Description of two different patients with abetalipoproteinemia: synthesis of a normal-sized apolipoprotein B-48 in intestinal organ culture

We describe here two patients, M. P. and S. L., with recessive abetalipoproteinemia. Analysis of restriction fragments of DNA from both patients using cDNA probes spanning the entire apolipoprotein B gene revealed no major insertions or deletions. Further, as defined by restriction fragment length polymorphism, abetalipoproteinemia, in these patients, did not appear associated with particular alleles of apolipoprotein B. Northern and dot blot analysis of intestinal mRNA of one patient (M. P.) revealed a normal-sized apolipoprotein B mRNA which was present in slightly reduced amounts. At the cellular level apolipoprotein B was detected in both intestinal and hepatic biopsies, of one patient (S. L.), by immunoenzymatic techniques using polyclonal and monoclonal antibodies to apolipoprotein B-48 and/or B-100. The level of apolipoprotein B-48 appeared to increase in the intestine after a fatty meal. In the other patient (M. P.), although no apolipoprotein B was detected in the enterocytes using similar immunoenzymatic techniques, organ culture experiments using [35S]methionine demonstrated the synthesis of a normal-sized apolipoprotein B-48 which appeared to be normally glycosylated. The glycosylation and processing of two intestinal membrane enzymes, sucrase-isomaltase and aminopeptidase N, were also normal. Although lipids and apolipoprotein B-48 were present intracellularly, no lipoprotein-like particles were observed by electron microscopy in the endoplasmic reticulum, the Golgi apparatus, or in the intercellular spaces of intestinal biopsies obtained in the fasted (M. P. and S. L.) or fed state (S. L.). The defect in these cases of abetalipoproteinemia, therefore, does not appear to involve the apolipoprotein B gene nor the synthesis or the glycosylation of the apolipoprotein but instead appears to involve some aspect of lipoprotein assembly or secretion.     

Proteins in cerebrospinal fluid and plasma of the pouch young tammar wallaby (Macropus eugenii) during development

The concentrations of total protein and albumin in cerebrospinal fluid (CSF) and plasma of tammar wallaby pouch young (Macropus eugenii) from birth until leaving the pouch have been measured. Total protein in CSF increased from birth (about 240 mg/100 ml) to 15-20 days postnatal (about 400 mg/100 ml) after which it declined. Albumin showed a proportionately greater increase from around 40 mg/100 ml to over 130 mg/100 ml, followed by decline after 75 days. Total protein and albumin in plasma increased throughout the period studied. Other proteins identified in CSF and plasma were: fetuin, alpha 2-macroglobulin, transferrin, alpha-lipoprotein, beta-lipoprotein, immunoglobin G and fibrinogen. One protein was only present in early pouch young (up to about 40 days) and was presumed to be the tammar equivalent of alpha-fetoprotein.     

Importance of cholesterol-phospholipid interaction in determining dynamics of normal and abetalipoproteinemia red blood cell membrane

Acanthocytic red blood cells in patients with abetalipoproteinemia have a decrease membrane fluidity that is associated with increased sphingomyelin/phosphatidylcholine (SM/PC) ratios. Here we describe studies designed to gain better insight into (i) the interrelationship between the composition of lipoprotein and red blood cell membrane in abetalipoproteinemia patients and normal controls; and (ii) how the differences in lipid composition of the red blood cell membrane affect its fluidity. The increased SM/PC ratio found in abetalipoproteinemia plasma high density lipoproteins (HDL) (3 times greater than controls) was paralleled by an increase in this ratio in acanthocytic red cells, but to a lesser degree (almost twice greater than control red cells). Cholesterol/phospholipid mole ratios (C/P) were increased 3-fold in abetalipoproteinemia HDL, but only slightly increased in red cells compared to controls values. As in the controls, 80-85% of abetalipoproteinemia red cell sphingomyelin was found to be in the outer half of the erythrocyte membrane. Membrane fluidity was defined in terms of microviscosity (eta) between 5 and 42 degrees C by the fluorescent polarization of 1,6-diphenylhexatriene (DPH) present in erythrocyte ghost membranes. At all temperatures, membrane microviscosity was higher in abetalipoproteinemia ghosts than controls, but these differences decreased at higher temperatures (12.34 vs 9.79 poise, respectively at 10 degrees C; 4.63 vs 4.04 poise at 37 degrees C). These differences were eliminated after oxidation of all membrane cholesterol to cholest-4-en-3-one by incubation with cholesterol oxidase. Following cholesterol oxidation, the membrane microviscosity decreased in patient ghosts more than in normal red blood cells so that at all temperatures no significant differences were present relative to control ghosts, in which the apparent microviscosity was also diminished but to a lesser degree. Therefore, although increased SM/PC ratios in abetalipoproteinemia may be responsible for decreased erythrocyte membrane fluidity, these effects are dependent upon normal interactions of cholesterol with red cell phospholipid.     

