Expression of Acsm2, a kidney-specific gene, parallels the function and maturation of proximal tubular cells.

The acyl-CoA synthetase medium-chain family member 2 (Acsm2) gene was first identified and cloned by our group as a kidney-specific "KS" gene. However, its expression pattern and function remain to be clarified. In the present study, we found that the Acsm2 gene was expressed specifically and at a high level in normal adult kidneys. Expression of Acsm2 in kidneys followed a maturational pattern: it was low in newborn mice and increased with kidney development and maturation. In situ hybridization and immunohistochemistry revealed that Acsm2 was expressed specifically in proximal tubular cells of adult kidneys. Data from the Encyclopedia of DNA Elements database revealed that the Acsm2 gene locus in the mouse has specific histone modifications related to the active transcription of the gene exclusively in kidney cells. Following acute kidney injury, partial unilateral ureteral obstruction, and chronic kidney diseases, expression of Acsm2 in the proximal tubules was significantly decreased. In human samples, the expression pattern of ACSM2A, a homolog of mouse Acsm2, was similar to that in mice, and its expression decreased with several types of renal injuries. These results indicate that the expression of Acsm2 parallels the structural and functional maturation of proximal tubular cells. Downregulation of its expression in several models of kidney disease suggests that Acms2 may serve as a novel marker of proximal tubular injury and/or dysfunction.

Characterization of a negative regulator of exopolysaccharide production by the plant-pathogenic bacterium Pseudomonas solanacearum.

Wild-type strains of the bacterial wilt pathogen Pseudomonas solanacearum exhibit reduced exopolysaccharide production and virulence when transformed with plasmids carrying the epsR locus. To understand the function of epsR, we used mutagenesis and DNA sequencing to identify the gene responsible for the shutoff of exopolysaccharide production. The epsR gene encodes a 236-amino-acid polypeptide that, based on polypeptide sequence homology, has significant similarity to other proteins of the luxR family of environmentally responsive, two-component regulatory systems. When a mutated copy of the epsR gene was marker-exchanged into the wild-type P. solanacearum chromosome, however, we observed no effect on growth in culture or on exopolysaccharide production. This suggests that the EpsR phenotype becomes apparent only via overproduction of the EpsR protein. By means of an antiserum directed against the EpsR protein, we detected the overproduction of EpsR in cell lysates of a strain of P. solanacearum harboring a multicopy plasmid with an active epsR gene but not in one harboring the same plasmid with a mutated epsR gene.

Bone strain and microcracks at stress fracture sites in human metatarsals.

Microcracks in bone have been implicated in the development of stress fractures. The goal of this study was to evaluate bone strain and microcracks at locations where stress fractures are common (second metatarsal diaphysis) and rare (fifth metatarsal diaphysis) in an attempt to increase our understanding of the pathogenesis of stress fractures. A dynamic gait simulator was used to simulate normal walking with cadaver feet. The feet were loaded over the entire stance phase of gait and diaphyseal strains were recorded in second and fifth metatarsals. Microcrack density (Cr.Dn) and surface density (Cr.S.Dn) were determined in metatarsal cross sections from the contralateral feet. Bone strain was significantly higher in second metatarsals (-1897 +/- 613 microstrain) than in fifth metatarsals (-908 +/- 503 microstrain). However, second metatarsal Cr.Dn (0.23 +/- 0.15 #/mm(2)) was not significantly different from fifth metatarsal Cr.Dn (0.35 +/- 0.19 #/mm(2)). There was also no significant difference between Cr.S.Dn in second (17.64 +/- 10.99 microm/mm(2)) and fifth (26.70 +/- 15.53 microm/mm(2)) metatarsals. There were no significant relationships between the microcrack parameters and peak strain in either metatarsal. Cracks that occurred in trabecular struts (92 +/- 33 microm) were significantly longer than those found ending at cement lines (71 +/- 15 microm) and within osteons (57 +/- 16 microm). There were no significant relationships between the microcrack parameters and age in either metatarsal. Peak strain was more than twofold greater in second metatarsals than in fifth metatarsals for simulations of normal walking; however, microcrack parameters were unable to explain the greater incidence of second metatarsal stress fractures.

[Nutritional status and land tenure. A study in adults of the rural area of the northeastern Brazil]. / Estado nutricional e posse da terra. Um estudo em adultos da área rural do nordeste brasileiro.

