Effect of cannabinoids on progesterone production by ovarian granulosa cells of pig and rat

Various cannabinoids inhibited both basal and FSH-stimulated progesterone synthesis by pig and rat granulosa cells $\text{in vitro}$ in a dose- (0.09 – 16 μM) and time- (4 – 24 h) dependent manner. The data present further evidence for a possible direct interference of cannabinoids with ovarian functions.     

Characterization of gluconeogenesis in hepatocytes isolated from the American eel,Anguilla rostrata LeSueur,

Summary Isolated hepatocyte preparations from fed immature American eels,Anguilla rostrata Le Sueur, were used to study gluconeogenic, lipogenic, glycogenic and oxidative rates of radioactively labelled lactate, glycerol, alanine and aspartate. Eel hepatocytes maintain membrane integrity and energy charge during a 2 h incubation period and are considered a viable preparation for studying fish liver metabolism.Incubating eel hepatocytes with 10 mM substrates, the following results were obtained: glycerol, alanine and lactate, in that order, were effective gluconeogenic substrates; these three substrates reduced glucose release from glycogen stores, while aspartate had no such effect; lactate, alanine and aspartate led to high rates of glycerol production, with subsequent incorporation into lipid; incorporation into glycogen was low from all substrates; and, alanine oxidation was seven times higher than that observed with other substrates.When eel hepatocytes were incubated with low or physiological substrate concentrations gluconeogenic rates from lactate were twice those from alanine; rates from aspartate were very low. Glucagon stimulated lactate gluconeogenesis, but not amino acid gluconeogenesis, and had no significant effect on glycogenolysis. Cortisol increased gluconeogenic rates from 1 mM lactate.Thus, in the presence of adequate substrate, eel liver gluconeogenesis is preferentially stimulated relative to glycogenolysis to produce plasma glucose. These data support three important roles for gluconeogenesis: the recycling of muscle lactate, the synthesis of glucose from dietary amino acids to supplement glucose levels, and the production of glycerol for lipogenesis.This work was supported from operating grants to TWM from the National Research Council of Canada (A6944)     

An anticomplementary agent, K-76 monocarboxylic acid: its site and mechanism of inhibition of the complement activation cascade.

A monocarboxylic acid derivative (K-76 COOH) of K-76, purified from the culture filtrate of Stachybotrys complement I nov. sp. K-76, inhibits complement (C) activity. Its inhibitory action is mainly on C5 step. It strongly inhibits the generation of EAC1,4b,2a,3b,5b from C5 and EAC1,4b,2a,3b, and accelerates the decay of EAC1,4b,2a,3b,5b. It also causes some inhibition of the reactions of the reactions of C2,C3,C6,C7 and C9 with their respective preceding intermediate cells. It has no effect on the generation of EAC1,4b from C4 and EAC1, or of EAC-8 from C8 and EAC-7, and apparently increases the generation of EAC1,4b from C1 and EAC4b probably by inhibiting transfer or turnover of C1. It does not affect the rate of decay of EAC1,4b,2a or the T max of generation of EAC1,4b,2a, and it inhibits immune adherence only at high concentration. K-76 COOH also strongly inhibits hemolysis through the alternative pathway of C activation by cobra venom factor, but it does not seem to inhibit the early steps of the alternative pathway, because it has little affect on the consumption of C3 or the conversion of beta 1C to beta 1A on treatment of C serum with zymosan. K-76 COOH probably combines with C5 molecules, forming the inactive complexes, or it causes the structural alteration of C5.     