Isolation of plasma lipoproteins by zonal ultracentrifugation in the B14 and B15 titanium rotors

Lipoproteins were isolated from plasma of man, dog, rabbit, rat, and chicken by ultracentrifugation in continuous density gradients using the B14 titanium and B15 titanium zonal rotors. Both the VLDL and the LDL of human plasma were separated easily from the HDL and from the other more plentiful plasma proteins by centrifugation for only 1 or 2 hr in the B14 or B15 rotor, respectively. Satisfactory separation of the HDL from the more dense plasma proteins was not achieved with these rotors. The human LDL achieved isopycnic equilibrium (d 1.04) on prolonged periods (> 24 hr) of centrifugation in a sucrose-KBr density gradient. The pattern of distribution of cholesterol and phospholipid throughout the density gradient coincided with the pattern of distribution of the lipoprotein-protein measured spectrophotometrically or chemically. The concentration of cholesterol and phospholipid in the lipoproteins isolated by zonal ultracentrifugation agreed with analyses reported for lipoproteins isolated by sequential centrifugation in solutions of increasing density. The lipoproteins isolated by zonal ultracentrifugation were characterized further by their electrophoretic behavior. The fractions which were identified as the LDL (d 1.04-1.05) from all species migrated on paper as a beta-globulin; the LDL from plasma of dogs contained an additional component which has been designated as an alpha(2)-globulin. The fractions which were identified as the HDL from all species migrated as an alpha(1)-globulin. Reaction of human LDL with either rabbit antihuman beta-lipoprotein or rabbit antihuman serum resulted in a single immunodiffusion band. The S(f, 1.063) of the human LDL was calculated to be 6.0. When plasma from humans or rabbits was centrifuged in the B15 rotor, the HDL was not visible as a distinct peak and was not separable from the bulk of the more dense plasma proteins; when plasma from dogs or chickens was centrifuged under identical conditions, the HDL was clearly detectable. Even though the mean density of the HDL from dogs or chickens was not different from that of man or rabbits, the visibility of this lipoprotein in dogs and chickens was probably due to its high concentration in the plasma of these species. When plasma from the rat was centrifuged under similar conditions, the HDL was also clearly in evidence. Although rat plasma contained a relatively small concentration of HDL, the lipoprotein had a lower mean density than did the HDL of the other species and was therefore more easily separable from the dense plasma proteins. The procedure of zonal ultracentrifugation for the isolation of lipoproteins by flotation is simultaneously preparative and analytical and should find useful application in the investigation of the soluble lipoproteins from plasma and tissues.     

Immunochemical studies of the plasma and cultured fibroblast media fractions containing the cystic fibrosis ciliary inhibitor.

