Characterization of Opioid Receptors in Cultured Neurons

The appearance of mu-, delta-, and kappa-opioid receptors was examined in primary cultures of embryonic rat brain. Membranes prepared from striatal, hippocampal, and hypothalamic neurons grown in dissociated cell culture each exhibited high-affinity opioid binding sites as determined by equilibrium binding of the universal opioid ligand (-)-[3H]bremazocine. The highest density of binding sites (per mg of protein) was found in membranes prepared from cultured striatal neurons (Bmax = 210 +/- 40 fmol/mg protein); this density is approximately two-thirds that of adult striatal membranes. By contrast, membranes of cultured cerebellar neurons and cultured astrocytes were devoid of opioid binding sites. The opioid receptor types expressed in cultured striatal neurons were characterized by equilibrium binding of highly selective radioligands. Scatchard analysis of binding of the mu-specific ligand [3H]D-Ala2,N-Me-Phe4,Gly-ol5-enkephalin to embryonic striatal cell membranes revealed an apparent single class of sites with an affinity (KD) of 0.4 +/- 0.1 nM and a density (Bmax) of 160 +/- 20 fmol/mg of protein. Specific binding of (-)-[3H]bremazocine under conditions in which mu- and delta-receptor binding was suppressed (kappa-receptor labeling conditions) occurred to an apparent single class of sites (KD = 2 +/- 1 nM; Bmax = 40 +/- 15 fmol/mg of protein). There was no detectable binding of the selective delta-ligand [3H]D-Pen2,D-Pen5-enkephalin. Thus, cultured striatal neurons expressed mu- and kappa-receptor sites at densities comparable to those found in vivo for embryonic rat brain, but not delta-receptors.     

Similarity and divergence among rodent repetitive DNA sequences

Summary We have analyzed the sequence of 63 B1 and 71 B2 repetitive elements from published data base sequences. The sequences conform to previously published consensus sequences, but are not identical to them. The B2 sequences show seven regions of high variability between family members, which we show points to the B2 family containing subfamilies; no similar evidence is found for subfamilies of the B1 family. The comparisons show no evidence for the emergence of species-specific variants of B1 or B2 sequences since the separation of murine and hamster lines of descent, nor of their concerted evolution within species in the last 10 million years.     

Linkage analysis of seven kindreds with the X-linked lymphoproliferative syndrome (XLP) confirms that the XLP locus is near DXS42 and DXS37

Summary Analysis of seven kindreds indicates that the XLP locus exhibits 1% recombination with DXS42 (lod = 17.5) and no recombination with DXS37 (lod = 13.3).     

A squid dynein isoform promotes axoplasmic vesicle translocation

Axoplasmic vesicles that translocate on isolated microtubules in an ATP-dependent manner have an associated ATP-binding polypeptide with a previously estimated relative molecular mass of 292 kD (Gilbert, S. P., and R. D. Sloboda. 1986. J. Cell Biol. 103:947-956). Here, data are presented showing that this polypeptide (designated H1) and another high molecular mass polypeptide (H2) can be isolated in association with axoplasmic vesicles or optic lobe microtubules. The H1 and H2 polypeptides dissociate from microtubules in the presence of MgATP and can be further purified by gel filtration chromatography. The peak fraction thus obtained demonstrates MgATPase activity and promotes the translocation of salt-extracted vesicles (mean = 0.87 microns/s) and latex beads (mean = 0.92 microns/s) along isolated microtubules. The H1 polypeptide binds [alpha 32P]8-azidoATP and is thermosoluble, but the H2 polypeptide does not share these characteristics. In immunofluorescence experiments with dissociated squid axoplasm, affinity-purified H1 antibodies yield a punctate pattern that corresponds to vesicle-like particles, and these antibodies inhibit the bidirectional movement of axoplasmic vesicles. H2 is cleaved by UV irradiation in the presence of MgATP and vanadate to yield vanadate-induced peptides of 240 and 195 kD, yet H1 does not cleave under identical conditions. These experiments also demonstrate that the actual relative molecular mass of the H1 and H2 polypeptides is approximately 435 kD. On sucrose density gradients, H1 and H2 sediment at 19-20 S, and negatively stained samples reveal particles comprised of two globular heads with stems that contact each other and extend to a common base. The results demonstrate that the complex purified is a vesicle-associated ATPase whose characteristics indicate that it is a squid isoform of dynein. Furthermore, the data suggest that this vesicle-associated dynein promotes membranous organelle motility during fast axoplasmic transport.     

