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Background and Purpose CYP2D6 metabolizes many centrally acting drugs, neurotoxins and endogenous neurochemicals, and differences in brain levels of CYP2D have been associated with brain function and drug response. Alcohol consumers and smokers have higher levels of CYP2D6 in brain, but not liver, suggesting ethanol and/or nicotine may induce human brain CYP2D6. We investigated the independent and combined effects of chronic ethanol self-administration and nicotine treatment on CYP2D expression in African green monkeys. Experimental Approach Forty monkeys were randomized into control, ethanol-only, nicotine-only and ethanol + nicotine groups. Two groups voluntarily self-administered 10% ethanol in sucrose solution for 4 h·day−1, whereas two groups consumed sucrose solution on the same schedule. Two groups received daily s.c. injections of 0.5 mg·kg−1 nicotine in saline bid, whereas two groups were injected with saline on the same schedule. Key Results Both nicotine and ethanol dose-dependently increased CYP2D in brain; brain mRNA was unaffected, and neither drug altered hepatic CYP2D protein or mRNA. The combination of ethanol and nicotine increased brain CYP2D protein levels to a greater extent than either drug alone (1.2–2.2-fold, P < 0.05 among the eight brain regions assessed). Immunohistochemistry revealed the induction of brain CYP2D protein within specific cell types and regions in the treatment groups. Conclusions and Implications Ethanol and nicotine increase brain CYP2D protein levels in monkeys, in a region and treatment-specific manner, suggesting that CNS drug responses, neurodegeneration and personality may be affected among people who consume alcohol and/or nicotine.

Cell adhesion to extracellular matrices involves specific bonds between cell membrane proteins and their extracellular ligands, less-defined interactions between macromolecules extending from the cell and matrix, and features of the matrix such as distribution of adhesion ligands, surface topography, electrostatic charge, and mechanical compliance. Many aspects of the cell phenotype are altered by changes in the matrix produced by other cells or environmental factors. This article summarizes how cell adhesion is altered by changes in the chemical or physical properties of the matrix and illustrates how matrix-dependent effects on cell function are associated with normal development or pathology.

Summary. Objective: The main objective of this study was to examine factors associated with utilization and costs for persons with haemophilia. Study design: Utilization data and patient characteristics were collected through medical record review of 336 patients receiving treatment for at least 90% of their haemophilia care at one of five comprehensive haemophilia treatment centres in California. Principal findings: The range of factor VIII deficiency in our sample was similar to the distribution among haemophilic patients in the Western United States; 215 (64%) had severe FVIII deficiency. The mean age in our sample was 21.4 (SD ¼ 16.2) years old and 114 (34%) were HIV-positive. In the multivariate model predicting the total cost of health care during 1995 (adjusted R 2 ¼ 0.40), total annual costs were significantly (P < 0.05) associated with being HIV-seropositive, infusing FVIII concentrate through a port vs. i.v. infusion, the number of comorbidities, moderate arthropathy (compared with no arthropathy), mild arthropathy, history of inhibitor to FVIII, and current prophylactic FVIII concentrate infusion. Conclusion: As expected, total health-care costs were correlated with comorbid medical conditions, such as HIV and sequelae of haemophilia such as arthropathy. Health policy should consider risk adjustment for the presence of complications such as arthropathy and HIV infection in the financing of haemophilia treatment to promote more equitable delivery of these services.

The purpose of this study was to assess the relationship between health care and utilization of that health care, and to provide a base measurement of health status in patients with haemophilia. Provider interview and retrospective chart review of 336 patients with haemophilia treated during 1995 at one of five comprehensive haemophilia treatment centres was conducted to measure patient health status characteristics and utilization of health care. Two health status scales were included. The first, the Self-Care Measure, was a four-point single item scale measuring the patient's ability for basic self-care, which was scored by a chart review and an interview with the health-care provider. The second, the Haemophilia Utilization Group Study (HUGS) Functional Status Measure, is a four-item, 10-point scale developed specifically for patients with haemophilia. Our sample represents 27% of actively treated patients in region IX. The mean score on the HUGS Functional Status Measure was 8.7 (SD=2.4). The HUGS scale exhibited a ceiling effect across all four scales: attitude (n=269, 80.1%), overall wellbeing (n=263, 78.3%), working (n=254, 75.6%) and orthopaedic status (n=195, 58.0%). Both higher total health-care costs and factor VIII annual costs were significantly associated with lower scores on the HUGS Functional Status Measure. Health status is a critical component in the assessment of the utilization and outcomes of care. In the absence of the availability of a patient interview, the HUGS Functional Status Measure can be used as one characteristic that explains the variation in the utilization of health care by patients with haemophilia.

Contractility of uterine smooth muscle is essential for the cyclic shedding of the endometrial lining and also for expulsion of the fetus during parturition. The nitric oxide (NO)-cGMP signaling pathway is involved in smooth muscle relaxation. The downstream target of this pathway essential for decreasing cytoplasmic calcium and muscle tone is the cGMP-dependent protein kinase (PKG). The present study was undertaken to localize expression of PKG in tissues of the female reproductive tract and to test the hypothesis that uterine smooth muscle PKG levels vary with the human menstrual cycle. Immunohistochemistry was used to localize PKG in myometrium, cervix, and endometrium obtained during proliferative and secretory phases. The PKG was localized to uterine and vascular smooth muscle cells in myometrium, stromal cells in endometrium, and a small percentage of cervical stromal cells. Using Western blot analysis and protein kinase activity assays, the expression of PKG was reduced significantly in progesterone-dominated uteri compared with myometrium from postmenopausal women or women in the proliferative phase. These findings support a role for PKG in the control of uterine and vascular smooth muscle contractility during the menstrual cycle.

