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Biomedical subjects

S D Hanes

Publications and source records attributed to S D Hanes.

At least 19 recordsLinked to original sources
PubMed2000

Identification of drosophila bicoid-interacting proteins using a custom two-hybrid selection.

Bicoid directs pattern formation in the developing Drosophila embryo, and does so by performing two seemingly unrelated tasks; it activates transcription and represses translation. To understand how Bicoid carries out this dual role, we sought to identify Bicoid-ancillary proteins that might mediate Bicoid's function in transcription or translation. We used a customized version of the two-hybrid method and found two Bicoid-interacting proteins, Bin1 and Bin3, both of which interact with Bicoid in vitro. Bin1 is similar to a human protein (SAP18) involved in transcription regulation, and Bin3, described in this paper, is similar to a family of protein methyltransferases that modify RNA-binding proteins. Given that Bicoid's role as a translation regulator requires RNA binding, we suggest that the Bicoid-interacting methyltransferase might be important for that role. The custom two-hybrid method we used, in which Bicoid is bound to DNA via its own DNA binding domain, rather than via a fusion-protein tether, should be generally applicable to other DNA binding proteins.

Amino Acid Sequence↗
PubMed1999

Prolonged opioid antagonism with naloxone in chronic renal failure.

Respiratory depression secondary to morphine intoxication occurred in an elderly patient with chronic renal failure (CRF). It was reversed with a continuous infusion of naloxone. Approximately 11 hours after the infusion was discontinued, the patient relapsed into respiratory depression consistent with opioid intoxication. He was rechallenged with a naloxone infusion with resolution of the opioid effects. This case suggests prolonged antagonism of opioid effects inconsistent with naloxone's reported pharmacologic effects. Serum naloxone concentrations measured after the end of the infusion suggest that the drug's pharmacokinetics were significantly altered. Further research is necessary to characterize pharmacokinetic changes that occur in CRF. In the absence of this information, similar patients should be closely monitored for relapse of respiratory depression after naloxone is discontinued.

Aged↗
PubMed1998

Alternative method for determination of ceftazidime in plasma by high-performance liquid chromatography.

A high-performance liquid chromatography procedure was developed to analyze ceftazidime concentrations in plasma. The procedure consisted of solid phase extraction followed by ion-pairing reverse-phase chromatography. An excellent linear relationship between ceftazidime peak height measurements and concentrations was demonstrated over the concentration range of 1-200 microg ml(-1). The advantage of this assay is the elimination of interference at the ceftazidime elution time that has been noted in previous studies and in our experience. Thus, this study describes an alternative, simple methodology that is clinically useful for analyzing ceftazidime in the research setting.

Ceftazidime↗
PubMed1998

Cooperative DNA-binding by Bicoid provides a mechanism for threshold-dependent gene activation in the Drosophila embryo.

The Bicoid morphogen directs pattern formation along the anterior-posterior (A-P) axis of the Drosophila embryo. Bicoid is distributed in a concentration gradient that decreases exponentially from the anterior pole, however, it transcribes target genes such as hunchback in a step-function-like pattern; the expression domain is uniform and has a sharply defined posterior boundary. A 'gradient-affinity' model proposed to explain Bicoid action states that (i) cooperative gene activation by Bicoid generates the sharp on/off switch for target gene transcription and (ii) target genes with different affinities for Bicoid are expressed at different positions along the A-P axis. Using an in vivo yeast assay and in vitro methods, we show that Bicoid binds DNA with pairwise cooperativity; Bicoid bound to a strong site helps Bicoid bind to a weak site. These results support the first aspect of the model, providing a mechanism by which Bicoid generates sharp boundaries of gene expression. However, contrary to the second aspect of the model, we find no significant difference between the affinity of Bicoid for the anterior gene hunchback and the posterior gene knirps. We propose, instead, that the arrangement of Bicoids bound to the target gene presents a unique signature to the transcription machinery that, in combination with overall affinity, regulates the extent of gene transcription along the A-P axis.

Animals↗
PubMed1998

Goosecoid-like, a gene deleted in DiGeorge and velocardiofacial syndromes, recognizes DNA with a bicoid-like specificity and is expressed in the developing mouse brain.