The cystic fibrosis ciliary inhibitor (CFCI) has been fractionated from plasma of cystic fibrosis (CF) homozygotes and from the media of cultured fibroblasts derived from CF homozygotes. Plasma and fibroblast media from normal controls have been fractionated in an identical manner. Fractions from plasma and fibroblast culture media that demonstrate ciliary inhibitory activity contain several proteins in a molecular weight range of approximately 5,000-11,000. These proteins have been partially characterized by immunochemical analysis with antisera to 33 human serum proteins. Immunological determinants of albumin, C3 (but not C3a), C4, C5, alpha1-lipoprotein, beta-lipoprotein, beta2-microglobulin and immunoglobulin light chains have been detected by hemagglutination in fractions of CF plasma that inhibited ciliary activity and in analogous fractions from normal sera. None of the proteins were detected in media of cultured fibroblasts from either genotype. Since the same proteins and protein fragments were identified in both CF and normal plasma fractions, and were not detected in CF fibroblast media, it appears that none of these proteins can be identified as the CFCI. Identification of these proteins will permit further purification of the CFCI by immunochemical methods.     

Structure-function analyses of microsomal triglyceride transfer protein missense mutations in abetalipoproteinemia and hypobetalipoproteinemia subjects

We describe two new hypolipidemic patients with very low plasma triglyceride and apolipoprotein B (apoB) levels with plasma lipid profiles similar to abetalipoproteinemia (ABL) patients. In these patients, we identified two previously uncharacterized missense mutations in the microsomal triglyceride transfer protein (MTP) gene, R46G and D361Y, and studied their functional effects. We also characterized three missense mutations (H297Q, D384A, and G661A) reported earlier in a familial hypobetalipoproteinemia patient. R46G had no effect on MTP expression or function and supported apoB secretion. H297Q, D384A, and G661A mutants also supported apoB secretion similarly to WT MTP. Contrary to these four missense mutations, D361Y was unable to support apoB secretion. Functional analysis revealed that this mutant was unable to bind protein disulfide isomerase (PDI) or transfer lipids. The negative charge at residue 361 was critical for MTP function as D361E was able to support apoB secretion and transfer lipids. D361Y most likely disrupts the tightly packed middle α-helical region of MTP, mitigates PDI binding, abolishes lipid transfer activity, and causes ABL. On the other hand, the hypolipidemia in the other two patients was not due to MTP dysfunction. Thus, in this study of five missense mutations spread throughout MTP's three structural domains found in three hypolipidemic patients, we found that four of the mutations did not affect MTP function. Thus, novel mutations that cause severe hypolipidemia probably exist in other genes in these patients, and their recognition may identify novel proteins involved in the synthesis and/or catabolism of plasma lipoproteins.     

Mutations of the Microsomal Triglyceride-Transfer–Protein Gene in Abetalipoproteinemia

Elevated plasma levels of apolipoprotein B (apoB)–containing lipoproteins constitute a major risk factor for the development of coronary heart disease. In the rare recessively inherited disorder abetalipoproteinemia (ABL) the production of apoB-containing lipoproteins is abolished, despite no abnormality of the apoB gene. In the current study we have characterized the gene encoding a microsomal triglyceride-transfer protein (MTP), localized to chromosome 4q22-24, and have identified a mutation of the MTP gene in both alleles of all individuals in a cohort of eight patients with classical ABL. Each mutant allele is predicted to encode a truncated form of MTP with a variable number of aberrant amino acids at its C-terminal end. Expression of genetically engineered forms of MTP in Cos-1 cells indicates that the C-terminal portion of MTP is necessary for triglyceride-transfer activity. Deletion of 20 amino acids from the carboxyl terminus of the 894-amino-acid protein and a missense mutation of cysteine 878 to serine both abolished activity. These results establish that defects of the MTP gene are the predominant, if not sole, cause of hereditary ABL and that an intact carboxyl terminus is necessary for activity.     

