The binding of nucleotides and bivalent cations to the calcium-and-magnesium ion-dependent adenosine triphosphatase from rabbit muscle sarcoplasmic reticulum.

The binding of MgATP to purified Ca2+Mg2+-dependent adenosine triphosphatase from rabbit muscle sarcoplasmic reticulum was studied by using a flow-dialysis method. Phosphoryl-enzyme formation and catalytic activity were also measured, and all three processes demonstrated negative co-operativity, with half-saturation of all three parameters at a MgATP concentration of 40-50muM, and a Hill coefficient (h) of 0.8. The variation of the binding constant with with pH was measured and showed tighter binding of MgATP with increasing pH over the range 6.8-8.5. Binding parameters for ATP analogues were also measured. The binding of Ca2+ in the presence and absence of ATP analogues gave half saturation at a Ca2+ concentration of 1.2-1.3muM. Hill plots of Ca2+-binding data gave a slope of 0.8. These results show that the binding of MgATP and Ca2+ can occur in a random manner, with neither substrate influencing the affinity of the enzyme for the other.     

Regulation of lymphocyte responses by human gangliosides. I. Characteristics of inhibitory effects and the induction of impaired activation

Gangliosides obtained from normal human brain were found to inhibit the in vitro activation of human lymphocytes by nonspecific mitogens and allogeneic cells at concentrations between 3 to 50 microgram/1.5 to 1.7 X 10(5) lymphocytes/0.2 ml culture. Ganglioside inhibition did not represent cytotoxic effects or altered lectin binding and was independent of the mitogen concentration. In addition to concentration, the degree of inhibition was dependent on the mode of presentation to lymphocytes, since gangliosides incorporated within liposomal membranes displayed a synergistic inhibitory effect greater than predicted from the cultures receiving either gangliosides or liposomes alone. In binding experiments, radiolabeled ganglioside GM1 became associated with human lymphocytes within 10 min. However, approximately 72 hr pre-exposure of human lymphocytes to gangliosides was required to induce impaired lymphocyte responses to mitogens and allogeneic cells. Thus, concentrations of human gangliosides equivalent to the levels occurring in the sera of patients with certain malignancies are capable of actively inhibiting lymphocyte stimulation in addition to inducing impaired lymphocyte responses.     

New methods for the demonstration of lysosomal hydrolases by the formation of osmium blacks

Summary Methods are described for the direct cytochemical demonstration of the enzymes nonspecific esterase and acid phosphatase based on synthetic substrates which initially deposit Hatchett's brown (cupric ferrocyanide, Cu₂Fe(CN)₆·7 H₂O) at their subcellular sites. The small amounts of Hatchett's brown deposited as a result of the enzyme's activity may be intensified by bridging to osmium through thiocarbohydrazide. Alternatively, even greater amplification of the sites of activity may be attained by utilizing the Hatchett's brown as a catalyst to effect the oxidative coupling of 3,3-diaminobenzidine resulting in the formation of an osmiophilic indamine-type polymer.One of the major advantages of this new approach is that it permits the study of acid hydrolase localization without lead in the incubation medium. Studies were performed with these methods having identical incubation media except for synthetic substrate in many different cell types and tissues. They verify a frequent nonlysosomal localization for acid phosphatase and the heterogeneity of lysosomes and lysosomal populations with respect to hydrolase content.These methods give information obtained by direct cytochemical observation an advantage not previously held, in comparison with information from cell-fractionation cytochemical or biochemical studies. Initial studies with these methods on many tissues reinforce previous suggestions of the involvement of acid hydrolases in extralysosomal sites in subcellulur anabolic processes.Supported by U.S.P.H.S. Grant DE-02668.Dr. Anderson's work was performed at the Department of Anatomy of the University of Chicago and was supported by Research Grant No. M 71-077C from the Population Council.     

Community ecology of small mammal populations in Panamá following an outbreak of Hantavirus pulmonary syndrome.

