Modulation of oocyte maturation by cyclic adenosine 3', 5'-pyrophosphate

The germinal vesicle (GV) of follicle-enclosed oocytes in mammals remains arrested at the dictyate state of meiosis. Upon releasing the oocytes from the follicles, the meiotic process resumes, leading to dissolution of the GV (GVBD), suggesting that factors in the follicular fluid sustain the meiotic arrest of oocytes. In the present study the spontaneous resumption of meiosis was blocked by the addition of cyclic adenosine 3', 5'-pyrophosphate (cAPP) plus dibutyryl cyclic adenosine 3',5'-monophosphate (dbcAMP), at final concentrations of 25 and 50 microM, respectively. These compounds were ineffective when added separately at these concentrations. None of the other related compounds tested with dbcAMP blocked GVBD. Bovine follicular fluid (BFF) was analyzed for inhibitors of GVBD. BFF was extracted with 70% ethanol and the ethanolic extract chromatographed on Dowex 1-X8 column. The fraction eluted with 0.1 N HCl markedly inhibited GVBD of isolated mouse oocytes in combination with dbcAMP. The active BFF substance and cAPP block spontaneous GVBD of mouse oocytes and may be related substances. The present study supports the thesis that meiotic arrest at the dictyate stage in oocytes is sustained by factors present in follicular fluid and may act in association with cAMP.     

An Overlapping Region between the Two Terminal Folding Units of the Outer Surface Protein A (OspA) Controls Its Folding Behavior

Although many naturally occurring proteins consist of multiple domains, most studies on protein folding to date deal with single-domain proteins or isolated domains of multi-domain proteins. Studies of multi-domain protein folding are required for further advancing our understanding of protein folding mechanisms. Borrelia outer surface protein A (OspA) is a β-rich two-domain protein, in which two globular domains are connected by a rigid and stable single-layer β-sheet. Thus, OspA is particularly suited as a model system for studying the interplays of domains in protein folding. Here, we studied the equilibria and kinetics of the urea-induced folding–unfolding reactions of OspA probed with tryptophan fluorescence and ultraviolet circular dichroism. Global analysis of the experimental data revealed compelling lines of evidence for accumulation of an on-pathway intermediate during kinetic refolding and for the identity between the kinetic intermediate and a previously described equilibrium unfolding intermediate. The results suggest that the intermediate has the fully native structure in the N-terminal domain and the single layer β-sheet, with the C-terminal domain still unfolded. The observation of the productive on-pathway folding intermediate clearly indicates substantial interactions between the two domains mediated by the single-layer β-sheet. We propose that a rigid and stable intervening region between two domains creates an overlap between two folding units and can energetically couple their folding reactions.     

Microtubule Stabilization in Pressure Overload Cardiac Hypertrophy

Increased microtubule density, for which microtubule stabilization is one potential mechanism, causes contractile dysfunction in cardiac hypertrophy. After microtubule assembly, α-tubulin undergoes two, likely sequential, time-dependent posttranslational changes: reversible carboxy-terminal detyrosination (Tyr-tubulin ↔ Glu-tubulin) and then irreversible deglutamination (Glu-tubulin → Δ2-tubulin), such that Glu- and Δ2-tubulin are markers for long-lived, stable microtubules. Therefore, we generated antibodies for Tyr-, Glu-, and Δ2-tubulin and used them for staining of right and left ventricular cardiocytes from control cats and cats with right ventricular hypertrophy. Tyr- tubulin microtubule staining was equal in right and left ventricular cardiocytes of control cats, but Glu-tubulin and Δ2-tubulin staining were insignificant, i.e., the microtubules were labile. However, Glu- and Δ2-tubulin were conspicuous in microtubules of right ventricular cardiocytes from pressure overloaded cats, i.e., the microtubules were stable. This finding was confirmed in terms of increased microtubule drug and cold stability in the hypertrophied cells. In further studies, we found an increase in a microtubule binding protein, microtubule-associated protein 4, on both mRNA and protein levels in pressure-hypertrophied myocardium. Thus, microtubule stabilization, likely facilitated by binding of a microtubule-associated protein, may be a mechanism for the increased microtubule density characteristic of pressure overload cardiac hypertrophy.     

Spontaneous establishment of an Epstein-Barr virus-infected fibroblast line from the synovial tissue of a rheumatoid arthritis patient.

An Epstein-Barr virus (EBV)-infected fibroblast line, designated DSEK, was spontaneously established from synovial tissue of a patient with rheumatoid arthritis (RA). DSEK cells expressed EBV nuclear antigens EBNA-1 and EBNA-2 and latent membrane protein LMP-1. Cell surface markers of DSEK cells were similar to those of EBV-negative fibroblast clones derived from synoviocytes and were negative for lymphocyte and macrophage markers. DSEK cells expressed CD44, CD58, and HLA-DR antigens and spontaneously produced interleukin-10 basic fibroblast growth factor and transforming growth factor beta1. These results indicate that rheumatoid synoviocytes can be a target for EBV infection and suggest that EBV may play a role in the pathogenesis of RA.     