Serum cholinesterase isoenzymes and the WHHL rabbit: the relationship between the activity of cholinesterase not bound to low-density-lipoprotein and lipoprotein titer

Summary Serum cholinesterase has been previously shown to complex with beta-lipoprotein in the plasma. Since serum cholinesterase exists as isoenzymes in plasma, the relationship between the activity of these isoenzymes (unbound to beta-lipoprotein) and lipoprotein titer was investigated. The results indicated that the total of C2, C3, and C4 isoenzyme activities were expressed within a narrow range and independent of low density lipoprotein titer. These findings may indicate that unbound plasma cholinesterase may undergo autoregulation independent of cholinesterase bound to beta-lipoprotein.Abbreviations WHHL rabbit Watanabe Heritable Hyperlipidemic rabbit - LDL low-density lipoprotein - VLDL very low-density lipoprotein - HDL high-density lipoprotein - C(1–4) serum cholinesterase isoenzyme (1–4)     

Importance of cholesterol-phospholipid interaction in determining dynamics of normal and abetalipoproteinemia red blood cell membrane

Acanthocytic red blood cells in patients with abetalipoproteinemia have a decreased membrane fluidity that is associated with increased sphingomyelin/phosphatidylcholine (SM/PC)§ ratios. Here we describe studies designed to gain better insight into (i) the interrelationship between the composition of lipoprotein and red blood cell membrane in abetalipo-proteinemia patients and normal controls; and (ii) how the differences in lipid composition of the red blood cell membrane affect its fluidity. The increased SM/PC ratio found in abetalipoproteinemia plasma high density lipoproteins (HDL) (3 times greater than controls) was paralleled by an increase in this ratio in acanthocytic red cells, but to a lesser degree (almost twice greater than control red cells). Cholesterol/phospholipid mole ratios (C/P) were increased 3-fold in abetalipoproteinemia HDL, but only slightly increased in red cells compared to controls values. As in the controls, 80–85% of abetalipo-proteinemia red cell sphingomyelin was found to be in the outer half of the erythrocyte membrane. Membrane fluidity was defined in terms of microviscosity ({ie116-1}) between 5 and 42°C by the fluorescent polarization of 1,6-diphenylhexatriene (DPH) present in erythrocyte ghost membranes. At all temperatures, membrane microviscosity was higher in abetalipoproteinemia ghosts than controls, but these differences decreased at higher temperatures (12.34 vs 9.79 poise, respectively, at 10°C; 4.63 vs 4.04 poise at 37°C). These differences were eliminated after oxidation of all membrane cholesterol to cholest-4-en-3-one by incubation with cholesterol oxidase. Following cholesterol oxidation, the membrane microviscosity decreased in patient ghosts more than in normal red blood cells so that at all temperatures no significant differences were present relative to control ghosts, in which the apparent microviscosity was also diminished but to a lesser degree. Therefore, although increased SM/PC ratios in abetalipoproteinemia may be responsible for decreased erythrocyte membrane fluidity, these effects are dependent upon normal interactions of cholesterol with red cell phospholipid.     

Studies on cholesterol accumulation in radicular cyst fluid--origin of heat-stable cholesterol-binding protein

1. The amount of apolipoprotein B (apo B) was measured using slit-immunoblotting in 20 specimens of radicular cyst fluids. Apo B was detected in all the cyst fluids with varying amounts. 2. Relationship between the amounts of apo B and free cholesterol or activity of heat-stable cholesterol-binding protein (HCBP) were examined. The amount of apo B was correlated well with the activity of HCBP (n = 20, r = 0.72, P less than 0.01) and with the amount of free cholesterol (n = 20, r = 0.45, P less than 0.05). 3. Anti-human apo B antibody inhibited cholesterol-binding activity in radicular cyst fluid. 4. When human-serum was chromatographed on a HPLC ion-exchange column, both cholesterol-binding activity and apo B had exactly the same retention time. 5. These results suggest that HCBP originates from beta-lipoprotein, and beta-lipoprotein may have an important role in cholesterol accumulation on radicular cysts.     

