Biomedical subjects
Publications and source records attributed to W Burgess.
A novel Quantitative Nucleic Acid Test (Q-NAT) technology has been developed to demonstrate, quantify and verify pathogen inactivation by methods that break pathogen nucleic acids, specifically, gamma irradiation. The Q-NAT technology provides significant advantages in cost, efficiency and broad applicability compared with traditional methods for pathogen inactivation detection and quantification such as cell culture.
Bone regeneration promoted by acidic recombinant human fibroblast growth factor (rhFGF-1), rabbit demineralized bone matrix (rDBM), and a fibrin (f) delivery system was measured in critical-sized defects in rabbits' radii. A unilateral segmental defect 20 mm in length was prepared in radii of 48 skeletally mature New Zealand White rabbits divided equally between 4- and 8-week cohorts. The temporal cohorts were divided equally among four treatment groups: rDBM, rDBM/f, rDBM/rhFGF-1/f, and rhFGF-1/f. Data for the fifth group, untreated critical-sized defects, were exploited from previous published reports from this laboratory. In response to experimental treatments, radiomorphometric and histomorphometric methods were used to derive quantitative outcome data that were tested by analysis of variance and post hoc multiple comparison tests (significance p </= 0.05). Radiomorphometric data (percentage of radiopacity of defect) were acquired at the day of the operation and every 2 weeks thereafter, whereas histomorphometric data (square millimeters of new bone formation) were determined at term. The objective for the study was to develop candidate bone regenerative therapies. Therefore, the hypotheses were that experimental treatments would promote bone formation within critical-sized defects and that one treatment would be superior to the rest. Testing hypotheses was achieved with quantitative methodology, and data were subjected to statistical models. Radiopacity at each 2-week period was greater in treated defects than in untreated critical-sized defects. The amount of radiopacity promoted by rDBM/f and rhFGF-1/f at 8 weeks was equivalent and was greater than antecedent times. Histomorphometric data analyses indicated that rDBM/f and rDBM evoked the same quantity of new bone formation at 4 weeks; by 8 weeks, all treatments except rDBM/f had more new bone within the critical-sized defects in comparison to untreated defects. That rDBM/f promoted less new bone than rDBM alone may suggest fibrin decreases bone formation, perhaps by impeding local solubility of endogenous and rDBM-containing signaling molecules. However, rhFGF-1/f promoted a significant and unexpected increase in bone formation response that could refute the previous notion. In conclusion, the combination of rDBM/rhFGF-1/f may represent a significant, new osteogenic therapeutic regimen. Additional assessments in higher order species must be accomplished to corroborate efficacy.
A novel series of mutilin 14-carbamates has been discovered as a result of structure-activity studies on the naturally occurring antibiotic pleuromutilin (1). In particular, the 4-methoxybenzoylcarbamate, SB-222734 (15o) displays potent antibacterial activity against a number of bacterial pathogens which are resistant to currently used agents and shows enhanced metabolic stability when compared to earlier pleuromutilin derivatives. Such derivatives therefore have the potential to provide a new class of antibacterial agents for human therapy which address the threat of bacterial resistance.
Interleukin-1beta (IL-1beta) is expressed in the mouse brain after intracerebroventricular injection of lipopolysaccharide (LPS) and is thought to be responsible for many of the behavioral and neuroendocrine changes that occur during inflammation. In this study we show that LPS in the brain also induces expression of interleukin-1beta converting enzyme (ICE) and that ICE is important for the characteristic anorectic response of mice to intracerebroventricular LPS. Specifically, mice that were deficient in ICE (ICE(-/-)) resisted the anorexia caused by intracerebroventricular injection of LPS but were sensitive to the anorectic properties of recombinant IL-1beta. The typical anorectic response seen in wild-type (WT) mice after LPS was restored in ICE(-/-) mice by intracerebroventricular administration of the ICE analog cathepsin G. Conversely, anorexia induced by intracerebroventricular injection of LPS in WT mice was blocked by prior intracerebroventricular injection of the ICE antagonist YVAD. CMK. Furthermore, in situ hybridization immunohistochemistry revealed intense expression of ICE mRNA in the hippocampus and dorsomedial hypothalamus of WT mice after intracerebroventricular injection of LPS. Thus ICE mRNA is expressed in brain after intracerebroventricular injection of LPS and is important for induction of anorexia, presumably because it generates mature IL-1beta. These results suggest that preventing generation of mature IL-1beta can inhibit anorexia induced by LPS in the brain and, therefore, reveal ICE as a potential target for regulating food intake during brain inflammation.
