Linkage of tyrosine hydroxylase to four other markers on the short arm of chromosome 11.

Tyrosine hydroxylase is the rate-limiting enzyme in catecholamine synthesis; the gene has previously been cloned and localised to the short arm of chromosome 11. Because of the interest in tyrosine hydroxylase as a candidate gene for manic-depressive psychosis and other affective disorders, we carried out family studies to determine the linkage of tyrosine hydroxylase with insulin, beta-globin, D11S12 and Harvey-ras 1, members of a linkage group which has previously been localised to 11p. Using DNA from the Centre d'Etude du Polymorphisme Humain (CEPH) and from two large British pedigrees, we show that tyrosine hydroxylase is closely linked to these four loci (z = 7.36, theta = 0.04 for linkage to insulin) and suggest a gene order based on multipoint mapping.     

Short- and Long-Term Alterations of Gene Expression in Limbic Structures by Repeated Electroconvulsive-Induced Seizures

Rats were submitted to a series of 10 daily electroconvulsive shocks (ECS). A first group of animals was killed 1 day after the last seizure and a second group 30 days later. Tyrosine hydroxylase (TH) activity was measured using an in vitro assay in the nucleus caudatus, anterior cortex, amygdala, substantia nigra, ventral tegmental area, and locus ceruleus. The mRNA corresponding to this enzyme (TH-mRNA) was evaluated using a cDNA probe at the cellular level in the ventral tegmental area, substantia nigra, and locus ceruleus. Met-enkephalin (MET)-immunoreactivity and the mRNA coding for the preproenkephalin (PPE-mRNA) were assayed in striatum and the central nucleus of the amygdala. The day after the last ECS an increase of TH activity was observed in the ventral tegmental area, locus ceruleus, and substantia nigra in parallel with a similar increase in the amygdala and striatum; in the anterior cortex TH activity remained unchanged. TH-mRNA was increased in the locus ceruleus, evidencing the presence in this structure of a genomic activation. The amounts of MET and PPE-mRNA were unaffected in the striatum but increased in the amygdala. Thirty days after the last ECS we observed a decrease of TH activity in the amygdala and of TH-mRNA amount in the ventral tegmental area. In the locus ceruleus TH-mRNA remained higher in treated animals than in controls whereas TH activity returned to control levels. These results demonstrate that a series of ECS induces an initial increase of the activity of mesoamygdaloid catecholaminergic neurons followed by a sustained decrease through alterations of TH gene expression which could mediate the clinical effect of the treatment.     

Identification of a unicellular,non-pigmented alga that mediates growth inhibition in anuran tadpoles: a new species of the genus Prototheca (Chlorophyceae: Chlorococcales)

A non-pigmented, unicellular alga isolated from the faeces of British anuran tadpoles and which is associated with growth inhibition in these tadpoles, was described and identified using cytological, ultrastructural, nutrient assimilation and immunological studies. The alga possessed all the distinctive morphological features of the genus Prototheca, it grew weakly on Prototheca Isolation Medium (PIM), it required thiamine for continued growth and replication, and it could assimilate the five major substrates used to speciate the protothecans. All of these characteristics, together with previous nucleic acid hybridisation studies, indicated that the microorganism belonged to the genus Prototheca. There are currently five species recognised as valid (Pore, 1985 & 1986): Prototheca zopfii Kruger, 1884, P. wickerhamii Tubaki & Soneda, 1959, P. moriformis Kruger, 1884, P. stagnora Cooke, 1968 and P. ulmea Pore, 1986.The immunology showed that the new species was related to two of the protothecans, but overall it showed that the alga was antigenically distinct from the other protothecans tested in the immunoassay. This, together with its inability to grow strongly on PIM, its ability to assimilate a wide rage of carbon substrates and its ability to mediate growth inhibition in anuran tadpoles, indicated a new species of Prototheca. We therefore propose the name Prototheca richardsi sp. n.     

Use of synthetic lethal mutants to clone and characterize a novel CTP synthetase gene in Saccharomyces cerevisiae

In the pyrimidine biosynthetic pathway, CTP synthetase catalyses the conversion of uridine 5-triphosphate (UTP) to cytidine 5-triphosphate (CTP). In the yeast Saccharomyces cerevisiae, the URA7 gene encoding this enzyme was previously shown to be nonessential for cell viability. The present paper describes the selection of synthetic lethal mutants in the CTP biosynthetic pathway that led us to clone a second gene, named URA8, which also encodes a CTP synthetase. Comparison of the predicted amino acid sequences of the products of URA7 and URA8 shows 78% identity. Deletion of the URA8 gene is viable in a haploid strain but simultaneous presence of null alleles both URA7 and URA8 is lethal. Based on the codon bias values for the two genes and the intracellular concentrations of CTP in strains deleted for one of the two genes, relative to the wild-type level, URA7 appears to be the major gene for CTP biosynthesis. Nevertheless, URA8 alone also allows yeast growth, at least under standard laboratory conditions.     

