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R D Smith

Publications and source records attributed to R D Smith.

At least 271 records · Page 15Linked to original sources
PubMed1991

Tandem mass spectrometry of very large molecules: serum albumin sequence information from multiply charged ions formed by electrospray ionization.

Serum albumin proteins, Mr approximately 66 kDa, from 10 different species (bovine, human, rat, horse, sheep, goat, rabbit, dog, porcine, and guinea pig) have been studied by electrospray ionization mass spectrometry (ESI-MS) and tandem MS using a triple-quadrupole instrument. The effectiveness of collisional activation for the multiply charged albumin ions greatly exceeds that for singly charged ions, allowing an extension by a factor of at least 20 to the molecular mass range for obtaining sequence-specific product ions by tandem MS. Efficient dissociation is largely attributed to "preheating" in the interface Coulombic instability and the large number of collisions. Increasing the electric field in the intermediate pressure region, between the nozzle-skimmer elements of the atmospheric pressure/vacuum interface, allows fragmentation of the multiply protonated (to 96+) molecules produced by ESI. The most abundant dissociation product ions assigned have a low charge state (2+ to 5+) and are attributed to "bn" mode species from cleavage of the -CO-N- peptide backbone bonds. Particularly abundant dissociation products originate from regions near residues n = 20-25 from the NH2 terminus for parent ions of moderate charge (approximately 50+). Collisionally activated dissociation (CAD) mass spectra from porcine serum albumin, in contrast to the other albumins, also gave prominent singly charged "yn" fragments formed from cleavages near the COOH terminus. Tandem mass spectrometry (MS/MS) of the multiply charged molecular ions, and of fragment species produced by dissociation in the interface (i.e., effective MS/MS/MS), produced similar "bn" species and served to confirm spectral assignments. We also show that ESI mass spectra allow a qualitative assessment of protein microheterogeneity and, in some cases, resolution of major contributions. The physical and analytical implications of the results are discussed, including the identification of possible errors in previously published sequences.

Amino Acid Sequence↗
PubMed1991

Inhibition of the thrombin-platelet reactions by DuP 714.

The efficacy and specificity of a novel synthetic thrombin inhibitor, DuP 714, on thrombin-induced elevation of cytoplasmic calcium, fibrinogen binding and aggregation in human platelets were examined. Thrombin (0.5 U/ml) stimulated an increase in [125I]fibrinogen binding in gel-filtered platelets which was blocked by DuP 714 with an IC50 value of 2 nM. Thrombin (1 U/ml)-induced elevation of intracellular [Ca2+]i was also blocked by DuP 714 with an IC50 value of 67 nM. A much higher concentration of thrombin (25 U/ml) was used to stimulate aggregation with heparinized platelet-rich plasma. Under these conditions, micromolar concentrations of DuP 714 were needed to inhibit thrombin. In all of these preparations, DuP 714 at concentrations as high as 10(-5) M had no intrinsic effects and did not affect the responses induced by arachidonate, ADP, collagen, epinephrine, vasopressin and serotonin. These data indicate that DuP 714 is a potent and specific thrombin inhibitor capable of arresting the actions of thrombin on human fibrin formation and platelet aggregation and secretion. It may serve as a potential antithrombotic agent for various forms of thrombotic disorders.

Adenosine Diphosphate↗
PubMed1991

DuP 532: a second generation of nonpeptide angiotensin II receptor antagonists.

