Genetics of high level penicillin resistance in clinical isolates of Streptococcus pneumoniae

Abstract Mosaic penicillin-binding proteins (PBP) 1A, 2X and 2B genes were cloned from four clinical isolates of Streptococcus pneumoniae with levels of susceptibility to penicillin ranging from 1.5 to 16 μg benzylpenicillin ml⁻¹. In each instance it was possible to transform either the penicillin-sensitive laboratory strain R6 or a sensitive clinical isolate 110K/70 to the full level of penicillin resistance with these three penicillin-binding proteins alone. Until now it has not been possible to clearly determine whether alterations to PBP1A, 2X and 2B alone were sufficient to attain high level penicillin resistance.     

Detection by electron spin resonance of an exchange-coupled Cob(II)alamin...free radical pair species generated by anaerobic photolysis of polycrystalline adenosylcobalamin

Exposure to nitrous oxide (N₂O) $\text{in vivo}$ is accompanied by oxidation of cob[I]alamin to the inactive cob[III]alamin [1] and to loss of methionine synthetase activity [2]. There is a steady increase in thymidylate synthetase activity in marrow collected from rats exposed to N₂O and this returns to normal on restoring the animals to an air environment.     

Heterotopic Renal Autotransplantation in a Porcine Model: A Step-by-Step Protocol

Kidney transplantation is the treatment of choice for patients suffering from end-stage renal disease. It offers better life expectancy and higher quality of life when compared to dialysis. Although the last few decades have seen major improvements in patient outcomes following kidney transplantation, the increasing shortage of available organs represents a severe problem worldwide. To expand the donor pool, marginal kidney grafts recovered from extended criteria donors (ECD) or donated after circulatory death (DCD) are now accepted for transplantation. To further improve the postoperative outcome of these marginal grafts, research must focus on new therapeutic approaches such as alternative preservation techniques, immunomodulation, gene transfer, and stem cell administration.Experimental studies in animal models are the final step before newly developed techniques can be translated into clinical practice. Porcine kidney transplantation is an excellent model of human transplantation and allows investigation of novel approaches. The major advantage of the porcine model is its anatomical and physiological similarity to the human body, which facilitates the rapid translation of new findings to clinical trials. This article offers a surgical step-by-step protocol for an autotransplantation model and highlights key factors to ensure experimental success. Adequate pre- and postoperative housing, attentive anesthesia, and consistent surgical techniques result in favorable postoperative outcomes. Resection of the contralateral native kidney provides the opportunity to assess post-transplant graft function. The placement of venous and urinary catheters and the use of metabolic cages allow further detailed evaluation. For long-term follow-up studies and investigation of alternative graft preservation techniques, autotransplantation models are superior to allotransplantation models, as they avoid the confounding bias posed by rejection and immunosuppressive medication.     

Protein kinase D: activation for Golgi carrier formation

Protein kinase D regulates fission at the trans-Golgi network (TGN) of transport carriers that deliver cargo to the plasma membrane. PKD is first recruited to the TGN through interaction with diacylglycerol and is subsequently activated by phosphorylation to promote carrier fission. In a recent study, the relevant upstream kinase at the TGN was identified as the novel protein kinase C isoform PKCeta, which in turn is activated in response to heterotrimeric G-protein activation. These findings indicate the existence of a kinase signaling cascade at the TGN that regulates carrier fission and suggest a mechanism by which cargo might direct the formation of its transport carriers.     

An unusual presentation of a malignant jejunal tumor and a different management strategy

BACKGROUND: Malignant small bowel tumors are very rare and leiomyosarcoma accounts for less than 15% of the cases. Management of these tumors is challenging in view of nonspecific symptoms, unusual presentation and high incidence of metastasis. In this case report, an unusual presentation of jejunal sarcoma and management of liver metastasis with radiofrequency ablation (RFA) is discussed. CASE PRESENTATION: A 45-year-old male presented with anemia and features of small bowel obstruction. Operative findings revealed a mass lesion in jejunum with intussusception of proximal loop. Resection of bowel mass was performed. Histopathological findings were suggestive of leiomyosarcoma. After 3-years of follow-up, the patient developed recurrence in infracolic omentum and a liver metastasis. The omental mass was resected and liver lesion was managed with radiofrequency ablation. CONCLUSION: Jejunal leiomyosarcoma is a rare variety of malignant small bowel tumor and a clinical presentation with intussusception is unusual. We suggest that an aggressive management approach using a combination of surgery and a newer technique like RFA can be attempted in patients with limited metastatic spread to liver to prolong the long-term survival in a subset of patients.     

