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.     

Photo-inhibition and the Effects of Protein Phosphorylation on Electron Transport in Wheat Thylakoids

The kinetics of changes in photosystem I (PSI), photosystemII (PSII), and whole chain (PSII and PSI) electron transport,chlorophyll fluorescence parameters, the capacity to bind atrazineand the polypeptide profiles of thylakoids isolated from wheatleaves on exposure to a photon flux density of 2000 µmolm^–2 s^–1 were determined. Severe and similar levelsof photo-inhibitory damage to both PSII and whole chain electrontransport occurred and were correlated with decreases in theratio of variable to maximal fluorescence, the proportionalcontribution of the rapid a phase of the fluorescence kineticsand the capacity to bind atrazine. Severe photo-inhibition ofelectron transport was not associated with a major loss of chlorophyllor total thylakoid protein. However, a small decrease in a 70kDa polypeptide together with increases in a number of low molecularmass polypeptides (8–24 kDa) occurred. Phosphorylation of thylakoid polypeptides alleviated photo-inhibitionof PSII electron transport but stimulated photoinhibitory damageto whole chain electron transport. The consequences of suchphosphorylation-induced effects on photoinhibition in vivo areconsidered. Key words: Chlorophyll fluorescence, electron transport, photo-inhibition, protein phosphorylation, thylakoid membranes, wheat (Triticum aestivum)     

Genetic analysis of the relationship between interleukin production and worm rejection in Trichinella spiralis-infected inbred mice

The production of interleukin 1 (IL-1), IL-2, and IL-3 by peritoneal macrophages, mesenteric lymph node (MLN), or spleen cells from inbred strains of mice infected with Trichinella spiralis was examined. The mice belonged to the worm rejection phenotypes previously characterized as strong (NFS), intermediate (C3H, BUB, DBA/1, SWR, CBA, etc.), or weak (B10.Q, B10.BR, etc.). Strong responder NFS mice produced approximately twice as much IL-1 as intermediate responder C3Heb/Fe or weak responder B10.BR mice. IL-3 production varied slightly among strains but did not show any relationship to the phenotype of rejection (highest: C3Heb/Fe, B10.BR; lowest: B10.Q). Of 16 strains of inbred mice and 6 F1 hybrid crosses assessed, marked variations occurred in IL-2 production from MLN cells in response to T. spiralis antigen challenge in vitro. When 16 mouse strains were compared IL-2 production ranged from 5.1 units/ml (A/J) to 29.8 (NFS). Variations in IL-2 production among mouse strains did not relate directly to MHC haplotype, and the capacity of an individual strain to release IL-2 or IL-3 did not correlate with adult worm rejection phenotype. Genetic linkage studies proved that the gene(s) regulating IL-2 production in T. spiralis infection were not linked to the gene(s) regulating adult worm rejection. Regression analysis showed a weak correlation of high IL-2 production with weak worm rejection suggesting that IL-2 production or an associated process is a negative factor in primary worm rejection.     

Expression and biochemical characterization of human immunodeficiency virus type 1 nef gene product.

nef genes from human immunodeficiency virus type 1 isolates BH10 and LAV1 (lymphadenopathy-associated virus type 1) were expressed in Escherichia coli under the deo operon promoter. The two proteins found in the soluble compartment of the bacterial lysate were purified by ion-exchange column chromatography to apparent homogeneity. Determination of the amino-terminal sequence revealed glycine as the first amino acid in the Nef protein, indicating removal of the initiator methionine during expression in E. coli. Under native conditions, the recombinant Nef protein is a monomer of 23 kilodaltons. In denaturing polyacrylamide gels, however, BH10 and LAV1 Nef proteins migrate as 28 and 26 kilodaltons, respectively. GTP binding and GTPase activity were monitored during Nef protein purification. These activities did not copurify with the recombinant Nef protein from either the BH10 or the LAV1 isolate. Purified recombinant BH10 Nef protein was used as an immunogen to elicit mouse monoclonal antibodies. A series of monoclonal antibodies were obtained which reacted with sequences at either the amino or carboxy terminus of Nef. In addition, a conformational epitope reacting with native BH10, but not LAV1, Nef was isolated.     

