Biomedical subjects

D M Hunt

Publications and source records attributed to D M Hunt.

At least 109 records · Page 6Linked to original sources

PubMed1995

Adaptive evolution of color vision genes in higher primates.

The intron 4 sequences of the three polymorphic alleles at the X-linked color photo-pigment locus in the squirrel monkey and the marmoset reveal that the alleles in each species are exceptionally divergent. The data further suggest either that each triallelic system has arisen independently in these two New World monkey lineages, or that in each species at least seven deletions and insertions (14 in the two species) in intron 4 have been transferred and homogenized among the alleles by gene conversion or recombination. In either case, the alleles in each species apparently have persisted more than 5 million years and probably have been maintained by overdominant selection.

Alleles↗

PubMed1995

Cloning vectors for the production of proteins in Dictyostelium discoideum.

We constructed and tested a series of cloning vectors designed to facilitate protein production and purification in Dictyostelium discoideum (Dd). These vectors carry the origin of replication of the Dd high-copy-number plasmid Ddp2, expression cassettes consisting of the strong, constitutive actin (act15) or the inducible discoidin (disI gamma) promoters, a translational start codon upstream from a multiple cloning site and sequences for the addition of epitope or affinity tags at the N- or C-termini of any protein. The affinity tag used corresponds to 7 (N-terminal fusion) or 8 (C-terminal fusion) His residues. The epitope tags correspond to an 11-amino-acid sequence from human c-myc, recognised by monoclonal antibody (mAb) 9E10, and the Glu-Glu-Phe sequence recognised by mAb YL1/2 to alpha-tubulin. Both these mAb are commercially available. The YL1/2 epitope offers a second affinity tag for the purification of proteins under native conditions. The functional competence of the vectors was tested by determining their ability to promote the expression of various Dd myosin constructs. High synthesis levels were obtained for each vector; up to 1 mg of homogenous, functional protein per g of cells was obtained after purification of the recombinant products.

Amino Acid Sequence↗

PubMed1995

Sequence and evolution of the blue cone pigment gene in Old and New World primates.

The sequences of the blue cone photopigments in the talapoin monkey (Miopithecus talapoin), an Old World primate, and in the marmoset (Callithrix jacchus), a New World monkey, are presented. Both genes are composed of 5 exons separated by 4 introns. In this respect, they are identical to the human blue gene, and intron sizes are also similar. Based on the level of amino acid identity, both monkey pigments are members of the S branch of pigments. Alignment of these sequences with the human gene requires the insertion/deletion of two separate codons in exon 1. The silent site divergence between these primate blue genes indicates a separation of the Old and New World primate lineages around 43 million years ago.

Amino Acid Sequence↗

PubMed1995

The chemistry of John Dalton's color blindness.

John Dalton described his own color blindness in 1794. In common with his brother, he confused scarlet with green and pink with blue. Dalton supposed that his vitreous humor was tinted blue, selectively absorbing longer wavelengths. He instructed that his eyes should be examined after his death, but the examination revealed that the humors were perfectly clear. In experiments presented here, DNA extracted from his preserved eye tissue showed that Dalton was a deuteranope, lacking the middlewave photopigment of the retina. This diagnosis is shown to be compatible with the historical record of his phenotype, although it contradicts Thomas Young's belief that Dalton was a protanope.

Base Sequence↗

PubMed1995

Overexpression of myosin motor domains in Dictyostelium: screening of transformants and purification of the affinity tagged protein.

The eukaryotic organism Dictyostelium discoideum has become one of the organisms of choice for the overexpression of recombinant myosins and myosin fragments. Here, we describe a protocol that facilitates the screening of cells that have been transformed with myosin expression constructs and allows the rapid purification of recombinant myosins. Depletion of cellular ATP is used to recruit most of the endogenous and recombinant myosin into a rigor-like complex with actin. Following cell lysis the insoluble actomyosin complex is precipitated by centrifugation, washed, and Mg(2+)-ATP is added to extract the recombinant protein from the pellet. More than 90% of the protein in the resulting supernatant corresponds to actin, myosin, and the recombinant myosin fragments. Therefore, it is easy to detect any differences in expression level between individual myosin constructs on SDS-polyacrylamide gels. Additionally, the dependence of expression on external factors, such as cell density, can be readily determined. Furthermore, the presence of a band corresponding to the recombinant protein indicates that the overexpressed protein has at least some of the functional properties that are characteristic for a myosin motor. This rapid and selective extraction protocol can also be utilized to facilitate the purification of recombinant myosin motors on a preparative scale and has proved particularly useful in the purification of myosin head fragments, that are tagged with histidine residues, by Ni(2+)-chelate affinity chromatography.

