Physical performance and soleus muscle fiber composition in wild-derived and laboratory inbred mouse strains.

We compared four inbred mouse strains in their physical performance, measured as a maximal treadmill running time, characteristics of soleus muscle, anatomic character, and growth. The strains used were Mus musculus domesticus [C57BL/6 (B6) and BALB/c], Mus musculus molossinus (MSM/Ms), and Mus spretus. Maximal running time was significantly different among these four mouse strains. Running time until exhaustion was highest in MSM/Ms and lowest in M. spretus. Maximal times for the laboratory mouse strains were nearly identical. Soleus muscle fiber type and cross-sectional area also differed significantly among the species. In particular, M. spretus was significantly different from the other inbred mouse strains. Growth in the wild-derived inbred mice appeared to be complete earlier than in the laboratory mice, and the body size of the wild strains was about half that of the laboratory strains. From these results, we propose that wild-derived inbred mouse strains are useful models for enhancing phenotypic variation in physical performance and adaptability.     

Acyl-(acyl-carrier protein) hydrolase from squash cotyledons specific to long-chain fatty acids: purification and characterization

Acyl-(acyl-carrier-protein) hydrolase (EC 3.1.2.14) releases fatty acids from the end-product of fatty acid synthesis in plastids for the subsequent synthesis of glycerolipids in the cytoplasm. Isoelectric focusing of chloroplast stroma proteins from squash cotyledons suggested that there were at least three isomeric forms of acyl-(acyl-carrier-protein) hydrolase having pI values of 4.5, 5.3 and 7.8. The pI 4.5 and pI 5.3 forms showed maximum activity at pH 9.8 whereas the activity of the pI 7.8 form increased within the range 6.2 to 10.2 but no optimum was seen. The pI 4.5 form was purified 100 000-fold from squash cotyledons. The highly purified fraction contained two polypeptides, whose molecular masses were estimated to be 35 kDa and 33 kDa by SDS-PAGE. It is suggested that the 33 kDa polypeptide was a degradation product of the 35kDa polypeptide. Oleoyl-(acyl-carrier protein) was the preferred substrate of this enzyme over palmitoyl- and stearoyl-(acyl-carrier protein), whereas lauroyl-(acyl-carrier protein) was nearly inactive. These results indicate the enzyme is specific for long-chain acyl-(acyl-carrier protein).     

Site-directed mutagenesis in hemoglobin: functional and structural role of inter- and intrasubunit hydrogen bonds as studied with 37 beta and 145 beta mutations.

In order to clarify the functional and structural role of intra- and intersubunit hydrogen bonds in human hemoglobin (Hb A), we prepared two artificial beta chain mutant hemoglobins by site-directed mutagenesis. The mutant Hb Phe-37 beta, in which Trp-37 beta is replaced by Phe to remove the intersubunit hydrogen bond between Asp-94 alpha and Trp-37 beta at the alpha 1-beta 2 interface in deoxy Hb A, showed a markedly increased oxygen affinity and almost completely diminished Bohr effect and cooperativity. However, 1H-NMR data indicated that the structure of deoxy Hb Phe-37 beta is rather similar to that of deoxy Hb A. The enhanced tetramer-to-dimer dissociation previously observed in Hb Hirose (Trp-37 beta----Ser) together with our observation of the effects of organic phosphate on the structure and function of Hb Phe-37 beta suggested that a large part of the abnormal properties of Hb Phe-37 beta observed for dilute solutions appears to result from partial dissociation into alpha beta dimers rather than direct destabilization of the T-quaternary structure in the deoxygenated state. Thus, the primary and direct role of the hydrogen bond between Asp-94 alpha and Trp-37 beta is to stabilize the tetrameric assembly, and thereby this hydrogen bond indirectly contributes to stabilization of the T-quaternary structure. The other mutant Hb Phe-145 beta has a Phe residue at the 145 beta site and lacks the intrasubunit hydrogen bond formed between Tyr-145 beta and the carbonyl group of Val-98 beta in deoxy Hb A.(ABSTRACT TRUNCATED AT 250 WORDS)     

Schizosaccharomyces pombe sxa1+ and sxa2+ encode putative proteases involved in the mating response.

The Schizosaccharomyces pombe sxa1 and sxa2 mutants showed an exaggerated response to mating pheromones, producing excessively long conjugation tubes and exhibiting mating deficiency. This phenotype was similar to phenotypes of cells bearing an activated allele of ras1, such as ras1Val-17 or ras1Leu-66, and phenotypes of cells defective in gap1. However, genetic evidence suggested that the sxa1 and sxa2 gene products are not directly involved in the Ras1 pathway. The gene products of sxa1 and sxa2, as deduced from their nucleotide sequences, were homologous to aspartyl proteases and serine carboxypeptidases, respectively. The sxa1 gene function was required for efficient mating only in h+ cells, although even disruption of sxa1 did not completely abolish the mating ability. Conversely, the sxa2 gene function was required only in h- cells. Wild-type cells produced a diffusible substance, which may be the sxa2 gene product itself, that could confer fertility to sxa2 mutant cells placed at a distance. These observations are consistent with the possibility that the sxa gene products are involved in degradation or processing of the mating pheromones and that their loss cause a persistent response to the pheromones.     

