Loss of adenylyl cyclase I activity disrupts patterning of mouse somatosensory cortex.

The somatosensory (SI) cortex of mice displays a patterned, nonuniform distribution of neurons in layer IV called the 'barrelfield' (ref. 1). Thalamocortical afferents (TCAs) that terminate in layer IV are segregated such that each barrel, a readily visible cylindrical array of neurons surrounding a cell-sparse center, represents a distinct receptive field. TCA arbors are confined to the barrel hollow and synapse on barrel-wall neurons whose dendrites are oriented toward the center of the barrel. Mice homozygous for the barrelless (brl) mutation, which occurred spontaneously in ICR stock at Université de Lausanne (Switzerland), fail to develop this patterned distribution of neurons, but still display normal topological organization of the SI cortex. Despite the absence of barrels and the overlapping zones of TCA arborization, the size of individual whisker representations, as judged by 2-deoxyglucose uptake, is similar to that of wild-type mice. We identified adenylyl cyclase type I (Adcy1) as the gene disrupted in brl mutant mice by fine mapping of proximal chromosome 11, enzyme assay, mutation analysis and examination of mice homozygous for a targeted disruption of Adcy1. These results provide the first evidence for involvement of cAMP signalling pathways in pattern formation of the brain.

Minimally invasive surgical wound infections: laparoscopic surgery decreases morbidity of surgical site infections and decreases the cost of wound care.

AIM: The morbidity of surgical site infections (SSIs) were compared in patients who underwent open (OS) vs laparoscopic (LS) colorectal surgery. METHOD: Data from 603 consecutive LS patients and 2246 consecutive OS patients were prospectively recorded. Morbidity of SSIs was assessed by the need for emergency department (ED) evaluation, subsequent hospital re-admission and re-operation. The cost of wound care was measured by the need for home healthcare, wound vacuum assisted closure (VAC) or independent patient wound care. RESULTS: SSIs were identified in 5.8% (n = 25) of LS patients and 4.8% (n = 65) of OS patients. ED evaluation for the infection was needed in 24% of the LS group and 42% of the OS group. Hospital re-admission was needed in one LS patient and in 52% OS patients. No LS patient needed re-operation compared with 12% of OS patients. HHC ($162/dressing change) was required in 63% of the OS group compared with 8% of LS group. A home wound VAC system ($107/day) was utilized in 12% of the OS patients but in none of the LS patients. Dressing changes were managed independently by the patient in 92% of the LS compared with 37% of the OS patients. CONCLUSION: Laparoscopic colorectal surgery patients experience less morbidity when they develop SSIs incurring less cost compared with open colorectal surgery patients.

High sodium and low potassium intake in patients with Type 2 diabetes.

AIMS: To document dietary sodium and potassium intake and adherence to the Australian National Heart Foundation (NHF) guidelines in patients with Type 2 diabetes mellitus attending an Australian tertiary referral and university teaching hospital. METHODS: In a longitudinal study, 24h urinary sodium (uNa), potassium (uK), creatinine (uCr), urea (uUrea) and glucose (uGlu) excretions, urine volume (uVol) and body mass index were recorded in 122 regular attenders over an 8 year period (2001-2008; mean of 1.9 samples/patient/year). In a cross-sectional study, the same measurements were recorded in patients providing urine samples in the month of June from 2001 to 2009 (782 patients, averaging 87/year). RESULTS: In the longitudinal study, uNa (mmol/24 h) was 170 ± 53 (mean ± SD) in males and 142 ± 51 in females, whereas uK (mmol/24 h) was 75 ± 22 in males and 62 ± 18 in females. Once adjusted for insensible losses, only 3% of males and 14% of females met the NHF dietary sodium intake guidelines, and 14% of males and 3% of female patients met the NHF dietary potassium guidelines. Body mass index, uUrea, uVol and uGlu were independent predictors of uNa (adjusted r(2) =0.57, P<0.0001). The mean intra-individual coefficient of variation of the corrected uNa was 21 ± 1%. The cross-sectional study confirmed these findings, and no temporal trends were observed. There was no correlation with glycated haemoglobin to suggest natriuresis with hyperglycaemia. CONCLUSIONS: Most patients with Type 2 diabetes mellitus do not meet NHF sodium or potassium intake guidelines. A diet high in sodium and low in potassium may contribute to the development of hypertension and to resistance to blood-pressure-lowering therapies.

Partitioning of the 2-microm circle plasmid of Saccharomyces cerevisiae. Functional coordination with chromosome segregation and plasmid-encoded rep protein distribution.

