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Features of primary vesicoureteric reflux detected by investigation of foetal hydronephrosis.

Primary vesicoureteric reflux (VUR) diagnosed on investigation of foetal hydronephrosis accounts for many antenatally detected uropathies. In order to study foetal VUR and its consequences, newborns with foetal hydronephrosis were investigated by ultrasound, micturating cystourethrogram and 99mTechnetium-dimercapto-succinic acid (DMSA), after beginning of chemoprophylaxis. Twenty-eight infants with VUR (bilateral in 15 cases) were identified giving a total 43 renal units for study. There was a predominance of males (86%), moderate/severe reflux (84%) and renal damage (51%). Presence of renal damage was correlated with the severity of reflux. VUR should be investigated in cases of foetal hydronephrosis and our results support that renal damage is frequently congenital and not secondary to urinary tract infection.

Female↗

Biological activity of secondary metabolites produced by a strain of Pseudomonas fluorescens.

Biological activity of secondary metabolites produced by a plant-growth-promoting Pseudomonas fluorescens was evaluated. The strain produced antibiotics phenazine (PHE), 2,4-diacetylphloroglucinol (PHL) and siderophore pyoverdin (PYO) in standard King's B and succinic acid media, respectively. After extraction, PYO was identified by comparing the UV-spectra and moss-green color development after 'diazotized sulfanilic acid' (DSA) spray in TLC. PHE and PHL were identified by comparing standard compounds on TLC and orange-color development immediately after DSA spray. In vitro antibiosis study of the metabolites revealed their antibacterial and antifungal activity against bacterial test organisms Corynebacterium sp., Mycobacterium phlei and M. smegmatis and test fungi Fusarium moniliforme, F. oxysporum, F. semitectum, F. solani and Rhizoctonia solani. A statistically significantly higher plant growth was recorded in siderophore-amended plantlets under gnotobiotic conditions whereas PHE and PHL did not show any plant-growth-promoting activity. These results support the importance of the secondary metabolites produced by the strain P. fluorescens in enhancing plant growth and in controlling fungal and bacterial pathogens.

Anti-Bacterial Agents↗

Pyruvate inhibits zinc-mediated pancreatic islet cell death and diabetes.

AIMS/HYPOTHESIS: We have shown that zinc ion (Zn2+) in secretory granules of pancreatic beta cells could act as a paracrine death effector in streptozotocin-induced diabetes. As Zn2+ has been reported to perturb glycolysis, we studied if pyruvate could inhibit Zn(2+)-mediated islet cell death in vitro and streptozotocin-induced diabetes in vivo by normalizing intracellular energy metabolism. METHODS: Cell death was measured by quantitative viable cell staining and Hoechst/propidium iodide staining. ATP was measured by bioluminescence determination. Pyruvate was infused through the tail vein 1 h before streptozotocin administration. Beta-cell volume was measured by point counting of the insulin-containing cells. RESULTS: Zn2+ induced classical necrosis on MIN6N8 insulinoma cells which was associated with a rapid decline of intracellular ATP levels. Pyruvate inhibited Zn(2+)-induced necrosis of insulinoma cells and depletion of intracellular ATP by Zn2+. Pyruvate did not inhibit other types of necrosis or apoptosis. Energy substrates such as oxaloacetate, alpha-ketoglutarate and succinic acid dimethylester also attenuated Zn(2+)-induced insulinoma cell death. Methylpyruvate that does not generate NAD+ in the cytoplasm or alpha-ketoisocaproate that stimulates ATP generation exclusively in mitochondria also protected insulinoma cells from Zn(2+)-induced necrosis. Pyruvate infusion inhibited the development of diabetes by protecting beta-cell mass after streptozotocin administration. CONCLUSION/INTERPRETATION: These results indicate that pyruvate inhibits Zn(2+)-induced necrosis of beta cells in vitro by protecting intracellular ATP levels and also streptozotocin-induced diabetes in vivo where Zn2+ has been reported to act as a paracrine death effector.

Adenosine Triphosphate↗

On the track to the beta-cell.

