Human and mouse homologues of the Drosophila melanogaster tweety (tty) gene: a novel gene family encoding predicted transmembrane proteins.

We have cloned cDNA for TTYH1, a human homologue of the Drosophila melanogaster tweety (tty) gene. The 450-residue predicted protein shows 27% amino acid sequence identity (51% similarity) to the Drosophila protein, which contains an additional C-terminal repetitive region. A second Drosophila homologue exhibits 42% identity (65% similarity) to the tty protein. Mouse (Ttyh1), macaque, and Caenorhabditis elegans homologues were also identified, and the complete coding sequence for the mouse gene was determined. The mouse protein is 91% identical to the human protein. Hydrophobicity analysis of the tty-related proteins indicates that they represent a new family of membrane proteins with five potential membrane-spanning regions. The yeast FTR1 and FTH1 iron transporter proteins and the mammalian neurotensin receptors 1 and 2 have a similar hydrophobicity profile, although there is no detectable sequence homology to the tty-related proteins. This suggests that the tweety-related proteins could be involved in transport of iron or other divalent cations or alternatively that they may be membrane-bound receptors. TTYH1 was mapped to chromosome 19q13.4 by FISH and by radiation hybrid mapping using the Stanford G3 panel.

Cloning, characterization, and chromosomal location of a novel human K+-Cl- cotransporter.

Differential display polymerase chain reaction has been used to isolate genes regulated in vascular endothelial cells by the angiogenic factor vascular endothelial cell growth factor (VEGF). Analysis of one of the bands consistently up-regulated by VEGF led us to the identification of a cDNA from a human umbilical vein endothelial cell library that is 77% identical to the human K+-Cl- cotransporter1 (KCC1). We have referred to the predicted protein as K+-Cl- cotransporter 3 (KCC3). Hydrophobicity analysis of the KCC3 amino acid sequence showed an almost identical pattern to KCC1, suggesting 12 membrane-spanning segments, a large extracellular loop with potential N-glycosylation sites, and cytoplasmic N- and C-terminal regions. The KCC3 mRNA was highly expressed in brain, heart, skeletal muscle, and kidney, showing a distinct pattern and size from KCC1 and KCC2. The KCC3 mRNA level in endothelial cells increased on treatment with VEGF and decreased with the proinflammatory cytokine tumor necrosis factor alpha, whereas KCC1 mRNA levels remained unchanged. Stable overexpression of KCC3 cDNA in HEK293 cells produced a glycoprotein of approximately 150 kDa, which was reduced to 120 kDa by glycosidase digestion. An increased initial uptake rate of 86Rb was seen in clones with high KCC3 expression, which was dependent on extracellular Cl- but not Na+ and was inhibitable by the loop diuretic agent furosemide. The KCC3 genomic localization was shown to be 15q13 by fluorescence in situ hybridization. Radiation hybrid analysis placed KCC3 within an area associated with juvenile myoclonic epilepsy. These results suggest KCC3 is a new member of the KCC family that is under distinct regulation from KCC1.

GPR56, a novel secretin-like human G-protein-coupled receptor gene.

A novel gene product, GPR56, with homology to the seven transmembrane-domain receptor superfamily, has been cloned by PCR amplification using degenerate oligonucleotide primers and subsequent screening of a human heart cDNA library. The isolated 2.8-kb cDNA clone encodes a protein of 693 amino acids that shows highest identity (32%) to HE6, a member of a subclass of the class B secretin-like G-protein-coupled receptors. Northern analysis of various human tissues revealed a wide distribution of the transcript with highest levels found in thyroid gland, brain, and heart. In situ hybridization analysis of human thyroid gland as well as rat heart and brain tissue confirms these results and identifies the hippocampus and hypothalamic nuclei as brain areas with particularly high expression of GPR56 mRNA. The high level of mRNA expression, its wide distribution, and the mucin-like extracellular domain of the receptor protein suggest a possible role for this receptor in cell-cell interaction processes. The human gene for GPR56 has been isolated and its exon-intron structure determined. The total length of the human GPR56 gene is approximately 15 kb, and it consists of 13 exons. Fluorescence in situ hybridization, PCR analysis of somatic cell hybrids, and interspecific mouse backcross mapping have localized the genes to human chromosome 16q13 and mouse chromosome 8.

