Addition of doxycycline to ciprofloxacin for infection prophylaxis during autologous stem cell transplants for multiple myeloma.

BACKGROUND: The most commonly used antibacterial prophylaxis during autologous stem cell transplants (ASCT) for multiple myeloma (MM) involves a fluoroquinolone, such as ciprofloxacin or levofloxacin. We assessed the impact of adding doxycycline to ciprofloxacin as routine antibacterial prophylaxis in these patients. METHODS: We retrospectively reviewed electronic medical records and our ASCT database to analyze rates and types of bacterial infections in MM patients who underwent ASCT in our institution. RESULTS: Among 419 patients, 118 received ciprofloxacin alone (cipro group), and 301 ciprofloxacin and doxycycline (cipro-doxy group). Neutropenic fever (NF) developed in 63 (53%) and 108 (36%) patients of the cipro and cipro-doxy groups, respectively (p = 0.010). The number of documented bacteremic episodes was 13 (11%) and 14 (4.7%) in the two groups, respectively (p = 0.017). Antimicrobial resistance and Clostridium difficile infections were uncommon. Transplant-related mortality was 1% in both groups. CONCLUSIONS: The addition of doxycycline to standard prophylaxis with ciprofloxacin seems to reduce the number of NF episodes and documented bacterial infections in patients with MM undergoing ASCT, without increasing rate of serious complications.

Conceptualising production, productivity and technology in pharmacy practice: a novel framework for policy, education and research.

CONTEXT AND BACKGROUND: People and health systems worldwide face serious challenges due to shifting disease demographics, rising population demands and weaknesses in healthcare provision, including capacity shortages and lack of impact of healthcare services. These multiple challenges, linked with the global push to achieve universal health coverage, have made apparent the importance of investing in workforce development to improve population health and economic well-being. In relation to medicines, health systems face challenges in terms of access to needed medicines, optimising medicines use and reducing risk. In 2017, the International Pharmaceutical Federation (FIP) published global policy on workforce development ('the Nanjing Statements') that describe an envisioned future for professional education and training. The documents make clear that expanding the pharmacy workforce benefits patients, and continually improving education and training produces better clinical outcomes. AIMS AND PURPOSE: The opportunities for harnessing new technologies in pharmacy practice have been relatively ignored. This paper presents a conceptual framework for analysing production methods, productivity and technology in pharmacy practice that differentiates between dispensing and pharmaceutical care services. We outline a framework that may be employed to study the relationship between pharmacy practice and productivity, shaped by educational and technological inputs. METHOD AND RESULTS: The analysis is performed from the point of view of health systems economics. In relation to pharmaceutical care (patient-oriented practice), pharmacists are service providers; however, their primary purpose is not to deliver consultations, but to maximise the quantum of health gain they secure. Our analysis demonstrates that 'technology shock' is clearly beneficial compared with orthodox notions of productivity or incremental gain implementations. Additionally, the whole process of providing professional services using 'pharmaceutical care technologies' is governed by local institutional frames, suggesting that activities may be structured differently in different places and countries. DISCUSSION AND CONCLUSION: Addressing problems with medication use with the development of a pharmaceutical workforce that is sufficient in quantity and competence is a long-term issue. As a result of this analysis, there emerges a challenge about the profession's relationship with existing and emerging technical innovations. Our novel framework is designed to facilitate policy, education and research by providing an analytical approach to service delivery. By using this approach, the profession could develop examples of good practice in both developed and developing countries worldwide.

Unsuccessful treatment of methicillin-resistant Staphylococcus aureus endocarditis with dalbavancin.

WHAT IS KNOWN AND OBJECTIVE: Limited evidence describes dalbavancin use in infective endocarditis (IE). CASE DESCRIPTION: A 27-year-old pregnant female received 4 weeks of dalbavancin for methicillin-resistant Staphylococcus aureus (MRSA) bacteraemia and tricuspid valve IE after conventional therapy was no longer an option due to non-compliance. Despite having a smaller cardiac vegetation following dalbavancin, she was bacteraemic <2 weeks later with vancomycin-intermediate (VISA) and telavancin-non-susceptible S. aureus. WHAT IS NEW AND CONCLUSION: This is the first report of unsuccessful IE treatment with dalbavancin. Blood cultures grew VISA and lipoglycopeptide-non-susceptible S. aureus <2 weeks following dalbavancin. Both outcomes raise concerns about using dalbavancin for IE.

