High-capacity cation-exchange column for enhanced resolution of adjacent peaks of cations in ion chromatography.

One of the advantages of ion chromatography [Anal Chem. 47 (1975) 1801] as compared to other analytical techniques is that several ions may be analyzed simultaneously. One of the most important contributions of cation-exchange chromatography is its sensitivity to ammonium ion, which is difficult to analyze by other techniques [J. Weiss, in: E.L. Johnson (Ed.), Handbook of Ion Chromatography, Dionex, Sunnyvale, CA, USA]. The determination of low concentrations of ammonium ion in the presence of high concentrations of sodium poses a challenge in cation-exchange chromatography [J. Weiss, Ion Chromatography, VCH, 2nd Edition, Weinheim, 1995], as both cations have similar selectivities for the common stationary phases containing either sulfonate or carboxylate functional groups. The task was to develop a new cation-exchange stationary phase (for diverse concentration ratios of adjacent peaks) to overcome limitations experienced in previous trails. Various cation-exchange capacities and column body formats were investigated to optimize this application and others. The advantages and disadvantages of two carboxylic acid columns of different cation-exchange capacities and different column formats will be discussed.

Ap4A and ADP-beta-S binding to P2 purinoceptors present on rat brain synaptic terminals.

1. Diadenosine tetraphosphate (Ap4A) a dinucleotide stored and released from rat brain synaptic terminals presents two types of affinity binding sites in synaptosomes. When [3H]-Ap4A was used for binding studies a Kd value of 0.10 +/- 0.014 nM and a Bmax value of 16.6 +/- 1.2 fmol mg-1 protein were obtained for the high affinity binding site from the Scatchard analysis. The second binding site, obtained by displacement studies, showed a Ki value of 0.57 +/- 0.09 microM. 2. Displacement of [3H]-Ap4A by non-labelled Ap4A and P2-purinoceptor ligands showed a displacement order of Ap4A > adenosine 5'-O-(2-thiodiphosphate) (ADP-beta-S) > 5'-adenylyl-imidodiphosphate (AMP-PNP) > alpha,beta-methylene adenosine 5'-triphosphate (alpha,beta-MeATP) in both sites revealed by the Ki values of 0.017 nM, 0.030 nM, 0.058 nM and 0.147 nM respectively for the high affinity binding site and values of 0.57 microM, 0.87 microM, 2.20 microM and 4.28 microM respectively for the second binding site. 3. Studies of the P2-purinoceptors present in synaptosomes were also performed with [35S]-ADP-beta-S. This radioligand showed two binding sites the first with Kd and Bmax values of 0.11 +/- 0.022 nM and 3.9 +/- 2.1 fmol mg-1 of protein respectively for the high affinity binding site obtained from the Scatchard plot. The second binding site showed a Ki of 0.018 +/- 0.0035 microM obtained from displacement curves. 4. Competition studies with diadenosine polyphosphates of [35S]-ADP-beta-S binding showed a displacement order of Ap4A > Ap5A > Ap6A in the high affinity binding site and Ki values of 0.023 nM, 0.081 nM and 5.72 nM respectively. The second binding site potency order was Ap5A> Ap4A > Ap6A,with Ki values of 0.28 microM, 0.53 microM and 5.32 microM respectively.5. Displacement studies of [35S]-ADP-beta-S with P2-purinoceptor agonists showed the following potency pattern: ADP-beta-S > AMP-PNP >alpha,beta-MeATP with Ki values of 0.021 nM, 0.029 nM 0.215 nM respectively in the high affinity binding site. 2-Methylthio-adenosine 5'-triphosphate (2MeSATP) was unable to displace [35S]-ADP-beta-S in this binding site. The second binding site showed a profile of ADP-beta-S> a,beta-MeATP> AMP-PNP > 2MeSATP and Ki values of 0.0 18 microM, 0.212 microM, 0.481 microM and 18.04 microM respectively.6. These studies suggest the presence of a new P2-purinoceptor in rat brain synaptosomes with high affinity for diadenosine polyphosphates which we tentatively designate as P2d.

Presence of diadenosine polyphosphates--Ap4A and Ap5A--in rat brain synaptic terminals. Ca2+ dependent release evoked by 4-aminopyridine and veratridine.

The study of the adenine nucleotides in middle brain synaptosomes from rat showed the presence of two diadenosine polyphosphates, Ap4A and Ap5A. HPLC techniques and phosphodiesterase digestion were employed in order to characterize and quantify the dinucleotides. The Ap4A content per mg of protein was 169 +/- 25 pmol and 159 +/- 22 pmol for Ap5A. The study of the exocytotic release of these compounds was carried out with 100 microM 4-aminopyridine or 10 microM veratridine in the presence and in the absence of calcium. 4-Aminopyridine released 14.5 +/- 3.0 pmol/mg protein of Ap4A and 11.6 +/- 2.4 pmol/mg protein of Ap5A in a calcium dependent process. Veratridine in the presence of calcium released 19.9 +/- 3.0 and 16.6 +/- 2.8 pmol/mg of protein of Ap4A and Ap5A respectively. The ratios of exocytosis were close to 7-9% and 10-12% of the total synaptosomal content in the presence of 4-aminopyridine and veratridine, respectively.

