Sensitivity of C-Reactive Protein and Procalcitonin Measured by Point-of-Care Tests to Diagnose Urinary Tract Infections in Nursing Home Residents: A Cross-Sectional Study.

BACKGROUND: Diagnosing urinary tract infections (UTIs) in nursing home residents is complex, as specific urinary symptoms are often absent and asymptomatic bacteriuria (ASB) is prevalent. The aim of this study was to assess the sensitivity of blood C-reactive protein (CRP) and procalcitonin (PCT), measured by point-of-care tests (PoCTs), to diagnose UTIs in this setting. METHODS: Elderly residents (≥65 years old) with a suspected UTI were recruited from psychogeriatric, somatic, or rehabilitation wards across 13 participating nursing homes. CRP and PCT were tested simultaneously in the same study participants. To assess the tests' sensitivities, a stringent definition of "true" UTI was used that included the presence of symptoms, urinary leucocytes, a positive urine culture, and symptom resolution during antibiotic treatment covering isolated uropathogen(s). The original sample size was 440 suspected UTI episodes, in order to detect a clinically relevant sensitivity of at least 65% when calculated using the matched analysis approach to compare both PoCTs. RESULTS: After enrollment of 302 episodes (68.6% of the planned sample size), an unplanned and funder-mandated interim analysis was done, resulting in premature discontinuation of the study for futility. For 247 of 266 eligible episodes, all mandatory items required for the true UTI definition (92.9%) were available. In total, 49 episodes fulfilled our stringent UTI definition (19.8%). The sensitivities of CRP (cut-off, 6.5 mg/L) and PCT (cut-off, 0.025 ng/mL) were 52.3% (95% confidence interval [CI], 36.7-67.5%) and 37.0% (95% CI, 23.2-52.5%), respectively. CONCLUSIONS: Our results indicate that CRP and PCT are not suitable tests for distinguishing UTI and ASB in nursing home residents. CLINICAL TRIALS REGISTRATION: Netherlands Trial Registry NL6293.

Impact of a vancomycin loading dose on the achievement of target vancomycin exposure in the first 24 h and on the accompanying risk of nephrotoxicity in critically ill patients.

BACKGROUND: The advocated pharmacokinetic/pharmacodynamic (PK/PD) target for vancomycin, AUC/MIC ≥ 400 mg·h/L, may not be reached with a conventional fixed starting dose of 1000 mg in critically ill patients, but increasing the dose may cause nephrotoxicity. OBJECTIVES: To evaluate the effect of a weight-based loading dose of 25 mg/kg vancomycin on PK/PD target attainment in the first 24 h (AUC0-24) in critically ill patients and to evaluate whether this increases the risk of acute kidney injury (AKI). PATIENTS AND METHODS: A prospective observational before/after study was performed in ICU patients, comparing the percentage of vancomycin courses with AUC0-24 ≥ 400 mg·h/L and the incidence of AKI, defined as worsening of the risk, injury, failure, loss of kidney function and end-stage kidney disease (RIFLE) score. The conventional dose group received 1000 mg of vancomycin as initial dose; the loading dose group received a weight-based loading dose of 25 mg/kg. A population PK model developed using non-linear mixed-effects modelling was used to estimate AUC0-24 in all patients. RESULTS: One hundred and four courses from 82 patients were included. With a loading dose, the percentage of courses achieving AUC0-24 ≥ 400 mg·h/L increased significantly from 53.8% to 88.0% (P = 0.0006). The percentage of patients with new-onset AKI was not significantly higher when receiving a 25 mg/kg loading dose (28.6% versus 37.8%; P = 0.48). However, the risk of AKI was significantly higher in patients achieving AUC0-24 > 400 mg·h/L compared with patients achieving AUC < 400 mg·h/L (39.0% versus 14.8%; P = 0.031). CONCLUSIONS: A weight-based loading dose of 25 mg/kg vancomycin led to significantly more patients achieving AUC0-24 ≥ 400 mg·h/L without increased risk of AKI. However, some harm cannot be ruled out since higher exposure was associated with increased risk of AKI.