Effects of needs-based patient education on self-efficacy and health outcomes in people with rheumatoid arthritis: a multicentre, single blind, randomised controlled trial.

OBJECTIVES: The Educational Needs Assessment Tool (ENAT) is a self-completed questionnaire, which allows patients with arthritis to prioritise their educational needs. The aim of this study was to evaluate the effects of needs-based patient education on self-efficacy, health outcomes and patient knowledge in people with rheumatoid arthritis (RA). METHODS: Patients with RA were enrolled into this multicentre, single-blind, parallel-group, pragmatic randomised controlled trial. Patients were randomised to either the intervention group (IG) where patients completed ENAT, responses of which were used by the clinical nurse specialist to guide patient education; or control group (CG) in which they received patient education without the use of ENAT. Patients were seen at weeks 0, 16 and 32. The primary outcome was self-efficacy (Arthritis Self Efficacy Scale (ASES)-Pain and ASES-Other symptoms). Secondary outcomes were health status (short form of Arthritis Impact Measurement Scale 2, AIMS2-SF) and patient knowledge questionnaire-RA. We investigated between-group differences using analysis of covariance, adjusting for baseline variables. RESULTS: A total of 132 patients were recruited (IG=70 and CG=62). Their mean (SD) age was 54 (12.3) years, 56 (13.3)  years and disease duration 5.2 (4.9) years, 6.7 (8.9) years for IG and CG, respectively. There were significant between-group differences, in favour of IG at week 32 in the primary outcomes, ASES-Pain, mean difference (95% CI) -4.36 (1.17 to 7.55), t=-2.72, p=0.008 and ASES-Other symptoms, mean difference (95% CI) -5.84 (2.07 to 9.62), t=-3.07, p=0.003. In secondary outcomes, the between-group differences favoured IG in AIMS2-SF Symptoms and AIMS2-SF Affect. There were no between-group differences in other secondary outcomes. CONCLUSIONS: The results suggest that needs-based education helps improve patients' self-efficacy and some aspects of health status. TRIAL REGISTRATION NUMBER: ISRCTN51523281.

Papular acantholytic dyskeratosis of the vulva associated with familial Hailey-Hailey disease.

Papular acantholytic dyskeratosis (PAD) of the vulva is a rare, chronic disorder first described in 1984. It presents in young women as white to skin-coloured smooth papules over the vulva, which are persistent but asymptomatic. Histologically, there is hyperkeratosis and focal parakeratosis with acantholytic and dyskeratotic cells forming corps ronds and grains, placing PAD within Ackerman's spectrum of focal acantholytic dyskeratoses with Hailey-Hailey disease (HHD) and Darier disease. There have been 17 previous reports of PAD of the vulva, to our knowledge. Only one demonstrated a familial pattern, and none of the cases was associated with a family history of HHD. This is the first report of PAD and HHD in a single family, suggesting that PAD and HHD lie on a spectrum of disease and are genetically linked.

The effect of addition of liraglutide to high-dose intensive insulin therapy: a randomized prospective trial.

