Out of synch: Clock mutation causes obesity in mice

The Clock gene encodes an essential component of the "master clock" driving circadian rhythm in the hypothalamic suprachiasmatic nucleus (SCN). New evidence that Clock mutant mice are hyperphagic and obese suggests a previously unrecognized link between molecular controls of circadian rhythm and energy homeostasis.     

Methotrexate binding causes structural and functional changes in lung cystatin

Regulation of cysteine proteinases and their inhibitors is of utmost importance in diseases like lung cancer, chronic inflammatory conditions such as asthma, emphysema, and idiopathic pulmonary fibrosis. Protease-antiprotease imbalance accelerates disease progression. In the present study, the effect of antineoplastic and antirheumatic drug methotrexate (MTX) on lung cystatin (a cysteine protease inhibitor) was studied to explore drug induced changes in functional and structural integrity of the protein. The basic binding interaction was studied by UV-absorption, FT-IR and fluorescence spectroscopy. The quenching of protein fluorescence confirmed the binding of MTX with goat lung cystatin (GLC-I). Stern-Volmer analysis of MTX-GLC-I system at different temperatures indicates the presence of static component in the quenching mechanism. The thermodynamic parameters ΔH? and ΔS? were -3.8 kJ/mol and 94.97 J?mol?1?K?1, respectively, indicating that both hydrogen bonds and hydrophobic interactions played a major role in the binding of MTX to GLC-I. Methotrexate (7 μM) caused complete inactivation of lung cystatin after 6 hours. The results of FT-IR spectroscopy reflect perturbation of the goat lung cystatin on interaction with MTX. Methotrexate induced loss of function change in the inhibitor could provide a rationale for the off target tissue injury caused by the drug and for the design of agents against such an injury.     

Reduced pH causes structural changes in the potent mitogenic toxin of Pasteurella multocida

Pasteurella multocida toxin is a potent mitogen that is believed to act intracellularly. On transverse urea gradient gels at pH 8.0 the toxin displayed one major unfolding transition at 4 M urea. However, at pH 6.1 the unfolding transition took place at 3.5 M urea. Circular dichroism spectra also indicated that a structural change took place at acidic pH. In addition it was found that the toxin that had been denatured in 8 M urea refolded in solution with a high recovery of biological activity. These findings are discussed in terms of the likely domain structure of the P. multocida toxin.     

A homozygous mutation in LTBP2 causes isolated microspherophakia

Microspherophakia is an autosomal-recessive congenital disorder characterized by small spherical lens. It may be isolated or occur as part of a hereditary systemic disorder, such as Marfan syndrome, autosomal dominant and recessive forms of Weill-Marchesani syndrome, autosomal dominant glaucoma–lens ectopia–microspherophakia–stiffness–shortness syndrome, autosomal dominant microspherophakia with hernia, and microspherophakia-metaphyseal dysplasia. The purpose of this study was to map and identify the gene for isolated microspherophakia in two consanguineous Indian families. Using a whole-genome linkage scan in one family, we identified a likely locus for microspherophakia (MSP1) on chromosome 14q24.1–q32.12 between markers D14S588 and D14S1050 in a physical distance of 22.76 Mb. The maximum multi-point lod score was 2.91 between markers D14S1020 and D14S606. The MSP1 candidate region harbors 110 reference genes. DNA sequence analysis of one of the genes, LTBP2, detected a homozygous duplication (insertion) mutation, c.5446dupC, in the last exon (exon 36) in affected family members. This homozygous mutation is predicted to elongate the LTBP2 protein by replacing the last 6 amino acids with 27 novel amino acids. Microspherophakia in the second family did not map to this locus, suggesting genetic heterogeneity. The present study suggests a role for LTBP2 in the structural stability of ciliary zonules, and growth and development of lens.     

A PRKAG2 mutation causes biphasic changes in myocardial AMPK activity and does not protect against ischemia

Dominant mutations in the gamma2 regulatory subunit of AMP-activated protein kinase (AMPK), encoded by the gene PRKAG2, cause glycogen storage cardiomyopathy. We sought to elucidate the effect of the Thr400Asn (T400N) human mutation in a transgenic mouse (TGT400N) on AMPK activity, and its ability to protect the heart against ischemia-reperfusion injury. TGT400N hearts had markedly vacuolated myocytes, excessive accumulation of glycogen, hypertrophy, and preexcitation. Early activation of myocardial AMPK, followed by depression, and then recovery to wild-type levels was observed. AMPK activity correlated inversely with glycogen content. Partial rescue of the phenotype was observed when TGT400N mice were crossbred with TGalpha2DN mice, which overexpress a dominant negative mutant of the AMPK alpha2 catalytic subunit. TGT400N hearts had greater infarct sizes and apoptosis when subjected to ischemia-reperfusion. Increased AMPK activity is responsible for glycogen storage cardiomyopathy. Despite high glycogen content, the TGT400N heart is not protected against ischemia-reperfusion injury.     