[The usefulness of determining antigliadin IgA antibodies for the detection and follow-up of adult celiac disease]. / Utilidad de la determinación de los anticuerpos antigliadina IgA séricos para la detección y seguimiento de la enfermedad celiaca del adulto.

Antibodies to gliadin IgA (IgA-AGA) were detected by enzyme-linked-immunosorbent-technique (ELISA) in 30 healthy controls, 20 coeliac patients and 25 patients with non coeliac malabsorption. All the controls had levels of IgA-AGA in the normal range (25AV). Three of the 25 patients with non coeliac malabsorption presented high titres of IgA-AGA. Thirty six determinations of IgA-AGA were made on the coeliac patients. All but one of the determinations made during the symptomatic phase of the disease were 25AV. The highest titres corresponded either to untreated patients or patients with complications (cirrhosis, lymphoma). In our study the sensitivity and the specificity of the test for symptomatic patients were 94.4% and 94.5% respectively.

Transmembrane envelope glycoproteins of human immunodeficiency virus type 2 and simian immunodeficiency virus SIV-mac exist as homodimers.

An 80-kilodalton glycoprotein (gp80) was produced in human immunodeficiency virus type 2 (HIV-2)-infected cells along with three envelope glycoproteins that we have recently reported: the extracellular glycoprotein (gp125), the envelope glycoprotein precursor (gp140), and the transient dimeric form of the precursor (gp300). gp125 and gp80 were detectable after the synthesis of gp140 and the formation of gp300. Using a specific monoclonal antibody, we showed here that gp80 is a dimeric form of the transmembrane glycoprotein gp36 of HIV-2. Dimerization of the envelope glycoprotein precursor and dimeric forms of the transmembrane glycoproteins were also observed in cells infected with simian immunodeficiency virus (SIV-mac), a virus closely related to HIV-2. Under routine conditions of our experiments (i.e., extraction by 1% Triton X-100 before polyacrylamide gel electrophoresis in sodium dodecyl sulfate [SDS]), monomeric forms of the transmembrane glycoprotein of HIV-2 and SIV-mac were only seldomly observed. Dimeric forms of the envelope precursors and the transmembrane glycoproteins are probably stabilized by extraction in the nonionic detergent Triton X-100 since such dimeric forms resist dissociation during subsequent electrophoresis in the presence of the ionic detergent SDS. However, the dissociation of these dimeric forms might occur when samples are prepared by extraction directly in 1% SDS or by incubation of the purified dimers at acidic pH. Dimerization of the envelope precursor might be required for its processing to give the mature envelope proteins, whereas the transmembrane dimer might be essential for optimal structure of the virion and thus its infectivity.

Characterization of an HIV-2-related virus with a smaller sized extracellular envelope glycoprotein.

A new isolate of the human immunodeficiency virus (HIV) related to the HIV-2 strain was isolated from peripheral blood lymphocytes of an Ivory Coast patient with AIDS. This isolate referred to as HIV-2 EHO could be differentiated by its envelope precursor and external glycoprotein which are 20-kDa smaller than those of HIV-2 ROD isolate. Furthermore, the apparent molecular weight of the major core protein of HIV-2 EHO is 27 kDa instead of 26 kDa as in HIV-2 ROD isolate. In addition, the product of the vpx gene which is a characteristic feature of the HIV-2 strain, is 14 kDa in HIV-2 EHO compared with 16 kDa in HIV-2 ROD. In contrast to these, the envelope precursor of HIV-2 EHO forms a transient dimer its maturation as is the case for HIV-2 ROD. In both cases the transmembrane proteins are 36 kDa and exists as homodimers of 80 kDa. Endoglycosidase H digestion experiments indicated that the 20-kDa difference between the two HIV-2 isolates is not due to a difference in the number of N-linked oligosaccharide chains per polypeptide, since deglycosylated envelope precursors of HIV-2 ROD and EHO have an apparent molecular weight of 80 and 60 kDa, respectively. Partial proteolysis of the envelope precursors from the two isolates with Staphylococcus aureus V8 protease gave a distinct pattern of polypeptides. These results suggest that the differences between the external envelope proteins of the two HIV-2 isolates are due to their amino acid composition. Accordingly, polyclonal antibodies raised against HIV-2 ROD envelope do not recognize the corresponding envelope proteins of HIV-2 EHO by immunoblotting and immunoprecipitation assays. These data illustrate that analysis of viral proteins could be useful for a rapid characterization of new viral isolates and show the heterogeneity of HIV-2 isolates in West Africa.

Cell surface expression of several species of human immunodeficiency virus type 1 major core protein.

The major core protein (p25) of the human immunodeficiency virus type 1 (HIV-1) was characterized by two-dimensional-gel isoelectric focusing. The p25 detectable in HIV-1-infected cells is composed of four species with related isoelectric points. This is due in part to the phosphorylated state of p25. The four species of p25 are expressed on the cell surfaces of infected cells, but only the two most basic species are incorporated into the HIV-1 virion. These findings emphasize the importance of p25 in understanding infection with HIV and might have implications for the development of vaccines.

