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Biomedical subjects

D H Robinson

Publications and source records attributed to D H Robinson.

At least 19 recordsLinked to original sources
PubMed1995

Porcine brain microvascular endothelial cells support the in vitro expansion of human primitive hematopoietic bone marrow progenitor cells with a high replating potential: requirement for cell-to-cell interactions and colony-stimulating factors.

Primary autologous as well as allogeneic and xenogeneic stroma will support human stem cell proliferation and differentiation for several months. In the present study, we investigated the capacity of porcine microvascular endothelial cells (PMVECs) together with combinations of cytokines (granulocyte-macrophage colony-stimulating factor [GM-CSF] + stem factor [SCF], interleukin-3 [IL-3] + SCF + IL-6, and GM-CSF + IL-3 + SCF + IL-6) to support the expansion and development of purified human CD34+ bone marrow cells. In short-term cultures (7 days), the greatest expansion of nonadherent hematopoietic cells and clonogenic progenitors was seen with CD34+ cells in direct contact with PMVEC monolayers (PMVEC contact), followed by PMVEC noncontact and liquid suspension cultures, respectively. Maximal expansion of nonadherent cells (42-fold) and total CD34+ cells (12.6-fold) occurred in PMVEC contact cultures treated with GM-CSF + IL-3 + SCF + IL-6, with similar increases in the number of granulocyte-macrophage colony-forming units (CFU-GM), CFU-mix, erythroid burst-forming units (BFU-E), CFU-blast and CFU-megakaryocyte (CFU-Mk) progenitor cells. Moreover, the number of CD34+ CD38- and CD34+ CD38+ cells increased 148.1-fold and 8.0-fold, respectively. Replating studies show that cells from day 7 dispersed blast cell colonies generated on cytokine-treated PMVEC monolayers have a high replating potential for multilineage progenitor cells. In long-term PMVEC contact cultures, CD34+ cells seeded onto PMVEC monolayers with GM-CSF + IL-3 + SCF + IL-6 showed a total calculated expansion of over 5,000,000-fold of nonadherent cells over 35 days in culture. Maximal clonogenic cell production was observed at day 28, with 6,353-fold for total CFC and comparable increases for CFU-GM, CFU-mix, CFU-blast, BFU-E, and CFU-Mk. The total number of CD34+ cells increased 2,584-fold at day 28. Furthermore, the extended growth kinetics of these cultures indicates that these phenotypically primitive progenitor cells are also functionally expanded on PMVEC monolayers. These results support the hypothesis that direct contact with a PMVEC monolayer supports the initial expansion of hematopoietic progenitor cells with a high replating potential and, possibly, a more primitive phenotype (CD34+, CD34+/CD38-).

Animals↗
PubMed1995

Expression of the mouse testis-determining gene Sry in male preimplantation embryos.

The testis-determining factor in the mouse is encoded by the Sry gene on the Y chromosome. Transcripts of this gene have been shown previously to be present in the genital ridge at the beginning of gonadal differentiation (11.5 days post coitum) and in adult testis. In this study, RNA transcripts of the Sry gene are also detected in male blastocyst-stage embryos (3.5 days post coitum) at approximately 40-100 copies per cell, long before overt sex differentiation. These results indicate that preimplantation mouse embryos have sexually dimorphic gene expression at least with respect to Sry transcripts. In addition, at least some of the Sry RNA transcripts in blastocysts are circular, as has been reported for Sry transcripts from adult testis. The appearance of Sry transcripts in blastocysts at this level raises the possibility that sex determination begins earlier during embryonic development than previously thought.

Animals↗
PubMed1995

Response to intracrevicular controlled delivery of 25% tetracycline from poly(lactide/glycolide) film strips in SPT patients.

