Biomedical subjects
M M Green
Publications and source records attributed to M M Green.
Cytokine-receptor complexes as chaperones for nuclear translocation of signal transducers.
A variety of ligands that include interleukins, interferons, and growth hormones activate STAT transcriptions factors. When activated, STATs are translocated to the nucleus apparently through the well described importin/Ran system where they activate target genes. Molecules utilizing this nuclear import system require specific nuclear localization sequences (NLSs). Paradoxically, such NLSs are not identifiable on STATs, raising the question of how they are imported into the nucleus. Surprisingly, most ligands and/or receptors that signal through STATs contain putative NLSs, and where examined either ligand or receptor undergo nuclear translocation. We hypothesize that these ligands and/or their receptors serve as chaperones in the nuclear translocation of STATs, and that they may be directly involved in signal transduction. Using IFN gamma as a model system we provide a possible mechanism for how this direct role is fulfilled. A C-terminal domain of IFN gamma has been identified that contains a functional NLS. Besides the fact that this domain, and the NLS in particular, is crucial for the biological properties of IFN gamma, a peptide encompassing this domain is sufficient to induce an antiviral state. Moreover, this domain interacts exclusively with an internal cytoplasmic domain of a subunit of the receptor complex in a region that is directly involved in the recruitment and activation of the elements of the JAK/STAT pathway. We suggest that this novel mode of receptor recognition and activation may be a driving force for nuclear translocation of molecules like STATs that are associated with the ligand-receptor complex.
Human IFN gamma receptor cytoplasmic domain: expression and interaction with HuIFN gamma.
To investigate the structural basis for human interferon gamma (huIFN gamma) binding to intracellular regions of the human IFN gamma receptor (huIFN gamma R), we have subcloned and expressed the huIFN gamma R free of fusion proteins in the yeast strain Pichia pastoris. HuIFN gamma bound to the cytoplasmic domain of the receptor via the IFN gamma C-terminus. Binding was inhibited by both human and mouse C-terminus peptides. N-terminus peptides failed to inhibit cytoplasmic binding. Thus, while extracellular receptor domain binding is species specific, binding to the cytoplasmic domain of the receptor is species non-specific. In solid-phase binding assays, IFN gamma had a Kd of 3.7 x 10(-8) M for the newly expressed cytoplasmic domain. Peptide competitions showed that IFN gamma bound to a receptor site corresponding to the membrane proximal residues 253-287, which is adjacent to the site of binding of the tyrosine kinase JAK2. The cytoplasmic binding affinity and binding site specificity suggest that the huIFN gamma R cytoplasmic domain can function independent of the extracellular domain to bind huIFN gamma and induce the biological activity previously associated with internalized huIFN gamma.
Molecular cloning, sequence analysis, expression, and tissue distribution of suppressin, a novel suppressor of cell cycle entry.
Suppressin (SPN) is an inhibitor of cell proliferation that was originally identified and purified to homogeneity from bovine pituitaries (LeBoeuf, R. D., Burns, J. N., Bost, K. L., and Blalock, J. E. (1990) J. Biol. Chem. 265, 158-165). In this report we have cloned the full-length cDNA encoding rat SPN and have identified the tissue distribution of SPN expression. The cDNA of SPN is 1882 nucleotides with a 1488-base coding region and 55 and 339 nucleotides of 5'- and 3'-untranslated sequences, respectively. Northern gel analysis of rat pituitary mRNA showed a single hybridizing species at approximately 2 kilobases. Sequence analyses showed that the nucleotide and deduced amino acid sequences of SPN are novel and unrelated to any known vertebrate inhibitors of proliferation. However, the deduced amino acid sequence of SPN contains two domains that have extensive sequence identity with a recently cloned transcription activator in Drosophila, deformed epidermal autoregulatory factor-1 (DEAF-1, see Gross, C. T., and McGinnis, W. (1996) EMBO J. 15, 1961-1970) suggesting that SPN represents a vertebrate cognate of deformed epidermal autoregulatory factor-1. Reverse transcriptase-polymerase chain reaction and immunohistochemical analyses showed that the SPN mRNA and the SPN protein are expressed in every tissue examined including testis, spleen, skeletal muscle, liver, kidney, heart, and brain suggesting that SPN may be involved in the control of proliferation in a variety of cell types.
DNA sequence analysis proves Hb M-Milwaukee-2 is due to beta-globin gene codon 92 (CAC-->TAC), the presumed mutation of Hb M-Hyde Park and Hb M-Akita.
Among the causes of congenital methemoglobinemia, Hb M-Milwaukee-2 was one of the earliest described, in a patient who also had Hb E trait. The structure of Hb M-Milwaukee-2 has been elusive. DNA sequence analysis, as here reported, proves that this hemoglobin variant is due to the mutation CAC-->TAC at codon 92 of the beta-globin gene, corresponding to the substitution of tyrosine for histidine. This mutation is identical with that presumed to be the cause of Hb M-Hyde Park and Hb M-Akita. In addition, the DNA mutation of Hb E, GAG-->AAG at codon 26, was confirmed in this case.
