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

Reviews in Endocrine and Metabolic Disorders -     

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.

---

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.

     

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.     

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.     

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.     

Analysis of the levels of endotoxin and β-d-glucan in the synovial fluid of hemodialysis patients

Abstract

We analyzed the levels of endotoxin and β-d-glucan, which possibly induce cytokine production, in the synovial fluid of patients on long-term hemodialysis and compared the results to those in patients with osteoarthritis and rheumatoid arthritis. We studied 42 knees in 42 hemodialysis patients, 21 in 21 osteoarthritis patients, and 26 in 26 rheumatoid arthritis patients. The mean ages were 60.7, 63.2, and 59.7 years, respectively. The duration of hemodialysis in the long-term hemodialysis group averaged 14.0 years. The concentrations of endotoxin and β-d-glucan in the synovial fluid of these three groups were measured. The concentration of endotoxin was the same in the three groups. However, the concentration of β-d-glucan was significantly higher in long-term hemodialysis patients. This finding suggests that β-d-glucan may have some relation to the pathogenesis of the synovitis which exists in the hydrarthrosis of long-term hemodialysis patients.     

Distribution of β-Thalassemia Mutations in the Northern Provinces of Iran

β-Thalassemia (thal) is one of the most common autosomal recessive disorders in Iran. There are more than two million carriers of β-thal and over 15,000 people affected with β-thal major who live in Iran. Prevalent mutations were identified by examining genomic DNAs isolated from 392 blood samples of β-thal carriers from three northern provinces of Iran. Furthermore, 172 pregnant women were analyzed from the 196 couples who requested pregnant diagnosis for β-thal. Allele identification was carried out using routine reverse dot-blot, amplification refractory mutation system (ARMS), and genomic sequencing. The most common mutation, IVS-II-1 (G→A), is followed, in order of frequency, by codon 30 (G→C), frameshift codons (FSC) 8,9 (+G), FSC 22/23/24 (?AAGTTGG), IVS-I-110 (G→A), IVS-I-5 (G→C), IVS-II-745 (C→G), IVS-I-2 (T→C), FSC 8 (?AA), IVS-I,3′-end (?25 bp), IVS-I-1 (G→A), FSC 36/37 (?T), IVS-I-6 (T→C), FSC 5 (?CT), ?28 (A→C), codon 37 (G→A), IVS-II-2,3 (+11/?2), ?30 (T→A), and ?88 (C→A). We have also revealed the existence of five new mutations from northern Iran, one of which (codon 37) is the first reported for Iran. Furthermore, the rate of unknown mutations is significantly reduced in our study (about 6%). These results could help with establishing a center for prenatal diagnosis, prevention, and control of thalassemia in the northern provinces of Iran.     

Molecular Characterization of β-Thalassemia in the Dohuk Region of Iraq

β-Thalassemia (thal) is an important health problem in the Dohuk region of northern Iraq because of its high carrier rate and the frequency of consanguineous marriages. Thus, the need to establish an effective preventative program is paramount. As part of this effort, we initiated this study to determine the molecular basis of this disorder in the region. For the latter purpose, either parent of 104 registered β-thal major/intermedia patients had their full blood counts, hemoglobin (Hb) electrophoresis, Hb A₂ and Hb F quantitation performed. Their DNA was extracted, amplified and reverse hybridized to specific oligonucleotide probes to detect 20 β-thal mutations. The testing detected 12 β-thalassemic mutations. The eight most frequent were: IVS-II-1 (G→A), codon 44 (–C), codon 5 (–CT), IVS-I-1 (G→A), codon 39 (C→T), IVS-I-6 (T→C), codons 8/9 (+G) and IVS-I-5 (G→C). These mutations accounted for 81.7% of the thalassemic defects in the studied individuals. The less frequent mutations were: codon 8 (–AA), IVS-I-110 (G→A), codon 30 (G→C) and codon 22 (?7 bp), and the β-thalassemic defects remained uncharacterized in 11.5% of cases. This is the first study of β-thal mutations from Iraq, and shows a frequency of thalassemic defects different from those reported in surrounding countries. It provides a foundation for prenatal genetic testing that will be part of a thalassemia prevention program in the Dohuk region.     