AIMS: Patients with type 2 diabetes and insulin resistance may require high insulin doses to control hyperglycaemia. The addition of glucagon-like peptide-1 receptor agonists (GLP-1 RAs) to basal insulin therapy has been shown to reduce insulin requirement while reducing insulin-associated weight gain [1,2]. The effect of GLP-1 RA therapy added to intensive (basal/bolus) insulin therapy has not been studied in a prospective trial. This trial evaluated the effect of the addition of liraglutide to high-dose intensive insulin therapy compared with standard insulin up-titration in obese insulin-resistant patients with type 2 diabetes requiring high-dose insulin therapy. METHODS: Thirty-seven subjects with type 2 diabetes requiring >100 units of insulin daily administered either by continuous subcutaneous insulin infusion (CSII) or by multiple daily injections (MDIs) with or without metformin were randomized to receive either liraglutide plus insulin (LIRA) or intensive insulin only (controls). Liraglutide was initiated at 0.6 mg subcutaneously (sq) per day and increased to either 1.2 or 1.8 mg daily in combination with intensive insulin therapy. Controls received intensive insulin up-titration only. RESULTS: At 6 months, subjects receiving liraglutide plus insulin experienced statistically significant reductions in HbA1c, weight, insulin dose and glycaemic variability (GV) by continuous glucose monitor (CGM) compared with the control group receiving insulin only. CONCLUSIONS: Adding liraglutide to intensive high-dose (basal/bolus) insulin therapy results in greater improvement in glycaemic control than insulin therapy alone, with additional benefits of weight loss and reduced GV.

Genetic diversity and population structure of a rare flowering tree endemic to Appalachia, Stewartia ovata.

Stewartia ovata (cav.) Weatherby, commonly known as mountain stewartia, is an understory tree native to the southeastern United States (U.S.). This relatively rare species occurs in isolated populations in Virginia, Kentucky, Tennessee, North Carolina, South Carolina, Georgia, Alabama, and Mississippi. As a species, S. ovata has largely been overlooked, and limited information is available regarding its ecology, which presents obstacles to conservation efforts. Stewartia ovata has vibrant, large white flowers that bloom in summer with a variety of filament colors, suggesting potential horticultural traits prized by ornamental industry. However, S. ovata is relatively slow growing and, due to long seed dormancy, propagation is challenging with limited success rates. This has created a need to assess the present genetic diversity in S. ovata populations to inform potential conservation and restoration of the species. Here, we employ a genotyping-by-sequencing (GBS) approach to characterize the spatial distribution and genetic diversity of S. ovata in the southern Appalachia region of the eastern United States. A total of 4475 single nucleotide polymorphisms (SNPs) were identified across 147 individuals from 11 collection sites. Our results indicate low genetic diversity (He = 0.216), the presence of population structure (K = 2), limited differentiation (F ST = 0.039), and high gene flow (Nm = 6.16) between our subpopulations. Principal component analysis corroborated the findings of STRUCTURE, confirming the presence of two distinct S. ovata subpopulations. One subpopulation mainly contains genotypes from the Cumberland Plateau, Tennessee, while the other consists of genotypes present in the Great Smoky Mountain ranges in Tennessee, North Carolina, and portions of Nantahala, Chattahoochee-Oconee national forests in Georgia, highlighting that elevation likely plays a major role in its distribution. Our results further suggested low inbreeding coefficient (F IS = 0.070), which is expected with an outcrossing tree species. This research further provides necessary insight into extant subpopulations and has generated valuable resources needed for conservation efforts of S. ovata.

Immunological regulation of experimental cutaneous leishmaniasis. III. Nature and significance of specific suppression of cell-mediated immunity in mice highly susceptible to Leishmania tropica.

BALB/c mice have been an exceptional susceptibility to Leishmania tropica infection such that cutaneous lesions grow without restraint in all cases leading to fatal metastasis and visceralization in normal and x-irradiated, bone-marrow reconstituted (XBM) animals. Adult thymectomized, x-irradiated, bone marrow-reconstituted (ATxXBM) BALB/c mice, however, show pronounced retardation of lesion growth leading to some survival and even cures. A similar trend was also found in moderately susceptible (BALB/c X C57BL/6)F1 mice, in contrast with the "resistant" CBA strain, in which, as previously known, ATxXBM animals showed impairment of normal, spontaneous self-healing. These convere effects are paralleled by respective leishmania-specific delayed-type hypersensitivity (DTH) reactivities, prior thymectomy leading to diminution in CBA and augmentation in BALB/c and (BALB/c X C57BL/6)F1. Anti-leishmanial DTH responses, amplfiable by cyclophosphamide pretreatment, can be detected in BALB/c mice within 10 d of infection with 2 X 10(7) promastigotes, but becomes near-totally suppressed by day 25-35. No such suppressin is found in CBA, C57BL/6, or (BALB/c X C57BL/6)F1 mice together with varying degrees of immune control of lesion development or regression. Suppression of DTH in BALB/c mice is leishmania specific and does not extent to 2,4-dinitrofluorobenzene (DNFB) or sheep erythrocytes specificities. Spleen cells from suppressed L. tropica-infected mice when transferred to normal BALB/c mice impaired the induction of DTH to leishmanial antigen. This property resided in the T cell-enriched fraction and not in the T cell-depleted fraction. It is concluded that a major component of the striking inability of BALB/c mice to control L. tropica infection involves profound impairment of a potentially curative cell-mediated immune response by suppressor T cell generation. The possibility is discussed that this may be secondary to rapid amastigote (antigen) accumulation in macrophages expressing the primary genetic "defect."

