In vitro fertilization of water buffalo follicular oocytes and their ability to cleave in vitro

Water buffalo (Murrah) oocytes were collected from ovaries obtained from a local slaughterhouse. They were classified according to the character of the cumulus cells under a stereomicroscope and then cultured in 25 mM Hepes buffered tissue culture medium-199 (TCM-199) supplemented with 5% estrous water buffalo serum in an atmosphere containing 5% CO(2) in air at 39 degrees C. After 20 to 24 hours of in vitro maturation, the oocytes were cultured at 38.5 degrees C in TCM-199 supplemented with 1% estrous water buffalo serum and in an atmosphere containing 5% CO(2) in air. Oocytes with compact and dense cumulus cells cleaved significantly further (P<0.01, 67.3%, 33 49 ) than those with fair, partially denuded oocytes with thin cumulus layers (27.5%, 25 91 ) or small remnants of cumulus cells and poor naked oocytes (3 100 ). A substantial variation in fertilization and developmental rates (16.0 to 43.8%) was observed among 4 different bulls. Late morulae were transferred nonsurgically into 14 buffalo recipients on Day 6 or 7 of their estrous cycle. One recipient was diagnosed to be pregnant by palpation per rectum on Day 60 and delivered a calf in October 1991.     

Developmental profiles of arylsulfatases A and B in rat cerebral cortex and spinal cord.

Arylsulfatases A (EC 3.1.6.1) and B (EC 3.1.6.12) are lysosomal enzymes that can remove sulfate groups from sulfatides and sulfo-glycosaminoglycans, respectively. The activities of these enzymes in cerebral cortex and in spinal cord of developing rat pups were measured. The tissues were homogenized and the arylsulfatases A and B in the soluble fraction were separated from each other by anion exchange chromatography on DE-52 cellulose. Subsequently, the enzyme activities were assayed with p-nitrocatechol sulfate as substrate at 37 degrees C and pH 5.6. We observed a developmental profile of arylsulfatase A, similar to that previously reported for cerebroside sulfatase (EC 3.1.6.8; (Van der Pal et al. (1990) Biochim. Biophys. Acta 1043, 91-96]. The activity of arylsulfatase A increased gradually during development, whereas arylsulfatase B rose more steeply, peaked around day 15 and declined thereafter. As a consequence the ratio between B and A forms of arylsulfatase dropped from about 4 in 1-week-old pups to 2.2 (cortex) and 0.7 (cord) in 7-week-old rat pups.     

Embryo culture of Costus speciosus (Koen.) Sm. to regenerate variable diosgenin yielding clones

Mature embryos of Costus speciosus were excised and cultured on Schenk and Hildebrandt's (1972) nutrient medium containing auxins and cytokinins either alone or in combination. Multiple shoots were obtained when kinetin and indole-3-butyric acid were supplemented each at 0.1 mg 1^–1 concentration. Embryo-derived plantlets were multiplied through propagation of rhizomes and the propagules derived from a single embryo were designated as an embryoclone. Twenty such embryo-clones were maintained in the field. Variations in rhizome biomass yield and diosgenin contents of these embryoclones were noted. Thirty-six percent of the embryo-clones studied were high diosgenin yielding types. Diosgenin contents at the intraclonal level were uniform. The in vitro raised plants were morphologically uniform and indistinguishable from their parent.Abbreviations SH Schenk and Hildebrandt (1972) medium - Kn kinetin - BAP 6-benzylaminopurine - 2,4-D 2,4-dichlorophenoxyacetic acid - NAA 1-naphthaleneacetic acid - IBA indole-3-butyric acid - IAA indole-3-acetic acid - CA casaminoacids (vitamin free) - TLC thin layer chromatography     

Hemoglobins,XLVIIII

Summary Hagfish hemoglobin has three main components, one of which is Hb III. It is monomeric and consists of 148 amino acid residues (M = 17 350). Its complete primary structure, previously published, is discussed here. The proximal amino acid (F8) of the heme linkage is histidine as always in the hemoglobins, but the regularly expected distal histidine E7 is substituted by glutamine. This substitution, leading to a new kind of heme linkage, has hitherto only been demonstrated in opossum hemoglobin. It is suggested that E7, Gln, is directed out of the heme pocket, and that the adjacent Ell, Ile, is directed toward the inside of the pocket, giving the distal heme contact instead of histidine.Myxine Hb III has an additional tail of 9 amino acid residues at its N-terminal end, as has the hemoglobin ofLampetra fluviatilis. The genetic codes ofMyxine andLampetra hemoglobins show 117 differences, in spite of many morphological resemblances between hagfish and lamprey. Their primary hemoglobin structures show differences substantial enough to bo compatible with the divergence of the two families some 400–500 million years ago.     