Solubilizing water involved in protein extraction using reversed micelles

The extraction of protein using reversed micelles was investigated in relation to the amount of solubilizing water in the reversed micellar organic phase. The minimal concentration of amphiphilic molecule di-2-ethylhexyl sodium sulfosuccinate (C(20)H(37)O(7)Na) (AOT) required for 100% cytochrome c extraction was recognized. This critical AOT concentration increased with protein concentration in the aqueous phase. On this minimal AOT condition, the molar ratio of solubilizing water to extracted protein was found to be a constant of 3500 under C(KCI) = 1.0 x 10(2) mol . m(-3) in this system. This ratio means the hydrophillic surroundings required for extracting one protein molecule into the micellar organic phase under the suitable pH and salt concentration for the forward extraction. In this regard, AOT molecules seemed to take the part of water solubilizing agent in the reversed micellar extraction. This role of AOT is important to extract protein under the suitable pH and salt concentration. The amount of solubilizing water in the protein-containing system was larger than in the protein-free system. This difference shows that the water molecules accompany the extracted protein into the reversed micellar organic phase at constant ratio 2200 under C(KCI) = 1.0 x 10(2) mol . m(-3), i.e., accompanying water molecules per one extracted protein. The minimal AOT concentration increased with ionic strength. On this minimal AOT condition, the molar ratio of solubilizing water to extracted protein also increased with ionic strength, so that in higher ionic strength, more solubilizing water was required. Then more AOT was required to provide the hydrophillic surroundings for protein. The pH affected the minimal AOT concentration required for 100% protein extraction.     

Molecular cloning and functional expression of rat leukotriene A4 hydrolase using the polymerase chain reaction.

We isolated a cDNA encoding rat leukotriene A4 (LTA4) hydrolase from mesangial cells by the polymerase chain reaction according to the human amino acid sequence. The deduced amino acid sequence shows that rat LTA4 hydrolase is a 609 amino acid protein with an Mr 69 kDa. Comparison of human LTA4 hydrolase revealed 93% homology, and include zinc-binding motifs of aminopeptidases. COS-7 cells transfected with the cDNA revealed substantial LTA4 hydrolase activity, and their activities were abolished by preincubation with captopril, representing the first reported cDNA expression of recombinant enzyme in mammalian cells. RNA blot analysis indicated that LTA4 hydrolase was expressed in glomerular endothelial, epithelial and mesangial cells.     

Production and secretion in Escherichia coli of hepatitis B virus pre-S2 antigen as fusion proteins with beta-lactamase

The diagnostically important surface antigen pre-S2 of hepatitis B virus was produced in large amounts in the periplasmic space of Escherichia coli. The DNA fragments (pre-S2) coding the pre-S2 antigen were tandemly duplicated or triplicated and ligated in the same reading frame to a fragment containing the promoter and the signal sequence of the alkaline phosphatase-coding gene (phoA) of E. coli. Further, a DNA fragment (bla) coding mature beta-lactamase was joined to the region coding the C terminus of the pre-S2 repeat to stabilize the gene product. Upon induction of the phoA-(pre-S2)3-bla fusion gene, the fusion protein was produced at up to 30% of the total cellular protein. Fractionation of the cellular components and trypsin accessibility of the product showed that the antigen was secreted in the periplasm and formed inclusion bodies there. The signal sequence of alkaline phosphatase was found to be correctly processed in E. coli.     

Inhibitory action of estradiol on growth promoting activity in extract from uterine cancers

Extracts from uterine cervical and body cancers, but not from benign tumor or intact tissues tested, were found to contain a growth-promoting activity which induced the proliferation of human endometrial fibroblasts. Exposure of cultured fibroblasts to the cancer extracts increased the rate of [³H]thymidine incorporation in a dose-dependent manner. The activity was heat-labile, and not inactivated by removal of lipid-soluble material suggesting that the activity is associated with a protein. When the fibroblasts were preincubated with estradiol for 12 hours, but not for 1 hour, the extract-induced fibroblast proliferation was suppressed. The inhibitory effect of estradiol was dose related (EC₅₀: 10 nM) and non-competitive, suggesting that the steroid may reduce the sensitivity of fibroblasts to the extracts. This is the first report to provide direct evidence that estradiol may play an inhibitory role in the action of growth factor-like peptide produced from malignant tumors.