Free and polymerized tubulin in cultured bone cells and Chinese hamster ovary cells: the influence of cold and hormones

A low pH method of liposome-membrane fusion (Schneider et al., 1980, Proc. Natl. Acad. Sci. U. S. A. 77:442) was used to enrich the mitochondrial inner membrane lipid bilayer 30-700% with exogenous phospholipid and cholesterol. By varying the phospholipid-to- cholesterol ratio of the liposomes it was possible to incorporate specific amounts of cholesterol (up to 44 mol %) into the inner membrane bilayer in a controlled fashion. The membrane surface area increased proportionally to the increase in total membrane bilayer lipid. Inner membrane enriched with phospholipid only, or with phospholipid plus cholesterol up to 20 mol %, showed randomly distributed intramembrane particles (integral proteins) in the membrane plane, and the average distance between intramembrane particles increased proportionally to the amount of newly incorporated lipid. Membranes containing between 20 and 27 mol % cholesterol exhibited small clusters of intramembrane particles while cholesterol contents above 27 mol % resulted in larger aggregations of intramembrane particles. In phospholipid-enriched membranes with randomly dispersed intramembrane particles, electron transfer activities from NADH- and succinate-dehydrogenase to cytochrome c decreased proportionally to the increase in distance between the particles. In contrast, these electron- transfer activities increased with decreasing distances between intramembrane particles brought about by cholesterol incorporation. These results indicate that (a) catalytically interacting redox components in the mitochondrial inner membrane such as the dehydrogenase complexes, ubiquinone, and heme proteins are independent, laterally diffusible components; (b) the average distance between these redox components is effected by the available surface area of the membrane lipid bilayer; and (c) the distance over which redox components diffuse before collision and electron transfer mediates the rate of such transfer.     

Molecular dynamics in mouse atrial tumor sarcoplasmic reticulum.

We have determined directly the effects of the inhibitory peptide phospholamban (PLB) on the rotational dynamics of the calcium pump (Ca-ATPase) of cardiac sarcoplasmic reticulum (SR). This was accomplished by comparing mouse ventricular SR, which has PLB levels similar to those found in other mammals, with mouse atrial SR, which is effectively devoid of PLB and thus has much higher (unregulated) calcium pump activity. To obtain sufficient quantities of atrial SR, we isolated the membranes from atrial tumor cells. We used time-resolved phosphorescence anisotropy of an erythrosin isothiocyanate label attached selectively and rigidly to the Ca-ATPase, to detect the microsecond rotational motion of the Ca-ATPase in the two preparations. The time-resolved phosphorescence anisotropy decays of both preparations at 25 degrees C were multi-exponential, because of the presence of different oligomeric species. The rotational correlation times for the different oligomers were similar for the two preparations, but the total decay amplitude was substantially greater for atrial tumor SR, indicating that a smaller fraction of the Ca-ATPase molecules exists as large aggregates. Phosphorylation of PLB in ventricular SR decreased the population of large-scale Ca-ATPase aggregates to a level similar to that of atrial tumor SR. Lipid chain mobility (fluidity), detected by electron paramagnetic resonance of stearic acid spin labels, was very similar in the two preparations, indicating that the higher protein mobility in atrial tumor SR is not due to higher lipid fluidity. We conclude that PLB inhibits by inducing Ca-ATPase lateral aggregation, which can be relieved either by phosphorylating or removing PLB.     

Movements and associations of ribosomal subunits in a secretory cell during growth inhibition by starvation

In Chironomus tentans salivary gland cells, the cytoplasm can be dissected into concentric zones situated at increasing distances from the nuclear envelope. After RNA labeling, the newly made ribosomal subunits are found in the cytoplasm mainly in the neighborhood of the nucleus with a gradient of increasing abundance towards the periphery of the cell. The gradient for the small subunit lasts for a few hours and disappears entirely after treatment with puromycin. The large subunit also forms a gradient but one which is only partially abolished by puromycin. The residual gradient which which is resistant to the addition of the drug is probably due to the binding of some large ribosomal units to the membranes of the endoplasmic reticulum (J.-E. Edstrom and u. Lonn. 1976. J. Cell Biol. 70:562-572, and U. Lonn and J.-E. Edstrom. 1976. J. Cell. Biol. 70:573-580). If growth is inhibited by starvation, only the puromycin-sensitive type gradient is observed for the large subunit, suggesting that the attachment of these newly made subunits to the endoplasmic reticulum membranes will not occur. If, on the other hand, the drug-resistant gradient is allowed to form in feeding animals, it is conserved during a subsequent starvation for longer periods than in control feeding animals. This observation provides a further support for an effect of starvation on the normal turnover of the large subunits associated with the endoplasmic reticulum. These results also indicate a considerable structural stability in the cytoplasm of these cells worth little or no gross redistribution of cytoplasmic structures over a period of at least 6 days.     