Molecular basis of β-thalassemia in the western province of Saudi Arabia: identification of rare β-thalassemia mutations

This study aimed at the identification of the spectrum of mutations in patients with β-thalassemia (β-thal) in the western province of Saudi Arabia. Screening for the mutations was done using the polymerase chain reaction-amplification refractory mutation system (PCR-ARMS) technique to test for 12 mutations, and direct automated DNA sequencing for the unknown samples. The study included 172 patients; of these 15 patients had sickle cell anemia and one Hb S [β6(A3)Glu→Val, GAG>GTG]/β-thal. A total of 23 mutations were identified to cause the disease in the western area. Seven common mutations were responsible for the β-thal alleles in 78% of patients and could be detected by the ARMS technique: IVS-II-1 (G>A), IVS-I-110 (G>A), IVS-I-5 (G>C), codon 39 (C>T), codon 26 (G>A) [Hb E or β26(B8)Glu→Lys, GAG>AAG], frameshift codons (FSC) 8/9 (+G), and IVS-I-1 (G>A). DNA sequencing of uncharacterized alleles detected eight less common mutations: FSC 41/42 (-TCTT), IVS-I 25 bp deletion, codon 37 (G>A), FSC 44 (-C), Cap site +1 (A>C), IVS-I-6 (T>C), FSC 5 (-CT) and IVS-I-1 (G>T), and eight rare mutations: -87 (C>G), initiation codon -1 (T>G), codon 15 (G>A), FSC 16 (-C), FSC 20/21 (+G), codon 27 (G>A), IVS-I-130 (G>C) and IVS-II-837 (A>C). Four alleles were normal by DNA sequencing. Genetic heterogeneity was observed in this study, 10 mutations were of Asian or Asian/Indian origin, two were Kurdish, one Chinese, one Turkish, one Saudi, and the remainder were of Mediterranean origin. The presence of a large population of immigrants in the western province is responsible for the great heterogeneity at the molecular level, and for the difference observed in the frequencies of mutations from those reported in the eastern province of Saudi Arabia. Screening for β-thal mutations using PCR-ARMS for the seven most frequent mutations in the Saudi population followed by DNA sequencing of the unknown alleles could be useful for the implementation of a strategy for carrier detection and preimplantation genetic diagnosis in high risk families.     

Characterization of Two Novel Deletions Involving the 5′ Region of the β-Globin Gene

We report two novel β-thalassemia (β-thal) deletions involving the 5′ region of the β-globin gene (HBB). The first deletion spans 538?bp and removes the β-globin promoter, 5′ untranslated region (5′UTR) and most of exon 1. This deletion was identified in a 3-year-old Vietnamese boy with non transfusion dependent Hb E (HBB: c.79G>A)/β⁰-thal. The second deletion spans 1517?bp and removes the β-globin gene promoter, 5′UTR, and exons 1 and 2. This deletion was identified in two unrelated adults of European descent who had β-thal trait with unusually high Hb A₂ levels. Deletions such as these are generally associated with higher levels of Hb A₂ and Hb F than typical β-thal alleles, which may ameliorate the severity of the disease.     

Spectrum of β-thalassemia mutations in the eastern province of Saudi Arabia

β-Thalassemias comprise a group of heterogeneous hemoglobin (Hb) disorders characterized by the absence or reduced synthesis of the β-globin chain with a variable clinical presentation. The Al-Qatif and Al-Ahsa oases in the Eastern Province of Saudi Arabia are regions known for the high prevalence of these disorders. This study was conducted to provide a more precise picture of the β-thalassemia (β-thal) mutations prevalent in these regions and to estimate their frequencies. One hundred and 96 subjects with transfusion-dependent β-thalassemia (β-thal) disease were included in this study. A total of 14 β-thal mutations were identified with five mutations accounting for more than 80% of the total β-thal mutations identified. Of the 196 patients, 164 were homozygous for a β-thal mutation, while 32 were compound heterozygotes. We report here the novel identification of two mutations, namely, the Tunisian splice site IVS-I-130 (G→C) and the Mediterranean cryptic splice site IVS-I-110 (G→A), which have not been previously reported in the population of the Eastern Province. However, 15 patients (46.9%) with compound heterozygosities carried one of the β-thal mutations and the sickle cell mutation [Hb S or β6(A3)Glu→Val]. These patients were less frequently transfused than the patients who were homozygous for the β-thal mutations and presented with fewer complications. A more comprehensive overview of the genetic heterogeneity of the β-thal mutations in the Eastern Province of Saudi Arabia is presented in this article. This study will contribute to the establishment of an effective prevention program, including premarital screening.     