"A false sense of confidence" The perceived role of inflammatory point-of-care testing in managing urinary tract infections in Dutch nursing homes: a qualitative study.

BACKGROUND: Diagnosing urinary tract infections (UTI) in nursing home residents is complex, due to frequent non-specific symptomatology and asymptomatic bacteriuria. The objective of this study was to explore health care professionals' perceptions of the proposed use of inflammatory marker Point-Of-Care Testing (POCT) in this respect. METHODS: We conducted a qualitative inquiry (2018-2019) alongside the multicenter PROGRESS study (NL6293), which assessed the sensitivity of C-reactive protein and procalcitonin POCT in UTI. We used semi-structured face-to-face interviews. The participants were physicians (n = 12) and nurses (n = 6) from 13 nursing homes in the Netherlands. Most respondents were not familiar with inflammatory marker POCT, while some used POCT for respiratory tract infections. Both the interview guide and the analysis of the interview transcripts were based on the Consolidated Framework for Implementation Research. RESULTS: All respondents acknowledged that sufficiently sensitive POCT could decrease diagnostic uncertainty to some extent in residents presenting with non-specific symptoms. They primarily thought that negative test results would rule out UTI and justify withholding antibiotic treatment. Secondly, they described how positive test results could rule in UTI and justify antimicrobial treatment. However, most respondents also expected new diagnostic uncertainties to arise. Firstly, in case of negative test results, they were not sure how to deal with residents' persisting non-specific symptoms. Secondly, in case of positive test results, they feared overlooking infections other than UTI. These new uncertainties could lead to inappropriate antibiotics use. Therefore, POCT was thought to create a false sense of confidence. CONCLUSIONS: Our study suggests that inflammatory marker POCT will only improve UTI management in nursing homes to some extent. To realize the expected added value, any implementation of POCT requires thorough guidance to ensure appropriate use. Developing UTI markers with high negative and positive predictive values may offer greater potential to improve UTI management in nursing homes.

Changes in microbiota during experimental human Rhinovirus infection.

BACKGROUND: Human Rhinovirus (HRV) is responsible for the majority of common colds and is frequently accompanied by secondary bacterial infections through poorly understood mechanisms. We investigated the effects of experimental human HRV serotype 16 infection on the upper respiratory tract microbiota. METHODS: Six healthy volunteers were infected with HRV16. We performed 16S ribosomal RNA-targeted pyrosequencing on throat swabs taken prior, during and after infection. We compared overall community diversity, phylogenetic structure of the ecosystem and relative abundances of the different bacteria between time points. RESULTS: During acute infection strong trends towards increases in the relative abundances of Haemophilus parainfluenzae and Neisseria subflava were observed, as well as a weaker trend towards increases of Staphylococcus aureus. No major differences were observed between day-1 and day 60, whereas differences between subjects were very high. CONCLUSIONS: HRV16 infection is associated with the increase of three genera known to be associated with secondary infections following HRV infections. The observed changes of upper respiratory tract microbiota could help explain why HRV infection predisposes to bacterial otitis media, sinusitis and pneumonia.

Preparedness for admission of patients with suspected Ebola virus disease in European hospitals: a survey, August-September 2014.

In response to the Ebola virus disease (EVD) outbreak in West Africa, the World Health Organization has advised all nations to prepare for the detection, investigation and management of confirmed and suspected EVD cases in order to prevent further spread through international travel. To gain insights into the state of preparedness of European hospitals, an electronic survey was circulated in August­September 2014 to 984 medical professionals representing 736 hospitals in 40 countries. The survey addressed the willingness and capacity to admit patients with suspected EVD as well as specific preparedness activities in response to the current Ebola crisis. Evaluable responses were received from representatives of 254 (32%) hospitals in 38 countries, mostly tertiary care centres, of which 46% indicated that they would admit patients with suspected EVD. Patient transfer agreements were in place for the majority of hospitals that would not admit patients. Compared with non-admitting hospitals, admitting hospitals were more frequently engaged in various preparedness activities and more often contained basic infrastructural characteristics such as admission rooms and laboratories considered important for infection control, but some gaps and concerns were also identified. The results of this survey help to provide direction towards further preparedness activities and prioritisation thereof.