Immunological regulation of experimental cutaneous leishmaniasis. IV. Prophylactic effect of sublethal irradiation as a result of abrogation of suppressor T cell generation in mice genetically susceptible to Leishmania tropica.

The overwhelming susceptibility of BALB/c mice to infection with Leishmania tropica can be substantially reversed by immediately prior sub-lethal irradiation. This is related to radiation dosage, and at 550 rad, causes 60 percent complete cures and only 19 percent (instead of 100 percent) incidence of progressive disease. Irradiation 10 d before infection is only weakly prophylactic, whereas 10 d after is without effect. Control of lesion development is only apparent after the first 30 d, coincident with the analogous onset previously found in resistant strains and adult thymectomized, x-irradiated, bone marrow-reconstituted BALB/c mice. Instead of the specific suppression of DTH characteristic of L. tropica infection in the BALB/c strain, healed irradiated mice express strong anti-leishmanial DTH reactivity and resistance to reinfection. T cells from these mice transfer DTH reactivity which is suppressed by admixture with cells from nonhealed, nonreactive donors. Irradiated BALB/c mice again develop inexorable disease progression, after its transient arrest, when they are reconstituted with normal T cells. When the T cells are derived from uncontrollably-infected donors, the susceptibility regained is indistinguishable from that of normal mice. B cells do not modify the prophylactic effect of 550 rad, whereas T cells from healed mice confer strong protective immunity throughout the initial phase. Regression or progression of disease correlates completely with DTH reactivity in all these groups. Although BALB/c mice express an extreme level of genetic susceptibility to L. tropica infection, they are nevertheless capable of mounting a curative cell mediated immune response. That this is ineffective during pathogenesis of the disease was previously associated correlatively with potent specific suppressor T cell generation, which is now shown to be preventable by prior irradiation. Most important, however, a causal role for these cells in vivo has been demonstrated directly by reconstitution.

Time of the day for 11beta-HSD1 inhibition plays a role in improving glucose homeostasis in DIO mice.

AIMS: The physiological effects of glucocorticoids in a given tissue are driven by the local level of the active glucocorticoid, which is determined by two sources: the plasma cortisol in human (or corticosterone in rodents) and the cortisol produced locally through 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1) activity. Because of the circadian variation of plasma glucocorticoids, the pharmacological efficacy of 11beta-HSD1 inhibition may depend on the time of the day for inhibitor administration. METHODS: The circadian profile of corticosterone was established in lean and diet-induced obesity (DIO) C57BL/6 mice from blood collected at different time of the day. 11beta-HSD1 enzyme activity was also measured throughout the day in DIO mice. To determine the optimal timing for administration of an 11beta-HSD1 inhibitor to obtain maximum efficacy, we used a DIO mouse model and a small molecule inhibitor of 11beta-HSD1 from our thiazolinone series. Based on the circadian profile of corticosterone obtained, we administered the 11beta-HSD1 inhibitor to these animals at different times of the day and evaluated the effects on plasma glucose levels and glucose tolerance. RESULTS: We report that corticosterone circadian rhythm was similar between lean and DIO C57BL/6 mice, and 11beta-HSD1 enzyme activity undergoes minimal variations throughout the day. Interestingly, the compound exhibited maximum efficacy if dosed in the afternoon when plasma corticosterone is high; the morning dosing when plasma corticosterone is low did not lead to efficacy. CONCLUSION: These data suggest that because of the circadian rhythm of circulating glucocorticoids, the time of the day for 11beta-HSD1 inhibitor administration is important in achieving efficacy.

