Cefetamet pivoxil treatment causes loss of carnitine reserves that can be prevented by exogenous carnitine administration

Two groups of pediatric patients receiving cefetamet pivoxil treatment (3 x 500 mg daily) for 7 days were studied. In the first group (Group A) the drug was administered alone; in the second group (Group B) the drug was given in combination with a molar excess of carnitine (3 x 1 g). Medication with cefetamet pivoxil alone was associated with a large urinary excretion of pivaloylcarnitine: Approximately 71% of the daily pivalate intake could be eliminated as carnitine ester in the urine. In this group, the plasma level and the urinary output of free carnitine decreased. By contrast, in Group B, the administration of molar excess of carnitine aided stochiometric elimination of pivalate as carnitine ester, and the plasma levels and carnitine-free urinary output were unchanged. The data show that medication of cefetamet pivoxil results in the formation of pivaloylcarnitine in children; the sustained loss of carnitine esters can ultimately lead to carnitine deficiency. Molar excess of exogenous carnitine aids in the elimination of pivalate derived from cefetamet pivoxil therapy and helps to maintain the carnitine reserves.     

Maximizing recovery of native protein from aggregates by optimizing pressure treatment

Recovering native protein from aggregates is a common obstacle in the production of recombinant proteins. Recent reports have shown that hydrostatic pressure is an attractive alternative to traditional denature-and-dilute techniques, both in terms of yield and process simplicity. To determine the effect of process variables, we subjected tailspike aggregates to a variety of pressure-treatment conditions. Maximum native tailspike yields were obtained with only short pressure incubations (<5 min) at 240 MPa. However, some tailspike aggregates were resistant to pressure, despite multiple cycles of pressure. Extending the postpressure incubation time to 4 days improved the yield of native protein from aggregates from 19.4 +/- 0.9 to 47.4 +/- 19.6 microg/mL (approximately 78% yield of native trimer from nonaggregate material). The nearly exclusive conversion of monomer to trimer over the time scale of days, when combined with previous kinetic data, allows for the identification of three postpressure kinetic phases: a rapid phase consisting of structured dimer conversion to trimer (30 min), an intermediate phase consisting of monomer conversion to aggregate (100 min), and a slow phase consisting of conversion of monomer to trimer (days). Optimizing the production of structured dimer can yield the highest level of folded protein. Typical refolding additives, such as glycerol, or low-temperature incubation did not improve yields.     

Evolution: sexual arms races

The reproductive interests of males and females usually differ, resulting in sexual conflict. Recent studies in which experimental selection trials were carried out under conditions of either 'high' or 'low' sexual conflict show that conflict can promote speciation and reduce female reproductive success.     

Spermatozoal traits and sperm competition in Atlantic salmon: relative sperm velocity is the primary determinant of fertilization success

Sperm competition occurs when sperm from more than one male compete for fertilizations. This form of post-copulatory sexual selection is recognized as a significant and widespread force in the evolution of male reproductive biology and as a key determinant of differential male reproductive success. Despite its importance, however, detailed mechanisms of sperm competition at the gamete level remain poorly understood. Here, we use natural variation in spermatozoal traits among wild Atlantic salmon (Salmo salar), a species naturally adapted to sperm competition, to examine how the relative influences of sperm (i) number, (ii) velocity, (iii) longevity, and (iv) total length determine sperm competition success. Atlantic salmon fertilize externally, and we were therefore able to conduct controlled in vitro fertilization competitions while concurrently measuring spermatozoal traits within the aqueous micro-environment to which salmon gametes are naturally adapted. Microsatellite DNA fingerprinting revealed that a male's relative sperm velocity was the primary determinant of sperm competition success. There was no significant relationship between fertilization success and either relative sperm number or total length; sperm longevity showed an inverse relationship with competition success. These relationships were consistent for two experimental repeats of the in vitro fertilization competitions. Our results therefore show, under the natural microenvironment for salmon gametes, that relative sperm velocity is a key spermatozoal component for sperm competition success. Atlantic salmon sperm can be considered to enter a competition analogous to a race in which the fastest sperm have the highest probability of success.     

The evolution of human phenotypic plasticity: age and nutritional status at maturity

Several evolutionary optimal models of human plasticity in age and nutritional status at reproductive maturation are proposed and their dynamics examined. These models differ from previously published models because fertility is not assumed to be a function of body size or nutritional status. Further, the models are based on explicitly human demographic patterns, that is, model human life-tables, model human fertility tables, and, a nutrient flow-based model of maternal nutritional status. Infant survival (instead of fertility as in previous models) is assumed to be a function of maternal nutritional status. Two basic models are examined. In the first the cost of reproduction is assumed to be a constant proportion of total nutrient flow. In the second the cost of reproduction is constant for each birth. The constant proportion model predicts a negative slope of age and nutritional status at maturation. The constant cost per birth model predicts a positive slope of age and nutritional status at maturation. Either model can account for the secular decline in menarche observed over the last several centuries in Europe. A search of the growth literature failed to find definitive empirical documentation of human phenotypic plasticity in age and nutritional status at maturation. Most research strategies confound genetics with phenotypic plasticity. The one study that reports secular trends suggests a marginally insignificant, but positive slope. This view tends to support the constant cost per birth model.     

