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At least 397 records · Page 22Linked to original sources
PubMed2001

Genetic and functional characterization of dpp genes encoding a dipeptide transport system in Lactococcus lactis.

The genes encoding a binding-protein-dependent ABC transporter for dipeptides (Dpp) were identified in Lactococcus lactis subsp. cremoris MG1363. Two (dppA and dppP) of the six ORFs (dppAdppPBCDF) encode proteins that are homologous to peptide- and pheromone-binding proteins. The dppP gene contains a chain-terminating nonsense mutation and a frame-shift that may impair its function. The functionality of the dpp genes was proven by the construction of disruption mutants via homologous recombination. The expression of DppA and various other components of the proteolytic system was studied in synthetic and peptide-rich media and by using isogenic peptide-transport mutants that are defective in one or more systems (Opp, DtpT, and/or Dpp). In peptide-rich medium, DppA was maximally expressed in mutants lacking Opp and DtpT. DppA expression also depended on the growth phase and was repressed by tri-leucine and tri-valine. The effect of tri-leucine on DppA expression was abolished when leucine was present in the medium. Importantly, the Dpp system also regulated the expression of other components of the proteolytic system. This regulation was achieved via the internalization of di-valine, which caused a 30-50% inhibition in the expression of the proteinase PrtP and the peptidases PepN and PepC. Similar to the regulation of DppA, the repressing effect was no longer observed when high concentrations of valine were present. The intricate regulation of the components of the proteolytic system by peptides and amino acids is discussed in the light of the new and published data.

Amino Acid Sequence↗
PubMed1992

Sympathoadrenal function in genetically obese Zucker rats.

The effects of genetic obesity on the actions and alterations of the sympathetic nervous system were studied in 10-12-month-old obese (fa/fa) and lean (Fa/-) Zucker rats. Blood glucose, plasma insulin, epinephrine (E), norepinephrine (NE), and free fatty acids (FFA) concentrations were measured in blood samples taken through a permanent heart catheter before, during, and after exercise or intravenous infusion of E and NE. Baseline plasma FFA and insulin levels were markedly increased in the obese animals. Exercise, i.e., strenuous swimming against a counter current for 15 min, led to reduction of plasma insulin concentrations and an increase of all other blood components in lean Zucker rats. In obese animals, an exaggerated increase of blood glucose and a large suppression of plasma insulin occurred. Plasma FFA levels tended to decline during exercise. Plasma catecholamine patterns in the exercising fatty Zuckers were not different to those of the lean animals. Infusion of E caused an increase of blood glucose and a decrease of plasma insulin concentrations in both groups of animals. The increase in blood glucose in the obese animals was significantly larger compared to the changes in the lean animals. Infusion of NE significantly reduced plasma insulin concentration in obese but not in lean animals. The results revealed that activation of the sympathetic system, expressed as exercise-induced alterations in plasma E and NE levels, is normal in obese Zucker rats. However, postsynaptic receptor effects of catecholamines on glycogenolysis and lipolysis are different in obese and lean animals, which points to permanent changes in adrenoceptor mechanisms on adipocytes, hepatocytes, and muscle cells in obesity.

Adrenal Glands↗
PubMed1991

Point mutations in human keratin 14 genes of epidermolysis bullosa simplex patients: genetic and functional analyses.

Previously we demonstrated that transgenic mice expressing mutant basal epidermal keratin genes exhibited a phenotype resembling a group of autosomal dominant human skin disorders known as epidermolysis bullosa simplex (EBS). EBS diseases affect approximately 1: 50,000 and are of unknown etiology, although all subtypes exhibit blistering arising from basal cell cytolysis. We now demonstrate that two patients with spontaneous cases of Dowling-Meara EBS have point mutations in a critical region in one (K14) of two basal keratin genes. To demonstrate function, we engineered one of these point mutations in a cloned human K14 cDNA, and showed that a K14 with an Arg-125----Cys mutation disrupted keratin network formation in transfected keratinocytes and perturbed filament assembly in vitro. Since we had previously shown that keratin network perturbation is an essential component of EBS diseases, these data suggest that the basis for the phenotype in this patient resides in this point mutation.

Amino Acid Sequence↗
PubMed1989

The Na-K-Cl cotransport in essential hypertension: cellular functions and genetic environment interactions.

