In Vitro Formation of Nitrate Reductase Using Extracts of the Nitrate Reductase Mutant of Neurospora crassa, nit-1, and Rhodospirillum rubrum

In vitro formation of reduced nicotinamide adenine dinucleotide phosphate (NADPH)-nitrate reductase (NADPH: nitrate oxido-reductase, EC 1.6.6.2) has been attained by using extracts of the nitrate reductase mutant of Neurospora crassa, nit-1, and extracts of either photosynthetically or heterotrophically grown Rhodospirillum rubrum, which contribute the constitutive component. The in vitro formation of NADPH-nitrate reductase is characterized by the conversion of the flavin adenine dinucleotide (FAD) stimulated NADPH-cytochrome c reductase, contributed by the N. crassa nit-1 extract from a slower sedimenting form (4.5S) to a faster sedimenting form (7.8S). The 7.8S NADPH-cytochrome c reductase peak coincides in sucrose density gradient profiles with the NADPH-nitrate reductase, FADH(2)-nitrate reductase and reduced methyl viologen (MVH)-nitrate reductase activities which are also formed in vitro. The constitutive component from R. rubrum is soluble (both in heterotrophically and photosynthetically grown cells), is stimulated by the addition of 10(-4) M Na(2)MoO(4) and 10(-2) M NaNO(3) to cell-free preparations, and has variable activity over the pH range from 3.0 to 9.5. The activity of the constitutive component in some extracts showed a threefold stimulation when the pH was lowered from 6.5 to 4.0. The constitutive activity appears to be associated with a large molecular weight component which sediments as a single peak in sucrose density gradients. However, the constitutive component from R. rubrum is dialyzable and is insensitive to trypsin and protease. These results demonstrate that R. rubrum contains the constitutive component and suggests that it is a low molecular weight, trypsin- and protease-insensitive factor which participates in the in vitro formation of NADPH nitrate reductase.     

The regulatory control of β-receptor dependent adenylate cyclase

The characteristics of the β-receptor in turkey erythrocyte adenylate cyclase were studied using both kinetics of enzyme activation and direct binding measurement of the β-agonists and antagonists to the β-receptor. The regulatory ligands Gpp(NH)p and Ca²⁺ do not have any direct effect on the β-receptor, but modulate the enzyme activity through the interaction with specific regulatory sites. It was found that the role of the catecholamine hormone is to facilitate the activation of the enzyme by the guanyl nucleotide. The regulatory guanyl nucleotide binds to its allosteric site in the absence of hormone, but the activation of the enzyme is slow in the absence of hormone. This role of the hormone can be described by the scheme: Where R is the receptor, E the enzyme, G the guanyl nucleotide, H the hormone, and E′ the activated form of the enzyme. The binding steps are fast and reversible but the conversion of the inactive enzyme E to its active form occurs with a k～1.0 min^?1 In the absence of the β-agonist (l-catecholamine) at the β-receptor and at physiological free Mg²⁺ concentrations, the activation of the enzyme is insignificant. Thus the presence of a guanyl nucleotide at the allosteric site is obligatory but not sufficient to induce the conversion of the inactive enzyme to its active form. At high (nonphysiological) Mg²⁺ concentration the conversion of E to E′ occurs slowly in the absence of hormone probably by another pathway. There are two classes of Gpp(NH)p regulatory sites: tight sites and loose sites, both of which can be identified kinetically. We have also identified the tight sites by direct binding studies using ³H-Gpp(NH)p. It is not clear, however, whether these are two distinct classes of sites or whether their existence reflects the presence of negative cooperativity among the guanyl nucleotide regulatory sites. Calcium was found to be a negative allosteric inhibitor of adenylate cyclase. The inhibitory effect of Ca²⁺ is exerted on the nonactivated enzyme as well as on the Gpp(NH)p preactivated enzyme.     

DNA deposition through laser induced forward transfer

Laser induced forward transfer (LIFT) is a laser direct write technique that appears to be specially adequate for the production of biosensors, since it permits to deposit patterns of biomolecules with high spatial resolution. In the LIFT technique, a laser pulse is focused on a thin film of the material to be transferred through a transparent support, and under the action of the laser pulse, a small fraction of the film is transferred to a receptor substrate that is placed parallel to the film-support system. In the case of biomolecules transfer, the thin film consists in a liquid solution containing the biomolecules. In this work, microarrays of two different cDNAs have been both spotted by LIFT and pin microspotting onto a poly-L-lysine treated glass slide. Once transferred, all the microarrays have been submitted to hybridization with the complementary strands of the spotted cDNAs, each one tagged with a different fluorochrome. Comparative fluorescence scanner analyses have revealed that the microarrays transferred through LIFT are equivalent to those transferred through pin microspotting in terms of signal intensity and gene discrimination capacity, and that the action of the laser pulse does not result in significant damage of the transferred DNA.     