Controlled local delivery of antibiotics has been shown to reduce periodontopathic micro-organisms with minimal side-effects. Clinical studies in our laboratory have shown that 25% tetracycline HCl delivered from poly(D,L-lactide/glycolide) film strips (25 TTC-PLGA) released therapeutic concentrations of tetracycline for 10 days. The present pilot study compared the intracrevicular delivery of 25% tetracycline HCl incorporated in these biodegradable film strips to scaling and root planing (SRP) in 10 adult periodontitis patients, who in spite of therapy and regular supportive periodontal treatment (SPT), continued to possess 5 bleeding periodontal pockets at least 5 mm deep. Sites were randomly selected to receive the following treatments: (1) 25 TTC-PLGA, (2) control strips without TTC (PLGA), (3) SRP, and (4) untreated control. Film-strip retention was augmented with a suture/cement technique, followed by strip removal after 2 weeks. Clinical parameters and subgingival bacterial morphotypes (darkfield analysis) were evaluated over time (0, 2.4, 8, 12, 26 weeks). Results indicated that, compared to baseline, 25 TTC-PLGA film strips caused significant (p < or = 0.01): (1) probing depth reduction for 26 weeks, (2) a clinical attachment level gain for 12 weeks, (3) lower %s of spirochetes for 4 weeks and motile rods for 8 weeks (p < or = 0.05), and (4) an accompanying increase in cocci for 4 weeks. In the scaled and root planed sites, probing depth was the only finding that demonstrated a significant change from baseline (p < or = 0.01). Controls and PLGA showed isolated reductions in probing depth and % of motile organisms. From these findings, applications of intracrevicular 25 TTC-PLGA, when compared to scaling and root planing, appears to have an enhanced antibacterial effect and a similar clinical effect in SPT patients. The results of this study indicate further investigation of 25 TTC-PLGA film strips should be undertaken using more subjects and sophisticated microbiological and clinical measurements.

Adult↗
PubMed1994

Oxidative stress-induced apoptosis prevented by Trolox.

The ability of oxidative stress to induce apoptosis (programmed cell death), and the effect of Trolox, a water soluble vitamin E analog, on this induction were studied in vitro in mouse thymocytes. Cells were exposed to oxidative stress by treating them with 0.5-10 microM hydrogen peroxide (H2O2) for 10 min, in phosphate-buffered saline supplemented with 0.1 mM ferrous sulfate. Cells were resuspended in RPMI 1640 medium with 10% serum and incubated at 37 degrees C under 5% CO2 in air. Electron microscopic studies revealed morphological changes characteristic of apoptosis in H2O2-treated cells. H2O2 treatment fragmented the DNA in a manner typical of apoptotic cells, producing a ladder pattern of 200 base pair increments upon agarose gel electrophoresis. The percentage of DNA fragmentation (determined fluorometrically) increased with increasing doses of H2O2 and postexposure incubation times. Pre- or posttreatment of cells with Trolox reduced H2O2-induced DNA fragmentation to control levels and below. The results indicate that oxidative stress induces apoptosis in thymocytes, and this induction can be prevented by Trolox, a powerful inhibitor of membrane damage.

Animals↗
PubMed1994

Shelf-lives and factors affecting the stability of morphine sulphate and meperidine (pethidine) hydrochloride in plastic syringes for use in patient-controlled analgesic devices.

Published reports regarding the stability of morphine are at variance, especially in syringes used in patient-controlled analgesia (PCA) devices. In addition to the effects of container type and vehicle, reasons for this variation include the effect of excipients temperature and light during storage. Furthermore, the literature varies regarding the mechanisms of decomposition for morphine. To our knowledge, the stability of meperidine (pethidine) stored in plastic syringes has not been reported. The purposes of this study were to investigate the stability of morphine sulphate (1 and 5 mg/ml) and meperidine hydrochloride (5 and 10 mg/ml) in plastic syringes for use in PCA devices for a duration of 12 weeks, and evaluate the influence of light (240 foot-candles), temperature (-20, 4 and 23 degrees C), diluent (5% dextrose or normal saline), and drug concentration on the stability of these narcotic analgesics. Samples were taken bi-weekly for solutions protected from light and weekly for solutions exposed to light. Morphine sulphate and meperidine hydrochloride concentrations were quantified using independent, stability-indicating, high performance liquid chromatographic assays. The within-day and between-day coefficients of variation for these assays were < or = 4% over each of the concentration ranges studied. Under the conditions of this study, it is proposed that although decomposition of morphine to its main product, pseudomorphine, can be interpreted using first-order kinetics, consecutive (to form the N-oxide) and parallel mechanisms (to form apomorphine) exist. Morphine solutions were more stable in normal saline than in 5% dextrose. Shelf-life data indicate that morphine is stable for at least 6 weeks when protected from light.(ABSTRACT TRUNCATED AT 250 WORDS)

Analgesics, Opioid↗
PubMed1993

Pharmaceutical properties, biodistribution, and imaging characteristics of manganese-mesoporphyrin. A potential hepatobiliary contrast agent for magnetic resonance imaging.