DuP 532 is a novel nonpeptide angiotensin II (AII) receptor antagonist under development for the treatment of hypertension. DuP 532 is a more potent antihypertensive agent in renal hypertensive rats (ED30 = 0.042 mg/kg, i.v.) and displays a similar or longer duration of action than the previously described AII antagonist, DuP 753. DuP 532, in contrast to DuP 753, is a noncompetitive antagonist of AII-induced contractions of rabbit aortic strips (KB = 1.1 x 10(-10) M). However, the inhibition of AII binding by DuP 532 in rat adrenal cortex does not correlate with either the aortic contractile response or with the hypotensive response. Assay conditions were evaluated and the presence or absence of BSA was shown to markedly affect the apparent binding affinity of DuP 532 and other 5-carboxylic acid derivatives. DuP 753 and other compounds were much less affected. The IC50 for DuP 532 was 4.7 x 10(-6) M with and 3 x 10(-9) M without BSA. The IC50s for DuP 753 were 1.7 x 10(-8) M with and 5 x -9 M without BSA. Both compounds with or without BSA did not completely inhibit AII binding which is characteristic of AT1 selectivity. BSA also reduced the effect of DuP 532 on the AII-induced contractions of rat main pulmonary artery preparations and the AII-induced Ca2+ mobilization in rat aortic smooth muscle cells. DuP 532 was very specific for AT1 receptors and did not interfere with receptors associated with neurotensin, prazosin, bradykinin, nitrendipine, or vasopressin. It is concluded that DuP 532 represents a new class of specific, but noncompetitive. AII receptor antagonists whose binding characteristics may provide new insight into AII receptor function.

Adrenal Glands↗
PubMed1991

Regional distribution of the two subtypes of angiotensin II receptor in rat brain using selective nonpeptide antagonists.

We have demonstrated the existence and localization of two angiotensin II (AII) receptor subtypes in different regions of the rat brain using competitive radioligand binding technique. The identification of the subtypes was made by the reciprocal selectivity of DuP 753 and PD123177 for the AII-1 and AII-2 receptors, respectively. In the pituitary gland, DuP 753 inhibited 95% of the specific AII binding with a Ki value of 1.85 x 10(-9) M while PD123177 had little effect. In the whole brain, thalamus-septum and midbrain, PD123177 inhibited 90% of the specific binding with Ki value of 7.77 x 10(-8) M, 8.21 x 10(-8) M and 4.93 x 10(-8) M, respectively while DuP 753 had little effect. In the hypothalamus, DuP 753 and PD123177 had Ki values of 5.67 x 10(-8) M and 1.60 x 10(-7) M, respectively, for their respective receptor subtypes. In the cerebellum and cerebral cortex, the AII specific binding was low. The data suggest there are at least two subtypes of AII receptor in the rat brain and that they are not uniformly distributed.

Angiotensin II↗
PubMed1991

Cooxidation of cyclophosphamide as an alternative pathway for its bioactivation and lung toxicity.

A single i.p. dose of cyclophosphamide produces lung cell injury and fibrosis in mice. Although cyclophosphamide is activated by the cytochrome P-450 mixed function oxidase (MFO) system, a role for this system in the development of lung injury has not been established. The involvement of other metabolic pathways, such as cooxidation via prostaglandin H synthase, in the toxicity of cyclophosphamide has not been studied. The objectives of the current study were to assess the effects of various inhibitors of MFO and prostaglandin H synthase activity on the development of cyclophosphamide-induced lung damage and fibrosis in mice, to determine whether arachidonic acid as well as NADPH could support the activation of cyclophosphamide to an alkylating metabolite, and to assess the capacity of cyclophosphamide to serve as a reducing cosubstrate. In addition, the ability of a low dose of cyclophosphamide to prevent the lung injury from a later higher dose was determined. Treatment with SKF 525A, piperonyl butoxide, or 1-benzylimidazole, followed by a single 200 mg/kg dose of cyclophosphamide, did not diminish pulmonary thymidine incorporation (an index of cell division after injury) or hydroxyproline content (an indicator of fibrosis), compared to mice treated with cyclophosphamide alone. Pretreatment with 1-aminobenzotriazole reduced the incorporation of thymidine into lung DNA on days 3 and 10, but not on day 7, and also reduced lung hydroxyproline accumulation. Treatment with indomethacin, nordihydroguiaretic acid, or aspirin prior to cyclophosphamide greatly reduced levels of pulmonary thymidine incorporation and/or hydroxyproline content, compared to cyclophosphamide alone. Low dose pretreatment with cyclophosphamide did not prevent the lung injury or fibrosis from a subsequent higher dose. NADPH supported greater production of alkylating metabolites in liver than in lung microsomes. In contrast, the arachidonic acid-supported production of alkylating metabolites was greater in lung microsomes. No NADPH- or arachidonate-supported alkylating activity was evident in lung or liver cytosol. SKF 525A and 1-aminobenzotriazole inhibited the NADPH-supported reaction in liver, but not lung, while indomethacin and nordihydroguiaretic acid inhibited the arachidonic acid-supported reaction in lung but not liver. Cyclophosphamide was a moderately active reducing cosubstrate for 5-phenyl-4-pentenyl hydroperoxide in both lung and liver microsomes. These results demonstrate that pathways in lung tissue unrelated to MFOs can metabolize cyclophosphamide to an alkylating compound and that MFO-mediated activation of cyclophosphamide may not be essential for the development of the pulmonary toxicity associated with this drug.

