A comparative hospital-based observational study of mono- and co-infections of malaria, dengue virus and scrub typhus causing acute undifferentiated fever.

Positive serology for dengue and/or scrub typhus infection with/without positive malarial smear (designated as mixed or co-infection) is being increasingly observed during epidemics of acute undifferentiated febrile illnesses (AUFIs). We planned to study the clinical and biochemical spectrum of co-infections with Plasmodium sp., dengue virus and scrub typhus and compare these with mono-infection by the same organisms. During the period from December 2012 to December 2013, all cases presenting with AUFIs to a single medical unit of a referral centre in Garhwal region of the north Indian state of Uttarakhand were retrospectively selected and categorised aetiologically as co-infections, malaria, dengue or scrub typhus. The groups thus created were compared in terms of demographic, clinical, biochemical and outcome parameters. The co-infection group (n = 49) was associated with milder clinical manifestations, fewer, milder and non-progressive organ dysfunction, and lesser need for intensive care, mechanical ventilation and dialysis as compared to mono-infections. When co-infections were sub-grouped and compared with the relevant mono-infections, there were differences in certain haematological and biochemical parameters; however, this difference did not translate into differential outcomes. Scrub typhus mono-infection was associated with severe disease in terms of both morbidity and mortality. Malaria, dengue and scrub typhus should be routinely tested in all patients with AUFIs. Co-infections, whether true or due to serological cross-reactivity, appear to be a separate entity so far as presentation and morbidity is concerned. Further insight is needed into the mechanism and identification of the protective infection.

Stylomandibular tunnel widening versus narrowing: a useful tool in evaluating suprahyoid mass lesions.

AIM: To evaluate whether qualitative and quantitative assessments of stylomandibular tunnel asymmetry are useful in lesion localization and differentiation. MATERIALS AND METHODS: The stylomandibular tunnel was measured in 60 control patients at CT to determine normal side-to-side variation. Twenty-one patients in the study group with suprahyoid neck masses were divided into two subgroups, those with widening and those with narrowing of the pathological side. Surgical and pathological findings in these subgroups were compared for site of origin and histology. RESULTS: Stylomandibular tunnel diameters in the control group had a mean variation of 0.9 mm (range: 0-3 mm, SD: 0.83 mm). Two-tailed t-test yielded a p-value of 0.018 for a variation of 3 mm and this was chosen as the threshold for disease. The widened stylomandibular tunnel group all had parotid gland lesions extending into the pre-styloid parapharyngeal space. The narrowed stylomandibular tunnel group had adenopathy, schwannomas, and paragangliomas/glomus vagale tumours arising from the post-styloid parapharyngeal space. CONCLUSION: Qualitative assessment for asymmetry of the stylomandibular tunnel surpass the 3 mm threshold for pathology. Widening of the stylomandibular tunnel is primarily from deep lobe parotid lesions extending into the pre-styloid parapharyngeal space. Narrowing of the stylomandibular tunnel can be from adenopathy, schwannomas, and paragangliomas arising from the post-styloid parapharyngeal space.

Contribution of medical colleges to tuberculosis control in India under the Revised National Tuberculosis Control Programme (RNTCP): lessons learnt & challenges ahead.

Medical college faculty, who are academicians are seldom directly involved in the implementation of national public health programmes. More than a decade ago for the first time in the global history of tuberculosis (TB) control, medical colleges of India were involved in the Revised National TB Control Programme (RNTCP) of Government of India (GOI). This report documents the unique and extraordinary course of events that led to the involvement of medical colleges in the RNTCP of GOI. It also reports the contributions made by the medical colleges to TB control in India. For more than a decade, medical colleges have been providing diagnostic services (Designated Microscopy Centres), treatment [Directly Observed Treatment (DOT) Centres] referral for treatment, recording and reporting data, carrying out advocacy for RNTCP and conducting operational research relevant to RNTCP. Medical colleges are contributing to diagnosis and treatment of human immunodeficiency virus (HIV)-TB co-infection and development of laboratory infrastructure for early diagnosis of multidrug-resistant and/or extensively drug-resistant TB (M/XDR-TB) and DOTS-Plus sites for treatment of MDR-TB cases. Overall, at a national level, medical colleges have contributed to 25 per cent of TB suspects referred for diagnosis; 23 per cent of 'new smear-positives' diagnosed; 7 per cent of DOT provision within medical college; and 86 per cent treatment success rate among new smear-positive patients. As the Programme widens its scope, future challenges include sustenance of this contribution and facilitating universal access to quality TB care; greater involvement in operational research relevant to the Programme needs; and better co-ordination mechanisms between district, state, zonal and national level to encourage their involvement.