The effect of age, acquired resistance, pregnancy and lactation on some reactions of cattle to infection with Ostertagia ostertagi

An experiment is described in which the effects of age, previous infection, pregnancy and lactation on some reactions of cattle to infection with Ostertagia ostertagi were studied. It was found that an acquired resistance to the establishment of worms developed more rapidly in 20-month-old heifers than in calves, that it was unaffected by pregnancy of the host but that it was largely lost by heifers in early lactation. The rate at which populations were turned over, i.e. the mean life-span of worms through the late 4th and 5th stages was unaffected by the factors studied. Although, in the conditions of the experiment, development of the worms was not arrested in susceptible calves, both age of the host and its previous experience of infection were significant causes of arrest, and in previously infected 20-month-old cattle 86% of the worms of a challenge infection were arrested. Pregnancy did not affect the proportion of worms that was arrested but in lactating heifers only marginally more worms were arrested than in calves. Worms that were not arrested grew more rapidly in calves and in lactating heifers than in empty heifers or those in mid-pregnancy.     

Studies onSargassum

Summary Cytological comparisons of homologous tissues in blades and stipes by stereological analysis have shown differences exist between blade and stipe organs inSargassum. Based on measurements of total thylakoid and cristae membrane surface areas in these organs blades were found to contain 61% more thylakoid membrane surface and 65% more cristae membrane than stipes on a per unit volume basis. Assuming photosynthesis and respiration are directly related to the surface area of the internal membranes in the respective organelles it is possible to predict that blades will have a 61% greater photosynthetic and a 65% greater respiratory potential. Photosynthetic and respiratory rates for blades and stipes were determined manometrically and show a 62% greater photosynthetic and 59% greater respiratory rates for the blade tissues agreeing very well with predicted values.Present evidence indicates that photosynthetic and respiratory rate differences observed in the blades and stipes inSargassum are the result of increased membrane surface areas in the larger cells of the tissues which make up the blade. The basic cell structure,i.e., the percent volume of cell cytoplasm occupied by each organelle, is similar in homologous tissues of both organs regardless of cell size. Therefore physiological differences between the two organs are primarily due to changes in cell size and not in basic cell construction. This provides an interesting mechanism for producing physiological differences without changing basic cell structure in the organs of this plant.     

Fine structure and metabolism of multiply innervated fast muscle fibres in teleost fish

Summary Both the fast and slow muscle fibres of advanced teleost fish are multiply innervated. The fraction of slow-fibre volume occupied by mitochondria is 31.3%, 25.5% and 24.6%, respectively, for the myotomal muscles of brook trout (Salvelinus fontinalis), crucian carp (Carassius carassius), and plaice (Pleuronectes platessa), respectively. The corresponding figures for the fast muscles of these species are 9.3%, 4.6% and 2.0%, respectively. Cytochrome-oxidase and citrate-synthetase activities in the fast muscles of 9 species of teleost range from 0.20–0.93 moles substrate utilised, g wet weight muscle^-1 min^-1 (at 15° C) or around 4–17% of that of the corresponding slow fibres. Ultrastructural analyses reveal a marked heterogeneity within the fast-fibre population. For example, the fraction of fibres with <1% or >10% mitochondria is 0,4,42% and 36, 12 and 0%, respectively, for trout, carp and plaice. In general, small fibres (<500 m²) have the highest and large fibres (>1,500 m²) the lowest mitochondrial densities. The complexity of mitochondrial cristae is reduced in fast compared to slow fibres.Hexokinase activities range from 0.4–2.5 in slow and from 0.08–0.7 moles, g wet weight^-1 min^-1 in fast muscles, indicating a wide variation in their capacity for aerobic glucose utilisation. Phosphofructokinase activities are 1.2 to 3.6 times higher in fast than slow muscles indicating a greater glycolytic potential. Lactate dehydrogenase activities are not correlated with either the predicted anaerobic scopes for activity or the anoxic tolerances of the species studied. The results indicate a considerable variation in the aerobic capacities and principal fuels supporting activity among the fast muscles of different species. Brook trout and crucian carp are known to recruit fast fibres at low swimming speeds. For these species the aerobic potential of the fast muscle is probably sufficient to meet the energy requirements of slow swimming.     

Characterization of human glucocerebrosidase from different mutant alleles.

