A mouse model of type B cystinuria due to spontaneous mutation in FVB/NJcl mice.

Cystinuria is an autosomal metabolic disorder caused by mutations in the SLC3A1 and SLC7A9 genes, encoding the amino acid transporter proteins rBAT and b0,+AT, respectively. Based on the causative gene, cystinuria is classified into 3 types: type A (SLC3A1), type B (SLC7A9), and type AB (SLC3A1 and SLC7A9). Patients with cystinuria exhibit hyperexcretion of cystine and dibasic amino acids in the urine and develop cystine crystals due to its low solubility in the urine, often resulting in calculus formation. In this study, we present an inbred strain FVB/NJcl mice affected with cystinuria. In the affected mouse kidney, Slc7a9 expression was completely abolished because of a large sequence deletion in the promoter region of the Slc7a9 mutant allele. Slc7a9-deficient mice with FVB/NJcl genetic background developed cystine calculi in the bladder with high penetrance, as compared to the previously reported mouse models of cystinuria. This model may be useful to understand the determinants of crystal aggregation, affecting calculus formation.

Brevibacterium linens RS16 confers salt tolerance to Oryza sativa genotypes by regulating antioxidant defense and H+ ATPase activity.

Soil salinity is one of the major limitations that affects both plant and its soil environment, leading to reduced agricultural production. Evaluation of stress severity by plant physical and biochemical characteristics is an established way to study plant-salt stress interaction, but the halotolerant properties of plant growth promoting bacteria (PGPB) along with plant growth promotion is less studied till date. The aim of the present study was to elucidate the strategy, used by ACC deaminase-containing halotolerant Brevibacterium linens RS16 to confer salt stress tolerance in moderately salt-tolerant (FL478) and salt-sensitive (IR29) rice (Oryza sativa L.) cultivars. The plants were exposed to salt stress using 0, 50, and 100 mM of NaCl with and without bacteria. Plant physiological and biochemical characteristics were estimated after 1, 5, 10 days of stress application. H+ ATPase activity and the presence of hydroxyectoine gene (ectD) that is responsible for compatible solute accumulation were also analyzed in bacteria. The height and dry mass of bacteria inoculated plants significantly increased compared to salt-stressed plants, and the differences increased in time dependent manner. Bacteria priming reduced the plant antioxidant enzyme activity, lipid peroxidation and it also regulated the salt accumulation by modulating vacuolar H+ ATPase activity. ATPase activity and presence of hydroxyectoine gene in RS16 might have played a vital role in providing salt tolerance in bacteria inoculated rice cultivars. We conclude that dual benefits provided by the halotolerant plant growth promoting bacteria (PGPB) can provide a major way to improve rice yields in saline soil.

A cancer-associated mutation in atypical protein kinase Cι occurs in a substrate-specific recruitment motif.

Atypical protein kinase Cι (PKCι) has roles in cell growth, cellular polarity, and migration, and its abundance is frequently increased in cancer. We identified a protein interaction surface containing a dibasic motif (RIPR) that bound a distinct subset of PKCι substrates including lethal giant larvae 2 (LLGL2) and myosin X, but not other substrates such as Par3. Further characterization demonstrated that Arg471 in this motif was important for binding to LLGL2, whereas Arg474 was critical for interaction with myosin X, indicating that multiple complexes could be formed through this motif. A somatic mutation of the dibasic motif (R471C) was the most frequent mutation of PKCι in human cancer, and the intact dibasic motif was required for normal polarized epithelial morphogenesis in three-dimensional cysts. Thus, the R471C substitution is a change-of-function mutation acting at this substrate-specific recruitment site to selectively disrupt the polarizing activity of PKCι.

Biosynthesis of the osmoprotectant ectoine, but not glycine betaine, is critical for survival of osmotically stressed Vibrio parahaemolyticus cells.