Hypothesis: ligand/receptor-assisted nuclear translocation of STATs.
The STAT transcription factors are mediators of signal transduction of a variety of factors, including interferons (IFNs), interleukins, growth factors, and peptide hormones. Subsequent to activation, STATs are translocated to the nucleus apparently through the well-described importin/Ran system, where they activate target genes. Molecules utilizing this nuclear import system require specific nuclear localization sequences (NLSs). Paradoxically, such NLSs are not identifiable on STATs, thus raising the question of how they are imported into the nucleus. Of considerable interest is the observation that ligands and/or receptors that signal through STATs contain putative NLSs and, where examined, either ligand or receptor undergoes nuclear translocation. We hypothesize that ligands and/or their receptors serve as vehicles for the nuclear translocation of STATs, and that they may be directly involved in signal transduction. Using IFNgamma as a model system, we provide a possible mechanism for how this direct role is fulfilled. A functional NLS has been identified in a C-terminal domain of IFNgamma. This domain and the NLS contained within are crucial for the biological properties of IFNgamma in that a peptide encompassing this domain is sufficient to induce an antiviral state. Further, this domain binds specifically to a membrane-proximal region internal cytoplasmic domain of the alpha subunit of the receptor complex in a region that is directly involved in the recruitment and activation of the JAK/STAT pathway. We suggest that this novel mode of receptor recognition and activation may be a driving force for nuclear translocation of molecules like STATs that are associated with the ligand-receptor complex.
Hb T-Cambodia, a beta chain variant with the mutations of Hb E and Hb D-Punjab, confirmed by DNA analysis.
Hemoglobin variants with two amino acid substitutions affecting one globin chain are relatively rare. Hb T-Cambodia, a doubly substituted beta-globin variant, was characterized previously by amino acid sequencing as having sequence alterations in beta 26 (beta 8)Glu-->Lys and beta 121(GH4) Glu-->Gln (1). It is a variant that migrates cathodic to Hb A2 on alkaline electrophoresis and with Hb A on acid citrate agar electrophoresis. We report here the mutations of Hb T-Cambodia at the nucleotide level using DNA sequencing, in beta-globin gene codon 121 (GAA-->CAA) and in codon 26 (GAG-->AAG). These are the mutations of Hb D-Punjab and Hb E, respectively.
Hemoglobin S/hemoglobin Osler: a case with 3 beta globin chains. DNA sequence (AAT) proves that Hb Osler is beta 145 Tyr-->Asn.
A 13-year-old African-American female with erythrocytosis and three different beta globins on electrophoresis beta A, beta S, and beta Osler, raised the possibility that one chromosome 11 might contain a duplicated beta globin gene, since there are normally only 2 beta globin genes. DNA sequence analysis showed GTG at codon 6 in exon 1, corresponding to Hb S and AAT at codon 145 in exon 3, indicating a substitution of Asn for Tyr. Thus, Hb Osler undergoes spontaneous post-translational deamidation, beta 145 Asn-->beta 145 Asp. Unmodified Hb Osler (Asn) co-migrates with Hb A on electrophoresis and co-elutes with Hb A on HPLC; therefore it has not been identified previously. All previous studies have incorrectly identified the mutation as being beta 145 (HC 2) Tyr-->Asp.
The "Genesis of the White-Eyed Mutant" in Drosophila melanogaster: A Reappraisal.
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Hb Cook [beta 132(H10)Lys-->Thr]: a new hemoglobin variant in a southeast Asian family.
Approximately 700 hemoglobin variants have been reported, causing a variety of clinical manifestations, with the majority being clinically silent. We report a new hemoglobin variant, Hb Cook, that was found in combination with Hb E in a child of Thai origin. DNA sequencing of the beta-globin gene showed that the mutation is AAA-->ACA in codon 132, corresponding to beta 132 (H10)Lys-->Thr.
Molecular characterization of spontaneous mutations at the scarlet locus of Drosophila melanogaster.
Six spontaneous mutations of the scarlet (st) locus of Drosophila melanogaster have been studied at the molecular level. Two of the mutants (st1 and stsp) arose in laboratory populations, while the other four (stcob, stct89, stdct and stdv) were isolated from natural populations. In five of these there is a DNA insertion within the st region and in four cases the insertion has been identified as being a transposable element; these include the retrotransposons 412 and B104/roo, and also jockey a member of the LINE family. In the other case (stdct), the insertion appears to consist of partially duplicated st sequences. In two of the mutants (st1 and stdv) the same transposable element (412) has inserted in the same orientation at exactly the same site within the st gene. The transposable element insertions are found in intron and exon regions of the st gene and also in the putative upstream regulatory region; insertions located in introns or exons result in the production of truncated st transcripts. The results show that the same types of transposable elements that cause spontaneous mutation in laboratory stocks of D. melanogaster also cause mutation in the wild.
The silver gene of Drosophila melanogaster encodes multiple carboxypeptidases similar to mammalian prohormone-processing enzymes.