Molecular characterization, pharmacological properties and chromosomal localization of the human GALR2 galanin receptor.

The neuropeptide galanin mediates a diverse spectrum of biological activities by interacting with specific G protein-coupled receptors. We have used homology genomic library screening and polymerase chain reaction (PCR) techniques to isolate both genomic and cDNA clones encoding the human homolog of the recently cloned rat GALR2 galanin receptor. By fluorescence in situ hybridization, the gene encoding human GALR2 (GALNR2) has been localized to chromosome 17q25.3. The two coding exons of the human GALNR2 gene, interrupted by an intron positioned at the end of transmembrane domain III, encode a 387 amino acid G protein-coupled receptor with 87% overall amino acid identity with rat GALR2. In HEK-293 cells stably expressing human GALR2, binding of [125I]porcine galanin is saturable and can be displaced by galanin, amino-terminal galanin fragments and chimeric galanin peptides but not by carboxy-terminal galanin fragments. In HEK-293 cells, human GALR2 couples both to Galphaq/11 to stimulate phospholipase C and increase intracellular calcium levels and to Galphai/o to inhibit forskolin-stimulated intracellular cAMP accumulation. A wide tissue distribution is observed by reverse transcriptase (RT)-PCR analysis, with human GALR2 mRNA being detected in many areas of the human central nervous system as well as in peripheral tissues.

Structure and chromosomal localization of the genomic locus encoding the Kiz1 LIM-kinase gene.

We have cloned and characterized the mouse gene encoding Kiz1/Limk1, a new member of the zinc-finger LIM family that also has a kinase domain. The gene encompasses 25 kb of the mouse genome, and the organization of its 16 exons does not correlate with its functional domains. The promoter region of Kiz1/Limk1 was identified by cloning a 1.06-kb genomic fragment upstream from the first ATG in a promotorless CAT vector. This construct was demonstrated to drive CAT expression in Jurkat cells. The promoter sequence lacks conventional TATA and CAAT motifs but contains consensus binding sequences for several transcriptional regulators implicated in control of transcription in many different cell types, including Sp1, Ets, and E2A. Analysis of the chromosomal localization of KIZ1/LIMK1 indicates that it lies on human chromosome 17 in the region 17q25 and on mouse Chromosome 5, band G2.

Latissimus dorsi muscle: assessment of stimulation after cardiomyoplasty with Doppler US tissue imaging.

PURPOSE: To evaluate Doppler ultrasound (US) tissue imaging for assessment of stimulated skeletal muscle contraction. MATERIALS AND METHODS: Seven patients were studied after left latissimus dorsi cardiomyoplasty. Myograft contraction programmed on alternate cardiac cycles was assessed with Doppler US tissue imaging. Beat-to-beat variation in inferior wall motion was assessed by examining peak myograft velocities during 10 muscle-assisted and 10 nonassisted cardiac cycles. The temporal relationship between electrostimulation and myograft contraction, changes in cardiac geometry, and the effect of alterations in stimulation voltage and muscle synchronization were assessed. RESULTS: Significant beat-to-beat variation in velocity profile could be detected in the proximal myograft in six patients (P < .05). Potentiation of infero-posterior wall motion was measurable in five patients (mean peak systolic wall velocity: nonassisted, 2.5 cm.sec-1 +/- 0.5 [standard deviation]; assisted, 7.8 cm.sec-1 +/- 6.3). The response between stimulation voltage and inferoposterior wall velocity was sigmoid. CONCLUSION: Doppler US tissue imaging depicted the effects of myostimulator programming on muscle contraction and ventricular wall motion.

The human glycine receptor beta subunit: primary structure, functional characterisation and chromosomal localisation of the human and murine genes.