Mapping of B-cell epitopes of the human hepatitis B virus X protein.

The immune response to the X protein of human hepatitis B virus (HBV) was studied by epitope mapping by using a set of MS2-HBx fusion proteins and synthetic peptides. Antibodies in sera of patients with acute and chronic HBV infection showed a multispecific immune response. Each serum contained antibodies to a different set of epitopes, which taken together cover most of the HBx sequence. Some of the epitopes were detectable only by immunoblotting with fusion proteins; others were detectable only by an enzyme-linked immunosorbent assay (ELISA) with synthetic peptides. The carboxy-terminal half of the HBx protein was preferentially recognized by antibodies from patients with chronic hepatitis and contained a short immunodominant antigenic region with at least two major nonoverlapping epitopes. Anti-HBx antibody titers as revealed by peptide ELISAs were highest and most frequent in patients with chronic hepatitis and usually low in acutely infected patients and asymptomatic carriers. The data demonstrate a remarkable qualitative and quantitative heterogeneity of the humoral HBx immune response which can be monitored by HBx-specific peptide ELISAs. Such tests may become useful diagnostic tools.     

The viral envelope gene is involved in macrophage tropism of a human immunodeficiency virus type 1 strain isolated from brain tissue.

Human immunodeficiency virus type 1 (HIV-1) strains isolated from the central nervous system (CNS) may represent a subgroup that displays a host cell tropism different from those isolated from peripheral blood and lymph nodes. One CNS-derived isolate, HIV-1SF128A, which can be propagated efficiently in primary macrophage culture but not in any T-cell lines, was molecularly cloned and characterized. Recombinant viruses between HIV-1SF128A and the peripheral blood isolate HIV-1SF2 were generated in order to map the viral gene(s) responsible for the macrophage tropism. The env gene sequences of the two isolates are about 91.1% homologous, with variations scattered mainly in the hypervariable regions of gp120. Recombinant viruses that have acquired the HIV-1SF128A env gene display HIV-1SF128A tropism for macrophages. Furthermore, the gp120 variable domains, V1, V2, V4, and V5, the CD4-binding domain, and the gp41 fusion domain are not directly involved in determining macrophage tropism.     

CD4-independent, productive human immunodeficiency virus type 1 infection of hepatoma cell lines in vitro.

Five hepatoma cell lines, including CZHC/8571, PLC/PRF/5, Hep3B, HepG2, and HUH7, were inoculated with three diverse isolates of human immunodeficiency virus type 1 (HIV-1). Productive infection was noted in all hepatoma cell lines, and expression of viral p24 antigen lasted for over 3 months, but its level decreased in proportion to the number of viable cells. HIV-1 antigens were also found in the cells by immunohistochemical staining and radioimmunoprecipitation assay, as were viral RNA by in situ hybridization and HIV-1-like particles by electron microscopy. Virus yield assays were also positive on supernatant fluids collected from hepatoma cultures inoculated with HIV-1. Despite their susceptibility to infection, all five hepatoma cell lines were negative for CD4 by immunofluorescence and for CD4 mRNA by slot-blot hybridization. In addition, HIV-1 infection of hepatoma cell lines was not blocked by anti-CD4 monoclonal antibody or soluble CD4. Together, these findings clearly demonstrate that all five hepatoma cell lines were susceptible to productive infection by HIV-1 in vitro via a CD4-independent mechanism.     

Regulation of bile acid synthesis. III. Correlation between biliary bile salt hydrophobicity index and the activities of enzymes regulating cholesterol and bile acid synthesis in the rat

