A model for the catabolism of rhizopine in Rhizobium leguminosarum involves a ferredoxin oxygenase complex and the inositol degradative pathway

Rhizopines are nodule-specific compounds that confer an intraspecies competitive nodulation advantage to strains that can catabolize them. The rhizopine (3-O-methyl-scyllo-inosamine, 3-O-MSI) catabolic moc gene cluster mocCABRDE(F) in Rhizobium leguminosarum bv. viciae strain 1a is located on the Sym plasmid. MocCABR are homologous to the mocCABR gene products from Sinorhizobium meliloti. MocD and MocE contain motifs corresponding to a TOL-like oxygenase and a [2Fe-2S] Rieske-like ferredoxin, respectively. The mocF gene encodes a ferredoxin reductase that would complete the oxygenase system, but is not essential for rhizopine catabolism. We propose a rhizopine catabolic model whereby MocB transports rhizopine into the cell and MocDE and MocF (or a similar protein elsewhere in the genome), under the regulation of MocR, act in concert to form a ferredoxin oxygenase system that demethylates 3-O-MSI to form scyllo-inosamine (SI). MocA, an NAD(H)-dependent dehydrogenase, and MocC continue the catabolic process. Compounds formed then enter the inositol catabolic pathway.     

The location of the carboxy-terminal region of gamma chains in fibrinogen and fibrin D domains

Elongated fibrinogen molecules are comprised of two outer "D" domains, each connected through a "coiled-coil" region to the central "E" domain. Fibrin forms following thrombin cleavage in the E domain and then undergoes intermolecular end-to-middle D:E domain associations that result in double-stranded fibrils. Factor XIIIa mediates crosslinking of the C-terminal regions of gamma chains in each D domain (the gammaXL site) by incorporating intermolecular epsilon-(gamma-glutamyl)lysine bonds between amine donor gamma406 lysine of one gamma chain and a glutamine acceptor at gamma398 or gamma399 of another. Several lines of evidence show that crosslinked gamma chains extend "transversely" between the strands of each fibril, but other data suggest instead that crosslinked gamma chains can only traverse end-to-end-aligned D domains within each strand. To examine this issue and determine the location of the gammaXL site in fibrinogen and assembled fibrin fibrils, we incorporated an amine donor, thioacetyl cadaverine, into glutamine acceptor sites in fibrinogen in the presence of XIIIa, and then labeled the thiol with a relatively small (0.8 nm diameter) electron dense gold cluster compound, undecagold monoaminopropyl maleimide (Au11). Fibrinogen was examined by scanning transmission electron microscopy to locate Au11-cadaverine-labeled gamma398/399 D domain sites. Seventy-nine percent of D domain Au11 clusters were situated in middle to proximal positions relative to the end of the molecule, with the remaining Au11 clusters in a distal position. In fibrin fibrils, D domain Au11 clusters were located in middle to proximal positions. These findings show that most C-terminal gamma chains in fibrinogen or fibrin are oriented toward the central domain and indicate that gammaXL sites in fibrils are situated predominantly between strands, suitably aligned for transverse crosslinking.     

Crystal structures of fragment D from human fibrinogen and its crosslinked counterpart from fibrin

In blood coagulation, units of the protein fibrinogen pack together to form a fibrin clot, but a crystal structure for fibrinogen is needed to understand how this is achieved. The structure of a core fragment (fragment D) from human fibrinogen has now been determined to 2.9 A resolution. The 86K three-chained structure consists of a coiled-coil region and two homologous globular entitles oriented at approximately 130 degrees to each other. Additionally, the covalently bound dimer of fragment D, known as 'double-D', was isolated from human fibrin, crystallized in the presence of a Gly-Pro-Arg-Pro-amide peptide ligand, which simulates the donor polymerization site, and its structure solved by molecular replacement with the model of fragment D.     