Actinin↗

PubMed1995

CAG repeat expansions and schizophrenia: association with disease in females and with early age-at-onset.

An increase in the severity of schizophrenia through consecutive generations (anticipation) has been found in some studies of families with affected members. Anticipation in five neurological disorders is known to arise from the expansion of CAG repeats between generations of affected individuals. The 'repeat expansion detection' method was used to screen individual genomes for the size of such expansions in a sample of schizophrenic and normal subjects. Comparison of the frequency distribution of CAG expansions observed in schizophrenic patients to that for normal subjects, showed that there are significantly more expansions in patients (p = 0.048). When male and female subjects are considered separately, there is a highly significant difference in the distribution of repeat sizes found between affected and normal females (p = 0.0023) but no significant difference between affected and normal males. Overall there is a 28% excess of expansions observed in affected versus normal females, and their presence confers a relative risk of 4.12 (p < 0.005). In contrast, the frequency distribution of age-at-onset with respect to repeat size is nearly the same in male and female patients and, when the sexes are combined, the larger (CAG)69-136 expansions are associated with a younger age-at-onset (p = 0.02).

Adolescent↗

PubMed1995

Localization of the gene for progressive bifocal chorioretinal atrophy (PBCRA) to chromosome 6q.

Progressive bifocal chorioretinal atrophy (PBCRA) is a rare, autosomal dominant congenital chorioretinal dystrophy. We have performed genetic linkage analysis on a five-generation British pedigree. Two-point linkage analysis showed significant linkage with nine microsatellite marker loci mapping to chromosome 6q. Multipoint analysis gave a maximum lod score of 11.8 (theta = 0.05) between D6S249 and D6S283. This region overlaps with that to which the gene for North Carolina macular dystrophy (MCDR1) has been assigned. However, given the range of differences in phenotype between these two retinal disorders, it is likely that different mutation mechanisms are responsible for each disease.

Atrophy↗

PubMed1995

Questionnaire format bias: when are juxtaposed scales appropriate: a call for further research.

This study examined the use of juxtaposed scales, a type of self-report response scale used to study numerous attitudinal and behavioral concepts. This research is an extension of the exploratory work on the bias associated with juxtaposed scales begun by Hunt and Miller in 1983. Findings support the hypothesis that juxtaposed scales will elicit different responses than separate scales. When are these differences explicit and when are they an unwanted bias? Results and attendant discussion and recommendations are used to call attention to the importance of planning for the method bias which can be created by the use of juxtaposed scales.

Attitude↗

PubMed1995

Visual responses in the lateral geniculate nucleus of dichromatic and trichromatic marmosets (Callithrix jacchus).

New-world primates such as the marmoset (Callithrix jacchus) show polymorphism for the middle- to long-wavelength sensitive cone pigments. Each X-chromosome carries a gene for only one of three possible pigments. All males are thus dichromats, but some females will be trichromats. We have investigated the responses of cells of the parvocellular (PC) and magnocellular (MC) systems within animals from a single marmoset family. The middle- to long-wavelength pigment of dichromats was identified physiologically. Trichromats could readily be distinguished from dichromats by the presence of a high proportion of red-green opponent PC-cells. The physiological classification of phenotypes was confirmed with genetic analysis. The pattern of inheritance was consistent with current genetic models. In trichromatic females, the properties of cells resembled in detail those of cells from the PC- and MC-pathways of the macaque. In dichromats, cell responses resembled those of trichromats (except for the lack of opponency in PC-cells); PC-cells showed sustained and MC-cells transient responses, with a lower contrast gain for the former type. One difference was that a proportion of PC-cells in dichromats showed strong rod input even at high levels of retinal illuminance. Thus, in trichromatic marmosets the presence of two middle- to long-wave pigments appears to permit the elaboration of all the physiological properties associated with trichromacy.

Animals↗

PubMed1994

Localisation of the human blue cone pigment gene to chromosome band 7q31.3-32.