Isoproteins of human apolipoprotein A-II: isolation and characterization

In human serum, polymorphism of apoA-II predominantly in HDL3 could be demonstrated. HDL3-apoA-II was composed of four isoproteins, each with a molecular weight of 8600 (reduced form) and identical immunological properties. The isoproteins are designated apoA-II-1 (pI 5.16), apoA-II-2 (pI 4.89) corresponding to the already known apoA-II monomer band, apoA-II-3 (pI 4.58), and apoA-II-4 (pI 4.31). The amino acid compositions of the A-II isoproteins were virtually identical with the published data for apoA-II. Treatment with acid phosphatase, alkaline phosphatase, or neuraminidase before electrophoresis did not alter the apoA-II pattern. The apoA-II isoprotein pattern was studied in ten male and ten female normolipidemic volunteers, in two patients with Tangier disease, and in three patients with abetalipoproteinemia. The isoelectric focusing patterns of apoA-II appeared virtually identical in all subjects. However, in Tangier disease, due to the low apo-A-II concentration, only apoA-II-1 and apoA-II-2 were detectable, and in abetalipoproteinemia a different relative distribution pattern of the individual isoforms was found as compared to normal HDL3. Our studies indicate that apoA-II, similar to apoA-I, exists in several isoforms. The relationship of these isoforms to each other is at present unclear. They may originate from relatively basic isoproteins that are modified in charge by post-translational processes such as proteolytic cleavage, sequential deamidation, or other mechanisms.     

Genetic mechanism of blood group (ABO)-expression

It has generally been believed that human blood group ABO is controlled by allelic ABO genes. However, this hypothesis has not yet been experimentally proven, and other possibilities such as the non-allelic gene model and the regulatory gene model for ABO locus have also been proposed. The genetic mechanisms of many unusual blood group expressions remain unanswered. Purification of human blood group N-acetylgalactosyltransferase (A-enzyme) which synthesizes A-substance, and blood group galactosyltransferase which is responsible for synthesis of B-substance, allows us to resolve these problems from an immuno-biochemical approach. It was found that rabbit antibody against-A-enzyme completely neutralized not only A-enzyme but also B-enzyme activity. Moreover, plasma from blood type O subjects contained an enzymatically inactive but immunologically cross-reactive material (CRM). Plasma from heterozygous AO and BO subjects also contained CRM, but plasma from homozygous AA and BB subjects did not contain CRM. These facts led us to conclude that the ABO genes are allelic in the strict sense, refuting other genetic models for ABO locus. Genotypes of phenotype A and B subjects can be unequivocally determined by examining the presence or absence of CRM in their plasma. Mechanism of the unusual blood group inheritance of Cis-AB (i.e., AB and/or O childbirth from AB X O parent) was elucidated by examining properties of the A and B enzymes, CRM in their plasma, and separation of active enzymes and CRM by affinity chromatography. It became clear that Cis-AB expressions in one family was due to unequal chromosomal crossing-over producing a single chromosome with the genes for A and B enzymes. In contrast, in the other two unrelated families, the Cis-AB expression was due to a structural mutation in A or B gene producing a single abnormal enzyme which was capable of transferring both GalNAc and Gal to H-substance. Mechanism of very weak B expression in a family with A1Bm character was studied. Plasma enzyme activity and kinetic characteristics of B-enzyme from the subjects was not different from that of normal. However, the A1Bm red cells contained a large amount of unoccupied H-sites which can be galactosylated in vitro and become B active. Examination of membrane components by isoelectric focussing revealed that blood group components of the A1Bm membranes were distinctively different from that of the usual membranes. Consequently, the weak B expression is not due to direct mutation of ABO locus, but due to a secondary consequence of genetic abnormality of a membrane component (or components) associated with blood group substances.     