Evaluation of the antiviral response to zanamivir administered intravenously for treatment of critically ill patients with pandemic influenza A (H1N1) infection.

A retrospective nationwide study on the use of intravenous (IV) zanamivir in patients receiving intensive care who were pretreated with oseltamivir in the Netherlands was performed. In 6 of 13 patients with a sustained reduction of the viral load, the median time to start IV zanamivir was 9 days (range, 4-11 days) compared with 14 days (range, 6-21 days) in 7 patients without viral load reduction (P = .052). Viral load response did not influence mortality. We conclude that IV zanamivir as late add-on therapy has limited effectiveness. The effect of an immediate start with IV zanamivir monotherapy or in combination with other drugs need to be evaluated.

Risk of SARS-CoV-2 transmission upon return to work in RNA-positive healthcare workers.

BACKGROUND: Healthcare workers (HCWs) are at risk for coronavirus disease 2019 (COVID-19), and for spreading severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) amongst colleagues and patients. AIM: To study the presence of SARS-CoV-2 RNA and possible onward transmission by HCWs upon return to work after COVID-19, and association with disease severity and development of antibodies over time. METHODS: Unvaccinated HCWs with positive SARS-CoV-2 reverse transcriptase polymerase chain reaction (RT-PCR) were recruited prospectively. Data on symptoms were collected via telephone questionnaires on days 2, 7, 14 and 21 after a positive test. Upon return to work, repeat SARS-CoV-2 RT-PCR was performed and serum was collected. Repeat serum samples were collected at weeks 4, 8, 12 and 16 to determine antibody dynamics over time. Phylogenetic analysis was conducted to investigate possible transmission events originating from HCWs with a positive repeat RT-PCR. FINDINGS: Sixty-one (84.7%) participants with mild/moderate COVID-19 had a repeat SARS-CoV-2 RT-PCR performed upon return to work (median 13 days after symptom onset), of which 30 (49.1%) were positive with a median cycle threshold (Ct) value of 29.2 (IQR 26.9-29.9). All HCWs developed antibodies against SARS-CoV-2. No significant differences in symptomatology and presence of antibodies were found between repeat RT-PCR-positive and -negative HCWs. Eleven direct colleagues of six participants with a repeat RT-PCR Ct value <30 tested positive after the HCW returned to work. Phylogenetic and epidemiologic analysis did not indicate onward transmission through HCWs who were SARS-CoV-2 RNA positive upon return to work. CONCLUSIONS: HCWs regularly return to work with substantial SARS-CoV-2 RNA loads. However, this study found no evidence for subsequent in-hospital transmission.

mTOR-driven glycolysis governs induction of innate immune responses by bronchial epithelial cells exposed to the bacterial component flagellin.

Human bronchial epithelial (HBE) cells play an essential role during bacterial infections of the airways by sensing pathogens and orchestrating protective immune responses. We here sought to determine which metabolic pathways are utilized by HBE cells to mount innate immune responses upon exposure to a relevant bacterial agonist. Stimulation of HBE cells by the bacterial component flagellin triggered activation of the mTOR pathway resulting in an increased glycolytic flux that sustained the secretory activity of immune mediators by HBE cells. The mTOR inhibitor rapamycin impeded glycolysis and limited flagellin-induced secretion of immune mediators. The role of the mTOR pathway was recapitulated in vivo in a mouse model of flagellin-triggered lung innate immune responses. These data demonstrate that metabolic reprogramming via the mTOR pathway modulates activation of the respiratory epithelium, identifying mTOR as a potential therapeutic target to modulate mucosal immunity in the context of bacterial infections.

