Effect of fatigue on hamstring coactivation during isokinetic knee extensions

We examined the effect of fatigue of the quadriceps muscles on coactivation of the hamstring muscles and determined if the response is different between two isokinetic speeds in ten males and ten females with no history of knee pathology. Electromyographic data were recorded from the vastus lateralis and biceps femoris muscles during 50 maximal knee extensions at isokinetic speeds of 1.75 rad · s⁻¹ (100° · s⁻¹) and 4.36 rad · s⁻¹ (250° · s⁻¹). A greater degree of coactivation was apparent at the higher speed, but the increase in coactivation of the hamstring muscles was similar at both speeds. The results revealed that: (1) coactivation is greater at a higher isokinetic speed, and (2) coactivation increases during fatigue, but the rate of increase is independent of contraction velocity. Accepted: 15 June 1998     

Distribution of amiloride-sensitive sodium channels in the oral cavity of the hamster

The distribution of amiloride-sensitive sodium channels (ASSCs) in taste buds isolated from the oral cavity of hamsters was assessed by patch clamp recording. In contrast to the case for rats, taste cells from the fungiform, foliate and vallate papillae and from the soft palate all contain functional ASSCs. The differential distribution of ASSCs between the hamster and the rat may be important for understanding the physiology underlying the differing behavioral responses of these species to sodium salts.      

Induction of drug metabolism enzymes and transporters by oltipraz in rats

Coordinate regulation of Phase-I and -II enzymes with xenobiotic transporters has been shown after treatment with microsomal enzyme inducers. The chemopreventive agent oltipraz (OPZ) induces Phase-I and -II drug-metabolizing enzymes such as CYP2B and NQO1. The purpose of this study was to examine the regulation of drug-metabolizing enzymes and transporters in response to OPZ treatment and to investigate a potential role for constitutive androstane receptor (CAR) in OPZ-mediated induction. Sprague-Dawley rats treated with OPZ exhibited increased mRNA and protein levels of both Nqo1 and Cyp2b1/2 by 24 h. To examine whether OPZ activates transporter gene expression via CAR, sexually dimorphic male and female Wistar-Kyoto (WKY) rats were treated with OPZ and mRNA levels quantified by bDNA signal amplification. OPZ induced Ugt1a6 and Ugt2b1 in males significantly higher than in females, indicating a CAR-dependent mechanism of induction. However, OPZ induced microsomal epoxide hydrolase, NAD(P)H quinone oxidoreductase, and Cyp3a1/23 equally in both genders, indicating a CAR-independent mechanism of induction of these genes. Similarly, the transporters Mdr1a, Mdr1b, Mrp3, and Mrp4 were induced by OPZ without any apparent difference between genders. In summary, OPZ coordinately increases multiple hepatic xenobiotic transporter mRNA levels, along with Phase-I and -II enzymes some of which may occur through CAR-dependent mechanisms.     

Nitric oxide-deficiency regulates hepatic heme oxygenase-1.

Nitric oxide plays a crucial role in the maintenance of liver function and integrity. During stress, the inducible heme oxygenase-1 protein and its reaction products, including carbon monoxide, also exert potent hepatoprotective effects. We investigated a potential relationship between endogenous nitric oxide synthesis and the hepatic regulation of heme oxygenase-1. Inhibition of nitric oxide synthesis in vivo by injection of l-NAME led to a dose-dependent induction of heme oxygenase-1 mRNA, protein and activity in the rat liver, whereas did not affect the expression of other heat shock proteins. The effect of l-NAME was demonstrated by hemodynamic changes within the liver circulation as measured by ultrasonic flow probes. Inhibition of nitric oxide synthase led to a decline in hepatic arterial and portal venous blood flow, and subsequently caused liver cell damage. In contrast, the combined administration of l-NAME and the nitric oxide-independent intestinal vasodilator dihydralazine completely restored portal venous flow, abolished the liver cell damage, and prevented the upregulation of heme oxygenase-1, despite inhibition of nitric oxide production. In conclusion, nitric oxide deficiency upregulates hepatic heme oxygenase-1, which is reversible by maintaining hepatic blood flow. This interdependence has important implications for the development of therapeutic strategies aimed at modulating the activity of these hepatoprotective mediator systems.     

