Muscle temperature transients before, during, and after exercise measured using an intramuscular multisensor probe.

Seven subjects (1 woman) performed an incremental isotonic test on a Kin-Com isokinetic apparatus to determine their maximal oxygen consumption during bilateral knee extensions (Vo(2 sp)). A multisensor thermal probe was inserted into the left vastus medialis (middiaphysis) under ultrasound guidance. The deepest sensor (tip) was located approximately 10 mm from the femur and deep femoral artery (T(mu 10)), with additional sensors located 15 (T(mu 25)) and 30 mm (T(mu 40)) from the tip. Esophageal temperature (T(es)) was measured as an index of core temperature. Subjects rested in an upright seated position for 60 min in an ambient condition of 22 degrees C. They then performed 15 min of isolated bilateral knee extensions (60% of Vo(2 sp)) on a Kin-Com, followed by 60 min of recovery. Resting T(es) was 36.80 degrees C, whereas T(mu 10), T(mu 25), and T(mu 40) were 36.14, 35.86, and 35.01 degrees C, respectively. Exercise resulted in a T(es) increase of 0.55 degrees C above preexercise resting, whereas muscle temperature of the exercising leg increased by 2.00, 2.37, and 3.20 degrees C for T(mu 10), T(mu 25), and T(mu 40), respectively. Postexercise T(es) showed a rapid decrease followed by a prolonged sustained elevation approximately 0.3 degrees C above resting. Muscle temperature decreased gradually over the course of recovery, with values remaining significantly elevated by 0.92, 1.05, and 1.77 degrees C for T(mu 10), T(mu 25), and T(mu 40), respectively, at end of recovery (P < 0.05). These results suggest that the transfer of residual heat from previously active musculature may contribute to the sustained elevation in postexercise T(es).     

Ultrastructure of Porocephalus crotali (Pentastomida) cuticle with phylogenetic implications.

The cuticle of P. crotali is pro-arthropodan, composed of an epi-, exo-, and endocuticle. The exo- and endocuticles are separated by a 600-A intermediate cuticular zone. The epicuticle is homogeneous and varies from 100 to 350 A in thickness. The exocuticle varies from 2 to eight mu in thickness and is divided into superficial and deep exocuticular zones. The endocuticle is lamellate and varies from 8 to 30 mu in thickness. Lamellae result from ordered parabolic orientations of 40-A chitin fibrils. Underlying cells lack a basement membrane. Subcuticular muscle cells insert tonofibrils directly into the adjacent endocuticle. No apodemes or apophyses occur.     

Formation of 30- to 40-Micrometer-Thick Laminations by High-Speed Marine Bacteria in Microbial Mats

The precision with which motile heterotrophic bacteria could position themselves in microbial mats was determined. This required the development of a technique to view motile bacteria in situ. This was successfully achieved by replacing a 1-cm-diameter minicore from the mat sediment with 210- to 300-(mu)m-diameter glass beads or sieved agar. After allowing 3 days for regrowth of the mat into the transparent medium, a cross section showed that bacteria formed a layer as thin as 30 to 40 (mu)m at a depth of 500 (mu)m below the surface. Bacterial concentrations in this microlamination were 20 times above background. Mean speeds were 200 (mu)m s(sup-1) inside and 60 (mu)m s(sup-1) outside the microlamination. The percentages of bacteria turning per 30 s were 93% inside and 10% outside the microlamination. Artificial chemical gradients were unsuccessful in stimulating microlamination formation or in eliciting the same extent of speed and turning responses. The significance of the results is that it is now possible to microscopically examine sedimentary bacteria in situ. Our first examination indicates that some bacteria form chemotactic microlaminations by increasing their turning frequency. This behavior is opposite that described in the enteric-based model of chemotactic movement, in which positive chemotaxis is achieved by decreasing the turning frequency.     