OBJECTIVES: Manganese (III) mesoporphyrin (Mn-mesoporphyrin) was investigated for its pharmaceutical properties and magnetic resonance imaging characteristics as a potential hepatobiliary contrast agent. METHODS: Solubility, partition coefficient, plasma binding, proton relaxation enhancement, biodistribution, biliary excretion, liver extraction ratio, and liver enhancement were measured in various in-vitro and in-vivo systems. RESULTS: Mn-mesoporphyrin was soluble and stable at moderate alkaline pH in phosphate buffer. The octanol/water coefficient was 25.98, and the compound was highly protein bound. R1 for water and plasma were 1.94 and 2.35 L/mmol sec, respectively. R1 in liver was calculated to be 15.72 L/mmol sec. Biodistribution studies in rats and mice confirmed hepatotrophic properties and biliary excretion was 65% over 24 hours. First pass liver uptake was 15%. Magnetic resonance imaging studies showed persistent liver enhancement at 0.05 mmol/kg. CONCLUSION: Mn-mesoporphyrin is a lipophilic compound that shows potential as a hepatobiliary magnetic resonance contrast agent.

Animals↗
PubMed1993

Complement activation during saturation diving.

In this study, the levels of activated complement fragments C3a and C5a were measured on 11 U.S. Navy divers as they performed a 28-day saturation dive to a pressure equivalent of 1,000 feet of seawater (fsw, 31.3 atm abs). Two subjects developed symptoms consistent with the high pressure nervous syndrome (HPNS) and three were treated for type I DCS (joint pain only). These events allowed us to test two hypotheses: a) alterations in C3a or C5a levels during compression are related to the occurrence of HPNS and b) increases in complement fragments are an indicator of decompression stress associated with type I DCS. There was no correlation between changes in C3a and C5a levels during compression and the diagnosis of HPNS. Our results suggest that an increase in C3a and C5a levels during saturation diving correlates with decompression stress and the clinical diagnosis of type I DCS.

Analysis of Variance↗
PubMed1992

The binding of amide substrate analogues to phospholipase A2. Studies by 13C-nuclear-magnetic-resonance and infrared spectroscopy.

(R)-(2-dodecanamidoisohexyl)phosphocholine (DAHPC), labelled with 13C at the amide carbonyl group, has been synthesized and its binding to bovine pancreatic phospholipase A2 (PLA2) studied by n.m.r. and i.r. spectroscopy. Two-dimensional 1H-n.m.r. spectra show that, in the presence of Ca2+, DAHPC binds to the active site of the enzyme in a similar manner to other phospholipid amide substrate analogues. The environment of the labelled carbonyl group has been investigated by a combination of 13C n.m.r. and difference-Fourier-transform i.r. spectroscopy. The carbonyl resonance shifts 3 p.p.m. downfield on the binding of DAHPC to PLA2. The carbonyl absorption frequency decreases by 14-18 cm-1, accompanied by a marked sharpening of the absorption band. These results indicate that the carbonyl bond undergoes significant polarization in the enzyme-ligand complex, facilitated by the enzyme-bound Ca2+ ion. This suggests that ground-state strain is likely to promote catalysis in the case of substrate binding. Simple calculations, based on the i.r. data, indicate that the carbonyl bond is weakened by 5-9 kJ.mol-1. This is the first report of observation of the amide vibration of a bound ligand against the strong background of protein amide vibrations.

Animals↗
PubMed1992

Design and synthesis of some substrate analogue inhibitors of phospholipase A2 and investigations by NMR and molecular modeling into the binding interactions in the enzyme-inhibitor complex.

A series of substrate analogue inhibitors of pancreatic phospholipase A2 has been designed and synthesized. The compounds were tested in a novel dual-screening system based on parallel assays with monomeric and micellar substrates. Intermolecular nuclear Overhauser effects between vinylic protons on one inhibitor and identified active site residues on the bovine pancreatic enzyme have been observed in solution NMR studies of the enzyme-inhibitor complex. It can be deduced from both the biochemical results and the NMR data that the mode of interaction between this type of inhibitor and the active site of phospholipase A2 is essentially the same, irrespective of the presence or absence of an aggregated phospholipid surface. A model of the binding between the enzyme and inhibitor which incorporates the two-dimensional NMR data has been developed. The model can account for the activity of modified inhibitor structures and can be extrapolated to an assessment of the mode of binding of the natural substrate itself.

Animals↗
PubMed1991

Retroviral transformation of cerebral microvascular endothelial cells: macrophage-like and microvascular endothelial cell properties.