The efficient partitioning of the 2-microm plasmid of Saccharomyces cerevisiae at cell division is dependent on two plasmid-encoded proteins (Rep1p and Rep2p), together with the cis-acting locus REP3 (STB). In addition, host encoded factors are likely to contribute to plasmid segregation. Direct observation of a 2-microm-derived plasmid in live yeast cells indicates that the multiple plasmid copies are located in the nucleus, predominantly in clusters with characteristic shapes. Comparison to a single-tagged chromosome or to a yeast centromeric plasmid shows that the segregation kinetics of the 2-microm plasmid and the chromosome are quite similar during the yeast cell cycle. Immunofluorescence analysis reveals that the plasmid is colocalized with the Rep1 and Rep2 proteins within the yeast nucleus. Furthermore, the Rep proteins (and therefore the plasmid) tend to concentrate near the poles of the yeast mitotic spindle. Depolymerization of the spindle results in partial dispersion of the Rep proteins in the nucleus concomitant with a loosening in the association between plasmid molecules. In an ipl1-2 yeast strain, shifted to the nonpermissive temperature, the chromosomes and plasmid almost always missegregate in tandem. Our results suggest that, after DNA replication, plasmid distribution to the daughter cells occurs in the form of specific DNA-protein aggregates. They further indicate that the plasmid partitioning mechanism may exploit at least some of the components of the cellular machinery required for chromosomal segregation.

Dissociation of intracellular lysosomal rupture from the cell death caused by silica.

The relationship between intracellular lysosomal rupture and cell death caused by silica was studied in P388d(1) macrophages. After 3 h of exposure to 150 mug silica in medium containing 1.8 mM Ca(2+), 60 percent of the cells were unable to exclude trypan blue. In the absence of extracellular Ca(2+), however, all of the cells remained viable. Phagocytosis of silica particles occurred to the same extent in the presence or absence of Ca(2+). The percentage of P388D(1) cells killed by silica depended on the dose and the concentration of Ca(2+) in the medium. Intracellular lyosomal rupture after exposure to silica was measured by acridine orange fluorescence or histochemical assay of horseradish peroxidase. With either assay, 60 percent of the cells exposed to 150 mug silica for 3 h in the presence of Ca(2+) showed intracellular lysosomal rupture, was not associated with measureable degradation of total DNA, RNA, protein, or phospholipids or accelerated turnover of exogenous horseradish peroxidase. Pretreatment with promethazine (20 mug/ml) protected 80 percent of P388D(1) macrophages against silica toxicity although lysosomal rupture occurred in 60-70 percent of the cells. Intracellular lysosomal rupture was prevented in 80 percent of the cells by pretreatment with indomethacin (5 x 10(-5)M), yet 40-50 percent of the cells died after 3 h of exposure to 150 mug silica in 1.8 mM extracellular Ca(2+). The calcium ionophore A23187 also caused intracellular lysosomal rupture in 90-98 percent of the cells treated for 1 h in either the presence or absence of extracellular Ca(2+). With the addition of 1.8 mM Ca(2+), 80 percent of the cells was killed after 3 h, whereas all of the cells remained viable in the absence of Ca(2+). These experiments suggest that intracellular lysosomal rupture is not causally related to the cell death cause by silica or A23187. Cell death is dependent on extracellular Ca(2+) and may be mediated by an influx of these ions across the plasma membrane permeability barrier damaged directly by exposure to these toxins.

A common origin of rickettsiae and certain plant pathogens.

On the basis of ribosomal RNA sequence comparisons, the rickettsia Rochalimaea quintana has been found to be a member of subgroup 2 of the alpha subdivision of the so-called purple bacteria, which is one of about ten major eubacterial divisions. Within subgroup alpha-2, R. quintana is specifically related to the agrobacteria and rhizobacteria, organisms that also have close associations with eukaryotic cells. This genealogical grouping of the rickettsiae with certain plant pathogens and intracellular symbionts suggests a possible evolution of the rickettsiae from plant-associated bacteria.

N-break states in a chain of nonlinear oscillators.

In the present work we explore a prestretched oscillator chain where the nodes interact via a pairwise Lennard-Jones potential. In addition to a homogeneous solution, we identify solutions with one or more (so-called) "breaks," i.e., jumps. As a function of the canonical parameter of the system, namely, the precompression strain d, we find that the most fundamental one-break solution changes stability when the monotonicity of the Hamiltonian changes with d. We provide a proof for this (motivated by numerical computations) observation. This critical point separates stable and unstable segments of the one-break branch of solutions. We find similar branches for two- through five-break branches of solutions. Each of these higher "excited state" solutions possesses an additional unstable pair of eigenvalues. We thus conjecture that k-break solutions will possess at least k-1 (and at most k) pairs of unstable eigenvalues. Our stability analysis is corroborated by direct numerical computations of the evolutionary dynamics.