Compromising the phosphodependent regulation of the GABAAR β3 subunit reproduces the core phenotypes of autism spectrum disorders

Alterations in the efficacy of neuronal inhibition mediated by GABA_A receptors (GABA_ARs) containing β3 subunits are continually implicated in autism spectrum disorders (ASDs). In vitro, the plasma membrane stability of GABA_ARs is potentiated via phosphorylation of serine residues 408 and 409 (S408/9) in the β3 subunit, an effect that is mimicked by their mutation to alanines. To assess if modifications in β3 subunit expression contribute to ASDs, we have created a mouse in which S408/9 have been mutated to alanines (S408/9A). S408/9A homozygotes exhibited increased phasic, but decreased tonic, inhibition, events that correlated with alterations in the membrane stability and synaptic accumulation of the receptor subtypes that mediate these distinct forms of inhibition. S408/9A mice exhibited alterations in dendritic spine structure, increased repetitive behavior, and decreased social interaction, hallmarks of ASDs. ASDs are frequently comorbid with epilepsy, and consistent with this comorbidity, S408/9A mice exhibited a marked increase in sensitivity to seizures induced by the convulsant kainic acid. To assess the relevance of our studies using S408/9A mice for the pathophysiology of ASDs, we measured S408/9 phosphorylation in Fmr1 KO mice, a model of fragile X syndrome, the most common monogenetic cause of ASDs. Phosphorylation of S408/9 was selectively and significantly enhanced in Fmr1 KO mice. Collectively, our results suggest that alterations in phosphorylation and/or activity of β3-containing GABA_ARs may directly contribute to the pathophysiology of ASDs.GABA_A receptors (GABA_ARs) are Cl⁻ selective ligand-gated ion channels that mediate phasic and tonic inhibition in the adult brain. Consistent with their roles in limiting neuronal excitability, benzodiazepines, barbiturates, general anesthetics, and neurosteroids exert their anxiolytic, anticonvulsant, hypnotic, and sedative effects via potentiating GABA_AR activity (1). GABA_ARs are heteropentamers constructed from α1–6, β1–3, γ1–3, δ, ε, θ, and π subunits. Phasic inhibition is principally mediated by receptors assembled from α1–3, β1–3, and γ2 subunits, whereas those receptors that mediate tonic inhibition contain α4–6, β1–3, and δ subunits (2). Studies using KO mice have shown that the β3 subunit is an essential component of receptor subtypes that mediate phasic and tonic inhibition (3). Together with the Fmr1 gene (Fragile X mental retardation), mutations to the 15q11–13 locus, where the GABA_AR β3 gene resides, are the leading monogenetic causes of autism spectrum disorders (ASDs) (4). Moreover, β3 subunit mutations have been described in seizure disorders, and alterations in subunit expression levels have also been reported in ASDs (3, 5).In vitro studies have revealed that the β3 subunit plays a critical role in regulating the plasma membrane accumulation and synaptic targeting of GABA_ARs via phosphorylation of the intracellular serine residues 408 and 409 (S408/9) (6, 7). S408/9 are substrates of cAMP-dependent PKA, PKC, Ca²⁺-calmodulin type 2-dependent protein kinases (Cam KIIs), and cGMP-dependent protein kinase, and they are principally dephosphorylated by protein phosphatase 2A (8). S408/9 are the principal mediators of high-affinity binding to the clathrin adaptor molecule AP2 within the β3 subunit, and thereby facilitate GABA_AR endocytosis (9). Phosphorylation of S408/9 reduces the affinity of the β3 subunit for AP2 by 100-fold, and mutation of S408/9 to alanine residues (S408/9A) has been shown to mimic the effects of phosphorylation on AP2 binding to the β3 subunit (9, 10). Accordingly, overexpression of the mutant β3 S408/9A subunit in cultured hippocampal neurons leads to an increase in the number and size of inhibitory synapses (7).Studies in animal models of ASDs have reported modifications in the expression levels of some GABA_AR mRNAs and proteins (11, 12). However, the mechanisms underlying these alterations in subunit expression and if they contribute to ASDs remain to be addressed. Therefore, in this study, we have analyzed the role that modified β3 subunit phosphorylation may play in the pathophysiology of ASDs. To test this role, we created a mouse in which the principal sites of phosphodependent regulation within the receptor β3 subunit, S408/9, have been mutated to S408/9A, a mutation that mimics the effects of their phosphorylation. S408/9A mice exhibited increased phasic but decreased tonic inhibition events, which correlated with alterations in the membrane stability of the receptor subtypes that mediate these distinct forms of inhibition. S408/9A mice exhibited alterations in dendritic spine structure, increased repetitive-like behavior, and decreased social interaction, which are hallmarks of ASDs. Therefore, our results provide evidence that alterations in the activity of GABA_ARs containing β3 subunits directly contribute to ASDs.     

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.