The effect of Escherichia coli endotoxin on luteal function in Holstein heifers

This study was undertaken to elucidate the possible role of endotcxin in mediating premature luteolysis in the well- documented phenomenon of short estrous cycles in postpartum dairy cows. Four groups of Holstein heifers (n = 4 to 6 each) received either intrauterine infusion of sterile culture medium (Group I); intrauterine infusion of Escherichia coli (E. coli ) endotoxin (5 mug/kg) in sterile culture medium (Group II); intrauterine administration of 10 ml of a 24-h culture of a strain of E. coli isolated from the uterus of a cow with metritis (approximately 10(9) colony forming units/ml; Group III); or intravenous administration of E. coli endotoxin (5 mug/kg; Group IV) on Day 7-9 of the estrous cycle. Blood samples were collected every 48 h during the pretreatment estrous cycle and up to the administration of the experimental treatment, thereafter 4-h samples were collected for 5 d. Sample collection was then performed every 48 h for the remainder of the treatment cycle and the post treatment cycle. Serum concentrations of progesterone and plasma concentrations of 15-keto-13, 14-dihydroprostaglandin F(2alpha) (PGFM) were determined by radionmmunoassay. Intrauterine infusion of endotoxin had no effect on the cycle length or on hormone concentrations, while infusion of viable E. coli organisms tended to shorten the estrous cycle. Intravenous administration of endotoxin produced a sharp increase in both progesterone and PGFM concentrations, followed by a transient decrease in progesterone concentrations. Cycle length remained unchanged. It was concluded that the intact endometrium prevents the uptake of endotoxin although pathogenic E. coli organisms may disrupt the endometrial integrity sufficiently to shorten the estrous cycle by premature luteolysis. It is postulated that intravenous administration of endotoxin influences luteal function by the activation of the arachidonic acid cascade, by a direct effect on the corpus luteum, or via other mediators.     

Down''s Syndrome Individuals Begin Life with Normal Levels of Brain Cholinergic Markers

We measured the activities of the cholinergic marker enzymes choline acetyltransferase (ChAT) and acetylcholinesterase (AChE) in autopsied brains of seven infants (age range 3 months to 1 year) with Down's syndrome (DS), a disorder in which virtually all individuals will develop by middle age the neuropathological changes of Alzheimer's disease accompanied by a marked brain cholinergic reduction. When compared with age-matched controls cholinergic enzyme activity was normal in all brain regions of the individuals with infant DS with the exception of above-normal activity in the putamen (ChAT) and the occipital cortex (AChE). Our neurochemical observations suggest that DS individuals begin life with a normal complement of brain cholinergic neurons. This opens the possibility of early therapeutic intervention to prevent the development of brain cholinergic changes in patients with DS.     

Identification of the thymidine kinase gene of feline herpesvirus: use of degenerate oligonucleotides in the polymerase chain reaction to isolate herpesvirus gene homologs.

Feline herpesvirus 1 (FHV) is the causative agent of viral rhinotracheitis in cats. Current vaccination programs employing attenuated live and killed FHV vaccines have been effective in reducing the incidence of this disease. As an initial step in the development of recombinant FHVs for use in the vaccination of cats, we have identified the thymidine kinase (TK) gene of this feline-specific alphaherpesvirus. Comparisons of the amino acid sequences of other herpesvirus TK proteins have shown that these proteins are highly divergent, sharing only short regions of imperfect amino acid identity. We have used the polymerase chain reaction method of DNA amplification to increase the specificity associated with the use of short, highly degenerate oligonucleotide probes derived from regions of imperfect amino acid conservation. These methods were used to isolate the TK gene of FHV and should prove to be useful in the identification of new members of other viral and cellular gene families. A recombinant FHV bearing a deletion in the identified TK gene was constructed and shown to possess the expected TK- phenotype. The FHV TK gene is located at a position of approximately 40% in the long unique component of the FHV genome. The location of the TK gene and the location and orientation of flanking FHV genes, homologs of herpes simplex virus type 1 UL24 and UL22, are conserved among alphaherpesviruses.     