Requirements for maximal enrichment of viable intraerythrocytic Plasmodium falciparum rings by saponin hemolysis

The purpose of the present study was to confirm the effectiveness of saponin hemolysis for concentrating ring-infected erythrocytes in Plasmodium falciparum cultures and to determine the actual numbers of the enriched parasites, not just percentage parasitemia. This is important because various molecular biology and vaccine development against malaria require useable quantities of pure culture with minimal number of uninfected erythrocytes at all stages. Synchronized cultures of three P. falciparum strains were exposed to 0.015% isotonic saponin solution for 30 minutes on ice. They were centrifuged and the pellets were treated again with saponin solution for 3-7 minutes. Initially, most of the cultures contained approximately 10(10) erythrocytes and 1-7% parasitemia, but at the end of the enrichment up to 10(8) of erythrocytes containing 90-99.8% parasitemia were recovered (maximal enrichment). From microscopic examination of the cells it was calculated that the hemolysis rate of uninfected and infected erythrocytes was circa 27 to 1, which could account for the enrichment. Studies by other investigators have suggested that P. falciparum merozoite invasion decreases erythrocyte membrane lipids, and it has been reported that reduction of membrane cholesterol could make erythrocytes saponin-resistant. The possibility that merozoite invasion made erythrocytes partially resistant to saponin hemolysis was strengthened by the observation that the proportions of multiple infections increased significantly in the enriched cultures. However, mature asexual parasites could not be concentrated by this method, suggesting possible differences between the membranes of erythrocytes containing ring forms and those of trophozoites and schizonts. Ring-infected erythrocytes freshly from malaria patients could also not be concentrated by the method described here, suggesting that the ability to induce saponin resistance in erythrocytes was acquired by the parasites in vitro.     

Guidelines for the use and interpretation of assays for monitoring autophagy in higher eukaryotes

Research in autophagy continues to accelerate,(1) and as a result many new scientists are entering the field. Accordingly, it is important to establish a standard set of criteria for monitoring macroautophagy in different organisms. Recent reviews have described the range of assays that have been used for this purpose.(2,3) There are many useful and convenient methods that can be used to monitor macroautophagy in yeast, but relatively few in other model systems, and there is much confusion regarding acceptable methods to measure macroautophagy in higher eukaryotes. A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers of autophagosomes versus those that measure flux through the autophagy pathway; thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from fully functional autophagy that includes delivery to, and degradation within, lysosomes (in most higher eukaryotes) or the vacuole (in plants and fungi). Here, we present a set of guidelines for the selection and interpretation of the methods that can be used by investigators who are attempting to examine macroautophagy and related processes, as well as by reviewers who need to provide realistic and reasonable critiques of papers that investigate these processes. This set of guidelines is not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to verify an autophagic response.     

Developmental expression patterns of cuticular protein genes with the R&R Consensus from Anopheles gambiae

CPR proteins are the largest cuticular protein family in arthropods. The whole genome sequence of Anopheles gambiae revealed 156 genes that code for proteins with the R&R Consensus and named CPRs. This protein family can be divided into RR-1 and RR-2 subgroups, postulated to contribute to different regions of the cuticle. We determined the temporal expression patterns of these genes throughout post-embryonic development by means of real-time qRT-PCR. Based on expression profiles, these genes were grouped into 21 clusters. Most of the genes were expressed with sharp peaks at single or multiple periods associated with molting. Genes coding for RR-1 and RR-2 proteins were found together in several co-expression clusters. Twenty-five genes were expressed exclusively at one metamorphic stage. Five out of six X-linked genes showed equal expression in males and females, supporting the presence of a gene dosage compensation system in A. gambiae. Many RR-2 genes are organized into sequence clusters whose members are extremely similar to each other and generally closely associated on a chromosome. Most genes in each sequence cluster are expressed with the same temporal expression pattern and at the same level, suggesting a shared mechanism to regulate their expression.     

Imaging activity of neuronal populations with new long-wavelength voltage-sensitive dyes

We have assessed the utility of five new long-wavelength fluorescent voltage-sensitive dyes (VSD) for imaging the activity of populations of neurons in mouse brain slices. Although all the five were capable of detecting activity resulting from activation of the Schaffer collateral-CA1 pyramidal cell synapse, they differed significantly in their properties, most notably in the signal-to-noise ratio of the changes in dye fluorescence associated with neuronal activity. Two of these dyes, Di-2-ANBDQPQ and Di-1-APEFEQPQ, should prove particularly useful for imaging activity in brain tissue and for combining VSD imaging with the control of neuronal activity via light-activated proteins such as channelrhodopsin-2 and halorhodopsin.