Hepatic bile acid synthesis is thought to be under negative feedback control by bile salts in the enterohepatic circulation, acting at the level of cholesterol 7 alpha-hydroxylase (C7 alpha H), the initial and rate-limiting step in the bile acid biosynthetic pathway. Bile salts also suppress the activity of the rate-limiting enzyme for cholesterol synthesis, 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMG-CoA-R). The mechanisms of these regulatory effects are poorly understood, and one or both may be indirect. Previous data suggest that the hydrophilic-hydrophobic balance of bile salts, a major determinant of their cholesterol solubilizing properties, also determines their potency as regulators of bile acid and cholesterol synthesis. To further evaluate the relationship between the physicochemical and regulatory properties of bile acids, we altered the composition of the bile salt pool of rats by feeding one or more of seven different bile acids (1% w/w for 14 days). We then determined the mean hydrophilic-hydrophobic balance (hydrophobicity index) of the bile salts in bile, and correlated this with the specific activities of C7 alpha H and HMG-CoA-R, and of acyl-CoA:cholesterol acyltransferase (ACAT), a third hepatic microsomal enzyme which regulates cholesterol esterification. In all instances following bile acid feeding, conjugates of the fed bile acid(s) became the predominant bile salts in bile. Highly significant negative linear correlations (each P less than 0.0001) were found between the hydrophobicity indices of biliary bile salts and the activities of C7 alpha H (r = 0.79) or HMG-CoA-R (r = 0.63). By contrast, no significant correlation could be demonstrated between ACAT activity and the hydrophobicity index of biliary bile salts. The correlation between activities of HMG-CoA-R and C7 alpha H was also highly significant (r = 0.81; P less than 0.0001). No significant correlation existed between ACAT and either HMG-CoA-R or C7 alpha H. Microsomal free cholesterol was not consistently altered by bile acid feeding. Thus, the potency of circulating bile salts as suppressors of the enzymes regulating bile acid and cholesterol synthesis increases with increasing hydrophobicity. The hydrophobic-hydrophilic balance of the bile salt pool may play an important role in the regulation of cholesterol and bile acid synthesis.     

Combined in-beam electron impact-B/E-linked scan mass spectrometry of oxazoline derivatives for the structure determination of long-chain unsaturated fatty acids

Long-chain unsaturated fatty acids (UFA) having up to six double bonds are derivatized to 2-substituted 4,4-dimethyloxazolines (DMOX) and then analyzed by combined in-beam electron impact (IBEI)-B/E-linked scan mass spectrometry. This technique provides highly characteristic mass spectra and may serve as an auxiliary means for direct structure determination of individual UFA in mixtures.     

Origin of ultraviolet damage in DNA

A novel ultraviolet (u.v.) footprinting technique has been used to analyze the formation of u.v. photoproducts at 250 bases of a 5 S rRNA gene under conditions where the gene is either double or single-stranded. Because many more types of u.v. damage can be detected by the u.v. footprinting technique than has been previously possible, we have been able to examine in detail why certain bases in DNA are damaged by u.v. light while others are not. Our measurements demonstrate that the ability of u.v. light to damage a given base in DNA is determined by two factors, the sequence of the DNA in the immediate vicinity of the photoproduct, and the flexibility of the DNA at the site of the photoproduct. For pyrimidines, the predominant photoreaction in double-stranded DNA involves covalent dimerization between adjacent pyrimidine residues. Dimerization is much easier in melted DNA because the geometrical changes required for adjacent pyrimidine residues to dimerize are easier in single-stranded DNA. The absorption of a u.v. photon cannot simultaneously induce the geometrical changes required for adjacent pyrimidines or other bases to dimerize with one another. Rather, upon the absorption of a u.v. photon, only those thermally excited bases that are in a geometry capable of easily forming a photodimer during excitation, can photoreact. In contrast to adjacent pyrimidines, non-adjacent pyrimidines (pyrimidines flanked on either side by a purine) do not readily form u.v. photoproducts in double-stranded DNA. Because photoreactions at non-adjacent pyrimidine residues are greatly enhanced in single-stranded DNA, their failure to form in double-helical DNA is attributed to torsional constraints imposed by the double helix which make it difficult for non-adjacent pyrimidines to adopt a geometry necessary for photoreaction. Although purines are believed to be resistant to u.v. damage, our measurements demonstrate that at moderate u.v. dosages purines which are flanked on their 5' side by two or more contiguous pyrimidines readily form u.v. photoproducts in double-stranded DNA. Flanking pyrimidines appear to activate purine photoreactions by transferring triplet excitation energy to the purine. Melting of the DNA helix greatly inhibits the ability of flanking pyrimidines to activate purine photoreactions, presumably by disrupting intimate orbital overlap required for triplet transfer.