Developing leadership interventions for black and minority ethnic staff: A case study of the National Health Service (NHS) in the U.K.

PURPOSE: The National Health Service (NHS) is the largest employer in the U.K. but, despite decades of equal opportunities legislation, its senior management workforce does not reflect the diversity of either the wider NHS workforce or the U.K. population. The aim of the paper is to consider the range of management interventions available to organisations like the NHS to deliver change in the area of promotion of Black and minority ethnic staff. DESIGN/METHODOLOGY/APPROACH: Intervention programmes in a range of public and private organisations are reviewed and the nature of barriers to promotion and the range of interventions to overcome these are explored. The paper uses the paradigm of institutional racism to examine the ways in which the NHS discriminates against certain sections of its workforce. The methods used include a literature review combined with key stakeholder interviews. A comparative dimension which involved a review of research on leadership initiatives in the U.S.A. was also undertaken. FINDINGS: The literature review found that there were a range of initiatives which could be implemented by public organisations such as the NHS to increase the presence of Black and Minority Ethnic (BME) staff in senior management positions. Most of these interventions were largely focused on the individual. Much more progress on institutional or organisational change needed to be made before the NHS could be perceived as a model employer in this area. The literature review also indicated that there is little published research on such initiatives within other European Union countries. ORIGINALITY/VALUE: The paper is targeted at both policy makers and human resource officers responsible for equality and diversity issues within large organisations, who have a remit to improve the career pathways of staff. The analysis provided offers a set of critical tools and interventions that have not hitherto been well examined in the U.K. context.

Emergence of G12 rotavirus strains in Delhi, India, in 2000 to 2007.

The prospect that rotavirus diarrhea in children may soon be prevented by vaccines has placed a new priority on understanding the diversity of rotavirus strains and the mechanism by which these strains evolve over time. We have characterized a total of 465 rotavirus strains collected in North India from 2000 to 2007 for G and P types by reverse transcription-PCR and sequencing. The novel G12 rotavirus strains recently detected in other countries were first detected in India in 2001 and have emerged as the predominant strains in Delhi, India, during 2005 to 2007. While the VP7 sequence was highly homologous among G12 strains isolated in Delhi, suggesting recent emergence from a common ancestor, the strains had a diverse constellation of other gene segments, demonstrating substantial reassortment. For the entire period, the common rotavirus G types G1 (26%), G2 (25%), and G9 (14%) comprised 65% of the strains, and common P types, P[4] (19%), P[6] (22%), and P[8] (35%), comprised 76% of the total P types. Of note, we detected a high percentage of unusual (17%) strains and fecal specimens with mixed (12% G and 15% P) rotavirus infections having a variety of genomic constellations. For the first time, we identified two novel rotavirus strains with unusual G/P combinations, G2P[11] and G3P[11], in patients with diarrhea. The study highlights the great diversity among rotaviruses isolated from Indian children, the opportunity for genetic reassortment between strains, and the emergence of a novel G12 strain in our country. Due to the demonstrated effect of antigenic diversity on rotavirus vaccines, it will be important to continue careful monitoring of these strains as rotavirus vaccine programs are implemented in India.

Gadolinium DTPA Enhancement Characteristics of the Rat Sciatic Nerve after Crush Injury at 4.7T.

