Temperature dependence of capacitation in bat sperm monitored by zona-free hamster ova

The temperature dependence of capacitation in bat sperm (Myotis lucifugus lucifugus) was studied by monitoring fertilizations rates of zona-free hamster ova at different temperatures. Spermatozoa were cultured in BWW medium at temperatures 4°C, 24°C, 32°C, 42°C, and 55°C from 0–24 hr. Activation of sperm could be determined visually due to the change in movement seen through light microscopy. Activation was later confirmed by higher rates of fertilization. Preincubation of the bat sperm was found to have a direct effect on the success of penetration of the zona-free hamster ova. Holding bat spermatozoa at low temperature for long intervals allowed them to remain motile but unable to fertilize. Sperm are not irreversibly damaged, however, and activation, when the temperature is increased to 32°C, is faster than when sperm are intitially put at 32°C, resulting in good fertilization rates.     

Binding of magnesium ions to cell walls of Bacillus subtilis W23 containing teichoic acid or teichuronic acid.

When grown in a chemostat under various nutritional conditions, cells of Bacillus subtilis W23 produce walls containing teichoic acid or teichuronic acid. The binding of Mg2+ to these walls and to the isolated anionic polymers in solution was measured by equilibrium dialysis. In solution the ribitol teichoic acid bound Mg2+ in the molar ratio Mg2+/P=1:1 with an apparent association constant (Kassoc.) of 0.61 X 10(3)M-1, and the teichuronic acid bound Mg2+ in the ratio Mg2+/CO2-=1.1, Kassoc.=0.3 X 10(3)M-1. Cell walls containing teichuronic acid exhibited closely similar binding properties to those containing teichoic acid; in both cases Mg2+ was bound in the ratio Mg/P or Mg/CO2- of 0.5:1 and with a greater affinity than displayed by the isolated polymers in solution. It was concluded that Mg2+ ions are bound bivalently between anionic centres in the walls and that the incorporation of teichoic acid or teichuronic acid into the walls gives rise to similar ion-binding and charged properties. The results are discussed in relation to the possible functions of anionic polymers in cell walls.     

Genetically mediated induction of drug-metabolizing enzymes associated with congenital defects in the mouse.

Various polycyclic aromatic compounds induce certain monooxygenase activities, including aryl hydrocarbon (benzo[a]pyrene) hydroxylase (EC 1.14.14.2), and cytochrome P1-450 in the liver and many nonhepatic tissues of the mouse. This induction process is controlled by the Ah locus. Genetic differences that have been shown in the past to be associated with the Ah locus include an increased susceptibility to chemical carcinogenesis, mutagenicity in vitro, and drug toxicity--manifested as hepatic necrosis, aplastic anemia, or shortened survival time. Pregnant mice received a single injection of 3-methylcholanthrene or 7,12-dimethylbenz[a] anthracene between day 5 and day 13 of gestation, and the uterine contents were examined on day 18. Striking increases were observed in the incidence of MC-1 and DMBA-induced resorptions and congenital malformations in the aromatic hydrocarbon "responsive" C57BL/6N inbred strain, and of DMBA-induced resorptions in the "responsive" C3H/HeN and BALB/cAnN strains--when compared with the similarly treated genetically "nonresponsive" AKR/N strain. These data suggest but do not prove that an association exists between the Ah locus and developmental toxicity, i.e., teratogenesis. Although numerous teratogenic differences among inbred mouse strains have previously reported, this study is unique in that the genetic differences in teratogenicity observed were predicted in advance on the basis of known differences among these strains in polycyclic hydrocarbon metabolism regulated by the Ah locus.     

The uptake of choline by Streptococcus pneumoniae.

Uptake of choline, a structural component of pneumococcal C- and F-teichoic acids, into bacteria growing in a defined medium was very efficient with an uptake constant ([S]10 5) of 3.2 microns. It was inhibited by iodoacetate, dinitrophenol and oligomycin but not by structural analogues of choline. Ethanolamine, however, was transported in the absence of choline but with a reduced affinity ([S]0.5 71.4 microns). The same constitutive system was probably used by both ethanolamine and choline. It is suggested that this system required ATP and probably involved choline kinase.     

