Binding of CFA/I Pili of Enterotoxigenic Escherichia coli to Asialo-GM1 Is Mediated by the Minor Pilin CfaE.

CFA/I pili are representatives of a large family of related pili that mediate the adherence of enterotoxigenic Escherichia coli to intestinal epithelial cells. They are assembled via the alternate chaperone-usher pathway and consist of two subunits, CfaB, which makes up the pilus shaft and a single pilus tip-associated subunit, CfaE. The current model of pilus-mediated adherence proposes that CFA/I has two distinct binding activities; the CfaE subunit is responsible for binding to receptors of unknown structure on erythrocyte and intestinal epithelial cell surfaces, while CfaB binds to various glycosphingolipids, including asialo-GM1. In this report, we present two independent lines of evidence that, contrary to the existing model, CfaB does not bind to asialo-GM1 independently of CfaE. Neither purified CfaB subunits nor CfaB assembled into pili bind to asialo-GM1. Instead, we demonstrate that binding activity toward asialo-GM1 resides in CfaE and this is essential for pilus binding to Caco-2 intestinal epithelial cells. We conclude that the binding activities of CFA/I pili for asialo-GM1, erythrocytes, and intestinal cells are inseparable, require the same amino acid residues in CfaE, and therefore depend on the same or very similar binding mechanisms.

Colonization factors of enterotoxigenic Escherichia coli.

Enterotoxigenic Escherichia coli (ETEC) is a major cause of life-threatening diarrheal disease around the world. The major aspects of ETEC virulence are colonization of the small intestine and the secretion of enterotoxins which elicit diarrhea. Intestinal colonization is mediated, in part, by adhesins displayed on the bacterial cell surface. As colonization of the intestine is the critical first step in the establishment of an infection, it represents a potential point of intervention for the prevention of infections. Therefore, colonization factors (CFs) have been important subjects of research in the field of ETEC virulence. Research in this field has revealed that ETEC possesses a large array of serologically distinct CFs that differ in composition, structure, and function. Most ETEC CFs are pili (fimbriae) or related fibrous structures, while other adhesins are simple outer membrane proteins lacking any macromolecular structure. This chapter reviews the genetics, structure, function, and regulation of ETEC CFs and how such studies have contributed to our understanding of ETEC virulence and opened up potential opportunities for the development of preventive and therapeutic interventions.

Genome Sequence of Enterotoxigenic Escherichia coli Strain B2C.

Enterotoxigenic Escherichia coli (ETEC) is a major cause of diarrheal disease around the globe, causing an estimated 380,000 deaths annually. The disease is caused by a wide variety of strains. Here, we report the genome sequence of ETEC strain B2C, which was isolated from an American soldier in Vietnam.

N-terminal disordered domain of Saccharomyces cerevisiae Hop1 protein is dispensable for DNA binding, bridging, and synapsis of double-stranded DNA molecules but is necessary for spore formation.

The cytological architecture of the synaptonemal complex (SC), a meiosis-specific proteinaceous structure, is evolutionarily conserved among eukaryotes. However, little is known about the biochemical properties of SC components or the mechanisms underlying their roles in meiotic chromosome synapsis and recombination. Functional analysis of Saccharomyces cerevisiae Hop1, a key structural component of SC, has begun to reveal important insights into its function in interhomolog recombination. Previously, we showed that Hop1 is a structure-specific DNA-binding protein, exhibits higher binding affinity for the Holliday junction, and induces structural distortion at the core of the junction. Furthermore, Hop1 promotes DNA condensation and intra- and intermolecular synapsis between duplex DNA molecules. Here, we show that Hop1 possesses a modular domain organization, consisting of an intrinsically disordered N-terminal domain and a protease-resistant C-terminal domain (Hop1CTD). Furthermore, we found that Hop1CTD exhibits strong homotypic as well as heterotypic protein-protein interactions, and its biochemical activities were similar to those of the full-length Hop1 protein. However, Hop1CTD failed to complement the meiotic recombination defects of the Δhop1 strain, indicating that both N- and C-terminal domains of Hop1 are essential for meiosis and spore formation. Altogether, our findings reveal novel insights into the structure-function relationships of Hop1 and help to further our understanding of its role in meiotic chromosome synapsis and recombination.

Cloning, overexpression and purification of functionally active Saccharomyces cerevisiae Hop1 protein from Escherichia coli.

One of the major limitations to the application of high-resolution biophysical techniques such as X-crystallography and spectroscopic analyses to structure-function studies of Saccharomyces cerevisiae Hop1 protein has been the non-availability of sufficient quantities of functionally active pure protein. This has, indeed, been the case of many proteins, including yeast synaptonemal complex proteins. In this study, we have performed expression screening in Escherichia coli host strains, capable of high-level expression of soluble S. cerevisiae Hop1 protein. A new protocol has been developed for expression and purification of S. cerevisiae Hop1 protein, based on the presence of hexa-histidine tag and double-stranded DNA-Cellulose chromatography. Recombinant S. cerevisiae Hop1 protein was >98% pure and exhibited DNA-binding activity with high-affinity to the Holliday junction. The availability of the recombinant HOP1 expression vector and active Hop1 protein would facilitate structure-function investigations as well as the generation of appropriate truncated and site-directed mutant proteins, respectively.

Factors influencing the expectations of parents for their mentally retarded children.

