Cisplatin: nephrotoxic action in vertebrates and its prevention

1. The kidney of frog and black sculpin appeared to be much less sensitive to the toxic action of CP in comparison with rat and pigeon. 2. The impairment of renal function after CP administration resulted in increased serum urea in rat, uric acid in pigeon and magnesium in black sculpin. 3. Kidney swelling is important feature of CP nephrotoxicity in rat and pigeon but not in frog and fish. 4. Pretreatment with choline chloride, PAH, furosemide and ethacrynic acid reduced the nephrotoxic action of CP in rat but did not prevent the accumulation of platinum in renal tissue which appeared to be a function of the dose injected to investigated animals.     

Temperature-dependent variations and intraspecies diversity of the structure of the lipopolysaccharide of Yersinia pestis

Yersinia pestis spread throughout the Americas in the early 20th century, and it occurs predominantly as a single clone within this part of the world. However, within Eurasia and parts of Africa there is significant diversity among Y. pestis strains, which can be classified into different biovars (bv.) and/or subspecies (ssp.), with bv. orientalis/ssp. pestis most closely related to the American clone. To determine one aspect of the relatedness of these different Y. pestis isolates, the structure of the lipopolysaccharide (LPS) of four wild-type and one LPS-mutant Eurasian/African strains of Y. pestis was determined, evaluating effects of growth at mammalian (37 degrees C) or flea (25 degrees C) temperatures on the structure and composition of the core oligosaccharide and lipid A. In the wild-type clones of ssp. pestis, a single major core glycoform was synthesized at 37 degrees C whereas multiple core oligosaccharide glycoforms were produced at 25 degrees C. Structural differences occurred primarily in the terminal monosaccharides. Only tetraacyl lipid A was made at 37 degrees C, whereas at 25 degrees C additional pentaacyl and hexaacyl lipid A structures were produced. 4-Amino-4-deoxyarabinose levels in lipid A increased with lower growth temperatures or when bacteria were cultured in the presence of polymyxin B. In Y. pestis ssp. caucasica, the LPS core lacked D-glycero-D-manno-heptose and the content of 4-amino-4-deoxyarabinose showed no dependence on growth temperature, whereas the degree of acylation of the lipid A and the structure of the oligosaccharide core were temperature dependent. A spontaneous deep-rough LPS mutant strain possessed only a disaccharide core and a slightly variant lipid A. The diversity and differences in the structure of the Y. pestis LPS suggest important contributions of these variations to the pathogenesis of this organism, potentially related to innate and acquired immune recognition of Y. pestis and epidemiologic means to detect, classify, control and respond to Y. pestis infections.     

Structural diversity and endotoxic activity of the lipopolysaccharide of Yersinia pestis

The endotoxic activity of the lipopolysaccharides (LPS) with defined chemical structure from Yersinia pestis strains of various subspecies differing in their epidemic potential was studied. The LPS of two strains of Y. pestis ssp. caucasica and ssp. altaica, whose structures have not been studied earlier, were analyzed by high-resolution mass spectrometry. In addition to reported structural changes, an increase in the degree of LPS phosphorylation was observed when strain I-2377 (ssp. altaica) was cultivated at an elevated temperature. A high tumor necrosis factor alpha(TNF-alpha)-inducing activity observed for LPS samples from Y. pestis cultures grown at 25 degrees C correlated with an increased degree of lipid A acylation, particularly, with the presence of the hexaacyl form of lipid A, which was absent from the LPS when bacteria were cultivated at 37 degrees C. No correlation was found between the lethal toxicity of the LPS in vivo and its ability to induce TNF-alpha production in vitro.     

Pharmacological blockade of the nephrotoxicity of cisplatin in rats

Cisplatin injection (0.5 mg/100 g body weight) induced 5 days later an increase in serum urea concentration from 3.31 +/- 0.67 to 47.1 +/- 8.68 mg/ml and diuresis decrease following water loading test. Ethacrynic acid, furosemide, paraaminohippurate pre-injections prevented cisplatin nephrotoxicity. The decrease in cisplatin nephrotoxicity took place when furosemide was administered before or 3 h after cisplatin injection; 6 h after cisplatin injection furosemide was not effective. The results suggest that pharmacological blockade of cisplatin nephrotoxicity depends on the drug penetration into secreting cells of proximal tubules.     

