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Biomedical subjects

D L Smith

Publications and source records attributed to D L Smith.

At least 91 records · Page 5Linked to original sources
PubMed2000

Holins: the protein clocks of bacteriophage infections.

Two proteins, an endolysin and a holin, are essential for host lysis by bacteriophage. Endolysin is the term for muralytic enzymes that degrade the cell wall; endolysins accumulate in the cytosol fully folded during the vegetative cycle. Holins are small membrane proteins that accumulate in the membrane until, at a specific time that is "programmed" into the holin gene, the membrane suddenly becomes permeabilized to the fully folded endolysin. Destruction of the murein and bursting of the cell are immediate sequelae. Holins control the length of the infective cycle for lytic phages and so are subject to intense evolutionary pressure to achieve lysis at an optimal time. Holins are regulated by protein inhibitors of several different kinds. Holins constitute one of the most diverse functional groups, with >100 known or putative holin sequences, which form >30 ortholog groups.

Amino Acid Sequence↗
PubMed2000

Characteristics of veal calves upon arrival, at 28 and 84 days, and at end of the production cycle.

Holstein bull calves arriving at five special-fed veal farms (eight production groups) were scored for various physical condition traits and blood-sampled within 2 h after arrival and at 28 d, 84 d, and 1 wk prior to slaughter at 116 to 143 d. Of 1179 total calves in the production groups, 758 calves were scored and evaluated. Blood cell analyses (red and white blood cell counts, hemoglobin, and packed cell volume) were conducted at all four sampling times; total serum protein concentration was obtained at 0 and 28 d. The study was initiated in autumn and ended the following autumn. Mean initial and final body weights were 46.3+/-0.17 and 209.7+/-0.77 kg; mean mortality was 2.5%. Average daily gain of the eight groups ranged from 1.23 to 1.70 kg/d. Subjective scores of 5 = excellent to 1 = very poor condition were used to evaluate 16 different physical condition characteristics. With the exception of leg joint, hoof, and foot scores, most of the physical condition scores exhibited improvement during the first 28 d. Foot and leg impairments did not appear to hinder the ambulatory ability of the calves during the production period. Physical condition scores at d 0 and 28 were generally not related to numbers or types of medical treatments (enteric, respiratory, other, or total) or to average daily gain during the production period. Means for most erythrocytic and leukocytic traits upon arrival (d 0) were within normal ranges, although 27.4% of the calves were clinically or marginally anemic. Final mean hemoglobin and packed cell volume were 8.53 g/dl and 26.1%. Forty-three percent of the calves at d 0 were colostral deficient, assuming that total serum protein concentrations of <5.5 g/dl indicate colostral deficiency. No blood trait was consistently correlated with body weight gain when gain during the production period was divided into quartiles and the blood traits were averaged by gain quartile. Calves in the lowest serum total protein quartile (mean 4.58 g/dl) had more respiratory and total medical treatments than quartiles with higher total protein means. Dairy bull calves arriving at veal production units after transporting from the dairy farm to the auction market (or other collection facility) have several physical impairments. However, most of these physical impairments are improved early in the veal feeding period and are not generally related to subsequent growth rate or medical treatment.

Aging↗
PubMed2000

No map, no guide. Family caregivers' perspectives on their journeys through the system.

Most program planning and research on case management and service integration has been framed within the perspectives of professionals, program providers or system managers. This qualitative study focused on the perspectives of family caregivers of survivors of traumatic brain injury, a client group that typically has complex and long-term needs, placing them "at risk" for discontinuity in care planning and integration. Four main themes were identified: the search for information, trust, and understanding; the search for support; the need to speak on behalf of the survivor; and navigating the system. Although faced with overwhelming obstacles and irreversible changes in their own lives, the caregivers in this study did not seek to relinquish their role as primary caregiver. However, it was apparent that these individuals urgently needed support to enable them to continue to bear the physical, social, and financial costs of their caregiving responsibilities. By focusing on the caregiver/client perspective, this study addresses a gap in the research literature. If replicated with other groups who are "at risk" for discontinuity of care, this research approach can be used to identify areas in which professional roles, programs and funding arrangements could be redesigned to more fully acknowledge and respond to the perspectives and needs of family caregivers.

Adaptation, Psychological↗
PubMed2000

Characterization of a phospholipase Dalpha cDNA from tomato fruit.