BACKGROUND AND PURPOSE: Traumatic peripheral nerve injury is common and results in loss of function and/or neuropathic pain. MR neurography is a well-established technique for evaluating peripheral nerve anatomy and pathology. However, the Gd-DTPA enhancement characteristics of acutely injured peripheral nerves have not been fully examined. This study was performed to determine whether acutely crushed rat sciatic nerves demonstrate Gd-DTPA enhancement and, if so, to evaluate whether enhancement is affected by crush severity. MATERIALS AND METHODS: In 26 rats, the sciatic nerve was crushed with either surgical forceps (6- to 20-N compressive force) or a microvascular/microaneurysm clip (0.1-0.6 N). Animals were longitudinally imaged at 4.7T for up to 30 days after injury. T1WI, T2WI, and T1WI with Gd-DTPA were performed. RESULTS: Forceps crush injury caused robust enhancement between days 3 and 21, while clip crush injury resulted in minimal-to-no enhancement. Enhancement after forceps injury peaked at 7 days and was seen a few millimeters proximal to, in the region of, and several centimeters distal to the site of crush injury. Enhancement after forceps injury was statistically significant compared with clip injury between days 3 and 7 (P < .04). CONCLUSIONS: Gd-DTPA enhancement of peripheral nerves may only occur above a certain crush-severity threshold. This phenomenon may explain the intermittent observation of Gd-DTPA enhancement of peripheral nerves after traumatic injury. The observation of enhancement may be useful in judging the severity of injury after nerve trauma.

Prospective research on infants with mild encephalopathy: the PRIME study.

OBJECTIVE: To determine short-term outcomes of infants with evidence of hypoxia-ischemia at birth and classified as mild neonatal encephalopathy (NE) at <6 h of age. STUDY DESIGN: Prospective multicenter study. Mild NE was defined as ⩾1 abnormal category in modified Sarnat score. Primary outcome was any abnormality on early amplitude integrated electroencephalogram (aEEG) or seizures, abnormal brain magnetic resonance imaging (MRI) or neurological exam at discharge. RESULTS: A total of 54/63 (86%) of enrolled infants had data on components of the primary outcome, which was abnormal in 28/54 (52%): discontinuous aEEG (n=4), MRI (n=9) and discharge exam (n=22). Abnormal tone and/or incomplete Moro were the most common findings. MRI abnormalities were confined to cerebral cortex but two infants had basal ganglia and/or thalamus involvement. The 18 to 24 months follow-up is ongoing. CONCLUSIONS: A larger than expected proportion of mild NE infants with abnormal outcomes was observed. Future research should evaluate safety and efficacy of neuroprotection for mild NE.

Multi-minicore disease: a rare form of myopathy.

BACKGROUND: Multi-minicore disease is a rare form of myopathy characterized by slowly progressive or nonprogressive muscle weakness and characteristic multiple cores within the muscle fibers. To the best of our knowledge, this is first documentation of the clinicopathological features of this rare entity from India. MATERIALS AND METHODS: A ll cases of multi-minicore disease diagnosed in our laboratory were retrieved. Clinical and pathological features were reviewed. RESULT: During a period of two years (January 2004 to December 2005), we received 985 muscle biopsies for various reasons. Of which, 15 were diagnosed as myopathies and four of which were of multi-minicore disease. Thus, multi-minicore disease comprises 0.40% of all muscle diseases and 26.6% of all myopathies. All were male and presented in early childhood (first decade of life) with generalized hypotonia and muscle weakness. All of them had dysmorphic facies and three had high arched palate. CPK levels were normal and EMG was myopathic except in one patient. Microscopic examination revealed minimal changes with Type I fibers' predominance but characteristic multiple cores in the myofibers. Ultrastructural examination showed both structured and unstructured cores. CONCLUSIONS: Multi-minicore disease, although a rare form of myopathies, should be suspected in children who present with generalized hypotonia and slowly progressive muscle weakness along with dysmorphic facies.

Nemaline rod myopathy: a rare form of myopathy.