The present study was carried out in the rural areas of four municipalities in the North-East of Brazil as part of a broader survey which covered a sample of families living in the area. A subsample of adults (453 women and 126 men) was obtained from individuals who attended the survey's post for clinical and anthropometric evaluation. According to land tenure, they were stratified into four categories: those with-without land (W.L.); small land owner (S.O.); medium land owners (M.O.); and large land owners (L.O.). Means for anthropometric variables (height, weight, middle arm circumference and skinfold thickness) were calculated for each stratum. The differences between groups were statistically significant (p less than 0.05). Findings revealed that the larger the property, the greater the mean value for height; extreme values attained a difference of 7 and 6 cm in men and women, respectively. Mean weight increased as land ownership grew reaching a difference of 9 and 11 kg in men and women, respectively (p less than 0.01). Skinfold thickness and middle arm circumference showed significant differences between strata. To evaluate their present nutritional status, the adequacy of weight to height was obtained. In spite of the low proportion of individuals who exhibited less than 90%, adequate improvement was detected as land tenure increased. The study discusses the precariousness of criteria and patterns for the nutritional evaluation of adults, and suggests the existence of a relationship between nutritional status and land ownership.

[Anemias in preschool children: diagnosis, treatment and evaluation, Recife-PE, Brazil]. / Anemias em pre-escolares: diagnostico, tratamento e avaliação Recife-PE, Brasil.

The diagnosis and the effects of treatment of anemia were assessed in children aged 6-71 months. A total of 1,161 preschool children from a health center of INAMPS (Instituto Nacional de Assistência Médica e Previdência Social) in Recife, Pernambuco, was studied. Hemoglobin was determined by the method described by Hainline. WHO criteria were used to identify anemia. According to their age, nutritional status and family income, the children were divided into groups, and those with anemia were treated with ferrous sulphate and an anti-helminthic (mebendazole). Anemia prevalence was substantially higher in children aged 2 years, and a statistically significant association (0.01 level) was found between anemia and nutritional status and family income. After treatment, hemoglobin values were normal in 40% of the anemic children; simultaneously, mean hemoglobin values increased from 9.11 to 10.3 g/dl which was statistically significant (p less than 0.001). This investigation is part of a collaborative study performed in four Brazilian states to offer "know-how" to a national program for combating iron deficiency anemia.

Surface components involved in bacterial pathogen-plant host recognition.

During their initial association with plant hosts, pathogenic bacteria interact with plant cell walls. The results of this interaction appear to determine whether bacterial multiplication will take place. With one group of bacterial plant pathogens (e.g. Agrobacterium tumefaciens), attachment to the host surface appears essential for pathogenesis. With another group (e.g. Pseudomonas solanacearum), only those strains that do not attach to the host cell wall are able to multiply in the intercellular spaces. Attachment of many incompatible strains to tobacco mesophyll cell walls leads to a rapid hypersensitive response (HR) and a drastic reduction in bacterial multiplication. Our working hypothesis is that these differences in host response to strains of P. solanacearum are the result of a recognition response in which surface components of both host and pathogen play important roles. Our approach is based on the use of spontaneous or transposon (Tn5)-generated mutants of strains K60 (virulent) and B1 (avirulent) that differ in surface components and in their ability to attach to host cells and to induce the HR. A study of the surface components of bacterial and tobacco cell walls has led to the tentative conclusion that bacterial lipopolysaccharide (LPS) and plant hydroxyproline-rich glycoproteins mediate initial attachment, apparently as a result of charge-charge interaction. This initial attachment is reversed by high salt concentrations during the first 15 min, but not thereafter. Firm attachment appears to depend on hydrophobic interactions mediated by bacterial pili. At the normal ionic strength of intercellular fluids, extracellular polysaccharide (EPS) appears to inhibit only the pili-mediated attachment. Several HR- mutants of strain B1 have been obtained by Tn5 insertion, but they remain avirulent on tobacco. We are examining the EPS, LPS and pili production, and the attachment characteristics of these strains.

The use of subtractive hybridization to obtain a DNA probe specific for Pseudomonas solanacearum race 3.

