Stress hypERactivation in the β-cell

In pancreatic β-cells, the endoplasmic reticulum (ER) is the crucial site for insulin biosynthesis, as this is where the protein-folding machinery for secretory proteins is localized. Perturbations to ER function of the β-cell, such as a high demand for insulin secretion, can lead to an imbalance in protein homeostasis and lead to ER stress. This stress can be mitigated by an adaptive, cellular response, the unfolded protein response (UPR). UPR activation is vital to the survival of β-cells, as these cells represent one of the most susceptible tissues for ER stress, due to their highly secretory function. However, in some cases, this response is not sufficient to relieve stress, leading to apoptosis and contributing to the pathogenesis of diabetes. Recent evidence shows that ER stress plays a significant role in both type 1 and type 2 diabetes. In this review, we outline the mechanisms of ER stress-mediated β-cell death and focus on the role of ER stress in various forms of diabetes, particularly a genetic form of diabetes called Wolfram syndrome.     

Susceptibility of new β-lactams to the expanded-spectrum β-lactamase CTX-1

Summary Forty-three clinical isolates of enterobacteria were selected for the production of the new plasmid-mediated expanded-spectrum -lactamase CTX-1. The geometric means of MICs were ranged as follows: ticarcillin, >4096 mg/l; ticarcillin + clavulanic acid (2 mg/l), 64–87 mg/l; LY 163892, 8.0–69.1 mg/l; cefotaxime, 5.7–26.4 mg/l; temocillin, 8.0–21.8 mg/l; Ro 158074, 4.0–18.7 mg/l aztreonam, 1.0–14.4 mg/l and BMY 28142, 1.4–2.8 mg/l. Moxalactam, imipenem and CM 40876 were resistant to hydrolysis and MICs were lower than 2.0 mg/l. A high protective effect on cefotaxime (MIC0.5 mg/l) was obtained by sulbactam (4 mg/l).Escherichia coli transconjugants from each species showed similar levels of MICs.

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Empfindlichkeit neuer -Laktame gegenüber der Breitspektrum -Laktamase CTX-1

Zusammenfassung 43 Isolate von Enterobakterien wurden im Hinblick auf die Bildung der neuen, plasmidkodierten Breitspektrum--Laktamase CTX-1 ausgewählt. Die geometrischen Mittel der MHK-Werte lagen in folgenden Bereichen: Ticarcillin >4096 mg/l; Ticarcillin plus Clavulansäure (2 mg/l) 64–87 mg/l; LY 163892, 8,0–69,1 mg/l; Cefotaxim 5,7–26,4 mg/l; Temocillin, 8,0–21,8 mg/l; Ro 158074, 4,0–18,7 mg/l; Aztreonam, 1,0–14,4 mg/l und BMY 28142, 1,4–2,8 mg/l. Moxalactam, Imipenem und CM 40876 wurden nicht hydrolysiert, die MHK-Werte lagen unter 2,0 mg/l. Sulbactam (4 mg/l) hatte eine hohe protektive Wirkung auf Cefotaxim (MHK0,5 mg/l).Escherichia coli-Transkonjuganten von jeder Spezies wiesen ähnliche MHK-Werte auf.

     

Autonomic Dysfunction of the β-Cell and the Pathogenesis of Obesity

Reviews in Endocrine and Metabolic Disorders -     

Reelin signaling antagonizes β-amyloid at the synapse

Abnormal processing of the amyloid precursor protein (APP) and β-amyloid (Aβ) plaque accumulation are defining features of Alzheimer disease (AD), a genetically complex neurodegenerative disease that is characterized by progressive synapse loss and neuronal cell death. Aβ induces synaptic dysfunction in part by altering the endocytosis and trafficking of AMPA and NMDA receptors. Reelin is a neuromodulator that increases glutamatergic neurotransmission by signaling through the postsynaptic ApoE receptors Apoer2 and Vldlr and thereby potently enhances synaptic plasticity. Here we show that Reelin can prevent the suppression of long-term potentiation and NMDA receptors, which is induced by levels of Aβ comparable to those present in an AD-afflicted brain. This reversal is dependent upon the activation of Src family tyrosine kinases. At high concentrations of Aβ peptides, Reelin can no longer overcome the Aβ induced functional suppression and this coincides with a complete blockade of the Reelin-dependent phosphorylation of NR2 subunits. We propose a model in which Aβ, Reelin, and ApoE receptors modulate neurotransmission and thus synaptic stability as opposing regulators of synaptic gain control.     