The vast majority of patients with DiGeorge syndrome (DGS) and velocardiofacial syndrome (VCFS) have deletions of chromosomal region 22q11.2. These patients exhibit broad and variable phenotypes that include conotruncal cardiac defects, hypocalcemia, palatal and facial anomalies and developmental delay. Most of these abnormalities are thought to be due to defects in neural crest cell migration or differentiation. We have identified a homeobox-containing gene, Goosecoid-like (GSCL), that is in the region within 22q11 that is deleted most consistently in patients with DGS/VCFS. The GSCL gene is expressed in a limited number of adult tissues as well as in early human development, and is a member of a family of homeobox genes in vertebrates that includes Goosecoid and GSX. In this report, we present functional studies of the GSCL protein and determine the expression pattern of the GSCL gene in mouse embryos. We demonstrate that GSCL exhibits DNA sequence-specific recognition of sites bound by the Drosophila anterior morphogen, Bicoid. Several of these sites (TAATCCC) were found in the 5' upstream region of the GSCL gene itself, and we present evidence suggesting that GSCL might regulate its own transcription. In situ hybridization revealed that the mouse ortholog of GSCL, Gscl, is expressed in the brain starting as early as embryonic day 9.5, and expression continues in adults. This expression pattern is consistent with GSCL having either an indirect role in the development of neural crest-derived structures or a direct role in a subset of the phenotype observed in DGS/VCFS, such as learning disorders or psychiatric disease.

Abnormalities, Multiple↗
PubMed1998

Fluconazole pharmacokinetics in burn patients.

The pharmacokinetics of fluconazole in nine adult patients with severe (30 to 95% total body surface area) burns were studied. There was no significant difference in half-life (t1/2), clearance (CL), or volume of distribution (V) over time in five patients on days 3 and 8 of the study (P > 0.05). Combined parameter estimates (means +/- standard deviations) for all nine patients for the two study periods were as follows: t1/2, 24.4 +/- 5.8 h; CL, 0.36 +/- 0.09 ml/min/kg; and V, 0.72 +/- 0.12 liters/kg. These estimates of t1/2 and CL in burn patients were approximately 13% shorter and 30% more rapid, respectively, than the most extreme estimates reported for other populations.

Adult↗
PubMed1998

Pharmacokinetic alterations after severe head injury. Clinical relevance.

Pharmacological therapy, present and future, will undoubtedly continue to play a large role within the overall management of patients with severe head injury. Nevertheless, limited clinical data are available to evaluate the effect of severe head injury on pharmacokinetics. The disruption of the blood-brain barrier secondary to trauma and/or subsequent hyperosmolar therapy can be expected to result in higher than expected brain drug concentrations. Aggressive dietary protein supplementation may result in increased oxidative drug metabolism. These effects may counterbalance inhibitory influences on drug metabolism secondary to cytokine release during the acute phase response. Alterations in protein binding can also be anticipated with the hypoalbuminaemia and increases in alpha 1-acid glycoprotein typically observed in these patients. Based on studies in other patient populations, moderate hypothermia, a treatment strategy in patients with head injury, can decrease drug metabolism. The pharmacokinetics of the following drugs in patients with severe head injury have been studied: phenytoin, pentobarbital (pentobarbitone), thiopental (thiopentone), tirilazad, and the agents used as marker substrates, antipyrine, lorazepam and indocynanine green (ICG). Several studies have documented increase in metabolism over time with phenytoin, pentobarbital, thiopental, antipyrine and lorazepam. Increases in tirilazad clearance were also observed but attributed to concurrent phenytoin therapy. No changes in the pharmacokinetics of ICG were apparent following head injury. With the frequent use of potent inhibitors of drug metabolism (e.g., cimetidine, ciprofloxacin) the potential for drug interaction is high in patients with severe head injury. Additional pharmacokinetic investigations are recommended to optimise pharmacological outcomes in patients with severe head injury.

Aminopyrine↗
PubMed1997

The dodo gene family encodes a novel protein involved in signal transduction and protein folding.

Recent studies in yeast, Drosophila and humans have revealed the existence of a highly conserved gene encoding a novel protein, Dodo, comprised of four modules: a WW domain, involved in protein-protein interactions, a peptidyl-prolyl cis-trans isomerase (PPIase) domain belonging to a recently described third family of PPIases involved in protein folding and unfolding, a nuclear localization motif and finally, a long, surface-exposed alpha-helix that is likely to be involved in binding to a cell cycle serine/threonine kinase. The genetic, molecular, biochemical and structural data are reviewed in the context of the potential biological properties of this new protein family.