Phospholipase D (PLD) initiates phospholipid (PL) catabolism in plant cells and is also involved in signal transduction and retailoring of membrane PL. Total PL declines and phosphatidic acid increases in pericarp tissue during tomato fruit ripening, suggesting that increased PLD activity alters membrane structure. To assess the role of PLD in tomato ripening, we have begun a molecular genetic approach. Using a castor bean PLDalpha cDNA as a probe, a PLDalpha cDNA (LEPLD2) was isolated from our tomato fruit library. It has an open reading frame of 2421 nucleotides, encoding a polypeptide of 807 amino acids with a molecular mass of 92 kDa. The deduced LEPLD2 PLDalpha shares >75% sequence identity with PLDalphas from castor bean, tobacco and tomato. LEPLD2 transcript, detected by RNA gel-blot analysis, was very low in roots, low in stems, moderate in leaves, high in flowers, and increased in fruit during development and ripening. Expression of LEPLD2 in Escherichia coli yielded active enzyme, and a FLAG-PLDalpha fusion protein produced by transformed E. coli migrated close to the calculated 94 kDa on SDS/PAGE.

3' Untranslated Regions↗
PubMed1999

Rate and equilibrium constants for protein unfolding and refolding determined by hydrogen exchange-mass spectrometry.

Studies of protein unfolding and refolding may help us understand the more general problem of protein folding. Recent studies from this laboratory demonstrated that the unfolding and refolding of a large protein, rabbit muscle aldolase (M(r) 157 kDa), can be studied by combining amide hydrogen exchange and mass spectrometry. Results of these studies indicated that aldolase has three unfolding domains which likely unfold sequentially. Urea was used to increase the populations of partially unfolded states which were labeled with deuterium following a brief exposure to D(2)O. Electrospray ionization mass spectra of both the intact protein and its peptic fragments had multiple envelopes of isotope peaks from which the populations of unfolded forms were determined. The present study extends the previous investigations to include different urea concentrations and kinetic modeling of data taken as the system approaches equilibrium. Analysis of these results gives rate and equilibrium constants describing the unfolding and refolding processes characteristic of aldolase destabilized in urea. The change in solvent-accessible surface, which has been used as a reaction coordinate for protein folding, is estimated from the dependence of the equilibrium and rate constants on the concentration of urea.

Animals↗
PubMed1999

Identification of a mammalian angiopoietin-related protein expressed specifically in liver.

Based on searches of EST databases for signal sequences and amphipathic helices, we have identified and cloned an angiopoietin-like gene, ANGPTL3. Multiple tissue Northern blots show that ANGPTL3 is expressed principally in the liver. ANGPTL3 is expressed early during liver development, and expression is maintained in adult liver. Human ANGPTL3 is a 460-amino-acid polypeptide with the characteristic structure of angiopoietins: a signal peptide, an extended helical domain predicted to form dimeric or trimeric coiled-coils, a short linker peptide, and a globular fibrinogen homology domain (FHD). Murine ANGPTL3 is a 455-acid polypeptide encoded by seven exons on mouse chromosome 4, spanning about 11 kb of DNA. ANGPTL3 contains the four conserved cysteines implicated in the intramolecular disulfide bonds within the FHD, but it does not contain two other cysteines that are found within the FHD of angiopoietins 1, 2, and 4. ANGPTL3 also does not contain the characteristic calcium binding motif found in the other angiopoietins. By radiation hybrid mapping and the use of surrounding genes, human ANGPTL3 maps to the 1p31 region.

Amino Acid Motifs↗
PubMed1999

Hydrogen exchange demonstrates three domains in aldolase unfold sequentially.

Rabbit muscle aldolase is a homotetramer in which the subunits have a classical alpha/beta-barrel structure and Mr 39,212 Da. We have previously reported that aldolase incubated in 3 M urea has three unfolding domains distinguished by their different unfolding rates. The unfolding rates of these domains were determined from isotope patterns in the mass spectra of peptic fragments derived from aldolase incubated in 3 M urea and pulse labeled in (2)H2O. The present study extends this investigation to more thoroughly characterize the structures of these unfolding intermediates. Mass spectra of intact monomers had four envelopes of isotope peaks corresponding to four structural forms of aldolase. Analysis of the present results suggests that these structural forms consist of native aldolase and three forms that have one to three domains unfolded. The molecular masses of these four structural forms indicate that there are 107 residues in each of the three unfolding domains of aldolase. Present results also show that aldolase remains a tetramer in 4 M urea, even though hydrogen exchange and circular dichroism indicate that it has lost most of its secondary and tertiary structure. The abundances of unfolded domains, which were determined from mass spectra of either intact aldolase or its peptic fragments, were used to determine the abundances of specific, partially unfolded forms of aldolase. Kinetic modeling of the abundances of these structures suggests that these structures are formed sequentially as aldolase unfolds in urea.

Animals↗
PubMed1999

Genetic selection of peptide inhibitors of biological pathways.

