The response to short-term intensive insulin therapy in type 2 diabetes.

AIM: Although a short course of intensive insulin therapy (IIT) can improve beta-cell function and glycaemic control in most patients with newly diagnosed type 2 diabetes (T2DM), the impact of this intervention in diabetes of longer duration has not been carefully studied. Thus, we sought to evaluate the effect of short-term IIT in patients with established T2DM. METHODS: Thirty-four patients, with diabetes of mean 5.9 +/- 6.6 years duration, underwent 4-8 weeks of IIT, with 4-h meal test administered at baseline and at 1 day post-IIT. A positive clinical response was defined as fasting glucose < 7.0 mmol/l off any antidiabetic therapy at the latter test. RESULTS: A positive response was achieved in 68% (n = 23) of the subjects. At baseline meal test, the responders had lower glucose levels than the non-responders from 120 to 240 min (all timepoints p < or = 0.0008) and higher late incremental area-under-the-C-peptide-curve (AUC(Cpep)), particularly from 60 to 150 min (all p < 0.005). Beta-cell function (ratio of AUC(Cpep) to AUC(gluc) divided by HOMA-IR) was similar between the groups at baseline (median 54.1 vs. 51.3, p = 0.62) but after IIT was significantly higher in the responders (109.3 vs. 57.4, p = 0.009). At baseline, the strongest predictors of the change in beta-cell function were glucose levels between 180 and 240 min (all r = -0.5, p = 0.005) and incremental AUC(Cpep) from 120 to 180 min (all r > or = 0.66, p < or = 0.0001), both reflecting late-phase insulin secretion. CONCLUSIONS: The clinical response to short-term IIT is variable, consistent with the heterogeneity of T2DM. However, preserved late-phase insulin secretion may identify those patients who can benefit from this intervention with improved beta-cell function.

Changes in neuropil ultrastructure in hippocampal field CA1 in rat pups after application of hyaluronidase.

Electron microscopy and electrophysiological studies were performed on cross-sectional slices of the hippocampus of four-week-old male rat pups (n = 35) to detect ultrastructural changes in hippocampal field CA1 and the characteristics of the formation of excitatory postsynaptic potentials in this area of the brain after incubation of slices in hyaluronidase solution (10 U/ml), whose specific substrate is the extracellular matrix glycosaminoglycan hyaluronic acid. At 1.5 min after enzyme application, there were reductions in synaptic cleft widths in axodendritic contacts of the striatum radiatum of hippocampal field CA1 by 15-25%, which were consistent with increases seen in the amplitudes of excitatory postsynaptic potentials. At 4.5 min of incubation, the lumens of synaptic clefts decreased by 45-55%: during this time there was blockade of signal transmission via Schäffer collaterals to hippocampal field CA1. Thus, the structural-functional state of glycosaminoglycans is among the factors determining the efficiency of synaptic transmission in the brain.

Generation of excitatory postsynaptic potentials in the hippocampus after functional modification of glycosaminoglycans.

Experiments on hippocampal slices form 4-week-old rats (n=28) showed that addition of lidase (1.0 and 10.0 U/ml) to the perfusion solution (artificial cerebrospinal fluid) was accompanied by the impaired generation or blockade of excitatory postsynaptic potentials and population spikes in the hippocampal CA1 region during stimulation of Schaffer collaterals. Removal of lidase from this solution normalized the amplitude of evoked responses. Hence, lidase in these concentrations produced a reversible effect on synaptic transmission. Our results indicate that structure and function of glycosaminoglycans in the extracellular matrix determine signal transduction in the nervous tissue.

Three cases of cytomegalovirus infection following pancreatic islet transplantation.

Transmission of cytomegalovirus (CMV) is uncommon in patients transplanted with CMV-mismatched pancreatic islets, and CMV-seropositive recipients rarely experience reactivation of the virus or reinfection from a CMV-positive graft. This study describes three cases of CMV infection following islet transplantation for type 1 diabetes despite prophylaxis with valganciclovir. Further studies are needed to evaluate risk factors for CMV transmission and reactivation in this patient population.

Dependence of electrical activity of neurons in rostral parts of spinal trigeminal nucleus on functional state of trigeminal Gasser ganglion.

Unilateral injection of 100 microl 1% lidocaine into the trigeminal Gasser ganglion of narcotized rats produced a long-term moderation of the discharge rate of neurons in the ipsilateral (relative to the side of injection) rostral area of the spinal trigeminal nucleus. Activity of neurons in the contralateral rostral area of the spinal trigeminal nucleus was not blocked. Functional state of neurons in the trigeminal ganglion determines discharge activity of ipsilateral neurons of the spinal trigeminal nucleus. Activity of neurons in the contralateral rostral area of spinal trigeminal nucleus was not inhibited. Functional state of the cells in the trigeminal ganglion determines the character of electrical activity of neurons in the ipsilateral rostral area of spinal trigeminal nucleus.

Metabolic activity of macrophages infected with hantavirus, an agent of hemorrhagic fever with renal syndrome.

Monocytes/macrophages are thought to play an important role in pathogenesis of viral infections. These cells are involved in distribution and persistence of viruses in the organism and also influence the regulation of immune reactions. The functional and enzymatic activities of macrophages infected with an agent of hemorrhagic fever with renal syndrome were analyzed for the first time. This disease is caused by a virus of the Hantavirus genus, the Bunyaviridae family. Activities of ectoenzymes 5 -nucleotidase and ATPase of the plasma membrane of the hantavirus-infected macrophages decreased along with the antigen accumulation in the infected cells. The contact of phagocytes with hantavirus resulted in activation in the cells of the oxygen-dependent metabolism and NO-synthase. The NO-synthase-dependent system of the infected macrophages was activated earlier than their oxygen-dependent system. The intracellular contents of acid and alkaline phosphatases increased within the first hours after the infection. The bactericidal activity of the hantavirus-infected macrophages relatively to Staphylococcus aureus increased during the specific antigen accumulation in the phagocytes. Thus, the infection of macrophages with hantavirus was associated with intracellular metabolic changes.