Dental agenesis in the Dariusleut Hutterite Brethren: comparisons to selected Caucasoid population surveys

We report the results of a study of the prevalence of nonsyndromic dental agenesis among a sample of 208 individuals (105 females, 103 males) between the ages of 15 and 29 years from a North American religious and genetic isolate, the Dariusleut Hutterites of Western Canada. Direct examination of dental casts, oral examination reports, dental treatment records, and a limited number of dental radiographs reveals congenital absence and/or obvious morphometric reduction of at least one tooth (excluding third molars) in 98 subjects (55 females, 43 males), yielding a prevalence estimate of approximately 47%. This estimate is nearly four times those reported for nonisolate Caucasoid populations of European descent and substantially higher than the elevated prevalences observed in several other isolated populations. Although the prevalence of dental agenesis in the Dariusleut is indeed high, neither the incidence of bilateral agenesis (exhibited at least once in 58% of affected dentitions), number of affected teeth per person (mean, 2.4), morphologic tooth classes affected, or combinations of tooth classes affected ostensibly distinguish them from other populations with similar geographic origins. We conclude that the dental agenesis observed in this North American genetic isolate does not represent a private polymorphism or rare developmental variant. Consequently, the results of further study in these Dariusleut Brethren will be directly relevant to critically testing as yet unresolved hypotheses for the mode of gene action and the relative contributions of hereditary and environmental factors to the reduction of tooth numbers in human dentitions.     

Reassessment of the Microbial Role in Mn-Fe Nodule Genesis in Andean Paleosols

The presence of Mn-Fe nodules in the epipedons (surface horizons) of paleosols of presumed Upper Neogene age in the northwestern Venezuelan Andes have been interpreted as products of inorganic oxidation and reduction processes operating over the full range of glacial and interglacial cycles that affected paleosol morphogenesis. New microscopic/chemical data from combined SEM-EDS-FIB analyses of representative Mn-Fe nodules indicate microbes play an important role in Mn/Fe precipitation leading to their genesis in alpine Mollisols (Argiustolls). Although the prevailing new data are based mainly on fossil forms of filamentous bacteria and fungi and other biogenic pseudomorphs that may represent the former resident bacteria, the presence of extant microbes must await field experiments/collection, followed by a molecular microbiology approach to determine the biological drivers of metal precipitation. As in other terrestrial niche environments, microbes are seen here to play a role, perhaps a key one, in the morphogenesis of paleosols of importance in upper Neogene paleoenvironmental reconstruction.     

Hematology and blood biochemistry in infant baboons (Papio hamadryas)

Although published normative reference standards for hematologic and clinical chemistry measures are available for adult baboons, their applicability to infants has not been addressed. We analyzed these measures in 110 infant baboons (55 females and 55 males) from a large breeding colony at the Southwest Regional Primate Research Center in San Antonio, Texas. The sample consists of olive baboons and olive/yellow baboon hybrids, 1 week to 12 months of age. We produced cross-sectional reference values and examined the effects of age, sex, and subspecies on these variables. Hematology reference ranges for infant baboons are similar to, but wider than, those for adults. Reference ranges for blood biochemistry measures are generally more dissimilar to adults, indicating that for many variables, reference ranges for adult baboons are not adequate for infants. Although sex and subspecies differences are rare, age accounts for more than 10% of the variance in many of the variables.     

Pleiotropic effects on cardiovascular risk factors within and between the fourth and sixth decades of life: implications for genotype x age interactions

We used an approach for detecting genotype x environment interactions to detect and characterize genotype x age interaction in longitudinal measures of three well known cardiovascular risk factors: total plasma cholesterol (TC), systolic blood pressure (SBP), and body weight (Wgt). Our objectives were to determine if the same gene or suite of genes influences quantitative variation in each of these phenotypes in the 4th and 6th decades of life, to assess the impact of additive gene effects in these two decades, and to evaluate the stability of pleiotropic relationships among these phenotypes. Using the Framingham Heart Study data, we constructed two cross-sectional samples comprising individuals on whom these phenotypes were measured at ages 30-39 years (Original Cohort: exam 1, Offspring Cohort: exam 2) and at ages 50-59 years (Original Cohort: exam 11, Offspring Cohort: exam 5). We also constructed a longitudinal sample from the cross-sectional sample members for whom measures on these traits were available at both ages (i.e., 4th and 6th decades of life). Patterns of pleiotropy, inferred from genetic correlations between traits, differ between the two age classes. Further, additive genetic variance in SBP during the 4th decade of life is attributable to a different gene or suite of genes than during the 6th. The magnitude of the effect increases for SBP. Variation in TC and Wgt appear to be influenced by the same gene or genes in both decades. The magnitude of the effect is stable for TC, but increases dramatically with age for Wgt.     