Crystal and molecular structure of the sulfhydryl protease calotropin DI at 3·2 Å resolution

The three-dimensional structure of the sulfhydryl protease calotropin DI from the madar plant, Calotropis gigantea, has been determined at 3·2 Å resolution using the multiple isomorphous replacement method with five heavy atom derivatives. A Fourier synthesis based on protein phases with a mean figure of merit of 0·857 was used for model building. The polypeptide backbone of calotropin DI is folded to form two distinct lobes, one of which is comprised mainly of α-helices, while the other is characterized by a system of all antiparallel pleated sheets. The overall molecular architecture closely resembles those found in the sulfhydryl proteases papain and actinidin.Despite the unknown amino acid sequence of calotropin DI a number of residues around its active center could be identified. These amino acid side-chains were found in a similar arrangement as the corresponding ones in papain and actinidin. The polypeptide chain between residues 1 and 18 of calotropin DI folds in a unique manner, providing a possible explanation for the unusual inability of calotropin DI to hydrolyze those synthetic substrates that papain and actinidin act upon.     

Pardaxin, a hydrophobic toxin of the Red Sea flatfish, disassembles the intact membrane of vesicular stomatitis virus

Reaction of vesicular stomatitis virus with pardaxin, the hydrophobic toxin of the Red Sea flatfish, resulted in a profound morphological change of many virions and dissociation of their membrane and nucleocapsid into components readily separable by density gradient centrifugation. The basic matrix protein and acidic pardaxin segregated largely with the high density nucleocapsid. The dissociated virion membrane formed lipoprotein vesicles which retained glycoprotein spikes and a certain amount of N protein but no appreciable amounts of other nucleocapsid proteins and little if any RNA. Iodination of the tyrosine residue of the glycoprotein tail fragment provided supporting evidence that the COOH terminus of the glycoprotein extends beyond the inner layer of the membrane into the interior of the virion. These data indicate that pardaxin may serve as a probe for studying the organization of viral membranes, and, hopefully, other biological membranes.     

Monoamine oxidase of rat skeletal muscle: substrate specificity and half-life

The substrate specificity of rat skeletal muscle MAO has been studied. By the use of clorgyline as a MAO A inhibitor, it is found that 5-hydroxytryptamine, tryptamine, and kynuramine are deaminated by MAO A whereas benzylamine is a substrate for both forms of MAO. Phenethylamine displays a concentration-dependent preference for the two forms of MAO. These substrate specificies of the two forms of MAO in skeletal muscle are different from those observed in liver and brain but resemble closely that seen with heart. The half-lives of MAO A and MAO B in muscle estimated by rate of recovery from pargyline inhibition are 6.9 and 6.4 days, respectively.     

The circular dichroism of lysozyme.

The circular dichroism spectra of hen egg white lysozyme, and of lysozyme derivatives in which tryptophan residues 62 or 108, or both, are selectively oxidized, have been measured as a function of pH over the range of 200 to 310 nm. Neither Trp-62 nor Trp-108 is principally responsible for the positive rotational strength in the 280 to 300 nm region. The spectrum in the 200 to 230 nm region is nearly the same in the native protein and in the derivatives, and is little affected by binding of saccharide. These results are used to reinterpret the circular dichroism spectra of the lysozymes and alpha-lactalbumins.     

Affinity labeling of the inhibitory DPNH site of bovine liver glutamate dehydrogenase by 5'-fluorosulfonylbenzoyl adenosine.

A new adenosine analogue has been synthesized, 5'-fluorosulfonylbenzoyl adenosine, which reacts covalently with bovine liver glutamate dehydrogenase with the incorporation of approximately 1 mol of 5'-sulfonylbenzoyl adenosine per peptide chain. Native glutamate dehydrogenase is known to be inhibited by relatively high concentrations of DPNH by binding to a second noncatalytic site; the major change in the kinetic characteristics of the modified enzyme is a total loss of this inhibition by DPNH. The modified enzyme retains full catalytic activity as measured in the absence of allosteric ligands, is still inhibited more than 90% by GTP, and is activated normally by ADP. These results demonstrate that the catalytic as well as the GTP and ADP regulatory sites are distinct from the inhibitory DPNH site. The rate constant for reaction of 5'-fluorosulfonylbenzoyl adenosine is decreased by high concentrations of DPNH alone or by DPNH plus GTP, but not by the substrate alpha-ketoglutarate, the coenzymes DPN or TPNH, or the regulators ADP or GTP alone. These observations are consistent with the postulate that the 5'-fluorosulfonylbenzoyl adenosine attacks exclusively the second inhibitory DPNH site. The DPNH inhibition is abolished when an average of only 0.5 mol of 5'-sulfonylbenzoyl adenosine per peptide chain has been incorporated. The structure of 5'-fluorosulfonylbenzoyl adenosine is critical in determining the course of the modification reaction. The smaller compound p-fluorosulfonylbenzoic acid does not affect the kinetic characteristics of the enzyme, and the isomeric compound 3'-fluorosulfonylbenzoyl adenosine produces a different pattern of changes in the regulatory properties (Pal. P. K., Wechter, W. J., and Colman, R. F. (1975) Biochemistry 14, 707-715). Indeed, enzyme which has combined stoichiometrically with 5'-fluorosulfonylbenzoyl adenosine is still able to react with 3'-fluorosulfonylbenzoyl adenosine; thus, the two adenosine analogues appear to react at distinct sites on glutamate dehydrogenase. It is proposed that 5'-fluorosulfonylbenzoyl adenosine will be complementary to 3'-fluorosulfonylbenzoyl adenosine as a general affinity label for dehydrogenases as well as other classes of enzymes which use adenine nucleotides as substrates or regulators.