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A decline in plasma concentrations of both growth hormone and IGF-I occurs during aging of humans and rodents, and this is accompanied by involution of the thymus gland. Exogenous growth hormone induces the synthesis of IGF-I, which acts on bone marrow-derived hematopoietic progenitors of the myeloid and lymphoid lineages to promote their replication and survival. The increase in survival of these cells is caused by the ability of IGF-I to inhibit their apoptotic death. In contrast to the multipotential colony-stimulating-factor IL-3, inhibition of apoptosis by IGF-I requires the activation of the critical intracellular effector PI 3-kinase. These data establish that hematopoietic progenitors can use more than one intracellular signaling pathway in order to maintain their survival. The data also extend the original hypothesis that IGF-I shares with the colony-stimulating factors the properties of promoting DNA synthesis and inhibiting programmed cell death. Collectively, these data establish that hematopoietic progenitor cells are important targets for IGF-I, and this is likely to be important in understanding thymic aging.
Interleukin-1beta (IL-1beta) mediates many of the behavioral responses to infection and inflammation, and IL-1beta-converting enzyme (ICE) processes intracellular IL-1beta, leading to its maturation and secretion. Here we demonstrate that intracerebroventricular injections of lipopolysaccharide (LPS) produced a greater reduction in both food intake and food-motivated behavior in wild-type compared with ICE-deficient (ICE -/-) mice. This defect occurred although ICE -/- mice were able to fully respond to intracerebroventricular injections of IL-1beta. In contrast, ICE -/- mice remained fully responsive to intraperitoneal injections of LPS. These results indicate that brain, but not peripheral, IL-1beta plays a critical role in the depression in food intake that occurs during inflammation.
The development of managed behavioral health care carve-out contracts covering a discrete subset of benefits available for use by persons with mental health and/or substance abuse disorders poses major challenges for public purchasers. This Issue Brief explores several key issues that arise when drafting such agreements. Many of the issues that arise in the drafting of carve-out agreements will require the public purchaser to resolve basic policy questions well before the drafting of requests for proposals or contracts can proceed. Analyses of public sector managed behavioral health care contracts by attorneys at the Center for Health Policy Research suggest that there are four essential areas that must be addressed if mental health and substance abuse services are carved-out (either by the purchaser or by a comprehensive managed health care entity): (1) what population is eligible for enrollment; (2) what services is the contractor expected to furnish; (3) what triggers a duty on the part of the mental health or substance abuse carve-out contractor to provide services; and (4) how are services furnished by the managed behavioral health care contractor integrated with or coordinated with services furnished by a beneficiary's primary health care provider, with pharmaceutical benefits, and with other services that may be available to a beneficiary through a fee-for-service or other mechanism. However a purchaser chooses to resolve these four issues, it is essential that parallel clarifying clauses are also built into the contracts of primary health care providers and other entities providing needed services for persons whose mental health and substance abuse service needs are covered by the carve-out. Underlying all of these issues is the fact that ambiguity, vagueness, or failure to define terms and responsibilities can create unexpected and unwelcome clinical and financial liabilities to purchasers.