Activation mechanism of the β2-adrenergic receptor

A third of marketed drugs act by binding to a G-protein-coupled receptor (GPCR) and either triggering or preventing receptor activation. Although recent crystal structures have provided snapshots of both active and inactive functional states of GPCRs, these structures do not reveal the mechanism by which GPCRs transition between these states. Here we propose an activation mechanism for the β(2)-adrenergic receptor, a prototypical GPCR, based on atomic-level simulations in which an agonist-bound receptor transitions spontaneously from the active to the inactive crystallographically observed conformation. A loosely coupled allosteric network, comprising three regions that can each switch individually between multiple distinct conformations, links small perturbations at the extracellular drug-binding site to large conformational changes at the intracellular G-protein-binding site. Our simulations also exhibit an intermediate that may represent a receptor conformation to which a G protein binds during activation, and suggest that the first structural changes during receptor activation often take place on the intracellular side of the receptor, far from the drug-binding site. By capturing this fundamental signaling process in atomic detail, our results may provide a foundation for the design of drugs that control receptor signaling more precisely by stabilizing specific receptor conformations.     

Changing patterns in the epidemiology of β-thalassemia

β-thalassemia major is an inherited hemoglobinopathy that requires lifelong red blood cell transfusions and iron chelation therapy to prevent complications due to iron overload. Traditionally, β-thalassemia has been more common in certain regions of the world such as the Mediterranean, Middle East, and Southeast Asia. However, the prevalence of β-thalassemia is increasing in other regions, including Northern Europe and North America, primarily due to migration. This review summarizes the available data on the changing incidence and prevalence of β-thalassemia as well as factors influencing disease frequency. The data suggest that the epidemiology of β-thalassemia is changing: Migration has increased the prevalence of the disease in regions traditionally believed to have a low prevalence, while, at the same time, prevention and screening programs in endemic regions have reduced the number of affected individuals. Various approaches to prevention and screening have been used. Region-specific prevention and treatment programs, customized to align with local healthcare resources and cultural values, have been effective in identifying patients and carriers and providing information and care. Significant challenges remain in universally implementing these programs.     

Newer Developments in the Identification of β-Thalasseuia

One of the easiest and most sensitive methods of detecting mutations in the β-globin gene leading to β-thalassemia is by the use of oligonucleotide probes. The current method involves digestion of 5–10 μg of genomic DNA followed by gel electrophoresis, and blotting onto nitrocellulose. The membrane is then hybridized with a 32P-radiolabeled oligonucleotide probe containing the specific point mutation of interest. Finally, the membrane is subjected to X-ray film for 3–10 days. We wish to report a method for detecting these mutations which involves 1 μg of genome DNA or less. The method involves the use of a gene amplification technique. A series of primers are synthesized which span the β-globin gene. In each primer set, one primer is complementary to the β-gene and the other primer is complementary to the non-coding strand. The suspected mutation point is located between these two primers. With the use of this primer set, the β-globin gene region is amplified by denaturing, annealing, and DNA synthesis. The amplification cycle is repeated 25 to 30 times. The amplification is conducted using the Klenow fragment of DNA polymerase I or Taq polymerase in the presence of all four deoxynucleotide triphosphates. The resulting amplified DNA is applied to a nylon membrane with the aid of a dot-blot apparatus and directly hybridized with normal and mutant deoxynucleotide probes. The entire process requires one to two days. More than 300 β-thalassemia homozygotes have been identified in our laboratories; over 20 different mutations have been observed.     

The Frequency of the AγT Gene in the Presence and Absence of the βs or βC Gene in the Black Poplilation of the Southeastern USA

Quantitative information about the three types of γ chains (^Aγ^{T, A}γ^{I, C}γ) has been obtained for the Hb F from 285 normal Black babies, 172 babies with a Hb S or Hb C heterogeneity, and from 150 babies and older patients with the SS, SC, or CC conditions by means of a high pressure liquid chromatographic micro-procedure. The frequency of the ^Aγ^T gene in the AA babies was 0.1035, while that in the SS patients was a low 0.0362. This low ^Aγ frequency in the SS POulation adequately explains the lower percentage (13.2%) of Aγ^T heterozygotes among AS newborn babies as compared to 17.9% among AA Babies. The genotype with the A_yT mutant in trans to the β^S mutation is presumed to occur about three times more frequently than that in which the A_yT mutant is in cis to the β^S mutation. A study of family members of one SS patient who has an A_yT homozygosity provided data supporting linkage of the A_yT and β^S anomalies in some Hb S heterozygotes.     

