Lactobacillus rhamnosus LRB mediated inhibition of oral streptococci.

Lactobacillus rhamnosus is a lactic acid bacterium with a diverse ecological habitat. We recently isolated a L. rhamnosus strain (LRB) from a healthy baby-tooth that had naturally fallen out. We determined the whole genome sequence of LRB and found that the isolate is closely genetically related to an intestinal isolate, L. rhamnosus GG (ATCC 53103). However, the LRB genome had lost about a 75-kb segment and undergone a genomic rearrangement. We assessed LRB's capacity to survive in the gut environment, at least temporarily. We found that LRB, like the intestinal isolate ATCC 53103, showed resistance to low pH but sensitive to bile salt. Surprisingly, we found that this oral isolate LRB showed strong antimicrobial activity against a variety of oral streptococci including Streptococcus mutans. The production of antimicrobial activity is dependent on media composition since some media supported the production while others did not. The production of antimicrobial activity is also dependent on growth temperature, with optimal production at 37°C. The antimicrobial activity was not restricted to streptococci, but effective against a variety of organisms, including ESKAPE pathogens.

Surgical outcome of laparoscopic and open surgery of pediatric inguinal hernia.

Inguinal hernia repair is one of the most frequently performed surgical procedures in infants and young children. This prospective comparative study was conducted with initial experience in the department of pediatric surgery, Dhaka Shishu (children) hospital during the period of July 2007 to August 2008. We enrolled 62 children undergoing surgery for inguinal hernia, of which 30 underwent laparoscopic procedure (bilateral in 21, unilateral 9) and 32 open procedures (bilateral in 5, unilateral in 27). Mean±SD patient age was 5.92±2.11 months in laparoscopic group and 6.63±2.64 months in open group (p=0.264), 3 months to 5 years in both groups. Patients were studied under variables of operative time, duration of postoperative hospital stay & post operative complications. During laparoscopy a contralateral patent processus vaginalis of ≥2cm was noted and repaired peroperatively in 18 out of 27 children (66%), who were initially diagnosed as unilateral hernia. For unilateral repair mean±SD operative time was significantly longer in Group A (62.63±52.75) minutes compares to the Group B (29.37±9.40), p<0.001. On the contrary, for bilateral repair Mean±SD operative time was comparable between the two groups (64.65±49.70) minutes for laparoscopy & (35.65±11.53 minutes) for open herniotomy & P=0.01, that was not remarkably significant. The mean±SD post operative length of hospital stay (in hours) 36.00±32.7 hours in Group A compared to 29.97±11.82 hours in Group B which was not statically significant (p=0.342). The mean±SD follow up was 24.5±10.5 months in laparoscopic group (Group A) & 22.5±10.5 months in open group (Group B), p=0.251. Regarding post operative complication, in this study, contra lateral metachronous inguinal hernia (CMIH) manifested in none of the patient out of 27 (total unilateral repaired number) patients in laparoscopic group but contrary to this in open group 2 patients out of 27 had developed CMIH & p value was <0.05, which is statistically significant. There were 2 cases of scrotal hydrocele out of 30, observed in Group A whereas 1 case out of 32 in Group B, p=0.49, which was statistically insignificant. The scrotal hydrocele was lasted only for 2 days & resolved spontaneously. About recurrence after operation, our study noted that, 1 case (3.3%) out of 30 in laparoscopic group and 2 cases (6%) out of 32 in open surgery group had developed recurrent inguinal hernia in about one year follow up where p value was 0.459, & it was statistically insignificant. In this study, none of the patient had developed post operative testicular atrophy (due to any vas or vascular injury) or testicular ascend. So, overall this study result implies that, Laparoscopic herniotomy might be a safe and effective option as open herniotomy for the treatment of inguinal hernia in children but which one would be superior or best option it requires a large series of randomized trial.

Island shapes and aggregation steered by the geometry of the substrate lattice.

We find that island shapes and aggregation in diindenoperylene deposited on Au(100), Au(110), and Au(111) single crystals are steered by the anisotropy due to the lattice geometry of the substrate. This phenomenon may be exploited as a tool for molecular patterning of surfaces.

[A method for differential identification of group C and G streptococci with PCR].

