Molecular cloning of bovine actin-like protein, actin2.

Actins are major cytoskeletal components and highly conserved in evolution. In mammals, there are six actin isoforms, a pair of which shows at least 93% identity in the amino acid sequence. We have cloned cDNA for a bovine protein that is distantly related to members of the mammalian actin isotypes. The predicted amino acid sequence (418 residues long, calculated molecular mass 47369) shows that this protein, which we have named actin2, exhibits 36% identity to mammalian actins and 60% identity to the yeast actin-like protein, act2. We have concluded that actin2 defines a new class of mammalian actin-like proteins. It was also revealed that actin2 messenger RNA is expressed in a broad range of tissues.     

Luteinizing hormone requirements for ovulation in the rat.

Luteinizing hormone requirements for ovulation induction were studied in proestrous rats through detailed observation of the preovulatory surge, through various forms of LH injection under sodium pentobarbital blockade, and through estimation of LH uptake by the ovary. Blood LH levels in individual proestrous rats were obtained every 30 min and grouped according to their peak time (designated 0 h); mean LH levels higher than 7 and 5 ng/ml continued for 30 min and 2.5 h, respectively, the pituitary LH contents at 1400 and 2000 h on the day of proestrus were 2.1 and 0.7 micrograms, respectively, indicating that the amount of LH secreted during the surge was at least 1.4 micrograms. Single intravenous injections of 2 micrograms and 1 micrograms of pure rat LH (NIDDK-rLH-I-7; FSH and prolactin contaminations: 0.02% and less than 0.01%, respectively) to sodium pentobarbital-blocked rats induced ovulation in 4 out of 4 rats and 4 out of 6 rats, respectively, while 500 ng failed to induce ovulation in any (out of 7) rats. Two injections of 300 ng each with an interval of 20 min induced ovulation in 3 out of 8 rats, but if the interval was prolonged to between 30 and 120 min, 100% ovulation was obtained. Blood LH levels in these experiments indicated that a lower long-lasting LH level (about 5 ng/ml blood) is more important than a short, high level for ovulation induction. It was also shown that this level of LH could be given in separate doses if the interval was 30-120 min.(ABSTRACT TRUNCATED AT 250 WORDS)     

Recombinant mouse ZP3 inhibits sperm binding and induces the acrosome reaction.

Mammalian fertilization involves interactions of sperm surface receptors with ligands of the zona pellucida, an extracellular matrix surrounding the ovulated egg. In mouse, the zona is composed of three glycoproteins. One of them, ZP3, participates in primary sperm binding and in the subsequent triggering of the sperm's acrosome reaction. Considerable evidence suggests that carbohydrate determinants of ZP3 are responsible for binding to sperm and may be important for acrosomal exocytosis. A full-length cDNA encoding mouse ZP3 was assembled and cloned into expression vectors that contained either a cytomegalovirus (CMV) or a vaccinia (P11) promoter. Mouse L-929 cells were stably transformed with the pZP3-CMV constructs, and green monkey CV-1 cells were infected with a recombinant vaccinia virus containing ZP3. rZP3 was affinity purified from culture media and detected on Western blots as a single 60- to 70-kDa band, which differed in molecular weight from native ZP3 (mean, 83 kDa). Nevertheless, rZP3 is biologically active. rZP3 decreases sperm-zona binding with a potency equivalent to that of native zona pellucida and, like native ZP3, rZP3 triggers acrosomal exocytosis in capacitated mouse sperm. Thus, rZP3 isolated from both rodent and primate cells appears to contain those carbohydrate and protein structures necessary for ZP3's dual role in fertilization.     

Unique structure in the intergenic and 5' external transcribed spacer of the ribosomal RNA gene from the pea aphid Acyrthosiphon pisum.

We analyzed the DNA sequence of the 5' external transcribed spacer (ETS) and part of the intergenic transcribed spacer (IGS) of the aphid ribosomal RNA gene (rDNA). The 5' ETS of aphid rDNA consists of 843 nucleotides with a G/C content of 69 mol/100 mol, far higher than that of any other known 5' ETS for insect rDNA. The IGS of aphid rDNA contained a characteristic array of repeated sequences of 247 nucleotides. The repeated sequences were identical. It was shown that the number of the repeating sequence is heterogeneous.     

Identification of an alpha 2-adrenoceptor in human coronary arteries by radioligand binding assay.

A single high affinity binding site for an alpha 2-adrenoceptor in human coronary arteries was identified by radioligand binding assay. Human coronary arteries were obtained at autopsy within 6 hours of death. A crude membrane solution was incubated with (3H)-rauwolscine at 25 degrees C for 30 min. The binding of (3H)-rauwolscine was rapidly saturable and reversible. Kd was 1.2 +/- 0.2 (SE) nM and Bmax 22 +/- 3 fmol/mg protein. This is the first study which has shown the presence of an alpha 2-adrenoceptor in human coronary arteries using a radioligand binding assay method.     

Unique DNA plasmid pRS64 associated with chromosomal DNAs of the plant pathogenic fungus Rhizoctonia solani.

Unique DNA sequences homologous to the linear DNA plasmid pRS64 were investigated in chromosomal DNAs of isolates belonging to anastomosis group 4 (AG-4) of the plant pathogenic fungus Rhizoctonia solani. Chromosome-sized DNAs of isolates RI-64 and 1271 of AG-4 were separated into six bands by orthogonal-field-alternation gel electrophoresis and hybridized to a cloned segment of pRS64. A small chromosome-sized DNA band of approximately 1.1 Mb carried the sequences homologous to pRS64 DNA. Sequences homologous to pRS64 were also maintained within the chromosomal DNA of isolate 127.1 of AG-4 which does not possess the plasmid. The plasmid showed no homology to the mitochondrial DNA of isolate 1271. The possibility that the linear plasmid pRS64 may act as a transposable genetic element is discussed.     

Studies on the relationship between the pathogenicity of Paracoccidioides brasiliensis in mice and its growth rate under different oxygen atmospheres

We performed comparative studies of the pathogenicity of six strains of Paracoccidioides brasiliensis (Bt-9, Bt-4, Pb-9, Pb-18, Bt-7 and B-1183) for young adult male ddY mice and the growth rate of each strain under different oxygen atmospheres (aerobic, micro-aerobic and anaerobic atmospheres) at 37 °C. 10⁶ units of yeast cells were intravenously injected into each mouse. The pathogenicity of each isolate was determined by a scoring system based on organ culture and histopathological findings. The growth rates under different oxygen atmospheres were determined by a scoring system in which 300 fungal units per strain were counted. The strain Bt-9 showed the greatest pathogenicity, followed by Bt-4. Pb-9 and Pb-18 had on intermediate rank of pathogenicity. Bt-7 and B-1183 were the least pathogenic of the strains tested. Except for strain Bt-7 all strains showed an excellent growth under an aerobic atmosphere. Bt-4 and Bt-9 also showed excellent growth under a micro-aerobic atmosphere, followed by Pb-9, whereas the growth of Pb-18, Bt-7 and B-1183 was limited. There was a correlation between the growth rate under a micro-aerobic atmosphere and the pathogenicity of a strain. The growth rate of P. brasiliensis under a micro-aerobic atmosphere strongly correlated to its pathogenicity.