Neurofibromatosis type 2 appears to be a genetically homogeneous disease

Neurofibromatosis type 2 (NF2) is an autosomal dominant syndrome characterized by the development of vestibular schwannomas and other tumors of the nervous system, including cranial and spinal meningiomas, schwannomas, and ependymomas. The presence of bilateral vestibular schwannomas is sufficient for the diagnosis. Skin manifestations are less common than in neurofibromatosis type 1 (NF1; von Recklinghausen disease). The apparent clinical distinction between NF1 and NF2 has been confirmed at the level of the gene locus by linkage studies; the gene for NF1 maps to chromosome 17, whereas the gene for NF2 has been assigned (in a single family) to chromosome 22. To increase the precision of the genetic mapping of NF2 and to determine whether additional susceptibility loci exist, we have performed linkage analysis on 12 families with NF2 by using four polymorphic markers from chromosome 22 and a marker at the NF1 locus on chromosome 17. Our results confirm the assignment of the gene for NF2 to chromosome 22 and do not support the hypothesis of genetic heterogeneity. We believe that chromosome 22 markers can now be used for presymptomatic diagnosis in selected families. The NF2 gene is tightly linked to the D22S32 locus (maximum lod score 4.12; recombination fraction 0). A CA-repeat polymorphism at the CRYB2 locus was the most informative marker in our families (lod score 5.99), but because the observed recombination fraction between NF2 and CRYB2 was 10 cM, predictions using this marker will need to be interpreted with caution.     

A microdeletion of less than 250 kb, including the proximal part of the FMR-1 gene and the fragile-X site, in a male with the clinical phenotype of fragile-X syndrome

A gene designated "FMR-1" has been isolated at the fragile-X locus. One exon of this gene is carried on a 5.1-kb EcoRI fragment that exhibits length variation in fragile-X patients because of amplification of or insertion into a CGG-repeat sequence. This repeat probably represents the fragile site. The EcoRI fragment also includes an HTF island that is hypermethylated in fragile-X patients showing absence of FMR-1 mRNA. In this paper, we present further evidence that the FMR-1 gene is involved in the clinical manifestation of the fragile-X syndrome and also in the expression of the cellular phenotype. A deletion including the HTF island and exons of the FMR-1 gene was detected in a fragile X-negative mentally retarded male who presented the clinical phenotype of the fragile-X syndrome. The deletion involves less than 250 kb of genomic DNA, including DXS548 and at least five exons of the FMR-1 gene. These data support the hypothesis that loss of function of the FMR-1 gene leads to the clinical phenotype of the fragile-X syndrome. In the fragile-X syndrome, there are pathogenetic mechanisms other than amplification of the CGG repeat that do have the same phenotypic consequences.     

Different requirements for productive interaction between the active site of HIV-1 proteinase and substrates containing -hydrophobic*hydrophobic- or -aromatic*pro- cleavage sites.

The sequence requirements for HIV-1 proteinase catalyzed cleavage of oligopeptides containing two distinct types of junctions (-hydrophobic*hydrophobic- or -aromatic*Pro-) has been investigated. For the first type of junction (-hydrophobic*hydrophobic-) the optimal residues in the P2 and P2' positions were found to be Val and Glu, respectively, in accord with recent statistical analysis of natural cleavage sites [Poorman, R. A., Tomasselli, A. G., Heinrikson, R. L., & Kézdy, F. J. (1991) J. Biol. Chem. 266, 14554-14561]. For the -aromatic*Pro- type of junction, in the specific sequence context studied here, the value of Glu in the P2' position was again observed. An explanation for the inefficient cleavage observed for peptides with the sequence -Val-Tyr*Pro- has been provided from molecular modeling of the putative enzyme-substrate complex. A significant effect upon cleavage rates due to the amino acid in the P5 position has also been documented. While lysine in the P5 position in one sequence of the -hydrophobic*hydrophobic- type produces a peptide cleaved very efficiently (kcat greater than 15 s-1 for Lys-Ala-Arg-Val-Nle*p-nitrophenylalanine-P2'-Ala-Nle-NH2, for P2' = Glu, Gln, Ile, Val, or Ala), for substrates of the -aromatic*Pro- type, the P5 residue can exert either a positive or negative effect on cleavage rates. These results have again been interpreted in light of molecular modeling. We suggest that interaction of the substrate sequence on the periphery of the active site cleft may influence the match of the enzyme-substrate pair and, hence, control the efficiency of catalysis.(ABSTRACT TRUNCATED AT 250 WORDS)