The paper describes a rapid method for PCR identification of Groups C and G streptococci (Streptococcus dysgalactiae subspecies equisimilis, Streptococcus constellatus, and Streptococcus anginosus) that cause human disease. Species-specific regions of the cpn60 gene encoding heat shock protein GroEL (HSP60) were chosen as markers for PCR diagnosis; three pairs of primers were constructed for these regions, each of which was peculiar to the specific type. The method was tested on a large collection of pathogenic streptococci of different serogroups isolated from man and animals; its specificity was shown to identify S. dysgalactiae subsp. equisimilis, S. constellatus, and S. anginosus. The proposed method has all benefits of PCR-based techniques, which enables it to be used for the purposes of molecular epidemiology.

Unusual energy shifts in resonant photoemission spectra of organic model molecules.

We study the electronic structure of zinc phthalocyanine (ZnPc) and 1,4-octa-decyl substituted zinc phthalocyanine [(Dec)(8)PcZn] thin films (approximately 6-15 nm) using resonant photoemission spectroscopy and X-ray absorption spectroscopy (XAS) at room temperature and at liquid He temperature. From XAS we conclude that the probability amplitude of the lowest unoccupied molecular orbital is located predominantly at the inner C and N atoms of the molecules. Nonlinear energy shifts in resonant photoemission were observed; large shifts are explained by reduced electrical conductivity of inhomogeneously oriented molecules.

From interfaces to surfaces: soft x-ray spectromicroscopy investigations of diindenoperylene thin films on gold.

We present the results of photoemission electron microscopy investigations on diindenoperylene (DIP) thin films deposited on polycrystalline gold, prepared in order to have a roughness much larger than the molecular size. Our investigations revealed the ability of the DIP molecule to form well-organized films, exhibiting a different molecular orientation with respect to the already known λ and σ phases. In locally thicker film regions, the energy of the films is minimized by a molecular arrangement that has an asymptotic tendency to the σ phase.

Tetra-t-butyl magnesium phthalocyanine on gold: electronic structure and molecular orientation.

In this work we have investigated the electronic structure and the molecular orientation of (t-Bu)(4)PcMg (tetra-t-butyl magnesium phthalocyanine) on polycrystalline and single crystalline gold substrates using photoemission spectroscopy and x-ray absorption spectroscopy, and we compare the results to the unsubstituted PcCu (copper phthalocyanine). The C 1s photoemission spectrum is described similar to unsubstituted relatives with an additional component for the aliphatic substituents. The variation of the excitation energy causes distinct differences in the shape of the C 1s spectrum, which is very useful for the analysis of the molecular orientation in the uppermost layer. It is shown that despite of the sterically demanding substituents, ordered sublimed films of (t-Bu)(4)PcMg are accessible, the orientation of the molecules, however, is different from the orientation of the unsubstituted relatives.

Generation and surface localization of intact M protein in Streptococcus pyogenes are dependent on sagA.

The M protein is an important surface-located virulence factor of Streptococcus pyogenes, the group A streptococcus (GAS). Expression of M protein is primarily controlled by Mga, a transcriptional activator protein. A recent report suggested that the sag locus, which includes nine genes necessary and sufficient for production of streptolysin S, another GAS virulence factor, is also needed for transcription of emm, encoding the M protein (Z. Li, D. D. Sledjeski, B. Kreikemeyer, A. Podbielski, and M. D. Boyle, J. Bacteriol. 181:6019-6027, 1999). To investigate this in more detail, we constructed an insertion-deletion mutation in sagA, the first gene in the sag locus, in the M6 strain JRS4. The resulting strain, JRS470, produced no detectable streptolysin S and showed a drastic reduction in cell surface-associated M protein, as measured by cell aggregation and Western blot analysis. However, transcription of the emm gene was unaffected by the sagA mutation. Detailed analysis with monoclonal antibodies and an antipeptide antibody showed that the M protein in the sagA mutant strain was truncated so that it lacks the C-repeat region and the C-terminal domain required for anchoring it to the cell surface. This truncated M protein was largely found, as expected, in the culture supernatant. Lack of surface-located M protein made the sagA mutant strain susceptible to phagocytosis. Thus, although sagA does not affect transcription of the M6 protein gene, it is needed for the surface localization of this important virulence factor.

In vivo evidence for two active nuclease motifs in the double-strand break repair enzyme RexAB of Lactococcus lactis.

