Fasting induces ketoacidosis and hypothermia in PDHK2/PDHK4-double-knockout mice

The importance of PDHK (pyruvate dehydrogenase kinase) 2 and 4 in regulation of the PDH complex (pyruvate dehydrogenase complex) was assessed in single- and double-knockout mice. PDHK2 deficiency caused higher PDH complex activity and lower blood glucose levels in the fed, but not the fasted, state. PDHK4 deficiency caused similar effects, but only after fasting. Double deficiency intensified these effects in both the fed and fasted states. PDHK2 deficiency had no effect on glucose tolerance, PDHK4 deficiency produced only a modest effect, but double deficiency caused a marked improvement and also induced lower insulin levels and increased insulin sensitivity. In spite of these beneficial effects, the double-knockout mice were more sensitive than wild-type and single-knockout mice to long-term fasting, succumbing to hypoglycaemia, ketoacidosis and hypothermia. Stable isotope flux analysis indicated that hypoglycaemia was due to a reduced rate of gluconeogenesis and that slightly more glucose was converted into ketone bodies in the double-knockout mice. The findings establish that PDHK2 is more important in the fed state, PDHK4 is more important in the fasted state, and survival during long-term fasting depends upon regulation of the PDH complex by both PDHK2 and PDHK4.     

Mechanism of Hyperinsulinism in Short-chain 3-Hydroxyacyl-CoA Dehydrogenase Deficiency Involves Activation of Glutamate Dehydrogenase

The mechanism of insulin dysregulation in children with hyperinsulinism associated with inactivating mutations of short-chain 3-hydroxyacyl-CoA dehydrogenase (SCHAD) was examined in mice with a knock-out of the hadh gene (hadh^−/−). The hadh^−/− mice had reduced levels of plasma glucose and elevated plasma insulin levels, similar to children with SCHAD deficiency. hadh^−/− mice were hypersensitive to oral amino acid with decrease of glucose level and elevation of insulin. Hypersensitivity to oral amino acid in hadh^−/− mice can be explained by abnormal insulin responses to a physiological mixture of amino acids and increased sensitivity to leucine stimulation in isolated perifused islets. Measurement of cytosolic calcium showed normal basal levels and abnormal responses to amino acids in hadh^−/− islets. Leucine, glutamine, and alanine are responsible for amino acid hypersensitivity in islets. hadh^−/− islets have lower intracellular glutamate and aspartate levels, and this decrease can be prevented by high glucose. hadh^−/− islets also have increased [U-¹⁴C]glutamine oxidation. In contrast, hadh^−/− mice have similar glucose tolerance and insulin sensitivity compared with controls. Perifused hadh^−/− islets showed no differences from controls in response to glucose-stimulated insulin secretion, even with addition of either a medium-chain fatty acid (octanoate) or a long-chain fatty acid (palmitate). Pull-down experiments with SCHAD, anti-SCHAD, or anti-GDH antibodies showed protein-protein interactions between SCHAD and GDH. GDH enzyme kinetics of hadh^−/− islets showed an increase in GDH affinity for its substrate, α-ketoglutarate. These studies indicate that SCHAD deficiency causes hyperinsulinism by activation of GDH via loss of inhibitory regulation of GDH by SCHAD.     

In vitro and in vivo evaluation of microbial-enriched compost tea on the development of powdery mildew on melon

This study evaluated the effects of microbial-enriched compost tea (CT) on the conidial germination of Golovinomyces cichoracearum DC. and development of powdery mildew on melons in a time-dependent manner. In vitro conidial germination was significantly reduced by 94?% and 85?% upon treatment with Daconil^? (fungicide) or microbial-enriched CT, respectively, 96?h after incubation (hai). Morphological analysis under light microscopy demonstrated that conidia co-incubated with microbial-enriched CT at 48?hai appeared ruptured, which contributed to higher inhibition of conidial germination, increased cell permeability and leakage of cellular contents. These observations may be explained by antibiosis. Moreover, different application time of microbial-enriched CT on melons significantly affected disease development. There was a delay in disease development by 12?days in plants treated with Daconil^?, microbial-enriched CT applied 24?h after inoculation and microbial-enriched CT applied simultaneously with inoculation when compared to the control treatment. Curative application of microbial-enriched CT (24?h after inoculation) delayed the onset of disease, and the efficiency of inhibition was comparable to a fungicidal spray (Daconil^?). Hence, microbial-enriched CT may be used to inhibit the development of powdery mildew on melons, thus reducing the dependency on chemical fertilisers.     