Cost-effective production of 13C, 15N stable isotope-labelled biomass from phototrophic microalgae for various biotechnological applications

The present study outlines a process for the cost-effective production of 13C/15N-labelled biomass of microalgae on a commercial scale. The core of the process is a bubble column photobioreactor with exhaust gas recirculation by means of a low-pressure compressor. To avoid accumulation of dissolved oxygen in the culture, the exhaust gas is bubbled through a sodium sulphite solution prior to its return to the reactor. The engineered system can be used for the production of 13C, 15N, and 13C-15N stable isotope-labelled biomass as required. To produce 13C-labelled biomass, 13CO2 is injected on demand for pH control and carbon supply, whereas for 15N-labelled biomass Na15NO3 is supplied as nitrogen source at the stochiometric concentration. The reactor is operated in semicontinuous mode at different biomass concentrations, yielding a maximum mean biomass productivity of 0.3 gL(-1) day(-1). In order to maximize the uptake efficiency of the labelled substrates, the inorganic carbon is recovered from the supernatant by acidification/desorption processes, while the nitrate is delivered at stochiometric concentration and the harvesting of biomass is performed when the 15NO3- is depleted. In these conditions, elemental analysis of both biomass and supernatant shows that 89.2% of the injected carbon is assimilated into the biomass and 6.9% remains in the supernatant. Based on elemental analysis, 97.8% of the supplied nitrogen is assimilated into the biomass and 1.3% remains in the supernatant. Stable isotope-labelling enrichment has been analysed by GC-MS results showing that the biomass is highly labelled. All the fatty acids are labelled; more than 96% of the carbon present in these fatty acids is 13C. The engineered system was stably operated for 3 months, producing over 160 g of 13C and/or 15N-labelled biomass. The engineered bioreactor can be applied for the labelling of various microalgae.     

Track structure in DNA irradiated with heavy ions

The spatial properties of trapped radicals produced in heavy-ion-irradiated solid DNA at 77 K have been probed using pulsed electron paramagnetic double resonance (PELDOR or DEER) techniques. Salmon testes DNA hydrated to 12 water molecules per nucleotide was irradiated with 40Ar ions of energy 100 MeV/nucleon and LET ranging from 300 to 400 keV/microm. Irradiated samples were maintained at cryogenic temperature at all times. PELDOR measurements were made using a refocused echo detection sequence that allows dipolar interaction between trapped radicals to be observed. The EPR spectrum is attributed to electron loss/gain DNA base radicals and neutral carbon-centered radicals that likely arise from sugar damage. We find a radical concentration of 13.5 x 10(18) cm(-3) in the tracks and a track radius of 6.79 nm. The cross section of these tracks is 144 nm2, yielding a lineal radical density of 2.6 radicals/nm. Based on the yields determined previously for particles having calculated LET values of 300-400 keV/microm and our measured lineal density, we obtain an LET of 270 keV/microm, which is in good agreement with the calculated range of values. These measurements of radical density and spatial extent provide the first direct experimental determination of track characteristics in irradiated DNA.     

Sugar radicals in DNA: isolation of neutral radicals in gamma-irradiated DNA by hole and electron scavenging

In this investigation of the radical formation and the reaction of radicals in gamma-irradiated DNA, we report the isolation of putative neutral radicals by the scavenging of holes by Fe(CN)6(4-) and of electrons by Fe(CN)6(3-). Experiments are performed under conditions that emphasize direct and quasi-direct effects (collectively called direct-type effects.) Samples containing Fe(CN)6(4-) show effective scavenging of holes and the ESR spectra obtained arise principally from DNA anion radicals and neutral radicals. On the other hand, for samples containing Fe(CN)6(3-), electron scavenging is highly efficient, and the resulting spectra arise principally from guanine cation radicals and neutral radicals. When both Fe(CN)6(4-) and Fe(CN)6(3-) are present, a near complete scavenging of cation radicals and anion radicals is observed at 77 K, and the ESR spectra that result originate predominantly with neutral radicals which are assigned predominantly to radicals on the sugar phosphate backbone. A notable finding is the presence of spectral components that indicate the formation, through the rupture of the C3'-O bond, of a neutral deoxyribose radical; a concurrent strand break must accompany formation of this radical. This radical was previously reported in argon-ion-irradiated DNA and now, for the first time, is reported in DNA irradiated with low-LET radiation.     

