Regulation of vascular tone in animals overexpressing the sarcolemmal calcium pump

The mechanisms governing vascular smooth muscle tone are incompletely understood. In particular, the role of the sarcolemmal calcium pump PMCA (plasma membrane calmodulin-dependent calcium ATPase), which extrudes Ca2+ from the cytosol, and its importance compared with the sodium/calcium exchanger remain speculative. To test whether the PMCA is a regulator of vascular tone, we generated transgenic mice overexpressing the human PMCA4b under control of the arterial smooth muscle-specific SM22alpha promoter. This resulted in an elevated systolic blood pressure compared with littermate controls. In PMCA-overexpressing mice, endothelium-dependent relaxation of norepinephrine-preconstricted aortic rings to acetylcholine did not differ from wild type controls (76 +/- 8% versus 79 +/- 8% of maximum relaxation; n = 12, n.s.). De-endothelialized aortas of transgenic mice exhibited stronger maximum contraction to KCl (100 mmol/liter) compared with controls (86 +/- 6% versus 68 +/- 7% of reference KCl contraction at the beginning of the experiment; p <0.05). Preincubation of de-endothelialized vessels with the nitric oxide synthase (NOS) inhibitor l-NAME (l-N(G)-nitroarginine methyl ester) (10-5 mol/liter) resulted in a stronger contraction to KCl (p <0.05 versus without l-NAME), thus unmasking vasodilatory effects of inherent NO production. Maximum contraction to KCl after preincubation with l-NAME did not differ between PMCA mice and controls. In analogy to the results in PMCA-overexpressing mice, contractions of de-endothelialized aortas of neuronal NOS-deficient mice to KCl were significantly increased compared with controls (151 +/- 5% versus 131 +/- 6% of reference KCl contraction; p <0.05). In conclusion, our data suggest a model in which the sarcolemmal Ca2+ pump down-regulates activity of the vascular smooth muscle Ca2+/calmodulin-dependent neuronal NOS by a functionally relevant interaction. Therefore, the PMCA represents a novel regulator of vascular tone.     

Is there resetting of central venous pressure in microgravity?

In the early phase of the Space Shuttle program, NASA flight surgeons implemented a fluid-loading countermeasure in which astronauts were instructed to ingest eight 1-g salt tablets with 960 ml of water approximately 2 hours prior to reentry from space. This fluid loading regimen was intended to enhance orthostatic tolerance by replacing circulating plasma volume reduced during the space mission. Unfortunately, fluid loading failed to replace plasma volume in groundbased experiments and has proven minimally effective as a countermeasure against post-spaceflight orthostatic intolerance. In addition to the reduction of plasma volume, central venous pressure (CVP) is reduced during exposure to actual and groundbased analogs of microgravity. In the present study, we hypothesized that the reduction in CVP due to exposure to microgravity represents a resetting of the CVP operating point to a lower threshold. A lower CVP 'setpoint' might explain the failure of fluid loading to restore plasma volume. In order to test this hypothesis, we conducted an investigation in which we administered an acute volume load (stimulus) and measured responses in CVP, plasma volume and renal functions. If our hypothesis is true, we would expect the elevation in CVP induced by saline infusion to return to its pre-infusion levels in both HDT and upright control conditions despite lower vascular volume during HDT. In contrast to previous experiments, our approach is novel in that it provides information on alterations in CVP and vascular volume during HDT that are necessary for interpretation of the proposed CVP operating point resetting hypothesis.     

Alpha- and beta-Proteobacteria control the consumption and release of amino acids on lake snow aggregates

We analyzed the composition of aggregate (lake snow)-associated bacterial communities in Lake Constance from 1994 until 1996 between a depth of 25 m and the sediment surface at 110 m by fluorescent in situ hybridization with rRNA-targeted oligonucleotide probes of various specificity. In addition, we experimentally examined the turnover of dissolved amino acids and carbohydrates together with the microbial colonization of aggregates formed in rolling tanks in the lab. Generally, between 40 and more than 80% of the microbes enumerated by DAPI staining (4',6'-diamidino-2-phenylindole) were detected as Bacteria by the probe EUB338. At a depth of 25 m, 10.5% +/- 7.9% and 14.2% +/- 10.2% of the DAPI cell counts were detected by probes specific for alpha- and beta-Proteobacteria. These proportions increased to 12.0% +/- 3.3% and 54.0% +/- 5.9% at a depth of 50 m but decreased again at the sediment surface at 110 m to 2.7% +/- 1.4% and 41.1% +/- 8.4%, indicating a clear dominance of beta-Proteobacteria at depths of 50 and 110 m, where aggregates have an age of 3 to 5 and 8 to 11 days, respectively. From 50 m to the sediment surface, cells detected by a Cytophaga/Flavobacteria-specific probe (CF319a) comprised increasing proportions up to 18% of the DAPI cell counts. gamma-Proteobacteria always comprised minor proportions of the aggregate-associated bacterial community. Using only two probes highly specific for clusters of bacteria closely related to Sphingomonas species and Brevundimonas diminuta, we identified between 16 and 60% of the alpha-Proteobacteria. In addition, with three probes highly specific for close relatives of the beta-Proteobacteria Duganella zoogloeoides (formerly Zoogloea ramigera), Acidovorax facilis, and Hydrogenophaga palleroni, bacteria common in activated sludge, 42 to 70% of the beta-Proteobacteria were identified. In the early phase (<20 h) of 11 of the 15 experimental incubations of aggregates, dissolved amino acids were consumed by the aggregate-associated bacteria from the surrounding water. This stage was followed by a period of 1 to 3 days during which dissolved amino acids were released into the surrounding water, paralleled by an increasing dominance of beta-Proteobacteria. Hence, our results show that lake snow aggregates are inhabited by a community dominated by a limited number of alpha- and beta-Proteobacteria, which undergo a distinct succession. They successively decompose the amino acids bound in the aggregates and release substantial amounts into the surrounding water during aging and sinking.     