The renal clearance of free and conjugated pyridinium cross-links of collagen

We developed a sensitive reversed-phase high performance liquid chromatography (HPLC) assay to measure free and total Pyridinoline (Pyr) and Deoxypyridinoline (Dpyr) in serum. The method was used to measure total serum levels in seven premenopausal women (ages 52.2 +/- 2.4 years) and to investigate the renal clearance of free and conjugated Pyr and Dpyr in two groups of children (group 1: 13 girls, ages 11-13 years; group 2: 18 older children [2 male, 16 female], ages 13-18 years). Blood and 24-h urine samples were collected from the younger group to measure the renal clearance, and blood and 2-h morning urine samples from the older group were collected to investigate the fractional clearance. Total Pyr and Dpyr in the premenopausal women was 4.08 +/- 0.91 and 1.18 +/- 0.39 nmol/l, respectively. Free and total Pyr and Dpyr in serum and urine was elevated in both groups of children. The free serum levels were 16 and 18% in young and older children, respectively, compared with 40 and 46% in the urine. The percentage of free Dpyr in serum decreased with total urinary Pyr excretion (r = -0.56, p < 0.005, n = 31). The renal clearance of the free cross-link fraction in both groups was 4-fold higher than the conjugated fraction. The fractional clearance of the free fraction was greater than 1 (p < 0.001) and the conjugated fraction less than 1 (p < 0.001). The fractional excretion of free Dpyr increased with total urinary Pyr excretion (r = 0.66, p < 0.005, n = 13). We conclude that HPLC can be used to measure free and total Pyr and Dpyr in serum and that some free Pyr and Dpyr excreted in urine is produced by the kidney.     

The mechanism of increased renal clearance of amylase in acute pancreatitis

Penicillium urticae (NRRL 2159A) was grown in culture broth containing 1 muCi of [1-14C-A1acetate to produce [14C]patulin. [14C]patulin was purified from the broth and added to apple cider. After the patulin concentration of the cider was adjusted to 30 mug/ml with unlabeled patulin, the cider was subjected to various charcoal treatments. [14C]patulin was completely removed by shaking the cider with 20 mg of activated charcoal per ml and by eluting the cider through a 40- to 60-mesh charcoal column. Activated charcola at 5 mg/ml reduced patulin in naturally contaminated cider to nondetectable levels.     

Dependence of renal clearance on urine flow: a mathematical model and its application

A mathematical model is developed to explain the dependence of renal clearance on urine flow rate. The model is tested using human data from the literature on compounds that are neither secreted nor reabsorbed by active or pH-sensitive mechanisms. The physiologically derived model explains and predicts the relationship between renal clearance and urine flow for a broad spectrum of compounds (i.e., butabarbital, chloramphenicol, creatinine, ethanol, theophylline, and urea) for which appropriate data are available.     

Structural and functional characterization of proteolytic fragments derived from the C-terminal regions of bovine fibrinogen

A number of new as well as previously described fragments derived from the D region of bovine fibrinogen by limited proteolysis have been characterized by sequence analysis, differential scanning calorimetry and circular dichroism. Determination of the extremities of the polypeptide chains forming individual fragments allowed the scheme of proteolysis and the borders between domains in the D region of fibrinogen to be established. It was also found that the most thermostable region of the D fragment (TSD) can be substantially reduced in size without loss of its compact structure. The alpha-helical content of the newly prepared 21-kDa TSD2 and 16-kDa TSD3 fragments were 82% and 75%, respectively, strongly supporting a coiled-coil structure for this region of the fibrinogen molecule. The DX and DZ fragments, prepared from a chymotryptic digest of the DLA fragment, were found to be similar to the DL and DY fragments, respectively, except for an internal cleavage at K393-T394 in their beta chains. This cleavage leads to destabilization of all thermolabile domains, indicating interaction between them. The DL and DY fragments, containing only one polymerization site in their beta chains, were able to inhibit fibrin polymerization at high concentration. However, these same fragments failed to bind to fibrin-Sepharose under conditions where their structural analogues, DX and DZ, were tightly bound, indicating that cleavage after K393 substantially increases the affinity of this site.     

Determination of the renal clearance in combination with isotope nephrography

The possibilities of a determination of the renal clearance including the separation of sides with the help of simple nuclear-medical examination and measuring techniques are described. In 50 patients the determination of the effective flow of renal plasma was carried out with 131J-labelled iodohippuric acid. Methodic variants were compared. The examinations of the patient last one hour. They can be carried out in the outpatient department within the isotopic nephrography. In addition to this only several takings of blood samples are necessary. A catheterization of the urinary bladder as it is necessary for the classical determinations of the clearance is no more to be done. The results show that with the help of the methods described valuable quantitative informations about the function of the kidneys are to be got.     

