Biomedical subjects
Publications and source records attributed to M Goodman.
To appreciate what illness means to an individual, physicians must understand that person's attitudes, views, and style of life. People react uniquely and creatively to illness and disability. Some incorporate the illness into their lives and use it to help them achieve goals that might not be obvious to the doctor. A case study presents diagnostic and therapeutic problems familiar to family physicians.
We recently isolated two orphan human G protein-coupled receptor genes designated GPR1 and GPR6. The gene GPR1 was shown to be transcribed abundantly but only in the hippocampus. Here we report the cloning of the rat GPR1 gene and report the absence of expression in hippocampus, demonstrating a functional variation for this receptor in these two species. The evolutionary history of an important sequence difference in the gene GPR1 in primate and rodent species has been examined. In contrast extensive mapping of gene GPR6 mRNA in rat brain was in keeping with the described distribution in human brain.
The use of the ammonia deprotection step to introduce (15)N labels at specific exocyclic amino positions of adenine, cytosine, guanine or 2-aminopurine of oligodeoxynucleotides is described.
Expression of the anthropoid (simian) gamma gene in fetal life contrasts with the exclusively embryonic expression pattern of the gamma-like genes of other eutherian mammals. To elucidate the factors responsible for this change in expression pattern, we utilized a strategy called differential phylogenetic footprinting (DPF). This strategy entails the following: (a) identification, within regulatory regions, of the gamma promoter, of individual nucleotides that differ between human (fetal expression), and galago (embryonic expression) gamma genes, (b) analysis of the effect of these nucleotide differences on the binding of nuclear proteins to human and galago sequences, and (c) assessment of the functional consequences of these binding changes in expression assays. The DPF analysis revealed several proteins that bind upstream from the CCAAT motif in the galago gamma promoter but do not bind to the corresponding region of the human gamma promoter. In transfection assays, binding of these proteins is associated with erythroid-specific repression of promoter strength. Binding sites for these proteins also occur near the CCAAT box of other embryonically expressed genes, including rabbit, mouse, and dwarf lemur gamma genes and the human epsilon globin gene. These data are consistent with the hypothesis that sequence changes near the proximal CCAAT box in the ancestral simian gamma gene may have facilitated a novel expression pattern by reducing the binding of repressors that act in the fetal stage.
Evidence from DNA sequences on the phylogenetic systematics of primates is congruent with the evidence from morphology in grouping Cercopithecoidea (Old World monkeys) and Hominoidea (apes and humans) into Catarrhini, Catarrhini and Platyrrhini (ceboids or New World monkeys) into Anthropoidea, Lemuriformes and Lorisiformes into Strepsirhini, and Anthropoidea, Tarsioidea, and Strepsirhini into Primates. With regard to the problematic relationships of Tarsioidea, DNA sequences group it with Anthropoidea into Haplorhini. In addition, the DNA evidence favors retaining Cheirogaleidae within Lemuriformes in contrast to some morphological studies that favor placing Cheirogaleids in Lorisiformes. While parsimony analysis of the present DNA sequence data provides only modest support for Haplorhini as a monophyletic taxon, it provides very strong support for Hominoidea, Catarrhini, Anthropoidea, and Strepsirhini as monophyletic taxa. The parsimony DNA evidence also rejects the hypothesis that megabats are the sister group of either Primates or Dermoptera (flying lemur) or a Primate-Dermoptera clade and instead strongly supports the monophyly of Chiroptera, with megabats grouping with microbats at considerable distance from Primates. In contrast to the confused morphological picture of sister group relationships within Hominoidea, orthologous noncoding DNA sequences (spanning alignments involving as many as 20,000 base positions) now provide by the parsimony criterion highly significant evidence for the sister group relationships defined by a cladistic classification that groups the lineages to all extant hominoids into family Hominidae, divides this ape family into subfamilies Hylobatinae (gibbons) and Homininae, divides Homininae into tribes Pongini (orangutans) and Hominini, and divides Hominini into subtribes Gorillina (gorillas) and Hominina (humans and chimpanzees). A likelihood analysis of the largest body of these noncoding orthologues and counts of putative synapomorphies using the full range of sequence data from mitochondrial and nuclear genomes also find that humans and chimpanzees share the longest common ancestry.