The present paper describes the functional characterization of two human aryl sulphotransferase (HAST) cDNAs, HAST1 and HAST3, previously isolated by us from liver and brain, respectively [Zhu, Veronese, Sansom, and McManus (1993) Biochem. Biophys. Res. Commun. 192, 671-676; Zhu, Veronese, Bernard, Sansom and McManus (1993) Biochem. Biophys. Res. Commun. 195, 120-127]. These appear to encode the two major forms of phenol sulphotransferase (PST) characterized in a number of human tissue cytosols, these being the phenolsulphating (P-PST) and monoamine-sulphating (M-PST) forms of phenol sulphotransferase. HAST1 and HAST3 cDNAs were functionally expressed in COS-7 cells and kinetically characterized using the model substrates for P-PST and M-PST, p-nitrophenol and dopamine (3,4-dihydroxyphenethylamine) respectively. COS-expressed HAST1 was shown to be enzymatically active in sulphating p-nitrophenol with high affinity (Km 0.6 microM), whereas dopamine was the preferred substrate for HAST3 (Km 9.7 microM). HAST1 could also sulphate dopamine, as could HAST3 sulphate p-nitrophenol, but the Km for these reactions were at least two orders of magnitude greater than for the preferred substrates. COS-expressed HAST1 and HAST3 displayed inhibition profiles with the ST inhibitor 2,6-dichloro-4-nitrophenol (DCNP), identical with human liver cytosolic P-PST and M-PST activities respectively. Thermal-stability studies with the expressed enzymes showed that HAST1 was considerably more thermostable (TS) than HAST3, which is consistent with P-PST being termed the TS PST and M-PST being termed the thermolabile (TL) PST. Western immunoblot analyses of the expressed PST proteins using an antibody generated to a bacterially expressed rat liver aryl/phenol ST showed that HAST1 and HAST3 migrated as single proteins with different electrophoretic mobilities (32 versus 34 kDa). This is consistent with the differences in electrophoretic mobilities observed for P-PST and M-PST in a variety of tissues reported by other workers. This report on the functional characterization of P-PST and M-PST cDNAs provides important information on the structural as well as functional relationships of human PSTs, which sulphate a vast array of exogenous and endogenous compounds.
This paper describes the use of both mammalian and bacterial expression systems as tools to study the structural and functional relationships of proteins encoded by cDNAs to both rat and human aryl sulfotransferases. In particular, we describe the use of the mammalian COS cell system for functional expression studies, and the use of Escherichia coli for the expression and purification of a sulfotransferase fusion protein suitable as an antigen for the generation of sulfotransferase antibodies.
Sperm were obtained via electroejaculation from Domestic ferret, (Mustela putorius furo), Siberian ferret (M. eversmanni), Black-footed ferret (M. nigripes), and a hybrid between Siberian and Domestic, called the Fitch ferret (M. sp.). Comparisons of sperm were made by four different microscopy techniques to determine whether differences exist among species. First, Nomarski differential interference microscopy could be used to distinguish domestic ferret sperm from the others on the basis of the structure of the posterior part of the acrosome. Second, both silver staining, which demonstrates argentophilic protein distribution, and scanning electron microscopy (SEM), revealed differences among the morphology of sperm for each species; variation in the unique appearance of the acrosome in ferret sperm was detected especially well by SEM. To quantify differences in morphology, five sperm head parameters were measured using image analysis; light microscopy produced significantly larger values than did SEM (all parameters and all species but Fitch), and there were significant differences owing to species for all parameters but one. Generally, our data demonstrate the value of complementary techniques to distinguish among sperm of closely related species and more specifically may help establish evolutionary relationships among the ferret species studied. In addition, they provide baseline data important for the captive breeding of the endangered Black-footed ferret.