Molecular analysis of Francisella tularensis subspecies tularensis and holarctica.

Rapid methods are needed for public health and military applications to specifically identify Francisella tularensis, the causative agent of tularemia in humans. A comparative analysis of the capabilities of multiple technologies was performed using a well-defined set of organisms to determine which approach would provide the most information in the shortest time. High-resolution molecular techniques, including pulsed-field gel electrophoresis, amplified fragment length polymorphism, and ribotyping, provided subspecies level identification within approximately 24 hours after obtaining an isolate, whereas multilocus variable number tandem repeat analysis with 8 or 25 targets provided strain level discrimination within about 12 hours. In contrast, Raman spectroscopy provided species level identification in 10 minutes but could not differentiate between subspecies tularensis and holarctica.

Training the trainers.

Our understanding of how adults learn has undergone many advances in the last few years. This information needs to be used to build more effective training in anaesthesia throughout the world, especially in those countries where the need to train large numbers is critical to the development of effective medical services. Training a new generation of teachers is a key part of this.

Impact of Helicobacter pylori on the management of dyspepsia in primary care.

BACKGROUND: It is unclear what impact Helicobacter pylori infection has had on the management of dyspepsia in primary care and to what extent published guidelines on H. pylori are implemented in routine clinical practice. AIM: To assess the impact of H. pylori infection on the management of dyspepsia in primary care. METHODS: Patients referred by primary care doctors to an open-access 13-carbon urea breath test service over a 2-year period for their first urea breath test were included in the study. Individual breath results were linked with data on prescribing obtained from the General Medical Services prescription database. RESULTS: Of 805 patients, 374 (47%) had a positive urea breath test and 431 (54%) a negative urea breath test. Of positive urea breath test patients, only 245 (64%) were prescribed eradication therapy in the 3 months after the breath test and only 43% were referred back for re-testing. In the year after the urea breath test, there was a significant fall in prescribing of antisecretory therapy which was greatest in the patients who received H. pylori therapy (P < 0.001). CONCLUSIONS: There appears to be under and inappropriate treatment of H. pylori infection in primary care, and a low rate of re-testing after eradication, indicating that current guidelines are not well implemented in practice.

The N363S polymorphism of the glucocorticoid receptor: potential contribution to central obesity in men and lack of association with other risk factors for coronary heart disease and diabetes mellitus.

Considerable evidence suggests that diabetes mellitus and hypertension are influenced by genetic factors. Studies in humans have associated glucocorticoid receptor (GR) polymorphisms with high blood pressure, insulin sensitivity, body mass index, increased visceral fat, and variations in tissue-specific steroid sensitivity. The N363S polymorphism of the GR results in an asparagine to serine amino acid substitution in a modulatory region of the receptor. Phosphorylation of serine residues in this region has been shown to enhance transactivation of GR responsive genes. The aim of this study was to investigate the association between the 363S allele and risk factors for coronary heart disease and diabetes mellitus in a population of European origin living in the northeast of the United KINGDOM: Blood samples from 135 males and 240 females were characterized for 363 allele status. The overall frequency of the 363S allele was 3.0%, 23 heterozygotes (7 males and 16 females) but no 363S homozygotes were identified. The data show a significant association of the 363S allele with increased waist to hip ratio in males but not females. This allele was not associated with blood pressure, body mass index, serum cholesterol, triglycerides, low-density lipoprotein and high-density lipoprotein cholesterol levels, and glucose tolerance status. The results of this study suggest that this GR polymorphism may contribute to central obesity in men. Further studies are required to elucidate the properties of GR(363S) at a molecular level.

Sequence variation in the LEU2 region of the saccharomyces cerevisiae genome.

The LEU2 regions present on yeast plasmid vectors come from two sources, a series of strains derived from S288c and strain M127. The LEU2 region from the S288c series contains a Tyl-17 element with its associated delta sequences and a small repetitive RNA gene while the LEU2 region from M127 which is present on pJDB248, lacks the Tyl-17 element, but carries a delta sequence and a small RNA gene. The various LEU2 plasmids currently in use vary with respect to these sequences depending on which restriction fragment from the region is present on the recombinant molecule. In addition, strain M127 contains three LEU2 homologous sequences that are represented by different EcoRI fragments and which segregate independently at meiosis. Therefore, there are at least four forms of the centromere-distal EcoRI fragment of the LEU2 locus in the Saccharomyces cerevisiae gene pool; these are 7.1 kb, 1.9 kb, 1.48 kb and 1.15 kb long.