Steroids protect the β-adrenergic system during reperfusion after ischemia: Effects of methylprednisolone on the β-adrenergic response system

Methylprednisolone sodium succinate (MPSS) administered during reperfusion may improve myocardial function. These effects have been related to adrenergic stimulation. The present study investigated (1) the effects of ischemia and reperfusion on the β-adrenergic response system and (2) the ability of MPSS to modify the ischemic effects on the β-adrenergic system. Isolated perfused rat hearts were used. The ischemic protocol consisted of aerobic perfusion (20 minutes) followed by total, global normothermic (37°C) ischemia (30 minutes) and reperfusion (30 minutes) with MPSS (0, 100, 500, or 1,000 mg/L). The non-ischemic protocol consisted of aerobic perfusion (20 minutes) followed by aerobic perfusion (20 minutes) with MPSS (0, 100, 500, or 1,000 mg/L). At the end of the experiments all hearts were rapidly frozen in liquid nitrogen. Crude sarcolemmal membranes were prepared and stimulated at the β-receptor, at the coupling (G_s- or N-) protein, or directly at the adenylate cyclase enzyme (AC). Results were assessed by cyclic adenosine monophosphate (cAMP) production. Tissue specimens were analyzed for myocardial content of cAMP and methylprednisolone (MP). In the ischemic protocol, the responsiveness of the β-adrenergic system was significantly reduced at the G_s-protein level. The treatment with MPSS (100 or 500 mg/L) during reperfusion preserved the β-adrenergic response. MPSS (1,000 mg/L) offered no protection. In the non-ischemic protocol, MPSS reduced the response of the β-adrenergic system in a dose-dependent manner at the same level. The hearts in the ischemic protocol had significantly higher contents of MP than the hearts in the non-ischemic protocol at corresponding concentrations of MPSS. The present study suggests that postischemic cardiac failure may result in part from β-adrenergic dysfunction. This loss of function, probably at the level of the protein connecting the receptor and AC, can successfully be prevented by an optimal dose of MPSS during reperfusion after ischemia.     

Assessing the severity of the small inframe deletion mutation in the α-subunit of β-hexosaminidase A found in the Turkish population by reproducing it in the more stable β-subunit

GM(2) gangliosidoses are a group of panethnic lysosomal storage diseases in which GM(2) ganglioside accumulates in the lysosome due to a defect in one of three genes, two of which encode the alpha- or beta-subunits of beta- N -acetylhexosaminidase (Hex) A. A small inframe deletion mutation in the catalytic domain of the alpha-subunit of Hex has been found in five Turkish patients with infantile Tay-Sachs disease. To date it has not been detected in other populations and is the only mutation to be found in exon 10. It results in detectable levels of inactive alpha-protein in its precursor form. Because the alpha- and beta-subunits share 60% sequence identity, the Hex A and Hex B genes are believed to have arisen from a common ancestral gene. Thus the subunits must share very similar three-dimensional structures with conserved functional domains. Hex B, the beta-subunit homodimer is more stable than the heterodimeric Hex A, and much more stable than Hex S, the alpha homodimer. Thus, mutations that completely destabilize the alpha-subunit can often be partially rescued if expressed in the aligned positions in the beta-subunit. To better understand the severity of the Turkish HEXA mutation, we reproduced the 12 bp deletion mutation (1267-1278) in the beta-subunit cDNA. Western blot analysis of permanently transfected CHO cells expressing the mutant detected only the pro-form of the beta-subunit coupled with a total lack of detectable Hex B activity. These data indicate that the deletion of the four amino acids severely affects the folding of even the more stable beta-subunit, causing its retention in the endoplasmic reticulum and ultimate degradation.     

RFX6 is needed for the development and maintenance of the β-cell phenotype

RFX6 has recently been found to be essential for the development of the endocrine pancreas through studies in both humans and mice. Interestingly, this gene maintains a specific expression in the postnatal hormone producing cells of the pancreas. Moreover in humans, different types of diabetes mellitus affect obligate carriers of RFX6 mutations. Therefore, RFX6 appears to have a pivotal role in the maintenance of the phenotype of the β-cells in addition to their development. Extensive research is needed to specify this role and explore its significance in the efforts to treat diabetes with medications, or more importantly, through β-cell replacement.     

Deletion of the β2-adrenergic receptor prevents the development of cardiomyopathy in mice