The present paper examines factors involved in the expression of the Na-K-Cl cotransport system present in erythrocytes and in vascular cells. This transport system is modulated by vasoactive peptides such as atrial natriuretic factor and bradykinin in vascular smooth muscle and endothelial cells. The Vmax of the Na-K-Cl in human red cells displays large interindividual differences which can be mainly accounted for by genetic factors. Elevation of the Km for Na of the outward Na cotransport is found in red cells of some Caucasian hypertensives and in Black normotensives born of hypertensive parents whose blood pressure increases with salt loading. Reduction of Na intake from 200 to 10 mEq/day does not influence the activity of the cotransport in normotensive individuals but decreases the Km for Na of hypertensive subjects to values similar to those of the normotensives. These findings indicate that the Na-K-Cl cotransport is an important probe of genetic and environmental factors in the hypertensive process.

Animals↗
PubMed2005

Genetic and functional association of the immune signaling molecule 4-1BB (CD137/TNFRSF9) with type 1 diabetes.

Idd9.3, a locus that determines susceptibility to the autoimmune disease type 1 diabetes (T1D) in the nonobese diabetic (NOD) mouse, has been mapped to the distal region of chromosome 4. In the current report we reduce the size of the Idd9.3 interval to 1.2Mb containing 15 genes, including one encoding the immune signaling molecule, 4-1BB, which shows amino acid variation between diabetes sensitive and resistant strains. 4-1BB, a member of the TNF receptor superfamily expressed by a variety of immune cells, mediates growth and survival signals for T cells. Functional analyses demonstrate that purified T cells from NOD congenic mice with the C57BL/10 (B10) allele at Idd9.3 produce more IL-2 and proliferate more vigorously in response to anti-CD3 plus immobilized 4-1BB ligand than T cells from NOD mice with the NOD allele at Idd9.3. In contrast, the response to anti-CD3 plus anti-CD28 costimulation was indistinguishable between the congenic strains, pinpointing the differences in NOD versus NOD.B10 Idd9.3 T cell responses to the 4-1BB costimulatory pathway. These data provide evidence in support of Idd9.3 as the locus encoding 4-1BB and suggest that the 4-1BB signaling pathway could have a primary function in the etiology of autoimmune disease.

Animals↗
PubMed2004

Genetic and functional analyses of the øX174 DNA binding protein: the effects of substitutions for amino acid residues that spatially organize the two DNA binding domains.

The øX174 DNA binding protein contains two DNA binding domains, containing a series of DNA binding basic amino acids, separated by a proline-rich linker region. Within each DNA binding domain, there is a conserved glycine residue. Glycine and proline residues were mutated and the effects on virion structure were examined. Substitutions for glycine residues yield particles with similar properties to previously characterized mutants with substitutions for DNA binding residues. Both sets of mutations share a common extragenic second-site suppressor, suggesting that the defects caused by the mutant proteins are mechanistically similar. Hence, glycine residues may optimize DNA-protein contacts. The defects conferred by substitutions for proline residues appear to be fundamentally different. The properties of the mutant particles along with the atomic structure of the virion suggest that the proline residues may act to guide the packaged DNA to the adjacent fivefold related asymmetric unit, thus preventing a chaotic packaging arrangement.

Amino Acid Sequence↗
PubMed2024

Genetic and functional analysis of Raynaud's syndrome implicates loci in vasculature and immunity.

Raynaud's syndrome is a dysautonomia where exposure to cold causes vasoconstriction and hypoxia, particularly in the extremities. We performed meta-analysis in four cohorts and discovered eight loci (ADRA2A, IRX1, NOS3, ACVR2A, TMEM51, PCDH10-DT, HLA, and RAB6C) where ADRA2A, ACVR2A, NOS3, TMEM51, and IRX1 co-localized with expression quantitative trait loci (eQTLs), particularly in distal arteries. CRISPR gene editing further showed that ADRA2A and NOS3 loci modified gene expression and in situ RNAscope clarified the specificity of ADRA2A in small vessels and IRX1 around small capillaries in the skin. A functional contraction assay in the cold showed lower contraction in ADRA2A-deficient and higher contraction in ADRA2A-overexpressing smooth muscle cells. Overall, our study highlights the power of genome-wide association testing with functional follow-up as a method to understand complex diseases. The results indicate temperature-dependent adrenergic signaling through ADRA2A, effects at the microvasculature by IRX1, endothelial signaling by NOS3, and immune mechanisms by the HLA locus in Raynaud's syndrome.

Raynaud Disease↗
PubMed2003

RPE CD14 immunohistochemical, genetic, and functional expression.