Solution structure of mu-conotoxin PIIIA,a preferential inhibitor of persistent tetrodotoxin-sensitive sodium channels

Mu-conotoxins are peptide inhibitors of voltage-sensitive sodium channels (VSSCs). Synthetic forms of mu-conotoxins PIIIA and PIIIA-(2-22) were found to inhibit tetrodotoxin (TTX)-sensitive VSSC current but had little effect on TTX-resistant VSSC current in sensory ganglion neurons. In rat brain neurons, these peptides preferentially inhibited the persistent over the transient VSSC current. Radioligand binding assays revealed that PIIIA, PIIIA-(2-22), and mu-conotoxins GIIIB discriminated among TTX-sensitive VSSCs in rat brain, that these and GIIIC discriminated among the corresponding VSSCs in human brain, and GIIIA had low affinity for neuronal VSSCs. (1)H NMR studies found that PIIIA adopts two conformations in solution due to cis/trans isomerization at hydroxyproline 8. The major trans conformation results in a three-dimensional structure that is significantly different from the previously identified conformation of mu-conotoxins GIIIA and GIIIB that selectively target TTX-sensitive muscle VSSCs. Comparison of the structures and activity of PIIIA to muscle-selective mu-conotoxins provides an insight into the structural requirements for inhibition of different TTX-sensitive sodium channels by mu-conotoxins.     

Control of inducer accumulation plays a key role in succinate-mediated catabolite repression in Sinorhizobium meliloti

The symbiotic, nitrogen-fixing bacterium Sinorhizobium meliloti favors succinate and related dicarboxylic acids as carbon sources. As a preferred carbon source, succinate can exert catabolite repression upon genes needed for the utilization of many secondary carbon sources, including the alpha-galactosides raffinose and stachyose. We isolated lacR mutants in a genetic screen designed to find S. meliloti mutants that had abnormal succinate-mediated catabolite repression of the melA-agp genes, which are required for the utilization of raffinose and other alpha-galactosides. The loss of catabolite repression in lacR mutants was seen in cells grown in minimal medium containing succinate and raffinose and grown in succinate and lactose. For succinate and lactose, the loss of catabolite repression could be attributed to the constitutive expression of beta-galactoside utilization genes in lacR mutants. However, the inactivation of lacR did not cause the constitutive expression of alpha-galactoside utilization genes but caused the aberrant expression of these genes only when succinate was present. To explain the loss of diauxie in succinate and raffinose, we propose a model in which lacR mutants overproduce beta-galactoside transporters, thereby overwhelming the inducer exclusion mechanisms of succinate-mediated catabolite repression. Thus, some raffinose could be transported by the overproduced beta-galactoside transporters and cause the induction of alpha-galactoside utilization genes in the presence of both succinate and raffinose. This model is supported by the restoration of diauxie in a lacF lacR double mutant (lacF encodes a beta-galactoside transport protein) grown in medium containing succinate and raffinose. Biochemical support for the idea that succinate-mediated repression operates by preventing inducer accumulation also comes from uptake assays, which showed that cells grown in raffinose and exposed to succinate have a decreased rate of raffinose transport compared to control cells not exposed to succinate.     

Insertional inactivation of the methionine s-methyltransferase gene eliminates the s-methylmethionine cycle and increases the methylation ratio

Methionine (Met) S-methyltransferase (MMT) catalyzes the synthesis of S-methyl-Met (SMM) from Met and S-adenosyl-Met (Ado-Met). SMM can be reconverted to Met by donating a methyl group to homocysteine (homo-Cys), and concurrent operation of this reaction and that mediated by MMT sets up the SMM cycle. SMM has been hypothesized to be essential as a methyl donor or as a transport form of sulfur, and the SMM cycle has been hypothesized to guard against depletion of the free Met pool by excess Ado-Met synthesis or to regulate Ado-Met level and hence the Ado-Met to S-adenosylhomo-Cys ratio (the methylation ratio). To test these hypotheses, we isolated insertional mmt mutants of Arabidopsis and maize (Zea mays). Both mutants lacked the capacity to produce SMM and thus had no SMM cycle. They nevertheless grew and reproduced normally, and the seeds of the Arabidopsis mutant had normal sulfur contents. These findings rule out an indispensable role for SMM as a methyl donor or in sulfur transport. The Arabidopsis mutant had significantly higher Ado-Met and lower S-adenosylhomo-Cys levels than the wild type and consequently had a higher methylation ratio (13.8 versus 9.5). Free Met and thiol pools were unaltered in this mutant, although there were moderate decreases (of 30%-60%) in free serine, threonine, proline, and other amino acids. These data indicate that the SMM cycle contributes to regulation of Ado-Met levels rather than preventing depletion of free Met.