Nemaline rod myopathy (NM) is a rare form of congenital myopathy characterized by slowly progressive or nonprogressive muscle weakness and pathognomonic rod-like structures within the muscle fibers. To the best of our knowledge, this is first documentation of the clinicopathological features of this rare entity from India. All cases of NM diagnosed in our laboratory were retrieved. Clinical and pathological features were reviewed. During a period of 1.5 years (Jan 2004 to June 2005), we received 750 muscle biopsies for various reasons. Of which, 15 were diagnosed as congenital myopathies and four as nemaline rod myopathies. Thus, NM comprises 0.53% of all muscle diseases and 22.6% of all congenital myopathies. All of them presented in childhood (first five years of life) with generalized hypotonia, feeding problems, repeated respiratory infections and muscle weakness. Both males and females were equally affected. The CPK levels were normal and EMG was myopathic. Microscopic examination revealed minimal changes but characteristic red-colored material was seen on modified Gomori trichrome staining which was immunopositive to alpha actinin. Ultrastructural examination confirmed this material to be nemaline rods. NM, although a rare form of congenital myopathies, should be suspected in children who present with generalized hypotonia, repeated chest infections and slowly progressive muscle weakness. This report highlights the importance of histochemistry and ultrastructural examination in the diagnosis of this entity, in the absence of the availability of methodology for genetic studies.

Awareness about taeniasis and neurocysticercosis among municipal schoolteachers in Delhi.

Taenia solium is the commonest parasitic infection of CNS and an important cause of new-onset seizures and epilepsy in children and adults. Human activities impact on almost every one of the stages of the lifecycle of the worm as man is responsible for dispersion of the parasite's egg through outdoor defecation and indiscriminate disposal of feces. Health education to cause behavioral changes in these practices can therefore be an effective intervention strategy. We conducted a study to assess KAP regarding taeniasis and neurocysticercosis among municipal school teachers in Delhi. The findings are presented in this communication. The study revealed that, general information related to personal food hygiene was known to majority of the teachers but core information in the context of taeniasis/cysticercosis and seizure prevention was lacking.

Evaluation for Blunt Cerebrovascular Injury: Review of the Literature and a Cost-Effectiveness Analysis.

BACKGROUND AND PURPOSE: Evaluation for blunt cerebrovascular injury has generated immense controversy with wide variations in recommendations regarding the need for evaluation and the optimal imaging technique. We review the literature and determine the most cost-effective strategy for evaluating blunt cerebrovascular injury in trauma patients. MATERIALS AND METHODS: A comprehensive literature review was performed with data extracted to create a decision-tree analysis for 5 different strategies: anticoagulation for high-risk (based on the Denver screening criteria) patients, selective DSA or CTA (only high-risk patients), and DSA or CTA for all trauma patients. The economic evaluation was based on a health care payer perspective during a 1-year horizon. Statistical analyses were performed. The cost-effectiveness was compared through 2 main indicators: the incremental cost-effectiveness ratio and net monetary benefit. RESULTS: Selective anticoagulation in high-risk patients was shown to be the most cost-effective strategy, with the lowest cost and greatest effectiveness (an average cost of $21.08 and average quality-adjusted life year of 0.7231). Selective CTA has comparable utility and only a slightly higher cost (an average cost of $48.84 and average quality-adjusted life year of 0.7229). DSA, whether performed selectively or for all patients, was not optimal from both the cost and utility perspectives. Sensitivity analyses demonstrated these results to be robust for a wide range of parameter values. CONCLUSIONS: Selective CTA in high-risk patients is the optimal and cost-effective imaging strategy. It remains the dominant strategy over DSA, even assuming a low CTA sensitivity and irrespective of the proportion of patients at high-risk and the incidence of blunt cerebrovascular injury in high-risk patients.

Interaction of lanthanide cations and uranyl ion with the calcium/proton antiport system in Mycobacterium phlei.