Blue cone pigment (BCP) is one of three types of cone photoreceptors responsible for normal colour vision. In this study, the BCP gene has been localised to chromosome 7q31.3-32 by fluorescent in situ hybridisation of cosmid clones containing the gene. This is consistent with previous mapping of the BCP gene to chromosome 7q31-35.

Cells, Cultured↗

PubMed1994

Visual pigments and the photic environment: the cottoid fish of Lake Baikal.

The endemic cottoid fish of Lake Baikal in Eastern Siberia offer a singular opportunity for examining within a number of closely related species, the relationships of visual pigments, photoreceptor complements and depth within a deep freshwater environment. The lake, the deepest (1600 m) and one of the largest and most ancient in the world, is unique in that the oxygen levels at the bottom are only reduced to about 80% of the surface levels. We have studied, by light microscopy, microspectrophotometry and visual pigment extraction, the retinas from 17 species of Baikal cottoids that live at different depths within the lake. Generally the retinas contain, in addition to rods, large green-sensitive double cones and small blue-sensitive single cones: surprisingly for freshwater fish, the visual pigments are based on Vitamin A1. The lambda max of both rods and cones are displaced to shorter wavelengths with increasing depth. Surface species have cones with lambda max at about 546, 525 and 450 nm and rods at 523 nm, deeper living species retain cones, but with lambda max shifting towards 500 and 425 nm and with rods at 480 nm, whereas the deepest living fish possess only rods (lambda max 480-500 nm). These data clearly show a correlation between photoreceptor complement, visual pigment lambda max and depth, but question the hypothesis that there is a correlation of pigment lambda max with water colour since, in contrast to oceanic waters, the maximum transmission of Baikal water is between 550 and 600 nm.

Animals↗

PubMed1994

Sequence divergence, polymorphism and evolution of the middle-wave and long-wave visual pigment genes of great apes and Old World monkeys.

In man, the spectral shift between the middle-wave (MW) and long-wave (LW) visual pigments is largely achieved by amino acid substitution at two codons, both located in exon 5. A third amino acid site coded by exon 3 is polymorphic between pigments. We have studied the equivalent regions of the cone opsin genes in two members of the Hominidea (the gorilla, Gorilla gorilla and the chimpanzee, Pan troglodytes) and in three members of the Cercopithecoidea family of Old World primates (the diana monkey, Cercopithecus diana, the talapoin monkey, Miopithecus talapoin, and the crab-eating macaque, Macaca fascicularis). No variation in the codons that specify the amino acids involved in spectral tuning were found. We predict therefore that the MW and LW pigments of gorilla and chimpanzee have similar spectral characteristics to those of man. Multiple copies of the same opsin gene sequence were identified in the chimpanzee, talapoin and macaque and we also show that non-human Old World primates are similar to man in showing a bunching of polymorphic sites in exon 3. We discuss the ancestry of the separate MW and LW genes of Old World primates and the equivalent polymorphic gene of the marmoset, a New World primate.

Amino Acid Sequence↗

PubMed1994

Effectiveness of transnasal butorphanol for the treatment of musculoskeletal pain.

A prospective, open-label study of the effectiveness of transnasal butorphanol in the treatment of pain resulting from musculoskeletal injuries. Twenty-eight patients with strains (n = 20), fractures (n = 6), contusions (n = 1), and stab wounds (n = 1) were included. All patients were examined by an attending level emergency medicine physician and deemed to have pain severe enough to warrant parenteral narcotic analgesia. All patients received an initial 1-mg dose of transnasal butorphanol. Subsequent dosing was flexible depending on response to the initial dose. All patients received pain relief from transnasal butorphanol, and only one requested alternative analgesic medication. Fifty-seven percent (n = 16) of patients noticed at least a little relief of pain within 5 minutes of administration and 93% (n = 26) received at least a little relief within 15 minutes. Seventy-one percent of the patients received a 50% reduction of pain within 60 minutes. No serious side effects were noted, but drowsiness occurred in 82% (n = 23) and dizziness in 54% (n = 15) of the patients. One patient discontinued participation in the study because of nausea. In this limited trial transnasal butorphanol proved to be a rapidly effective opioid analgesic. Further controlled studies comparing transnasal butorphanol with standard parenteral narcotics are needed.

Administration, Intranasal↗

PubMed1994

Genetic linkage of cone-rod retinal dystrophy to chromosome 19q and evidence for segregation distortion.