Longitudinal sampling is required to maximize detection of intrahost A/H3N2 virus variants.

Seasonal human influenza viruses continually change antigenically to escape from neutralizing antibodies. It remains unclear how genetic variation in the intrahost virus population and selection at the level of individual hosts translates to the fast-paced evolution observed at the global level because emerging intrahost antigenic variants are rarely detected. We tracked intrahost variants in the hemagglutinin and neuraminidase surface proteins using longitudinally collected samples from 52 patients infected by A/H3N2 influenza virus, mostly young children, who received oseltamivir treatment. We identified emerging putative antigenic variants and oseltamivir-resistant variants, most of which remained detectable in samples collected at subsequent days, and identified variants that emerged intrahost immediately prior to increases in global rates. In contrast to most putative antigenic variants, oseltamivir-resistant variants rapidly increased to high frequencies in the virus population. Importantly, the majority of putative antigenic variants and oseltamivir-resistant variants were first detectable four or more days after onset of symptoms or start of treatment, respectively. Our observations demonstrate that de novo variants emerge, and may be positively selected, during the course of infection. Additionally, based on the 4-7 days post-treatment delay in emergence of oseltamivir-resistant variants in six out of the eight individuals with such variants, we find that limiting sample collection for routine surveillance and diagnostic testing to early timepoints after onset of symptoms can potentially preclude detection of emerging, positively selected variants.

Fluoroquinolone resistance detection in Mycobacterium tuberculosis with locked nucleic acid probe real-time PCR.

SETTING: Pham Ngoc Thach Hospital for Tuberculosis and Lung Diseases, Ho Chi Minh City, Vietnam. OBJECTIVE: Fluoroquinolones (FQs) are increasingly used in the treatment of tuberculosis (TB) and are the second-line drugs of choice for treatment of multidrug-resistant TB. We aimed to set up a polymerase chain reaction (PCR) based assay to detect the most common FQ-resistance-associated mutations in gyrase A (gyrA) of Mycobacterium tuberculosis. DESIGN: A total of 42 FQ-resistant and 40 FQ-susceptible isolates were collected in 2005-2006 and sequenced in gyrA. Using sequencing results as gold standard, a real-time PCR using three locked nucleic acid probes (LNA-PCR) was designed to detect mutations at positions 90, 91 and 94 (97% of gyrA FQ-resistance-associated mutations) and evaluated. RESULTS: Sequencing of 42 FQ-resistant isolates revealed no gyrA mutations in 10 isolates, 20 isolates had a single mutation and 12 isolates showed double peaks at resistance-associated alleles, suggesting a heterogeneous population. With LNA-PCR, all wild-type and 19/20 mutant isolates were correctly identified. Eleven of 12 heterogeneous isolates were correctly identified as resistant mutants. Overall, 71% ([19 + 11]/42) of phenotypically FQ-resistant isolates were detected. Specificity was 100% on 40 FQ-susceptible isolates. CONCLUSION: This assay provides a simple and rapid means to reliably detect FQ-resistance-associated gyrA mutations in M. tuberculosis.

Selective inhibition of syncytium-inducing and nonsyncytium-inducing HIV-1 variants in individuals receiving didanosine or zidovudine, respectively.