Uranyl ions (UO2+(2)) and lanthanide cations (La3+, Nd3+, Sm3+, Eu3+, Tb3+ and Dy3+) at 100-200 microM concentration inhibited active transport of Ca2+, mediated by respiratory linked substrates as well as by ATP hydrolysis, without affecting respiration and membrane-bound ATPase activity, in inside-out membrane vesicles of Mycobacterium phlei. The extent of inhibition in the uptake of Ca2+, mediated by ATP hydrolysis, increased with increase in ionic radii of these cations. Lanthanide cations did not dissipate the formation of a proton gradient, as measured by determining the effect either on the uptake of [14C]methylamine or energy-linked quenching of the fluorescence of 9-aminoacridine. However, uranyl ion (UO2+(2+)) caused reversal of the energy-linked quenching of 9-aminoacridine. UO2+(2)) concentration yielding 50% of Vmax (S0.5) was approx. 15 microM. Kinetic studies revealed that inhibition in the uptake of Ca2+ was competitive with UO2+(2) while non-competitive with rare-earth metals. It is proposed that inhibition in the uptake of Ca2+ by uranyl ion occurs as a result of UO2+(2) transport into the interior of vesicles in exchange for protons, while lanthanide cations are not being transported but affect the binding of Ca2+ to the membrane, presumably to the Ca2+/H+ antiporter.

Purification and functional properties of the DCCD-reactive proteolipid subunit of the H+-translocating ATPase from Mycobacterium phlei.

Interaction of N,N'-dicyclohexylcarbodiimide (DCCD) with ATPase of Mycobacterium phlei membranes results in inactivation of ATPase activity. The rate of inactivation of ATPase was pseudo-first order for the initial 30-65% inactivation over a concentration range of 5-50 microM DCCD. The second-order rate constant of the DCCD-ATPase interaction was k = 8.5 X 10(5) M-1 X min(-1). The correlation between the initial binding of [14C]DCCD and 100% inactivation of ATPase activity shows 1.57 nmol DCCD bound per mg membrane protein. The proteolipid subunit of the F0F1-ATPase complex in membranes of M. phlei with which DCCD covalently reacts to inhibit ATPase was isolated by labeling with [14C]DCCD. The proteolipid was purified from the membrane in free and DCCD-modified form by extraction with chloroform/methanol and subsequent chromatography on Sephadex LH-20. The polypeptide was homogeneous on SDS-acrylamide gel electrophoresis and has an apparent molecular weight of 8000. The purified proteolipid contains phosphatidylinositol (67%), phosphatidylethanolamine (18%) and cardiolipin (8%). Amino acid analysis indicates that glycine, alanine and leucine were present in elevated amounts, resulting in a polarity of 27%. Cysteine and tryptophan were lacking. Butanol-extracted proteolipid mediated the translocation of protons across the bilayer, in K+-loaded reconstituted liposomes, in response to a membrane potential difference induced by valinomycin. The proton translocation was inhibited by DCCD, as measured by the quenching of fluorescence of 9-aminoacridine. Studies show that vanadate inhibits the proton gradient driven by ATP hydrolysis in membrane vesicles of M. phlei by interacting with the proteolipid subunit sector of the F0F1-ATPase complex.

Abnormal activation of sterol synthesis in lymphocytes of atherosclerotic-susceptible pigeons and heterozygous familial hypercholesterolemic patients.

Delipidated human sera enhances the incorporation of [2-14C]acetate, but not mevalonate, into digitonin-precipitable sterols of pigeon lymphocytes. Show Racer and White Carneau pigeons exhibit inherited differences in induction of sterol synthesis dissociated from inheritance of hypercholesterolemia. Moreover, lymphocytes of three familial hypercholesterolemic individuals, having serum cholesterol level in the normal range by drug therapy, exhibited a higher activation of sterol synthesis by delipidated sera when compared to cells of normal individuals. It is suggested that genetic abnormality in lymphocytes of familial hypercholesterolemic can be dissociated from hypercholesterolemia.

Studies on the mechanism of action of local anesthetics on proton translocating ATPase from Mycobacterium phlei.

We have measured the inhibitory potencies of local anesthetics (procaine, lidocaine, tetracaine and dibucaine) on ATP-mediated H+-translocation, Ca2+-transport and ATPase activity in membrane vesicles from Mycobacterium phlei. Procaine and lidocaine up to 1 mM concentration did not inhibit ATP-dependent H+-translocation, Ca2+-transport and ATPase activity. However, tetracaine and dibucaine at 0.2 mM concentration caused dissipation of the proton gradient, measured by the reversal of the quenching of fluorescence of quinacrine, and inhibition of active Ca2+-transport. Tetracaine (1 mM) inhibited membrane-bound ATPase activity without affecting solubilized F1-ATPase activity. Studies show that these local anesthetics do not prevent the inactivation of F0-F1 ATPase by dicyclohexylcarbodiimide (DCCD). Binding of [14C]DCCD to F0-proteolipid component remained unchanged in the presence of tetracaine indicating that DCCD and tetracaine do not share common binding sites on the F0-proteolipid sector. The inhibition of H+-translocation and membrane-bound ATPase activity by tetracaine was substantially additive in the presence of vanadate.