Antidiabetic effects of 11beta-HSD1 inhibition in a mouse model of combined diabetes, dyslipidaemia and atherosclerosis.

AIM: 11 beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1) is considered to contribute to the aetiology of the metabolic syndrome, and specific inhibitors have begun to emerge as treatments for insulin resistance and other facets of the syndrome, including atherosclerosis. Given the role of glucocorticoids and 11beta-HSD1 in the anti-inflammatory response and the involvement of inflammation in the development of atherosclerosis, 11beta-HSD1 inhibition may exacerbate atherosclerosis. Our aim was to investigate in vivo the effects of a specific 11beta-HSD1 inhibitor (2922) on atherosclerosis while assessing glucose homeostasis. METHODS: We conducted a 12-week study administering 2922 (at three doses, 3, 10 and 100 mg/kg body weight) in Ldlr 3KO (Ldlr(-/-)Apob(100/100)Lep(ob/ob)) mice, a genetic model of obesity, insulin resistance, dyslipidaemia and atherosclerosis. Rosiglitazone and simvastatin were used to test the responsiveness of our model in both types of therapy. RESULTS: 2922 was effective in reducing 11beta-HSD1 activity in inguinal adipose tissue (>90% for 100 mg/kg) and was efficacious in improving glucose homeostasis at doses > or =10 mg/kg. Plasma insulin, blood glucose, glucose tolerance and homeostatic model assessment indices were all improved in mice treated with 2922 (100 mg/kg) compared with control animals. Despite an improvement in these parameters, no differences were observed in body weight, adipose or lean tissue masses in the 2922-treated mice. Interestingly, circulating lipids, proinflammatory cytokines and atherosclerosis were unaltered in response to 2922, although a small reduction in LDL cholesterol was detected. CONCLUSIONS: Importantly, 11beta-HSD1 inhibition leads to improved glucose metabolism and does not result in a worsening of atherosclerotic lesion area, yet retained antidiabetic potential in the face of multiple severe metabolic aberrations. This study reinforces the potential use of 11beta-HSD1 inhibitors in patients with the metabolic syndrome without negatively impacting atherosclerosis.

Effect of an acidifying diet combined with zeolite and slight protein reduction on air emissions from laying hens of different ages.

The objectives of the study were to evaluate the effectiveness of a reduced-emission (RE) diet containing 6.9% of a CaSO(4)-zeolite mixture and slightly reduced CP to 21-, 38-, and 59-wk-old Hy-Line W-36 hens (trials 1, 2, and 3, respectively) on egg production and emissions of NH(3), H(2)S, NO, NO(2), CO(2), CH(4), and non-CH(4) total hydrocarbons as compared with feeding a commercial (CM) diet. At each age, 640 hens were allocated, randomly to 8 environmental chambers for a 3-wk period. On an analyzed basis, the CM diet contained 18.0, 17.0, and 16.2% CP and 0.25, 0.18, and 0.20% S in trials 1, 2, and 3, and the RE diet contained 17.0, 15.5, and 15.6% CP and 0.99, 1.20, and 1.10% S in trials 1, 2, and 3. Diets were formulated to contain similar Ca and P contents. Average daily egg weight (56.3 g), average daily egg production (81%), average daily feed intake (92.4 g), and BW change (23.5 g), across ages, were unaffected by diet (P > 0.05) over the study period. Age effects were observed for all performance variables and NH(3) emissions (P < 0.05). In trials 1, 2, and 3, daily NH(3) emissions from hens fed the RE diets (185.5, 312.2, and 333.5 mg/bird) were less than emissions from hens fed the CM diet (255.1, 560.6, and 616.3 mg/bird; P < 0.01). Daily emissions of H(2)S across trials from hens fed the RE diet were 4.08 mg/bird compared with 1.32 mg/bird from hens fed the CM diet (P < 0.01). Diet (P < 0.05) and age (P < 0.05) affected emissions of CO(2) and CH(4). A diet effect (P < 0.01) on NO emissions was observed. No diet or age effects (P > 0.05) were observed for NO(2) or non-CH(4) total hydrocarbons. Results demonstrated that diet and layer age influence air emissions from poultry operations.

