocytes to the protein phosphatase (PP)-1 (PP1) and PP2A inhibitor okadaic acid (OA) stimulates oocyte meiosis. However, treated oocytes do not develop beyond metaphase I (MI), and they display morphological aberrations. Experiments were conducted to define inhibitor treatment conditions for macaque oocytes that would result in germinal vesicle breakdown (GVB) stimulation and completion of meiosis without significant cytoplasmic abnormalities. As described above for OA, continual exposure of macaque oocytes to 50 nM calyculin-a (CL-A) significantly enhanced GVB at 24 h compared to that in controls, and the majority of the treated oocytes displayed cytoplasmic abnormalities. However, transient exposure (10 min) of rhesus macaque oocytes to either 50 nM CL-A or 1.0 microM OA enhanced GVB rates compared to that in controls and did not increase the incidence of cytoplasmic abnormalities. Meiotic maturation from germinal vesicle-intact oocytes to MII was enhanced following transient treatment with CL-A or OA compared to that in controls; however, development from MI to MII occurred at a similar frequency. In vitro-matured oocytes transiently exposed to OA and CL-A were capable of fertilization. In addition, ovarian immunohistochemical analysis revealed that both PP1 and PP2A were present in macaque oocytes. PP1 was localized throughout the cytoplasm with a predominance in the nucleus, whereas PP2A was evenly distributed throughout the cytoplasm with a reduction in the nuclear area. These results taken together-differential developmental responses to inhibitor treatment and intracellular enzyme localizations-may be indicative of multiple regulatory roles of PP1 and/or PP2A during meiosis.

Animals↗

PubMed1997

CpG-rich sequences close to the site of duplication within the human IGH constant region.

Human immunoglobulin heavy chains are encoded by a highly complex locus, IGH, which encompasses many transcriptional units, including nine alternative constant region genes. Much of the constant region gene cluster was duplicated during hominoid evolution. In rodents, IGH is known to be regulated by multiple elements, including several enhancers 3' of the alpha chain-encoding A constant region gene, CA, the last transcribed region. Sequences downstream of the two human CA genes, possibly containing homologous enhancer elements, have not yet been reported. By long-range mapping of genomic DNA, a cluster of unmethylated rare restriction sites, representing a potential CpG island, was previously reported downstream of each CA gene, close to the 3' end of the duplicated region. Such potential CpG islands are candidates for additional IGH regulatory elements. We isolated bacteriophage clones containing these clusters of methylation-sensitive restriction sites, which lie within short CpG-rich stretches. Some of these sites showed tissue-specific methylation. 3' of the unmethylated CpG-rich sequences, clones derived from the 5' (telomeric) copy of the duplicated region, contained, in order, endogenous retroviral sequences, a processed ELK1 pseudogene, and the pseudogene IGHGP. Comparison with the presumed 3' (centromeric) copy of the duplicated region showed that similarity was lost exactly at the end of the retroviral long terminal repeat sequences. These results imply that an endogenous retroviral integration was present immediately 3' of IGH in the common hominoid ancestor and suggest models for the duplication of the region.

Base Sequence↗

PubMed1997

BCL6 can repress transcription from the human immunodeficiency virus type I promoter/enhancer region.

The gene BCL6 encodes a zinc finger protein with similarities to transcription factors. We previously reported that a number of viral genomes, including human immunodeficiency virus type I (HIV-1), contain sequences which are similar to the BCL6 DNA-binding consensus in their promoter regions. Electrophoretic mobility shift assays showed that the full-length BCL6 protein extracted from transfected COS cells and a bacterially expressed truncated protein containing the BCL6 zinc fingers can bind specifically to DNA from the U3 promoter/enhancer region of HIV-1. Transient transfections were performed to analyze the effects of the BCL6 protein on luciferase expression driven by the HIV-1 long terminal repeat (LTR) sequences. Full-length BCL6 significantly repressed luciferase activity compared with multiple controls. We conclude that the BCL6 protein can bind to the HIV-1 promoter-enhancer region and contains a domain upstream of its zinc fingers that can repress transcription from the HIV-1 LTR.

Animals↗

PubMed1996

Steady-state luteinizing hormone receptor messenger ribonucleic acid levels and endothelial cell composition in bovine normal- and short-lived corpora lutea.