A multifamily sequence alignment of the rabbit CYP4A members with the known structure of CYP102 indicates amino acid differences falling within the so-called substrate recognition site(s) (SRS). Chimeric proteins constructed between CYP4A4 and CYP4A7 indicate that laurate activity is affected by the residues within SRS1 and prostaglandin activity is influenced by SRS2-3. Site-directed mutant proteins of CYP4A7 found laurate and arachidonate activity markedly diminished in the R90W mutant (SRS1) and somewhat decreased in W93S. While PGE(1) activity was only slightly increased, the mutant proteins H206Y and S255F (SRS2-3), on the other hand, exhibited remarkable increases in laurate and arachidonate metabolism (3-fold) above wild-type substrate metabolism. Mutant proteins H206Y, S255F, and H206Y/S255F but not R90W/W93S, wild-type CYP4A4, or CYP4A7 metabolized arachidonic acid in the absence of cytochrome b(5). Stopped-flow kinetic experiments were performed in a CO-saturated environment performed to estimate interaction rates of the monooxygenase reaction components. The mutant protein H206Y, which exhibits 3-fold higher than wild-type substrate activity, interacts with CPR at a rate at least 10 times faster than that of wild-type CYP4A7. These experimental results provide insight regarding the residues responsible for modulation of substrate specificity, affinity, and kinetics, as well as possible localization within the enzyme structure based on comparisons with homologous, known cytochrome P450 structures.

CYP2D6 metabolizes many centrally acting drugs, neurotoxins and endogenous neurochemicals, and differences in brain levels of CYP2D have been associated with brain function and drug response. Alcohol consumers and smokers have higher levels of CYP2D6 in brain, but not liver, suggesting ethanol and/or nicotine may induce human brain CYP2D6. We investigated the independent and combined effects of chronic ethanol self-administration and nicotine treatment on CYP2D expression in African green monkeys.Forty monkeys were randomized into control, ethanol-only, nicotine-only and ethanol + nicotine groups. Two groups voluntarily self-administered 10% ethanol in sucrose solution for 4 h·day(-1) , whereas two groups consumed sucrose solution on the same schedule. Two groups received daily s.c. injections of 0.5 mg·kg(-1) nicotine in saline bid, whereas two groups were injected with saline on the same schedule.Both nicotine and ethanol dose-dependently increased CYP2D in brain; brain mRNA was unaffected, and neither drug altered hepatic CYP2D protein or mRNA. The combination of ethanol and nicotine increased brain CYP2D protein levels to a greater extent than either drug alone (1.2-2.2-fold, P0.05 among the eight brain regions assessed). Immunohistochemistry revealed the induction of brain CYP2D protein within specific cell types and regions in the treatment groups.Ethanol and nicotine increase brain CYP2D protein levels in monkeys, in a region and treatment-specific manner, suggesting that CNS drug responses, neurodegeneration and personality may be affected among people who consume alcohol and/or nicotine.

Macrophage activation is deficient in the fetus and neonate when the serum concentrations of docosahexaenoic acid (DHA) are 150 microM, or 10-50-fold higher than in the adult. We now show that DHA inhibits production of nitric oxide (NO) by macrophages stimulated in vitro by IFNgamma plus LPS, or by IFNgamma plus TNFalpha. The half-maximal inhibitory activity of DHA was approximately 25 microM. There were strict biochemical requirements of the fatty acid for inhibition. Polyenoic fatty acids with 22 carbons were more inhibitory than those with 20 carbons. Among 22-carbon fatty acids, those with a greater number of double bonds and a double bond in the n-3 position were more inhibitory. DHA was the most inhibitory of the polyenoic acids we tested. Inducible nitric oxide synthase (iNOS) is the enzyme responsible for the production of NO by macrophages. NO production is initiated after new iNOS enzyme is synthesized following transcription of the iNOS gene. In macrophages stimulated by IFNgamma plus LPS, DHA inhibited accumulation of iNOS mRNA, as measured by Northern blotting, and iNOS transcription, as measured by nuclear run-on assays. We transfected RAW 264.7 macrophages with a construct containing the iNOS promoter fused to the chloramphenicol acetyl transferase gene. DHA inhibited activation of this promoter by IFN gamma plus LPS. By inhibiting iNOS transcription in the fetus and neonate, DHA may contribute to their increased susceptibility to infection.

Urinary diversion procedures in the dog have been described for both benign and malignant processes involving the bladder, urethra, or both. These procedures are performed rather infrequently, primarily because of the potential complications associated with urinary diversion into an intact gastrointestinal system. A case managed for five years following trigonal-colonic anastomosis for lymphocytic-plasmacytic urethritis is presented, along with a review of urinary diversion techniques. Postoperative management recommendations following urinary diversion are discussed.

5-(Glutathion-S-yl)-alpha-methyldopamine [5-(GSyl)-alpha-MeDA] is a putative metabolite of the serotonergic neurotoxicants 3,4-(+/-)-(methylenedioxy)amphetamine and 3,4-(+/-)-(methylenedioxy)methamphetamine. Glutathione (GSH) conjugates of several polyphenols are biologically (re)active. Therefore, as part of our studies on the role of 5-(GSyl)-alpha-MeDA in MDA-mediated neurotoxicity, we determined the regional brain metabolism of 5-(GSyl)-alpha-MeDA (720 nmol) following intracerebroventricular administration to male Sprague-Dawley rats. 5-(GSyl)-alpha-MeDA was rapidly cleared from all brain regions examined, and regional differences in the distribution of gamma-glutamyl transpeptidase (gamma-GT) correlated with the formation of 5-(cystein-S-yl)-alpha-methyldopamine (5-[CYS]-alpha-MeDA). We also observed the formation of 5-(N-acetyl-L-cystein-S-yl)-alpha-MeDA (5-[NAC]-alpha-MeDA) in all brain regions, indicating that the brain has the ability to synthesize mercapturic acids. Peak concentrations of 5-(NAC)-alpha-MeDA were found in the order: hypothalamus > midbrain/diencephalon/telencephalon > pons/medulla > hippocampus > cortex > striatum. In contrast to 5-(GSyl)-alpha-MeDA and 5-(CYS)-alpha-MeDA, 5-(NAC)-alpha-MeDA was eliminated relatively slowly from the brain. Differences were also found in cystein conjugate N-acetyltransferase activity in microsomes prepared from the various brain regions, but little difference was observed in brain cytosolic N-acetyl-L-cysteine conjugate N-deacetylase activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Changes in dietary acid and blood pH lead to adaptations in key enzymes and transport proteins of the renal proximal tubule that tend to return blood pH to normal. The mechanisms responsible, as well as acid-regulated signaling pathways that may effect these responses, are reviewed.