Abetalipoproteinemia with an ApoB-100-lipoprotein(a) glycoprotein complex in plasma. Indication for an assembly defect

Patients with autosomal recessive abetalipoproteinemia (ABL) lack in their plasma all lipoproteins containing apolipoprotein (apo)B-100 or B-48. Previous studies have suggested that this is due to the complete absence of apoB. We have investigated whether such patients (n = 10) are able to secrete the lipoprotein(a) (Lp(a] glycoprotein (apo(a] which, in normal plasma, exists as a complex with low density lipoproteins containing apoB-100 (Lp(a) lipoprotein). All 10 patients had reduced but detectable apo(a) levels in plasma (mean, 0.49 mg/dl; range, 0.2-2.03 mg/dl) but no Lp(a) lipoprotein. However, we also detected small amounts (0.2-2.8 mg/dl) of apoB in all patients with ABL. The apoB in the ABL patients had the size of apoB-100 and occurred as a lipid-poor complex with the Lp(a) glycoprotein in a fraction of density 1.22 g/ml. This material may represent partially assembled Lp(a) lipoprotein. There was also uncomplexed apo(a) and apoB-100 in the ABL plasma. The distribution and relative concentration of both proteins in the density fraction greater than 1.06 g/ml varied among patients. The data suggest that in ABL, the assembly of apoB-containing lipoproteins is defective and that apoB-100 may be secreted without its full lipid complement when complexed with apo(a).     

Alterations in lipid metabolism produced by ethyl trichloracetate.

Rats maintained on a choline deficient diet and treated with subcutaneous doses of ethyl trichloracetate responded by increasing plasma beta-lipoprotein and plasma triglyceride levels while excess triglyceride was being removed from the liver. There was a transient depression in plasma phospholipid at the beginning of the treatment. Continued administration of ethyl trichloracetate raised plasma triglyceride in choline depleted rats and raised hepatic phospholipid concentration in both choline deficient and supplemented rats. It is suggested that the lipotropic action of ethyl trichloracetate occurs through hepatic triglyceride being removed by the altered plasma lipids and not by inhibition of hepatic triglyceride synthesis.     

Safety of human blood products: inactivation of retroviruses by heat treatment at 60 degrees C

Acquired immune deficiency syndrome (AIDS) can be transferred to patients by blood transfusions or human blood preparations, such as cryoprecipitates or factor VIII concentrates. Retroviruses have been discussed as infectious AIDS agents and more recently human T-lymphotropic retroviruses designated as HTLV type III and LAV (lymphadenopathy-associated virus) have been isolated from AIDS patients. Whether heat treatment at 60 degrees C (pasteurization) of liquid human plasma protein preparations inactivates retroviruses was therefore investigated. Pasteurization had already been included in the routine manufacturing process of human plasma protein preparations in order to guarantee safety with regard to hepatitis B. Since high titer preparations of human retroviruses were not available, heat inactivation was studied using Rous sarcoma virus added to the various plasma protein preparations tested. This retrovirus which was obtained in preparations of 6.0 log10 FFU/ml was shown to be at least as heat stable as two mammalian retroviruses studied, i.e., feline and simian sarcoma virus. In all of eight different plasma protein preparations tested, Rous sarcoma virus was completely inactivated after a heat treatment lasting no longer than 4 hr. It is thus concluded that pasteurization of liquid plasma protein preparations at 60 degrees C over a period of 10 hr must confer safety to these products with respect to AIDS, provided that the AIDS agents are retroviruses of comparable heat stability as Rous sarcoma virus and the mammalian retroviruses tested.     

Mutational and biochemical analysis of plasma membrane targeting mediated by the farnesylated, polybasic carboxy terminus of K-ras4B