Effect of estradiol on the early incorporation of (3H) thymidine into uterine DNA on immature mouse.

The incorporation of [3H]thymidine into uterine DNA was markedly depressed within 10 to 30 minutes after intraperitoneal administration of 17beta-estradiol to immature mouse. Maximum inhibition occurred about 6 hours after the hormone was administered. Uterine DNA content and the amount of [3H]thymidine incorporated into the acid-soluble fraction was not affected during the period of hormone-induced inhibition. Moreover, the in vitro incorporation of [3H]thymidine by isolated estradiol-treated mouse uterus was blocked. In contrast to the uterus, 17beta-estradiol did not influence the incorporation of thymidine into mouse liver DNA. Evidence is presented to show that the incorporation of thymidine into uterine DNA was blocked initially by 17beta-estradiol.     

A study on the stability of an estrogen-protein conjugate in vivo and under in vitro conditions

[³ H] Estradiol-17β-succinyl bovine serum albumin conjugate ([³H]-E₂-BSA) was synthesized with a specific activity of 1.92 × 10⁷ cts/min/mg. The conjugate was administered iv to ovariectomized rats and the quantity of free [³H] steroid in the uterus was determined. Radioactive material was detected in all of the subcellular fractions of the uterus and identified as estradlol-17β. Similar subcellular distribution of the radioactivity was observed when [³H]E₂-BSA was added $\text{in vitro}$ to uterine homogenate. Free estradlo1-17β was released when the conjugate was Incubated with rat uterine homogenate or with serum. The results of the present study suggest that E₂-BSA is hydrolyzed $\text{in vivo}$ and under $\text{in vitro}$ conditions. It is recommended that the stability of a hormone-protein conjugate be established before use.     

Mouse oocyte maturation modulated by a granulosa cell factor and by heparin and heparan sulfate

Oocyte maturation-preventing factor (OMPF) was extracted from bovine granulosa cells with a buffer containing 1 M urea and 5 mM EDTA. OMPF was partially purified by gel filtration on Sephadex G25 and by reversed-phase high performance liquid chromatography. The maturation-preventing activity of purified fractions was determined by measuring their capacity to block the spontaneous dissolution of the germinal vesicle (GVBD) of isolated cumulus-enclosed mouse oocytes. Hyaluronic acid, chondroitin sulfate, heparin, heparan sulfate, keratan sulfate, and dextran sulfate at concentrations of 500 μg/ml did not affect the frequency of GVBD of isolated mouse oocytes. Heparin and heparan sulfate, however, blocked the inhibitory effect of OMPF, whereas the inhibition of GVBD induced with dibutyryl cAMP, forskolin, W7 (calmodulin antagonist), and 3-isobutyl-l-methylxanthine (phosphodiesterase inhibitor) was not blocked. OMPF was eluted in the adsorbed fraction when chromatographed on heparin-Agarose, showing interaction of OMPF with heparin. The present results suggest that the glycosaminoglycan matrix may influence OMPF action on oocytes.     

Inhibitory effects of 1 alpha, 25-dihydroxycholecalciferol on parathyroid hormone secretion in rats

In an attempt to study the influence of vitamin D metabolites on PTH secretion, serum calcium and urinary excretion of cAMP were sequentially measured in conscious perfused rats, and the effects of a single iv injection of the metabolites on these parameters were examined. Four hours after the administration of 0.25 microgram/kg (0.6 nmol/kg, probably a physiological dose) of 1 alpha, 25-dihydroxycholecalciferol [1 alpha, 25 (OH)2D3], the urinary excretion of cAMP decreased to a level compatible with that of parathyroidectomized rats (approximately 60% of the initial value; P less than 0.05) and this level was sustained for nearly 24 h. Serum concentrations of calcium (total and ionized) did not change. In parathyroidectomized rats which were continuously infused with bovine PTH (1 U/h), the vitamin D metabolite had no significant effect on the urinary excretion of cAMP. 24 R, 25-dihydroxcholecalciferol (12.5 microgram/kg) had no significant effect either on the urinary excretion of cAMP or on serum calcium. These results suggest that in rats, a physiological dose of 1 alpha, 25(OH)2D3 inhibits PTH secretion without causing a significant rise iu serum calcium, reflecting a feed-back mechanism between active vitamin D metabolite, 1 alpha, 25(OH)2D3 and the parathyroid glands.     

RNase and alkali sensitivity of closed circular mitochondrial DNA of rat ascites hepatoma cells

A preparation of the closed circular DNA duplex was obtained from whole rat ascites hepatoma cells, AH66, by lysis of cells with SDS and purification by CsCl-dye buoyant-density centrifugation. RNase A converted the closed circular mitochondrial DNA to open circular molecules. The closed circular DNA was also sensitive to alkali. The conversion to the open form was shown from the results of centrifugal analyses on neutral and alkaline sucrose density gradients and CsCl-ethidium bromide. These results indicate the presence of at least one RNA region in closed circular double stranded mitochondrial DNA.