Technical considerations for assessing alterations in skeletal muscle sarcoplasmic reticulum Ca++-sequestration functionin vitro

A multiple measurement system for assessing sarcoplasmic reticulum (SR) Ca⁺⁺-ATPase activity and Ca⁺⁺-uptake was used to examine the effects of SR fractionation and quick freezing on rat white (WG) and red (RG) gastrocnemius muscle.In vitro measurements were performed on whole muscle homogenates (HOM) and crude microsomal fractions (CM) enriched in SR vesicles before and after quick freezing in liquid nitrogen. Isolation of the CM fraction resulted in protein yields of 0.96±0.1 and 0.99±0.1 mg/g in WG and RG, respectively. The percent Ca⁺⁺-ATPase recovery for CM compared to HOM was 14.5% (WG) and 10.1% (RG). SR Ca⁺⁺-activated Ca⁺⁺-ATPase activity was not affected by quick freezing of HOM or CM, but basal ATPase was reduced (P<0.05) in frozen HOM (5.12±0.18–3.98±0.20 mole/g tissue/min in WG and from 5.39±0.20–4.48±0.24 mole/g tissue/min in RG). Ca⁺⁺-uptake was measured at a range of physiological free [Ca⁺⁺] using the Ca⁺⁺ fluorescent dye Indo-1. Maximum Ca⁺⁺-uptake rates when corrected for initial [Ca⁺⁺]_f were not altered in HOM or CM by quick freezing but uptake between 300 and 400nM free Ca⁺⁺ was reduced (P<0.05) in quick frozen HOM (1.30±0.1–0.66±0.1 mole/g tissue/min in WG and 1.04±0.2–0.60±0.1 mole/g tissue/min in RG). Linear correlations between Ca⁺⁺-uptake and Ca⁺⁺-ATPase activity measured in the presence of the Ca⁺⁺ ionophore A23187 were r=+0.25, (P<0.05) and r=+0.74 (P<0.05) in HOM and CM preparations, respectively, and were not altered by freezing. The linear relationships between HOM and CM maximum Ca⁺⁺-uptake (r=+0.44, P<0.05) and between HOM and CM Ca⁺⁺-ATPase activity (r=+0.34, P<0.05) were also not altered by tissue freezing. These data suggest that alterations in maximal SR Ca⁺⁺-uptake function and maximal Ca⁺⁺-ATPase activity may be measured in both HOM and CM fractions following freezing and short term storage. (Mol Cell Biochem139, 41–52, 1994)     

On protein solubility in organic solvent

Solubility of a model protein, hen egg-white lysozyme, was investigated in a wide range of neat nonaqueous solvents and binary mixtures thereof. All solvents that are protic, very hydrophilic, and polar readily dissolve more than 10 mg/mL of lysozyme (lyophilized from aqueous solution of pH 6.0). Only a marginal correlation was found between the lysozyme solubility in a non-aqueous solvent and the letter's dielectric constant or Hildebrand solubility parameter, and no correlation was observed with the dipole moment. Lysozyme dissolved in dimethyl sulfoxide (DMSO) could be precipitated by adding protein nondissolving co-solvents, although the enzyme had a tendency to form supersaturated solutions in such mixtures. The solubility of lysozyme, both in an individual solvent (1,5-pentanediol) and in binary solvent mixtures (DMSO/acetonitrile), markedly increased when the pH of the enzyme aqueous solution prior to lyophilization was moved away from the proteins's isoelectric point. (c) 1994 John Wiley & Sons, Inc.     

Photosynthetic Adaptation to High Temperature Associated with Thylakoid Membranes of Synechococcus PCC7002

Photosynthetic adaptation to high temperature was investigatedin intact cells and isolated thylakoid membranes of the cyanobacterium,Synechococcus PCC7002. In intact cells, the thermal stabilityof photosynthesis and photosystem 2-mediated electron transportfrom H₂O to 1,4-benzoquinone changed in concert with growthtemperature. The photosystem 2-mediated electron transport fromH₂O to phenyl-1,4-benzoquinone showed greater thermal stabilityin thylakoid membranes isolated from cells which had adaptedto high temperature than in those from non-adapted cells. Enhancedthermal stability was also observed in the thylakoid membranesin the transport of electrons from H₂O to 2,6-dichlorophenolindophenolbut not in the transport of electrons from diphenylcarbazideto 2,6-dichlorophenolindophenol. These observations suggestthat oxygen-evolving sites acquire enhanced thermal stability,and that factors which are responsible for thermal stabilityremain in isolated thylakoid membranes. (Received October 30, 1992; Accepted December 18, 1992)     

The Drosophila Meiotic Recombination Gene Mei-9 Encodes a Homologue of the Yeast Excision Repair Protein Rad1

Meiotic recombination and DNA repair are mediated by overlapping sets of genes. In the yeast Saccharomyces cerevisiae, many genes required to repair DNA double-strand breaks are also required for meiotic recombination. In contrast, mutations in genes required for nucleotide excision repair (NER) have no detectable effects on meiotic recombination in S. cerevisiae. The Drosophila melanogaster mei-9 gene is unique among known recombination genes in that it is required for both meiotic recombination and NER. We have analyzed the mei-9 gene at the molecular level and found that it encodes a homologue of the S. cerevisiae excision repair protein Rad1, the probable homologue of mammalian XPF/ERCC4. Hence, the predominant process of meiotic recombination in Drosophila proceeds through a pathway that is at least partially distinct from that of S. cerevisiae, in that it requires an NER protein. The biochemical properties of the Rad1 protein allow us to explain the observation that mei-9 mutants suppress reciprocal exchange without suppressing the frequency of gene conversion.     

