Cardiac contractile proteins and sarcoplasmic reticulum in hearts of rats trained by running

Rats were trained with two running protocols previously demonstrated to result in enhanced cardiac performance. Control groups included free-eating sedentary animals and food-restricted animals in which the body weights were the same as the runners. Calcium binding by isolated sarcoplasmic reticulum (SR) was slightly but significantly increased in SR from runners at low but not high calcium concentrations at 15 s and 1 min. Calcium uptake in the presence of 1 mM oxalate was increased in SR from runners. Actomyosin ATPase activity was increased by 10% (P less than 0.001) with one running protocol but not with the other. Myosin Ca2+ ATPase activity and actin-activated ATPase activity were also slightly increased in hearts of runners. In food-restricted cardiac actomyosin ATPase was significantly decreased. Actomyosin ATPase activity was found to be normal in hearts of sedentary animals subjected to water immersion without exercise. Therefore, physical training of rats by running, which produces a cardiac mechanical advantage similar to training by swimming, is not accompanied by cardiac biochemical changes of the same magnitude as in the hearts of swimmers.     

Response properties of thick myelinated group II afferents in the medial articular nerve of normal and inflamed knee joints of the cat.

This study investigated the responses to innocuous and noxious mechanical stimuli of joint mechanoreceptors with thick myelinated articular afferents (conduction velocities 21-65 m/sec) in the medial articular nerve of the cat's knee. In nine experiments, we examined whether acute arthritis would modify the discharge properties. The vast majority of the group II afferents were excited by gentle local stimuli and by movements in the working range of the knee. Although they encoded pressure and particular movement stimuli up to the noxious range, their responses were more closely related to the particular type of stimulus (e.g., a movement in a specific direction) than to its intensity (innocuous vs. noxious). In inflamed joints, the response patterns of group II units were similar with regard to local mechanical thresholds, thresholds for passive movements, and patterns of responses to passive movements. In both situations, most units had no resting activity. These results suggest that articular group II afferents do not play a significant role in nociception. Rather, they subserve proprioceptive functions such as deep pressure sensation and kinesthesia in normal as well as inflamed joints.     

The Paleobiosphere: a novel device for the in vivo testing of hydrocarbon producing-utilizing microorganisms

The construction and testing of a unique instrument, the Paleobiosphere, which mimics some of the conditions of the ancient earth, is described. The instrument provides an experimental testing system for determining if certain microbes, when provided an adequate environment, can degrade biological materials to produce fuel-like hydrocarbons in a relatively short time frame that become trapped by the shale. The conditions selected for testing included a particulate Montana shale (serving as the “Trap Shale”), plant materials (leaves and stems of three extant species whose origins are in the late Cretaceous), a water-circulating system, sterile air, and a specially designed Carbotrap through which all air was passed as exhaust and volatile were hydrocarbons trapped. The fungus for initial testing was Annulohypoxylon sp., isolated as an endophyte of Citrus aurantifolia. It produces, in solid and liquid media, a series of hydrocarbon-like molecules. Some of these including 1,8-cineole, 2-butanone, propanoic acid, 2-methyl-, methyl ester, benzene (1-methylethyl)-, phenylethyl alcohol, benzophenone and azulene, 1,2,3,5,6,7,8,8a-octahydro-1,4-dimethyl-7-(1-methylethenyl), [1S-(1α,7α,8aβ)]. These were the key signature compounds used in an initial Paleobiosphere test. After 3 weeks, incubation, the volatiles associated with the harvested “Trap Shale” included each of the signature substances as well as other fungal-associated products: some indanes, benzene derivatives, some cyclohexanes, 3-octanone, naphthalenes and others. The fungus thus produced a series of “Trap Shale” products that were representative of each of the major classes of hydrocarbons in diesel fuel (Mycodiesel). Initial tests with the Paleobiosphere offer some evidence for a possible origin of hydrocarbons trapped in bentonite shale. Thus, with modifications, numerous other tests can also be designed for utilization in the Paleobiosphere.     

Exclusion of galectin 9 as a candidate gene for hyperuricosuria in the Dalmatian dog

All Dalmatian dogs have an inherited defect in purine metabolism leading to high levels of uric acid excretion in their urine (hyperuricosuria) rather than allantoin, the normal end product of purine metabolism in all other breeds of dog. Transplantation experiments have demonstrated that the defect is intrinsic to the liver and not the kidney. Uricase, the enzyme involved in the breakdown of urate into allantoin, has been shown to function in Dalmatian liver cells. Therefore, candidate genes for this defect include transporters of urate, a salt of uric acid, across cell membranes. We excluded one such urate transporter candidate, galectin 9, using a Dalmatian x Pointer backcross in which hyperuricosuria was segregating.     

Protein identification and tracking in two-dimensional electrophoretic gels by minimal protein identifiers

Protein identification by matrix-assisted laser desorption/ionization mass-spectrometry peptide mass fingerprinting (MALDI-MS PMF) represents a cornerstone of proteomics. However, it often fails to identify low-molecular-mass proteins, protein fragments, and protein mixtures reliably. To overcome these limitations, PMF can be complemented by tandem mass spectrometry and other search strategies for unambiguous protein identification. The present study explores the advantages of using a MALDI-MS-based approach, designated minimal protein identifier (MPI) approach, for protein identification. This is illustrated for culture supernatant (CSN) proteins of Mycobacterium tuberculosis H37Rv after separation by two-dimensional gel electrophoresis (2-DE). The MPI approach takes into consideration that proteins yield characteristic peptides upon proteolytic cleavage. In this study, peptide mixtures derived from tryptic protein cleavage were analyzed by MALDI-MS and the resulting spectra were compared with template spectra of previously identified counterparts. The MPI approach allowed protein identification by few protein-specific signature peptide masses and revealed truncated variants of mycobacterial elongation factor EF-Tu, previously not identified by PMF. Furthermore, the MPI approach can be employed to track proteins in 2-DE gels, as demonstrated for the 14 kDa antigen, the 10 kDa chaperone, and the conserved hypothetical protein Rv0569 of M. tuberculosis H37Rv. Furthermore, it is shown that the power of the MPI approach strongly depends on distinct factors, most notably on the complexity of the proteome analyzed and accuracy of the mass spectrometer used for peptide mass determination.     

Apoptosis facilitates antigen presentation to T lymphocytes through MHC-I and CD1 in tuberculosis

Protective immunity against Mycobacterium tuberculosis involves major histocompatibility complex class I (MHC-I)- and CD1-restricted CD8 T cells, but the mechanisms underlying antigen delivery to antigen-presenting molecules remain enigmatic. Macrophages, the primary host cells for mycobacteria, are CD1-negative. Here we show that M. tuberculosis phagosomes are secluded from the cytosolic MHC-I processing pathway and that mycobacteria-infected cells lose their antigen-presenting capacity. We also show that mycobacteria induce apoptosis in macrophages, causing the release of apoptotic vesicles that carry mycobacterial antigens to uninfected antigen-presenting cells (APCs). Inhibition of apoptosis reduced transfer of antigens to bystander cells and activation of CD8 T cells. Uninfected dendritic cells, which engulfed extracellular vesicles, were indispensable for subsequent cross-presentation of antigens, through MHC-I and CD1b, to T cells from mycobacteria-sensitized donors. This new 'detour' pathway for presentation of antigens from a phagosome-contained pathogen shows the functional significance of infection-induced apoptosis in the activation of CD8 T cells specific for both protein and glycolipid antigens in tuberculosis.