Vibrio parahaemolyticus is a halophile present in marine and estuarine environments, ecosystems characterized by fluctuations in salinity and temperature. One strategy to thrive in such environments is the synthesis and/or uptake of compatible solutes. The V. parahaemolyticus genome contains biosynthesis systems for both ectoine and glycine betaine, which are known to act as compatible solutes in other species. We showed that V. parahaemolyticus had a 6% NaCl tolerance when grown in M9 minimal medium with 0.4% glucose (M9G) with a >5-h lag phase. By using (1)H nuclear magnetic resonance spectroscopy ((1)H-NMR) analysis, we determined that cells synthesized ectoine and glutamate in a NaCl-dependent manner. The most effective compatible solutes as measured by a reduction in lag-phase growth in M9G with 6% NaCl (M9G 6% NaCl) were in the order glycine betaine > choline > proline = glutamate > ectoine. However, V. parahaemolyticus could use glutamate or proline as the sole carbon source, but not ectoine or glycine betaine, which suggests that these are bona fide compatible solutes. Expression analysis showed that the ectA and betA genes were more highly expressed in log-phase cells, and expression of both genes was induced by NaCl up-shock. Under all conditions examined, the ectA gene was more highly expressed than the betA gene. Analysis of in-frame deletions in betA and ectB and in a double mutant showed that the ectB mutant was defective for growth, and this defect was rescued by the addition of glycine betaine, proline, ectoine, and glutamate, indicating that these compounds are compatible solutes for this species. The presence of both synthesis systems was the predominant distribution pattern among members of the Vibrionaceae family, suggesting this is the ancestral state.

[Clinico-morphological and molecular-genetic characteristic of the miometrium with failure of the uterine scar after the Cesarean section at women with undifferentiated forms of connective tissue dysplasia].

Connective tissue state, its strength, is actual problem especially in the moment of labor because the weakness of labor activity or accelerated labor can be in depending on its consistency and elasticity. In addition, connective tissue is very important in the involution of the uterus after labor or wounds healing. Clinico-morphological and molecular-genetic investigation of 90 patients with undifferentiated forms of connective tissue dysplasia (uCTD) has been done. Reparation of tissue at patients with uCTD had a number of peculiarities, such as fibromuscular scar formation by substitution mechanisms with the laminin deficiency in the basic capillar membrane and extracellular matrix, accumulation of III and IV types of collagen, low expression of VEGF in the stromal cell and polymorphism of the alpha estrogen receptor gene. This immunohistochemical changes correlated with focuses of connective tissue disorganization as mucoid swelling, fibrinoid changes and hyalinosis, as well pathology of the microvasculature, resulted in chronic ischemia of the tissue. The disorganization is connected with disturbed reparation as a result of the genetically determined polymorphism of alpha and beta estrogen receptors. uCTD in pregnant women is prognostically significant for selection of way of delivery.

Natural and engineered hydroxyectoine production based on the Pseudomonas stutzeri ectABCD-ask gene cluster.

We report on the presence of a functional hydroxyectoine biosynthesis gene cluster, ectABCD-ask, in Pseudomonas stutzeri DSM5190(T) and evaluate the suitability of P. stutzeri DSM5190(T) for hydroxyectoine production. Furthermore, we present information on heterologous de novo production of the compatible solute hydroxyectoine in Escherichia coli. In this host, the P. stutzeri gene cluster remained under the control of its salt-induced native promoters. We also noted the absence of trehalose when hydroxyectoine genes were expressed, as well as a remarkable inhibitory effect of externally applied betaine on hydroxyectoine synthesis. The specific heterologous production rate in E. coli under the conditions employed exceeded that of the natural producer Pseudomonas stutzeri and, for the first time, enabled effective hydroxyectoine production at low salinity (2%), with the added advantage of simple product processing due to the absence of other cosolutes.

Accumulation and role of compatible solutes in fast-growing Salinivibrio costicola subsp. yaniae.

The moderately halophilic bacterium Salinivibrio costicola subsp. yaniae showed an extremely fast growth rate. Optimal growth was observed in artificial seawater containing 1.4 mol/L NaCl and in MM63 media containing 0.6 mol/L NaCl. We analyzed a variety of compatible solutes that had accumulated in this strain grown in the media. The supplementation effect of the compatible solutes glycine betaine, glutamate, and ectoine to the growth of S. costicola subsp. yaniae was examined. Glycine betaine and glutamate had no supplementation effect on the fast growth rate. Growth of salt-sensitive mutants MU1 and MU2, both of which were defective in the ability to synthesize ectoine, was not observed in MM63 medium in the presence of more than 1.0 mol/L NaCl. From these data, we conclude that ectoine was the predominant compatible solute synthesized in this bacterium that effected an extremely fast growth rate.