Failure of short term stimulation to reduce sarcoplasmic reticulum Ca2+-ATPase function in homogenates of rat gastrocnemius

To examine the effect of short term intense activity on sarcoplasmic reticulum (SR) Ca²⁺ sequestering function, the gastrocnemius (G) muscles of 11 anaesthetized male rats (weight, 411±8 g,X±SE) were activated using supramaximal, intermittent stimulation (one train of 0.2 msec impulses per sec of 100 msec at 100 Hz). Homogenates were obtained from stimulated white (WG-S) and red (RG-S) tissues, assayed for Ca²⁺ uptake and maximal Ca²⁺ ATPase activity and compared to contralateral controls (WG-C, RG-C). Calcium uptake (nmoles/mg protein/min) determined using Indo-l and at [Ca²⁺]_f concentrations between 300–400 nM was unaffected (p>0.05) by activity in both WG (6.14+0.43 vs 5.37+0.43) and RG (3.21+0.18 vs 3.07+0.20). Similarly, no effect (p>0.05) of contractile activity was found for maximal Ca²⁺ ATPase activity (mole/mg protein/min) determined spectrophotometrically in RG (0.276+0.03 vs 0.278+0.02). In WG, Ca²⁺ ATPase activity was 15% higher in WG-S compared to WG-C (0.412+0.03 vs 0.385+0.04). Repetitive stimulation resulted in a reduction in tetanic tension of 74% (p<0.05) by 2 min in the G muscle. By the end of the stimulation period, ATP concentration was reduced (p<0.05) by 57% in the WG and by 47% in the RG. These results indicate that the repeated generation of maximal tetanic force, at least for short term periods, need not adversely affectin vitro homogenate determination of Ca²⁺ sequestering function in spite of severe alterations in energy potential and that some other mechanism must be involved to explain the depression in Ca²⁺ uptake and Ca²⁺ ATPase activity previously noted with short term intense exercise.     

Distribution of digestive proteinases in the alimentary tract of the european corn borer Ostrinia nubilalis (lepidoptera: Pyralidae)

The distribution of digestive proteinases in either the anterior and posterior midgut or between the midgut epithelium and ectoperitrophic and endo-peritrophic spaces in the midgut were examined in the European corn borer, Ostrinia nubilalis. Trypsin, chymotrypsin, elastase, and aminopeptidase activities were the same in the anterior and posterior halves of the midgut. Of the total aminopeptidase activity, 95% was located in the midgut epithelium, and 90% of the trypsin, 97% of chymotrypsin, and 93% of the elastase activity were found in the midgut lumen. Trypsin, measured by hydrolysis of benzoyl-L-arginine ethyl ester, and chymotrypsin levels were significantly higher in the ectoperitrophic space compared to the endoperitrophic space. Digestion in the midgut is proposed to be sequential with tryptic digestion occurring in the endoperitrophic space. Ingested protein is digested further in the ectoperitrophic space by the action of elastase, chymotrypsin, and a second trypsin. Final digestion occurs by an intracellular aminopeptidase. © 1995 Wiley-Liss, Inc.     

Taenia taeniaeformis: characterization of larval metabolic products and growth of host gastric cells in vitro

Development of larvae of the cestode parasite Taenia taeniaeformis in the liver of rats induces gross hyperplasia of the gastric mucosa and excessive mucus production in the stomach without any direct contact with the stomach. Because the taeniid larvae are known to elaborate excretory-secretory (E-S) product in vivo and in vitro, the product was analyzed further, and its effects on cultured rat and dog stomach cells were investigated. In vitro E-S product contained less negatively charged glycosaminoglycan than either heparin or chondroitin sulfate, and proteins of various molecular weights. It stimulated the growth of both rat and dog stomach cells at concentrations of 3-9 micrograms protein/ml culture medium. At a concentration of 30 micrograms protein/ml culture medium, it stimulated hexosamine production in the cells up to 20 times, and multiple intracytoplasmic granules were found in both rat and dog cultured cells by light and electron microscopy. These results suggest that larval E-S product may be involved in the induction of gastric hyperplasia and hypermucus secretion.     

Aromatic origin of cyclopentenoid metabolites

Oxidative cleavage of aromatic compounds is often part of a degradative process and is widely observed in nature. The immediate catabolic products can sometimes cyclize or rearrange to new secondary metabolites. The enzymatic contraction of a dehydroisocoumarin to yield cyclopentenoid metabolites in Cryptosporiopsis sp. is reported. The label distribution of (+) cryptosporiopsin, a chlorinated cyclopentenone, was determined by analysis of the [¹³C]nmr of [1-¹³C] and [2-¹³C]acetate enriched-cryptosporiopsin. The putative aromatic precursor of cyclopentenoid metabolites, 2,3-dihydro-6,8-dihydroxy-2-methylisocoumarin (6), was isolated from Aspergillus terreus. This metabolite (6) was prepared doubly labeled ($\text{T}{}^{14}\text{C}$). The aromatic origin of the Cryptosporiopsis chlorinated cyclopentenoid metabolites was rigorously proven from feeding experiments with doubly labeled compound 6. A related but nonchlorinated metabolite, terrein, was isolated from A. terreus and was also shown to be derived from [$\text{T}{}^{14}\text{C}\text{]-2,3-dihydro-6,8-dihydroxy-2-methylisocoumarin}$.