A mutation in the pericentrin gene causes abnormal interneuron migration to the olfactory bulb in mice

Precise control of neuronal migration is essential for proper function of the brain. Taking a forward genetic screen, we isolated a mutant mouse with defects in interneuron migration. By genetic mapping, we identified a frame shift mutation in the pericentrin (Pcnt) gene. The Pcnt gene encodes a large centrosomal coiled-coil protein that has been implicated in schizophrenia. Recently, frame shift and premature termination mutations in the pericentrin (PCNT) gene were identified in individuals with Seckel syndrome and microcephalic osteodysplastic primordial dwarfism (MOPD II), both of which are characterized by greatly reduced body and brain sizes. The mouse Pcnt mutant shares features with the human syndromes in its overall growth retardation and reduced brain size. We found that dorsal lateral ganglionic eminence (dLGE)-derived olfactory bulb interneurons are severely affected and distributed abnormally in the rostral forebrain in the mutant. Furthermore, mutant interneurons exhibit abnormal migration behavior and RNA interference knockdown of Pcnt impairs cell migration along the rostal migratory stream (RMS) into the olfactory bulb. These findings indicate that pericentrin is required for proper migration of olfactory bulb interneurons and provide a developmental basis for association of pericentrin function with interneuron defects in human schizophrenia.     

Human alpha B-crystallin mutation causes oxido-reductive stress and protein aggregation cardiomyopathy in mice

The autosomal dominant mutation in the human alphaB-crystallin gene inducing a R120G amino acid exchange causes a multisystem, protein aggregation disease including cardiomyopathy. The pathogenesis of cardiomyopathy in this mutant (hR120GCryAB) is poorly understood. Here, we show that transgenic mice overexpressing cardiac-specific hR120GCryAB recapitulate the cardiomyopathy in humans and find that the mice are under reductive stress. The myopathic hearts show an increased recycling of oxidized glutathione (GSSG) to reduced glutathione (GSH), which is due to the augmented expression and enzymatic activities of glucose-6-phosphate dehydrogenase (G6PD), glutathione reductase, and glutathione peroxidase. The intercross of hR120GCryAB cardiomyopathic animals with mice with reduced G6PD levels rescues the progeny from cardiac hypertrophy and protein aggregation. These findings demonstrate that dysregulation of G6PD activity is necessary and sufficient for maladaptive reductive stress and suggest a novel therapeutic target for abrogating R120GCryAB cardiomyopathy and heart failure in humans.     

GABA-shunt enzymes activity in GH3 cells with reduced level of PMCA2 or PMCA3 isoform

GABA (γ-aminobutyric acid) is important neurotransmitter and regulator of endocrine functions. Its metabolism involves three enzymes: glutamate decarboxylase (GAD65 and GAD67), GABA aminotransferase (GABA-T) and succinic semialdehyde dehydrogenase (SSADH). As many cellular processes GABA turnover can depend on calcium homeostasis, which is maintained by plasma membrane calcium ATPases (PMCAs). In excitable cells PMCA2 and PMCA3 isoforms are particularly important. In this study we focused on GABA-metabolizing enzymes expression and activity in rat anterior pituitary GH3 cells with suppressed expression of PMCA2 or PMCA3. We observed that PMCA3-reduced cells have increased GAD65 expression. Suppression of PMCA2 caused a decrease in total GAD and GABA-T activity. These results indicate that PMCA2 and PMCA3 presence may be an important regulatory factor in GABA metabolism. Results suggest that PMCA2 and PMCA3 function is rather related to regulation of GABA synthesis and degradation than supplying cells with metabolites, which can be potentially energetic source.     

Rod photoreceptor ribbon synapses in DBA/2J mice show progressive age-related structural changes

The DBA/2J mouse is a commonly used animal model in glaucoma research. The eyes of DBA/2J mice show severe age-related changes that finally lead to the degeneration of retinal ganglion cells and the optic nerve. Recent electroretinogram studies identified functional deficits, which suggest that also photoreceptor cells are involved in the pathological processes occurring in the DBA/2J mouse retina. In a comparative study, we examined anatomical and molecular changes in the retinae of DBA/2J and C57BL/6 control mice with light and electron microscopy and with PCR analyses. In the retina of the DBA/2J mouse, we found a thinning of the outer plexiform layer, the first synaptic layer in the transfer of visual signals, and age-dependent and progressive degenerative structural changes at rod photoreceptor ribbon synapses. The structural ribbon changes represent a photoreceptor synaptic phenotype that has not yet been described in this animal model of secondary angle-closure glaucoma. Furthermore, genes of the classical complement cascade were upregulated in the photoreceptor cells of aging DBA/2J mice, suggesting a putative link between ribbon synapse degradation and the innate immune system.     

Novel mutation that causes a structural change in a lipoprotein in the outer membrane of Escherichia coli.

