Improved developmental potential of rabbit oocytes fertilized by sperm microinjection into the perivitelline space enlarged by hypertonic media

The objectives of the present study were: 1) to develop a simple and more efficient technique for sperm microinjection than is currently available, using the rabbit as a model, and 2) to evaluate the development of rabbit oocytes fertilized by single or multiple sperm microinjection. Hyperosmotic sucrose in phosphate-buffered saline (SPBS) was employed to dehydrate oocytes to increase the perivitelline space for sperm microinjection and prevent possible injury to the vitellus. In the first experiment, 58% (n = 29) oocytes treated with 0.5 M SPBS developed to morulae following multiple sperm microinjection compared, respectively, to 47% (n = 34) and 60% (n = 15) for control IVF with or without sucrose exposure (P greater than 0.05). Blastocyst development from microinjected oocytes, however, was much lower (P less than 0.05) than that of controls (14% vs. 42% and 40%, respectively). Sham operation by puncturing the zona pellucida of the sucrose-treated oocytes with the microinjection pipette did not increase parthenogenesis (P greater than 0.05). In Experiment 2 a smaller-size injection pipette and shorter sucrose exposure time after sperm microinjection resulted in 41% (n = 42) of the oocytes developing into blastocysts for the microinjection group, whereas only 21% (n = 24) developed to blastocysts in the control IVF group (P less than 0.05). When relatively older oocytes (17 hr post ovulation injection) were used to test if microinjection could reduce the time to fertilization and cleavage (Expt. 3), an average of 27% (n = 63) blastocysts resulted from microinjection vs. 0% (n = 28) for the control IVF group.     

Early biochemical events after MHC class II-mediated signaling on human B lymphocytes

These studies examined the role of the MHC class II Ag in signal transduction using human B lymphocytes. Early events in signal transduction were considered including the intracellular calcium [Ca2+)i) flux, the activation of phospholipase C, and induction of protein phosphorylation. The (Ca2+)i was enhanced after incubation of B lymphocytes with several mAb anti-HLA class II and cross-linking with rabbit anti-mouse-F(ab')2. We have also demonstrated an enhancement of the (Ca2+)i in response to a suboptimal concentration of a monoclonal anti-IgM either in the presence of or after preincubation with a mAb anti-HLA class II. The activation of phospholipase C was assessed by measuring the generation of inositol phosphates in permeabilized B lymphocytes. mAb anti-HLA-class II of two different epitopes were used to demonstrate both the (Ca2+)i flux and the generation of inositol phosphates. Two-dimensional gel electrophoresis was used to investigate the phosphorylation pattern of resting B lymphocytes and the changes in the pattern after stimulation with soluble mAb anti-HLA-DR, immobilized mAb anti-HLA-DR, and PMA. In addition to the augmentation of phosphorylation observed with regard to phosphoproteins already present in resting B lymphocytes, new phosphorylations were observed after stimulation by any one of the reagents. Furthermore, stimulation by PMA did not result in an identical pattern to that observed after stimulation by mAb anti-HLA class II. An inhibition of the proliferative response to PMA was demonstrated after prestimulation of cells with immobilized mAb anti-HLA-DR, supporting the notion of a shared pathway of activation. In summary, these data demonstrate signal transduction via MHC class II Ag as assessed by three different measures of early events in human B lymphocyte activation and suggest that a protein kinase C pathway is at least partly involved.     

ELISA measurement of LDL receptors

An enzyme-linked immunosorbent assay was developed for measurement of low density lipoprotein (LDL) receptors. A monospecific polyclonal antibody to LDL receptor purified from rat liver that reacted with rat, mouse, canine, and human LDL receptor was used. With this assay, LDL receptors could be measured on 2-4 x 10(5) adherent cells and 1.0 x 10(5) cells in suspension, although results were more variable with cell suspensions. Membranes from a variety of receptor-rich and receptor-poor tissues could be assayed directly after adherence of the membranes to the ELISA plate by an overnight incubation. In some instances, the quality of the assay was improved by first solubilizing the membranes. The sensitivity of the assay is such that between 0.15 and 2 micrograms of membrane protein is required. This could be obtained from leukocytes in a modest (20-30 ml) quantity of human blood. The assay was used to demonstrate the rapid down-regulation of LDL receptors in human mononuclear leukocytes in response to a cholesterol-containing meal. Overall, the results support the use of ELISA technology to measure LDL receptors, particularly for physiologic studies.     