Factors affecting heterologous gene expression in Saccharomyces cerevisiae.

The 'promoter' fragment from the yeast phosphoglycerate kinase (PGK) gene has been used to direct the expression of human interferon-alpha-2 (IFN alpha 2) on a high-copy-number plasmid in yeast. The yields of IFN alpha 2 are only 1-3% of yeast total protein, whereas the maximum yield of PGK produced by the PGK gene on a high-copy-number plasmid is at least 50%. IFN alpha 2 is turned over more rapidly than PGK but in addition a major reason for the relatively low level of IFN alpha 2 is that IFN-specific RNA levels are much lower. This does not reflect differences in plasmid copy number or integrity, or differences in the 5' and 3' untranslated regions of the transcripts or DNA flanking regions. It appears that the presence of heterologous coding sequences, or the absence of specific yeast sequences causes a reduction in heterologous RNA levels in yeast.

Efficient synthesis of enzymatically active calf chymosin in Saccharomyces cerevisiae.

We have constructed a high-efficiency expression vector to direct the synthesis of heterologous polypeptides in yeast. The vector is termed a sandwich expression vector as the heterologous gene is inserted between the 5' and 3' control regions of the efficiently expressed yeast PGK gene. We have used this vector to direct the expression of three derivatives of the calf chymosin cDNA gene; preprochymosin, prochymosin and chymosin. Prochymosin is synthesised to at least 5% of total yeast-cell protein and furthermore, it can be readily activated to produce an enzyme which has milk-clotting activity.

Differential effects of retinoic acid isomers on the expression of nuclear receptor co-regulators in neuroblastoma.

Retinoic acid modulates growth and induces differentiation and apoptosis of neuroblastoma cells in vitro, with the all-trans and 9-cis isomers having different biological properties. Transcriptional activation in response to retinoic acid isomers is mediated by retinoic acid receptors and retinoid X receptors. The differential expression of co-activators and co-repressors which preferentially interact with retinoic acid receptors or retinoid X receptors may be a mechanism leading to different cellular responses to 9-cis and all-trans retinoic acid. To test this hypothesis, we have studied the expression of the nuclear receptor co-regulators TIF1alpha, TIF1beta, SUG1 and SMRT in the N-type and S-type neuroblastoma cell lines SH SY 5Y and SH S EP. Transcripts for all four co-regulators were expressed in these neuroblastoma cells. The expression of TIF1alpha, TIF1beta and SUG1 did not change in response to retinoic acid; however, SMRT was induced in both neuroblastoma cell lines, but particularly by all-trans retinoic acid in SH S EP cells. An additional co-activator, Trip3, was isolated by differential mRNA display and shown to be preferentially induced by 9-cis retinoic acid in SH SY 5Y and SH S EP cells. These data suggest that retinoic acid isomer-specific induction of nuclear receptor co-regulators may determine, in part, the differential biological effects of retinoic acid isomers.

Sublethal gamma irradiation increases IL-1alpha, IL-6, and TNF-alpha mRNA levels in murine hematopoietic tissues.

The effects of sublethal (7.75 Gy) 60Co gamma radiation exposure on endogenous bone marrow and splenic interleukin-1alpha (IL-1alpha), IL-6, and tumor necrosis factor-alpha (TNF-alpha) mRNA and protein levels were assayed in B6D2F1 female mice. Bone marrow and spleen were harvested from normal and irradiated mice on days 2, 4, 7, 10, and 14 postexposure, and cytokine mRNA levels were determined by reverse transcription polymerase chain reaction (RT-PCR) and Southern blot analysis. IL-1alpha mRNA levels were significantly increased in bone marrow at days 2 and 4 postirradiation and at day 7 in spleen compared with controls. Postirradiation IL-6 mRNA levels showed a significant increase at day 2 in bone marrow and at days 7 and 10 in spleen. TNF-alpha mRNA levels exhibited a significant increase at day 2 postirradiation in bone marrow, but in spleen no difference between control and irradiated samples was observed on any day postirradiation. Interestingly, there were no significant differences in the cytokine protein levels in postirradiation bone marrow, spleen, or serum when compared with normal controls.