Nutrient digestibility and mass balance in laying hens fed a commercial or acidifying diet.

The objectives of the current study were to evaluate the effect of an acidifying diet (gypsum) combined with zeolite and slightly reduced crude protein (R) vs. a control diet (C) on nutrient retention in laying hens and compare 3 approaches to estimating nutrient excretion from hens: 1) mass balance calculation (feed nutrients - egg nutrient), 2) use of an indigestible marker with analyzed feed and excreta nutrient content, and 3) an environmental chamber that allowed for capturing all excreted and volatilized nutrients. Hens (n = 640) were allocated randomly to 8 environmental chambers for 3-wk periods. Excreta samples were collected at the end of each trial to estimate apparent retention of N, S, P, and Ca. No diet effects on apparent retention of N were observed (53.44%, P > 0.05). Apparent retention of S, P, and Ca decreased in hens fed R diet (18.7, - 11.4, and 22.6%, respectively) compared with hens fed the C diet (40.7, 0.3, and 28.6%, respectively; P < 0.05). Total N excretion from hens fed the C and R diet was not different (1.16 g/hen/d); however, mass of chamber N remaining in excreta following the 3-wk period was less from hens fed the C diet (1.27 kg) than from hens fed the R diet (1.43 kg). Gaseous emissions of NH(3) over the 3-wk period from hens fed the C diet (0.74 kg per chamber) were greater than emissions from hens fed the R diet (0.45 kg). The 3-wk S excretion mass (estimated using the calculation, indigestible marker, and environmental chamber methods, respectively) was greater from hens fed the R diet (1.85, 1.54, and 1.27 kg, respectively) compared with hens fed the C diet (0.24, 0.20, and 0.14 kg, respectively). The 3-wk P excretion was similar between diets (0.68 kg). Results demonstrate that feeding the acidified diet resulted in decreased N emissions, but because of the acidulant fed, greatly increased S excretion and emissions.

Hyperhaploidy is a novel high-risk cytogenetic subgroup in multiple myeloma.

Hyperhaploid clones (24-34 chromosomes) were identified in 33 patients with multiple myeloma (MM), demonstrating a novel numerical cytogenetic subgroup. Strikingly, all hyperhaploid karyotypes were found to harbor monosomy 17p, the single most important risk stratification lesion in MM. A catastrophic loss of nearly a haploid set of chromosomes results in disomies of chromosomes 3, 5, 7, 9, 11, 15, 18, 19 and 21, the same basic set of odd-numbered chromosomes found in trisomy in hyperdiploid myeloma. All other autosomes are found in monosomy, resulting in additional clinically relevant monosomies of 1p, 6q, 13q and 16q. Hypotriploid subclones (58-68 chromosomes) were also identified in 11 of the 33 patients and represent a duplication of the hyperhaploid clone. Analysis of clones utilizing interphase fluorescence in situ hybridization (iFISH), metaphase FISH and spectral karyotyping identified either monosomy 17 or del17p in all patients. Amplification of 1q21 was identified in eight patients, demonstrating an additional high-risk marker. Importantly, our findings indicate that current iFISH strategies may be uninformative or ambiguous in the detection of these clones, suggesting this patient subgroup maybe underreported. Overall survival for patients with hyperhaploid clones was poor, with a 5-year survival rate of 23.1%. These findings identify a distinct numerical subgroup with cytogenetically defined high-risk disease.

Purification of a basic fibroblast growth factor-binding proteoglycan from bovine cardiac plasma membrane.

