The effect of thawing velocity on survival and acrosomal integrity of ram spermatozoa frozen at optimal and suboptimal rates in straws

The effect of various thawing velocities on the motility and acrosomal maintenance of ram spermatozoa frozen at 20 degrees C/min (optimal) or 2 degrees C/min (suboptimal) was studied. The freeze-thaw motility and the percentage of intact acrosomes of spermatozoa frozen at 20 degrees C/min increased progressively with the thawing velocity. In semen frozen at 2 degrees C/min, motility of spermatozoa and the percentage of intact acrosomes declined drastically when the thawing velocity obtained in air at 20 degrees C was increased by thawing in water at 20 degrees C. Thawing at higher temperatures markedly increased both motility and acrosomal preservation, but the best results with semen frozen at 2 degrees C/min were lower than those obtained with semen frozen at 20 degrees C/min. The optimal freeze-thaw conditions for semen protected by 4% glycerol were freezing at 20 degrees C/min and thawing in water at 60 or 80 degrees C for 8 or 5 sec, respectively. Semen collected from rams exposed to a decreasing photoperiod exhibited higher motility after freezing and thawing than those exposed to an increasing photoperiod. However, there was no effect on acrosomal preservation after freezing at 20 degrees C/min.     

Simultaneous determination of histamine and N tau-methylhistamine with high-performance liquid chromatography using electrochemical detection

We have developed a liquid chromatographic method which uses electrochemical detection for the simultaneous quantitation of histamine and N tau-methylhistamine in rat brain. The amines are derivatized with the water-soluble Bolton-Hunter reagent (sulfo B-H). Perchloric acid extracts of rat brains are chromatographed on a strong cation-exchange resin. The eluate is evaporated and allowed to react with sulfo B-H at pH 9.8 at room temperature. The derivatization is complete after 30 s vortexing. The derivatives are purified using a cellulose-phosphate fibrous cation exchanger. They are quantified with an electrochemical detector at a potential of 0.56 V after preoxidizing the sample at 0.47 V. The derivatives of histamine, N tau-methylhistamine, and N alpha-methylhistamine are completely separated without interfering peaks. Since no N alpha-methylhistamine was detected in rat brain it was used as an internal standard. The detection limits are 0.1 pmol of histamine and 0.2 pmol of N tau-methylhistamine. The precision of this method is high, with within-run and between-run coefficients of variation of 2-7% and linearity of 0.999. Both histamine and N tau-methylhistamine peak heights increased significantly and selectively after treatment with pargyline. Because of the high sensitivity, accuracy, and precision, the histamine and N tau-methylhistamine contents of single nuclei of the rat hypothalamus can be routinely quantified.     

Neurites show pathway specificity but lack directional specificity or predetermined lengths in Xenopus embryos

The earliest outgrowth of nerve fibers from identified spinal neurons labeled with horseradish peroxidase (HRP) was traced along surgically rearranged pathways in the central nervous system (CNS) of Xenopus embryos. Parts of the CNS were misaligned or inverted rostrocaudally by grafting a segment of labeled spinal cord in place of the same or different spinal cord segment of an unlabeled embryo or by joining two rostral half embryos (head-to-head) or two caudal half embryos (tail-to-tail), one half of which was derived from a labeled embryo in each combination. Donor embryos were labeled by injection of HRP into a selected blastomere at the 16- or 32-cell stage. Host embryos were unlabeled. Grafts from labeled donors to unlabeled host embryos were made at early neural tube stages before outgrowth of any nerve fibers had started (Jacobson and Huang, 1985). Routes taken by labeled nerve fibers growing into unlabeled CNS were observed at later stages, and the rates of nerve fiber elongation were calculated. Labeled nerve fibers were normal in appearance, and elongated without branching, at normal rates (22-71 micron/h). In head-to-head and tail-to-tail embryos and in embryos with inverted spinal cord grafts, nerve fibers continued elongating without branching in the direction opposite to normal in the CNS. Many fibers reached lengths that were far greater than normal. No reorientation of such maldirected nerve fibers was seen. These results indicate that nerve fiber elongation is not guided by axially polarized pathway cues or markers and that nerve fibers do not grow to predetermined lengths. However, neurites preferred to grow along stereotyped nerve fiber pathways even when forced to grow in the wrong direction or when confronted with nonneural tissue.     