Genotyping of <Emphasis Type="Italic">Bacillus anthracis</Emphasis> and Closely Related Microorganisms

Bacillus cereus sensu lato group, which includes the causative agent of anthrax Bacillus anthracis, has a high degree of genetic similarity. However, in recent years, the development of molecular genetics has made it possible to find some molecular markers for identifying Bacillus anthracis in order to differentiate it from the closely related bacilli, get the clustering of the collection of strains, and describe the phylogenetic relationships between the clusters. This article deals with up-to-date methods of genotyping of Bacillus anthracis; it also evaluates its discrimination ability, informational value, validity of the results, efficiency, simplicity, and ease of use. In addition, international online resources and the possibility of their use for comparing collections of the B. anthracis strains isolated from various parts of the world are discussed.     

The presence of nitric oxide synthase in mucosal epithelial cells of the frog urinary bladder

In experiments on frog Rana temporaria L. urinary bladder, we investigated localization of NO-synthase (NOS) in urinary bladder slices and measured NOS activity in the suspension of mucosal epithelial cells. Intensive NADPH-diaphorase staining which is widely used as an indicator of NOS activity was found in mucosal epithelium. Almost all mucosal epithelial cells isolated in Ca2+ -free conditions demonstrated positive NADPH-diaphorase reactivity. Direct measurement of NOS activity in suspension of mucosal cells determined by the rate of conversion of L-arginine to L-citrullin showed that the enzyme activity was reduced in absence of external Ca2+ and was inhibited by L-NAME: non-specific NOS inhibitor, and 1400 W: a highly selective iNOS inhibitor (control: 754 +/- 184; L-NAME, 1 mM 329 +/- 87; 1400 W, 20 mM: 547 +/- 25; Ca2+ -free/EDTA: 490 +/- 184 cpm [3H]-citrullin/10(6) cells per 45 min, p < 0.05, n = 7-8). The data obtained demonstrate that frog urinary bladder mucosa epithelial cells provided antidiuretic hormone-induced increase of osmotic water permeability contain nitric oxide synthase. The presence of inducible (iNOS) as well as constitutive isoform(s) revealed in these cells allows to suggest involvement of NOS in intracellular signaling pathways regulated water transport across the epithelium.     

Effect of cGMP-dependent signaling system in regulation of osmotic permeability in the frog urinary bladder

In frogs' isolated urinary bladders, contribution of cytosolic guanylate cyclase and cGMP-dependent protein kinase to regulation of osmotic permeability was studied. ODQ (25-100 microM), an inhibitor of cytosolic guanylate cyclase induced an increase of vasotocin-activated osmotic permeability but had no effect on the hormone-activated transepithelial urea transport. In isolated mucosal epithelial cells ODQ (50 microM) decreased the concentration of intracellular cGMP. In these cells L-NAME (0.5 nM), an inhibitor of NO synthase, also decreased the level of cGMP whereas cAMP was significantly increased. 8-pCPT-cGMP (25 and 50 microM), a permeable cGMP analogue which selectively activates protein kinase G, inhibited vasotocin-induced increase of water transport along osmotic gradient indicating that protein kinase G is involved in regulation of water reabsorption. The data obtained show that NO/cGMP signalling system in the frog urinary bladder appears to be a negative modulator of vasotocin-activated increase of osmotic permeability.     

Atrial natriuretic factor stimulates the frog urinary bladder osmotic permeability in presence of a cyclic nucleotide phosphodiesterase inhibitor