Amino Acid Sequence↗
PubMed1997

Incidence and risk factors of thrombocytopenia in critically ill trauma patients.

OBJECTIVE: To determine the incidence of thrombocytopenia (< 100 platelets x 10(3)/mm3) and potential risk factors, including medications, associated with the development of thrombocytopenia in critically ill trauma patients. DESIGN: Prospective, observational study. SETTING: A 20-bed trauma intensive care unit (ICU) at a university hospital. PATIENTS: Sixty-three critically ill trauma patients without baseline thrombocytopenia admitted to the trauma ICU for at least 48 hours. INTERVENTIONS: Patients were followed for up to 14 days. Platelet counts were determined daily. The following data were collected and analyzed as potential risk factors for the development of thrombocytopenia: medications, age, sex, race, trauma score, mode and type of injury, alcohol history, units of packed red blood cells (PRBC) and platelets transfused, surgical procedures, duration of ICU stay, and the development of systemic inflammatory response syndrome or disseminated intravascular coagulation. RESULTS: Thrombocytopenia occurred in 26 (41%) of the patients. Among risk factors studied, nonhead injury, age, trauma score, duration of ICU stay, and the number of PRBC transfusions were significantly associated with the development of thrombocytopenia (p < 0.05). However, nonhead injury, age, and trauma score were useful variables in predicting the development of thrombocytopenia by using multivariate analysis. Medications were not associated with the development of thrombocytopenia. CONCLUSIONS: The type of injury sustained, the quantity of platelet-deficient, transfusions, and age are the greatest risk factors associated with the development of thrombocytopenia in critically ill trauma patients. Drug-induced thrombocytopenia appears to play a minor role in the development of thrombocytopenia; therefore, medications should not be automatically discontinued or substituted when thrombocytopenia occurs.

Adolescent↗
PubMed1996

A human peptidyl-prolyl isomerase essential for regulation of mitosis.

The NIMA kinase is essential for progression through mitosis in Aspergillus nidulans, and there is evidence for a similar pathway in other eukaryotic cells. Here we describe the human protein Pin1, a peptidyl-prolyl cis/trans isomerase (PPIase) that interacts with NIMA. PPIases are important in protein folding, assembly and/or transport, but none has so far been shown to be required for cell viability. Pin1 is nuclear PPIase containing a WW protein interaction domain, and is structurally and functionally related to Ess1/Ptf1, an essential protein in budding yeast. PPIase activity is necessary for Ess1/Pin1 function in yeast. Depletion of Pin1/Ess1 from yeast or HeLa cells induces mitotic arrest, whereas HeLa cells overexpressing Pin1 arrest in the G2 phase of the cell cycle. Pin1 is thus an essential PPIase that regulates mitosis presumably by interacting with NIMA and attenuating its mitosis-promoting activity.

Amino Acid Isomerases↗
PubMed1996

The Drosophila melanogaster dodo (dod) gene, conserved in humans, is functionally interchangeable with the ESS1 cell division gene of Saccharomyces cerevisiae.

We have sequenced the region of DNA adjacent to and including the flightless (fli) gene of Drosophila melanogaster and molecularly characterized four transcription units within it, which we have named tweety (twe), flightless (fli), dodo (dod), and penguin (pen). We have performed deletion and transgenic analysis to determine the consequences of the quadruple gene removal. Only the flightless gene is vital to the organism; the simultaneous absence of the other three allows the overriding majority of individuals to develop to adulthood and to fly normally. These gene deletion results are evaluated in the context of the redundancy and degeneracy inherent in many genetic networks. Our cDNA analyses and data-base searches reveal that the predicted dodo protein has homologs in other eukaryotes and that it is made up of two different domains. The first, designated WW, is involved in protein-protein interactions and is found in functionally diverse proteins including human dystrophin. The second is involved in accelerating protein folding and unfolding and is found in Escherichia coli in a new family of peptidylprolyl cis-trans isomerases (PPIases; EC 5.2.1.8). In eukaryotes, PPIases occur in the nucleus and the cytoplasm and can form stable associations with transcription factors, receptors, and kinases. Given this particular combination of domains, the dodo protein may well participate in a multisubunit complex involved in the folding and activation of signaling molecules. When we expressed the dodo gene product in Saccharomyces cerevisiae, it rescued the lethal phenotype of the ESS1 cell division gene.