Characterization of salt tolerance in ectoine-transformed tobacco plants (Nicotiana tabaccum): photosynthesis, osmotic adjustment, and nitrogen partitioning.

Ectoine (1,4,5,6-tetrahydro-2-methyl-4-pyrimidinecarboxylic acid) biosynthetic genes (ect. ABC) from Halomonas elongata were introduced to tobacco plants using an Agrobacterium-mediated gene delivery system. The genes for ectoine biosynthesis were integrated in a stable manner into the tobacco genome and the corresponding transcripts were expressed. The concentration of ectoine under salt-stress conditions was higher in the roots than in leaves. A close relationship was found between stomatal conductance and the amount of transported nitrogen, suggesting that water transport through the xylem in the stem and transpiration may be involved in nitrogen transport to leaves. The data indicate that the turgor values of the ectoine transgenic lines increased with increasing salt concentration. The data revealed two ways in which ectoine enhanced salinity tolerance of tobacco plants. First, ectoine improved the maintenance of root function so that water is taken up consistently and supplied to shoots under saline conditions. Second, ectoine enhanced the nitrogen supply to leaves by increasing transpiration and by protecting Rubisco proteins from deleterious effects of salt, thereby improving the rate of photosynthesis.

Removing a bottleneck in the Bacillus subtilis biotin pathway: bioA utilizes lysine rather than S-adenosylmethionine as the amino donor in the KAPA-to-DAPA reaction.

In biotin biosynthesis, DAPA aminotransferase encoded by the bioA gene catalyzes the formation of the intermediate 7,8-diaminopelargonic acid (DAPA) from 7-keto-8-aminopelargonic acid (KAPA). DAPA aminotransferases from Escherichia coli, Serratia marcescens, and Bacillus sphaericus use S-adenosylmethionine (SAM) as the amino donor. Our observation that SAM is not an amino donor for B. subtilis DAPA aminotransferase led to a search for an alternative amino donor for this enzyme. Testing of 26 possible amino acids in a cell-free extract assay revealed that only l-lysine was able to dramatically stimulate the in vitro conversion of KAPA to DAPA by the B. subtilis DAPA aminotransferase. The K(m) for lysine and KAPA was estimated to be between 2 and 25 mM, which is significantly higher than the K(m) of purified E. coli BioA for SAM (0.15 mM). This higher requirement for lysine resulted in accumulation of KAPA during fermentation of B. subtilis biotin producing strains. However, this pathway bottleneck could be relieved by either addition of exogenous lysine to the medium or by introduction of lysine deregulated mutations into the production strains.

The Arg617-Arg618 cleavage site in the C-terminal domain of PC1 plays a major role in the processing and targeting of the enzyme within the regulated secretory pathway.

The C-terminal domain of the prohormone convertase PC1 is involved in targeting of the enzyme to secretory granules in neuroendocrine cells and is subsequently processed in this compartment at an Arg617-Arg618 site. Three other dibasics are found in the C-terminal domain of mouse PC1. Here, we examined the role of the four dibasics in targeting PC1 to secretory granules. All 15 possible combinations of dibasic mutations were performed. Wild-type (WT) and mutant PC1 were stably expressed in neuroendocrine PC12 cells that lacked endogenous PC1. Processing, secretion and intracellular localization of PC1 and its mutants were analyzed. Leaving intact Arg617-Arg618 and mutating any combination of the three other dibasics yielded proteins that were stored and processed in secretory granules, similarly to WT PC1. Mutating Arg617-Arg618 alone or with any one of the three remaining dibasics generated proteins that were efficiently stored in secretory granules but were not processed further. Mutating Arg617-Arg618 with more than one of the remaining dibasics produced proteins that reached the TGN but were not stored in secretory granules and exited the cells through the constitutive secretory pathway. These data demonstrate that the Arg617-Arg618 plays a prominent role in targeting PC1 to secretory granules.

Analysis of putative heparin-binding domains of fibroblast growth factor-1. Using site-directed mutagenesis and peptide analogues.