Amlodipine decreases fibrosis and cardiac hypertrophy in spontaneously hypertensive rats: persistent effects after withdrawal

Our objective was to examine the effect of chronic treatment with amlodipine on blood pressure, left ventricular hypertrophy, and fibrosis in spontaneously hypertensive rats and the persistence of such an effect after drug withdrawal. We investigated the effects of treatment with 2, 8 and 20 mg/kg/day of amlodipine given orally for six months and at three months after drug withdrawal. Systolic blood pressure was measured using the tail-cuff method. At the end of the study period, the heart was excised, the left ventricle was isolated, and the left ventricle weight/body weight ratio was calculated as a left ventricular hypertrophy index. Fibrosis, expressed as collagen volume fraction, was evaluated using an automated image-analysis system on sections stained with Sirius red. Age-matched untreated Wistar-Kyoto and SHR were used as normotensive and hypertensive controls, respectively. Systolic blood pressure was reduced in the treated SHR in a dose-dependent way and after amlodipine withdrawal it increased progressively, without reaching the values of the hypertensive controls. Cardiac hypertrophy was reduced by 8 and 20 mg/kg/day amlodipine, but when treatment was withdrawn only the group treated with 8 mg/kg/day maintained significant differences versus the hypertensive controls. All three doses of amlodipine reduced cardiac fibrosis and this regression persisted with the two highest doses after three months without treatment. We concluded that antihypertensive treatment with amlodipine is accompanied by a reduction in left ventricular hypertrophy and regression in collagen deposition. Treatment was more effective in preventing fibrosis than in preventing ventricular hypertrophy after drug withdrawal.     

Seroprevalence of Helicobacter pylori in Hispanics living in Puerto Rico: A population‐based study

Background

Helicobacter pylori is an important etiologic factor for peptic ulcers and gastric cancer, one of the top ten leading causes of cancer death in Puerto Rico. However, the prevalence of H. pylori infections in this population was previously unknown. The aim of this study was to examine the seroprevalence of H. pylori and its associated risk factors in Puerto Rico.

Materials and Methods

A cross‐sectional study was designed using an existing population‐based biorepository. Seropositivity was determined using the Premier^? H. pylori immunoassay. Helicobacter pylori seroprevalence was estimated with 95% confidence using marginal standardization following logistic regression. To assess the risk factors associated with H. pylori seropositivity, a multivariable log‐binomial model was fitted to estimate the prevalence ratio (PR) and its 95% confidence interval (95% CI).

Results

A total of 528 population‐based serum samples were analyzed. The mean age of the study population was 41 ± 12 years, of whom 55.3% were females. The overall seroprevalence of H. pylori was 33.0% (95% CI = 28.3%‐38.1%). Increasing age and having <12 years of education were significantly (P < .05) associated with H. pylori seropositivity in the multivariable model; however, residing in counties with low population density reached marginal significance (P = .085).

Conclusions

We report that H. pylori infection is common among Hispanics living in Puerto Rico. The H. pylori seroprevalence observed in Puerto Rico is similar to the seroprevalence reported in the overall population of the United States. The association between H. pylori seroprevalence and the risk factors analyzed offers insight into the epidemiology of gastric cancer in Puerto Rico and warrants further investigation.

     

Two Differential Binding Mechanisms of FG-Nucleoporins and Nuclear Transport Receptors

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Inhibition of proliferation of T47D human breast cancer cells: Alterations in progesterone receptor and p53 tumor suppressor protein

We have investigated the influence of three structurally different but functionally related compounds [1, 10 ortho-phenanthroline (phenanthroline), Rifampicin and aurin tricarboxylic acid (ATA)] on the rate and the extent of proliferation of progesterone-responsive T47D human breast cancer cells. These compounds have previously been used in this laboratory and have been shown to modulate properties of nucleic acid binding proteins. Because p53 and the progesterone receptor (PR) are both DNA binding proteins that appear to regulate proliferation of breast cells, alterations in T47D cell p53 and PR levels were examined to determine their relevance in cell proliferation. T47D cells were grown in the absence of phenol red and in the presence of 5% fetal calf serum with or without charcoal stripping in the presence of the inhibitors. The rate of proliferation of cells grown in Rifampicin containing medium exhibited nearly 70% inhibition. Phenanthroline, a known metal chelator, was an effect ive inhibitor of proliferation at 3 mM reducing the cell number by more than 75%. ATA (0.24–2.4 µg/ml) inhibited the growth of the cells by nearly 50%. Analysis of the mechanism of action of these compounds revealed that treatment with these compounds caused specific changes in the molecular composition of T47D cell PR. Whereas ATA caused increased stability of PR isoforms, Rifampicin induced a upshift in the mobility of PR in SDS gels – a phenomenon associated with hyperphosphorylation of steroid receptors (SRs). Phenanthroline treatment (> 2 mM) caused a complete down-regulation of PR and the tumor suppressor protein, p53. The downregulation of p53 paralleled the changes in the molecular composition of PR. We propose that the inhibition of T47D cell proliferation by phenanthroline, Rifampicin and ATA results from a number of cellular changes that include regulation of p53 and PR. (Mol Cell Biochem 175: 81–89, 1997)