Amino Acid Sequence↗
PubMed1994

Backward bicoid?

Explore the source record for details and available documents.

Base Sequence↗
PubMed1994

Specific DNA recognition and intersite spacing are critical for action of the bicoid morphogen.

We examined DNA site recognition by Bicoid and its importance for pattern formation in developing Drosophila embryos. Using altered DNA specificity Bicoid mutants and appropriate reporter genes, we show that Bicoid distinguishes among related DNA-binding sites in vivo by a specific contact between amino acid 9 of its recognition alpha-helix (lysine 50 of the homeodomain) and bp 7 of the site. This result is consistent with our earlier results using Saccharomyces cerevisiae but differs from that predicted by crystallographic analysis of another homeodomain-DNA interaction. Our results also demonstrate that Bicoid binds directly to those genes whose transcription it regulates and that the amino acid 9 contact is necessary for Bicoid to direct anterior pattern formation. In both Drosophila embryos and yeast cells, Bicoid requires multiple binding sites to activate transcription of target genes. We find that the distance between binding sites is critical for Bicoid activation but that, unexpectedly, this critical distance differs between Drosophila and S. cerevisiae. This result suggests that Bicoid activation in Drosophila might require an ancillary protein(s) not present in S. cerevisiae.

Animals↗
PubMed1991

A genetic model for interaction of the homeodomain recognition helix with DNA.

The Bicoid homeodomain protein controls anterior development in the Drosophila embryo by binding to DNA and regulating gene expression. With the use of genetic assays in yeast, the interaction between the Bicoid homeodomain and a series of mutated DNA sites was studied. These experiments defined important features of homeodomain binding sites, identified specific amino acid-base pair contacts, and suggested a model for interaction of the recognition alpha-helices of Bicoid and Antennapedia-class homeodomain proteins with DNA. The model is in general agreement with results of crystallographic and magnetic resonance studies, but differs in important details. It is likely that genetic studies of protein-DNA interaction will continue to complement conventional structural approaches.

Amino Acid Sequence↗
PubMed1989

DNA specificity of the bicoid activator protein is determined by homeodomain recognition helix residue 9.

Formation of anterior structures in the Drosophila embryo requires the product of the gene bicoid. The bicoid protein contains a homeodomain and may exert its effects in early development by regulating transcription of the gap gene, hunchback (hb). Consistent with this view, we have demonstrated that DNA-bound Bicoid fusion proteins stimulate gene expression. We used the gene activation phenotype in yeast to study DNA recognition by the Bicoid homeodomain. We found that a single amino acid replacement at position 9 of the recognition helix was sufficient to switch the DNA specificity of the Bicoid protein. The altered specificity Bicoid mutants recognized DNA sites bound by Ultrabithorax, fushi tarazu, and other related homeo-domain proteins. Our results suggest that DNA specificity in Bicoid and Antennapedia class proteins is determined by recognition helix residue 9.

Animals↗
PubMed1986

Expression of a cDNA derived from the yeast killer preprotoxin gene: implications for processing and immunity.

The type I killer strains of Saccharomyces cerevisiae secrete a dimeric 19-kDa protein that kills sensitive cells by disrupting cytoplasmic membrane function. This toxin is encoded by the double-stranded RNA plasmid M1-dsRNA, which also determines specific immunity to toxin. A preprotoxin, the 35-kDA in vitro translation product of denatured M1-dsRNA, is presumed to be the primary in vivo gene product. To facilitate studies on preprotoxin structure and maturation, we have inserted a partial cDNA copy of M1-dsRNA into the yeast vector p1A1, bringing it under control of the phosphate-repressible PHO5 promoter. This in-frame gene fusion encodes all of the preprotoxin sequence except for its N-terminal secretion leader, which is replaced by the leader sequence of PHO5. Transformation of sensitive yeast strains lacking M1-dsRNA with such fusion plasmids converts them to phosphate-repressible, immune killers, demonstrating that both toxin and immunity determinants are contained within the preprotoxin molecule. L-1-Tosylamido-2-phenylethyl chloromethyl ketone retards glycosylation of preprotoxin to toxin, facilitating size comparisons and indicating that processing of the normal precursor involves three glycosylation events but does not involve cotranslational leader peptidase action. In contrast, the PHO5 leader is apparently removed from the fusion preprotoxin.

DNA↗