Heterogeneity of bone marrow lymphocytes: radioautographic detection of pre-B cells bearing cytoplasmic mu chains, and of B and T lymphocytes, and characterization of null lymphoid cells

A radioautographic immunolabeling technique has been developed to detect pre-B cells bearing cytoplasmic mu chains among populations of bone marrow lymphoid cells identified by conventional hematologic stains. 125I-Anti-mu antibody was applied either to fixed marrow smears, labeling total mu chains both in the cytoplasm (c mu) and at the cell surface (s mu), or to cell suspensions, labeling s mu alone. In stained radioautographs the incidence of c mu+ s mu- pre-B cells was derived both indirectly by subtracting values for s mu+ cells from those for total mu+ cells of various sizes in normal mice and directly by the total mu chain labeling in mice depleted of s mu+ cells by anti-IgM treatment in vivo. Binding specificity was demonstrated by the displacement of labeling by nonradioactive anti-mu antibody. The c mu+ s mu- cells showed a bimodal size distribution. They accounted for 40% of the large lymphoid cells and 30% of the small lymphocytes in the marrow. A further 50% of the small lymphocytes were B lymphocytes (s mu+) and 8% were T lymphocytes (Thy 1.2+). Thus, the technique demonstrates the presence of c mu+ s mu- pre-B cells among both proliferating large lymphoid cells and nondividing small lymphocytes, as classically defined in marrow smears. In addition, the results reveal a broad size distribution of mu- lymphoid cells, including a subset of small lymphocytes which lack c mu, s mu, and Thy 1.2 and thus cannot be assigned to either B or T lineage by these criteria. The findings suggest that in addition to B cells the marrow may produce other types of lymphoid cells, yet to be defined.     

Internalization and recycling of human mu opioid receptors expressed in Sf9 insect cells

Internalization and recycling of G protein-coupled receptors (GPCRs), such as the mu-opioid receptor, largely depend on agonist stimulation. Agonist-promoted internalization of some GPCRs has been shown to mediate receptor desensitization, resensitization, and down-regulation. In this study, we investigated whether different mu opioid agonists displayed different effects in receptor internalization and recycling, the potential mechanisms involved in ohmefentanyl-induced internalization process. In transfected Sf9 insect cells expressing 6His-tagged wild type mu opioid receptor, exposure to 100 nM ohmefentanyl caused a maximum internalization of the receptor at 30 min and receptors seemed to reappear at the cell membrane after 60 min as determined by radioligand binding assay. Ohmefentanyl-induced human mu opioid receptor internalization was concentration-dependent, with about 40% of the receptors internalized following a 30-min exposure to 1 microM ohmefentanyl. 10 microM morphine and 1 microM DAMGO could also induce about 40% internalization. The antagonist naloxone and pretreatment with pertussis toxin both blocked ohmefentanyl-induced internalization without affecting internalization themselves. Incubation with sucrose 0.45 M significantly inhibited ohmefentanyl-induced internalization of the mu receptor. The removal of agonists ohmefentanyl and morphine resulted in the receptors gradually returning to the cell surface over a 60 min period, while the removal of agonist DAMGO only partly resulted in the receptor recycling. The results of this study suggest that ohmefentanyl-induced internalization of human mu opioid receptor in Sf9 insect cells occurs via Gi/o protein-dependent process that likely involves clathrin-coated pits. In addition, the recycling process displays the differential modes of action of different agonists.     

Correction

Phosphoribosylpyrophosphate synthetase (PRS; EC 2.7.6.1) from Hevea brasiliensis Mull. Arg. latex was located in the cytosol. After purification, its apparent molecular weight under nondenaturing conditions was estimated at 200,000 [plus or minus] 10,000; a single band at 57,000 [plus or minus] 3,000 was detected after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme seemed to be a homotetramer. Its affinity constants were estimated at 200 [plus or minus] 30 [mu]M for adenosine triphosphate and 40 [plus or minus] 2 [mu]M for ribose-5-phosphate. The purified enzyme proved to be functional in a paraphysiological medium (cytosol deproteinized by ultrafiltration). Optimum pH was 7.5 in buffer and 6.5 in a paraphysiological medium. No PRS activity was detected in the absence of the Mg2+ ion. Of the numerous compounds tested, only Mn2+, phosphoribosylpyrophosphate and inorganic phosphate affected the enzymatic reaction. Mn2+ (inhibitor constant = 20 [mu]M) and phosphoribosylpyrophosphate (inhibitor constant = 30 [mu]M) were inhibitors. PRS responded allosterically (Hill's coefficient = 2.3) to ribose-5-phosphate in the presence of a physiological concentration of inorganic phosphate (10 mM). These results are set in the physiological context of laticifers.     