High CO(2) Requiring Mutant of Anacystis nidulans R(2)

Some physiological characteristics of a mutant (E₁) of Anacystis nidulans R₂, incapable of growing at air level of CO₂, are described. E₁ is capable of accumulating inorganic carbon (C_i) internally as efficiently as the wild type (R₂). The apparent photosynthetic affinity for C_i in E₁, however, is some 1000 times lower than that of R₂. The kinetic parameters of ribulose 1,5-bisphosphate carboxylase/oxygenase from E₁ are similar to those observed in R₂. The mutant appears to be defective in its ability to utilize the intracellular C_i pool for photosynthesis and depends on extracellular supply of Ci in the form of CO₂. The very high apparent photosynthetic K_m (CO₂) of the mutant indicate a large diffusion resistance for CO₂. Data obtained here are used to calculate the permeability coefficient for CO₂ between the bulk medium and the carboxylation site of cyanobacteria.     

The complete nucleotide sequence of the group II RNA coliphage GA

The complete nucleotide sequence of the RNA coliphage GA, a group II phage, is presented. The entire genome comprises 3466 bases. Three large open reading frames were identified, which correspond to the maturation protein gene (390 amino acids), the coat protein gene (129 amino acids) and the replicase beta-subunit protein gene (531 amino acids). In addition, untranslated regions occur at the 5' (135 bases) and 3' (122 bases) ends of the molecule. Two intercistronic untranslated regions occur between the cistrons for the maturation and coat proteins, and between the coat and beta-subunit proteins. We have compared the nucleotide sequence of GA RNA with the published sequence of MS2 RNA, and show that they are related. The comparative structures of two important regulatory regions are presented; the coat protein binding site which is involved in translational repression of the replicase beta-subunit protein gene, and a hairpin in a region proximal to the lysis protein gene.     

N-acetyl-L-glutamate synthase of Neurospora crassa. Characteristics, localization, regulation, and genetic control

N-Acetylglutamate synthase, an early enzyme of the arginine pathway, provides acetylglutamate for ornithine synthesis in the so-called "acetylglutamate cycle." Because acetylglutamate is regenerated as ornithine is formed, the enzyme has only a catalytic or anaplerotic role in the pathway, maintaining "bound" acetyl groups during growth. We have detected this enzyme in crude extracts of Neurospora crassa and have localized it to the mitochondria along with other ornithine biosynthetic enzymes. The enzyme is bound to the mitochondrial membrane. The enzyme has a pH optimum of 9.0 and Km values for glutamate and CoASAc of 6.3 and 1.6 mM, respectively. It is feedback-inhibited by L-arginine (I0.5 = 0.16 mM), and its specific activity is augmented 2-3-fold by arginine starvation of the mycelium. Mutants of the newly recognized arg-14 locus lack activity for the enzyme. Because these mutants are complete auxotrophs, we conclude that N-acetylglutamate synthase is an indispensible enzyme of arginine biosynthesis in N. crassa. This work completes the assignment of enzymes of the arginine pathway of N. crassa to corresponding genetic loci. The membrane localization of the enzyme suggests a novel mechanism by which feedback inhibition might occur across a semipermeable membrane.     

Genetic and physiological parameters associated with cadmium toxicity in Drosophila melanogaster

Two strains of Drosophila melanogaster represent the extremes in resistance and sensitivity to the lethal effects of CdCl₂. The strain containing the mutations vermilion and brown (v; bw) and the strain Austin had LC₅₀'s of 3.3 and 1.3mm CdCl₂, respectively. The three major chromosomes from these two strains were assorted genetically into the six possible combinations. The measured LC₅₀'s for CdCl₂ for these six genotypes fell into two groups according to the X chromosome; those containing the X chromosome from v; bw had LC₅₀'s 0.5–1.0mm greater than those in which the X chromosome was from Austin. Since the parent strains differed by 2mm, we suggest that the X chromosome is a major, but not the sole, site of genes to produce resistance to CdCl₂. When ¹⁰⁹Cd was in the diet the uptake by v; bw and Austin over 2 days was the same. After 4 days of uptake, the Austin strain excreted the ¹⁰⁹Cd five times faster than v; bw but the six genotypes did not differ appreciably in excretion rate from one another and resembled the sensitive parent Austin more than the resistant one. Thus a second process is indicated that distinguishes resistance to CdCl₂ that apparently is not associated with the X chromosome.This research was sponsored by the Office of Health and Environmental Research, U.S. Department of Energy, under Contract DE-AC05-84OR21400 with Martin Marietta Energy Systems, Inc.     

