Uninterrupted translation through putative 12-nucleotide coding gap in sequence of carA: business as usual

Previous work of others reported an untranslated stretch of 12 nucleotides in the 5' coding sequence of carA from Pseudomonas aeruginosa. However, N-terminal protein sequencing of carA-lacZ translational fusions shows that these 12 nucleotides are normally translated in a continuous triplet manner, both in P. aeruginosa and in Escherichia coli.     

Cloning and nucleotide sequence of a complementary DNA encoding Xenopus laevis metallothionein: mRNA accumulation in response to heavy metals

This investigation studied the effect of 50 Hz electric and magnetic fields on the human heart. The electrocardiograms of 27 transmission-line workers and 26 male volunteers were recorded with a Holter recorder both in and outside the fields. The measurements took from half an hour to a few hours. The electric field strength varied from 0.14 to 10.21 kV/m and the magnetic flux density from 1.02 to 15.43 microT. Analysis of the ECG recordings showed that extrasystoles or arrhythmias were as frequent outside the field as in the field. In some cases a small decrease in heart rate was observed after field exposure.     

Mutagenic effect of small doses of diethylsulfate in laboratory mice studied by specific locus method]

Diethylsulphate solution was injected intraperitoneally twice a week to male mice B10.C3H having the wild-type colour at a 5 mg/kg dose. The treatment lasted 10 weeks, so that the total dose administered was 5-20 = 100 mg/kg. In fifth week after the end of the treatment these males were crossed to YT females homozygous for seven recessive mutant genes. In the control group all the F1 descendants were of wild-type colour, while in the experimental group among 5042 F1 mice derived from DES-treated spermatogonia there was one mutant d and 4 mosaics (3 with respect to the locus c and 1 with respect to the locusa). These results are interpreted as the evidence of the mutagenic effect of DES.     

Opioid regulation of gonadotropin-releasing hormone gene expression in the male rat brain as studied by in situ hybridization

It is well documented that gonadotropin-releasing hormone (GnRH) release is negatively regulated by opiates. In order to investigate the role of opiates in the regulation of GnRH gene expression in the rat brain, we studied the effects of chronic administration of the opioid drug morphine and an opiate receptor antagonist naloxone on GnRH mRNA levels as measured by in situ hybridization. Four-day treatment with morphine (40 mg kg day-2) produced a 44% decrease in the number of silver grains overlying GnRH neurones. Conversely, naloxone (4 mg kg day-2) also administered during 4 days increased by hybridization signal by 22%. The concomitant administration of morphine and naloxone completely prevented the effect of morphine on GnRH gene expression indicating the inhibitory influence of morphine is likely to be mediated by opioid receptors. The present data clearly indicate that opiates can inhibit not only the release of GnRH and consequently LH secretion but also the biosynthesis of the neuropeptide as evaluated by mRNA level measurements.     

Subcellular distribution of zinc in rat prostate studied by x-ray microanalysis: I. Normal prostate

Zinc is distributed subcellularly throughout the lateral prostate of the rat in both the stromal and epithelial elements. The connective tissue appears to be a major store of zinc. Within the epithelium, the highest concentrations of the element are found in the lysosomes, nucleoli, nuclear chromatin, secretory granules and luminal secretion. Histochemical studies indicate that the metal is bound relatively tightly within the nucleoli (associated with RNA) and in the secretory products of the cytoplasm. Changes in tissue zinc concentration, observed by other workers, following changes in various external stimuli, may not necessarily be reflected by proportionate changes in epithelial concentrations. The role of zinc in the epithelium is considered to be at least two-fold: firstly, for incorporation into vital cellular mechanisms necessary for cell maintenance and, secondly, for involvement in secretory products. It is also possible that the metal participates in the physiology of the sub-epithelial stroma.     

Tumor angiogenesis as a prognostic factor in ovarian carcinoma: quantification of endothelial immunoreactivity by image analysis

BACKGROUND: The growth of a malignant tumor requires the formation of new capillaries. Quantification of these microvessels is difficult. The purpose of this study was to establish an objective technique for quantifying angiogenesis and to evaluate whether microvessel quantity may predict tumor aggressiveness in patients with ovarian carcinoma. METHODS: Endothelial area was used to quantify microvessel density in immunohistochemically stained sections of 28 International Federation of Gynecology and Obstetrics Stage IIIC ovarian carcinomas. The endothelial area was measured with a computer-aided image analysis system in the subepithelial stroma of highest vascularization. The endothelial area in the specimens of 14 patients who survived for > or =6 years was compared with that of 14 patients matched for stage and treatment who died of the disease. RESULTS: The mean tumor area analyzed was 5.04 +/- 0.23 mm2. The mean endothelial area per mm2 of stroma from survivors and dead patients was 0.038 +/- 0.026 mm2 and 0.110 +/- 0.034 mm2, respectively (P < 0.0001). No significant differences were found in histology, tumor grade, status of lymph nodes, and amount of residual tumor. CONCLUSIONS: Image analysis was used to overcome the potential subjectivity of manual counts. Computer-assisted image analysis can evaluate accurately the angiogenic potential in ovarian carcinomas. Tumor angiogenesis may prove to be a prognostic factor in patients with ovarian carcinoma. This study suggests that the measurement of the endothelial area would be clinically useful in determining microvessel density [See editorial on pages 2219-21, this issue.]     

