Dynamics of double and single Wolbachia infections in Drosophila simulans from New Caledonia

The bacterial symbiont Wolbachia can cause cytoplasmic incompatibility in Drosophila simulans flies: if an infected male mates with an uninfected female, or a female with a different strain of Wolbachia, there can be a dramatic reduction in the number of viable eggs produced. Here we explore the dynamics associated with double and single Wolbachia infections in New Caledonia. Doubly infected females were compatible with all males in the population, explaining the high proportion of doubly infected flies. In this study, males that carry only wHa or wNo infections showed reduced incompatibility when mated to uninfected females, compared with previous reports. These data suggest that either the DNA of these bacterial isolates have diverged from those previously collected, or the genetic background of the host has lead to a reduction in the phenotype of incompatibility. Mitochondrial sequence polymorphism at two sites within the host genome was assayed to investigate population structure related to infection types. There was no correlation between sequence polymorphism and infection type suggesting that double infections are the stable type, with singly infected and uninfected flies arising from stochastic segregation of bacterial strains. Finally, we discuss the nomenclature of Wolbachia strain designation.     

Sleep, respiratory physiology, and nocturnal asthma.

The nocturnal worsening of asthma is a common feature of this disease that recently has received extensive investigation. Most recent efforts have focused on the role of circadian biorhythms that could promote a nocturnal increase in airway inflammation, leading to a subsequent increase in airflow obstruction and asthma symptoms. However, definitive studies remain lacking. As discussed in this review, there is also substantial evidence that sleep itself may play a direct role in the nocturnal worsening of asthma. Potential mechanisms for such a sleep-related effect could include the supine posture, alterations in sympathetic and parasympathetic "balance," sleep-associated reductions in lung volume, intrapulmonary pooling of blood, and sleep-associated upper airway narrowing, both with and without snoring and obstructive sleep apnea (OSA). These potential contributors to this troublesome phenomenon deserve further consideration when investigating mechanisms of nocturnal asthma.     

Differential effects of furosemide on porcine bronchial arterial and airway smooth muscle.

Furosemide attenuates airway obstruction in asthmatic subjects when administered as an aerosol pretreatment. This protective effect of furosemide could be related to relaxation of bronchial smooth muscle or to increased bronchial blood flow. To determine whether furosemide dilates bronchial smooth muscle, isometric contractile responses in distal bronchi from young pigs were studied. In bronchial smooth muscle rings that were precontracted with 10(-5) M acetylcholine, significant relaxation occurred with 10(-8) to 3 x 10(-6) M isoproterenol but not with 10(-8) to 10(-3) M furosemide. In contrast, bronchial arteries that were precontracted with either 10(-4) M norepinephrine or 10(-8) M vasopressin significantly relaxed in response to 10(-4) to 3 x 10(-3) M and 10(-3) to 3 x 10(-3) M furosemide, respectively. We conclude that furosemide, under the described experimental conditions, relaxes airway vascular smooth muscle but not bronchial smooth muscle. These results are consistent with previous suggestions that inhaled furosemide increases blood flow to airway tissues (Gilbert IA, Lenner KA, Nelson JA, Wolin AD, and Fouke JM. J Appl Physiol 76: 409-415, 1994).     

Is there a link between impaired glucose metabolism and protein kinase C activity in the diabetic heart?

The activity of the isoform of protein kinase C (PKC) is reduced in the diabetic heart. Since this isozyme has been implicated in insulin action, we tested the hypothesis that PKC contributes to the development of impaired glucose metabolism by the noninsulin-dependent diabetic heart. Exposure of the diabetic heart to buffer containing the protein kinase C activator, phorbol myristate acetate, increased PKC activity in the membrane. Associated with the improvement in PKC activity was a biphasic change in glucose metabolism. The initial phase was characterized by a breakdown in glycogen stores, a stimulation in glucose oxidation and a decrease in endogenous fatty acid oxidation. This was followed by a second phase in which the uptake of glucose was modestly stimulated. Nonetheless, since the phorbol ester did not overcome the diabetes-linked defect in pyruvate dehydrogenase, the increase in glycolytic flux was not associated with a rise in glucose oxidation. Consequently, nearly 50% of the triose units were diverted into lactate and pyruvate production and the generation of ATP from glucose was restricted. Since insulin promotes not only glucose uptake, but also glycogen synthesis and glucose oxidation, the phorbol ester and insulin effects are very different. Thus, the data do not support a role for PKC in the development of glucose metabolic defects in the hearts of noninsulin-dependent diabetic rats.     