Purification and Characterization of Phosphoribosylpyrophosphate Synthetase from Rubber Tree Latex

Phosphoribosylpyrophosphate synthetase (PRS; EC 2.7.6.1) from Hevea brasiliensis Mull. Arg. latex was located in the cytosol. After purification, its apparent molecular weight under nondenaturing conditions was estimated at 200,000 [plus or minus] 10,000; a single band at 57,000 [plus or minus] 3,000 was detected after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme seemed to be a homotetramer. Its affinity constants were estimated at 200 [plus or minus] 30 [mu]M for adenosine triphosphate and 40 [plus or minus] 2 [mu]M for ribose-5-phosphate. The purified enzyme proved to be functional in a paraphysiological medium (cytosol deproteinized by ultrafiltration). Optimum pH was 7.5 in buffer and 6.5 in a paraphysiological medium. No PRS activity was detected in the absence of the Mg2+ ion. Of the numerous compounds tested, only Mn2+, phosphoribosylpyrophosphate, and inorganic phosphate affected the enzymatic reaction. Mn2+ (inhibitor constant = 20 [mu]M) and phosphoribosylpyrophosphate (inhibitor constant = 30 [mu]M) were inhibitors. PRS responded allosterically (Hill's coefficient = 2.3) to ribulose-5-phosphate in the presence of a physiological concentration of inorganic phosphate (10 mM). These results are set in the physiological context of laticifers.     

Cercospora beticola Toxins (X. Inhibition of Plasma Membrane H+-ATPase by Beticolin-1)

Influxes of 13NH4+ across the root plasmalemma were measured in intact seedlings of Picea glauca (Moench) Voss. Two kinetically distinct uptake systems for NH4+ were identified. In N-deprived plants, a Michaelis-Menten-type high-affinity transport system (HATS) operated in a 2.5 to 350 [mu]M range of external NH4+ concentration ([NH4 +]o). The Vmax of this HATS was 1.9 to 2.4 [mu]mol g-1 h-1, and the Km was 20 to40 [mu]M. At [NH4+]o from 500 [mu]M to 50 mM, a linear low-affinity system (LATS) was apparent. Both HATS and LATS were constitutive. A time-dependence study of NH4+ influx in previously N-deprived seedlings revealed a small transient increase of NH4+ influx after 24 h of exposure to 100 [mu]M [NH4+]o. This was followed by a decline of influx to a steady-state value after 4 d. In seedlings exposed to 100 [mu]M external NO3- concentration for 3 d, the Vmax for NH4+ uptake by HATS was increased approximately 30% compared to that found in N-deprived seedlings, whereas LATS was down-regulated. The present study defines the much higher uptake capacity for NH4+ than for N03- in seedlings of this species.     

Structural and immunochemical studies of carp (Cyprinus carpio L.) immunoglobulin. V. Tryptic fragments of carp (Cyprinus carpio L.) immunoglobulin M]

Carp IgM, isolated from normal serum is more sensitive to trypsinization compared to a human myeloma protein IgMGo. Under the same conditions (treatment with trypsin at 56 degrees C for 30 min) carp IgM was degraded to small, mostly dialysable peptides to a larger extent than IgMGo. In both cases the fragmentation resulted in immunoelectrophoretically pure Fab mu and Fc mu fragments. The Fab mu fragments of human IgM (yield: 20% of used IgM material) had a molecular weight of 54,000, the Fc mu fragments (yield: 30%) were a heterogenous mixture as far as molecular sizes concerned with values of about 300,000. For the corresponding fragments of carp IgM we could analyze a molecular weight of about 43,000 for Fab mu (yield: 8%) and for Fc mu (yield 10%) three fractions of 160,000, 130,000 and 90,000. The reductive subunits of Fc mu fragments showed different molecular weights: 39,000 for IgMGo and 45,000 for carp IgM. The anti-fragment antisera prepared in rabbits were monospecific as demonstrated by immunodiffusion.     