Copper sensing based on the far-red fluorescent protein, HcRed, from Heteractis crispa

In this article, we report for the first time on the copper (Cu(2+)) binding characteristics of the far-red fluorescent protein, HcRed, and its application in the development of a reagentless sensing system for copper. The far-red emission of HcRed (lambda(max) = 645 nm) where background cellular fluorescence is low should prove to be advantageous in the development of the sensing system. In the studies performed in our laboratory, we found that the fluorescence of HcRed is quenched in the presence of copper ions (Cu(2+)). The results obtained through UV-visible and circular dichroism spectra generated in the presence and absence of copper, as well as Stern-Volmer plots at different temperatures, indicate static quenching of HcRed fluorescence in the presence of copper, possibly through the formation of a copper-protein complex. On the basis of this observation, we developed a reagentless sensing system for the detection of copper(II) based on HcRed as the biosensing element. A detection limit for Cu(2+) in the nanomolar range was obtained. HcRed was found to bind copper ions selectively when compared with other divalent ions. A dissociation constant of 3.6muM was observed for copper binding. Histidine and cysteine residues are commonly involved in copper binding within proteins; therefore, to investigate the role of these amino acids present in HcRed, we chemically modified Cys and His residues using iodoacetamide and diethyl pyrocarbonate, respectively. The effect of copper addition on the fluorescence of the chemically modified HcRed was investigated. The His modification of HcRed substantially affected copper ion binding, pointing to histidine as the possible amino acid residue involved in the binding of copper ions in HcRed. A purification strategy for HcRed was also developed based on a copper immobilized affinity column without the addition of any affinity tag on the protein. The HcRed-based copper sensing system can potentially be employed to perform intracellular copper detection by genetically encoding the biosensing element or can be employed in environmental sensing.     

Influences on Trimerization and Aggregation of Soluble,Cleaved HIV-1 SOSIP Envelope Glycoprotein

We describe methods to improve the properties of soluble, cleaved gp140 trimers of the human immunodeficiency virus type 1 (HIV-1) envelope glycoproteins (Env) for use in structural studies and as immunogens. In the absence of nonionic detergents, gp140 of the KNH1144 genotype, terminating at residue 681 in gp41 (SOSIP.681), has a tendency to form higher-order complexes or aggregates, which is particularly undesirable for structure-based research. We found that this aggregation in the absence of detergent does not involve the V1, V2, or V3 variable regions of gp120. Moreover, we observed that detergent forms micelles around the membrane-proximal external region (MPER) of the SOSIP.681 gp140 trimers, whereas deletion of most of the MPER residues by terminating the gp140 at residue 664 (SOSIP.664) prevented the aggregation that otherwise occurs in SOSIP.681 in the absence of detergent. Although the MPER can contribute to trimer formation, truncation of most of it only modestly reduced trimerization and lacked global adverse effects on antigenicity. Thus, the MPER deletion minimally influenced the kinetics of the binding of soluble CD4 and a CD4-binding site antibody to immobilized trimers, as detected by surface plasmon resonance. Furthermore, the MPER deletion did not alter the overall three-dimensional structure of the trimers, as viewed by negative-stain electron microscopy. Homogeneous and aggregate-free MPER-truncated SOSIP Env trimers are therefore useful for immunogenicity and structural studies.     

Respiratory mechanics following chronic cigarette smoke exposure in the Apoe $$^{-/-}$$ mouse model

Biomechanics and Modeling in Mechanobiology - Even though cigarette smoking (CS) has been on the decline over the past 50 years, it is still the leading cause of preventable premature death in the...     

Structural Bioinformatics of <Emphasis Type="Italic">Neisseria meningitidis</Emphasis> LD-Carboxypeptidase: Implications for Substrate Binding and Specificity

Neisseria meningitidis, a gram negative bacterium, is the leading cause of bacterial meningitis and severe sepsis. Neisseria meningitidis genome contains 2,160 predicted coding regions including 1,000 hypothetical genes. Re-annotation of N. meningitidis hypothetical proteins identified nine putative peptidases. Among them, the NMB1620 protein was annotated as LD-carboxypeptidase involved in peptidoglycan recycling. Structural bioinformatics studies of NMB1620 protein using homology modeling and ligand docking were carried out. Structural comparison of substrate binding site of LD-carboxypeptidase was performed based on binding of tetrapeptide substrate ‘l-alanyl-d-glutamyl-meso-diaminopimelyl-d-alanine’. Inspection of different subsite-forming residues showed changeability in the S1 subsite across different bacterial species. This variability was predicted to provide a structural basis to S1-subsite for accommodating different amino acid residues at P1 position of the tetrapeptide substrate ‘l-alanyl-d-glutamyl-meso-diaminopimelyl-d-alanine’.