Fusion of human erythrocytes induced by uranyl acetate and rare earth metals.

Incubation of human erythrocytes with either uranyl ions (UO22+) or rare earth metals (La3+, Nd3+, Sm3+, Eu3+, Tb3+, Dy3+ and Yb3+) at 37 degrees C for 30-45 min resulted in the fusion of erythrocytes. Redistribution of membrane-associated particles was observed using colloidal-iron charge labelling and freeze-fracture electron microscopy. The fusion of erythrocytes induced by these agents, unlike Ca2+, did not exhibit the absolute requirement for phosphate. Moreover, agglutination and fusion by these agents was observed in neuraminidase-treated erythrocytes in contrast to Ca2+- and phosphate-induced fusion. Inhibitors of intrinsic transglutaminase activity partially inhibited (35-45%) the fusion induced by UO22+ suggesting that cross-linking of membrane proteins results in protein-free areas of lipid where fusion may be initiated.

Photoaffinity labeling of procaine-binding sites in normal and sickle cell membranes.

A photoaffinity probe, procaine azide, was employed to determine the sites of interaction of procaine in normal and sickle cell erythrocytes. Studies show that the number of binding sites and affinity of procaine to membranes derived from normal and sickled cell erythrocytes were similar, although procaine retards the in vitro formation of irreversibly sickled cells from cells, The results show that procaine azide, a photoaffinity analogue of procaine, is covalently incorporated into both protein (60--70%) and lipid (40--30%) components of the membrane. Sodium dodecyl sulfate-gel electrophoresis of the labeled ghosts show that procaine binds specifically to band 3 and periodic acid Schiff staining bands in membranes derived from labeled erythrocytes. Binding of procaine or covalent incorporation of procaine azide into membrane proteins does not affect the phosphate transport. Moreover, pre treatment of intact erythrocytes with 4,4-'diisothiocyano-2,2'-stilbene disulfonate, an anion transport inhibitor, did not affect either the binding or covalent incorporationof procaine azide into erythrocytes. These results indicate that the binding of procaine azide to Band 3 protein occurs at a locus different than that involved in anion translocation process.

Phospholipid methylation of kidney cortex brush border membranes. Effect on fluidity and transport.

Exposure of intact brush border membrane vesicles of hog kidney cortex to cholesterol oxidase resulted in 24% oxidation of membrane cholesterol compared with more than 95% oxidation of cholesterol in lipids isolated from membranes, showing that cholesterol is asymmetrically distributed in membranes. Phospholipase C, hydrolyzed 76% of phosphatidylcholine and 10-12% phosphatidylethanolamine while phosphatidylserine was not hydrolyzed, thus indicating that majority of phosphatidylcholine is present on the outer surface of these vesicles while phosphatidylethanolamine and phosphatidylserine are present on the inner surface. Methylation of phospholipids in brush border membrane with S-adenosyl-[methyl-3H]methionine resulted in the formation of phosphatidyl-N-monomethylethanolamine, phosphatidyl-N,N]dimethylethanolamine and phosphatidylcholine from endogenous phosphatidylethanolamine. The Km for S-adenosylmethionine was 1.10(-4) M with an optimum pH 9.0 for the formation of all three methyl derivatives. Mg2+ was without any effect between pH 5 to 10. Addition of exogenous mono- and dimethylphosphatidylethanolamine derivatives enhanced methyl group incorporation by 4-5-fold as compared to the addition of phosphatidylethanolamine. The conversion of endogenous phosphatidylethanolamine to phosphatidyl-N-monomethylethanolamine or addition of exogenous phosphatidylmonomethylethanolamine to brush border membrane did not result in a change in bulk membrane fluidity as determined by fluorescence polarization of diphenylhexatriene. Methylation of phosphatidylethanolamine in brush border membrane did not affect the Na+-dependent uptake of either D-glucose or phosphate, although the accessibility of cholesterol in membrane to cholesterol oxidase was diminished by 21%, presumably due to altered flip-flop movement of cholesterol in the membrane.