Kinetics studies of the cardiac Ca-ATPase expressed in Sf21 cells: new insights on Ca-ATPase regulation by phospholamban

Kinetics studies of the cardiac Ca-ATPase expressed in Sf21 cells (Spodoptera frugiperda insect cells) have been carried out to test the hypotheses that phospholamban inhibits Ca-ATPase cycling by decreasing the rate of the E1.Ca to E1'.Ca transition and/or the rate of phosphoenzyme hydrolysis. Three sample types were studied: Ca-ATPase expressed alone, Ca-ATPase coexpressed with wild-type phospholamban (the natural pentameric inhibitor), and Ca-ATPase coexpressed with the L37A-phospholamban mutant (a more potent monomeric inhibitor, in which Leu(37) is replaced by Ala). Phospholamban coupling to the Ca-ATPase was controlled using a monoclonal antibody against phospholamban. Gel electrophoresis and immunoblotting confirmed an equivalent ratio of Ca-ATPase and phospholamban in each sample (1 mol Ca-ATPase to 1.5 mol phospholamban). Steady-state ATPase activity assays at 37 degrees C, using 5 mM MgATP, showed that the phospholamban-containing samples had nearly equivalent maximum activity ( approximately 0.75 micromol. nmol Ca-ATPase(-1).min(-1) at 15 microM Ca(2+)), but that wild-type phospholamban and L37A-phospholamban increased the Ca-ATPase K(Ca) values by 200 nM and 400 nM, respectively. When steady-state Ca-ATPase phosphoenzyme levels were measured at 0 degrees C, using 1 microM MgATP, the K(Ca) values also shifted by 200 nM and 400 nM, respectively, similar to the results obtained by measuring ATP hydrolysis at 37 degrees C. Measurements of the time course of phosphoenzyme formation at 0 degrees C, using 1 microM MgATP and 268 nM ionized [Ca(2+)], indicated that L37A-phospholamban decreased the steady-state phosphoenzyme level to a greater extent (45%) than did wild-type phospholamban (33%), but neither wild-type nor L37A-phospholamban had any effect on the apparent rate of phosphoenzyme formation relative to that of Ca-ATPase expressed alone. Measurements of inorganic phosphate (P(i)) release concomitant with the phosphoenzyme formation studies showed that L37A-phospholamban decreased the steady-state rate of P(i) release to a greater extent (45%) than did wild-type phospholamban (33%). However, independent measurements of Ca-ATPase dephosphorylation after the addition of 5 mM EGTA to the phosphorylated enzyme showed that neither wild-type phospholamban nor L37A-phospholamban had any effect on the rate of phosphoenzyme decay relative to Ca-ATPase expressed alone. Computer simulation of the kinetics data indicated that phospholamban and L37A-phospholamban decreased twofold and fourfold, respectively, the equilibrium binding of the first Ca(2+) ion to the Ca-ATPase E1 intermediate, rather than inhibiting rate of the E.Ca to E'.Ca transition or the rate of phosphoenzyme decay. Therefore, we conclude that phospholamban inhibits Ca-ATPase cycling by decreasing Ca-ATPase Ca(2+) binding to the E1 intermediate.     

Multiplex Real-Time PCR Assay Using TaqMan Probes for the Identification of Trypanosoma cruzi DTUs in Biological and Clinical Samples

Background

Trypanosoma cruzi has been classified into six Discrete Typing Units (DTUs), designated as TcI–TcVI. In order to effectively use this standardized nomenclature, a reproducible genotyping strategy is imperative. Several typing schemes have been developed with variable levels of complexity, selectivity and analytical sensitivity. Most of them can be only applied to cultured stocks. In this context, we aimed to develop a multiplex Real-Time PCR method to identify the six T. cruzi DTUs using TaqMan probes (MTq-PCR).

Methods/Principal Findings

The MTq-PCR has been evaluated in 39 cultured stocks and 307 biological samples from vectors, reservoirs and patients from different geographical regions and transmission cycles in comparison with a multi-locus conventional PCR algorithm. The MTq-PCR was inclusive for laboratory stocks and natural isolates and sensitive for direct typing of different biological samples from vectors, reservoirs and patients with acute, congenital infection or Chagas reactivation. The first round SL-IR MTq-PCR detected 1 fg DNA/reaction tube of TcI, TcII and TcIII and 1 pg DNA/reaction tube of TcIV, TcV and TcVI reference strains. The MTq-PCR was able to characterize DTUs in 83% of triatomine and 96% of reservoir samples that had been typed by conventional PCR methods. Regarding clinical samples, 100% of those derived from acute infected patients, 62.5% from congenitally infected children and 50% from patients with clinical reactivation could be genotyped. Sensitivity for direct typing of blood samples from chronic Chagas disease patients (32.8% from asymptomatic and 22.2% from symptomatic patients) and mixed infections was lower than that of the conventional PCR algorithm.

Conclusions/Significance

Typing is resolved after a single or a second round of Real-Time PCR, depending on the DTU. This format reduces carryover contamination and is amenable to quantification, automation and kit production.     

Modeling <Emphasis Type="Italic">Neisseria meningitidis</Emphasis> metabolism: from genome to metabolic fluxes

Background  

Neisseria meningitidis is a human pathogen that can infect diverse sites within the human host. The major diseases caused by N. meningitidis are responsible for death and disability, especially in young infants. In general, most of the recent work on N. meningitidis focuses on potential antigens and their functions, immunogenicity, and pathogenicity mechanisms. Very little work has been carried out on Neisseria primary metabolism over the past 25 years.