The silver (svr) gene of Drosophila melanogaster is required for viability, and severe mutant alleles result in death prior to eclosion. Adult flies homozygous or hemizygous for weaker alleles display several visible phenotypes, including cuticular structures that are pale and silvery in color due to reduced melanization. We have identified and cloned the DNA encoding the svr gene and determined the sequence of several partially overlapping cDNAs derived from svr mRNAs. The predicted amino acid sequence of the polypeptides encoded by these cDNAs indicates that the silver proteins are members of the family of preprotein-processing carboxypeptidases that includes the human carboxypeptidases E, M, and N. One class of svr mRNAs is alternatively spliced to encode at least two polyproteins, each of which is composed of two carboxypeptidase domains.
The molecular analysis of brown eye color mutations isolated from geographically discrete populations of Drosophila melanogaster.
A large proportion of spontaneous mutations in Drosophila melanogaster strains of laboratory origin are associated with insertions of mobile DNA elements. As a first step toward determining whether spontaneous laboratory mutations are predictive for mutational events occurring in the wild, recessive brown (bw) eye color mutants were isolated. By inbreeding the progeny of wild-caught Drosophila melanogaster females, bw mutations were isolated from seven separate geographic sites distributed among Japan, California. Siberia and Hungary. Among a total of 14 mutations studied, no case of transposon mutagenesis was found. At least 4 mutations are associated with small deletions in the bw gene. The remainder are inseparable from wild-type bw by Southern analysis and are presumed to be basepair changes or very small indels. Although only two spontaneous bw mutants of laboratory origin have been analyzed molecularly, one is a mobile element insertion.
An intragenic tandem duplication of genomic DNA is responsible for the f3N mutation of Drosophila melanogaster.
Among the numerous X chromosome-linked forked bristle (f) mutations described in Drosophila melanogaster, one designated f3N exhibits the unusual property of reverting spontaneously to wild type at an inordinate frequency, a frequency that can be increased with x-ray irradiation. In contrast to the f mutants described thus far, all of which are associated with the insertion of mobile DNA elements, f3N is associated with an intragenic duplication of 2.8 kb of genomic DNA that resolves to the normal sequence when reversions occur. Consideration is given to intrachromosomal recombination as the mechanism of reversion and truncation of the forked protein as a cause for the mutant phenotype.
Good seroconversion after a fourth hepatitis B vaccination in aboriginal children.
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White as a reporter gene to detect transcriptional silencers specifying position-specific gene expression during Drosophila melanogaster eye development.
The white+ gene was used as a reporter to detect transcriptional silencer activity in the Drosophila genome. Changes in the spatial expression pattern of white were scored in the adult eye as nonuniform patterns of pigmentation. Thirty-six independent P[lacW] transposant lines were collected. These represent 12 distinct pigmentation patterns and probably 21 loci. The spatial pigmentation pattern is due to cis-acting suppression of white+ expression, and the suppression probably depends on cell position rather than cell type. The mechanism of suppression differs from inactivation by heterochromatin. In addition, activation of lacZ in P[lacW] occurs also in specific patterns in imaginal discs and embryos in many of the lines. The expression patterns of white+ and lacZ may reflect the activity of regulatory elements belonging to an endogenous gene near each P[lacW] insertion site. We speculate that these putative POSE (position-specific expression) genes may have a role in pattern formation of the eye as well as other imaginal structures. Three of the loci identified are optomotor-blind, engrailed and invected. teashirt is also implicated as a candidate gene. We propose that this "silencer trap"' may be an efficient way of identifying genes involved in imaginal pattern formation.
Undiagnosed post-traumatic stress disorder following motor vehicle accidents.
OBJECTIVES: To determine the pattern of emergence of post-traumatic stress disorder (PTSD) among motor vehicle accident victims and to examine the influence of PTSD on subsequent levels of disability. DESIGN: A longitudinal study of motor vehicle accident victims one month and 18 months after the accident. SUBJECTS: Twenty-four motor vehicle accident victims admitted by the trauma team at the Royal Adelaide Hospital. A 52% response rate was achieved. MAIN OUTCOME MEASURES: Post-traumatic stress disorder as diagnosed by the Diagnostic Interview Schedule and disability as measured with the Sickness Impact Profile. RESULTS: Eighteen months after their accidents, six of the 24 subjects had clinically significant PTSD and one was considered borderline. None had been previously diagnosed or treated. The group with PTSD had higher scores on all measures of psychological distress one month after the accident and were more likely to use immature psychological defences. There was no association between physical outcome (measured with the modified Glasgow Outcome Scale) at six months and subsequent diagnosis of PTSD. However, the group with PTSD had higher levels of disability on assessment with the Sickness Impact Profile, particularly in the domain of social functioning. The results suggest PTSD was associated with work-related dysfunction equal to that associated with severe physical handicap. CONCLUSION: The data from this pilot study suggest that PTSD after motor vehicle accidents is an important cause of disability, which may also become the focus for damages in litigation. Thus, there is a need for further investigation of the early patterns of distress and to design preventive programs for victims of road accidents.