The imaging and quantification of the endocrine pancreas by a non-invasive procedure remains a challenge. In the prolongation of prior work on selected succinic acid esters and monosaccharide esters, it is proposed that D-mannoheptulose, which might be transported across the plasma membrane at the intervention of GLUT-2, could be used to label preferentially the endocrine moiety of the pancreatic gland. This heptose is taken up more efficiently by hepatocytes and islet cells, than by erythrocytes, parotid cells, acinar pancreatic cells or tumoural islet cells of either the RINm5F or INS-1 line. Likewise, D-mannoheptulose only inhibits D-glucose metabolism in hepatocytes and isolated islets. Its hexaacetate ester, however, inhibits the catabolism of the hexose in all cell types. The uptake of D-mannoheptulose represents a carrier-mediated process. Human islets behave like rat islets in terms of D-mannoheptulose uptake and inhibition by the heptose of both D-glucose metabolism and insulinotropic action. The use of radiolabelled analogs of D-mannoheptulose suitable for imaging of the endocrine pancreas is discussed. In the same perspective, it is proposed that advantage could be taken of the much greater accumulation of glycogen in insulin-producing cells, as compared to other pancreatic cell types, in situations of sustained hyperglycaemia.

Animals↗

Micrococcus luteus -- survival in amber.

A growing body of evidence now supports the isolation of microorganisms from ancient materials. However, questions about the stringency of extraction methods and the genetic relatedness of isolated organisms to their closest living relatives continue to challenge the authenticity of these ancient life forms. Previous studies have successfully isolated a number of spore-forming bacteria from organic and inorganic deposits of considerable age whose survival is explained by their ability to enter suspended animation for extended periods of time. However, despite a number of putative reports, the isolation of non-spore-forming bacteria and an explanation for their survival have remained enigmatic. Here we describe the isolation of non-spore-forming cocci from a 120-million-year-old block of amber, which by genetic, morphological, and biochemical analyses are identified as belonging to the bacterial species Micrococcus luteus. Although comparison of 16S rRNA sequences from the ancient isolates with their modern counterparts is unable to confirm the precise age of these bacteria, we demonstrate, using complementary molecular and cell biological techniques, evidence supporting the view that these (and related modern members of the genus) have numerous adaptations for survival in extreme, nutrient-poor environments, traits that will assist in this bacteria's persistence and dispersal in the environment. The bacteria's ability to utilize succinic acid and process terpine-related compounds, both major components of natural amber, support its survival in this oligotrophic environment.

Amber↗

Biodegradation of sodium lauryl ether sulfate (SLES) by two different bacterial consortia.

Two bacterial consortia capable of degrading SLES were isolated from a wastewater treatment plant. The two consortia consisted of three members, Acinetobacter calcoacetiacus and Klebsiella oxytoca in one co-culture (A-K) and Serratia odorifera in the second co-culture (S-A), which contains Acinetobacter calcoacetiacus as well. In all experiments, cells were grown on SLES (1000-7000 ppm) containing the M9 minimal medium as sole carbon source. The co-culture A-K demonstrated a higher growth rate (0.26 h(-1)) and significant greater viability than that of the co-culture S-A (0.21 h(-1)). Glucose, sucrose, maltose, mannitol, and succinic acid as carbon sources produced the same degradation rate (approximately 100 ppm/h) and enhanced the SLES degradation rate by 3-fold upon the control (without an added carbon source). In the case of the co-culture S-A, the situation was different; all the carbon sources being tested except maltose caused a repression in the degradation ability in a range between 25-100%. Maltose causes an enhancement by almost fivefold, compared with the positive control.

Bacteria↗

Obstructive ureterocele-an ongoing challenge.

