Cell signalling-mediating insulin increase of mRNA expression for cationic amino acid transporters-1 and -2 and membrane hyperpolarization in human umbilical vein endothelial cells

Insulin induces vasodilatation in human subjects and increases l-arginine transport and NO synthesis in human umbilical vein endothelial cells (HUVEC). Cell signalling events associated with insulin effects on activity and mRNA expression of the human cationic amino acid transporters 1 (hCAT-1) and 2B (hCAT-2B) are unknown. l-Arginine transport and eNOS activity were determined in HUVEC exposed to insulin. mRNA levels for hCAT-1, hCAT-2B and eNOS were quantitated by real time RT-PCR and endothelial NO synthase (eNOS) protein was identified by Western blot analysis. Intracellular Ca²⁺, l-arginine and l-citrulline levels, l-[³H]citrulline formation from l-[³H]arginine, cGMP formation, nitrite level, ATP release and membrane potential were determined. Insulin increased l-arginine transport and the mRNA levels for hCAT-1 and hCAT-2B and eNOS expression and activity. Insulin also induced membrane hyperpolarization and increased intracellular Ca²⁺, l-[³H]citrulline, cGMP and nitrite formation. Insulin-mediated stimulation of the l-arginine/NO pathway is thus associated with increased hCAT-1 and hCAT-2B mRNA, and eNOS expression, via mechanisms involving membrane hyperpolarization, mitogen-activated protein kinases p42 and p44, phosphatidylinositol 3-kinase, NO and protein kinase C. We have characterized a cell signalling pathway by which hyperinsulinaemia could lead to vasodilatation in human subjects, and which could have implications in patients in whom plasma insulin levels are altered, such as in diabetes mellitus.     

Use of a non-radioactive hybridisation assay for direct detection of gram-negative bacteria carrying TEM beta-lactamase genes in infected urine

DNA in infected urines from 81 patients with urinary tract infection was hybridised directly with a non-radioactive DNA probe specific for bacterial genes coding for TEM-type beta-lactamase. The results were assessed by means of a computerised image analysis system and compared with those obtained following isolation of the infecting organism, conventional sensitivity testing and isoelectric focusing (IEF) procedures for the detection of TEM-type beta-lactamase. Of the 27 ampicillin-resistant gram-negative organisms isolated in pure culture from the urines, 14 were shown by both hybridisation and IEF to carry a gene for TEM beta-lactamase production. Only four discordant results were obtained: three "false positive" direct hybridisation results, one due to urine pigmentation, and one, possibly, to a TEM beta-lactamase gene which was not being expressed, and one "false negative" result due to insufficient cell numbers in the urine. The system is capable of screening large numbers of samples and is applicable to any gene for which a suitable DNA probe is available.     

Value of electron microscopy in diagnosis of renal disease.

AIMS--To assess the role and value of electron microscopy in the diagnosis of renal disease. METHODS--Retrospective evaluation of 88 renal biopsy specimens received for primary diagnosis by assessment of the contribution of electron microscopy to the final diagnosis in the knowledge of the light microscopy and immunofluorescence findings. RESULTS--Electron microscopy had an important diagnostic role in 75% of cases and was essential or necessary for diagnosis in 25%. In 25% of cases electron microscopy was considered unhelpful in diagnosis. CONCLUSION--Electron microscopy has an integral role in the diagnosis of renal disease, and tissue should be taken for electron microscopy in all cases if possible. In some selected cases once the light microscopy and immunofluorescence findings are known it may be possible to forego electron microscopic examination. Electron microscopy is particularly useful in the differential diagnosis of minimal change disease and the nephrotic syndrome.     