CD14 is the primary receptor for lipopolysaccharide (LPS)that plays important roles in host defense and subserves other host-related biological functions. We previously identified CD14 on cultured human retinal pigment epithelial (HRPE) cells using immunocytochemical techniques. In this study, we investigated immunoreactive HRPE CD14 expression by immunohistochemically staining HRPE cells and HRPE cells in sections of human eyes with anti-CD14 monoclonal antibodies (mAb). Constitutive HRPE gene and protein expression were confirmed by semiquantitative PCR and western blotting. ELISA for cell-associated and secreted (s) HRPE CD14 revealed that specific digestion by phosphoinositol-specific phospholipase C (PI-PLC) significantly reduced (P<0.01) cell-associated HRPE CD14 which was not modulated by LPS or gamma-IFN. ELISA of the conditioned media (CM) of HRPE cells treated with PI-PLC contained significantly more (P<0.001) sCD14, but sCD14 was not modulated by LPS or gamma-IFN. FACS analysis confirmed HRPE cell surface CD14. To show functional CD14, fluorescently-labelled LPS and CD14 were demonstrated to show significant co-localization on live, cultured HRPE cells in close proximity (<7A) as demonstrated by resonance energy transfer of the fluorescent ligands (P<0.0001). Significant inhibition (P<0.001) of LPS-induced IL-8 secretion, as measured by ELISA, occurred in the presence of function blocking anti-CD14 mAb. Significant inhibition of LPS-induced HRPE IL-8 secretion by PKC, PTK, PI3 kinase, and p38 kinase inhibitors indicated cell mediators responsible for LPS-induced HRPE chemokine secretion. This study demonstrates that HRPE cells express functional CD14 in vitro and in situ along at the outer blood-retina barrier.

Antibodies, Monoclonal↗
PubMed1996

New insights into the regulation of somatotrope function using genetic and transgenic models.

Growth hormone (GH) secretion is under the control of the hypothalamic hormones GH-releasing hormone (GHRH) and somatostatin (SRIF), and is regulated by feedback effects of GH and insulin-like growth factor (IGF-1). GHRH and SRIF act on somatotropes by binding to G-protein-coupled receptors. GHRH activates the stimulatory G protein (Gs), leading primarily to activation of adenylyl cyclase and protein kinase A. SRIF activates the inhibitory G protein (Gi). Several animal models enable the study of various disorders of GH secretion in vivo. Genetic models of impaired GH secretion include the little (lit) mouse, the dwarf (dw) rat, the fatty (fa) rat, and the high-growth (hg) mouse. Transgenic models of impaired and excessive GH secretion, respectively, include the tyrosine hydroxylase-human GH (TH-hGH) transgenic mouse and the metallothionein-human GHRH transgenic mouse. These models encompass a wide spectrum of disorders of GH secretion, involving defects of hypothalamic regulation, feedback control at the pituitary level, or the mechanism of GHRH action in the somatotrope. They may provide insights into our understanding of human GH secretory disorders.

Animals↗
PubMed2001

Genetic and functional analysis of PARP, a DNA strand break-binding enzyme.

Poly(ADP-ribose) polymerase (PARP) is a nuclear enzyme activated by binding to a single- or double-strand break of DNA and is one of the death substrates for caspase-3 in apoptosis. The nuclear function of PARP is well studied and recent PARP-knockout studies indicate that PARP takes part in chromosomal stability. To analyze the effect of PARP overexpression, or loss of function, we have cloned PARP cDNA and the gene from Drosophila melanogaster and studied its function in developmental stages. Organization of exons corresponds to the functional domains of PARP. An alternatively spliced form of PARP lacking exon 5, which encodes the auto-modification domain, is found in Drosophila. Expression of the PARP gene is at high levels in embryos at 0-6h after egg laying and gradually decreased. In situ mRNA hybridization indicates localization of PARP mRNA in cells along the central nervous system at a late stage of embryogenesis. Overexpression of the gene in the developing eye primordia of D. melanogaster is an excellent experimental model to analyze the cell cycle and programmed cell death. We introduced PARP expression vector overexpresses PARP in the eye discs of Drosophila, and established the PARP transgenic flies by P element-mediated germ line transformation. These flies showed mild roughening of the normally smooth ommatidial lattice involving tissue polarity disruption characterized by missrotation and incorrect chirality of ommatidia. Possible mechanisms of involvement of PARP in the development are discussed.

Animals↗