CAR undertook this investigation to better understand the costs and challenges of a local (state) regulation necessitating the implementation of alternative or advanced powertrain technology. CAR will attempt to add insight into the challenges that local regulations present to the automotive industry, and to contribute further to the discussion of how advanced powertrain technology may be used to meet such regulation. Any local law that (directly or indirectly) affects light duty motor vehicle fuel economy creates what in effect is a specialty market for powertrain technology. As such these small markets present significant challenges for automotive manufacturers. First, a small market with unique standards presents significant challenges to an industry that has sustained growth by relying on large volumes to achieve scale economies and deliver products at a cost acceptable to the consumer. Further, the challenges of the additional technology make it likely that any powertrain capable of meeting the stringent emissions standards will include costly additional components, and thus will be more costly to manufacture. It is likely that manufacturers would consider the following actions as steps to deliver products to meet the pending California regulatory requirements anticipated as a result of prior California legislation: (1) Substituting more fuelmore » efficient vehicles: Bring in more efficient vehicles from global operations, while likely dropping existing domestic products. (2) Substituting powertrains: Add existing downsized engines (i.e. turbocharged versions, etc.) into California market-bound vehicles. (3) Powertrain enhancements: Add technology to current engine and transmission offerings to improve efficiency and reduce emissions. (4) Incorporating alternative powertrains into existing vehicle platforms: Develop a hybrid or other type of powertrain for an existing vehicle. (5) New powertrains and new platforms: Develop vehicles specifically intended to incorporate new powertrain technologies, materials and/or design (e.g. the General Motors EV1 or the Toyota Prius). These five actions represent the gamut from the least complicated solution to the most complex. They also generally represent the least expensive response to the most expensive. It is possible that the least expensive responses may be least likely to meet market demands while achieving required GHG emission limits. At the same time, the most expensive option may produce a vehicle that satisfies the GHG reduction requirements and meets some consumer requirements, but is far too costly to manufacture and sell profitably. The response of a manufacturer would certainly have to take market size, consumer acceptance, technology implication and cost, as well as internal capacities and constraints, into consideration. It is important to understand that individual companies may respond differently in the short term. However, it is probable that there would be a more consistent industry-wide response in the longer term. Options 1 and 2 present the simplest responses. A company may reach into its global portfolio to deliver vehicles that are more fuel-efficient. These vehicles are usually much smaller and significantly less powerful than current U.S. offerings. Industry respondents indicated that such a strategy may be possible but would likely be met with less than positive reaction from the buying public. A general estimate for the cost to homologize a vehicle--that is, to prepare an existing vehicle for entry into the United States provided all business conditions were met (reasonable product, capacity availability, etc.), would be approximately $50 million. Assuming an estimated cost for homologation to meet U.S. standards of $50 million and a 20,000 vehicle per year sales volume in California, the company would then incur a $2,500 per-vehicle cost to bring them into the market. A manufacturer may also choose to incorporate a more efficient powertrain into a vehicle already sold in the market. The costs associated with such a strategy would include reengineering the vehicle engine compartment to accept the new powertrain, and developing, engineering and manufacturing those parts unique to the vehicle. Costs would also be incurred to achieve emission certification. Total costs per vehicle, if sold only in California would be similar to nationally averaged costs per vehicle when bringing a new vehicle into the national market. While companies may consider the importation of a more fuel-efficient vehicle from their current global portfolio, or the addition of a powertrain from another market, it is likely that these would be seen as stop-gap responses to the legislation. Many of the candidate vehicles and powertrains would likely not meet California consumer expectations, and may not provide enough fuel savings to achieve more severe emission regulations, thus offering only a step toward any solution.« less

An approach to the representation of individual amino acids using dihedral angle measurements, based on the maxi-min distance algorithm presented by J.T. Tou and R.C. Gonzalez (1974), is outlined. The methodology, based on maxi-min and data set partitions, generates 124 distinct cluster centers from 19121 total residues. While only 18 of the 124 capture 1% or more of the data set, the first cis-proline conformation is 49th in population and suggests that over fifty centers may be significant. Four applications of these centers are demonstrated: representation of individual proteins, tripeptide sequence dependency, analysis of neighboring centers, and a simple structure prediction algorithm. >

Based on the similarity in both structure and function of the reductase domain of neuronal nitric oxide synthase (nNOSred) to that of NADPH-cytochrome P450 reductase (CPR), we determined whether the characteristics of hydride transfer from NADPH to flavin adenine dinucleotide (FAD) were similar for both proteins. Secondly, we questioned whether hydride transfer from NADPH to either nNOSred or holo-nNOS was rate limiting for reactions catalyzed by these two proteins. Utilizing 500 MHz proton NMR and deuterated substrate, we determined that the stereospecificity of hydride transfer from NADPH and the conformation of the nicotinamide ring around the glycosidic bond were similar between CPR and nNOSred. Specifically, nNOSred abstracts the A-side hydrogen from NADPH, and the nicotinamide ring is in the anti conformation. We determined that the rate of hydride transfer to FAD appears to become partially rate limiting only for exceptionally good electron acceptors such as cytochrome c. Hydride transfer is not rate limiting for NO. production under any conditions used in this study. Interestingly, the deuterium isotope effect was decreased in the cytochrome c reductase assay with both nNOS and nNOSred when the assays were conducted in high ionic strength buffer, suggesting an increase in the rate of hydride transfer to FAD. These results are in stark contrast to results obtained with CPR (D. S. Sem and C. B. Kasper, 1995, Biochemistry 34, 3391-3398) whereby hydride transfer is partially rate limiting at high, but not at low, ionic strength. The seemingly opposite results in deuterium isotope effect observed with CPR and nNOSred, under conditions of high and low ionic strength, suggest differences in structure and/or regulation of these important flavoproteins.