Alkylation↗
PubMed1991

Solvent-induced conformational changes of polypeptides probed by electrospray-ionization mass spectrometry.

Electrospray-ionization (ESI) mass spectrometry is used to monitor higher order structural changes of polypeptides induced by alteration of the pH or organic solvent composition in the protein solution environment. A bimodal charge-state distribution is observed in the ESI mass spectrum of ubiquitin (relative molecular mass 8565) in solutions containing small amounts (less than 20%) of organic solvents. The distribution of peaks at high m/z (low-charge state) is found to represent the protein in its native, globular state; the higher-charge-state distribution is characteristic for a more extended conformation. Addition of methanol denaturant in excess of 40% v/v is needed to eliminate the low-charge-state distribution completely. Lesser amounts of acetonitrile, acetone, or isopropanol (approximately 20%) are required to denature the ubiquitin protein. Other proteins showing conformational effects in their ESI mass spectra are also illustrated. While the ESI spectra are related to solution phase structure, ESI-tandem mass spectrometry of multiply charged molecular ions of different conformation is suggested as a probe of gas-phase protein three-dimensional structure.

Animals↗
PubMed1991

Academic manpower survey of 1990: II. Kinetics in the United States (from the Joint Task Force on Pathology Manpower ASCP/CAP/APC).

Survey data obtained from chairmen of United States departments of pathology shows a continued shortage of faculty, especially those with the MD or MD/PhD degree. Losses of such faculty by responding departments during the 12 months prior to the survey were distributed as follows: retirement, 40 (15.6% of total); disability/death, 19 (7.4%); termination, 23 (8.9%); resignation, 172 (69.3%); and other, 3 (1.2%). Resignations were due primarily to decisions to join another university (41.4%) or to enter private practice (42.5%); a small proportion were due to individuals who left academe for a pathology position in government or industry, or left both academe and pathology for another field. Comparison with a similar survey performed in 1984 suggests that the manpower situation in academic pathology is worsening, especially with respect to physician faculty. An assessment of those factors known to influence manpower kinetics suggests that it will continue to deteriorate for the foreseeable future with an adverse impact on all sectors of pathology.

Education, Medical↗
PubMed1991

Academic manpower survey of 1990: I. Descriptors of departments of pathology in the United States (from the Joint Task Force on Pathology Manpower ASCP/CAP/APC).

A survey of chairmen of United States departments of pathology (97% response rate) augmented with data from the Association of American Medical Colleges shows that roughly two thirds (65%) of departmental faculty are physicians, the great majority of whom are pathologists. Most of the remainder are persons with solely the PhD degree (26.4% of faculty). The composition of departments of pathology in terms of age, gender, ethnic origin, academic rank, and tenure status is also reported. In contrast to a comparable survey performed 5 years previously, the present report shows that in 1989, departments of pathology contained (1) 20.6% more faculty positions, (2) a greater proportion of faculty members with a PhD degree, (3) greater numbers of open positions, and (4) a probable increase in the proportion of faculty that are female and that are of an ethnic minority.

Faculty, Medical↗
PubMed1991

Pathology trainee manpower: APC program directors' questionnaire, 1989 results.