By studying changes in the clonal composition of HIV-1 populations during the first weeks of zidovudine (ZDV) treatment before the development of ZDV resistance-conferring mutations, we demonstrated previously a selective inhibition of nonsyncytium-inducing (NSI) HIV-1, even when present as coexisting population in individuals also harboring syncytium-inducing (SI) HIV-1. In this study, we observed the opposite in individuals receiving didanosine (ddI) treatment. In these individuals (n = 7) a median -0.98 log change (range -1.55-0.08) in infectious cellular SI load was observed, whereas the coexisting NSI load was only minimally affected (median -0.15 log, range -1.27-0.50; P = 0.03). The virus phenotype-dependent treatment responses were independent of the clonal composition of HIV-1 populations at baseline. Individuals treated with a combination of ZDV and ddI revealed an equal decline of both NSI and SI infectious cellular load (n = 4; NSI: median -1.55 log, range -2.19 to -1.45; SI: median -1.47 log, range -1.81 to -0.86; P = 0.56). To test the hypothesis that the previously reported optimal activation of ZDV and ddI in activated and resting T cells, respectively, in combination with the differential T cell tropism of NSI and SI HIV-1 is the basis for the observed virus phenotype specific efficacy of nucleoside analogs, we studied the effect of treatment with a protease inhibitor that does not require intracellular activation. Individuals receiving ritonavir (n = 4) indeed showed equal declines in NSI and SI infectious cellular load (NSI: median -2.37 log, range -2.59 to -2.16; SI: median -2.82 log, range -3.14 to -2.50; P = 0.25). Our data suggest HIV-1 phenotype as an additional parameter in the design of optimal treatment regimens.

Interleukin-10 and soluble tumor necrosis factor receptor II are potential biomarkers of Plasmodium falciparum infections in pregnant women: a case-control study from Nanoro, Burkina Faso.

BACKGROUND: Diagnosis of malaria in pregnancy is problematic due to the low sensitivity of conventional diagnostic tests (rapid diagnostic test and microscopy), which is exacerbated due to low peripheral parasite densities, and lack of clinical symptoms. In this study, six potential biomarkers to support malaria diagnosis in pregnancy were evaluated. METHODS: Blood samples were collected from pregnant women at antenatal clinic visits and at delivery. Microscopy and real-time PCR were performed for malaria diagnosis and biomarker analyses were performed by ELISA (interleukin 10, IL-10; tumor necrosis factor-α, TNF-α; soluble tumor necrosis factor receptor II, sTNF-RII; soluble fms-like tyrosine kinase 1, sFlt-1; leptin and apolipoprotein B, Apo-B). A placental biopsy was collected at delivery to determine placental malaria. RESULTS: IL-10 and sTNF-RII were significantly higher at all time-points in malaria-infected women (p < 0.001). Both markers were also positively associated with parasite density (p < 0.001 and p = 0.003 for IL-10 and sTNF-RII respectively). IL-10 levels at delivery, but not during pregnancy, were negatively associated with birth weight. A prediction model was created using IL-10 and sTNF-RII cut-off points. For primigravidae the model had a sensitivity of 88.9% (95%CI 45.7-98.7%) and specificity of 83.3% (95% CI 57.1-94.9%) for diagnosing malaria during pregnancy. For secundi- and multigravidae the sensitivity (81.8% and 56.5% respectively) was lower, while specificity (100.0% and 94.3% respectively) was relatively high. Sub-microscopic infections were detected in 2 out of 3 secundi- and 5 out of 12 multigravidae. CONCLUSIONS: The combination of biomarkers IL-10 and sTNF-RII have the potential to support malaria diagnosis in pregnancy. Additional markers may be needed to increase sensitivity and specificity, this is of particular importance in populations with sub-microscopic infections or in whom other inflammatory diseases are prevalent.

Companion Animals as a Source of Viruses for Human Beings and Food Production Animals.