Ureterocele is a cystic dilatation of the intravesical ureter that is most commonly observed in females and children, and usually affects the upper moiety of a complete pyeloureteral duplication. According to their position, ureteroceles are divided into intravesical, when the ureterocele is completely contained inside the bladder, and extravesical when part of the cyst extends to the urethra or bladder neck. Most ureteroceles are diagnosed in utero or immediately after birth during an echographic screening of renal malformations. Severe, febrile urinary tract infection is the most common postnatal presentation of ureteroceles, but they may, rarely, prolapse and acutely obstruct the bladder outlet. Once an ureterocele is identified sonographically, a voiding cystourethrogram to detect vesicoureteral reflux (VUR) and a 99m-technetium dimercapto-succinic acid renal scan to evaluate the function of the different portions of the kidney are mandatory. VUR in the lower pole is observed in 50% of cases and in the contralateral kidney in 25%. Simple endoscopic puncture of the ureterocele has recently been advocated as an emergency therapy for infected or obstructing ureteroceles and as an elective therapy for intravesical ureteroceles. The rate of additional surgery after elective endoscopic puncture of an orthotopic ureterocele ranges from 7 to 23%. Treatment of ectopic ureteroceles is more challenging and both endoscopic puncture and upper pole partial nephrectomy frequently require additional surgery at the bladder level. The reoperation rate after endoscopic treatment varies from 48 to 100%. It is 15 to 20% after upper pole partial nephrectomy if VUR was absent before the operation, but is as high as 50-100% when VUR was present. Thus, endoscopic incision is appropriate as an emergency treatment or when dealing with a completely intravesical ureterocele. Upper pole partial nephrectomy is the elective treatment for an ectopic ureterocele without preoperative VUR. In an ectopic ureterocele with VUR, no matter which type of primary therapy has been chosen, a secondary procedure at the bladder level, involving ureterocele removal and reimplantation of the ureter(s), should be anticipated.

Child↗

Current roles and controversies in the imaging evaluation of acute renal infection.

The current roles and controversies in imaging of the kidneys for the evaluation of patients with acute renal infection are reviewed. The nomenclature used in describing the extent of the renal imaging findings in acute pyelonephritis suggested by the Society of Uroradiology to help avoid confusion in terminology in the past literature is briefly described. Computed tomography (CT) is superior to urography and renal sonography for the evaluation and management of adults with acute renal infection. [99mTc]-dimercapto succinic acid (DMSA) cortical scintigraphy is the imaging study for the evaluation of children with acute pyelonephritis investigated by some, although power Doppler ultrasound, and even CT, can be considered as a possible alternative.

Acute Disease↗

Studies on the extra- and intracellular acid-base status and its role on metabolic depression in the land snail Helix lucorum (L.) during estivation.

The aim of the present study was to examine the acid-base status of extra- and intracellular fluids and its possible role on the regulation of the metabolic rate of Helix lucorum during prolonged estivation. For this purpose, the rate of oxygen consumption for active and estivating snails was determined. The acid-base status was also examined in the hemolymph and tissues from active and estivating snails acclimated at 25 degrees C. In addition, the buffer values of hemolymph and tissues were determined in order to examine whether there is a change in the snails during estivation. The rate of oxygen consumption decreased significantly within the 1st 10 days of estivation from 122.51+/-10 microl.g(-1).h(-1) to 25.86+/-5.2 microl.g(-1).h(-1), indicating a marked decrease in metabolic rate. P(CO2)increased within the 1st 20 days of estivation from 13.52+/-0.68 mmHg to 25.09+/-2.05 mmHg, while the pH of hemolymph (pH(e)) decreased from 7.72+/-0.04 to 7.44+/-0.06. The level of bicarbonates decreased in the hemolymph of estivating snails, indicating a metabolic acidosis, which was moderate in extracellular fluids. In contrast to pH(e), the intracellular pH (pH(i)) was maintained in the tissues of estivating H. lucorum, indicating a regulation of pH(i) despite the developed hypercapnia. According to the results presented here, it seems that the timing of pH(e) changes does not correlate with the timing of metabolic rate reduction in estivating H. lucorum.

Acid-Base Equilibrium↗

Anionic polymers of the cell wall of Brevibacterium linens VKM Ac-2159.