We report that L-cell-conditioned medium (LCM) transforms porcine cerebral microvascular (PCMV) endothelial cells into cells with macrophage-like properties. LCM is known to contain both cytokine(s) and the L-cell virus, a murine retrovirus found in the L929 cell and LCM. Our evidence suggests that both LCM cytokine(s) and the L-cell virus are involved in this PCMV endothelial cell transformation. Criteria for transformation include focus formation, decreased serum requirements for growth, changes in morphology including nonadherence, propagation in suspension culture, and a decreased growth response to stimulation with a known endothelial cell mitogen. Macrophage-like characteristics of this transformed cell, designated as RVTE, include pinocytosis of low-density lipoprotein, Fc receptor-mediated phagocytosis, phagocytosis of bacteria and zymosan, the expression of macrophage enzyme markers, and constitutive production of colony-stimulating factor 1. However, the transformed cell retains several properties of the nontransformed cell including the expression of FVIII:RAg and in vitro self-organization into capillary-like structures. Cloning of RVTE cells clearly shows that both macrophage-like and cerebral microvascular endothelial cell properties are present in the same cell. During self-organization, nontransformed cells express morphologic and functional characteristics classically associated with the macrophage. These findings suggest that some brain capillary pathophysiologies could involve macrophage-like cerebral microvascular endothelial cells. Furthermore, the "reticuloendothelial" phenotypic repertoire expressed by this transformed cerebral microvascular endothelial cell may show that the cerebral capillary endothelial cell in vivo is derived from a hematopoietic and/or phagocytic precursor.

Animals↗
PubMed1991

Epithelial sodium conductance in rabbit preimplantation trophectodermal cells.

We examined the development of epithelial Na+ conductance in 6- and 7-day post coitus (p.c.) preimplantation rabbit embryos using the whole-cell patch-clamp technique on dissociated rabbit trophectodermal cells and by immunocytochemical localization using a polyclonal antibody directed against subunits of an apical epithelial Na+ channel on the intact blastocyst. In Day 6 and 7 p.c. trophectodermal cells, we observed an outwardly rectified whole-cell Na+ current. The current-voltage characteristics did not differ between the 6- and the 7-day p.c. cells. Replacement of Na+ with the impermeant cation N-methyl-D-glucamine in the pipette or bath reduced outward currents and inward currents, respectively, indicating that the current was Na(+)-dependent. Treatment of 7-day p.c. cells with 100 microM amiloride, benzamil, or ethylisopropyl amiloride (EIPA) blocked the whole-cell currents within 5 min. However, the current of the Day 6 p.c. embryo was not blocked by amiloride. The amiloride block at Day 7 p.c. was only partially reversible after 15 min of continuous perfusion of the bath with an amiloride-free solution. The apparent dissociation constant (Ki) for amiloride, benzamil, and EIPA was 12, 50, and 16 microM, respectively, when measured 5 min after drug addition. Immunolocalization studies of blastocysts with a polyclonal antibody raised against a high amiloride affinity Na+ channel isolated from bovine kidney revealed no specific binding to the trophectodermal cells at Day 6 p.c.(ABSTRACT TRUNCATED AT 250 WORDS)

Amiloride↗
PubMed1991

Design, synthesis, and physicochemical properties of a novel, conformationally restricted 2,3-dihydro-1,3,4-thiadiazole-containing angiotensin converting enzyme inhibitor which is preferentially eliminated by the biliary route in rats.

Two novel series of dihydrothiadiazole ring containing inhibitors of angiotensin converting enzyme have been designed and synthesized. The compounds are highly potent enzyme inhibitors and, as a consequence of conformational restriction, chemically stable with respect to undesirable cyclization reactions. The most interesting compound from this series, 5a (FPL 63547), is the monoethyl ester prodrug of the highly potent "aminocarboxy" inhibitor 5b (FPL 63674). It produces an antihypertensive effect of long duration in animal models after oral dosing. Unlike other ACE inhibitors, 5b is eliminated almost entirely by biliary clearance in the rat. The favorable pharmacological properties of 5a and 5b are rationalized in terms of their unique physicochemical profiles. The clear preference for biliary clearance seen with 5b is consistent with its lipophilicity and its high degree of net ionization at physiological pH, which results from the very low pKa of the C-terminus carboxylic acid function. FPL 63547 is presently undergoing clinical investigation in man.

Angiotensin-Converting Enzyme Inhibitors↗
PubMed1991

Glucose metabolism in the trophectoderm and inner cell mass of the rabbit embryo.