A heparan sulfate proteoglycan (HSPG) from bovine cardiac plasma membrane was purified to homogeneity using either isoelectric focusing or anion-exchange chromatography, followed by affinity chromatography on immobilized basic fibroblast growth factor (bFGF). Fractions were assayed for bFGF-binding activity using 125I-bFGF as a probe. Purified proteoglycan ran as a broad band on SDS-PAGE, spanning an apparent molecular mass range of 100-200 kDa, and could be incorporated into liposomes. Digestion of radioiodinated proteoglycan with heparitinase yielded a product of 73 kDa, while digestion with chondroitinase ABC did not change the apparent molecular mass. Monoclonal antibody directed against the ectodomain of another plasma membrane HSPG, syndecan, failed to recognize the purified cardiac proteoglycan on immunoblots. We conclude that adult bovine myocardium contains a membrane-associated bFGF-binding heparan sulfate proteoglycan containing little or no chondroitin sulfate and that this HSPG may be distinct from those of the syndecan family of heparan sulfate proteoglycans.

Characterization of cardiac Na+/Ca(2+)-exchange by site-directed polyclonal antibodies.

Cardiac Na+/Ca(2+)-exchange is an integral membrane protein consisting of approx. 970 amino acids with as many as 12 membrane-spanning and 11 extramembranal regions (Nicoll, D.A., Lognoni, S. and Philipson, K.D. (1985) Science 250, 562-565). Based upon primary sequence information, 3 amino-acid sequences located in either extramembranal segment a or f, consisting largely of acidic amino-acids, were selected for the production of synthetic peptides. The peptides were cross-linked to carrier ovalbumin and used to generate site-directed polyclonal antibodies (sd-Ab). Western blot analysis of bovine cardiac sarcolemmal (SL) proteins demonstrated that sd-Ab against segment a and 1 against loop f recognized a 70 kDa protein and a lower molecular mass band at 55 kDa under reducing conditions. A different loop f sd-Ab failed to recognize the 70 kDa protein but did associate with a 120, 65 and 55 kDa protein under the same conditions. Under non-reducing conditions, antibodies to all three peptides recognized the 65 kDa protein. All sd-Ab were blocked by addition of their respective peptides and were not inhibited by either of the other peptides. A sd-Ab against loop f was immobilized to an affinity support matrix and used to immunoprecipitate detergent solubilized cardiac SL vesicle protein. Immunoprecipitated protein was reconstituted into proteoliposomes which demonstrated Na+/Ca(2+)-exchange activity. Immunoprecipitated protein cross-reacted with sd-Ab against all three peptides with bands at 120, 70 and 55 kDa on Western blots. Tryptic digests of native SL vesicles abolished recognition of segment a sd-Ab for SL proteins while having little or no affect on reactivity to the protein by both sd-Ab against loop f. Digestion of the SL vesicle protein with endoproteinase Arg C did not alter sd-Ab recognition. The results suggest that specific domains of the cardiac Na+/Ca(2+)-exchanger depending upon the conformation of the protein, may not be available for antibody binding. The 70 kDa polypeptide appears to include the N-terminal region of the protein and what is believed to be a large cytoplasmic extramembranal loop. Limited proteolysis by trypsin and endoproteinase Arg C yielded results consistent with the model which places the N-terminus of the protein on the extracellular surface and a large extramembranal segment (loop f) on the cytoplasmic side of the SL membrane.

Kinetic characterization and radiation-target sizing of the glucose transporter in cardiac sarcolemmal vesicles.

Stereospecific glucose transport was assayed and characterized in bovine cardiac sarcolemmal vesicles. Sarcolemmal vesicles were incubated with D-[3H]glucose or L-[3H]glucose at 25 degrees C. The reaction was terminated by rapid addition of 4 mM HgCl2 and vesicles were immediately collected on glass fiber filters for quantification of accumulated [3H]glucose. Non-specific diffusion of L-[3H]glucose was never more than 11% of total D-[3H]glucose transport into the vesicles. Stereospecific uptake of D-[3H]glucose reached a maximum level by 20 s. Cytochalasin B (50 microM) inhibited specific transport of D-[3H]glucose to the level of that for non-specific diffusion. The vesicles exhibited saturable transport (Km = 9.3 mM; Vmax = 2.6 nmol/mg per s) and the transporter turnover number was 197 glucose molecules per transporter per s. The molecular sizes of the cytochalasin B binding protein and the D-glucose transport protein in sarcolemmal vesicles were estimated by radiation inactivation. These values were 77 and 101 kDa, respectively, and by the Wilcoxen Rank Sum Test were not significantly different from each other.