Companion animals comprise a wide variety of species, including dogs, cats, horses, ferrets, guinea pigs, reptiles, birds and ornamental fish, as well as food production animal species, such as domestic pigs, kept as companion animals. Despite their prominent place in human society, little is known about the role of companion animals as sources of viruses for people and food production animals. Therefore, we reviewed the literature for accounts of infections of companion animals by zoonotic viruses and viruses of food production animals, and prioritized these viruses in terms of human health and economic importance. In total, 138 virus species reportedly capable of infecting companion animals were of concern for human and food production animal health: 59 of these viruses were infectious for human beings, 135 were infectious for food production mammals and birds, and 22 were infectious for food production fishes. Viruses of highest concern for human health included hantaviruses, Tahyna virus, rabies virus, West Nile virus, tick-borne encephalitis virus, Crimean-Congo haemorrhagic fever virus, Aichi virus, European bat lyssavirus, hepatitis E virus, cowpox virus, G5 rotavirus, influenza A virus and lymphocytic choriomeningitis virus. Viruses of highest concern for food production mammals and birds included bluetongue virus, African swine fever virus, foot-and-mouth disease virus, lumpy skin disease virus, Rift Valley fever virus, porcine circovirus, classical swine fever virus, equine herpesvirus 9, peste des petits ruminants virus and equine infectious anaemia virus. Viruses of highest concern for food production fishes included cyprinid herpesvirus 3 (koi herpesvirus), viral haemorrhagic septicaemia virus and infectious pancreatic necrosis virus. Of particular concern as sources of zoonotic or food production animal viruses were domestic carnivores, rodents and food production animals kept as companion animals. The current list of viruses provides an objective basis for more in-depth analysis of the risk of companion animals as sources of viruses for human and food production animal health.

A molecular epidemiological perspective of rhinovirus types circulating in Amsterdam from 2007 to 2012.

Rhinoviruses (RVs) are frequently detected respiratory viruses that cause mild common cold symptoms, but may also lead to more severe respiratory tract infections. The large number of RV types, classified into species A, B and C, hampers clear insights into the epidemiology and clinical significance of each RV type. The aim of this study was to map the circulation of RV types in the Amsterdam area. RV-positive nasopharyngeal and oropharyngeal samples, collected from 2007 to 2012 in the Academic Medical Centre (Amsterdam, the Netherlands), were typed based on the sequence of the region coding for capsid proteins VP4 and VP2. RV-A, RV-B and RV-C were found in proportions of of 52.4% (334/637), 11.3% (72/637), and 36.2% (231/637), respectively. We detected 129 of the 167 currently classified types. RVs circulated throughout the entire year with a peak in the autumn and a decline in the summer. Some RV types were observed throughout the entire sampling period and others had a more seasonal pattern. Nine RV-A and four RV-B novel provisionally assigned types were identified. This study provides an insight into the molecular epidemiology of RVs in the Amsterdam area. The RVs circulating are diverse and include several provisionally new types.

Dissociation between Ca2+-ATPase and alkaline phosphatase activities in plasma membranes of rat duodenum.

The presence of Ca2+-ATPase activities with high-affinity sites for Ca2+ in brush border as well as basolateral plasma membranes of rat duodenal epithelium has been reported previously (Ghijsen, W.E.J.M. and van Os, C.H. (1979) Nature 279, 802-803). Since both plasma membranes contain alkaline phosphatase (EC 3.1.3.1), which also can be stimulated by Ca2+, the substrate specificity of Ca2+-induced ATP-hydrolysis has been studied to determine whether or not alkaline phosphatase and Ca2+-ATPase are two distinct enzymes. In basolateral fragments, the rate of Ca2+-dependent ATP-hydrolysis was greater than that of ADP, AMP and p-nitrophenylphosphate at Ca2+ concentrations below 25 muM. At 0.2 mM Ca2+ the rates of ATP, ADO, AMO and p-nitrophenylphosphate hydrolysis were not significantly different. In brush border fragments the rates of ATP, ADP and AMP hydrolysis were identical at low Ca2+, but at 0.2 mM Ca2+, Ca2+-induced hydrolysis of ADO and AMO was greater than either ATP or p-nitrophenylphosphate. Alkaline phsophatase in brush border and basolateral membranes was inhibited by 75% after addition of 2.5 mM theophylline. Ca2+-stimulated ATP hydrolysis at 1 muM Ca2+ was not sensitive to theophylline in basolateral fragments while the same activity in brush border fragments was totally inhibited. At 0.2 mM Ca2+, Ca2+-induced ATP hydrolysis in both basolateral and brush border membranes was sensitive to theophylline. Oligomycin and azide had no effect on Ca2+-stimulated ATP hydrolysis, either at low or at high Ca2+ concentrations. Chlorpromazine fully inhibited Ca2+-stimulated ATP hydrolysis in basolateral fragments at 5 muM Ca2+, while it had no effect in brush border fragments. From these results we conclude that, (i) Ca2+-ATPase and alkaline phosphatase are two distinct enzymes, (ii) high-affinity Ca2+-ATPase is exclusively located in basolateral plasma membranes, (iii) alkaline phosphatase activity, present on both sides of duodenal epithelium is stimulated slightly by low Ca2+ concentrations, but this Ca2+-induced activity is inhibited by theophylline and shows no specificity with respect to ATP, ADP or AMP.