The roles of accumbal dopamine D1 and D2 receptors in maternal memory in rats.

Female rats show enhanced maternal responsiveness toward their young if they have had maternal experiences before. This kind of maternal experience-based memory is critically dependent on the mesolimbic dopamine (DA) system, especially the nucleus accumbens (NA) shell. However, the relative contributions of the two main DA receptor systems (D? and D?) within the shell have not been delineated. This study investigates the roles of dopamine D? and D? receptors in maternal memory by infusing a selective D? antagonist, SCH-23390; a selective D? antagonist, sulpiride; or a combination D?/D? antagonist, cis-Z-flupenthixol, into the NA shell of postpartum female rats. Sulpiride-infused rats showed a significantly longer latency to exhibit full maternal behavior following a 10-day pup isolation period in comparison to the controls that received a vehicle. Cis-Z-flupenthixol disrupted maternal memory to a greater extent, as rats receiving this showed the longest latencies to express maternal behavior. SCH-23390 infusions had only marginal effects. These findings suggest that both the D? and the D? receptor subtypes play a role in the consolidation of maternal memory and they might do so by mediating the motivational salience of pup stimulation. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Probenecid inhibits the renal clearance and renal glucuronidation of nalidixic acid. A pilot experiment

The aim of this pilot study was to demonstrate the possible inhibitory effect of probenecid on the renal glucuronidation and on the renal clearance of nalidixic acid in a human volunteer. Under acidic urine conditions, hardly any nalidixic acid is excreted unchanged (0.2%). It is excreted as acyl glucuronide (53.4%), 7-hydroxymethylnalidixic acid (10.0%), the latter's acyl glucuronide 30.9%, and 7-carboxynalidixic acid (4.2%). Under probenecid co-medication the renal glucuronidation of nalidixic acid is reduced from 53% to 16%; the renal clearance of both nalidixic acid and 7-hydroxymethylnalidixic acid are reduced (p < 0.001); the intrinsic t1/2 of the metabolite 7-hydroxymethylnalidixic acid increased from 0.48 h to 4.24 h. The amount of acyl glucuronidation of 7-hydroxymethylnalidixic acid was not altered. The in vitro protein binding of both acyl glucuronides was increased, while no effect on the unconjugated compounds was seen. Nalidixic acid had no effect on the maximal renal excretion rate of probenecid acyl glucuronide.     

Depression and self-medication with nicotine: The modifying influence of the dopamine D4 receptor gene.

This study evaluated whether there are genetic subgroups of depressed individuals who are more or less predisposed to engage in self-medication smoking practices. Smokers (N?=?231) completed self-report questionnaires of depression and smoking practices and were genotyped for the dopamine D4 (DRD4) gene. A significant interaction (DRD4 Genotype?×?Depression) was found for stimulation smoking and negative-affect reduction smoking. Specifically, these smoking practices were significantly heightened in depressed smokers homozygous for the short alleles of DRD4 but not in those heterozygous or homozygous for the long alleles of DRD4. These preliminary results suggest that the rewarding effects of smoking and the beneficial effects of nicotine replacement therapy for depressed smokers may depend, in part, on genetic factors involved in dopamine transmission. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

The deiodination of thyroxine in hyperthyroid rats as determined by renal clearance of iodide

Other investigators have reported that whole body clearance of thyroxine (T4) is increased in hyperthyroid rats isotopically equilibrated with radioactive T4, using the 24 h post-injection serum T4 concentration in the clearance calculation. Data from this laboratory indicate that serum T4 concentration is lowest at this point yielding falsely high clearance values, particularly when high doses of T4 are injected. To investigate this problem further, two types of experiments were performed. First, rats were equilibrated with [125I]T4, 5 or 20 mug/day, and the urinary clearance of iodide derived from T4 (deiodinative clearance) was measured from 0-7 and 7-24 h after a T4 injection, using the T4 concentration in serum obtained at the midpoint of each urine collection period. Urine was then collected from the ureters for several 1 h periods during the 4th to 8th h following T4 injection, calculating clearances using the midpoint plasma T4 concentration. Second, normal rats were given a single dose of [125I]T4, 5 or 55 mug/rat, and deiodinative clearance was determined during the subsequent 0-7 and 7-24 h periods. The first experiment indicated that deiodinative clearance was significantly enhanced in rats equilibrated with the large dose of T4 under all conditions studied. In contrast, the clearance in normal rats given a single large dose of T4 was not significantly different from that of normal rats given a small dose of T4. These results support the view that T4 clearance is increased in hyperthyroidism, due in part to an increase in the deiodination of T4.     