Contractility of uterine smooth muscle is essential for the cyclic shedding of the endometrial lining and also for expulsion of the fetus during parturition. The nitric oxide (NO)-cGMP signaling pathway is involved in smooth muscle relaxation. The downstream target of this pathway essential for decreasing cytoplasmic calcium and muscle tone is the cGMP-dependent protein kinase (PKG). The present study was undertaken to localize expression of PKG in tissues of the female reproductive tract and to test the hypothesis that uterine smooth muscle PKG levels vary with the human menstrual cycle. Immunohistochemistry was used to localize PKG in myometrium, cervix, and endometrium obtained during proliferative and secretory phases. The PKG was localized to uterine and vascular smooth muscle cells in myometrium, stromal cells in endometrium, and a small percentage of cervical stromal cells. Using Western blot analysis and protein kinase activity assays, the expression of PKG was reduced significantly in progesterone-dominated uteri compared with myometrium from postmenopausal women or women in the proliferative phase. These findings support a role for PKG in the control of uterine and vascular smooth muscle contractility during the menstrual cycle.

Recently, we obtained x-ray crystallographic data showing the presence of a ZnS4 center in the structure of Escherichia coli-expressed bovine endothelial nitric-oxide synthase (eNOS) and rat neuronal nitric-oxide synthase (nNOS). The zinc atom is coordinated by two CXXXXC motifs, one motif being contributed by each NOS monomer (cysteine 326 through cysteine 331 in rat nNOS). Mutation of the nNOS cysteine 331 to alanine (C331A) results in the loss of NO. synthetic activity and also results in an inability to bind zinc efficiently. Although prolonged incubation of the C331A mutant of nNOS with high concentrations of L-arginine results in a catalytically active enzyme, zinc binding is not restored. In this study, we investigate the zinc stoichiometry in wild-type nNOS and eNOS, as well as in the C331A-mutated nNOS, using a chelation assay and electrothermal vaporization-inductively coupled plasma-mass spectrometry. The data reveal an approximate 2:1 stoichiometry of heme to zinc in (6R)-5,6,7,8-tetrahydro-L-biopterin-replete, wild-type nNOS and eNOS and show that the reactivated C331A mutant of nNOS has a limited ability to bind zinc. The present study substantiates that the zinc in NOS is structural rather than catalytic and is important for maintaining optimally functional, enzymatically active, constitutive NOSs.

A variety of monovalent anions and cations were effective in stimulating both calcium ion/calmodulin (Ca2+/CaM)-independent NADPH-cytochrome c reductase activity of, and Ca2+/CaM-dependent nitric oxide (NO.) synthesis by, neuronal nitric oxide synthase (nNOS). The efficacy of the ions in stimulating both activities could be correlated, in general, with their efficacy in precipitating or stabilizing certain proteins, an order referred to as the Hofmeister ion series. In the hemoglobin capture assay, used for measurement of NO. production, apparent substrate inhibition by L-arginine was almost completely reversed by the addition of sodium perchlorate (NaClO4), one of the more effective protein-destabilizing agents tested. Examination of this phenomenon by the assay of L-arginine conversion to L-citrulline revealed that the stimulatory effect of NaClO4 on the reaction was observed only in the presence of oxyhemoglobin or superoxide anion (generated by xanthine and xanthine oxidase), both scavengers of NO. Spectrophotometric examination of nNOS revealed that the addition of NaClO4 and a superoxide-generating system, but neither alone, prevented the increase of heme absorption at 436 nm, which has been attributed to the nitrosyl complex. The data are consistent with the release of autoinhibitory NO. coordinated to the prosthetic group of nNOS, which, in conjunction with an NO. scavenger, causes stimulation of the reaction.

The techniques described herein have added to our repertoire of experimental approaches for the characterization of the NOSs. These procedures have reinforced our conviction that the NOSs are structurally suited to perform unique functions in their cellular milieux and that these differences have physiological consequences.

The techniques described herein have added to our repertoire of experimental approaches for the characterization of the NOSs. These procedures have reinforced our conviction that the NOSs are structurally suited to perform unique functions in their cellular milieux and that these differences have physiological consequences.

To determine blood protein concentration, immunoglobulin concentration, and lymphocyte profiles in equine infectious anemia virus (EIAV) seropositive, naturally infected horses without clinical signs of disease.26 clinically normal seropositive horses, 6 febrile ponies with experimentally induced EIA, and 52 clinically normal seronegative horses and ponies.Serum and EDTA-anticoagulated blood were obtained from all horses and ponies, and total serum protein and albumin concentrations, immunoglobulin concentrations, and blood lymphocyte subset counts were determined.Compared with seronegative horses, EIAV seropositive inapparent carrier horses had no significant difference in serum reverse transcriptase activity, PCV, or platelet count. Inapparent carrier horses had increased plasma total solids and serum globulin concentrations and decreased serum albumin concentration and albumin-to-globulin ratio. Total serum immunoglobulin and serum IgM concentrations were increased. Inapparent carrier horses had significantly decreased percentages of CD5+ and CD4+ blood lymphocytes.Serum protein and lymphocyte subset changes in EIAV-infected inapparent carrier horses are consistent with immune activation or chronic inflammation, both of which may, in part, be the result of virus-induced polyclonal B-cell activation.EIAV seropositive horses have immune-related abnormalities consistent with ongoing viral activity regardless of the duration they have been infected, even when the usual signs of disease (anemia, fever, weight loss) are not apparent.