The short-lived corpus luteum (CL) contributes to reproductive inefficiency during the postpartum period in beef cows. The cause for the early demise of the short-lived CL is not fully understood but is believed to involve a premature release of prostaglandin F2 alpha. The objectives of this study were to evaluate norgestomet-hCG-induced normal-lived CL and hCG-induced short-lived CL in postpartum cows with respect to serum progesterone (P4) and 13,14-dihydro-15-keto, prostaglandin F2 alpha (PGFM) concentrations and luteal LH receptor (LH-R) concentrations, LH-R mRNA levels, and vascularity. Although serum P4 profiles from the time of hCG administration (Day 0) until luteectomy (Day 6, 7, or 8) were similar between CL life span groups, PGFM concentrations were elevated (p < 0.05) on Day 8 in cows expected to have short-lived CL compared to normal-lived CL. The LH-R concentrations were similar between normal- and short-lived CL on all days measured. Irrespective of luteal life span and day of luteectomy, all CL possessed a 4.4-kb LH-R transcript. Actin-normalized LH-R mRNA levels were similar between normal- and short-lived CL on Days 6 and 7; however, Day 8 short-lived CL contained less (p < 0.05) LH-R mRNA than Day 8 normal-lived CL. Although the area of luteal tissue occupied by capillaries in normal- and short-lived CL was similar on Days 6 and 7, the area occupied by capillaries in short-lived CL was lower (p < 0.05) than that for normal-lived CL on Day 8. Collectively, these results indicate that there is a decrease in steady-state LH-R mRNA and a reduction in luteal vascularity in CL expected to be short-lived. These changes occur concomitantly with a rise in serum PGFM, but prior to a decline in serum P4.

Animals↗

PubMed1995

BCL6 encodes a sequence-specific DNA-binding protein.

Chromosomal rearrangements of BCL6 are commonly associated with diffuse large-cell lymphomas. We set out to determine the DNA-binding site of a glutathione-S-transferase fusion protein containing the BCL6 zinc finger region by employing cyclic amplification and selection of targets (CASTing). From oligonucleotides containing 16 central random bases, sequences binding to the protein on glutathione-coated beads were repeatedly selected and amplified by polymerase chain reaction (PCR). The binding sites were cloned and sequenced. A consensus, TTTNNNGNNATNCTTT, was obtained. Protein binding studies of double-stranded oligomers containing point mutations within the 3' CTTT confirmed the binding specificity of this part of the consensus. In addition, evidence indicated that some of the base pairs held constant in the oligonucleotides used for CASTing also contributed to binding.

Base Sequence↗

PubMed1992

A kinetic study of the kinesin ATPase.

The mechanism of kinesin ATPase has been investigated by transient state kinetic analysis. The results satisfy the scheme [formula: see text] where T, D, and P(i) refer to nucleotide tri- and diphosphate and inorganic phosphate, respectively. The nucleotide-binding steps were measured by the fluorescence enhancement of mant (2'-(3')-O-(N-methylanthraniloyl)-ATP and mant-ADP. The initial rapid equilibrium binding steps (1) and (6) are followed by isomerizations (k2 = 170 +/- 30 s-1 at 20 degrees C, k-5 greater than 100 s-1). The increase in fluorescence is 20-25% larger for K.T** than K.D*. The rate constant of the hydrolysis step k3 is 6-7 s-1. The fluorescence decreases after formation of K.T** at a rate of 7-10 s-1. This change could occur in step 3 or in step 4 if k4 much greater than k3. The value of k4 is larger than 0.1 s-1. The steady state rate is 0.003 s-1 which agrees with the rate of ADP dissociation (k5). Step 5 is rate limiting in the scheme in agreement with the conclusion of Hackney (Hackney, D. D. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 6314-6318) that ADP dissociation is the rate-limiting step.

Adenosine Triphosphatases↗

PubMed1989

Role of second metal ion in establishing active conformations of concanavalin A.

The stoichiometry of Mn2+ binding to concanavalin A was found to be influenced by temperature, pH, and the presence or absence of saccharide. Demetalized concanavalin A binds one Mn2+ (S1 site) at 5 degrees C, pH 6.5, and two Mn2+ at 25 degrees C (S1 and S2 sites). The association constants for Mn2+ are 6.2 x 10(5) and 3.7 x 10(4) M-1 for the S1 and S2 sites, respectively, at 25 degrees C. Concanavalin A with one Mn2+ bound per monomer remains in an open conformation and exhibits a relatively high water proton relaxation rate. Concanavalin A with two Mn2+ ions remains in a closed conformation characterized by a lower relaxation rate. The rate of binding of the second Mn2+ to concanavalin A as determined by ESR and the rate of conversion of open form to closed form (folding over) as determined by proton relaxation rate measurements gave an identical rate constant of 80.0 +/- 5.8 M-1 h-1 at 17 degrees C. Ca2+, Sr2+, and high levels of methyl alpha-D-mannopyranoside also induce folding of concanavalin A. Ca2+ is not catalytic but stoichiometric in causing the folding. Mn2+ in the S1 site can be displaced by Ni2+, Co2+, and Zn2+, and Mn2+ in the S2 site can be displaced by Ca2+ and Sr2+. Concanavalin A with Ni2+, Co2+, Zn2+, or Mn2+ in the S1 site and Ca2+ or Sr2+ in the S2 site has a higher affinity for methylumbelliferyl alpha-D-mannopyranoside than Ni-Mn-, Co-Mn-, Zn-Mn-, and Cd-Cd-concanavalin A.

Binding Sites↗