Pseudomonas solanacearum, the causal agent of bacterial wilt, has been classified into three races based on host range and into five biovars based on physiological properties. Strains of race 3 belong exclusively to biovar 2 and primarily affect potatoes. Although this race is thought to have originated in the Andean highlands, it has unusual physiological properties that make it a potential threat to potatoes grown at the cooler latitudes worldwide. Consequently, there is need for a rapid and sensitive method for detection of race 3 strains. We have used subtractive hybridization to enrich for race 3-specific DNA sequences in total race 3 genomic DNA, and thereby obtained a 2 kb clone homologous to DNA from all 28 race 3 strains tested, but with only five of 90 non-race 3 strains. In addition, two larger regions of the genome, containing a minimum of 23 kb of DNA, are also specific for race 3. Deletion of this DNA did not affect virulence. This race 3-specific DNA is a potentially useful diagnostic tool for the detection of race 3 strains.

Genetic and biochemical characterization of a Pseudomonas solanacearum gene cluster required for extracellular polysaccharide production and for virulence.

Infection of host plants by Pseudomonas solanacerum results in wilting, which is thought to be due largely to the occlusion of xylem vessels by the P. solanacearum extracellular polysaccharide (EPS) that primarily consists of N-acetylgalactosamine (GalNAc). By means of Tn3 mutagenesis, we identified a 6.5-kb gene cluster that contains five complementation units required for EPS production and virulence in this bacterium. There was positive correlation between the amount of EPS produced in culture and (i) in planta growth and (ii) virulence. Based on analysis of beta-glucuronidase-gene fusions, these genes are expressed both in broth cultures and in planta and may be constitutive. Both wild-type and mutant strains contained similar amounts of UDP-GalNAc, the predicted primary substrate for EPS synthesis. Thus, the EPS mutants we obtained should be useful in the analysis of steps in the assembly of the polysaccharide and how this process is related to virulence.

Partial purification and kinetics of indoleacetic Acid oxidase from tobacco roots.

Extracts from roots of Nicotiana tabacum L var. Bottom Special contain oxidative enzymes capable of rapid degradation of indoleacetic acid (IAA) in the presence of Mn(2+) and 2, 4-dichlorophenol. Purification of IAA oxidase was attempted by means of ammonium sulfate fractionation and elution through a column of SE-Sephadex. Two distinct fractions, both causing rapid oxidation of IAA in the absence of H(2)O(2), were obtained. One fraction exhibited high peroxidase activity when guaiacol was used as the electron donor; the other did not oxidase guaiacol. Both enzyme fractions caused similar changes in the UV spectrum of IAA; absorption at 280 mmu was reduced, while major absorption peaks appeared at 254 and 247 mmu. The kinetics of IAA oxidation by both fractions were followed by measuring the increase in absorption at 247 mmu. The peroxidase-containing fraction showed no lag or a slight lag which could be eliminated by addition of H(2)O(2) (3 mumoles/ml). The peroxidase-free fraction showed a longer lag, but addition of similar amounts of H(2)O(2) inhibited the rate of IAA oxidation and did not remove the lag. With purified preparations, IAA oxidation was stimulated only at low concentrations of H(2)O(2) (0.03 mumole/ml). A comparison of K(m) values for IAA oxidation by the peroxidase-containing and peroxidase-free fractions suggests that tobacco roots contain an IAA oxidase which may have higher affinity for IAA and may be more specific than the general peroxidase system previously described from other plant sources. A similar oxidase is present in commercial preparations of horseradish peroxidase. It is suggested that oxidation of IAA by horseradish peroxidase may be due to a more specific component.

Identification of a locus that regulates multiple functions in Pseudomonas solanacearum.

When Pseudomonas solanacearum K60 carries a multicopy plasmid containing cosmid clone pE6C (from the wild-type strain K60) or pBE6 (from the nonpathogenic strain B1), several phenotypic changes are observed, including the following: loss of virulence, reduced extracellular polysaccharide production, and increased polygalacturonase activity. Both cosmids contain a common 8-kilobase DNA region that is required for the phenotype shift. Saturation mutagenesis of pBE6 with Tn3-gus suggested that a single transcriptional unit of at least 1 kilobase is responsible for the phenotype shift. In maxicell assays, subclones containing this transcriptional unit expressed a single protein of about 25 kilodaltons.