Effects of dimethylsulfoxide and mercurial sulfhydryl reagents on water and solute permeability of rat kidney brush border membranes.

The effects of dimethylsulfoxide, DMSO, and mercurial sulfhydryl reagents have been studied on water and small solute permeability of rat renal brush border membrane vesicles. Water and solute permeability was measured by mixing membrane vesicles with hypertonic solutions in a stopped-flow apparatus and following osmotically-induced changes in vesicular volume via changes in scattered light intensity. The rate constant of the fast osmotic shrinkage is proportional to the osmotic water permeability, while the rate constant of the slow reswelling phase is proportional to the solute permeability. Using mannitol as the osmotic agent, the osmotic shrinkage of rat renal brush border membrane vesicles followed a biphasic time course. 80% of the vesicles shrunk with a rate constant of approx. 50 s-1 and 20% with a rate constant of approx. 2 s-1. DMSO decreased dose-dependently the amplitude of the fast osmotic shrinkage, without affecting its rate constant. In contrast to DMSO, HgCl2 decreased the rate constant but not the amplitude of the fast osmotic shrinkage of renal brush border vesicles. Between 40-50 microM HgCl2, the inhibition of the fast osmotic shrinkage was completed. DMSO and HgCl2 increase the activation energy of water permeation in renal membranes from 3 to 12-15 kcal/mol. DMSO and HgCl2 did not affect the rate constant of the slow osmotic shrinkage of renal membrane vesicles and were also without effect on osmotic shrinkage of small intestinal brush border and pure phospholipid vesicles. In renal brush border membranes, HgCl2 at low concentrations (less than 10 microM) increased by 15-fold the permeability to NaCl and urea but not to mannitol, an effect which precedes the inhibition of water permeability at higher HgCl2 concentrations. The increase in small solute permeability was irreversible while the inhibition of water permeability could be reversed with cysteine and dithiothreitol. We conclude that water and small solute pathways in rat renal brush border membranes are completely separate entities, which are effected differently by DMSO and HgCl2. These pathways for water and solutes must be membrane proteins since neither DMSO nor HgCl2 affect the permeability properties of pure phospholipid vesicles.

ATP-dependent calcium transport and its correlation with Ca2+ -ATPase activity in basolateral plasma membranes of rat duodenum.

Isolated basolateral plasma membrane vesicles from rat duodenum epithelial cells exhibit ATP-dependent calcium-accumulation and Ca2+ -dependent ATPase activity. Calcium accumulation stimulated by ATP is prevented by the calcium ionophore A23187, inhibited 80% by 0.1 mM orthovanadate but is not effected by oligomycin. Calcium accumulation is not observed with the substrate beta-gamma-(CH2)-ATP, ADP and p-nitrophenyl phosphate. Kinetic studies reveal an apparent Km of 0.2 microM Ca2+ and a Vmax of 5.3 nmol Ca2+/min per mg protein for the ATP-dependent calcium-uptake system. Calmodulin and phenothiazines have no effect on calcium accumulation in freshly prepared membranes, but small effects are inducible after a wash with a 5 mM EGTA. The kinetic parameters of Ca2+ -ATPase are: Km = 0.25 microM Ca2+ and Vmax = 19.2 nmol Pi/min per mg protein. Three techniques, osmotic shock, treatment with Triton X-100 or the channel-forming peptide alamethicin, reveal that about 40% of the vesicles are resealed. Assuming that half of the resealed vesicles have an inside-out orientation, the Vmax of ATP-dependent calcium uptake amounts to 25 nmol Ca2+/min per mg protein and of the Ca2+ -ATPase to 23 nmol Pi/min per mg protein. The close correlation between kinetic parameters of Ca2+ -ATPase and ATP-dependent calcium-transport strongly suggests that both systems are expressions of a Ca2+ -pump located in duodenal basolateral plasma membranes.