Unsubstituted 1,3-poly(glycerol phosphate) and two sugar-1-phosphate polymers were identified in the cell wall of Brevibacterium linens VKM Ac-2159 by NMR spectroscopy and chemical methods. A monomer of one of the sugar-1-phosphate polymers has the branched repeating unit of the following structure: -4)-[beta-D-GlcpNAc-(1-->3)]-alpha-D-Glcp-(1-P-. The repeating unit of another sugar-1-phosphate polymer has a linear structure consisting of alternating beta- and alpha-N-acetylglucosamine residues: -4)-beta-D-GlcpNAc-(1-->6)-alpha-D-GlcpNAc-(1-P-. Some part of the beta-N-acetylglucosaminyl residues bear O-ester-bound succinic acid residues at C-3. The identified sugar-1-phosphate polymers have not been described earlier in cell walls of other bacteria.

Anions↗

Oxidative metabolism of Polytron versus Nagarse mitochondria in hearts of genetically diabetic mice.

We have shown previously that heart mitochondria obtained by the Nagarse method from genetically diabetic mice (C57BL/KsJ db/db) exhibit a defect in oxidizing NAD+-linked substrates (Kuo, T.H., Moore, K.H., Giacomelli, F. and Wiener, J. (1983) Diabetes 32, 781-787). In this study, the oxidative phosphorylation characteristics of cardiac mitochondria isolated by the Polytron method were compared with that of Nagarse mitochondria. Evidence is presented here that in the diabetic heart both Nagarse and Polytron mitochondria have defective pyruvate oxidation, whereas only the former exhibit impaired fatty acid oxidation. Assay of two rate-limiting beta-oxidation enzymes, namely beta-hydroxyacyl-CoA dehydrogenase and beta-ketothiolase, indicates no alteration in specific activities from diabetic mice vs. controls. The data suggest that two populations of mitochondria are present in myocardium and that the defective oxidative metabolism in the cardiac mitochondria of db/db mice may be linked to deficiencies in total NAD + NADH content.

3-Hydroxyacyl CoA Dehydrogenases↗

Free radical-mediated membrane depolarization in renal and cardiac cells.

Cell membrane potential was measured with a flow cytometer by quantitating the intracellular accumulation of a fluorescent cationic carbocyanine dye. We used this system to demonstrate depolarization upon the addition of hydrogen peroxide (10-1,000 microM) and ferrous chloride (25-100 microM) to cultures of either neonatal rat myocardial or LLC-PK1 renal epithelial cells. Ferrous chloride-induced depolarization was prevented by superoxide dismutase, catalase and dimethyl sulfoxide, suggesting roles for the superoxide anion, hydrogen peroxide and the hydroxyl radical in effecting this depolarization, possibly through a Fenton-type reaction mechanism. Supplementation of either cell type with 2 microM tocopherol acid succinate during growth in tissue culture, prior to exposure to the oxidizing agent, decreased the magnitude of the depolarization in both cell types. The results are consistent with a role for tocopherols in scavenging free radical species responsible for the depolarization of the cell membrane.

Animals↗

Increased therapeutic efficacy of zidovudine in combination with vitamin E.

Antiviral activity and bone marrow toxicity of 3'-azido-3'deoxythymidine (Zidovudine; AZT) was evaluated in the presence of alpha-D-tocopherol acid succinate (ATS) in the MT4 cell line and in murine hematopoietic progenitor cells, respectively. At varying concentrations (.016 to .125 microM) of AZT, addition of ATS (5 to 15 micrograms/ml) showed a dose-dependent increase in anti-HIV activity. The ED90 of AZT in this test system was 0.37 microM, whereas in the presence of ATS (15 micrograms/ml) it was 0.06 microM, thus producing an approximately 6-fold increase in anti-HIV activity. In contrast, in murine bone marrow cells, ATS (4 micrograms/ml) showed significant protection (p less than 0.05) against AZT-induced toxicity as measured by CFU-E and CFU-GM assays. The IC50 values in the presence and absence of ATS for CFU-E were 3.7 and 1.5 microM, whereas for CFU-GM were 6.0 and 2.7 microM, respectively. Overall, these data suggest that AZT in combination with ATS has greater therapeutic efficacy against HIV-1.