Two months after colic surgery, subcutaneous masses were found on the ventral and lateral portions of the thorax of a 3-year-old Hanoverian-cross filly. Six months later, the filly was admitted for evaluation of severe lameness. Arthrocentesis of the tarsocrural joint yielded clotted sanguineous material; however, unusual multinucleated giant cells were seen. Radiography of the right tarsus revealed soft tissue opacity and degenerative joint disease. The filly was euthanatized to prevent further suffering. At necropsy, multiple soft-tissue masses were located throughout the fascial planes of the tarsi and in the subcutis of the ventral and lateral portions of the thorax. Neoplasms consisted primarily of a large number of mature well-differentiated T lymphocytes. On the basis of these findings, the diagnosis was cutaneous lymphoma with unusual involvement of periarticular tissues. Severe degenerative joint disease in the right tarsus did not appear to be associated with the tumors.

To describe the clinical and radiographic features of septic and aseptic failure of two femoral endoprostheses and their successful revision.Case report.Two skeletally mature male research dogs.An uncemented porous-coated anatomic (PCA) endoprosthesis was implanted in a single-stage revision procedure after thorough debridement and lavage of the femoral canal. An autogenous cancellous bone graft was used in dog 2 (aseptic loosening). Serial clinical and radiographic examinations were performed postoperatively. The dogs were euthanatized 1 year (dog 1) and 2 years (dog 2) after revision surgery, and necropsy was performed. High-resolution contact radiographs and histopathologic evaluation of femoral sections were obtained.The cause of implant failure was septic loosening in dog 1 and aseptic loosening in dog 2. In both dogs, clinical function returned to normal after revision. Serial radiographic assessment after revision documented disappearance of the bone pedestal and the periprosthetic lucency. Cancellous hypertrophy seen adjacent to the proximal porous-coated region of the implants provided radiographic evidence of bony fixation. Histological evaluation of femoral sections documented successful implant integration with bone and fibrous tissue.Revision with an uncemented implant in a single-stage procedure was successful in the two dogs described in this report.This report provides a detailed description of the clinical course and serial radiographic assessment of septic and aseptic loosening of two femoral endoprostheses. Single-stage revision is a potential treatment for either condition as demonstrated by the successful outcome in these two dogs.

Neuronal nitric oxide synthase (nNOS) is a modular enzyme which consists of a flavin-containing reductase domain and a heme-containing oxygenase domain, linked by a stretch of amino acids which contains a calmodulin (CaM) binding site. CaM binding to nNOS facilitates the transfer of NADPH-derived electrons from the reductase domain to the oxygenase domain, resulting in the conversion of L-arginine to L-citrulline with the concomitant formation of a guanylate cyclase activating factor, putatively nitric oxide. Numerous studies have established that peroxynitrite-derived nitrogen oxides are present following nNOS turnover. Since peroxynitrite is formed by the diffusion-limited reaction between the two radical species, nitric oxide and O2.-, we employed the adrenochrome assay to examine whether nNOS was capable of producing O2.- during catalytic turnover in the presence of L-arginine. To differentiate between the role played by the reductase domain and that of the oxygenase domain in O2.- production, we compared its production by nNOS against that of a nNOS mutant (CYS-331), which was unable to transfer NADPH-derived electrons efficiently to the heme iron under special conditions, and against that of a flavoprotein module construct of nNOS. We report that O2.- production by nNOS and the CYS-331 mutant is CaM-dependent and that O2.- production can be modulated by substrates and inhibitors of nNOS. O2.- was also produced by the reductase domain of nNOS; however, it did not display the same CaM dependency. We conclude that both the reductase and oxygenase domains of nNOS produce O2.-, but that the reductase domain is both necessary and sufficient for O2.- production.

Cell injury frequently occurs in the setting of tissue destruction and inflammation and is associated with a rise in intracellular calcium (Cai) and increased NO production. The mechanisms that trigger rises in Cai and NO during cell injury are not fully defined, but they may involve activation of G protein-coupled receptors for substances such as bradykinin, Ang II, thromboxane, and thrombin. These receptors act through G proteins from different families that have distinct functions. Receptors for bradykinin and Ang II act through members of the G alpha i and G alpha q families, whereas receptors for thrombin and thromboxane act through members of the G alpha i, G alpha q, and G alpha 12/13 families. These G proteins cooperate to regulate Cai and NO in epithelial cells through distinct mechanisms. In a number of experimental settings, activators of the adenylyl cyclase system reduce the severity of cell injury. To understand the mechanisms by which G protein-dependent signaling systems may contribute to cell injury and to define the role of adenylyl cyclase in ameliorating cell injury, the effects of adenylyl cyclase on bradykinin-stimulated Ca influx and NO in cultured renal epithelial cells that stably overexpress G alpha q and G alpha 13 were studied. This system allowed for the separation of different components of the signals initiated by receptors for thromboxane and thrombin. G alpha 13 increased bradykinin-stimulated Ca influx by a mechanism that depends on NO and cGMP. The increased Ca influx was blocked by inhibitors of NO synthase and guanylyl cyclase and by activation of adenylyl cyclase. NO production was inhibited by activators of cAMP-dependent protein kinase, which indicated that cAMP blocks Ca influx by inhibiting NO production. Expression of G alpha q, the G protein that regulates phospholipase C, also increased bradykinin-stimulated Ca influx, but by an NO, cGMP-independent mechanism that was insensitive to inhibition by adenylyl cyclase. The authors conclude that Ca influx is modulated by NO-dependent and independent mechanisms, and that to the extent that increased NO production contributes to increased Ca influx and cell injury, cell injury may be reduced by agents that activate adenylyl cyclase.