One-dimensional nmr exchange spectroscopy was carried out to determine thermodynamic parameters of cyclophilin-induced cis-trans isomerization of succinyl-Ala-Phe-Pro-Phe-p-nitroanilide. Rate measurements were possible at physiological temperatures. The kc/Km of rat cyclophilin was found to be 12.8 (+/- 0.5) s-1 microM-1 at 37 degrees C, intermediate to previously reported values that used a coupled enzyme assay extrapolated to this temperature. Activation energies (delta G not equal to) for the uncatalyzed and catalyzed reaction at 37 degrees C were found to be 19.7 and 17.1 kcal/mol, respectively, and were primarily due to an enthalpic barrier.
This is a single case report of a 56-year-old Caucasian female referred for biofeedback by her dermatologist after seven years of failed standard medical treatment for psoriasis. Patient's presenting complaint was the embarrassing psoriasis lesions on her arms. Following 13 weekly one-hour finger/hand thermal biofeedback treatments, all 11 presenting psoriasis lesions (2-6 cm) had disappeared. Interestingly, any new psoriasis lesions that surfaced during our treatment disappeared without leaving palpable or visible scarring, unlike lesions that were present prior to biofeedback treatment. Patient was unmedicated for psoriasis during our treatment and continues to be unmedicated and asymptomatic at 12-month follow-up.
Articulation norms are typically used to measure sound acquisition and mastery in children with Down syndrome. The present longitudinal study examines the clinical records of 60 children with Down syndrome to evaluate phoneme acquisition and emergence. Results document the order of emergence of sounds in children with Down syndrome. Factors affecting emergence of sounds (e.g. oral motor skills) and difficulties in using a mastery model to evaluate speech sound acquisition in children with Down syndrome are explored. Clinical as well as research implications of the data are presented.
BACKGROUND: Amide bonds in peptides and proteins typically adopt planar cis or trans conformations. Conversions between cis and trans amide conformations are necessary for protein folding and for many other processes, but are difficult to achieve since they involve disruption of the planarity of the bond. As a first step to understanding cis-trans isomerization, we set out to synthesize and characterize peptides that mimic the tilted or twisted amide structures that are postulated to form the intermediate states in this process. RESULTS: We have synthesized a model amino acid and four dipeptide derivatives containing a methyl-substituted aziridine residue. Single crystals of phenacyl (2R, 3R)-benzyloxycarbonyl-3-methyl-2-aziridinecarboxylate and phenacyl (2R, 3R)-acetyl-glycyl-3-methyl-2-aziridine-carboxylate were obtained. Using X-ray diffraction analysis, we determined that the amide nitrogens of the aziridine rings have tetrahedral sp3-like geometry with tilt angles in the range of 37-38 degrees. The 13C-NMR spectra indicate that the amide carbonyl is dramatically shifted downfield as a consequence of the tilt. CONCLUSIONS: In peptides containing a substituted aziridine ring, the orbitals of the amide nitrogen are constrained into a tilted configuration. These peptides may mimic the transition state between cis and trans amide conformations. This technique thus provides a novel strategy for the study of isomerization and other biorecognition processes.
The concentrations of inhibin and follistatin in amniotic fluid and in tissue extracts from the placenta, gonads and adrenals of fetal sheep were measured using radioimmunoassays. These tissue extracts were from whole fetuses from days 16 to 45 and from the individual organs from day 46 to 145 (term) and were assayed at multiple dilutions. The capacity of these extracts to alter FSH production of rat anterior pituitary cells in culture was also assessed at multiple dilutions. Immunoactive inhibin concentrations in amniotic fluid from both sexes increased during gestation and levels were significantly greater in males than females. Peak concentrations of immunoreactive inhibin of 11.2 +/- 1.9 ng/ml were found in males at 116-125 days of gestation. Follistatin concentrations did not change throughout gestation and no significant difference was noted between sexes. Mean follistatin levels throughout gestation were 3.0 +/- 0.9 ng/ml for males and 3.7 +/- 0.9 ng/ml for females. Despite the potential for FSH inhibition by inhibin and follistatin, amniotic fluid from both sexes at all stages of gestation stimulated FSH secretion in the pituitary cell bioassays, suggesting the presence of activin which was confirmed by the measurement of immunoactive activin (13.3 +/- 2.5 ng/ml) in a specific radioimmunoassay. Maximum