Non-toxigenic Escherichia coli O157:H7 strain NCTC12900 causes attaching-effacing lesions and eae-dependent persistence in weaned sheep

Ruminants are regarded as a primary reservoir for Escherichia coli O157:H7, an important human pathogen. Intimin, encoded by the Locus of Enterocyte Effacement by E. coli O157:H7 organisms, has been cited as one bacterial mechanism of colonisation of the gastrointestinal tract. To confirm this and to test whether a non-toxigenic E. coli O157:H7 strain would colonise and persist in a sheep model, E. coli O157:H7 strain NCTC12900, that lacks Shiga toxin (stx) genes, was evaluated for use in a sheep model of persistence. Following oral inoculation of six-week-old sheep, persistent excretion of NCTC12900 was observed for up to 48 days. E. coli O157-associated attaching-effacing (AE) lesions were detected in the caecum and rectum of one six-week-old lamb, one day after inoculation. This is the first recorded observation of AE lesions in orally inoculated weaned sheep. Also, mean faecal excretion scores of NCTC12900 and an isogenic intimin (eae)-deficient mutant were determined from twenty-four six-week-old orally inoculated sheep. The eae mutant was cleared within 20 days and had lower mean excretion scores at all time points after day one post inoculation compared with the parental strain that was still being excreted at 48 days. Tissues were collected post mortem from animals selected at random from the study groups over the time course of the experiment. The eae mutant was detected in only 1/43 samples but the parental strain was recovered from 64/140 samples primarily from the large bowel although rumen, duodenum, jejunum, and ileum were culture positive especially from animals that were still excreting at and beyond 27 days after inoculation.     

Adaptive changes in locomotor activity following botulinum toxin injection in ankle extensor muscles of cats.

The present study investigated the adaptations made in motor behavior following a temporary reduction in ankle extensor activity in the walking cat. Temporary muscle weakness was induced by injecting botulinum toxin into the lateral gastrocnemius (LG), plantaris (PL), and soleus (SOL) muscles, or SOL alone. The medial gastrocnemius (MG) muscle was not injected. Adaptations in the level of muscle activity were recorded using chronically implanted electromyographic (EMG) electrodes. Serial recordings were made prior to botulinum toxin injections and for several days following the injections. Kinematic analysis of ankle joint movements was made from video records to assess the impact of the botulinum toxin injections on the function of the ankle joint during walking. Following injection of the LG, PL, and SOL muscles with botulinum toxin, the amplitude of the MG burst increased over a period of a few days to a week. This increase was similar to the previously reported changes produced in MG following transection of the nerves serving LG, PL, and SOL. Following the weakening of the ankle extensor muscles, there was a temporary deficit in ankle function during walking as evidenced by a marked increase in the amount of ankle flexion that occurred at stance onset. This functional deficit recovered relatively quickly and was not associated with a return of the EMG pattern to the preinjection pattern. After recovery from the initial injections, a second injection of botulinum toxin into SOL alone was performed. No functional deficits were observed in the ankle movements during walking following this second injection. However, weakening SOL produced increases in the burst amplitudes of the MG, LG, and PL muscles over a period of a few days. This suggests that normal movements at the ankle during walking can be generated with more than one pattern of ankle extensor activity and that there is flexibility in how the necessary torque is produced. A final procedure, transection of the nerves serving LG, PL, and SOL, failed to produce any functional deficits in ankle movements. The implication is that adaptations to the neural control of ankle extensor activity that were induced by the initial procedure persisted after the recovery of the injected muscles and were sufficient to compensate for the subsequent challenges.     