Effect of coitus on sperm transport and fertilization in rabbits

The number of spermatozoa into the Fallopian tubes and levels of in vivo fertilization were measured following different treatment. The results show that copulation by the artificially inseminated animal has a strong influence on the transport of the rabbit spermatozoa and "in vitro" fertilization. This constitutes some more evidence about the physiological role played by the utero-tubal junction.     

Latent fibronectin-degrading serine proteinase activity in N-terminal heparin-binding domain of human plasma fibronectin

The N-terminal 70-kDa fragment of human plasma fibronectin, purified from a cathepsin D digest, is characterized by lack of stability. It is processed proteolytically during incubation in the presence of Ca2+ into 27-kDa N-terminal heparin-binding and 45-kDa collagen-binding domains. The N-terminal residue in the 27-kDa fragment was blocked as in native fibronectin. The 45-kDa fragments began with the sequences AAVYQP, AVYQP and VYQP (residues 260, 261, 262-265 of fibronectin) that correspond to the beginning of the collagen-binding domain. In the presence of Ca2+ the purified 27-kDa fragment underwent further processing finally leading to the cleavage of the bond K85-D86 and to the simultaneous appearance of a specific proteolytic activity. Inhibition studies suggests that the newly generated enzyme is a Ca(2+)-dependent serine proteinase. Among all assayed matrix proteins, the newly generated enzyme cleaves native fibronectin and its fragments. It is proposed that this fibronectinase may originate from the N-terminal domain of fibronectin.     

Crystallization and preliminary X-ray investigation of Escherichia coli porphobilinogen deaminase.

Porphobilinogen deaminase, the polymerase that catalyses the synthesis of preuroporphyrinogen, the linear tetrapyrrole precursor of uroporphyrinogen III, has been crystallized from sodium acetate buffer with polyethylene glycol 6000 as precipitant. The crystals are orthorhombic and the space group is P2(1)2(1)2, with unit cell dimensions a = 88.01 A, b = 75.86 A, c = 50.53 A and alpha = beta = gamma = 90 degrees, indicating a single molecule of 34 kDa in the asymmetric unit. The crystals grow to dimensions of 1 mm x 2 mm x 0.5 mm within two weeks in the dark and are stable in the X-ray beam for at least 40 hours. Diffraction data beyond 1.7 A resolution, observed with a synchrotron radiation source, indicate that a high resolution structure analysis is feasible.     

Xeroderma pigmentosum endonuclease complexes show reduced activity on and affinity for psoralen cross-linked nucleosomal DNA.

Two DNA endonuclease complexes have been isolated from the chromatin of normal human and xeroderma pigmentosum, complementation group A (XPA), lymphoblastoid cells which are active on DNA damaged with psoralen plus long wavelength ultraviolet radiation (UVA). In both normal and XPA cells, one endonuclease complex, pI 4.6, recognizes the psoralen cross-link and the other endonuclease complex, pI 7.6, recognizes the psoralen monoadduct. The levels of activity of these complexes from both normal and XPA cells are similar on damaged naked DNA. Kinetic analysis of assays using graduated concentrations of substrate revealed that selective activity of these endonuclease complexes on 8-MOP plus UVA treated DNA correlates with a reduction in Km of these complexes, indicating an increased affinity for, or rate of association with, damaged naked DNA. When the damaged substrates were reconstituted into core nucleosomes (without histone H1), both normal endonuclease complexes showed a 2.5-fold enhancement of activity, which correlated kinetically with a further increase in affinity, or rate of association (decreased Km), for this damaged nucleosomal substrate. This increase in activity and in affinity was reduced but not eliminated when histone H1 was present. By contrast, neither XPA endonuclease complex showed this enhanced activity on, or affinity for, damaged core nucleosomal DNA, and actually showed decreased activity, and affinity, when histone H1 was present. Introduction, via electroporation, of either of the normal complexes into 8-MOP plus UVA treated XPA cells in culture corrected their DNA-repair defect, further confirming the role of these complexes in the repair process.     