The composition of coding joints formed in V(D)J recombination is strongly affected by the nucleotide sequence of the coding ends and their relationship to the recombination signal sequences

V(D)J recombination proceeds in two stages. Precise cleavage at the border of the conserved recombination signal sequences (RSSs) and the coding ends results in flush double-stranded signal ends and coding ends terminating in hairpins. In the second stage, the signal and coding ends are processed into signal and coding joints. Coding ends containing certain nucleotide homopolymers affect the efficiency of V(D)J recombination. In this study, we have tested the effect of small changes in coding-end nucleotide composition on the frequency of coding- and signal joint formation. Furthermore, we have determined the sequences of coding joints resulting from recombination of coding ends with different compositions. We found that the presence of two T nucleotides 5' of both RSSs, but not a single T, reduces the frequency of signal joint formation, i.e., interferes with the cleavage stage of V(D)J recombination. However, coding-joint processing is sensitive even to a single T. Both the sequence of the coding ends and the particular RSS (12-mer or 23-mer) with which the coding end is associated affect the final composition of the coding joints. Thus, the presence of P nucleotides, the conservation of one undeleted coding end, the formation of joints without any deletions, and the template-dependent insertion of nucleotides are strongly influenced by the coding-end nucleotide composition and/or RSS association. The implications of these results with respect to the processing of coding ends are discussed.     

Sensing by intrahepatic muscarinic nerves of a portal-arterial glucose concentration gradient as a signal for insulin-dependent glucose uptake in the perfused rat liver

In vivo, insulin increases net hepatic glucose uptake efficiently only in the presence of a portal-arterial glucose gradient. In isolated perfused rat livers supplied with a glucose gradient (portal 10 mM/arterial 5 mM) insulin-induced glucose uptake was blocked by atropine; in livers not supplied with the gradient (portal = arterial 5 mM) insulin-dependent glucose uptake was elicited by acetylcholine. Apparently, the gradient was sensed and transformed into a metabolic signal by intrahepatic nerves, releasing acetylcholine to muscarinic receptors.     

Inhibitory effect of TMK688 on skin tumor initiation caused by 7,12-dimethylbenz[a]anthracene in relation to inhibition of aryl hydrocarbon hydroxylase activity and Cyp1a1 mRNA induction

The effects of single and repeated (9 times) administration of two dihydropyridines (DHPs), nimodipine (NIM) and nifedipine (NIF) (5 mg/kg per 12 h and 2.5 mg/kg per 12 h, IP), on the behavior of male adult rats in the holeboard and in the plus-maze, were investigated. Besides, the effects of repeated administration of the drugs on the levels of dopamine (DA), serotonin (5-HT), and their respective major metabolites in several regions of the central nervous system (CNS) were also assessed. The effects of single and repeated administration of the drugs were similar. Both DHPs caused a significant decrease in general motor activity which was evident in both tests and more marked, with the higher doses. The two exploratory parameters measured in the holeboard, i.e. head-dipping frequency and duration, were dissociated under pharmacological treatment. The drug-treated animals did not show an increased emotionality in the holeboard. However, in the plus-maze, NIF (5 mg/kg) and to a lesser extent NIM, appeared to induce some anxiety-related responses which may be secondary, at least in part, to the depressing effect on activity and exploration. Repeated administration of NIM and NIF caused an increase in striatal DA and DOPAC levels, whilst no effects were found on serotonergic system in any of the regions of the CNS analyzed.     

Protective effect of amiloride against reperfusion damage as evidenced by inhibition of accumulation of free fatty acids in working rat hearts

To examine whether amiloride protects against ischemia-induced or reperfusion-induced damage to the heart, mechanical and metabolic studies were performed in the isolated, working rat heart. Ischemia decreased both mechanical function and the tissue levels of high-energy phosphates and increased the tissue levels of free fatty acids (FFAs). Reperfusion restored the levels of high-energy phosphates but further increased FFA accumulation. For this reason, accumulation of FFAs was used as an indicator of both ischemia-induced and reperfusion-induced damage. Drugs were added to the perfusion solution 5 min before ischemia until the end of ischemia (pre) or until 10 min after reperfusion (pre + post). Diltiazem (1 or 5 mumol/L pre) decreased the mechanical function of the non-ischemic heart and attenuated both ischemia-induced and reperfusion-induced accumulation of FFAs. Amiloride (50 mumol/L pre) did not affect the mechanical function of the non-ischemic heart or attenuate ischemia-induced or reperfusion-induced FFA accumulation effectively. However, amiloride (50 mumol/L pre + post) did markedly attenuate the reperfusion-induced accumulation of FFAs. In conclusion, diltiazem attenuates both ischemia-induced and reperfusion-induced myocardial damage, probably through its energy-sparing effect as a result of a decrease in mechanical function before ischemia. In contrast, amiloride attenuates only the reperfusion-induced myocardial damage through mechanisms other than the energy-sparing effect.     