Selectivity in the binding of hydroxylated benzo[a]pyrene derivatives to purified cytochrome P-450c

The interaction of rat liver microsomal cytochrome P-450c with potential benzo[a]pyrene (BP) metabolites has been compared with the binding of BP by optical and fluorescence spectroscopy. Fluorescence quenching of the phenolic derivatives of BP derives from 1:1 complex formation with P-450c, is a function of the position of the hydroxyl substituent, and correlates with the concomitant increase in high-spin cytochrome observed in parallel optical titrations. The proportion of high-spin cytochrome seen when P-450c was reconstituted in dilauroylphosphatidylcholine vesicles (60 micrograms/mL) ranged from about 7% for the 3- and 7-phenols to 75% for 11- and 12-phenols. BP and all 12 methyl-BP derivatives have comparable high affinities for P-450c (50-70% high spin). Kd determinations with purified P-450c indicated very strong binding of BP phenols that induce high-spin complexes (4-, 5-, 9-, 10-, 11-, and 12-phenols; Kd = 3-25 nM). Inhibition of n-octylamine binding by the 3- and 7-phenols indicated weak interactions (Kd = 80-90 nM), even though low-spin complexes were formed. Inhibition of BP metabolism catalyzed by P-450c with BP phenols correlated with their respective dissociation constants. These results suggest that phenolic substitution at certain positions on BP (1, 2, 3, 7, or 8) interferes with binding to the active site while substitutions at the other positions either enhance or have no effect on binding. BP dihydrodiols [including the (+)- and (-)-BP 7,8-dihydrodiols] were relatively ineffective in forming high-spin complexes (approximately 20%), and fluorescence quenching of dihydrodiols by P-450c also saturated at low levels.(ABSTRACT TRUNCATED AT 250 WORDS)     

Identification of a novel ganglioside on erythrocytes with blood group Cad specificity

The blood group Cad antigen is a carbohydrate structure well characterized on the sialoglycoproteins of the red cell membrane from some rare individuals (Blanchard, D., Cartron, J. P., Fournet, B., Montreuil, J., Van Halbeck, H., and Vliegenthart, J.F.G. (1983) J. Biol. Chem. 258, 7691-7695). However, protease treatment of whole cells did not destroy their antigenic activity which indicated that glycolipid might also be involved in the antigenic reaction. A crude ganglioside fraction was prepared from Cad cells and found to inhibit the hemagglutination reaction, whereas neutral glycolipids were inactive. Further analysis of the ganglioside extract from Cad erythrocytes by thin layer chromatography revealed an unusual profile characterized by a lower content of sialosylparagloboside and the presence of a novel ganglioside of slower mobility. Immunochemical studies demonstrate that this ganglioside binds Helix pomatia lectin and inhibits human anti-Sda antibody. In addition, a ganglioside with identical chromatographic mobility can be obtained by the enzymatic transfer of GalNAc from UDP-GalNAc to sialosylparagloboside using a microsomal preparation from human kidney. These results together with cell surface labeling experiments suggest that the major ganglioside of Cad erythrocytes might be derived from sialosylparagloboside by substitution with an additional N-acetylgalactosamine residue.     

Effect of spermidine on the conformation of bacteriophage MS2 RNA. Electron microscopy and computer modeling

The structure of single-stranded RNA from the bacteriophage MS2 has been examined by electron microscopy in the presence of the polyamine spermidine. The molecules are found in two alternate conformations. The first of these can be characterized as a cruciform structure composed of three large loops approximately 500 to 700 nucleotides in size. The interior of the molecule has extensive base-paired regions which connect distant regions of the molecule; the farthest being 2500 nucleotides apart. In the second conformation, the molecules appear rod-like. Two of the large loops disappear, and these regions form, instead, extensive long-range helices. Computer modeling has been employed to explore the base-pairing potential of the sequence of bacteriophage MS2 RNA. Double-stranded regions identified by electron microscopy are shown to occur in local G + C-rich stretches of the RNA. Detailed models have been calculated for two regions of long-range contact. One of these includes the ribosome-binding site for the viral coat protein gene. The results are discussed in the context of the known role of RNA structure in the regulation of viral gene expression.