An immunoaffinity process based on monoclonal antibody (MAb) to factor IX (FIX) has been developed. Initially, vitamin-K-dependent proteins from cryoprecipitate-poor plasma are isolated on DEAE-Sephadex. The eluate is applied to an immunoaffinity column that utilizes a divalent metal-ion-dependent MAb directed against FIX. After washing the column with high salt in the presence of magnesium ion, the FIX is eluted using a citrate- or EDTA-containing buffer. Coagulation assays and Western blots show no detectable amounts of any contaminating proteins. Purity of the FIX product is established using reduced and nonreduced Coomassie-stained SDS-PAGE and HPLC. The N-terminal 20 amino acids of the single peak of the HPLC were shown to be identical to those reported for FIX. The process shows no detectable leakage of monoclonal antibodies (MAb), efficient utilization of MAb, and provides yields greater than 95%. The use of solvent/detergent treatment as a potential viral inactivation methods is incorporated in the process. Studies with tritiated Triton X-100 indicate that the detergent can be washed out of the MAb column so that less than 1 ppm (total) Triton X-100 coelutes with the FIX.
The tumor inhibitory factor-2 from the conditioned medium of the human rhabdomyosarcoma cell line A673 was purified and sequenced. The 19 N-terminal amino acid residues were identical to those of human interleukin 1 (IL-1), corresponding to the residues 119-137 of the IL-1 alpha precursor. The purified material had an apparent molecular weight similar to that of the mature secreted form of IL-1 alpha (Mr 17,400). In addition, similarly to IL-1, it induced the production of IL-2 by T-cells. The purified protein inhibited the growth of the A673 cells from which it was derived, suggesting that it may act as an autocrine growth inhibitor. It also inhibited the growth of a human adenocarcinoma of the lung and three human mammary carcinomas, but not of two human melanoma cell lines. In contrast, it stimulated the proliferation of normal human fibroblasts. These biological activities, previously assigned to a putative tumor inhibitory factor molecule, are apparently due to the production by the tumor cells of IL-1 alpha.
Ejaculated sperm from the domestic ferret (Mustela putorius furo) and the black-footed ferret (Mustela nigripes) were compared for differences in morphological abnormalities and argentophilic protein distribution. Thawed domestic ferret sperm was also compared to fresh sperm to determine whether there were any effects on cell morphology due to cryopreservation. There were statistically significant differences between the two species of ferret in two of the categories scored. The domestic ferret had a higher frequency of cells that were bent in the midpiece and in the principal piece, and a higher frequency of headless and tailless cells when compared to the black-footed ferret. There were no statistically significant differences in cell morphology between the fresh and cryopreserved ejaculates of the domestic ferret employing a standard egg yolk cryoextender. Silver nitrate staining distribution was different between the two species in both the head and tail region.
This current study analyzed macrophage/biomaterial interactions as modulators of endothelial cell proliferation. Rabbit peritoneal macrophages were harvested and seeded (1 x 10(6) cells/ml) into culture flasks with Dulbecco's modified Eagle medium and 10% platelet-poor, plasma-derived equine serum. Macrophages were identified by morphologic characteristics, nonspecific esterase, and Fc (immunoglobulin G) receptors on the cell membranes. Culture conditions were (1) no prosthetic material, (2) Dacron, or (3) Polyglactin 910 (PG910) (Ethicon, Inc., Somerville, N.J.). Both prosthetic materials were finely shredded into the media. After 5 weeks in culture, PG910 inclusions were seen within macrophage cytoplasm. No intracytoplasmic Dacron was observed. Conditioned media from all three groups were collected weekly from week 5 to week 10, centrifuged, filtered, and added in serial dilutions to cultured quiescent murine capillary lung endothelial cells. Quiescence was achieved by serum deprivation and verified by [3H]thymidine incorporation. Sixteen hours after addition of conditioned media, [3H]thymidine was measured in and expressed as percent increase above quiescent levels. Mitogenic activity in the PG910 group progressively increased from weeks 6 to 10. At week 10, the PG910 group (1:10 dilution) yielded a 620% increase in DNA synthesis. The Dacron group never varied from the control group (no prosthetic). The mean increases in [3H]thymidine incorporations over weeks 7 to 10 were PG910, 540% +/- 65%; Dacron, 323% +/- 65%, and control, 343% +/- 26% (PG910 vs Dacron, p less than or equal to 0.004). These studies suggest macrophage activation by bioresorbable prostheses, yielding growth factor release with subsequent enhanced endothelial cell proliferation.