alpha-Methyldopamine (alpha-MeDA) is a metabolite of the serotonergic neurotoxicants 3,4-(+/-)-(methylenedioxy)amphetamine (MDA) and 3,4-(+/-)-(methylenedioxy)methamphetamine (MDMA). alpha-MeDA readily oxidizes, and in the presence of glutathione (GSH) it forms 5-(glutathion-S-yl)-alpha-methyldopamine [5-(glutathion-S-yl)-alpha-MeDA]. Since GSH conjugates of many polyphenols are biologically (re)active, we investigated the role of 5-(glutathion-S-yl)-alpha-MeDA in the acute and long-term neurochemical changes observed after administration of MDA. Intracerebroventricular (icv) administration of 5-(glutathion-S-yl)-alpha-MeDA (720 nmol) to male Sprague-Dawley rats produced behavioral changes similar to those reported after subcutaneous administration of MDA. Thus, animals became hyperactive and aggressive and displayed forepaw treading and Straub tails, behaviors usually seen after administration of serotonin (5-HT) releasers, and consistent with a role for 5-(glutathion-S-yl)-alpha-MeDA in some of the behavioral alterations seen after administration of MDA and MDMA. In addition to the behavioral changes, 5-(glutathion-S-yl)-alpha-MeDA also caused short-term alterations in the dopaminergic, serotonergic, and noradrenergic systems. An increase in dopamine synthesis appears to be a prerequisite for the long-term depletion of brain 5-HT following MDMA administration. However, although 5-(glutathion-S-yl)-alpha-MeDA reproduced some of the effects of MDA on the dopaminergic system and was capable of causing acute increases in 5-HT turnover, a single icv injection of 5-(glutathion-S-yl)-alpha-MeDA did not result in long-term serotonergic toxicity. Thus, although acute stimulation of dopamine turnover may be necessary for long-term serotonergic toxicity, such changes are not sufficient to produce these effects. The effects of a multiple dosing schedule of 5-(glutathion-S-yl)-alpha-MeDA will therefore require investigation before we can define a role for this metabolite in MDA and MDMA mediated neurotoxicity. MDA also produces a pressor response that is related to its ability to release neuronal norepinephrine stores, and 5-(glutathion-S-yl)-alpha-MeDA caused comparable depletions of brain norepinephrine concentrations, indicating that both compounds produce similar effects on the noradrenergic system.

Protein fold recognition has been approached by threading an amino acid sequence onto a library of folds, calculating a sequence-structure compatibility score, and ranking these scores. Due to imperfections in the empirically derived pairwise potential functions and the necessarily heuristic approach to the sequence-structure alignment problem, the method benefits from the assessment of threaded models to evaluate the most probable structures among the list of possible folds. THREADER and ANALYST, software tools available through the Internet, facilitate the alignment and assessment steps of a threading prediction. No process has been found to be universally reliable for the detection of folds related to the structure of a known input sequence, but several useful steps and approaches are discussed.

Opossum kidney OKP cells express an apical membrane Na+/H+ antiporter that is encoded by NHE-3 (for Na+/H+ exchanger 3) and is similar in many respects to the renal proximal tubule apical membrane Na+/H+ antiporter. Chronic incubation of OKP cells in acid medium for 24 hr increases Na+/H(+)-antiporter activity and NHE-3 mRNA abundance. The increase in Na+/H(+)-antiporter activity was not prevented by H7, a protein kinase C/protein kinase A inhibitor, but was prevented by herbimycin A, a tyrosine kinase inhibitor. Incubation of cells in acid medium increased c-src activity, and this was inhibited by herbimycin A. To determine the role of the src family of nonreceptor protein-tyrosine kinases, Csk (for carboxyl-terminal src kinase), a physiologic inhibitor of these kinases, was overexpressed in OKP cells. In three clones overexpressing csk, acid-induced increases in Na+/H(+)-antiporter activity and NHE-3 mRNA abundance were inhibited. In these clones, inhibition of acid activation of Na+/H(+)-antiporter activity paralleled inhibition of acid activation of c-src. Neither herbimycin A nor overexpression of csk inhibited dexamethasone-induced increases in Na+/H(+)-antiporter activity. These studies show that decreases in pH activate c-src and that the src family nonreceptor protein-tyrosine kinases play a key role in acid activation of NHE-3.

Continued, exposure-dependent proliferation of hepatocytes in basophilic proliferative lesions appears to be critical to the mechanism of peroxisome proliferator carcinogenesis in rodents. Identification of the growth regulatory pathway(s) involved in the exaggerated hepatocellular proliferation observed in these lesions is proceeding. So far, regulatory pathways involving cyclophilin and IGFII/M6P receptor have been implicated, while no evidence for involvement of HGF-R, TGF alpha, or PPAR is available. Clearly, this work is preliminary and additional information is needed. Exposure/dose response relationships for hepatocellular proliferation in the basophilic proliferative lesions, as well as species differences in regulation of hepatocellular proliferation, will be important information for development of realistic assessments of cancer risk in humans exposed to peroxisome proliferators. Given the traditional reliance on theoretical models of carcinogenesis that assume carcinogen-DNA interaction and mutation, utilization of newer information on hepatocellular growth and differentiation is expected to significantly change the estimated human risk of peroxisome proliferator-induced cancer.

Priming of macrophages with low concentrations of lipopolysaccharide (LPS) enhances the ability of substances that act through heterotrimeric G proteins to stimulate immune cell functions. Although LPS-induced alterations in the expression and functions of G proteins of the alpha i family have been reported in hematopoietic cells, their effects on subsequent steps in LPS priming of macrophages have not been defined. To study the role of G alpha i2 in priming of macrophages by LPS, we expressed a mutant, activated form of alpha i2 (alpha i2Q205L) in P388D1 cells, and compared its effects on PAF-dependent C alpha signalling and arachidonic acid release to those in cells treated with LPS. In control P388D1 cells, treatment with LPS (100 ng/ml) for 1 hr increased the amount of alpha i2 protein 2-fold. Both LPS treatment and expression of alpha i2Q205L increased the rate of PAF-induced C alpha influx across the cell membrane and arachidonic acid release, although neither altered release of C alpha from intracellular stores by PAF. Expression of alpha i2Q205L is sufficient to mimic the effects of LPS on the PAF-induced C alpha i signal and enhanced arachidonic acid release. Consequently, although increasing the expression of alpha i2 may not be the sole mechanism by which LPS enhances signalling by PAF, increased alpha i2 expression can account for the alterations in PAF-induced C alpha i regulation, and arachidonic acid release in LPS-primed P388D1 cells.