In late 1999 and early 2000, an outbreak of hantavirus pulmonary syndrome (HPS) occurred in and around Los Santos, on the Azuero Peninsula of southwestern Panamá. This HPS episode, resulting in 22% case fatality, was linked to the Costa Rican pigmy rice rat, Oligoryzomys fulvescens costaricensis, which harbored a then undescribed hantavirus, Choclo virus. In addition, Cherrie's cane rat, Zygodontomys brevicauda cherriei, was identified as carrying a distinct hantavirus, Calabazo virus with no known pathogenicity to humans. Herein we present the ecological results of the outbreak investigations in the Azuero region. A total of 164 animals were captured, of which 126 were potential small, non-volant mammal hosts of a hantavirus: rodents in the family Muridae. There were significant differences in small mammal community structure between case sites and a negative control site. Differences were manifest in ecological measures of species diversity and in species evenness and heterogeneity measures, as indicated by Pairwise Euclidian distances and Morisita indices of community similarity. Our analyses suggest that human activities (i.e., deforestation for cattle ranching) coupled with environmental factors (i.e., increased precipitation) may have synergistically coalesced for an increased risk of HPS to area residents.     

Transneuronal tracing of neural pathways that regulate hindlimb muscle blood flow

Despite considerable interest in the neural mechanisms that regulate muscle blood flow, the descending pathways that control sympathetic outflow to skeletal muscles are not adequately understood. The present study mapped these pathways through the transneuronal transport of two recombinant strains of pseudorabies virus (PRV) injected into the gastrocnemius muscles in the left and right hindlimbs of rats: PRV-152 and PRV-BaBlu. To prevent PRV from being transmitted to the brain stem via motor circuitry, a spinal transection was performed just below the L2 level. Infected neurons were observed bilaterally in all of the areas of the brain that have previously been shown to contribute to regulating sympathetic outflow: the medullary raphe nuclei, rostral ventrolateral medulla (RVLM), rostral ventromedial medulla, A5 adrenergic cell group region, locus coeruleus, nucleus subcoeruleus, and the paraventricular nucleus of the hypothalamus. The RVLM, the brain stem region typically considered to play the largest role in regulating muscle blood flow, contained neurons infected following the shortest postinoculation survival times. Approximately half of the infected RVLM neurons were immunopositive for tyrosine hydroxylase, indicating that they were catecholaminergic. Many (47%) of the RVLM neurons were dually infected by the recombinants of PRV injected into the left and right hindlimb, suggesting that the central nervous system has a limited capacity to independently regulate blood flow to left and right hindlimb muscles.     

Using crossover breakpoints in recombinant inbred lines to identify quantitative trait loci controlling the global recombination frequency

Recombination is a crucial component of evolution and breeding, producing new genetic combinations on which selection can act. Rates of recombination vary tremendously, not only between species but also within species and for specific chromosomal segments. In this study, by examining recombination events captured in recombinant inbred mapping populations previously created for maize, wheat, Arabidopsis, and mouse, we demonstrate that substantial variation exists for genomewide crossover rates in both outcrossed and inbred plant and animal species. We also identify quantitative trait loci (QTL) that control this variation. The method that we developed and employed here holds promise for elucidating factors that regulate meiotic recombination and for creation of hyperrecombinogenic lines, which can help overcome limited recombination that hampers breeding progress.     

Lead optimization of methionine aminopeptidase-2 (MetAP2) inhibitors containing sulfonamides of 5,6-disubstituted anthranilic acids

A series of aryl sulfonamides of 5,6-disubstituted anthranilic acids were identified as potent inhibitors of methionine aminopeptidase-2 (MetAP2). Small alkyl groups and 3-furyl were tolerated at the 5-position of anthranilic acid, while -OCH(3), CH(3), and Cl were found optimal for the 6-position. Placement of 2-aminoethoxy group at the 6-position enabled interaction with the second Mn(2+) but did not result in enhancement in potency. Introduction of a tertiary amino moiety at the ortho-position of the sulfonyl phenyl ring gave reduced protein binding and improved cellular activity, but led to lower oral bioavailability.