In bacteria, double-strand DNA break (DSB) repair involves an exonuclease/helicase (exo/hel) and a short regulatory DNA sequence (Chi) that attenuates exonuclease activity and stimulates DNA repair. Despite their key role in cell survival, these DSB repair components show surprisingly little conservation. The best-studied exo/hel, RecBCD of Escherichia coli, is composed of three subunits. In contrast, RexAB of Lactococcus lactis and exo/hel enzymes of other low-guanine-plus-cytosine branch gram-positive bacteria contain two subunits. We report that RexAB functions via a novel mechanism compared to that of the RecBCD model. Two potential nuclease motifs are present in RexAB compared with a single nuclease in RecBCD. Site-specific mutagenesis of the RexA nuclease motif abolished all nuclease activity. In contrast, the RexB nuclease motif mutants displayed strongly reduced nuclease activity but maintained Chi recognition and had a Chi-stimulated hyperrecombination phenotype. The distinct phenotypes resulting from RexA or RexB nuclease inactivation lead us to suggest that each of the identified active nuclease sites in RexAB is involved in the degradation of one DNA strand. In RecBCD, the single RecB nuclease degrades both DNA strands and is presumably positioned by RecD. The presence of two nucleases would suggest that this RecD function is dispensable in RexAB.

Distinctive features of homologous recombination in an 'old' microorganism, Lactococcus lactis.

Homologous recombination is needed to assure faithful inheritance of DNA material, especially under stress conditions. The same enzymes that repair broken chromosomes via recombination also generate biodiversity. Their activities may result in intrachromosomal rearrangements, assimilation of foreign DNA, or a combination of these events. It is generally supposed that homologous recombination systems are conserved, and function the same way everywhere as they do in Escherichia coli, the accepted paradigm. Studies in an 'older' microorganism, the gram-positive bacterium of the low GC branch Lactococcus lactis, confirm that many enzymes are conserved across species lines. However, the main components of the double strand break (DSB) repair system, an exonuclease/helicase (Exo/hel) and a short DNA modulator sequence Chi, differ markedly between bacteria, especially when compared to the gram-negative analogues. Based on our studies, a model is proposed for the functioning of the two-subunit Exo/hel of L. lactis and other gram-positive bacteria, which differs from that of the three-subunit E. coli enzyme. The differences between bacterial DSB repair systems may underlie a selection for diversity when dealing with DSB. These and other features of homologous recombination in L. lactis are discussed.

Disruption of the helix-u-turn-helix motif of MutS protein: loss of subunit dimerization, mismatch binding and ATP hydrolysis.

The DNA mismatch repair protein, MutS, is a dimeric protein that recognizes mismatched bases and has an intrinsic ATPase activity. Here, a series of Taq MutS proteins having C-terminal truncations in the vicinity of a highly conserved helix-u-turn-helix (HuH) motif are assessed for subunit oligomerization, ATPase activity and DNA mismatch binding. Those proteins containing an intact HuH region are dimers; those without the HuH region are predominantly monomers in solution. Steady-state kinetics of truncated but dimeric MutS proteins reveals only modest decreases in their ATPase activity compared to full-length protein. In contrast, disruption of the HuH region results in a greatly attenuated ATPase activity. In addition, only dimeric MutS proteins are proficient for mismatch binding. Finally, an analysis of the mismatch repair competency of truncated Escherichia coli MutS proteins in a rifampicin mutator assay confirms that the HuH region is critical for in vivo function. These findings indicate that dimerization is critical for both the ATPase and DNA mismatch binding activities of MutS, and corroborate several key features of the MutS structure recently deduced from X-ray crystallographic studies.

Requirement for Phe36 for DNA binding and mismatch repair by Escherichia coli MutS protein.

The MutS family of DNA repair proteins recognizes base pair mismatches and insertion/deletion mismatches and targets them for repair in a strand-specific manner. Photocrosslinking and mutational studies previously identified a highly conserved Phe residue at the N-terminus of Thermus aquaticus MutS protein that is critical for mismatch recognition in vitro. Here, a mutant Escherichia coli MutS protein harboring a substitution of Ala for the corresponding Phe36 residue is assessed for proficiency in mismatch repair in vivo and DNA binding and ATP hydrolysis in vitro. The F36A protein is unable to restore mismatch repair proficiency to a mutS strain as judged by mutation to rifampicin or reversion of a specific point mutation in lacZ. The F36A protein is also severely deficient for binding to heteroduplexes containing an unpaired thymidine or a G:T mismatch although its intrinsic ATPase activity and subunit oligomerization are very similar to that of the wild-type MutS protein. Thus, the F36A mutation appears to confer a defect specific for recognition of insertion/deletion and base pair mismatches.