concentrations of immunoactive and bioactive inhibin in placental extracts were observed in late gestation (2.2 +/- 0.6 and 3.8 +/- 1.6 ng/g respectively) and there was no significant difference between sexes. Follistatin concentrations in placental cotyledons ranged from 11.5 to 27.1 ng/g with no significant difference between sexes. In view of the higher follistatin concentrations compared with inhibin, it is likely that the capacity of placental extracts to suppress FSH production by pituitary cells in culture is due predominantly to follistatin. Immunoactive inhibin was observed in high concentrations in the fetal testis throughout gestation; with concentrations increasing to a maximum of 1993.0 +/- 519.7 ng/g at 126-135 days of gestation with a ratio of bioactive: immunoactive inhibin of 1:20. Although bioactive and immunoactive inhibin was also observed in fetal ovaries and adrenals from both male and female fetuses, concentrations were lower than those observed in fetal testes. Follistatin concentrations in the fetal testis were elevated between 70 and 95 days (97.6 ng/g) and then declined. Similar concentrations were found in the adrenal glands of both sexes (males 83.5-103.3 ng/g: females 55.3-95.8 ng/g).(ABSTRACT TRUNCATED AT 400 WORDS)
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To investigate structure-function relationships of erythropoietin (Epo), we have obtained cDNA sequences that encode the mature Epo protein of a variety of mammals. A first set of primers, corresponding to conserved nucleotide sequences between mouse and human DNAs, allowed us to amplify by polymerase chain reaction (PCR) intron 1/exon 2 fragments from genomic DNA of the hamster, cat, lion, dog, horse, sheep, dolphin, and pig. Sequencing of these fragments permitted the design of a second generation of species-specific primers. RNA was prepared from anemic kidneys and reverse-transcribed. Using our battery of species-specific 5' primers, we were able to successfully PCR-amplify Epo cDNA from Rhesus monkey, rat, sheep, dog, cat, and pig. Deduced amino acid sequences of mature Epo proteins from these animals, in combination with known sequences for human, Cynomolgus monkey, and mouse, showed a high degree of homology, which explains the biologic and immunological cross-reactivity that has been observed in a number of species. Human Epo is 91% identical to monkey Epo, 85% to cat and dog Epo, and 80% to 82% to pig, sheep, mouse, and rat Epos. There was full conservation of (1) the disulfide bridge linking the NH2 and COOH termini; (2) N-glycosylation sites; and (3) predicted amphipathic alpha-helices. In contrast, the short disulfide bridge (C29/C33 in humans) is not invariant. Cys33 was replaced by a Pro in rodents. Most of the amino acid replacements were conservative. The C-terminal part of the loop between the C and D helices showed the most variation, with several amino acid substitutions, deletions, and/or insertions. Calculations of maximum parsimony for intron 1/exon 2 sequences as well as coding sequences enabled the construction of cladograms that are in good agreement with known phylogenetic relationships.
The human epsilon-globin gene undergoes dramatic changes in transcriptional activity during development, but the molecular factors that control its high expression in the embryo and its complete repression at 6-8 weeks of gestation are unknown. Although a putative silencer has been identified, the action of this silencer appears to be necessary but not sufficient for complete repression of epsilon gene expression, suggesting that multiple control elements may be required. Phylogenetic footprinting is a strategy that uses evolution to aid in the elucidation of these multiple control points. The strategy is based on the observation that the characteristic developmental expression pattern of the epsilon gene is conserved in all placental mammals. By aligning epsilon genomic sequences (from -2.0 kb upstream to the epsilon polyadenylylation signal), conserved sequence elements that are likely binding sites for trans factors can be identified against the background of neutral DNA. Twenty-one such conserved elements (phylogenetic footprints) were found upstream of the epsilon gene. Oligonucleotides spanning these conserved elements were used in a gel-shift assay to reveal 47 nuclear binding sites. Among these were 8 binding sites for YY1 (yin and yang 1), a protein with dual (activator or repressor) activity; 5 binding sites for the putative stage selector protein, SSP; and 7 binding sites for an as yet unidentified protein. The large number of high-affinity interactions detected in this analysis further supports the notion that the epsilon gene is regulated by multiple redundant elements.