Transforming activity of E5a protein of human papillomavirus type 6 in NIH 3T3 and C127 cells.

Human papillomavirus type 6 (HPV-6) is the etiologic agent of genital warts and recurrent respiratory papillomatosis. We are investigating the mechanism by which this virus stimulates cell proliferation during infection. In this paper, we report that the E5a gene of HPV-6c, an independent isolate of HPV-11, is capable of transforming NIH 3T3 cells. The E5a open reading frame (ORF) was expressed under the control of the mouse metallothionein promoter in the expression vector pMt.neo.1, which also contains the gene for G418 resistance. Transfected cells were selected for G418 resistance and analyzed for a transformed phenotype. The transformed NIH 3T3 cells overgrew a confluent monolayer, had an accelerated generation time, and were anchorage independent. In contrast, E5a did not induce foci in C127 cells, but C127 cells expressing E5a did form small colonies in suspension. The presence of the 12-kilodalton E5a gene product in the transformed NIH 3T3 cells was shown by immunoprecipitation and was localized predominantly to nuclei by an immunoperoxidase assay. A mutation in the E5a ORF was engineered to terminate translation. This mutant was defective for transformation, demonstrating that translation of the E5a ORF is required for biological activity. This is the first demonstration of a transforming oncogene in HPV-6, and the differential activity of E5a in these two cell lines should facilitate future investigations on the mechanism of transformation.     

鲢、鳙在天然条件下的摄食强度(Ⅱ)武汉东湖鲢、鳙周年摄食强度的研究

武汉东湖的鲢(Hypophthalmichthy molitrix)鳙(Aristichthys nobilis)在天然条件下摄食强度具季节性变化。摄食强度高峰处于夏季,低谷处于冬季。在实验条件下,按周年采样期间水温变化范围,测定鱼的肠管排空率。食物通过鱼肠管时间(Y_p—h)与水温(X_t—℃)的关系为: 鲢Y_p=270.63 X_t~(0.6408) 鳙Y_p=280.46 X_t~(0.6642) 根据修正后Bajkov公式(D=C (24.A)/n),估算鱼的日粮。鱼日粮(Y_D)与水温(X_t)关系为: 鲢Y_D=0.2683e~(0.1503X_t) 鳙Y_D=0.0075X_t~(2.2715) 计算鱼在天然条件下周年月粮及年粮。鲢、鳙对天然饵料年消耗量分别为18.924公斤及17.39公斤,饵料系数分别为18.02及13.38。     

Hydroxyl radical production during oxidative deposition of iron in ferritin

The chemistry of oxidative deposition of iron(III) in ferritin and apoferritin is poorly understood. This study was undertaken to look for radicals formed as the hydrous ferric oxide core is developed from Fe(II) and O2. Radicals were observed indirectly by using the spin-trapping reagent N-tert-butyl-alpha-phenylnitrone (PBN) at room temperature and directly by measuring ESR spectra of frozen solutions at 77 K. In both instances, radical production was inhibited by the hydroxyl radical scavenging agents dimethyl sulfoxide, thiourea, and mannitol and enhanced by the addition of hydrogen peroxide. These findings strongly suggest that hydroxyl radical, produced from the iron-catalyzed Haber-Weiss reaction, is a by-product of core formation in ferritin and is a precursor to the observed radicals. The yield of ESR-observable and spin-trapped radicals is quite low, being at the micromolar level when millimolar concentrations of ferrous ion are employed. Furthermore, radical production appears to be confined to the interior of the ferritin molecule, where cellular components would be protected from the oxygen-derived toxic effects of iron. It is postulated that hydroxyl radical-medicated oxidative damage to the protein, a process that may contribute to the formation of hemosiderin from ferritin, leads to the observed radicals. By serving as a sink for hydroxyl radical, the protein shell may therefore efficiently minimize damage to other biomolecules in the cell.     