The shortage of pathologists is being created by a combination of three factors: inadequate trainee recruitment, high trainee attrition, and accelerating practitioner attrition. The current report provides comprehensive data on the first two factors. The third factor is, by every estimate, likely to become worse over the decade beginning in 1988. Demographics show that greater numbers of pathologists will be reaching retirement age every year. The trainee attrition problem appears to be very serious, and we currently do not have data on why residents leave pathology training, when they leave, or where they go (ie, to what other specialties, etc). Future APC program directors' questionnaires must address these issues. Yet, we must also solve a serious problem in recruitment. Currently, about 35% to 38% of all first-year pathology residents decide to enter pathology only after entering residency programs in other specialties with the intent to remain in that specialty. Therefore, we need to recognize both the problem of inadequate recruitment and the problem of high attrition in order to address the serious manpower shortage facing pathology. When shortages occur in specialties, practitioners tend to cease performing the most time-intensive tasks (eg, autopsy). However, shortages of the magnitude predicted for pathology suggest that many more tasks traditionally performed by pathologists may very well be in jeopardy. In such a setting, other specialties (especially subspecialties) are likely to fill the void. This is likely to be catalyzed by the emergence of the new resource-based relative-value scale. Subspecialties in medicine and surgery will be looking for additional ways to retain income when they cannot expect as much for their services as before. This scenario is especially worrisome given the necessity for the practice of pathology to expand into the domain of molecular biology. Unless problems causing the shortage in pathology manpower are addressed, not only are our traditional practices threatened, we are also likely to have inadequate manpower to take advantage of those areas crucial for the future of pathology.

Career Choice↗
PubMed1991

The Lupron screening test: tailoring the use of leuprolide acetate in ovarian stimulation for in vitro fertilization.

STUDY OBJECTIVE: To evaluate the prognostic and therapeutic value of a Lupron (leuprolide acetate; Tap Pharmaceuticals, North Chicago, IL) screening test before ovarian stimulation for in vitro fertilization (IVF). DESIGN: Prospective. SETTING: Outpatient IVF program. PATIENTS: Eighty patients exhibited four early estradiol (E2) patterns. Patients with pattern A and B remained on a flare-up ovarian stimulation protocol. Patients with pattern C were randomized to three ovarian stimulation protocols. Patients with pattern D were treated with the flare-up protocol using a high pure follicle-stimulating hormone (FSH) dose. RESULTS: Patterns, A, B, C, and D occurred in 44%, 16%, 25%, and 15% of the patients, respectively. The E2 pattern recurred in 77% of subsequent IVF cycles. Pattern A and B patients achieved a 41% (23/56) and 22% (5/23) ongoing pregnancy rate (PR) per stimulated cycle. An early luteal phase Lupron protocol had the best ongoing PR per stimulated cycle (10/27, 37%) in patients with a pattern C response. Pattern D patients had a 20% (5/25) ongoing PR per stimulated cycle. CONCLUSION: The Lupron screening test allows prospective selection of stimulation protocols in ovulatory patients undergoing IVF. Early E2 patterns A and B should be treated with the flare-up protocol. Pattern C patients benefit from the luteal phase Lupron protocol and pattern D patients benefit from a high pure FSH flare-up protocol.

Adult↗
PubMed1991

The Brief Social Phobia Scale.

An observer measure of social phobic symptoms, referred to as the Brief Social Phobia Scale, consists of 11 items, 7 evaluating commonly feared or avoided situations and 4 additional items measuring autonomic distress. Symptoms represented by the scale items are found frequently in social phobia, and the instrument demonstrates acceptable interrater and test-retest reliability, internal consistency, concurrent validity against other measures of social phobia, and the ability of patients to change as a result of treatment. It can also detect differences between active treatment and placebo treatment. A comparison of the sensitivity of this scale with other scales in detection of size of effect also is presented.

Humans↗
PubMed1991

Effects of DuP 753 on renal function of normotensive and spontaneously hypertensive rats.