Human cDNA was mutagenized to duplicate six naturally occurring mutations in the gene for glucocere-brosidase. The mutant genes were expressed in NIH 3T3 cells. The abnormal human enzymes were purified by immunoaffinity chromatography and characterized. The Asn370----Ser mutant protein differed from normal enzyme in its inhibition by both conduritol B epoxide and glucosphingosine demonstrating that the 370 mutant enzyme has an abnormal catalytic site. In addition, the 370 mutant enzyme is less activated by saposin C, but more stimulated by phosphatidylserine than the wild type enzyme. The Arg463----Cys mutant protein was normal with respect to conduritol B epoxide and glucosphingosine inhibition, but was less activated by both saposin C and phosphatidylserine. The Arg120----Gln mutant protein was catalytically inactive. The Leu444----Pro, the pseudopattern, and the Pro415----Arg mutants appear to have reduced amounts of enzyme protein in cells. The studies demonstrated that mutations in the gene for glucocerebrosidase have different effects on the catalytic activity and stability of the enzyme.     

An alternate method for estimation of cell growth kinetics from batch cultures

An alternative to estimation of cell growth kinetics via continuous culture experiments is proposed in this article. The method employed is based on batch culture experiments with very small inocula (initial cell concentrations being typically less than 5000 cells/mL). Such low initial cell concentrations result in extended exponential cell growth phase during which culture conditions remain unchanged, thereby permitting precise estimation of specific cell growth rates from batch experiments especially for fast-growing microorganisms such as Bacillus species. The effectiveness and utility of this approach are demonstrated via several experiments conducted with a wild-type strain (Bacillus subtilis TN106) and a recombinant strain (B. subtilis TN106[pAT5]). True establishment of exponential growth phase requires insignificant variance of most of the culture conditions during the initial growth phase. Satisfaction of this requirement is demonstrated for microbial systems investigated here. This approach is especially well suited for recombinant microorganisms containing segregationally unstable plasmids, since estimation of growth kinetics of these from continuous cultures is very difficult and highly unreliable due to continual reversion of recombinant ceils to plasmid-free host cells unless some selection pressure is applied at levels sufficient to keep the presence of plasmid-free cells minimal.     

Mutational specificity of the dnaE173 mutator associated with a defect in the catalytic subunit of DNA polymerase III of Escherichia coli.

We developed a system to examine forward mutations that occurred in the rpsL gene of Escherichia coli placed on a multicopy plasmid. Using this system we determined the mutational specificity for a dnaE173 mutator strain in which the editing function of DNA polymerase III is impeded. The frequency of rpsL- mutations increased 32,000-fold, due to the dnaE173 mutator, and 87 independent rpsL- mutations in the mutator strain were analyzed by DNA sequencing, together with 100 mutants recovered from dnaE+ strain, as the control. While half the number of mutations that occurred in the wild-type strain were caused by insertion elements, no such mutations were recovered from the mutator strain. A novel class of mutation, named "sequence substitution" was present in mutants raised in the dnaE173 strain; seven sequence substitutions induced in the mutator strain occurred at six sites, and all were located in quasipalindromic sequences, carrying the GTG or CAC sequence at one or both endpoints. While other types of mutation were found in both strains, single-base frameshifts were the most frequent events in the mutator strain. Thus, the mutator effect on this class of mutation was 175,000-fold. A total of 95% of the single-base frameshifts in the mutator strain were additions, most of which occurred at runs of A or C bases so as to increase the number of identical residues. Base substitutions, the frequency of which was enhanced 25,000-fold by the mutator effect, occurred primarily at several hotspots in the mutator strain, whereas those induced in the wild-type strain were more randomly distributed throughout the rpsL sequence. The dnaE173 mutator also increased the frequency of duplications 28,000-fold. Of the three duplications recovered from the mutator strain, one was a simple duplication, the region of which was flanked by direct repeats. The other duplications were complex, one half part of which was in the inverted orientation of a region containing two sets of inverted repeats. The same duplications were also recovered from the wild-type strain. The present data suggest that dnaE173 is a novel class of mutator that sharply induces sequence-directed mutagenesis, yielding high frequencies of single base frameshifts, duplications with inversions, sequence substitutions and base substitutions at hotspots.