The inhibitory glycine receptor (GlyR) is a pentameric receptor comprised of alpha and beta subunits, of which the beta subunit has not been characterised in humans. A 2106 bp cDNA, isolated from a human hippocampal cDNA library, contained an open reading frame of 497 amino acids which encodes the beta subunit of the human GlyR. The mature human GlyR beta polypeptide displays 99% amino acid identity with the rat GlyR beta subunit and 48% identity with the human GlyR alpha 1 subunit. Neither [3H]strychnine binding nor glycine-gated currents were detected when the human GlyR beta subunit cDNA was expressed in the human embryonic kidney 293 cell line. However, co-expression of the beta subunit cDNA with the alpha 1 subunit cDNA resulted in expression of functional GlyRs which showed a 4-fold reduction in the EC50 values when compared to alpha 1 homomeric GlyRs. Glycine-gated currents of alpha 1/beta GlyRs were 17-fold less sensitive than homomeric alpha 1 GlyRs to the antagonists picrotoxin, picrotoxinin and picrotin, providing clear evidence that heteromeric alpha 1/beta GlyRs were expressed. The beta subunit appears to play a structural rather than ligand binding role in GlyR function. Fluorescence in situ hybridisation was used to localise the gene encoding the human GlyR beta subunit (GLRB) to chromosome 4q32, a position syntenic with mouse chromosome 3. In situ hybridisation using the human GlyR beta subunit cDNA showed that the murine GlyR beta subunit gene (Glrb) maps to the spastic (spa) locus on mouse chromosome 3 at bands E3-F1. This is consistent with the recent finding that a mutation in the murine GlyR beta subunit causes the spa phenotype. It also raises the possibility that mutations in the human beta subunit gene may cause inherited disorders of the startle response.

Cloning, chromosomal mapping and characterization of the human metal-regulatory transcription factor MTF-1.

Metallothioneins (MTs) are small cysteine-rich proteins that bind heavy metal ions such as zinc, cadmium and copper with high affinity, and have been functionally implicated in heavy metal detoxification and radical scavenging. Transcription of metallothioneins genes is induced by exposure of cells to heavy metals. This induction is mediated by metal-responsive promoter elements (MREs). We have previously cloned the cDNA of an MRE-binding transcription factor (MTF-1) from the mouse. Here we present the human cDNA equivalent of this metal-regulatory factor. Human MTF-1 is a protein of 753 amino acids with 93% amino acid sequence identity to mouse MTF-1 and has an extension of 78 amino acids at the C-terminus without counterpart in the mouse. The factors of both species have the same overall structure including six zinc fingers in the DNA binding domain. We have physically mapped the human MTF-1 gene to human chromosome 1 where it localizes to the short arm in the region 1p32-34, most likely 1p33. Both human and mouse MTF-1 when produced in transfected mammalian cells strongly bind to a consensus MRE of metallothionein promoters. However, human MTF-1 is more effective than the mouse MTF-1 clone in mediating zinc-induced transcription.

Hydrogen ion compartmentation during and following cerebral ischemia evaluated by 31P NMR spectroscopy.

Two peaks were observed in the inorganic phosphate region in 31P NMR spectra obtained during and shortly after incomplete forebrain ischemia induced in rats under normoglycemic conditions. Pre-ischemia, a single Pi peak was observed at a chemical shift of 2.5 ppm (pH 7.2). This peak shifted upfield to 1.6 ppm (pH 6.5) and, following reperfusion, returned to its pre-ischemia value. During ischemia and for a short time following reperfusion, a second, smaller peak was observed which we assigned to a second pool of inorganic phosphate. The data support the proposal by Kraig and coworkers of pH compartmentation between neurons and glia during and following transient forebrain ischemia.

Localization of a human receptor tyrosine kinase (ETK1) to chromosome region 3p11.2.

We have recently described a human receptor tyrosine kinase (hek) that is expressed by some pre-B and thymic T cell lines, but is not detectable on normal adult human tissues. Gene cloning studies established that hek is a new member of the EPH family of receptor tyrosine kinases. The expression of hek may normally be developmentally regulated and inappropriate expression may contribute to oncogenesis. In the present study, we have used Southern blot analysis of somatic cell hybrids and fluorescence in situ hybridization to localize the hek gene to human chromosome region 3p11.2. Karyotype analysis of the cell lines that over-express hek showed no cytogenetically visible abnormality involving the hek locus.