Heteroduplex DNA and ATP induced conformational changes of a MutS mismatch repair protein from Thermus aquaticus.

ATP hydrolysis by MutS homologues is required for the function of these proteins in mismatch repair. However, the function of ATP hydrolysis in the repair reaction is not very clear. We have examined the role of ATP hydrolysis in oligomerization of Thermus aquaticus (Taq) MutS protein in solution. Analytical gel filtration and cross-linking of MutS protein with disuccinimidyl suburate suggest that TaqMutS is a dimer in the presence of ATP. ATP binding and hydrolysis by TaqMutS reduces the heteroduplex-DNA binding by the protein. Using limited proteolysis we detected extensive conformational changes of the TaqMutS protein in the presence of ATP and heteroduplex DNA. Heteroduplex-DNA binding is necessary for the observed conformational changes since F39A mutant protein defective in DNA binding does not display ATP-induced conformational changes. The implications of the observed conformational changes in the MutS protein are discussed with respect to two different models proposed for the role of ATP hydrolysis by MutS in DNA mismatch repair.

Oligomerization of a MutS mismatch repair protein from Thermus aquaticus.

The MutS DNA mismatch protein recognizes heteroduplex DNAs containing mispaired or unpaired bases. We have examined the oligomerization of a MutS protein from Thermus aquaticus that binds to heteroduplex DNAs at elevated temperatures. Analytical gel filtration, cross-linking of MutS protein with disuccinimidyl suberate, light scattering, and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry establish that the Taq protein is largely a dimer in free solution. Analytical equilibrium sedimentation showed that the oligomerization of Taq MutS involves a dimer-tetramer equilibrium in which dimer predominates at concentrations below 10 microM. The DeltaG(0)(2-4) for the dimer to tetramer transition is approximately -6.9 +/- 0.1 kcal/mol of tetramer. Analytical gel filtration of native complexes and gel mobility shift assays of an maltose-binding protein-MutS fusion protein bound to a short, 37-base pair heteroduplex DNA reveal that the protein binds to DNA as a dimer with no change in oligomerization upon DNA binding.

Branch migration through DNA sequence heterology.

Branch migration of a DNA Holliday junction is a key step in genetic recombination. Previously, it was shown that a single base-pair heterology between two otherwise identical DNA sequences is a substantial barrier to passage of a Holliday junction during spontaneous branch migration. Here, we exploit this inhibitory effect of sequence heterology to estimate the step size of branch migration. We also devise a simulation of branch migration through mismatched base-pairs to arrive at the underlying molecular basis for the block to branch migration imposed by sequence heterology. Based on the observation that two adjacent sequence heterologies exert their effects on branch migration more or less independently, we conclude that the step size of branch migration is quite small, of the order of one or two base-pairs per migratory step. Comparison of branch migration experiments through a single base-pair heterology with simulations of a random walk through sequence heterology suggests that the inhibition of branch migration is largely attributable to a thermodynamic barrier arising from the formation of unpaired or mispaired bases in heteroduplex DNAs.

Photocross-linking of the NH2-terminal region of Taq MutS protein to the major groove of a heteroduplex DNA.

The MutS DNA mismatch repair protein recognizes heteroduplex DNAs containing mispaired or unpaired bases. To identify regions of MutS protein in close proximity to the heteroduplex DNA, we have utilized the photoactivated cross-linking moiety 5-iododeoxyuridine (5-IdUrd). Nucleoprotein complexes of Thermus aquaticus MutS protein bound to monosubstituted 5-IdUrd-containing heteroduplex DNAs were cross-linked with long-wavelength ultraviolet light. Positioning of the 5-IdUrd moiety at one of three positions within the DNA bulge, two nucleotides upstream or three nucleotides downstream of the unpaired base, resulted in an identical subset of cross-linked peptides as determined by proteolytic fingerprinting. The tryptic peptide cross-linked to an unpaired 5-IdUrd residue was determined by peptide sequencing to correspond to a highly conserved region spanning residues 25-49. Cross-linking to the bulge nucleotide occurred at Phe-39, indicating that this residue contacts, or is in close proximity to, the unpaired base of a heteroduplex DNA. Site-directed mutagenesis resulting in the substitution of Ala for Phe-39 reduced the affinity of the mutant protein for heteroduplex DNA by roughly 3 orders of magnitude, but had no apparent effect on its ability to dimerize, its thermostability, or its ATPase activity. These results implicate the region in the vicinity of Phe-39 as being crucial for heteroduplex DNA binding by Taq MutS protein.