Expression of collagens I, III, IV and V mRNA in excimer wounded rat cornea: analysis by semi-quantitative PCR

A semi-quantitative polymerase chain reaction (PCR) methodology was used to evaluate the kinetic changes occurring in collagens I, III, IV and V mRNA in rat cornea following excimer laser keratectomy. cDNA was synthesized from RNA extracted from rat cornea at various times following excimer laser photoablative keratectomy. Collagen cDNA sequences were subsequently amplified using specific sets of oligonucleotide primers. Competitive PCR amplification was carried out using an internal standard so that a semi-quantitative analysis of message for synthesis of collagen types I, III, IV and V could be performed and time course dynamics of message for these collagens studied. There was a biphasic increase in the levels of collagens III, IV and V mRNA following excimer laser keratectomy. Collagen I mRNA levels demonstrated a more sustained increase and were still elevated at 6 weeks following wounding. Collagens IV and V mRNA showed the largest increase with an approximate three fold increase over controls between 4 days and 1 week. Our results demonstrate that upregulation of stromal collagens I, III, and V mRNA and basement membrane collagen IV mRNA occurs in rat cornea following excimer laser keratectomy.     

Pharmacological protection of NSAID-induced intestinal permeability in the rat: effect of tempo and metronidazole as potential free radical scavengers

Recently, NSAID-induced changes in both the structure and function of the distal intestine have been found to occur more frequently and with greater toxicological significance than previously thought. We have previously validated a suitable animal model to evaluate intestinal permeability changes using orally administered 51Cr-EDTA that correlates with intestinal ulceration. In this study we investigated the suitability of metronidazole and the nitroxide stable free radical scavenger (tempo) as protective agents against NSAID-induced intestinal permeability. Male Sprague-Dawley rats were dosed with two doses of metronidazole (50 mg/kg, 12 and 1 h pre-NSAID) or a single 100 mg/kg dose of tempo 1 h prior to NSAIDs. The urinary excretion of the orally administered marker 51Cr-EDTA was measured. Both tempo and metronidazole dramatically reduced indomethacin (20 mg/kg) and flurbiprofen (10 mg/kg)-induced intestinal permeability. All the animals exposed to indomethacin alone died within 48-96 h and presented with histological evidence of drug-induced enteropathy, ulceration and frank peritonitis. Protection by tempo and metronidazole suggests that free radicals and/or bacteria may be important mediators in the pathogenesis of intestinal mucosal damage induced by NSAIDs. Nitric oxide donor compounds used concomitantly with NSAIDs may protect gastrointestinal tract.     

Difference between human and guinea pig Hageman factors in activation by bacterial proteinases: cleavage site shift due to local amino acid substitutions may determine the activation efficiency of serine proteinase zymogens

Human and guinea pig Hageman factors have been subjected to the action of pseudomonal elastase and serratial E15 proteinase. The pseudomonal elastase cleaved 22-24% of the human molecule at Arg353-Val354, and the remainder at Gly357-Leu358 resulting in the generation of about 20% of potential activity as activated Hageman factor, compared with trypsin activation, while it hydrolyzed Arg340-Ile341 bond in guinea pig molecule and generated about 75% of activity as activated Hageman factor. The serratial proteinase did not hydrolyze the essential cleavage site (Arg353-Val354) of the human zymogen but Gly356-Gly357 (30%) and Gly357-Leu358 (70%) bonds. Both products showed no activity. The guinea pig zymogen, in contrast, was cleaved mostly at Arg340-Ile341 (70%) and less abundantly at Gly344-Leu345 (30%), generating about 85% of the whole potential activity as activated Hageman factor. From the high correspondence between the proportions of activation and of hydrolysis at the essential cleavage site in activation, it was concluded that hydrolysis of the bonds different from the essential bond did not cause activation, even when the spatial separation was only 3 or 4 residues. Considering the amino acid differences between human and guinea pig Hageman factors, -Met351-Thr-Arg-Val-Val-Gly-Gly-Leu-Val-Ala360- and -Leu338-Ser-Arg-Ile-Val-Gly-Gly-Leu-Val-Ala347-, respectively, it was realized that even the minor amino acid substitutions caused the cleavage site shift which resulted in significant differences in activation efficiency of the proteinase zymogens.