Infectivity of a mermithid nematode Romanomermis iyengari (Welch) in different conductivity levels under laboratory and field conditions

Infectivity of R. iyengari was examined by exposing mosquito (Culex quinquefasciatus) larvae to the preparasite at different conductivity levels. The preparasite infected 63.5, 30, 11, 1.5 and 0.5% of the mosquito larvae respectively at 2000, 2500, 3000, 3300 and 3600 mu ho/cm. Although, 62-69% of the preparasite survived at 4000-5400 mu ho/cm, it did not infect. Application of preparasite to tree-holes resulted in 53-63% infection of Aedes albopictus larvae initially. On 6th day the infection level was 40% which decreased further to 7% by 15th day. The infection reappeared on 38th day indicating that R. iyengari has not only infected mosquito larvae as soon as they were applied to tree-holes in which the conductivity was 600-2800 mu ho/cm but also got established there.     

Activation of the H(+)-ATP synthase in the photosynthetic bacterium Rhodobacter capsulatus.

The regulation of the membrane-bound H(+)-ATPase from the photosynthetic bacterium Rhodobacter capsulatus was investigated. In the presence of uncouplers the rate of ATP hydrolysis was about 40 mM ATP/M bacteriochlorophyll (Bchl)/s. Without uncouplers this rate increased and if, additionally, the chromatophores were illuminated, it was almost doubled. If uncouplers were added shortly after illumination, the rate increased to 300-350 mM ATP/M Bchl/s. Obviously, energization of the membrane leads to the formation of a metastable, active state of the H(+)-ATPase. The maximal rate of ATP hydrolysis can be measured only when first all H(+)-ATPases are activated by delta mu H+ and when the delta mu H+ is abolished in order to release its back pressure on the hydrolysis rate. The half-life time of the metastable state in the absence of delta mu H+ is about 30 s. It is increased by 3 mM Pi to about 80 s and it is decreased by 1 mM ADP to about 15 s. Quantitatively, the fraction of active H(+)-ATPases shows a sigmoidal dependence on pHin (at constant pHout) and the magnitude of delta psi determines the maximal fraction of enzymes which can be activated: delta pH and delta psi are not equivalent for the activation process.     

Solubilization in high yield of opioid receptors retaining high-affinity delta, mu and kappa binding sites

The binding sites for opiates (agonist and antagonist) and opioid peptides can be solubilized from rat brain membranes with digitonin in the presence of Mg2+ (10 mM). High affinity and high capacity binding to the soluble delta, mu, and kappa receptors is obtainable when the membranes are treated in Mg2+ (30 degrees C, 60 min) prior to solubilization. The yields of solubilized binding sites extracted with digitonin, 40-90%, are higher than those obtained from Mg2+-pretreated membranes with other detergents commonly used for receptor solubilization. The stability of the digitonin-soluble opioid receptor at room temperature makes it useful for purification and characterization.     

Nonviable mutants of simian virus 40 with deletions near the 3'' end of gene A define a function for large T antigen required after onset of viral DNA replication

Deletion mutants of simian virus 40 (SV40) with lesions at the three DdeI sites near the 3' end of the early region were constructed. Mutants with deletions at 0.203 and 0.219 map units (mu) which did not change the large T antigen reading frame were viable. This extends slightly the upstream boundary for the location of viable mutants with deletions in the 3' end of the A gene. Mutants with frameshift deletions at 0.193 and 0.219 mu were nonviable. These are the first nonviable mutants with deletions in this portion of the A gene. None of the three nonviable mutants with deletions at 0.219 mu produced progeny viral DNA. These three mutants all used the alternate reading frame located in this portion of the SV40 early region. The mutant with a deletion at 0.193 mu, dlA2459, was positive for viral DNA replication and was defective for adenovirus helper function. All of these mutations were located in the portion of the SV40 large T antigen which has no homology to the polyoma T antigens. These results indicate that this portion of large T antigen is required for some late step in the viral growth cycle and suggest that adenovirus helper function is required for productive infection by SV40.     