The metabolism of glucose in the intact Day-6 and -7 post coitum (p.c.) rabbit blastocyst and in the separated trophectoderm and inner cell mass (ICM) of the Day-7 p.c. embryo was investigated. At Day-6 p.c., glucose traversed the trophectoderm with a half-time of 39 +/- 9.3 min, and was metabolized to CO2 at a rate of 25.5 +/- 1.6 nmol.cm-2.h-1. Neither the Na+ ionophore, amphotericin B, nor cyclic AMP had an effect on glucose metabolism to CO2. Lactate production by the Day-6 blastocyst was largely independent of glucose. At Day-7 p.c. in the intact embryo, CO2 production from glucose significantly decreased to 7.76 +/- 2.8 nmol.cm-2.h-1. Per unit surface area, the metabolism of glucose to CO2 was similar in the separated Day-7 p.c. trophectoderm and ICM. We conclude that the rabbit blastocyst is not highly dependent on glucose, and that the ICM does not utilize glucose as a metabolite to a greater extent than does the trophectoderm, at least in the Day-7 p.c. embryo.

Animals↗
PubMed1991

Drug delivery systems.

New and emerging drug delivery systems for traditional drugs and the products of biotechnology are discussed, and the role of the pharmacist in ensuring the appropriate use of these systems is outlined. Advantages of advanced drug delivery systems over traditional systems are the ability to deliver a drug more selectively to a specific site; easier, more accurate, less frequent dosing; decreased variability in systemic drug concentrations; absorption that is more consistent with the site and mechanism of action; and reductions in toxic metabolites. Four basic strategies govern the mechanisms of advanced drug delivery: physical, chemical, biological, and mechanical. Oral drug delivery systems use natural and synthetic polymers to deliver the product to a specific region in the gastrointestinal tract in a timely manner that minimizes adverse effects and increases drug efficacy. Innovations in injectable and implantable delivery systems include emulsions, particulate delivery systems, micromolecular products and macromolecular drug adducts, and enzymatic-controlled delivery. Options for noninvasive drug delivery include the transdermal, respiratory, intranasal, ophthalmic, lymphatic, rectal, intravaginal, and intrauterine routes as well as topical application. Rapid growth is projected in the drug delivery systems market worldwide in the next five years. Genetic engineering has mandated the development of new strategies to deliver biotechnologically derived protein and peptide drugs and chemoimmunoconjugates. The role of the pharmacist in the era of advanced drug delivery systems will be broad based, including administering drugs, compounding, calculating dosages based on pharmacokinetic and pharmacodynamic monitoring, counseling, and research. The advent of advanced drug delivery systems offers pharmacists a new opportunity to assume an active role in patient care.

Administration, Oral↗
PubMed1990

Comparison of new and existing spectrophotometric methods for the analysis of tobramycin and other aminoglycosides.

New indirect spectrophotometric methods of analysis of tobramycin using the derivatizing agents o-phthaldialdehyde, fluorescamine, and dansyl chloride were developed and compared with existing methods. Optimum reaction conditions were established for the complex formation between tobramycin and each reagent. The sensitivity and reproducibility of these new procedures were compared with results obtained using published methods. Of the new methods, the o-phthaldialdehyde and fluorescamine procedures were more sensitive and reproducible than the dansyl chloride procedure. Further, these procedures were subsequently applied to the analysis of aqueous solutions of other aminoglycosides including amikacin, neomycin, gentamicin, and kanamycin.

Anti-Bacterial Agents↗
PubMed1990

Morphologic plasticity and periodicity: porcine cerebral microvascular cells in culture.

Porcine cerebral microvascular (PCMV) endothelial cell cultures and pericyte-endothelial cell cocultures were established and the self-organizational properties of the cells were examined in various culture conditions. Cultured PCMV endothelial cells were characterized by the capacity to produce prostacyclin in response to bradykinin. Cultured PCMV pericytes were identified with a smooth muscle actin-specific stain. PCMV endothelial cells organized into cord structures when left in culture for several weeks without passage. Lumina were observed in cross sections of these cords and appeared to form through a process of cell-selective autolysis. PCMV endothelial cells required three dimensions for self-organization, forming suspended cords in planes that either intersected or paralleled the culture vessel floor. After formation, suspended cords continued to exhibit a morphologic plasticity punctuated by the coordinated migrations of PCMV endothelial cells en masse. Sequential propagation of PCMV endothelial cell monolayers and development of suspended capillarylike cords recurred cyclically when cells were left in culture without passage for several weeks. Cord development was also observed in PCMV pericyte-endothelial cell cocultures with large proportions of pericytes. However, pericytes were not located in cross sections of suspended cords formed in coculture. Apparently, in some conditions of PCMV coculture, populations of PCMV endothelial cells and pericytes segregate. Retina-derived growth factor (RDGF) promoted this cell-type segregation and the subsequent formation of suspended cords in PCMV cocultures, although its exact mode of action is unclear. These results indicate that cultured cerebral microvascular endothelial cells and pericytes have capacities for complex, temporal self-organization that varies according to culture conditions.

Animals↗