Lipopolysaccharide can activate BK channels of arterial smooth muscle in the absence of iNOS expression.

The role of inducible nitric oxide synthase (iNOS) in the acute activation of large-conductance, Ca(2+)-dependent K(+) channels (BK channels) by Escherichia coli endotoxin (lipopolysaccharide, LPS) was studied in murine vascular smooth muscle cells. Confocal laser scanning microscopy and patch clamp recordings were utilised. Within 2 h of donor rat sacrifice, iNOS-like immunoreactivity could be detected in cerebrovascular smooth muscle cells (CVSMCs) enzymatically dispersed from rat cerebral arteries. This staining was absent in cells fixed immediately after donor rat sacrifice. LPS was then applied to the cytoplasmic face of inside-out membrane patches excised from rat CVSMCs within 2-4 h of donor rat sacrifice. It was found that LPS (10-100 microg/ml) rapidly and reversibly increased the open probability of BK channels in these patches. This LPS response was not altered in the presence of the non-isoform specific NOS inhibitor N(omega)-nitro-L-arginine. LPS responses were then compared in aortic smooth muscle (ASMC) BK channels derived from wild-type and iNOS-knockout (iNOS-KO) mice. LPS activated BK channels in inside-out patches of ASMC membrane derived from both wild-type and iNOS-knockout mice. These studies establish that LPS can activate BK channels by a mechanism quite independent of the well-established pathway mediated by iNOS in vascular smooth muscle cells.

S-phase function of Drosophila cyclin A and its downregulation in G1 phase.

BACKGROUND: Cyclin E is the normal inducer of S phase in G1 cells of Drosophila embryos. Stable G1 quiescence requires the downregulation both of cyclin E and of other factors that can bypass the normal regulation of cell cycle progression. RESULTS: High-level expression of cyclin A triggered the G1/S transition in wild-type embryos and in mutant embryos lacking cyclin E. Three types of control downregulated this activity of cyclin A. First, cyclin destruction limited the accumulation of cyclin A protein in G1. Second, inhibitory phosphorylation of cdc2, the kinase partner of cyclin A, reduced the S-phase promoting activity of cyclin A in G1. Third, rux, a protein with unknown biochemical function, limited cyclin A function in G1. Overexpression of rux blocked S phase induction by coexpressed cyclin A and promoted the degradation of cyclin A. Rux also prevented a stable cyclin A mutant from inducing S phase, indicating that inhibition does not require cyclin destruction, and drove the nuclear localization of cyclin A. CONCLUSIONS: Cyclin A can drive the G1/S transition, but this function is suppressed by three types of control: cyclin A destruction, inhibitory phosphorylation of cdc2, and inhibition by rux. The partly redundant contributions of these three inhibitory mechanisms safeguard the stability of G1 quiescence until the induction of cyclin E. The action of rux during G1 resembles the action of inhibitors of mitotic kinases present during G1 in yeast, although no obvious sequence similarity exists.

Cloning of Drosophila MCM homologs and analysis of their requirement during embryogenesis.

MCM (minichromosome maintenance) gene family of Saccharomyces cerevisiae encodes essential DNA replication factors that participate in the initiation of DNA replication. In addition, their localization to the nucleus in a mitosis-dependent manner fueled the hypothesis that MCMs also act to couple DNA replication to mitosis. We report the identification of a Drosophila gene family with extensive sequence identity to the MCM genes. Results from antibody injection experiments suggest that MCMs play an essential role in DNA replication during embryogenesis. Evolutionary conservation of MCM sequences and function in Drosophila could potentially facilitate studies of how initiation of DNA replication is regulated and coupled to mitosis during metazoan development.

Phasmids as effective and simple tools for construction and analysis of gene libraries.

Phasmid lambda pMYF131, a hybrid of phage lambda vectors and plasmid pUC19, was constructed. The phasmid and its derivatives were shown to be efficient vectors for construction and analysis of gene libraries in Escherichia coli cells. The lambda pMYF131 DNA molecule contains all the genes and regions essential for phage lytic development. The plasmid cannot be packaged either in the monomeric or the oligomeric form due to its specific length. Elongation of the DNA molecule by ligation with fragments of foreign DNA can make it packageable and this is easily detected by plaque formation. Hence, the procedures used to construct genomic libraries can be simplified by selection of only recombinant DNA molecules just at the time and on the basis of their packaging in vitro. The output of recombinant clones per vector molecule was several times higher for vector lambda pMYF131, compared to phage vector lambda L47.1AB, and attained 3 x 10(6) clones per micrograms DNA. Vector and recombinant phasmids can be obtained in large quantities in plasmid form. lambda pMYF131 contains nine unique restriction sites which allow the cloning of DNA fragments with blunt ends and of fragments with various types of cohesive ends, obtained by digestion with 14 prototype restriction enzymes. The maximal size of the cloned DNA fragments is approx. 20 kb for lambda pMYF131. Phasmid vectors were used to construct libraries of bovine, pig and quail genomes, and genomic libraries of 17 species of bacteria. Application of suitable methods allowed the identification 13 individual genes within these libraries.