Multiple biotin-containing proteins in 3T3-L1 cells.

Extracts of 3T3-L1 cells prepared after labelling the monolayer cultures with [3H]biotin contained numerous protein bands that were detected by fluorography of dried SDS/polyacrylamide electrophoresis gels. All labelled proteins in the extracts could be removed by avidin affinity chromatography. The biotin-containing subunits of acetyl-CoA carboxylase, pyruvate carboxylase, methylcrotonyl-CoA carboxylase and propionyl-CoA carboxylase, with molecular masses of approx. 220, 120, 75 and 72 kDa respectively, were detected together with minor bands at 100, 85 and 37 kDa that did not appear to be partial degradation products. Additional labelled bands increased in amount during incubation of cell extracts or did not occur in extracts prepared with trichloroacetic acid, 9.5 M-urea or proteolytic inhibitors, and were tentatively classified as partial degradation products. The unknown bands were not removed by incubation of cell monolayers for 24 h, a treatment that gave degradation rate constants of 0.47 day-1 for acetyl-CoA carboxylase and 0.28 day-1 for pyruvate carboxylase. Upon two-dimensional electrophoresis, pyruvate carboxylase, methylcrotonyl-CoA carboxylase and propionyl-CoA carboxylase had isoelectric points of 6.4, 7.2 and 6.4 respectively. Several additional discrete spots with isoelectric points below 6.2 were also present. All the unknown biotin-containing proteins banded with intact mitochondria during density-gradient centrifugation. We conclude that several unknown biotin-containing proteins are present in the mitochondria of 3T3-L1 cells, whereas others are partial breakdown products of mitochondrial proteolysis.     

Purification and partial sequence analysis of insulin-like growth factor-1 from bovine colostrum.

Growth-promoting activity in bovine colostrum has been detected as the capacity to stimulate protein synthesis in L6 myoblasts. By using this assay as a measure of bioactivity, a growth factor has been purified to near homogeneity from centrifuged colostrum by a series of steps including acid extraction, chromatography on sulphopropyl-Sephadex, followed by adsorption to, and elution from, C18 columns using acetonitrile and propan-1-ol gradients. The purified growth factor has a low solubility at neutral and alkaline pH and has an Mr of 7800 by gel-permeation chromatography. Sequence analysis of the first 30 amino acids from the N-terminus indicated complete identity in this region with human insulin-like growth factor-1. Accordingly we conclude that the purified growth factor is bovine insulin-like growth factor-1.     

Regulation of the breakdown rates of biotin-containing proteins in Swiss 3T3-L1 cells.

1. Degradation rate constants for individual biotin-labelled proteins were measured in Swiss 3T3-L1 adipocytes that had been incubated with inhibitors of autophagy or of lysosomal proteolysis. 2. Inhibitory effects produced by 10 mM-3-methyladenine and a combination of 5 mM-NH4Cl and leupeptin (50 micrograms/ml) were approximately equal. The inclusion of NH4Cl did not significantly enhance the responses to 3-methyladenine, suggesting that autophagy was already maximally inhibited. 3. The extent of inhibition by 3-methyladenine or by the NH4Cl/leupeptin mixture was similar for the cytosolic enzyme acetyl-CoA carboxylase and for the three mitochondrial carboxylases. This inhibition averaged 50%. The breakdown rate of a more-stable 38 kDa biotin-containing mitochondrial protein was more responsive to the inhibitory agents. These results are best explained by mitochondrial proteolysis occurring via a combination of the degradation of whole mitochondria within autophagic vacuoles, supplemented by the selective intramitochondrial breakdown of more labile proteins. 4. A number of intermediate products in the degradation of biotin-containing proteins were detected. Differences in the patterns of radioactivity between these peptides after incubation of cells in the presence of inhibitors of the breakdown process provided evidence that some peptides were produced before autophagy, others as a result of intralysosomal inhibition, while at least one was associated with intramitochondrial proteolysis.