These studies examined the effect of acidosis on immediate early (IE) gene expression in renal tubule cells. In MCT cells, an SV40 transformed mouse proximal tubule cell line, incubation in acid media led to transient increases in c-fos, c-jun, junB, and egr-1 mRNA abundance, peaking at 30 min to 1 h. In vivo metabolic acidosis caused more prolonged increases in these mRNA species in renal cortex. Nuclear runon studies demonstrated increased rates of transcription for these IE genes. In addition, pretreatment of cells with cycloheximide caused superinduction of these mRNA by acid incubation. These responses are similar to those elicited by growth factors. Inhibition of tyrosine kinase pathways prevented IE gene activation by acid, while inhibition of protein kinase C and/or increases in cell calcium had no effect. In 3T3 cells, acid activated IE genes by a different mechanism in that the increase in mRNA did not include c-jun, was more prolonged, and was blocked by cycloheximide. In summary, incubation of renal cells in acid media leads to activation of IE genes that is similar to growth factor-induced IE gene activation, and is likely mediated by tyrosine kinase pathways.

Specific antisera were produced to peptides representing the carboxyl terminus of alpha 13, a recently identified alpha subunit of the heterotrimeric guanine nucleotide-binding proteins (G proteins). Immunodetection with the antisera indicated that the 43-kDa protein is expressed ubiquitously at low levels (0.005-0.05% of membrane protein) in tissues and cultured cells. A combination of conventional and immunoaffinity chromatographic techniques was used to purify small quantities of alpha 13 from bovine brain. Quantities of protein sufficient for biochemical analysis could be produced by concurrent expression of alpha 13 with G protein beta 2 and gamma 2 subunits using a baculovirus system. The rate of dissociation of GDP from recombinant alpha 13 (r alpha 13) is slow (0.01-0.02 min-1 at 30 degrees C), and relatively high concentrations of guanosine 5'-3-O-(thio)triphosphate (GTP gamma S) are required to observe nucleotide binding. This binding was reduced significantly in the presence of 20 mM Mg2+. Rates of hydrolysis of GTP by alpha 13 were limited by nucleotide exchange; attempts to measure the intrinsic rate of hydrolysis indicate that it is greater than 0.2 min-1. Stoichiometric concentrations of beta gamma subunits inhibited binding of GTP gamma S to and hydrolysis of GTP by alpha 13. By reconstitution, the purified alpha 13 did not affect the activity of several known effector enzymes. The availability of purified r alpha 13 and knowledge of its biochemical properties will allow further characterization of its interactions with receptors and effectors.

To study signaling pathways regulated by alpha s and alpha i1 in renal epithelial cells, we expressed mutant, activated forms of alpha s and alpha i1 in a continuous proximal tubule cell line (MCT cells). alpha sQ227L increased cAMP production, and alpha ilQ204L reduced forskolin-sensitive cAMP production. alpha ilQ204L increased and alpha sQ227L decreased bradykinin-induced Ca influx across the cell membrane, but neither mutant affected bradykinin-stimulated intracellular Ca release or basal Ca influx. Bradykinin-stimulated Ca influx was reduced by dibutyryl cAMP, isoproterenol, and forskolin. Expression of a mutant regulatory type I subunit for cAMP-dependent protein kinase with reduced affinity for cAMP and treatment with KT-5720, a specific cAMP-dependent protein kinase inhibitor, enhanced Ca influx to a degree similar to that in cells expressing alpha ilQ204L. Bradykinin-stimulated c-fos mRNA expression is partially dependent on extracellular Ca. alpha sQ227L reduced and alpha ilQ204L enhanced bradykinin-stimulated c-fos expression. Consequently, in bradykinin-stimulated cells, the adenylyl cyclase system regulates Ca influx through cAMP-dependent protein kinase, but not intracellular Ca release. Furthermore, the Ca influx mechanism acts as an integrator of two signaling pathways such that Ca-dependent signals are damped by activators of adenylyl cyclase and enhanced by inhibitors of adenylyl cyclase.

Angiotensin II has been reported to stimulate the proximal tubule Na-H antiporter by inhibition of adenylyl cyclase, and possibly by an adenosine 3',5'-cyclic monophosphate (cAMP)-independent mechanism. We examined the effect of angiotensin II on Na-H antiporter activity (JNa-H) in opossum kidney (OKP) cells, a proximal tubule-like cell line, whose Na-H antiporter resembles that of the proximal tubule apical membrane. We found that angiotensin II regulates JNa-H in a concentration-dependent manner similar to the proximal tubule, with angiotensin II concentrations < 10(-8) M stimulating and > 10(-8) M inhibiting JNa-H. The stimulatory effect of angiotensin II was blocked by 10(-8) M losartan and was pertussis toxin sensitive, suggesting mediation through an angiotensin II (AT1) receptor coupled to a pertussis toxin-sensitive G protein. Acute treatment with 10(-4) M 8-bromoadenosine 3',5'-cyclic monophosphate (8-BrcAMP) inhibited JNa-H by 30% and blocked angiotensin II-induced stimulation. However, angiotensin II (10(-12)-10(-6) M) did not inhibit basal, dopamine-stimulated, or forskolin-stimulated cAMP production measured in the presence of 3-isobutyl-1-methylxanthine (IBMX). In addition, angiotensin II had no effect on cAMP levels measured in the absence of IBMX. We conclude that angiotensin II at physiological concentrations stimulates JNa-H in OKP cells via a cAMP-independent mechanism mediated by an AT1 receptor and a pertussis toxin-sensitive G protein.

Primary right atrial chondrosarcoma was diagnosed in a dog that had clinical signs of right-sided heart failure. The tumor was attached to the intra-atrial septum, dorsal to the septal leaf of the tricuspid valve. Tumor cells were not identified elsewhere.