Construction of the endoplasmic reticulum

To study the construction of the ER, we used the microtubule-disrupting drug nocodazole to induce the complete breakdown of ER structure in living cells followed by recovery in drug-free medium, which regenerates the ER network within 15 min. Using the fluorescent dye 3,3'-dihexyloxacarbocyanine iodide to visualize the ER, we have directly observed the network construction process in living cells. In these experiments, the ER network was constructed through an iterative process of extension, branching, and intersection of new ER tubules driven by the ER motility previously described as tubule branching. We have tested the cytoskeletal requirements of this process. We find that newly formed ER tubules are aligned with single microtubules but not actin fibers or vimentin intermediate filaments. Microtubule polymerization preceded the extension of ER tubules and, in experiments with a variety of different drugs, appeared to be a necessary condition for the ER network formation. Furthermore, perturbations of the pattern of microtubule polymerization with microtubule-specific drugs caused exactly correlated perturbations of the pattern of ER construction. Induction of abnormally short, nonintersecting microtubules with 20 microM taxol prevented the ER network formation; ER tubules only extended along the few microtubules contacting the aggregated ER membranes. This requirement for a continuous network of intersecting microtubules indicates that ER network formation takes place through the branching and movement of ER membranes along microtubules. Cytochalasin B had no apparent effect on the construction of the ER network during recovery, despite apparently complete disruption of actin fibers as stained by phalloidin. Blockage of protein synthesis and disorganization of intermediate filaments with cycloheximide pretreatment also failed to perturb ER construction.     

Age determination in the adult screwworm (Diptera: Calliphoridae) by pteridine levels

Fluorescence spectrophotometry was used to assess the possible use of pteridines in the compound eyes to estimate the age of adult screwworms, Cochliomyia hominivorax (Coquerel). Factors affecting the quantities of pteridines include temperature and head size. No difference in pteridine levels was found among flies fed protein or carbohydrate. A regression model for estimating the age of female screwworms was constructed. The model uses head capsule size and relative pteridine quantities and assumes a constant body temperature of 30 degrees C. This regression formula has an r2 of 0.74. Our study extends the use of pteridine accumulation for age determination from obligate sanguinivorous Diptera to an autogenous species that feeds facultatively on nectar and wound exudates. The technique appears to provide a valid means to determine age of these flies.     

Molecular characterization of a major autoantibody-associated cross-reactive idiotype in Sjogren's syndrome

Primary Sjogren's syndrome is an autoimmune disorder characterized by lymphocytic infiltration of the salivary and lacrimal glands, producing associated dry eyes (keratoconjunctivitis sicca), dry mouth, and intermittently swollen salivary glands. A high proportion of the infiltrating B lymphocytes express surface and cytoplasmic Ig bearing a kappa-L chain-associated CRI defined by reactivity with the murine mAb, 17.109. To determine the structural basis for CRI expression in this disease, we generated CRI+ lymphoblastoid cell lines and a cDNA library from lymphocytes extracted from Sjogren's syndrome patients' salivary gland biopsy specimens. Nucleic acid sequence analyses of the mRNA of one such 17.109-CRI+ lymphoblastoid cell line (NOV) reveals the expressed kappa light chain variable region gene (V kappa gene) to be homologous to Humkv325, a conserved V kappa gene used at relatively high frequency in certain B cell malignancies. In addition, synthetic oligonucleotides, corresponding to the first and third frameworks and the second complementarity determining region of the Humkv325 gene, were used to identify and isolate clones from a cDNA library generated from SS salivary gland lymphocytes. Clones annealing specifically with one or more of these oligonucleotide probes contained kappa light chain cDNA. The sequences corresponding to the variable region of two clones (Taykv320 and Taykv306) were homologous to Humkv325. The V kappa genes of four other cDNA clones (Taykv322, Taykv310, Taykv308, and Taykv312) most likely were generated somatically from the rearranged Humkv325 gene through a limited number of nucleic acid base substitutions. Our results suggest that the high frequency of 17.109-CRI expression in Sjogren's syndrome patients results from a multiclonal expansion of B cells using Humkv325, and that the expressed Humkv325 may undergo somatic diversification in an apparent Ag-driven response.