Cell Line↗

Phospholipid prodrug inhibitors of the HIV protease. Antiviral activity and pharmacokinetics in rats.

The aspartyl protease of the human immunodeficiency virus (HIV) is an important target for chemotherapeutic intervention because of its key role in cleaving the HIV gag-pol polyprotein during viral assembly and budding. Short peptides and peptidomimetics, which bind to the active site of the HIV aspartyl protease and inhibit processing of the polyprotein, have been synthesized. These compounds are active against HIV in vitro, but many face substantial development problems because of their rapid elimination from the body in bile and urine. Refinement of these agents appears to be necessary if they are to become useful clinically. Recently, we developed a novel chemical strategy for increasing plasma levels of HIV protease inhibitory peptides, which involves the attachment of a biodegradable phospholipid group to the C-terminus of a pentapeptide, iBOC-[L-Phe]-[D-beta-Nal]-Pip-[alpha-(OH)-Leu]-Val (7194). We coupled phosphatidylethanolamine to the C-terminal valine of 7194 to make a phospholipid prodrug (7196). In vitro assays in HT4-6C cells infected with HIV-1 showed that the antiviral activity of the C-terminal phospholipid prodrug, 7196, was equal to that of the free peptide, 7194. Similar results were obtained in vitro when a related pentapeptide (7140) was derivatized at the N-terminal with dipalmitoylphosphatidylethanolamine-succinic acid (7172). Tritium-labeled 7194 and 7196 were prepared and injected intravenously into rats at 3 mumol/kg; then the plasma was assayed for native compound and metabolites by HPLC radioactivity flow detection. The peak plasma level of the tritium-labeled lipid prodrug (7196) was 36 microM versus 1.6 microM for the free protease inhibitor pentapeptide (7194). The area under the curve of the phospholipid prodrug (7196) was 48-fold greater and its mean residence time was increased 43-fold versus the free peptide (7194). Phospholipid prodrugs appear to offer an alternative approach to optimizing in vivo performance of HIV protease inhibitors and other small peptides.

Amino Acid Sequence↗

Fasciola hepatica: inhibition of phosphoenolpyruvate carboxykinase, and end-product formation by quinolinic acid and 3-mercaptopicolinic acid.

Quinolinic acid and 3-mercaptopicolinic acid act as inhibitors of Fasciola hepatica phosphoenolpyruvate carboxykinase. Low concentrations of these compounds (0.1 mM quinolinate and 0.01 mM 3-mercaptopicolinate) resulted in noncompetitive inhibition, which became mixed inhibition at higher concentrations (1.5 and 0.15 mM, respectively). 3-mercaptopicolinic acid proved to be a much more potent effector than quinolinic acid. Both quinolinic acid and 3-mercaptopicolinic acid caused a significant reduction in the total amount of end product excreted, again 3-mercaptopicolinate being more effective than quinolinate. When glucose was present in the medium, both propionate and acetate levels fell significantly with both inhibitors; however, only 3-mercaptopicolinic acid caused an effect in the absence of glucose.

Acetates↗

Specific antibodies directed against toxins of Ptychodiscus brevis (Florida's red tide dinoflagellate).

Specific antibodies directed against Ptychodiscus brevis 'brevetoxins' have been produced in a goat. The haptenic toxin T34 was chemically reduced to toxin T17, covalently-linked to succinic acid via anhydride coupling, and coupled to bovine serum albumin using standard carbodiimide condensation procedures. The hapten coupling efficiency ranged from 10.4 to 13.5 moles of toxin bound per mole of protein. Antibody titers were directly related to the frequency of immunization, and weekly intervals appeared optimum for maintaining adequate titers. [3H]Brevetoxin T17 is displaced in a competitive manner from the antibody-antigen complexes by unlabeled toxin, but the antibodies do not distinguish between T17 and T34. The sensitivity achieved, using purified brevetoxins as competitive inhibitors of [3H]T17 binding, was 600 picograms. The assay linearity ranged from 1.5 to 48 ng.

Animals↗