Interaction of MutS protein with the major and minor grooves of a heteroduplex DNA.

Thermus aquaticus MutS protein is a DNA mismatch repair protein that recognizes and binds to heteroduplex DNAs containing mispaired or unpaired bases. Using enzymatic and chemical probe methods, we have examined the binding of Taq MutS protein to a heteroduplex DNA having a single unpaired thymidine residue. DNase I footprinting identifies a symmetrical region of protection 24-28 nucleotides long centered on the unpaired base. Methylation protection and interference studies establish that Taq MutS protein makes contacts with the major groove of the heteroduplex in the immediate vicinity of the unpaired base. Hydroxyl radical and 1, 10-phenanthroline-copper footprinting experiments indicate that MutS also interacts with the minor groove near the unpaired base. Together with the identification of key phosphate groups detected by ethylation interference, these data reveal critical contact points residing in the major and minor grooves of the heteroduplex DNA.

Gastropleural fistula: an unusual cause of intractable postoperative nausea and vomiting.

Gastropleural fistula is an uncommon finding (1). Gastropleural fistulae have been reported after pulmonary resection (1), perforated paraesophageal hernia (2), perforated malignant gastric ulcer at the fundus, and gastric bypass operation for morbid obesity. We present a case of gastropleural fistula that resulted acutely from intractable postoperative nausea and vomiting after ambulatory knee arthroscopic surgery under general anesthesia.

Identification and characterization of a thermostable MutS homolog from Thermus aquaticus.

Recognition of mispaired or unpaired bases during DNA mismatch repair is carried out by the MutS protein family. Here, we describe the isolation and characterization of a thermostable MutS homolog from Thermus aquaticus YT-1. Sequencing of the mutS gene predicts an 89.3-kDa polypeptide sharing extensive amino acid sequence homology with MutS homologs from both prokaryotes and eukaryotes. Expression of the T. aquaticus mutS gene in Escherichia coli results in a dominant mutator phenotype. Initial biochemical characterization of the thermostable MutS protein, which was purified to apparent homogeneity, reveals two thermostable activities, an ATP hydrolysis activity in which ATP is hydrolyzed to ADP and Pi and a specific DNA mismatch binding activity with affinities for heteroduplex DNAs containing either an insertion/deletion of one base or a GT mismatch. The ATPase activity exhibits a temperature optimum of approximately 80 degrees C. Heteroduplex DNA binding by the T. aquaticus MutS protein requires Mg2+ and occurs over a broad temperature range from 0 degrees C to at least 70 degrees C. The thermostable MutS protein may be useful for further biochemical and structural studies of mismatch binding and for applications involving mutation detection.

Entry of Listeria monocytogenes into hepatocytes requires expression of inIB, a surface protein of the internalin multigene family.

The intracellular bacterium Listeria monocytogenes can invade several types of normally non-phagocytic cells. Entry into cultured epithelial cells requires the expression of inIA, the first gene of an operon, comprising two genes: inIA, which encodes internalin, an 800-amino-acid protein, and inIB, which encodes a 630-amino-acid protein. Several genes homologous to inIA are detected in the genome of L. monocytogenes; InIB is one of them. We have assessed the role of inIB in invasiveness of L. monocytogenes by constructing isogenic chromosomal deletion mutants in the inIAB locus. Our findings indicate that: i) inIB is required for entry of L. monocytogenes into hepatocytes, but not into intestinal epithelial cells; ii) inIB encodes a surface protein; iii) internalin plays a role for entry into some hepatocyte cell lines. These results provide the first insight into the cell tropism displayed by L. monocytogenes.