Genetic selections were used to find peptides that inhibit biological pathways in budding yeast. The peptides were presented inside cells as peptamers, surface loops on a highly expressed and biologically inert carrier protein, a catalytically inactive derivative of staphylococcal nuclease. Peptamers that inhibited the pheromone signaling pathway, transcriptional silencing, and the spindle checkpoint were isolated. Putative targets for the inhibitors were identified by a combination of two-hybrid analysis and genetic dissection of the target pathways. This analysis identified Ydr517w as a component of the spindle checkpoint and reinforced earlier indications that Ste50 has both positive and negative roles in pheromone signaling. Analysis of transcript arrays showed that the peptamers were highly specific in their effects, which suggests that they may be useful reagents in organisms that lack sophisticated genetics as well as for identifying components of existing biological pathways that are potential targets for drug discovery.

Amino Acid Sequence↗
PubMed1999

Synthesis, biological activity, and absolute stereochemical assignment of NPS 1392: a potent and stereoselective NMDA receptor antagonist.

The synthesis, biological activity, and single crystal X-ray structure of NPS 1392, (R)-(-)-3,3-bis(3-fluorophenyl)-2-methylpropan-1-amine (3a), a potent, stereoselective antagonist of the NMDA receptor, are described. The NMDA receptor selectively bound the levo isomer (3a) over its enantiomer (3b), which prompted a rigorous absolute configuration assignment. NPS 1392 has the R configuration based on the single-crystal X-ray diffraction analysis of the hydroiodide salt of NPS 1392. This compound is a potential neuroprotective agent for use in the treatment of ischemic stroke.

Animals↗
PubMed1999

Hydrogen exchange shows peptide binding stabilizes motions in Hck SH2.

Src-homology-2 domains are small, 100 amino acid protein modules that are present in a number of signal transduction proteins. Previous NMR studies of SH2 domain dynamics indicate that peptide binding decreases protein motions in the pico- to nanosecond, and perhaps slower, time range. We suggest that amide hydrogen exchange and mass spectrometry may be useful for detecting changes in protein dynamics because hydrogen exchange rates are relatively insensitive to the time domains of the dynamics. In the present study, hydrogen exchange and mass spectrometry were used to probe hematopoietic cell kinase SH2 that was either free or bound to a 12-residue high-affinity peptide. Hydrogen exchange rates were determined by exposing free and bound SH2 to D(2)O, fragmenting the SH2 with pepsin, and determining the deuterium level in the peptic fragments. Binding generally decreased hydrogen exchange along much of the SH2 backbone, indicating a widespread reduction in dynamics. Alterations in the exchange of the most rapidly exchanging amide hydrogens, which was detected following acid quench and analysis by mass spectrometry, were used to locate differences in low-amplitude motion when SH2 was bound to the peptide. In addition, the results indicate that hydrogen exchange from the folded form of SH2 is an important process along the entire SH2 backbone.

Amino Acid Sequence↗
PubMed1999

Comparison of SH3 and SH2 domain dynamics when expressed alone or in an SH(3+2) construct: the role of protein dynamics in functional regulation.

Protein dynamics play an important role in protein function and regulation of enzymatic activity. To determine how additional interactions with surrounding structure affects local protein dynamics, we have used hydrogen exchange and mass spectrometry to investigate the SH2 and SH3 domains of the protein tyrosine kinase Hck. Exchange rates of isolated Hck SH3 and SH2 domains were compared with rates for the same domains when part of a larger SH(3+2) construct. Increased deuterium incorporation was observed for the SH3 domain in the joint construct, particularly near the SH2 interface and the short sequence that connects SH3 to SH2, implying greater flexibility of SH3 when it is part of SH(3+2). Slow cooperative unfolding of the SH3 domain occurred at the same rate in isolated SH3 as in the SH(3+2) construct, suggesting a functional significance for this unfolding. The SH2 domain displayed relatively smaller changes in flexibility when part of the SH(3+2) construct. These results suggest that the domains influence each other. Further, our results imply a link between functional regulation and structural dynamics of SH3 and SH2 domains.

Amino Acid Sequence↗
PubMed1999

Identification of a human 5-HT6 receptor variant produced by alternative splicing.