Mechanism of gamma-secretase cleavage activation: is gamma-secretase regulated through autoinhibition involving the presenilin-1 exon 9 loop?

Maturation of gamma-secretase requires an endoproteolytic cleavage in presenilin-1 (PS1) within a peptide loop encoded by exon 9 of the corresponding gene. Deletion of the loop has been demonstrated to cause familial Alzheimer's disease. A synthetic peptide corresponding to the loop sequence was found to inhibit gamma-secretase in a cell-free enzymatic assay with an IC(50) of 2.1 microM, a value similar to the K(m) (3.5 microM) for the substrate C100. Truncation at either end, single amino acid substitutions at certain residues, sequence reversal, or randomization reduced its potency. Similar results were also observed in a cell-based assay using HEK293 cells expressing APP. In contrast to small-molecule gamma-secretase inhibitors, kinetic inhibition studies demonstrated competitive inhibition of gamma-secretase by the exon 9 peptide. Consistent with this finding, inhibitor cross-competition kinetics indicated noncompetitive binding between the exon 9 peptide and L685458, a transition-state analogue presumably binding at the catalytic site, and ligand competition binding experiments revealed no competition between L685458 and the exon 9 peptide. These data are consistent with the proposed gamma-secretase mechanism involving separate substrate-binding and catalytic sites and binding of the exon 9 peptide at the substrate-binding site, but not the catalytic site of gamma-secretase. NMR analyses demonstrated the presence of a loop structure with a beta-turn in the middle of the exon 9 peptide and a loose alpha-helical conformation for the rest of the peptide. Such a structure supports the hypothesis that this exon 9 peptide can adopt a distinct conformation, one that is compact enough to occupy the putative substrate-binding site without necessarily interfering with binding of small molecule inhibitors at other sites on gamma-secretase. We hypothesize that gamma-secretase cleavage activation may be a result of a cleavage-induced conformational change that relieves the inhibitory effect of the intact exon 9 loop occupying the substrate-binding site on the immature enzyme. It is possible that the DeltaE9 mutation causes Alzheimer's disease because cleavage activation of gamma-secretase is no longer necessary, alleviating constraints on Abeta formation.     

Species loss of stoneflies (Plecoptera) in the Czech Republic during the 20th century

1. Rapid expansion and intensification of anthropogenic activities in the 20th century has caused profound changes in freshwater assemblages. Unfortunately, knowledge of the extent and causes of species loss (SL) is limited due to the lack of reliable historical data. An unusual data set allows us to compare changes in the most sensitive of aquatic insect orders, the Plecoptera, at some 170 locations in the Czech Republic between two time periods, 1955–1960 and 2006–2010. Historical data (1890–1911) on assemblages of six lowland rivers allow us to infer even earlier changes. 2. Regional stonefly diversity decreased in the first half of the 20th century. Streams at lower altitudes lost a substantial number of species, which were never recovered. In the second half of the century, large‐scale anthropogenic pressure caused SL in all habitats, leading to a dissimilarity of contemporary and previous assemblages. The greatest changes were found at sites affected by organic pollution and a mixture of organic pollution and channelisation or impoundment. Colonisation of new habitats was observed in only three of the 80 species evaluated. 3. Species of moderate habitat specialisation and tolerance to organic pollution were most likely to be lost. Those with narrow specialisations in protected habitats were present in both historical and contemporary collections. 4. Contemporary assemblages are the consequence of more than a 100 years of anthropogenic impacts. In particular, streams at lower altitude and draining intensively exploited landscapes host a mere fragment of the original species complement. Most stonefly species are less frequently present than before, although their assemblages remain almost intact in near‐natural mountain streams. Our analyses demonstrate dramatic restriction of species ranges and, in some cases, apparent changes in altitudinal preference throughout the area.     

Pseudomonads: major antagonistic endophytic bacteria to suppress bacterial wilt pathogen, <Emphasis Type="Italic">Ralstonia solanacearum</Emphasis> in the eggplant (<Emphasis Type="Italic">Solanum melongena</Emphasis> L.)