Inhibition, Inactivation, and Recovery of Ammonia-Oxidizing Activity in Cometabolism of Trichloroethylene by Nitrosomonas europaea

The kinetics of the cometabolism of trichloroethylene (TCE) by the ammonia-oxidizing soil bacterium Nitrosomonas europaea in short-term (<10-min) incubations were investigated. Three individual effects of TCE cometabolism on this bacterium were characterized. First, we observed that TCE is a potent competitive inhibitor of ammonia oxidation by N. europaea. The K(infi) value for TCE (30 (mu)M) is similar to the K(infm) for ammonia (40 (mu)M). Second, we examined the toxicity associated with TCE cometabolism by N. europaea. Stationary-phase cells of N. europaea oxidized approximately 60 nmol of TCE per mg of protein before ammonia-oxidizing activity was completely inactivated by reactive intermediates generated during TCE oxidation. At the TCE concentrations used in these experiments, ammonia did not provide significant protection against inactivation. Third, we have determined the ability of cells to recover ammonia-oxidizing activity after exposure to TCE. Cells recovering from TCE inactivation were compared with cells recovering from the specific inactivation of ammonia-oxidizing activity by light. The recovery kinetics were indistinguishable when 40% or less of the activity was inactivated. However, at increased levels of inactivation, TCE-inactivated cells did not recover as rapidly as light-inactivated cells. The kinetics of recovery appear to be dependent on both the extent of inactivation of ammonia-oxidizing activity and the degree of specificity of the inactivating treatment.     

Mu opioid receptor: a gateway to drug addiction

Mu opioid receptors mediate positive reinforcement following direct (morphine) or indirect (alcohol, cannabinoids, nicotine) activation, and our understanding of mu receptor function is central to the development of addiction therapies. Recent data obtained in native neurons confirm that mu receptor signaling and regulation are strongly agonist-dependent. Current functional mapping reveals morphine-activated neurons in the extended amygdala and early genomic approaches have identified novel mu receptor-associated proteins. A classification of about 30 genes either promoting or counteracting the addictive properties of morphine is proposed from the analysis of knockout mice data. The targeting of effectors or regulatory proteins, beyond the mu receptor itself, might provide valuable strategies to treat addictive disorders.     

The effect of delta mu H+ on the interaction of rotenone with complex I of submitochondrial particles.

The inhibition by rotenone of the forward (NADH-oxidase) and reverse (delta mu H(+)-dependent succinate-NAD+ reductase activities of submitochondrial vesicles was measured. The inhibition of NADH-oxidase, measured in the presence of uncoupler, followed a monophasic inhibition curve with Ki < or = 2 nM. The reverse electron flow was only partially (40%) inhibited at these rotenone concentrations. The rest of the activity was less sensitive to the inhibitor (Ki approximately 30 nM). The lower affinity for the inhibitor of the reverse electron flow is a consequence of enhanced rate of rotenone dissociation caused by the high delta mu H+ value required for this reaction. The analysis of the results indicates that the AS-SMP preparation consists of two subpopulations: one with a relatively low degree of coupling, which exhibits high sensitivity to rotenone and the other which is highly coupled with lower affinity to the inhibitor.     

Impact of Nutrient Composition on a Degradative Biofilm Community

A microbial community was cultivated in flow cells with 2,4,6-trichlorobenzoic acid (2,4,6-TCB) as sole carbon and energy source and was examined with scanning confocal laser microscopy and fluorescent molecular probes. The biofilm community which developed under these conditions exhibited a characteristic architecture, including a basal cell layer and conspicuous mounds of bacterial cells and polymer (approximately 20 to 30 (mu)m high and 25 to 40 (mu)m in diameter) occurring at 20- to 200-(mu)m intervals. When biofilms grown on 2,4,6-TCB were shifted to a labile, nonchlorinated carbon source (Trypticase soy broth), the biofilms underwent an architectural change which included the loss of mound structures and the formation of a more homogeneous biofilm. Neutrally charged fluorescent dextrans, which upon hydration become cationic, were observed to bind to mounds, as well as to the basal cell layer, in 14-day biofilms. In contrast, polyanionic dextrans bound only to the basal cell layer, indicating that this material incorporated sites with both positive and negative charge. The results from this study indicate that nutrient composition has a significant impact on both the architecture and the physicochemistry of degradative biofilm communities.