A novel mutation which caused a structural change in a lipoprotein in the outer-membrane has been found in Escherichia coli K-12. The lipoprotein of the wild-type strain is known to have a peculiar amino terminal structure: glycerylcysteine with two fatty acids attached by ester linkages and one fatty acid by an amide linkage. In contrast to the wild-type lipoprotein, the mutant lipoproteins is isolated from the E. coli envelope as a dimer of molecular weight of about 15,000. The dimer can be reduced by mercaptoethanol to the lipoprotein monomer of molecular weight of about 7,500. The monomer has a free thiol group which is susceptible to monoiodacetie mutant lipoprotein is extremely low in comparison with that into the wild-type lipoprotein. These results suggest that the mutant is defective in transferring a glycerol group to the thiol group of the amino terminal cysteine residue of the lipoprotein. The gene responsible for this modification reaction has been located at 36.5 min on the E. coli chromosome.     

Lack of Socs2 expression causes the high-growth phenotype in mice

Characterizing causal molecular defects in mouse models of overgrowth or dwarfism helps to identify the key genes and pathways that regulate the growth process. We report here the molecular basis for high growth (hg), a spontaneous mutation that causes a 30-50% increase in postnatal growth. We conclude that hg is an allele of the suppressor of cytokine signaling 2 (Socs2), a member of a family of regulators of cytokine signal transduction. We demonstrate mapping of Socs2 to the hg region, lack of Socs2 mRNA expression, a disruption of the Socs2 locus in high-growth (HG) mice, and a similarity of phenotypes of HG mice and Socs2(-/-) mice generated by gene targeting. Characteristics of the HG phenotype suggest that Socs2 deficiency affects growth prenatally and postnatally most likely through deregulating the growth hormone (GH)/insulin-like growth factor I (IGF1). These results demonstrate a critical role for Socs2 in controlling growth.     

Prenatal inflammation exacerbates hyperoxia-induced functional and structural changes in adult mice

Maternally derived inflammatory mediators, such as IL-6 and IL-8, contribute to preterm delivery, low birth weight, and respiratory insufficiency, which are routinely treated with oxygen. Premature infants are at risk for developing adult-onset cardiac, metabolic, and pulmonary diseases. Long-term pulmonary consequences of perinatal inflammation are unclear. We tested the hypothesis that a hostile perinatal environment induces profibrotic pathways resulting in pulmonary fibrosis, including persistently altered lung structure and function. Pregnant C3H/HeN mice injected with LPS or saline on embryonic day 16. Offspring were placed in room air (RA) or 85% O(2) for 14 days and then returned to RA. Pulmonary function tests, microCTs, molecular and histological analyses were performed between embryonic day 18 and 8 wk. Alveolarization was most compromised in LPS/O(2)-exposed offspring. Collagen staining and protein levels were increased, and static compliance was decreased only in LPS/O(2)-exposed mice. Three-dimensional microCT reconstruction and quantification revealed increased tissue densities only in LPS/O(2) mice. Diffuse interstitial fibrosis was associated with decreased micro-RNA-29, increased transforming growth factor-β expression, and phosphorylation of Smad2 during embryonic or early fetal lung development. Systemic maternal LPS administration in combination with neonatal hyperoxic exposure induces activation of profibrotic pathways, impaired alveolarization, and diminished lung function that are associated with prenatal and postnatal suppression of miR-29 expression.     

Losartan ameliorates progression of glomerular structural changes in diabetic KKAy mice

Pathological changes in glomerular structure are typically associated with the progression of diabetic nephropathy. The involvement of angiotensin II (AII) in pathogenesis of diabetic nephropathy has been extensively studied and the therapeutic advantages associated with blockade of renin-angiotensin system (RAS), primarily with angiotensin converting enzyme (ACE) inhibitors, has been well-documented. We studied the effect of RAS blockade with an AII receptor antagonist (losartan) vs. an ACE inhibitor (enalapril) on glomerular lesions in KKAy mice, a model of type 2 diabetes mellitus. Losartan was administered at 3 and 10 mg/kg/day and enalapril at 3 mg/kg/day for 14 weeks in the drinking water. The doses of losartan at 10 mg/kg/day was expected to be equivalent to 3 mg/kg/day of enalapril when considering clinical doses for lowering blood pressure. The dose of 3 mg/kg/day of losartan was selected to compare the efficacy at equivalent dose of enalapril. Histologic observation demonstrated suppression of glomerular mesangial expansion and glomerulosclerosis with exudative lesion in the 10 mg/kg/day losartan group when compared to the untreated diabetic controls. A lesser degree of glomerulosclerosis was also observed with losartan and enalapril treatment at 3 mg/kg/day. Ultrastructural examination of renal glomeruli from the high dose losartan group revealed a decreased degree of effacement and/or irregular arrangement of glomerular podocytic foot process. The beneficial effect of RAS inhibition with the AII receptor antagonist losartan on diabetic glomerular lesions was clearly demonstrated in this study. These findings, therefore, provide mechanistic explanation for the clinical utility of losartan for use in the treatment of diabetic nephropathy in man.