Mutational analysis and in vitro assays of membrane association have been combined to investigate the mechanism of plasma membrane targeting mediated by the farnesylated, polybasic carboxy-terminal sequence of K-ras4B in mammalian cells. Fluorescence-microscopic localization of chimeric proteins linking the enhanced green fluorescent protein (EGFP) to the K-ras4B carboxy-terminal sequence, or to variant forms of this sequence, reveals that the normal structure of this targeting motif can be greatly altered without compromising plasma membrane-targeting activity so long as an overall strongly polybasic/amphiphilic character is retained. An EGFP/K-ras4B(171-188) chimeric protein was readily abstracted from isolated cell membranes by negatively charged lipid vesicles, and this abstraction was markedly enhanced by the anionic lipid-binding agent neomycin. Our results strongly favor a mechanism in which at the plasma membrane the carboxy-terminal sequence of K-ras4B associates not with a classical specific proteinaceous receptor but rather with nonspecific but highly anionic 'sites' formed at least in part by the membrane lipid bilayer. Our findings also suggest that the recently demonstrated prenylation-dependent trafficking of immature forms of K-ras4B through the endoplasmic reticulum [Choy et al. (1999) Cell 98, 69-80], while required for maturation of the protein, beyond this stage may not be essential to allow the ultimate delivery of the mature protein to the plasma membrane.     

Serum IgG response to Burkholderia cepacia outer membrane antigens in cystic fibrosis: Assessment of cross-reactivity with Pseudomonas aeruginosa

Abstract Burkholderia cepacia (Pseudomonas cepacia) is now recognised as an important pathogen in cystic fibrosis patients, and several reports have suggested that sputum-culture-proven colonisation occurs despite the presence of specific antibody. In an attempt to establish the use of antibody studies as diagnostic and prognostic indicators of B. cepacia infection, we have examined the IgG response to B. cepacia outer membrane proteins and lipopolysaccharide in patients also colonised with P. aeruginosa . The B. cepacia strains were grown in a modified iron-depleted chemically defined medium and outer membrane components examined by SDS-PAGE and immunoblotting. IgG antibodies were detected against B. cepacia outer membrane antigens, which were not diminished by extensive preadsorption with P. aeruginosa . The response to B. cepacia O-antigen could be readily removed by adsorption of serum either with B. cepacia whole cells or purified LPS, whereas we were unable to adsorb anti-outer membrane protein antibodies using B. cepacia whole cells. The inability to adsorb anti-outer membrane protein antibodies using B. cepacia whole cells maybe due to non-exposed surface epitopes. Several B. cepacia sputum-culture negative patients colonised with P. aeruginosa had antibodies directed against B. cepacia outer membrane protein. This study suggests that there is a specific anti- B. cepacia LPS IgG response, which is not due to antibodies cross-reactive with P. aeruginosa . Our studies indicate that much of the B. cepacia anti-outer membrane protein response is specific and not attributable to reactivity against co-migrating LPS.     

A designed probe for acidic phospholipids reveals the unique enriched anionic character of the cytosolic face of the mammalian plasma membrane

It is generally accepted that the cytosolic face of the plasma membrane of mammalian cells is enriched in acidic phospholipids due to an asymmetric distribution of neutral and anionic phospholipids in the two bilayer leaflets. However, the phospholipid asymmetry across intracellular membranes is not known. Two models have been proposed for the selective targeting of K-Ras4B, which contains a C-terminal farnesyl cysteine methyl ester adjacent to a polybasic peptide segment, to the cytosolic face of the plasma membrane. One involves electrostatic interaction of the lipidated polybasic domain with anionic phospholipids in the plasma membrane, and the other involves binding of K-Ras4B to a specific protein receptor. To address this issue, we prepared by semi-synthesis a green fluorescent protein variant that is linked to a farnesylated, polybasic peptide corresponding to the K-Ras4B C terminus as well as a variant that contains an all-d amino acid version of the K-Ras4B peptide. As expected based on electrostatics, both constructs showed preferential in vitro binding to anionic phospholipid vesicles versus those composed only of zwitterionic phospholipid. Both constructs fully targeted to the plasma membrane when microinjected into live Chinese hamster ovary and Madin-Darby canine kidney cells. Because the all-d amino acid peptide should be devoid of binding affinity to a putative highly specific K-Ras membrane receptor, these results support an electrostatic basis for the targeting of K-Ras4B to the plasma membrane, and they support an intracellular landscape of phospholipids in which the cytosolic face of the plasma membrane is the most enriched in acidic phospholipids.