We have previously isolated a human gene from an endothelial cell cDNA library encoding a putative tyrosine kinase; we have designated this gene the fms-like gene (FLG). To analyze the gene product(s) of FLG, we have generated rabbit polyclonal antibodies directed against a synthetic peptide from FLG and used it to immunoprecipitate biosynthetically labeled FLG protein from a variety of human cell lines. These antibodies specifically recognized glycoprotein(s) of 100, 120, and 135 kDa with protein cores of 90 and 110 kDa. Acidic fibroblast growth factor (aFGF) stimulated tyrosine kinase activity of FLG in vitro and in living cells, suggesting that FLG encodes the membrane receptor for aFGF. Further supporting evidence came from cross-linking experiments on intact cells with the covalent cross-linking agent disuccinimidyl suberate and 125I-labeled aFGF as a specific probe. The cross-linked 125I-labeled aFGF-aFGF receptor complex was specifically immunoprecipitated with FLG antipeptide antibodies. It appears, therefore, that the receptor(s) for aFGF is related to the FLG gene product.
In an attempt to elucidate the mechanism(s) behind the susceptibility of tissues to the toxic effects of chemicals, NADPH-cytochrome P450 reductase IgG has been used to map the distribution and localization of the cytochrome P450 system in hepatic and extrahepatic human tissues. Employing the Western blot procedure this antibody recognized a single band in human liver microsomes which corresponded in mol. wt to the purified reductase. Immunoreactive NADPH-cytochrome P450 reductase staining was detected in all zones of the liver acinus, with maximal staining in hepatocytes adjacent to the terminal hepatic venules (zone 3). Considerable variation in the intensity of reductase staining was observed in different segments of the gastrointestinal tract. Staining was most intense in the enterocytes of the small intestine, with maximal staining at the tips of the villi. Colonic epithelial cells were variably positive while the rectum was negative. Pancreatic ductal cells were positive whereas exocrine cells were negative. In the lung, reductase was detected in bronchiolar and bronchial epithelial cells, Clara cells and alveolar lining cells. In the kidney, the proximal and distal convoluted tubules, the loops of Henle, the collecting ducts in the medulla and the transitional epithelium all stained positively for reductase. The results demonstrate specific cellular localization of NADPH-cytochrome P450 reductase, and hence the cytochrome P450 system, in human tissues. The differential distribution of the reductase within human tissues may lead to a better understanding of the mechanism underlining site-specific carcinogenesis.
1. The NADPH-cytochrome P450 reductases (EC 1.6.2.4) from human and rabbit liver have been purified to electrophoretic homogeneity. The human reductase had an apparent monomeric molecular weight of 77,500 and the rabbit enzyme of 76,500. 2. Both flavoproteins exhibited typical flavoprotein spectra and contained equimolar quantities of FAD and FMN. The two reductases were catalytically active in reducing cytochrome c, ferricyanide and dichlorophenolindophenol, and in supporting rabbit liver cytochrome P450 Form 4 metabolism of 2-acetylaminofluorene. 3. An antibody raised in the goat against the human enzyme formed a precipitin line with the human reductase in a double-diffusion assay, but did not react with the rabbit reductase. Similarly, an antibody raised in the goat against the rabbit reductase formed a precipitin line with the rabbit enzyme, but did not cross-react with the human reductase. 4. Both antibodies inhibited cytochrome c reduction by the two reductases suggesting some immunochemical recognition. 5. Immunochemical cross-reactivity was confirmed when both reductases were subjected to the more sensitive immunoblot technique using either anti-human or anti-rabbit reductase IgG. 6. The human and rabbit reductases are essentially similar in amino acid composition, except that the former has larger amounts of serine and glycine.