Faculty Bibliography

We developed a method for the biopsy of preimplantation mouse embryos (preembryos) at the four- to eight-cell stage, which uses partial zona pellucida dissection. The preembryos were collected in calcium- and magnesium-free phosphate-buffered saline solution with 0.01% ethylenediaminetetraacetic acid, 0.1 mol/L sucrose, and 4 mg/ml of bovine serum albumin to facilitate removal of blastomeres. This allows entry of a fine micropipette into the perivitelline cavity with subsequent removal of a single blastomere by gentle suction. The majority of embryos (75%) from which biopsy specimens were obtained in this fashion developed to the blastocyst stage. The blastomeres obtained were mainly intact and they were fixed to glass slides. After permeabilization, in situ hybridization was performed with chromosome X- and chromosome 3-specific probes. Human unfertilized eggs and blastomeres from human polyspermic embryos also have been analyzed by in situ hybridization with chromosome specific probes. The combination of nondestructive embryo biopsy and in situ hybridization is a possible approach for preimplantation genetic diagnosis

Human ovarian tissue and bovine ovarian stroma and follicles bound tritiated oxytocin and tritiated arginine vasopressin with similar affinity, whereas bovine corpora lutea bound tritiated oxytocin only. Competition for the binding of tritiated oxytocin by various agonists and antagonists was suggestive of receptor function. The number of oxytocin binding sites varied cyclically in all tissues. In bovine ovarian stroma and corpora lutea the concentrations were lowest on day 14 and highest on days 17, 19, and 21 after ovulation, with a striking peak in the luteal concentration on day 19. In human ovarian tissues the concentrations also were highest in samples obtained in late luteal phase. In large follicles the concentration of oxytocin binding sites was highest on the day of estrus and lowest on day 7. In bovine ovary the number of arginine vasopressin binding sites was approximately 50% lower than oxytocin binding sites and the cyclic variations were not significant. Human ovarian tissue had similar numbers of oxytocin and arginine vasopressin binding sites. Because bovine ovaries produce oxytocin and arginine vasopressin the results suggest a paracrine or autocrine role for these neuropeptides in luteolysis and ovulation. Although their synthesis in human ovaries is still controversial the presence of binding sites suggests a physiologic role in the regulation of human ovarian function as well.

Interstitial pregnancy which resulted in a term, live infant was found in association with pathologic torsion of the uterus. Antenatal sonograms revealed a hypervascular area anterior to the uterus

Serologic markers were evaluated to determine if they could aid in the differential diagnosis of pelvic inflammatory disease in 48 consecutive women seeking evaluation for pelvic pain. On the basis of clinical and microbiologic parameters, 29 patients (60.4%) were diagnosed as having pelvic inflammatory disease. Neisseria gonorrhoeae only was isolated from the cervix of eight (27.6%) patients with pelvic inflammatory disease, five (17.2%) had only Chlamydia, and two (6.9%) had Neisseria and Chlamydia, whereas in 15 (48.3%) patients no pathogen was isolated. Interferon-gamma was present in significantly more sera (p less than 0.025) from patients with pelvic inflammatory disease (65.5%) than from women without pelvic inflammatory disease (15.8%). Sera from 10 healthy women lacked detectable interferon-gamma. In patients with only Neisseria, seven (87.5%) had circulating interferon-gamma; three (60%) of the women with only Chlamydia, one (50%) woman with Neisseria and Chlamydia, and eight (57.1%) with no identified pathogens were also positive for interferon-gamma. Sera from 11 of 28 patients with pelvic inflammatory disease (39%) but only one of 19 sera from women without pelvic inflammatory disease (5%) also inhibited the Candida-induced proliferation of control lymphocytes. This immunosuppressive activity was prevented by immunoprecipitation of interferon-gamma by anti-interferon-gamma antibody but not by treatment with anti-interferon-alpha antibody. The persistence of interferon-gamma in the sera of patients with pelvic inflammatory disease may aid in the differential diagnosis of this disease and increase our understanding of the pathogenesis of microbial-mediated tubal damage

The effects of 5' proximal secondary structure in mRNA molecules on their translation and on their interaction with the eukaryotic initiation factors (eIF)-4F, eIF-4A, and eIF-4B have been examined. Secondary structures were generated in the 5' noncoding region of rabbit globin and reovirus mRNAs by means of hybridization with cDNA molecules. cDNAs hybridized to the first 15 bases downstream from the cap inhibited the translation of the mRNAs in both reticulocyte and wheat germ lysates. The degree of inhibition was directly related to the monovalent ion concentration and inversely related to reaction temperature. These hybrid structures also reduced the competitive ability of the messages. Hybrid structures beginning downstream from the first 15 bases did not inhibit the translation of beta-globin mRNA or reovirus s3 mRNA. None of the hybrid structures were detrimental to the interaction of the mRNAs with the 26-kDa cap binding protein of eIF-4F, as determined by chemical cross-linking assays. However, in the presence of ATP, hybrid structures immediately adjacent to the cap severely inhibited the cross-linking to the p46 subunit of eIF-4F or to additional eIF-4A or eIF-4B. In order to account for these observations, a two-step mechanism is proposed for the interaction of eIF-4F with the 5' end of an mRNA molecule. The first step involves a weak initial interaction of the p26 subunit with the cap. The second step requires the hydrolysis of ATP and results in the formation of a stable initiation factor-mRNA complex, which may involve eIF-4A and eIF-4B. This second step is inhibited by the presence of 5' proximal secondary structure. In any event, our results demonstrate that the effect of mRNA structure on translation rate depends strongly on its position with respect to the 5' end and that this effect is due at least in part to an inhibition of the action of initiation factors normally required for the unwinding of structure