Inherited retinal dystrophies are the most common cause of childhood blindness in the developed world. Cone-rod retinal dystrophies are severe examples of this group of disorders. Analysis of a large cone-rod dystrophy pedigree suggested that inheritance within the family was influenced by meiotic drive (p = 0.008), a rare segregation distortion in human genetics. Two-point linkage analysis showed significant linkage with three markers mapping to chromosome 19q. Multipoint analysis gave a maximum lod score of 10.08 (theta = 0.05) distal to D19S47. Cone-rod dystrophy is therefore assigned to 19q13.1-q13.2 and a new candidate locus for other retinal dystrophies is identified.

Child↗

PubMed1994

Identification of an amino acid change that affects N protein function in vesicular stomatitis virus.

TsW16B is a temperature-sensitive mutant of vesicular stomatitis virus. Others have shown that it is temperature-sensitive for replication in vivo and for transcription in vitro and that these phenotypes are probably due to mutation of the N (nucleocapsid) gene. Five independent revertants were isolated from tsW16B based on their ability to grow at 39 degrees C. The thermosensitivity of in vitro transcription by these revertants was similar to that of the wild-type virus [wt(HR)] from which tsW16B was derived. Fractionation-reconstitution studies of two revertants indicated that the reversion was in the N or P (phosphoprotein) gene. The N and P genes of wt(HR), tsW16B, and these two revertants were sequenced. There were no differences between the P genes. Comparison of the predicted N protein sequences of wt(HR), tsW16B and the two revertants indicated that the growth and in vitro transcription phenotypes of tsW16B were due to a change of amino acid residue 238 from threonine to isoleucine. The amino acid at position 238 in the other three revertants also showed an exact reversion to threonine. Amino acid residue 238 lies in a domain of the N protein which is highly conserved among vesiculoviruses.

Animals↗

PubMed1994

Localization of the gene encoding human phosphatidylinositol transfer protein (PITPN) to 17p13.3: a gene showing homology to the Drosophila retinal degeneration B gene (rdgB).

The human gene for phosphatidylinositol transfer protein (PITPN) has previously been shown to share sequence and functional homology to part of the Drosophila retinal degeneration B gene (rdgB). In view of the possible involvement of the PITPN locus in the etiology of retinal disease, the gene has been mapped to human chromosome 17p13.3 and mouse Chromosome 11.

Animals↗

PubMed1994

The changing relationship between physicians and hospitals.

There is a growing trend towards the unification of the practices of physicians under the umbrella of a hospital or group of hospitals. Prior to entering into such relationships, hospitals must test the relationship to be sure that it will not lead to a violation of the anti-kickback statute or, if the hospital is tax-exempt under section 501(c)(3), the prohibition against private inurement under the Internal Revenue Code. These are complex determinations and competent advice should be sought before entering into such relationships.

Hospital-Physician Joint Ventures↗

PubMed1993

The molecular basis of a spectral shift in the rhodopsins of two species of squid from different photic environments.

The molecular basis of spectral tuning of rhodopsin pigments in two squid species is examined. The absorbance spectra of rhodopsin extracts from Alloteuthis subulata (lambda max 499 nm) showed a 5 nm red shift compared with Loligo forbesi (lambda max 494 nm). The rhodopsin gene sequence of A. subulata opsin was determined from polymerase chain reaction (PCR) amplified fragments by using genomic DNA as template. The deduced amino acid sequence was compared with that obtained from the previously published cDNA sequence of Loligo forbesi. A total of 22 amino acid differences are present, although only seven can be considered to be non-homologous substitutions. Three of these changes occur in helical transmembrane regions but only one, the substitution of phenylalanine by serine at site 270 in Alloteuthis subulata, involves the replacement of an apolar with a hydroxyl-bearing residue at a site located near the centre of helix VI and on the inner face of the retinal-binding pocket. The equivalent site is also used for the spectral tuning of primate cone pigments, an example of convergent evolution. It is proposed that substitution at this site is responsible for the 5 nm red shift. The relation between this spectral shift and the maximum depth distribution of Alloteuthis subulata of about 200 m, compared with about 360 m for Loligo forbesi, is discussed. An unexpected finding was that the rhodopsin gene of both species appears to lack introns.

Amino Acid Sequence↗