Beta adrenergic receptor (β-AR) subtypes act through diverse signaling cascades to modulate cardiac function and remodeling. Previous in vitro studies suggest that β1-AR signaling is cardiotoxic whereas β2-AR signaling is cardioprotective, and may be the case during ischemia/reperfusion in vivo. The objective of this study was to assess whether β2-ARs also play a cardioprotective role in the pathogenesis of non-ischemic forms of cardiomyopathy. To dissect the role of β1 vs β2-ARs in modulating MLP (Muscle LIM Protein) cardiomyopathy, we crossbred MLP −/− with β1 −/− or β2 −/− mice. Deletion of the β2-AR improved survival, cardiac function, exercise capacity and myocyte shortening; by contrast haploinsufficency of the β1-AR reduced survival. Pathologic changes in Ca^2 + handling were reversed in the absence of β2-ARs: peak Ca^2 + and SR Ca^2 + were decreased in MLP −/− and β1 +/−/MLP −/− but restored in β2 −/− MLP −/−. These changes were associated with reversal of alterations in troponin I and phospholamban phosphorylation. Gi inhibition increased peak and baseline Ca^2 +, recapitulating changes observed in the β2 −/−/MLP −/−. The L-type Ca^2 + blocker verapamil significantly decreased cardiac function in β2 −/− MLP −/− vs WT. We next tested if the protective effects of β2-AR ablation were unique to the MLP model using TAC-induced heart failure. Similar to MLP, β2 −/− mice demonstrated delayed progression of heart failure with restoration of myocyte shortening and peak Ca^2 + and Ca^2 + release. Deletion of β2-ARs prevents the development of MLP −/− cardiomyopathy via positive modulation of Ca^2 + due to removal of inhibitory Gi signaling and increased phosphorylation of troponin I and phospholamban. Similar effects were seen after TAC. Unlike previous models where β2-ARs were found to be cardioprotective, in these two models, β2-AR signaling appears to be deleterious, potentially through negative regulation of Ca^2 + dynamics.     

Backtracking on the folding landscape of the β-trefoil protein interleukin-1β?

Interleukin-1β (IL-1β) is a cytokine within the β-trefoil family. Our data indicate that the folding/unfolding routes are geometrically frustrated. Follow-up theoretical studies predicted backtracking events that could contribute to the broad transition barrier and the experimentally observed long-lived intermediate. The backtracking route is attributed to the topological frustration introduced by the packing of the functional loop (the β-bulge, residues 47–53) to the nascent barrel. We used real-time refolding NMR experiments to test for the presence of backtracking events predicted from our theoretical studies. Structural variants of IL-1β, a β-bulge deletion, and a circular permutation that opens the protein in the middle of the experimentally observed kinetic intermediate, were also refolded and studied to determine the affects on the observed folding reactions. The functional loop deletion variant demonstrated less backtracking than in WT protein whereas the permutation still maintains backtracking in agreement with theoretical predictions. Taken together, these findings indicate that the backtracking results from geometric frustration introduced into the fold for functional purposes.     

Molecular basis of β-thalassemia in the United Arab Emirates

In an attempt to define the prevalence of β-thalassemia (β-thal) in the United Arab Emirates (UAE), we have conducted molecular studies on nearly 2000 randomly-selected adult UAE nationals. The results demonstrated that the prevalence of β-globin gene defects in the UAE was 8.5%. Among these anomalies were β-thal mutations, abnormal hemoglobin (Hb) variants, e.g., Hb S, Hb D-Punjab, Hb O-Arab, Hb C and Hb E. The sickle gene (β(S) or Hb S) contributed significantly to the molecular epidemiology of the hemoglobinopathies in the UAE. In this article, Hb S and other abnormal Hbs are excluded as they are comprehensively described by other contributors in this current issue. The molecular characterization and mutational analyses of all β-thal patients were carried out using current molecular techniques including amplification refractory mutation system (ARMS), restriction enzyme analysis (REA), dot-blot hybridization, β-strip hybridization, allele-specific oligonucleotide (ASO), polymerase chain reaction (PCR), gap-PCR and DNA Sequencing. Most of these techniques are now virtually obsolete. Almost all molecular characterizations are currently performed through PCR followed by DNA sequencing using a fully automated ABI PRISM? 3130 Genetic Analyzer. Our molecular studies showed that the majority of the β-thal mutations in the UAE are very severe; the most common allele was the IVS-I-5 (G>C). Although this allele is a β(+)-thal, its phenotype is very severe. All other mutations are also severe β(0)-thal. High frequency of moderate or severe β-thal mutations have implications in the wide spectrum of clinical manifestations seen in patients whose phenotypes vary from β-thal intermedia (β-TI) to severe transfusion-dependent β-thal major (β-TM). The molecular pathology of the β-thal patients demonstrated that a vast majority were homozygous. The most frequent homozygous mutation was the IVS-I-5(G>C)/IVS-I-5(G>C) (53.0%) followed by -25 bp del/-25 bp del (6.8%), codons 8/9(+G)/codons 8/9(+G) (2.8%) and codon 39(C>T)/codon 39(C>T) (2.4%). Four mutations accounted for 65.0% of the homozygous patient population. Remarkably, the two most prevalent mutations, IVS-I-5 and Hb S, accounted for 77% of all the homozygous β-thal patients from the UAE. We showed 13 discrete homozygosities in the UAE national patients in contrast to 23 homozygosities in the expatriate population. Since the number of homozygous mutations has a direct correlation with the degree of consanguinity, the data shown here corroborate the social tendency towards family planning. In fact, in the UAE, more than 50% of all marriages are between relatives and more than half of these are between first cousins.