This study examined the effects of a new, orally-active, nonpeptide angiotensin II (AII) receptor antagonist, 2-n-butyl-4-chloro-5-hydroxymethyl-1-[2'-(1H-tetrazol-5-yl)biph eny l-4- yl)methyl] imidazole, DuP 753, on renal function of anesthetized, volume-expanded Wistar Kyoto (WKY) and spontaneously hypertensive rats (SHR), and in a group of euvolemic Munich-Wistar (MW) rats. Plasma renin activities were similar and averaged 4.4 +/- 0.7 and 4.3 +/- 1.4 ng AI/mL.h, respectively, in the SHR and WKY rats. In WKY rats (n = 15), DuP 753 (2 or 10 mg/kg, intravenously) had no effect on urine flow, sodium excretion, renal blood flow (RBF), or glomerular filtration rate (GFR). Fractional excretion of lithium (FELi) rose from 32 +/- 5 to 40 +/- 4% of the filtered load and arterial pressure decreased slightly from 129 +/- 2 to 122 +/- 2 mm Hg. In SHR (n = 9), urine flow fell 24%, and FELi and RBF increased by 27% and 30%, respectively, after 2 mg/kg DuP 753, but sodium excretion, GFR, and arterial pressure were not significantly altered. A higher dose of DuP 753 (10 mg/kg; n = 8) reduced arterial pressure, urine flow, and sodium excretion in the SHR. RBF increased 34%, while GFR and FELi were not significantly altered. Similar effects were seen in SHR (n = 11), given an equivalent antihypertensive dose of captopril (20 mg/kg). In euvolemic MW rats in which plasma renin activity was elevated to 18.8 +/- 3.3 ng AI/mL.h, DuP 753 (2 mg/kg, n = 7) increased RBF, urine flow, and sodium excretion, while mean arterial pressure and GFR were unaltered.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1991

The efficacy of OKT3 in vascular rejection.

Sixty-six consecutive biopsies of renal allograft recipients treated with OKT3 monoclonal antibody were reviewed and placed into one of two groups. Group I (29 patients) had evidence of acute vascular and cellular rejection, while group II (32 patients) had cellular rejection but no vascular rejection. In 5 cases, the sample was inadequate to determine if vascular rejection was present or not. The severity of the cellular rejection was graded histologically as mild, moderate, or severe. The severity was equivalent when comparing group I with group II (mild, 17% vs. 10%; moderate, 52% vs. 59%; and severe, 31% vs. 31%). There was no difference in the rejection reversal rate between the two groups (86% vs. 91%). However, at 6 and 12 months there was a higher graft loss in the group with vascular rejection (graft survival 64% vs. 81%, P = 0.13, and 58% vs. 75%, P = 0.08, respectively). The poorest outcome was in those patients with both severe acute cellular rejection and acute vascular rejection (4/9, or 44%). The serum creatinine level was higher both pre- and post-OKT3 therapy and at 1, 6, and 12 months in the group with vascular rejection. In conclusion, OKT3 was equally successful in reversing acute cellular rejection and acute vascular rejection. However, increased graft loss occurred at 6 and 12 months in the group with vascular rejection.

Adult↗
PubMed1991

Isolation and expression of a maize type 1 protein phosphatase.

The dephosphorylation of phosphoproteins by protein phosphatases represents an important mechanism for regulating specific cellular processes in eukaryotic cells. The aim of the present study was to examine the structural and biochemical characteristics of a specific class of protein Ser/Thr phosphatases (type 1 protein phosphatases) which have received very little attention in higher plants. A cDNA clone (ZmPP1) was isolated from a maize (Zea mays L.) cDNA library. The deduced amino acid sequence is 80% identical with a 292-amino acid core region of rabbit and yeast type 1 protein phosphatase catalytic subunit. Southern blot analysis indicates that ZmPP1 may belong to a family of related genes in maize. ZmPP1 RNA was present in all maize tissues examined, indicating that it may play a fundamental role in cellular homeostasis. To demonstrate that ZmPP1 encodes an active protein phosphatase and, in an effort to characterize this gene product biochemically, high levels of ZmPP1 were expressed in Escherichia coli. Active ZmPP1 enzyme dephosphorylates rabbit phosphorylase a and is strongly inhibited by okadaic acid and by the mammalian inhibitor-2. These data show that ZmPP1 is structurally and biochemically very similar to the corresponding enzyme in animal cells. These results also suggest that the function and regulation of the higher plant type 1 protein phosphatases may be similar to the mammalian protein phosphatases.

Journal Article↗