The contribution of individual basic amino acids within three putative "consensus sequences" for heparin binding of fibroblast growth factor-1 have been examined by site-directed mutagenesis. The results indicate that a significant reduction in the apparent affinity of fibroblast growth factor-1 for heparin is only observed when basic residues in one of the three regions are mutated. Mutation in the other regions are without affect on heparin binding. The heparin binding properties of synthetic peptides based on the three "consensus sequences" paralleled the mutagenesis results. That is, synthetic peptides corresponding to regions of the protein that were affected by mutagenesis with respect to heparin binding exhibited a relatively high affinity for immobilized heparin, whereas those corresponding to regions of similar charge density that were unaffected by mutagenesis did not. In addition, amino acid substitution of a nonbasic residue in the heparin-binding peptide could abolish its heparin binding capacity. The heparin-binding peptide could antagonize the mitogenic activity of FGF-1, probably because of the heparin dependence of this activity. Together these data demonstrate that the heparin binding properties of fibroblast growth factor-1 are dictated by structural features more complex than clusters of basic amino acids. The results of these and other studies indicate that consensus motifs for heparin-binding require further definition. More importantly, the results provide a basis for the design of peptide-based inhibitors of FGF-1.

Interleukin-1 beta secretion. A possible multistep process that is regulated in a cell type-specific manner.

In a prior study, we found that the processed form of human interleukin-1 beta (mature IL-1 beta) is secreted to a significantly greater extent than the precursor form of the protein, indicating that the precursor domain acts in some manner to reduce the secretory potential of the protein. In view of this observation, we sought to define the sequence(s) in the IL-1 beta precursor that limit the secretion of the protein as well as the sequences in the mature protein that promote secretion. The P388D1 murine macrophage cell line and the Jurkat human T-cell line were transiently transfected with cDNA expression vectors encoding truncated forms of human precursor IL-1 beta proteins, lacking either the first 76, 94, 99, or 104 amino acids. The removal of increasing numbers of precursor amino acid residues resulted in a graded increase in the secretion of the truncated precursor IL-1 beta proteins from both cell lines. The minimal region of the precursor sequence required to inhibit the optimal secretion of IL-1 beta occurs between amino acids 100 and 104 for P388D1 cells and 95-99 for Jurkat cells. Deletion of the amino acids within these regions increased the secretion level of the truncated proteins to that of mature IL-1 beta. Mutagenesis of the mature IL-1 beta sequence revealed that a region of basic amino acids may play an important role in the optimal secretion of mature IL-1 beta in P388D1 cells, but not in Jurkat cells. Based on the differences in the structural requirements for IL-1 beta secretion in P388D1 and Jurkat cell lines, it is likely that the secretion of IL-1 beta may be subject to multiple levels of regulation that are differentially operative in different cell types.

Basic amino acids flanking the zinc finger of Moloney murine leukemia virus nucleocapsid protein NCp10 are critical for virus infectivity.

Nucleocapsid (NC) protein NCp10 of Moloney murine leukemia virus is encoded by the 3' domain of gag and contains a zinc finger surrounded by basic amino acids. During virion assembly, NC protein is necessary for core formation and the NC zinc finger is required for the packaging of the genomic RNA dimer. In vitro NCp10 has RNA-binding and -annealing activities critical for virus infectivity, since NCp10 promotes dimerization of viral RNA containing the Psi packaging element and annealing of replication primer tRNA(Pro) to the initiation site of reverse transcription (primer-binding site). To investigate the role of the basic amino acids flanking the NCp10 zinc finger, neutral residues were substituted for the basic amino acids and the effects of these mutations in vivo on virus assembly and infectivity and in vitro on the RNA-annealing activity of NCp10 were analyzed. Here we report that the substitution of 1 or 2 neutral amino acids for the basic residues did not impair the production of mature virions but that infectivity was either moderately or strongly attenuated. When more than 2 basic residues were replaced by neutral amino acids, viruses were poorly infectious because of a severe defect in genomic RNA dimer packaging and initiation of reverse transcription. In vitro NCp10-derived peptides with similar mutations were chemically synthesized and were found to be either fully or partially active or completely inactive. These data indicate that the basic residues flanking the zinc finger of NCp10 are required for the production of infectious Moloney murine leukemia virus virions.