The toxic component in the development of lung damages in uremia

Renal ischemia causes accumulation of middle-sized molecular components of peptide and non-peptide character in the blood. These substances, obtained from the blood by method of ultrafiltration are able to cause the marked lung injuries with formation of leukocyte and erythrocyte aggregations, endothelial lung cell injured and with the phenomenon of hyperhydration. The indicated changes are proportional by their expressiveness to the dose of administrated middle-sized molecules. The conclusion can be made that toxic component, associated with accumulation of biologically active middle-sized molecules in the blood plays a significant role in the development of nephrogenic pulmonary edema.     

Opposite roles of apolipoprotein E in normal brains and in Alzheimer's disease

We have characterized the interaction between apolipoprotein E (apoE) and amyloid beta peptide (Abeta) in the soluble fraction of the cerebral cortex of Alzheimer's disease (AD) and control subjects. Western blot analysis with specific antibodies identified in both groups a complex composed of the full-length apoE and Abeta peptides ending at residues 40 and 42. The apoE-Abeta soluble aggregate is less stable in AD brains than in controls, when treated with the anionic detergent SDS. The complex is present in significantly higher quantity in control than in AD brains, whereas in the insoluble fraction an inverse correlation has previously been reported. Moreover, in the AD subjects the Abeta bound to apoE is more sensitive to protease digestion than is the unbound Abeta. Taken together, our results indicate that in normal brains apoE efficiently binds and sequesters Abeta, preventing its aggregation. In AD, the impaired apoE-Abeta binding leads to the critical accumulation of Abeta, facilitating plaque formation.     

Free-water clearance and the early recognition of acute renal insufficiency after cardiopulmonary bypass

The predictive value of free-water clearance measurements for the early recognition of acute renal insufficiency was evaluated in 59 patients immediately following cardiopulmonary bypass. Blood urea nitrogen and serum creatinine measurements were taken before and after operation. Intraoperatively, immediately after completion of bypass, urine and serum samples were obtained for osmolality. Duration of bypass, urine output, degree of hemolysis, and quality of perfusion were recorded. Fifty-four patients developed no signs of renal insufficiency following bypass, and all had free-water clearance values equal to or less than -20 ml per hour. Five patients who had free-water clearance values equal to greater than -8 ml per hour developed manifestations of an acute renal insufficiency state. There were no false-negative or false-positive determinations. Consequently, free-water clearance measurements appear to be a reliable indicator of those patients who will develop renal insufficiency following cardiopulmonary bypass. Early recognition provides an opportunity immediately after operation for initiating treatment consisting of administration of diuretics, potassium restriction, and oliguric fluid regimens.     

Endothelial cell markers in chronic uremia: relationship with hemostatic defects and severity of renal failure

The Read Codes are a hierarchically-arranged controlled clinical vocabulary introduced in the early 1980s and now consisting of three maintained versions of differing complexity. The code sets are dynamic, and are updated quarterly in response to requests from users including clinicians in both primary and secondary care, software suppliers, and advice from a network of specialist healthcare professionals. The codes' continual evolution of content, both across and within versions, highlights tensions between different users and uses of coded clinical data. Internal processes, external interactions and new structural features implemented by the NHS Centre for Coding and Classification (NHSCCC) for user interactive maintenance of the Read Codes are described, and over 2000 items of user feedback episodes received over a 15-month period are analysed.     

The role of protein L16 and its fragments in the peptidyltransferase activity of 50-S ribosomal subunits

Two large polypeptide fragments of ribosomal protein L16 were obtained by limited hydrolysis with trypsin and chymotrypsin. The chymotryptic fragment, lacking nine N-terminal amino acids residues, is fully active in the restoration of the peptidyltransferase activity of the LiCl-stripped 50-S ribosomal subunits, whereas the tryptic fragment, lacking an additional six residues, is inactive. We also show that under the optimized ionic conditions protein L16 is not needed for the peptidyltransferase activity.