This review focuses on studies from our laboratory investigating the mechanisms of chronic regulation of the Na/H antiporter in renal and nonrenal cells. Tissue culture provides an ideal tool for investigating this problem because it avoids many complicating effects that would occur in an intact animal during a chronic study. Chronic decreases in extracellular fluid pH cause an increase in Na/H antiporter activity that is dependent on protein synthesis and associated with an increase in NHE-1 (isoform of the sodium-hydrogen antiporter) mRNA abundance. This effect is associated with acid-induced increases in a number of immediate early genes, including c-fos, c-jun, junB, and egr-1. In primary cultures of rabbit proximal tubule cells, activation of protein kinase C for 2 hours causes an increase in Na/H antiporter activity that persists 24 hours later, is dependent on transcription and translation, and is associated with an increase in NHE-1 mRNA abundance. Chronic activation of protein kinase A in opossum kidney (OKP) cells causes an increase in Na/H antiporter activity that persists 16 to 20 hours later and is dependent on protein synthesis. This latter effect is of particular interest because it is opposite in direction to the acute inhibitory effect of protein kinase A on the Na/H antiporter in these cells.

Estrogen receptor (ER) immunocytochemical assay (ICA) can be a useful procedure in patients with breast cancer. The authors sought to study the utility of monoclonal antibody D75P3 gamma (D75) in paraffin-embedded sections of breast cancers.Sixty-seven cases of breast cancer were studied by ICA for ER using monoclonal antibody D75 in routinely processed formaldehyde-fixed paraffin-embedded tissue, employing ficin predigestion of sections before application of D75. The results were tabulated using a modified histochemical score (HS). In 59 cases, frozen sections of tumors were also studied with D75. In 40 cases, paraffin-embedded sections were stained with both D75 and monoclonal antibody H222 (which is commercially available). Quantitative biochemical ER assays were performed by the standard dextran-coated charcoal method.Statistical analysis of the data showed a highly significant correlation (P0.0001) between biochemical ER and paraffin-embedded section HS using D75. The results of frozen section assay and paraffin-embedded section assay using D75 were also highly correlated (P0.0001). In addition, paraffin-embedded section HS using D75 correlated well with paraffin-embedded section HS using H222 (P0.0001). However, D75 HS generally were higher than H222 HS because of the more intense staining with D75, and this difference was statistically significant (P0.0001). Assuming that the biochemical ER data were "true," paraffin-embedded section ICA with D75 had a diagnostic sensitivity of 100%, a diagnostic specificity of 70%, a positive predictive value of 89%, and a negative predictive value of 100%. The results of biochemical ER status and paraffin-embedded section D75 ICA ER status agreed in 61 of 67 cases (91%). In all six cases where the ER status differed, the biochemical ER status was negative, and the ER status by ICA with D75 was positive, leading the authors to suspect that some of these cases may have been biochemical false-negative findings.It was concluded that ICA for ER with D75 using a 2-minute ficin predigestion of formaldehyde-fixed paraffin-embedded tissue is an acceptable alternative to frozen section ICA. In addition, paraffin-embedded section ICA with D75 is easier to interpret than frozen section ICA, and the results are superior to ICA with H222 on paraffin-embedded sections.

Mechanical properties of cells and extracellular matrices are critical determinants of function in contexts including oncogenic transformation, neuronal synapse formation, hepatic fibrosis and stem cell differentiation. The size and heterogeneity of biological specimens and the importance of measuring their mechanical properties under conditions that resemble their environments in vivo present a challenge for quantitative measurement. Centimeter-scale tissue samples can be measured by commercial instruments, whereas properties at the subcellular (nm) scale are accessible by atomic force microscopy, optical trapping, or magnetic bead microrheometry; however many tissues are heterogeneous on a length scale between micrometers and millimeters which is not accessible to most current instrumentation. The device described here combines two commercially available technologies, a micronewton resolution force probe and a micromanipulator for probing soft biological samples at sub-millimeter spatial resolution. Several applications of the device are described. These include the first measurement of the stiffness of an intact, isolated mouse glomerulus, quantification of the inner wall stiffness of healthy and diseased mouse aortas, and evaluation of the lateral heterogeneity in the stiffness of mouse mammary glands and rat livers with correlation of this heterogeneity with malignant or fibrotic pathology as evaluated by histology.

The information that cells require to grow and prosper comes from diverse sources, adjacent to them and from considerable distances. Through evolution, cells have developed multiple mechanisms to acquire and process information. When a signaling mechanism has been successful, it has been used repeatedly with relatively minor modifications, and consequently is seen throughout biology. Specificity comes from using precise combinations of signaling molecules to achieve different ends in different cell types. Among the signaling systems that fit this description include the receptor and nonreceptor tyrosine kinases, G protein-coupled receptors, cell attachment receptors, guanylate cyclases, ligand-gated ion channels, the second messenger dependent and independent protein kinases and phosphatases, and transcription factors. The consequence of this conservation of molecular mechanisms is that many basic regulatory systems, which cannot be dissected free of the complexity of mammals, can be understood in relatively simple, manipulable experimental systems. With information from simple systems and the tools of genetics and molecular biology, complex problems such as human physiology and disease may be understood and eventually controlled.

A 9-month-old sexually intact male longhair cat was examined because of lethargy, anorexia, cold intolerance, and failure to thrive since acquisition at an early age. Clinical signs of disease were less pronounced when the cat was fed a low-protein diet. Anemia, hypoglycemia, low total CO2 content, and hyperammonemia were detected. The cat was euthanatized. Urine obtained immediately before euthanasia contained a large amount of methylmalonic acid. Total serum cobalamin concentration was low. Hepatic methylmalonic-CoA mutase activity, with and without the addition of coenzyme adenosylcobalamin, was consistent with a cobalamin deficiency. Methylmalonic acidemia secondary to a putative defect in cobalamin absorption was diagnosed.