Conformational properties of Eu(III) complexes of 3,3′; 5,3″-polymethylene bridged derivatives of 2,2′; 6,2″-terpyridine

The complex [Eu(tpy)₃](ClO₄)₃ where TPY=2,2′; 6,2″-terpyridine, has been prepared and reexamined. The complex appears to be stable in acetonitrile solution with respect to decomplexation of the ligands but the addition of water does cause partial replacement of tpy. Analogous complexes have been prepared with 3,3′; 5,3″-polymethylene bridged derivatives of tpy having two or three carbons in the bridge. The bridging enforces a cisoid geometry of the ligand and prohibits its replacement by added water. An X-ray determination was carried out for [Eu(3b)₃](ClO₄)₃, where 3b=3,3′; 5,3″-dimethylene tpy, which crystallizes in the monoclinic space group P2₁/c with a=11.908(4), b=15.768(5), c=29.513(9) Å, β=93.60(2)°, μ=13.5 cm⁻¹ and Z=4. The complex forms a tricapped trigonal prism with each of the ligands adopting the same dl conformation. Variable temperature NMR analysis of the bridged ligand complexes indicates that conformational inversion of the bound ligand is not a concerted process and barriers for inversion of individual methylene units can be estimated from coalescence of the signals from the geminal methylene protons. The luminescence properties of the bridged tpy complexes are similar to the parent unbridged system.     

Is baldness a thermoregulatory adaptive process?

In 100 adult men the area of the face and neck where beard was growing was measured and compared to that of glabrous skin on the forehead and calvaria. In the population as a whole, forehead area was found to be proportional to bearded area. Forehead and calvaria sweat rate was measured on 10 baldheaded male subjects and compared with that of 10 hairy control subjects during mild hyperthermia. Bald skin was found to sweat more than twice as much as hairy skin. In the light of these results the hypothesis that baldness is a thermoregulatory adaptative process is proposed.     

Regulation of recombinant human tyrosine hydroxylase isozymes by catecholamine binding and phosphorylation. Structure/activity studies and mechanistic implications.

Three isozymes of human tyrosine hydroxylase (hTH1, hTH2 and hTH4) were expressed in Escherichia coli and purified to homogeneity. Natural catecholamines and related synthetic compounds were found to be potent inhibitors, competitive to the tetrahydrobiopterin cofactor, of all the isozymes. Combining visible spectroscopy and equilibrium-binding studies, it was found that catecholamines bind to hTH1 and hTH2 with a stoichiometry of about 1.0 mol/mol enzyme subunit, interacting with the catalytic iron at the active site. All the isozymes tested were excellent substrates for cAMP-dependent protein kinase (Km = 5 microM, Vmax = 9.5 mumol.min-1.mg kinase-1). The incorporation of about 1.0 mol phosphate/subunit at Ser40 decreased the affinity of dopamine binding by a factor of 10. Conversely, the addition of stoichiometric amounts of Fe(II) and dopamine to the apoenzymes reduced both the affinity and stoichiometry of phosphorylation by cAMP-dependent protein kinase by 2-3-fold. These data provide evidence for a mutual interaction between the presumed regulatory and catalytic domains of hTH, and show that activation of the enzyme by phosphorylation and inactivation by binding of catecholamines are related events, which probably represent important mechanisms for the regulation of the enzyme activity in vivo.     

Immunological studies on the Purkinje cells from rat and mouse cerebella. I. Evidence for antibodies characteristic of the Purkinje cells.

Antibodies have been raised against an enriched preparation of isolated rat cerebellar Purkinje cells. The immunoglobulins were labeled with ¹²⁵I and the strength and specificity of the serum determined by a direct binding assay on cerebellar membranes. About 2% of the ¹²⁵I-labeled IgG bound to an excess of cerebellar membranes. Absorption with heart and liver membranes removed 80.5% of the ¹²⁵I-labeled IgG binding to cerebellar membranes; absorption with cerebrum membranes removed 13% more; the remaining 6.5% were directed specifically against cerebellar membranes. An enriched ¹²⁵I-labeled anti-Purkinje antibody population was prepared by absorption and subsequent elution from cerebellar membranes. The absorption pattern with heart, liver, and cerebrum membranes resembled that found with the total population of IgG except that the nonspecific binding was significantly diminished. The Purkinje cell degeneration (pcd) mouse mutant was used to assess the specificity of the serum toward the Purkinje cells. After absorption of the enriched anti-Purkinje antibodies with heart, liver, and cerebrum membranes, the binding of labeled IgG to membranes prepared from pcd/pcd cerebella was about one-fourth that found with control cerebella. The direct immunoperoxidase technique performed on smears of purified Purkinje and granule cells shows that the unabsorbed serum stains both classes of cells, but that after absorption with liver, heart, and cerebrum membranes, only the Purkinje cells react positively.