Forebrain ischemia in diabetic and nondiabetic BB rats studied with 31P magnetic resonance spectroscopy.

In spontaneously diabetic BB rats, the effect of chronically maintained blood glucose levels on the degree of energy failure and brain pH change during an ischemic insult, and on subsequent recovery after reperfusion, was studied with in vivo 31P magnetic resonance spectroscopy. Short duration forebrain ischemia (10-min carotid occlusion plus hypotension of 50 mmHg) was induced in diabetic and nondiabetic male BB rats whose blood glucose levels were maintained with insulin. Spectra were obtained in 1-min blocks before, during, and for 1 h after ischemia. Before ischemia, hypoglycemic (blood glucose less than 3 mM) diabetic rats had an increased Pi peak intensity, with no significant pH change, compared with other groups. During ischemia, the rate and extent of hydrolysis of high-energy phosphate metabolites (as measured by an increase in Pi) decreased, and the severity of tissue acidosis increased as preischemia blood glucose concentration increased. Among hyperglycemic BB rats, similar ischemia-induced changes were found for subgroups with blood glucose levels of 13.7 +/- 1.2 and 20.3 +/- 0.6 mM, in keeping with the known decrease in hexose binding sites associated with chronic hyperglycemia. Decline in PCr level during ischemia was not significantly different between groups. With reperfusion, both Pi and pH values rapidly returned to preischemia values. PCr levels, however, did not recover in hyperglycemic diabetic animals, with the degree of residual impairment dependent on the preischemia glucose level. Results suggest that optimal management of diabetes may lessen the degree of injury within the ischemic penumbra in diabetic patients who suffer a stroke.

Prevention of rickets in Asian children: assessment of the Glasgow campaign.

In March 1979 the Greater Glasgow Health Board launched a campaign to reduce the high prevalence of rickets in Asian children in the city. A precampaign survey had shown that voluntary low dose vitamin D supplementation would reduce the prevalence of rickets in Asian children. A survey carried out two and three years after the launch of the official campaign also showed a reduction in the prevalence of rickets in children taking low dose supplements equivalent to about 2.5 micrograms (100 IU) vitamin D daily. There was a considerable reduction in the total prevalence of rickets in this survey compared with the precampaign survey. Hospital discharges of Asian children with rickets declined rapidly after the start of the campaign.

Relationships between radiological and biochemical evidence of rickets in Asian schoolchildren.

One hundred Asian schoolchildren provided evidence of the relationships between radiological and biochemical evidence of rickets in a vitamin D-deficient population. In a retrospective study of the X-rays of 56 children the variables serum alkaline phosphatase, inorganic phosphorus and age provided a discriminant function which correctly classified 10 of 11 children with radiological evidence of rickets and 44 of 45 children with negative or marginally abnormal X-rays. When the discriminant function was applied to a prospective study of 44 children, three children with radiological evidence of rickets were correctly classified together with 38 of the remaining 41 children with negative or marginally abnormal X-rays. Serum alkaline phosphatase was the most important variable in the discriminant analysis, followed by serum inorganic phosphorus and age. Low levels of serum 25-hydroxy vitamin D (25-OHD) are of little value in predicting the severity of radiological evidence of rachitic bone disease in a vitamin D-deficient population.

Policy for prevention of Asian rickets in Britain: a preliminary assessment of the Glasgow rickets campaign.

Evidence of continuing hospital admissions of patients with Asian rickets and osteomalacia led to a further attempt to provide more effective preventive measures for the Glasgow Asian community. Dose-response studies showed that the equivalent of 10 microgram of vitamin D daily would provide effective prophylaxis, and a general practice survey showed that self-administered vitamin D supplements would reduce the prevalence and severity of Asian rickets. A multidisciplinary working group devised a preventive campaign based on the free issue of vitamin D supplements on demand to children who required them. Supported by a health education programme for community health personnel and the Asian community, the first 16 months of the campaign produced an eight-fold rise in the issue of supplements to older Asian children and a 33% increase in their issue to infants of all ethnic groups. Because more children are receiving vitamin D supplementation the campaign seems likely to reduce the prevalence of Asian rickets in Glasgow.