Endophytic bacteria of eggplant, cucumber and groundnut were isolated from different locations of Goa, India. Based on in vitro screening, 28 bacterial isolates which effectively inhibited Ralstonia solanacearum, a bacterial wilt pathogen of the eggplant were characterized and identified. More than 50% of these isolates were Pseudomonas fluorescens in which a vast degree of variability was found to exist when biochemical characteristics were compared. In greenhouse experiments, the plants treated with Pseudomonas isolates (EB9, EB67), Enterobacter isolates (EB44, EB89) and Bacillus isolates (EC4, EC13) reduced the wilt incidence by more than 70%. All the selected isolates reduced damping off by more than 50% and improved the growth of seedlings in the nursery stage. Most of the selected antagonists produced an antibiotic, DAPG, which inhibited R. solanacearum under in vitro conditions and might have been responsible for reduced wilt incidence under in vivo conditions. Also production of siderophores and IAA in the culture medium by the antagonists was recorded, which could be involved in biocontrol and growth promotion in crop plants. From our study we conclude that Pseudomonas is the major antagonistic endophytic bacteria from eggplants which have the potential to be used as a biocontrol agent as well as plant growth-promoting rhizobacteria. Large scale field evaluation and detailed knowledge on antagonistic mechanism could provide an effective biocontrol solution for bacterial wilt of solanaceous crops.     

Moderate alcohol consumption is associated with better endothelial function: a cross sectional study

Background

Natural heterologous valved conduits with a diameter greater than 22 mm that can be used for right ventricular outflow tract reconstruction in adults are not commercially available. The purpose of this study was to measure by ultrasonography the maximum diameter of the distended jugular veins of horses and cattle, respectively, to identify a population of animals that would be suitable for post-mortem collection of jugular veins at sizes greater than 22 mm.

Methods

The study population included 60 Warmblood horses, 25 Freiberger horses, 20 Brown Swiss cows, and 20 Holstein cows (including 10 Holstein and 10 Red Holstein). The maximum cross-sectional diameter of the distended jugular veins was measured at a location half-way between the mandibular angle and the thoracic inlet. The thoracic circumference (heart girth length) was used as a surrogate of body size. The jugular vein diameters of the different populations were compared by analysis of variance and the association between heart girth length and jugular vein diameter was determined in each of the four study populations by linear regression analysis.

Results

There was considerable individual variation of jugular vein diameters within each of the four study populations. There was no statistically significant relationship between thoracic circumference and jugular vein diameter in any of the populations. The jugular vein diameters of Brown Swiss cows were significantly larger than those of any of the other populations. Warmblood horses had significantly larger jugular vein diameters compared to Freiberger horses.

Conclusion

The results of this study suggest that the production of bovine or equine xenografts with diameters of greater than 22 mm would be feasible. Differences between species and breeds need to be considered. However, prediction of the jugular vein diameter based on breed and heart girth length in an individual animal is inaccurate.     

Intragenic duplication and divergence in the spectrin superfamily of proteins

Many structural, signaling, and adhesion molecules contain tandemly repeated amino acid motifs. The alpha-actinin/spectrin/dystrophin superfamily of F-actin-crosslinking proteins contains an array of triple alpha-helical motifs (spectrin repeats). We present here the complete sequence of the novel beta-spectrin isoform beta(Heavy)- spectrin (beta H). The sequence of beta H supports the origin of alpha- and beta-spectrins from a common ancestor, and we present a novel model for the origin of the spectrins from a homodimeric actin-crosslinking precursor. The pattern of similarity between the spectrin repeat units indicates that they have evolved by a series of nested, nonuniform duplications. Furthermore, the spectrins and dystrophins clearly have common ancestry, yet the repeat unit is of a different length in each family. Together, these observations suggest a dynamic period of increase in repeat number accompanied by homogenization within each array by concerted evolution. However, today, there is greater similarity of homologous repeats between species than there is across repeats within species, suggesting that concerted evolution ceased some time before the arthropod/vertebrate split. We propose a two-phase model for the evolution of the spectrin repeat arrays in which an initial phase of concerted evolution is subsequently retarded as each new protein becomes constrained to a specific length and the repeats diverge at the DNA level. This evolutionary model has general applicability to the origins of the many other proteins that have tandemly repeated motifs.