Metabolic activation of AMP kinase in vascular smooth muscle.

AMP-activated kinase (AMPK) is a highly conserved heterotrimeric kinase that functions as a metabolic master switch to coordinate cellular enzymes involved in carbohydrate and fat metabolism that regulate ATP conservation and synthesis. AMPK is activated by conditions that increase AMP-to-ATP ratio, such as exercise and metabolic stress. In the present study, we probed whether AMPK was expressed in vascular smooth muscle and would be activated by metabolic stress. Endothelium-denuded porcine carotid artery segments were metabolically challenged with 2-deoxyglucose (10 mM) plus N(2) (N(2)-2DG). These vessels exhibited a rapid increase in AMPK activity by 1 min that was near maximal by 20 min. AMPK inactivation on return to normal physiological saline was approximately 50% in 1 min and fully recovered by 5 min. Immunoprecipitation of the alpha(1)- and alpha(2)-catalytic subunit followed by immunoblot analysis for [P]Thr(172)-AMPK indicates that alpha(1)-AMPK accounts for all activity. Little if any alpha(2)-AMPK was detected in carotid smooth muscle. AMPK activity was not increased by contractile agonist (endothelin-1) or by the reported AMPK activators 5-aminoimidazole-4-carboxamide ribofuranoside (2 mM), metformin (2 mM), or phenformin (0.2 mM). AMPK activation by N(2)-2DG was associated with a rapid and pronounced reduction in endothelin-induced force and reduced phosphorylation of Akt and Erk 1/2. These data demonstrate that AMPK expression differs in vascular smooth muscle compared with striated muscles and that activation and inactivation after metabolic stress occur rapidly and are associated with signaling pathways that may regulate smooth-muscle contraction.

Conditional-ready mouse embryonic stem cell derived macrophages enable the study of essential genes in macrophage function.

The ability to differentiate genetically modified mouse embryonic stem (ES) cells into functional macrophages provides a potentially attractive resource to study host-pathogen interactions without the need for animal experimentation. This is particularly useful in instances where the gene of interest is essential and a knockout mouse is not available. Here we differentiated mouse ES cells into macrophages in vitro and showed, through a combination of flow cytometry, microscopic imaging, and RNA-Seq, that ES cell-derived macrophages responded to S. Typhimurium, in a comparable manner to mouse bone marrow derived macrophages. We constructed a homozygous mutant mouse ES cell line in the Traf2 gene that is known to play a role in tumour necrosis factor-α signalling but has not been studied for its role in infections or response to Toll-like receptor agonists. Interestingly, traf2-deficient macrophages produced reduced levels of inflammatory cytokines in response to lipopolysaccharide (LPS) or flagellin stimulation and exhibited increased susceptibility to S. Typhimurium infection.

Plasmalogen and anionic phospholipid dependence of the cardiac sarcolemmal sodium-calcium exchanger.

Although plasmalogens are the predominant phospholipids of cardiac sarcolemma, their physiological role has not been forthcoming. Since the cardiac sarcolemmal sodium-calcium exchanger has been proposed to be regulated by anionic phospholipids, the roles of plasmalogens and anionic phospholipids as regulators of the sodium-calcium exchanger were explored. Reconstituted sodium-calcium exchange activity in plasmalogen-containing proteoliposomes was 10-fold higher than that in control proteoliposomes comprised of only diacyl phospholipids. Additionally, exchange activity in plasmalogen-containing proteoliposomes was regulated by anionic phospholipids. Thus, plasmalogens provide a critical lipid environment in which anionic phospholipids serve as boundary lipids for the regulation of the trans-sarcolemmal sodium-calcium exchanger.