Genetic variation in ICF syndrome: evidence for genetic heterogeneity

ICF syndrome is a rare autosomal recessive immunoglobulin deficiency, sometimes combined with defective cellular immunity. Other features that are frequently observed in ICF syndrome patients include facial dysmorphism, developmental delay, and recurrent infections. The most diagnostic feature of ICF syndrome is the branching of chromosomes 1, 9, and 16 due to pericentromeric instability. Positional candidate cloning recently discovered the de novo DNA methyltransferase 3B (DNMT3B) as the responsible gene by identifying seven different mutations in nine ICF patients. DNMT3B specifically methylates repeat sequences adjacent to the centromeres of chromosome 1, 9, and 16. Our panel of 14 ICF patients was subjected to mutation analysis in the DNMT3B gene. Mutations in DNMT3B were discovered in only nine of our 14 ICF patients. Moreover, two ICF patients from consanguineous families who did not show autozygosity (i.e. homozygosity by descent) for the DNMT3B locus did not reveal DNMT3B mutations, suggesting genetic heterogeneity for this disease. Mutation analysis revealed 11 different mutations, including seven novel ones: eight different missense mutations, two different nonsense mutations, and a splice-site mutation leading to the insertion of three aa's. The missense mutations occurred in or near the catalytic domain of DNMT3B protein, indicating a possible interference with the normal functioning of the enzyme. However, none of the ICF patients was homozygous for a nonsense allele, suggesting that absence of this enzyme is not compatible with life. Compound heterozygosity for a missense and a nonsense mutation did not seem to correlate with a more severe phenotype.     

Strategic control and medial frontal negativity: beyond errors and response conflict

Errors in timed choice tasks typically produce an error-related negativity (ERN) in the event-related potential (ERP). The error specificity of the ERN has been challenged by studies showing a correct response negativity (CRN). Forty-five participants engaged in a flanker task in which both compatibility between flankers and target and the probability of compatible flankers were manipulated. Correct responses elicited a CRN, the amplitude of which increased with the degree of mismatch between the presence of conflict and conflict probability, even on low-conflict (compatible) trials. The fronto-central N2 component was larger on high-conflict (incompatible) correct response trials. However, in contrast to some recent accounts, this N2 was largest for highly probable stimuli. These findings suggest revision to models of the effects of conflict on response-related negativity to account for strategic adjustments made in preparation for the response.     

ImuVert activation of natural killer cytotoxicity and interferon gamma production via CD16 triggering

The effect and mechanism of action of ImuVert, a new biological response modifier consisting of ribosomes and natural membrane vesicles of Serratia marcescens, on endogenous natural killer (NK) cells and activated NK activity has been analyzed. The studies showed that endogenous NK activity of peripheral blood mononuclear cells (PBMC) from normal cell donors was significantly increased (P less than 0.03) against K562, U937, and Molt-4 target cells. PBMC from cord blood of newborn infants lacking NK activity were upregulated (1.5-4 fold over endogenous NK activity) by ImuVert. Other studies showed that the abnormal NK activity of PBMC from patients with the human immunodeficiency virus (HIV) infection was significantly augmented in vitro (P less than 0.01) by ImuVert. ImuVert strongly stimulated interferon gamma production and in combination with interleukin 2 produced synergistically enhanced interferon gamma production and greater cytotoxicity than that induced by either alone. Studies on lymphocyte differentiation antigen expression following treatment with ImuVert indicated that ImuVert triggers interferon gamma production through binding the low affinity IgG Fc receptor, type III, CD16. The studies suggest that ImuVert may trigger interferon gamma production by binding to the Fc receptor and that the amplitude of the ensuing reaction and the ability of ImuVert to induce cytotoxicity in a setting where this activity has been down regulated is based on the absence of suppressor activation or direct contra suppressor activity.     

Brain regions and their dynamics in prospective memory retrieval: a MEG study.

We measured brain activity using magnetoencephalography in five participants during ongoing tasks that included prospective memory, retrospective memory, and oddball trials. Sources were identified in the hippocampal formation and posterior parietal and frontal lobes. Posterior parietal cortex activation had an earlier onset in the prospective memory condition than retrospective memory or oddball conditions, a higher level of theta activity in the retrospective condition, and higher levels of upper alpha in the prospective and oddball conditions. Activation of the hippocampal formation had a longer duration in the retrospective memory and prospective memory conditions than the oddball condition, but prominent alpha and theta band activity was present in all three conditions. We interpret the early (87 ms) onset of activity in parietal cortex as evidence for an initial noticing of appropriate conditions for a PM response. Hippocampal activity may reflect a subsequent memory search for the intended action.