The role of atrial natriuretic factor (ANF) in regulation of osmotic water permeability was studied in isolated frog Rana temporaria L. urinary bladder. It was found that ANF (rANF, 1-28) added to the serosal solution at concentrations 5 x 10(-8) M and higher dosedependently stimulated the arginine-vasotocin (AVT)-induced increase of osmotic water permeability. The effect of ANF was revealed only in presence of 3-isobuthyl-1-methylxantine (180 microM) and was accompanied by significant elevation of cGMP level in urinary bladder homogenate and isolated mucosal epithelial cells. C-ANF (des[Gln18, Ser19, Gly20, Leu21, Gly22]-ANF-(4-23)-NH2), a specific agonist of NPR-C receptor, exerted no effect on osmotic water permeability. ANF induced a significant increase of cAMP in urinary bladder homogenates (AVT, 5 x 10(-11) M: 52.3 +/- 10.6; AVT + ANF, 10(-7) M: 114.2 +/- 26.9 pmol/mg protein, n = 5, p < 0.05). The activity of adenylate cyclase in crude plasmatic membrane fraction was not changed. Milrinone, a specific inhibitor of phosphodiesterase 3, at concentrations from 25 to 80 microM, enhanced both the hydroosmotic response to AVT and AVT-stimulated cAMP production. Altogether these data demonstrate that, in the frog urinary bladder, ANF stimulates the AVT-induced increase of osmotic water permeability acting probably through NPR-A receptor-coupled mobilization of cGMP and cGMP-dependent inhibition of phosphodiesterase 3.     

Various effects of prostaglandin E2 on reabsorption of water and urea in the amphibia osmosis-regulating epithelium

Principal similarities between molecular pathways providing the enhancement of water and urea reabsorption under the action of argininvasotocin (AVT) in amphibian urinary bladder suggest that prostaglandin E2 (PGE2) could be a negative regulator of urea transport. To analyse this hypothesis, the role of PGE2 in regulation of urea transport was studied in isolated frog (Rana temporaria L.) urinary bladder. The urea permeability (Pu) was determined from the rate of efflux of (14) Curea from mucosal to serosal solution in isoosmotic conditions. The water permeability was measured in separate experiments in presence of an osmotic gradient. In contrast to water permeability, we were unable to demonstrate any inhibitory effect of 10-1000 nM PGE2 on AVT-stimulated urea transport using a variety of protocols. It was found that basolateral PGE2 exposure (10 nM-1 microM) caused an increase in Pu with no effect on osmotic water flow. The PGE2 effect was markedly inhibited by phloretin, a specific inhibitor of urea transporter. Sulprostone, an EP1/EP3 prostaglandin E2 receptor agonist, had no effect on Pu suggesting the contribution of EP2/EP4 receptor subtypes. In presence of osmotic water flow, the AVT-induced urea transport was significantly higher. This water flow-dependent urea permeability was inhibited by PGE2 although the inhibitory effect was less pronounced in comparison to the action of PGE2 on osmotic water flow. On the basis of these results we can make a conclusion that PGE2 has different role in regulation of water and urea transport in the frog urinary bladder. PGE2 could be considered as a stimulator of urea transport and an inhibitor of osmotic water flow activated by the AVT. The ability of PGE2 to regulate various types of cAMP-dependent transport by different mechanisms seems to be based on the presence of multiple basolateral PGE2 receptor subtypes in amphibian osmosis-regulatory epithelium.     

Functional characterization and biological significance of Yersinia pestis lipopolysaccharide biosynthesis genes

In silico analysis of available bacterial genomes revealed the phylogenetic proximity levels of enzymes responsible for biosynthesis of lipopolysaccharide (LPS) of Yersinia pestis, the cause of plague, to homologous proteins of closely related Yersinia spp. and some other bacteria (Serratia proteamaculans, Erwinia carotovora, Burkholderia dolosa, Photorhabdus luminescens and others). Isogenic Y. pestis mutants with single or double mutations in 14 genes of LPS biosynthetic pathways were constructed by site-directed mutagenesis on the base of the virulent strain 231 and its attenuated derivative. Using high-resolution electrospray ionization mass spectrometry, the full LPS structures were elucidated in each mutant, and the sequence of monosaccharide transfers in the assembly of the LPS core was inferred. Truncation of the core decreased significantly the resistance of bacteria to normal human serum and polymyxin B, the latter probably as a result of a less efficient incorporation of 4-amino-4-deoxyarabinose into lipid A. Impairing of LPS biosynthesis resulted also in reduction of LPS-dependent enzymatic activities of plasminogen activator and elevation of LD(50) and average survival time in mice and guinea pigs infected with experimental plague. Unraveling correlations between biological properties of bacteria and particular LPS structures may help a better understanding of pathogenesis of plague and implication of appropriate genes as potential molecular targets for treatment of plague.