Levels of hypoxanthine phosphoribosyltransferase RNA in human cells

The gene for the purine salvage enzyme hypoxanthine phosphoribosyltransferase (HPRT) is expressed at a low level in many cells. As is the case with several other “housekeeping genes,” thorough studies of hprt gene regulation have been hampered by the low levels of its mRNA. We have used RNA/RNA hybridization in solution to determine the concentration of hprt-RNA in human cells. The sensitivity and specificity of the method have been validated, and it is shown that hprt-RNA can be accurately determined at a level of a few mRNA molecules per cell. As expected for a housekeeping gene, low and relatively constant hprt-RNA levels (0.3–0.8 pg/μg DNA) were found in primary cultures of normal amnion cells and fibroblasts, EBV-transformed lymphoblastoid cell lines, neuroblastoma, glioblastoma, and melanoma cell cultures. While resting lymphocytes were found to contain very low amounts of hprt-RNA, lymphocytes stimulated with phytohemagglutinin (PHA) showed a 10-fold increase to about 0.8–1.2 pg/μg DNA, which corresponds to 6–10 hprt-RNA molecules per cell. The level started to increase about 20 h after PHA stimulation, 5–10 h before the onset of DNA synthesis, and a steady-state level was reached after 2–3 days in culture. In PHA-stimulated lymphocytes from two brothers with inherited HPRT deficiency (LeschNyhans syndrome), the hprt-RNA level in PHA-stimulated lymphocytes was only about 25% of that in normal subjects. In T-cells selected for HPRT deficiency by growth in 6-thioguanine medium, the levels of hprt-RNA were either normal or very low, which probably reflects the different nature of the mutations involved. These results demonstrate the sensitivity of this method for determinations of low levels of RNA and clearly show induction of hprt-RNA after mitogenic stimulation of human lymphocytes.     

Cloning and expression of rat histidase. Homology to two bacterial histidases and four phenylalanine ammonia-lyases

Histidase (histidine ammonia-lyase, EC 4.3.1.3) catalyzes the deamination of histidine to urocanic acid. Apart from phenylalanine ammonia-lyase, which is not expressed in animals, histidase is the only enzyme known to have a dehydroalanine residue in its active site. The amino site precursor and the mechanism of formation of dehydroalanine are not known. As an initial step to determining the precursor of dehydroalanine in histidase, we have isolated a functional cDNA clone for histidase from a rat liver cDNA library using an affinity-purified antiserum. The 2.2-kilobase cDNA has a 1,971-base pair open reading frame coding for a 657-amino acid polypeptide with a predicted molecular mass of 72,165 Da. The cDNA has a rare polyadenylation signal (AAUACA) that appears to inefficiently direct polyadenylation in transfected COS monkey kidney cells. Conversion of this sequence to the consensus polyadenylation signal (AAUAAA) resulted in increased levels of stable mRNA. COS cells transfected with a histidase expression vector produce active histidase. The formation of active histidase in cells that have no endogenous histidase activity suggests either that the requisite modifying enzyme is present in these cells or that the dehydroalanine residue forms by an autocatalytic mechanism. Rat histidase was found to have 41 and 43% amino acid identity to Pseudomonas putida and Bacillus subtilis histidases, respectively. Phenylalanine ammonia-lyases from parsley, kidney bean, and two yeast strains were also found to have approximately 20% amino acid identity to rat histidase. On the basis of the similarity of function of histidase and phenylalanine ammonia-lyase, dehydroalanine at the active sites, and the sequence conservation over a large evolutionary distance (mammals, bacteria, yeast, and plants), we propose that the genes for histidase and phenylalanine ammonia-lyase have diverged from a common ancestral gene, of which the most conserved regions are likely to be involved in catalysis or dehydroalanine formation.