Left ventricular, systemic arterial, and baroreflex responses to ketamine and TEE in chronically instrumented monkeys

Effects of prescribed doses of ketamine five minutes after application and influences of transesophageal echocardiography (TEE) on left ventricular, systemic arterial, and baroreflex responses were investigated to test the hypothesis that ketamine and/or TEE probe insertion alter cardiovascular function. Seven rhesus monkeys were tested under each of four randomly selected experimental conditions: (1) intravenous bolus dose of ketamine (0.5 ml), (2) continuous infusion of ketamine (500 mg/kg/min), (3) continuous infusion of ketamine (500 mg/kg/min) with TEE, and (4) control (no ketamine or TEE). Monkeys were chronically instrumented with a high fidelity, dual-sensor micromanometer to measure left ventricular and aortic pressure and a transit-time ultrasound probe to measure aortic flow. These measures were used to calculate left ventricular function. A 4-element Windkessel lumped-parameter model was used to estimate total peripheral resistance and systemic arterial compliance. Baroreflex response was calculated as the change in R-R interval divided by the change in mean aortic pressure measured during administration of graded concentrations of nitroprusside. The results indicated that five minutes after ketamine application heart rate and left ventricular diastolic compliance decreased while TEE increased aortic systolic and diastolic pressure. We conclude that ketamine may be administered as either a bolus or continuous infusion without affecting cardiovascular function 5 minutes after application while the insertion of a TEE probe will increase aortic pressure. The results for both ketamine and TEE illustrate the classic "Hawthorne Effect," where the observed values are partly a function of the measurement process. Measures of aortic pressure, heart rate, and left ventricular diastolic pressure should be viewed as relative, as opposed to absolute, when organisms are sedated with ketamine or instrumented with a TEE probe.     

The matrix metalloproteinase 9 (mmp-9) hemopexin domain is a novel gelatin binding domain and acts as an antagonist

Matrix metalloproteinases (MMPs) are involved in the remodeling processes of the extracellular matrix and the basement membrane. Most MMPs are composed of a regulatory, a catalytic, and a hemopexin subunit. In many tumors the expression of MMP-9 correlates with local tumor growth, invasion, and metastasis. To analyze the role of the hemopexin domain in these processes, the MMP-9 hemopexin domain (MMP-9-PEX) was expressed as a glutathione S-transferase fusion protein in Escherichia coli. After proteolytic cleavage, the isolated PEX domain was purified by size exclusion chromatography. In a zymography assay, MMP-9-PEX was able to inhibit MMP-9 activity. The association and dissociation rates for the interaction of MMP-9-PEX with gelatin were determined by plasmon resonance. From the measured rate constants, the dissociation constant was calculated to be K(d) = 2,4 x 10(-8) m, demonstrating a high affinity between MMP-9-PEX and gelatin. In Boyden chamber experiments the recombinant MMP-9-PEX was able to inhibit the invasion of melanoma cells secreting high amounts of MMP-9 in a dose-dependent manner. These data demonstrate for the first time that the hemopexin domain of MMP-9 has a high affinity binding site for gelatin, and the particular recombinant domain is able to block MMP-9 activity and tumor cell invasion. Because MMP-9 plays an important role in metastasis, this antagonistic effect may be utilized to design MMP inhibition-based cancer therapy.     

Selected biostatistical aspects of the validation of in vitro toxicological assays

An overview is presented on selected biostatistical aspects of the validation of in vitro toxicological assays. Primarily, the statistical analysis of single assays is discussed. Several approaches are compared for the possible non-monotonic dose-response relationship with a priori unknown shapes. The use of confidence intervals instead of p values for toxicologically appropriate decision making is explained. New methods are discussed for demonstrating interlaboratory similarity for dose-response designs are discussed. For validation, the inappropriateness of the concordance coefficient is shown, and sensitive and specificity as well as predictive values are proposed as alternatives. The problem of the missing gold standard is highlighted.     

Characterization of the human adenylyl cyclase gene family: cDNA,gene structure,and tissue distribution of the nine isoforms

The membrane-bound adenylyl cyclases (ACs) represent one of the major families of effector enzymes for G protein-coupled receptors. Eight human AC isoforms, encoded by separate genes, have been identified up to now. However, in several cases only partial cDNA sequences are available (ADCY1,2,5). A ninth expected isoform, the human ortholog of rat ADCY4, has not been described yet. Using the high inter-species homology of mammalian AC isoforms, we searched the human genome and we succeeded to identify full-length coding sequences for all enzymes. Where required, missing sequence information was provided experimentally. Analysis of genomic sequences from the Celera database also allowed us to determine the exon-intron boundaries for ADCY1-9 and to establish the gene structures. We found that human AC genes comprise 11 to 26 exons, which are distributed over 16 to 430kb. We further report expression profiles for the nine ACs in a panel of 16 human tissues and in human embryonic kidney (HEK) cells.