A topochemical model to explain the bioactivity of morphiceptin (Tyr1-Pro2-Phe3-Pro4-NH2) was developed by taking account of accessible conformations around rotatable bonds which define relative spatial arrangements of opioid pharmacophores, the amine and phenolic groups of tyrosine and the aromatic ring of phenylalanine, necessary for receptor recognition. For this purpose, 1H-NMR measurements and computer simulations were extensively carried out on 10 stereoisomeric analogs related to morphiceptin: Tyr-Pro-(L and D)-Phe- (L and D)-Pro-NH2; Tyr-Pro-(L and D)-(NMe)Phe-(L and D)-Pro-NH2; Tyr-(NMe)Ala-Phe-D-Pro-NH2; and Tyr-Ala-Phe-D-Pro-NH2. These analogs are structurally close to one another but display various opiate potencies from highly active to inactive. The conformation of each rotatable bond has been specifically identified by measuring accessible space for the analogs, in which the difference in composition is observed in the specific site affecting only the conformation around the target bond. The most interesting characteristic of the model is a requirement of a cis amide bond linking residues 1 and 2. The model also requires the side chains in a trans conformation (chi 1 = 180 degrees) for the Tyr and Phe residues. The distances between the three pharmacophores, d1 (Tyr N to Tyr OH), d2 (Tyr N to the center of the aromatic ring of the third residue), and d3 (Tyr OH to the center of the aromatic ring of the third residue), were found to be approximately 8, approximately 7, and approximately 11-13 A, respectively. This model should aid in pharmaceutical design of peptide and nonpeptide ligands with opioid potencies.
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By sequencing extensive regions of the beta-globin gene cluster from capuchin monkey (New World monkey) and tarsier (prosimian) we confirmed that capuchin monkey and tarsier have two and one gamma-globin gene(s), respectively. These findings indicate that the ancestral anthropoid gamma-globin gene duplicated after anthropoids diverged from tarsier, but before they diverged into platyrrhines (New World monkeys) and catarrhines (Old World monkeys, apes, and human). The capuchin monkey gamma 1-globin gene promoter region accumulated many nucleotide substitutions, including a T to C substitution in the proximal CCAAT element. This adverse mutation, along with the previous finding that the gamma 1 locus in spider monkey is a pseudogene, suggests that in platyrrhines the gamma 2-globin gene may be the primary fetal beta-like globin gene. The aligned gamma gene sequences contain several conserved sequence elements (phylogenetic footprint) of 6 bp or longer in the 5' flanking region, but none in the 3' flanking region. Gene conversions frequently occurred in the 5' flanking and transcribed regions of the duplicated genes of anthropoids but rarely in the 3' flanking sequences. However, an absence of conversions within the capuchin and spider monkeys promoter regions suggests that in platyrrhines selection acted against conversions as they could decrease (or inactivate) gamma 2 expression.
Phylogenetic relationships among the 16 extant genera of Ceboidea (the New World monkeys) were examined using aligned epsilon-globin gene sequences from 19 New World monkeys (representing all 16 extant ceboid genera), and seven catarrhines (one Old World monkey and six hominoids) and tarsier as the outgroups. The consensus maximum parsimony tree found for these epsilon-globin sequences and the levels of support from parsimony and bootstrap analyses, for the clades in this tree, provided strong evidence for a cladistic classification with the following clusters. Subtribes Callitrichina (Callithrix, Cebuella), Callimiconina (Callimico), Leontopithecina (Leontopithecus), and Saguina (Saguinus) constitute subfamily Callitrichinae, and subfamilies Callitrichinae, Aotinae (Aotus), Saimiriinae (Saimiri), and Cebinae (Cebus) constitute family Cebidae. In turn, subtribes Chiropotina (Chiropotes, Cacajao) and Pitheciina (Pithecia) constitute tribe Pithecini, tribes Pitheciini and Callicebini (Callicebus) constitute subfamily Pitheciinae, tribes Atelini (Brachyteles, Lagothrix, Ateles) and Alouattini (Alouatta) constitute subfamily Atelinae, and subfamilies Pitheciinae and Atelinae constitute family Atelidae. The two families (Cebidae and Atelidae) constitute the Ceboidea, the only extant superfamily of infraorder Platyrrhini. The sister-group relationships of Brachyteles and Lagothrix, Saguinus and Leontopithecus, and Callimico with a Cebuella/Callithrix clade is not as well supported by the parsimony and bootstrap analyses. Therefore, these relationships are not incorporated in the proposed cladistic classification. On determining branch lengths for the ceboid phylogenetic tree from only the more freely evolving noncoding sequences at the epsilon-globin locus and taking the reference age of 35 million years ago (MYA) for the New World monkey-catarrhine branch point, we estimated the age of the atelid-cebid branch point as about 20 MYA, and the ages of the next branch points, those between the subfamilies in each family, as 19-16 MYA.