Effect of cell shape, membrane deformability and phospholipid organization on phosphate-calcium-induced fusion of erythrocytes.

Fusion of bovine and goat erythrocytes was studied using the phosphate-calcium protocol. Both bovine and goat red cells are resistant to fusion with phosphate and calcium, under conditions that promote fusion of normal human erythrocytes. Fusion resistance is not related to decreased (5%) membrane deformability of erythrocytes of these species, since chicken erythrocytes which are 40% less deformable than human erythrocytes undergo fusion with efficiency similar to human red blood cells. Incorporation of either phosphatidylcholine or phosphatidylserine into bovine erythrocytes mediated by lipid exchange/transfer protein, caused fusion of these erythrocytes. Fluorescence analysis of merocyanine 540 dye labeled erythrocytes, by flow cytometry, showed that the frequency of cells which exhibit dye binding was much less (35%) in dimyristoylphosphatidylcholine (DMPC) incorporated compared to untreated bovine erythrocytes (80%), indicating that incorporation of DMPC caused closed packing of lipids in the external leaflet of the bilayer. These studies show that fusion of bovine erythrocytes, mediated by phosphate and calcium, has a requirement for either specific phospholipids such as phosphatidylcholine, phosphatidylserine, or closed packing of lipids in the external leaflet of the bilayer.

Separation of luminal and abluminal membrane enriched domains from cultured bovine aortic endothelial cells: monoclonal antibodies specific for endothelial cell plasma membranes.

Two kinds of membrane (luminal and abluminal membrane domains) fractions have been isolated from bovine aortic endothelial cells by fractionation of whole cell homogenate on discontinuous sucrose density gradients. The luminal membrane domain was enriched 12-16-fold for angiotensin-converting enzyme activity and 8-10-fold in alkaline phosphatase activity. The abluminal membrane domain displayed an enrichment of 8-fold in (Na+ + K+)-ATPase activity. Both of the membrane domains were minimally contaminated with mitochondria, microsomes and Golgi bodies, as assessed by their corresponding marker enzyme activities. 125I-labeling of endothelial cell monolayers by the Enzymo-Bead lactoperoxidase-catalyzed iodination procedure, followed by isolation of membranes, revealed that the radioactivity was predominantly associated with membranes enriched in angiotensin-converting enzyme activity, corresponding to the luminal membrane domain. However, when cells were radioiodinated in suspension culture, radioactivity was found equally associated in both the luminal and abluminal membrane fractions. Electron microscopy of freeze-fractured and sectioned material showed both luminal and abluminal membrane domains to be in the form of vesicles varying in size from 100 to 400 nm in diameter. To characterize the separation of endothelial cell membrane domains, we have attempted to prepare monoclonal antibodies specific for endothelial cells. Several clones were obtained, producing antibodies which bound to endothelial cells of arterial, venous and capillary origin. Two antibodies of these clones, XIVC6 and XVD2, were studied in more detail. In the ELISA assay, these antibodies reacted with bovine vascular endothelial cells, but not with human umbilical cord endothelial cells, nor with bovine corneal endothelial cells, smooth muscle cells or fibroblasts. Both of these antibodies are directed against an antigen of approximately 130 kDa, under reducing and non-reducing conditions, as assayed by the immunoprecipitation method. Western blot analysis of luminal and abluminal membrane fractions revealed that only MAb XVD2 reacted with an antigen, indicating that the antibody XIVC6 is directed against an epitope which is denatured by SDS. Moreover, MAb XVD2 preferentially reacted with the luminal membrane compared to the abluminal membrane domain of the endothelial cell. These monoclonal antibodies do not react with platelet membrane proteins, indicating that this 130 kDa membrane antigen is not common to both endothelial cells and platelets.(ABSTRACT TRUNCATED AT 400 WORDS)