Interaction of protein synthesis initiation factors with mRNA has been studied in order to characterize early events in the eukaryotic translation pathway. Individual reovirus mRNAs labeled with 32P in the alpha position relative to the m7G cap and eukaryotic initiation factor (eIF)-4A, -4B, and -4F purified from rabbit reticulocytes were employed. It was found that eIF-4A causes a structural change in mRNA, as evidenced by a nuclease sensitivity test: addition of high concentrations of eIF-4A greatly increase the nuclease sensitivity of the mRNA, suggesting that this factor can melt or 'unwind' mRNA structure. ATP is required for this reaction. At low concentrations of eIF-4A, addition of eIF-4B is required for maximal unwinding activity. Thus eIF-4B enhances eIF-4A activity. Addition of eIF-4F also makes the mRNA sensitive to nuclease indicating a similar unwinding role to that of eIF-4A. Stoichiometric comparisons indicate that eIF-4F is more than 20-fold more efficient than eIF-4A in catalyzing this reaction. The unwinding activity of eIF-4F is inhibited by m7GDP, while that of eIF-4A is not. This suggests that eIF-4A functions independent of the 5' cap structure. Our results also suggest that the unwinding activity of eIF-4F is located in the 46,000-dalton polypeptide of this complex, which has shown by others to be similar or identical to eIF-4A

Studies were conducted to determine whether encephalomyocarditis virus infection causes proteolytic cleavage of any of the polypeptides which comprise eucaryotic initiation factor 4F. Since no such alterations in the components of the initiation factor were detected, these observations confirmed that the mechanisms whereby encephalomyocarditis virus and poliovirus shut off host translation are different

Previously, we have described an ATP-dependent recognition and binding of mRNA by eukaryotic initiation factors (eIF)-4A, eIF-4B, and eIF-4F (Grifo, J. A., Tahara, S. M., Leis, J. P., Morgan, M. A., Shatkin, A. J., and Merrick, W. C. (1982) J. Biol. Chem. 257, 5246-5252; Grifo, J. A., Tahara, S. M., Morgan, M. A., Shatkin, A. J., and Merrick, W. C. (1983) J. Biol. Chem. 258, 5804-5810). This finding was consistent with other studies which implicated eIF-4A and eIF-4B in binding mRNA to the 40 S ribosomal subunit, an ATP-requiring process. As part of ongoing studies of this step, and, in particular its ATP requirement, we have examined ATPase activity of various initiation factors. In this communication we describe an RNA-dependent ATP hydrolysis catalyzed by eIF-4A and eIF-4F. Although eIF-4B has little or no ATPase activity it can stimulate the RNA-dependent ATPase activity of either eIF-4A or eIF-4F. Similar to the ATP-dependent mRNA binding assay, the RNA-dependent ATPase activity is inhibited by the cap analogue m7GDP when globin mRNA is used as the activator. In addition, a variety of polynucleotides stimulate the ATPase activity of these factors including rRNA, tRNA, poly(U), and poly(A) but not poly(dA). Finally, an attempt has been made to discern whether phosphorylation or ATP hydrolysis is responsible for the ATP-stimulated binding of mRNA by eIF-4A and eIF-4B. We present evidence which is consistent with the interpretation that ATP hydrolysis and not protein phosphorylation correlates with ATP-stimulated binding of mRNA

The ability of homogeneous phospholipid-sensitive Ca2+-dependent protein kinase (PL-Ca-PK) from pig spleen to phosphorylate eukaryotic initiation factor 2 (eIF-2) was examined. PL-Ca-PK phosphorylated the beta-subunit of eIF-2, whereas myosin light chain kinase (MLCK) and cyclic AMP- and cyclic GMP-dependent protein kinases (cA-PK and cG-PK) did not. PL-Ca-PK could incorporate a maximum of 1.6 mol phosphate/mol eIF-2. The app. Km and Vmax for PL-Ca-PK phosphorylation of eIF-2 were 0.13 microM and 0.02 mumol.min-1.mg enzyme-1, respectively. Phosphoamino acid analysis revealed that incorporation of phosphate into eIF-2 occurred almost exclusively at serine residues. These findings indicate that eIF-2 was an effective substrate for PL-Ca-PK, suggesting that this enzyme may play a role in the regulation of protein synthesis

The translation of poliovirion RNA (polio RNA) in an in vitro fractionated system was much less efficient than that of encephalomyocarditis virion RNA (EMC RNA). In contrast, when polio and EMC RNAs were added to postmitochondrial cell lysates (S10), they were translated with equal efficiency. However, this equality was observed only when high concentrations of S10 were employed; at lower concentrations, polio RNA translation was reduced relative to that of EMC RNA. These results suggest that both the fractionated and S10 systems are limiting in a component that is required for the optimal translation of polio RNA. The elongation rates for EMC and polio RNA translation in the fractionated system were found to be similar, indicating that this component acts at an initiation step. Various components, including excess ribosomal salt wash and postribosomal supernatant of cell lysate, were added to the fractionated system in an effort to identify the slow step more precisely. Of these, only excess ribosomal salt wash specifically stimulated polio RNA translation, suggesting that one or more initiation factors is necessary in unusually large amounts for this mRNA. Various purified initiation factors were tested for the ability to enhance polio RNA translation. Of these, only purified eukaryotic initiation factor 4A had a specific effect. This suggests that polio RNA, in contrast to other mRNAs tested (EMC, reoviral, and globin), may have an unusually low affinity for this initiation factor. The significance of these results is discussed in terms of the methods picornaviruses have evolved for reprogramming the translational machinery of the host cell