The complexity of the 5-hydroxytryptamine (5-HT) (serotonin) receptor family has been increased by the findings that isoforms or splice variants exist for subtypes such as the 5-HT2B, 5-HT2C, 5-HT4 and 5-HT7 subtypes. Further molecular biological studies in our laboratory have demonstrated that a splice variant of the 5-HT6 receptor exists in the human brain. Experiments performed using a degenerate PCR approach from human caudate cDNA revealed a 5-HT6 receptor clone with a 289 bp deletion of the region coding for transmembrane IV through the third intracellular loop. This deletion produces a frameshift creating a downstream stop codon which results in a truncated protein containing 10 unique amino acids at its carboxyl end. The variant transcript occurs as a result of alternative splicing using an upstream donor site and the acceptor site from the first intron in the 5-HT6 receptor gene. The splicing pattern seen for this transcript was not detected in rat or mouse whole brain cDNA by PCR due to the lack of a consensus 5' donor site. Coexpression of the variant 5-HT6 transcript and the full length 5-HT6 transcript was observed in caudate and substantia nigra but not in hippocampus, cortex, cerebellum and thalamus. Transient transfection of a 5-HT6 variant construct into Cos-7 cells demonstrated that a truncated receptor was translocated to the membrane but appeared nonfunctional.

Alternative Splicing↗
PubMed1999

Antineoplastic urinary protein inhibits Kaposi's sarcoma and angiogenesis in vitro and in vivo.

Kaposi's sarcoma (KS) is the most common tumor in human immunodeficiency virus infection and acquired immune deficiency syndrome. Recent clinical trials with human chorionic gonadotropin (hCG) prepared from early pregnancy urine have shown encouraging results in the resolution of KS lesions. A urinary protein with antitumor activity, ANUP (antineoplastic urinary protein), a dimer of 32 kD, has previously been shown to inhibit the growth of various tumor cell lines in vivo. It was thus studied for its activity in KS cell lines in vitro and in vivo to determine whether it could be a source of the anti-KS activity observed in hCG preparations. ANUP is a strong growth inhibitor for KS cell lines, but has little or no effect on fibroblast, aortic smooth muscle, T- and B-lymphocyte, and monocyte cell lines. ANUP also inhibited the proliferation of endothelial cell lines, suggesting that the in vitro effects were endothelial cell lineage-specific. However, ANUP antibodies did not block the inhibitory effect of certain commercial preparations of hCG, previously shown to be active in KS. Thus, the active protein in these commercial preparations of hCG may be distinct from ANUP. The antitumor activity of ANUP was further confirmed in a chicken allantoic membrane (CAM) assay in which vascular endothelial growth factor (VEGF) and beta fibroblast growth factor (bFGF)-induced angiogenesis was inhibited by ANUP in a dose-dependent manner. In vivo activity of ANUP was demonstrated in the murine model of KS, where ANUP inhibited tumor growth. ANUP is thus a potential candidate for development in the treatment of KS and other diseases in which angiogenesis plays an important role.

Adult↗
PubMed1999

Vascular endothelial growth factor chimeric toxin is highly active against endothelial cells.

Angiogenesis is a critical step in a benign tumor's evolution toward malignancy and metastasis. Tumor cells acquire such a phenotype by their ability to secrete angiogenic factors such as vascular endothelial growth factor (VEGF). VEGF receptors (VEGFRs) flt-1/VEGFR-1 and Flk-1/ KDR/VEGFR-2 are restricted to activated endothelial cells, with the highest expression being in the tumor vasculature. The present study was undertaken to target the VEGFRs. Targeted toxins were developed by recombinant methods by fusing VEGF165 or VEGF121 to the diphtheria toxin (DT) translocation and enzymatic domain (DT390-VEGF165 or DT390-VEGF121). Both fusion proteins were found to be highly toxic to proliferating endothelial cells but not to vascular smooth muscle cells. The fusion protein is also active in Kaposi's sarcoma, a tumor type that expresses high levels of VEGFRs. These fusion proteins completely inhibit the basic fibroblast growth factor-induced growth of new blood vessels in the chick chorioallantoic membrane assay. Furthermore, the fusion toxin substantially retards the growth of Kaposi's sarcoma tumors in mice. Because nearly all tumors induce local angiogenesis with high VEGFR expression, VEGF-derived toxins may have wide application in cancer therapy.

Animals↗
PubMed1999

Getting our act together: lessons on meaningful psychotherapy research from the philosophy of science.

Psychotherapy research should concentrate on building up a scientifically validated, theoretical knowledge base by means of disciplined empirical research. The normal nested relationship between technology, theory, and research has not been the norm in the world of psychotherapy. Psychotherapy researchers should learn from the history of science and concentrate on building basic theory. Investigations into the causal relations underpinning psychotherapy is the best way that research can help us do psychotherapy better. These investigations must conform to the canons of inductive reasoning. Conventional use of clinical data to underwrite psychotherapeutic theory is vulnerable to Grünbaum's critique. A research proposal based on Langs' communicative approach to psychotherapy is presented predicting measurable unconscious responses to brief, time-limited psychotherapy.

Humans↗