Kinetic properties of Na+/Ca2+ exchange in basolateral plasma membranes of rat small intestine.

The presence of an Na+/Ca2+ exchange system in basolateral plasma membranes from rat small intestinal epithelium has been demonstrated by studying Na+ gradient-dependent Ca2+ uptake and the inhibition of ATP-dependent Ca2+ accumulation by Na+. The presence of 75 mM Na+ in the uptake solution reduces ATP-dependent Ca2+ transport by 45%, despite the fact that Na+ does not affect Ca2+-ATPase activity. Preincubation of the membrane vesicles with ouabain or monensin reduces the Na+ inhibition of ATP-dependent Ca2+ uptake to 20%, apparently by preventing accumulation of Na+ in the vesicles realized by the Na+-pump.l It was concluded that high intravesicular Na+ competes with Ca2+ from intravesicular Ca2+ binding sites. In the presence of ouabain, the inhibition of ATP-dependent Ca2+ transport shows a sigmoidal dependence on the Na+ concentration, suggesting cooperative interaction between counter transport of at least two sodium ions for one calcium ion. The apparent affinity for Na+ is between 15 and 20 mM. Uptake of Ca2+ in the absence of ATP can be enhanced by an Na+ gradient (Na+ inside greater than Na+ outside). This Na+ gradient-dependent Ca2+ uptake is further stimulated by an inside positive membrane potential but abolished by monensin. The apparent affinity for Ca2+ of this system is below 1 microM. In contrast to the ATP-dependent Ca2+ transport, there is no significant difference in Na+ gradient-dependent Ca2+ uptake between basolateral vesicles from duodenum, midjejunum and terminal ileum. In duodenum the activity of ATP-driven Ca2+ uptake is 5-times greater than the greater than the Na+/Ca2+ exchange capacity but in the ileum both systems are of equal potency. Furthermore, the Na+/Ca2+ exchange mechanism is not subject to regulation by 1 alpha, 25-dihydroxy vitamin D-3, since repletion of vitamin D-deficient rats with this seco-steroid hormone does not influence the Na+/Ca2+ exchange system while it doubles the ATP-driven Ca2+ pump activity.

Separation of basolateral plasma membranes from smooth endoplasmic reticulum of the rat enterocyte by zonal electrophoresis on density gradients.

Basolateral plasma membranes of rat small intestinal epithelium were purified by density gradient centrifugation followed by zonal electrophoresis on density gradients. Crude basolateral membranes were obtained by centrifugation in which the marker enzyme, (Na+ + K+)-ATPase, was enriched 10-fold with respect to the initial homogenate. The major contaminant was a membrane fraction derived from smooth endoplasmic reticulum, rich in NADPH-cytochrome c reductase activity. The crude basolateral membrane preparation could be resolved into the two major components by subjecting it to zonal electrophoresis on density gradients. The result was that (Na+ + K+)-ATPase was purified 22-fold with respect to the initial homogenate. Purification with respect to mitochondria and brush border membranes was 35- and 42-fold, respectively. Resolution of (Na+ + K+)-ATPase from NADPH-cytochrome c reductase by electrophoresis was best with membrane material from adult rats between 180 and 250 g. No resolution between the two marker enzymes occurred with material from young rats of 125 to 140 g. These results demonstrate that zonal electrophoresis on density gradients, a simple and inexpensive technique, has a similar potential to free-flow electrophoresis.