Technique for the Determination of the Rate of Ethylene Production by Pseudomonas solanacearum.

A tube culture system was designed for measurement of ethylene evolved by the phytopathogenic bacterium, Pseudomonas solanacearum. The system consisted of 10 glass tubes joined together in series and coated on the inside surface with a dextrose-peptone-casamino acids agar medium. The system provided a large surface for bacterial growth in relation to the volume of air. The system was seeded with a bacterial suspension (7 x 10(8) cells/ml) drawn through all the tubes by vacuum applied at one end and was then placed in a water bath at 30 C. Air was pumped through the system at 3 ml/min; the outlet was connected directly to the inlet port of a gas sampling loop and ethylene in the sample was determined by gas chromatography.Maximum rate of ethylene production for a fluidal, virulent isolate of P. solanacearum (K60) was 5.5 x 10(-9) moles/min and occurred at the end of lag phase and beginning of stationary phase. Three other fluidal isolates produced ethylene at relatively low rates (2.4-6.4% that of K60). Avirulent, butyrous variants of these isolates grew as well as the virulent forms in most cases, but ethylene production rates per cell were much lower for the avirulent than for the virulent forms. Loss of virulence appears to be accompanied by lower ethylene production.Peak CO(2) production (14.5 mumoles/min) and O(2) consumption (11.7 mumoles/min) for isolate K60 also occurred at the time when the bacterial culture was entering stationary phase. The concentrations of O(2) (11%) and CO(2) (11%) in air present at this time were thought to be neither limiting nor inhibitory to bacterial growth.

Evidence for the cotransfer of genetic markers in Pseudomonas solanacearum strain K60.

A procedure in which transfer of genetic information between genetically distinct derivatives of strain K60 of Pseudomonas solanacearum occurred is described. Genetical analysis of recombinants demonstrated the transfer of an unselected marker, and the linkage of genes determining resistance to streptomycin and rifampicin.

A New Bacterial Agglutinin from Soybean: II. EVIDENCE AGAINST A ROLE IN DETERMINING PATHOGEN SPECIFICITY.

The activity of a bacterial agglutinin from soybean seed [Glycine max (L.) Merrill cv. Clark] against two bacterial pathogens, Pseudomonas glycinea (causal agent of bacterial blight) and Xanthomonas phaseoli var. sojensis (causal agent of bacterial pustule) was determined. The agglutinin was active against several strains of X. phaseoli var. sojensis grown on nutrient agar, but there was no correlation between pathogenicity and agglutination. Agglutination was affected by the age of the bacterial cells and the growth medium used. None of seven strains of P. glycinea was agglutinated.Bacterial agglutination was inhibited by both purified lipopolysaccharide and extracellular polysaccharide from five strains of X. phaseoli var. sojensis. The lipopolysaccharides and extracellular polysaccharides from other species of bacteria were ineffective.Ultrastructural studies showed that an avirulent strain of X. phaseoli var. sojensis was attached to leaf mesophyll cell walls of the susceptible cultivar Clark by 34 hours after vacuum infiltration. Cells of this avirulent strain were enveloped by fibrillar and granular material at the mesophyll cell wall. In contrast, cells of a virulent strain were not attached or enveloped, and they remained free to multiply in the intercellular spaces.

A New Bacterial Agglutinin from Soybean: I. ISOLATION, PARTIAL PURIFICATION, AND CHARACTERIZATION.

A new bacterial agglutinin was isolated from seeds of the soybean cultivar Clark. Purification was carried out by ammonium sulfate precipitation and ion-exchange chromatography. The agglutinin is a heat-labile glycoprotein most active at pH 4.0. Addition of Ca(2+), Mn(2+) and Mg(2+) did not enhance the agglutinating activity of this glycoprotein. Gel electrophoresis in the presence of sodium dodecyl sulfate showed that the agglutinin is composed of two subunits of approximately 50,000 daltons each. In the undissociated state, it agglutinates Xanthomonas phaseoli var. sojensis, the causal agent of bacterial pustule disease of soybean, at concentrations as low as 10 micrograms protein per milliliter but has no hemagglutinating activity. The agglutinin could be distinguished from previously reported soybean lectins on the basis of solubility in ammonium sulfate, lack of hemagglutinating activity, molecular weight, hapten specificity, and immunological determinants.