In terms of treatment for cure, education, training and general information, parental expectations for their mentally retarded children are influenced by various factors such as the age and sex of the retarded child, the level of mental retardation, the education and occupation of the parents, and the socio-economic status and area of living. In the present study, an attempt has been made to find out the correlation of six variables namely, age, sex, literacy, locality of living, level of retardation and duration of follow-up on the expectation of one hundred parents of mentally retarded children who attend the home-based services at the National Institute for the Mentally Handicapped, Secunderabad, India. The intercorrelation among the six independent variables and the dependent variable revealed that age of the child and duration of follow-up have a high correlation (P < 0.01), and literacy of the parents and duration of follow-up were positively correlated (P < 0.05 level). It was also found that age and duration of follow-up have a positive correlation. Further detailed multiple regression analysis was conducted and the results are discussed.

Epilepsy and mental retardation.

Epilepsy is one of the most frequently associated conditions with mental retardation which interferes with the learning process. Vie present study investigates the 1207 cases (Male -8I4, Female-393) registered at NIMH, Secunderabad, over a period of two years. Vie factors studied were the prevalence of epilepsy, degree of mental retardation, aetiology and associated factors. Ten mentally retarded persons with epilepsy were followed up longitudinally to study the effect of epilepsy on learning. It was observed that an attack of seizure resulted in a setback in the learning of skills. The results are discussed.

Consanguinity and mental retardation.

Consanguinity among parents as a cause of mental retardation in their children is debatable. The present study was conducted to find out the effect of consanguinity on mental retardation where the causative factor is not established. A total of 517 mentally retarded persons and their families were studied out of which 160 were born of consanguineous marriage and 357 were of non-consanguineous marriage. The results indicated that, when there is a history of mental retardation in the family and if the parents are consanguineously married, the risk of mental retardation in the offspring is significantly high (chi 2 = 11.52; P less than 0.001). Among the consanguineously married families, the blood relationship of uncle-niece seems to have the highest risk of affecting the offsprings. The implications are discussed in detail.

Protein patterns, serotyping and plasmid DNA profiles in the epidemiologic fingerprinting of Pseudomonas aeruginosa.

The epidemiological typing schemes based on serotyping, antibiotic susceptibility pattern, plasmid DNA profile, and protein patterns determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) were evaluated for their usefulness in typing clinical isolates of Pseudomonas aeruginosa. The serotypability was lower (45%) than reported in the literature (85-95%). The most commonly found serotypes were O:1 (19%), O:11 (25%), O:6 (35%). The electrophoretic analysis of plasmid DNAs showed plasmids (molecular weight = 1 to greater than 40 megadaltons). Two plasmids of Mr 2 and 38 megadaltons were found in various serotypes. The restriction enzyme analysis of plasmid DNA showed identical DNA fragment patterns among distinct serotypes. The SDS-PAGE protein banding patterns of whole-cell proteins showed homogeneity among the strains. However, analysis of the soluble protein patterns of the strains showed sufficiently distinct protein profiles that can be used to differentiate between various strains. The results of this study demonstrate that the electrophoretic patterns of soluble proteins, in combination with plasmid DNA profile or serotyping, can be of value in the epidemiologic fingerprinting of clinical isolates of Pseudomonas aeruginosa.

Plasmid profiles and klebocin types in epidemiologic studies of infections by Klebsiella pneumoniae.

The epidemiological methods of klebocin typing, antibiogram and plasmid DNA profile were evaluated using organisms isolated from a suspected epidemic of gentamicin-resistant Klebsiella pneumoniae and unrelated strains from different geographical areas as controls. The electrophoretic analysis of plasmid DNAs from Klebsiella pneumoniae showed the presence of at least one and up to as many as seven plasmids in each strain. The molecular weight of plasmid DNAs ranged from 1 to greater than 70 mega daltons. While none of the control Klebsiella pneumoniae strains showed identical plasmid profiles, 63% of the epidemic-related Klebsiella pneumoniae strains did. Klebocin typing and plasmid DNA profile gave different results for the same strains. Plasmid DNA profile was found to be a more valuable method than klebocin typing alone or klebocin typing in combination with antibiogram for differentiating epidemiologically related from unrelated isolates. Both plasmid DNA profile and klebocin typing methods were superior to antibiogram.

Mesenteric chylous cyst associated with Crohn's disease.

Mesenteric cysts are rare, and mesenteric chylous cysts are extremely rare. A case report is presented of a patient with Crohn's disease and a mesenteric chylous cyst. Included in this case report is a full discussion of the preoperative radiological work-up (including barium enema, sonogram, and CT scan), the operative findings, and the pathological findings. It is suggested that there may be a causal relationship between the Crohn's disease and the mesenteric chylous cyst.

Characterization of self-transmissible plasmids determining lactose fermentation and multiple antibiotic resistance in clinical strains of Klebsiella pneumoniae.

The lactose fermentation (Lac+) and antibiotic resistance (R+) phenotypes were conjugally transferred from Klebsiella pneumoniae strains (K166, K182, K186, K218, and K220) to Salmonella typhi, S. typhimurium, Shigella flexneri, and Vibrio cholerae. The genes for lactose fermentation and antibiotic resistance were located on the plasmids. Further analysis of plasmid DNA from these isolates indicated the presence of multiple plasmids (Mr ranged less than 2.7 to 70 X 10(6)). The Lac+R+ plasmids p166 and p182 were members of the FII incompatibility group. The fertility inhibition property of plasmids, p182, p218, and p220 was fi+ type. Furthermore, phage typing experiments showed that plasmids p166 and p218 (Lac+R+) conferred the ability to inhibit the multiplication of bacteriophages 12 and 13 in S. typhimurium. However, the plasmids p182, p186, and p220 (Lac+R+) could inhibit the visible lysis of all the 30 phages in S. typhimurium. This study describes the characterization of Lac+R+ plasmids and the medical significance of an intergeneric transfer of lactose fermentation to non-lactose-fermenting pathogens.