Characterization of human immunodeficiency virus type 2 envelope glycoproteins: dimerization of the glycoprotein precursor during processing.

Four glycoproteins with apparent molecular weights of 300,000, 140,000, 125,000, and 36,000 (gp300, gp140, gp125, and gp36) were detectable in human immunodeficiency virus type 2 (HIV-2)-infected cells. gp125 and gp36 are the external and transmembrane components, respectively, of the envelope glycoproteins of HIV-2 mature virions. gp300 and gp140 are only detectable in virus-infected cells. They have identical isoelectric points, suggesting that gp300 might be a dimeric form of the immature precursor, gp140. The purified gp300 can be dissociated in a slightly acidic buffer to give rise to monomers of 140,000 molecular weight. Such dissociated monomers and the purified gp140 showed identical patterns of polypeptides after partial proteolysis with Staphylococcus aureus V8 protease. Pulse-chase experiments indicated that gp300 is formed after synthesis of gp140 and before the detection of the mature external envelope glycoprotein, gp125. These results were confirmed by using various inhibitors of glycosylation and inhibitors of trimming enzymes. Dimer formation of the envelope glycoprotein precursor was also observed in cells infected with simian immunodeficiency virus (SIV), a virus closely related to HIV-2. On the other hand, the envelope glycoprotein precursor of HIV-1 did not form a dimer during its processing. Therefore, dimer formation seems to be a specific property of HIV-2 and SIV envelope gene expression. Such transient dimerization of the glycoprotein precursor might be required for its efficient transport to the Golgi apparatus and for its processing.

Immunoglobulins and complement components in 37 patients infected by HIV-1 virus: comparison of general (systemic) and intrathecal immunity.

Intrathecal (IT) immunity was assessed by simultaneous analysis of paired cerebrospinal fluid (CSF) and sera of 37 patients infected by human immunodeficiency virus-1 (HIV-1). Only 8 of these 37 patients had no neurological or neuropsychiatric symptoms. There were 3 prominent abnormalities observed: (1) IT IgA production occurred in 15 patients, IT IgM production in 14 patients, and IT IgG production in 34 patients. (2) IT Anti-HIV-1 antibody specific activity (ASA) was higher than in serum in 33 of the 37 patients indicating that IT synthesis of antibody specific for HIV-1 occurs even in asymptomatic patients; IT anti-HIV-1 antibody synthesis was not correlated with clinical severity or neurological involvement. IT anti-herpes simplex ASA was also higher than serum ASA in 6 patients indicating a possible associated herpes simplex virus infection. (3) IT production of the complement component C4 was found frequently and was highly correlated with increased serum C4. IT C3 levels were decreased in 21 of 37 patients indicating that complement activation is a frequent accompaniment of the IT immune response in HIV-1-positive patients. These results indicate a unique and localized IT immune response which is different from the pattern observed in the systemic immune compartment in HIV-1-seropositive individuals and from the pattern common to the other CNS infectious diseases.

Prenatal diagnosis of HIV infection: two attempts using fetal blood sampling.

Blood samples were studied, prior to medical terminations of pregnancy, in two second trimester fetuses with HIV-positive mothers. Fetal blood was obtained from the umbilical vein under ultrasound guidance. No evidence of infection was found in either fetus. In particular, lymphocyte subpopulations were at normal levels and cultures yielded no reverse transcriptase activity. Postmortem findings were normal. Reliable means for prenatal diagnosis of HIV infection, with a minimal risk of false-negative results, have yet to be developed. However, further studies using fetal blood sampling should be useful for the management of HIV-positive pregnancies.

Prospective study of HIV I seropositive patients in hemodialysis centers.

A prospective multicenter study was undertaken between February 1985 and August 1986 in 4 hemodialysis centers in the Paris area (France) in order to assess the prevalence of HIV I infection and the risk of transmission of the virus within the centers. A four-month follow-up was carried out in 221 patients undergoing hemodialysis (HD) and in 40 staff members caring for the patients in 2 centers. 62 patients undergoing peritoneal dialysis (PD) and 126 hemodialysis patients who transited through a center (HDT) were screened once. A questionnaire exploring risk factors was completed for each patient and staff member. Sera were tested for HIV I antibodies by ELISA (ELAVIA) and confirmed by Western Blot. Of the 347 HD + HDT patients, 4 were found to be positive. Of the 221 HD patients, 1 multi-transfused hemophiliac and 1 multitransfused Nigerian without other risk factors were positive in the first screening. Another patient seroconverted after transfusion during the study; no other risk factors existed and the donor has not yet been found. One of the 126 HDT patients had received infected plasma. No staff members or PD patients were positive. No transmission within centers, from patient-to-patient or patient-to-staff was evidenced. Although HIV I seems to be less infectious than HBV, precautions to prevent transmission of HIV I in dialysis centers should be maintained.

[HIV1 infections in dialysis centers. Prospective studies in 4 centers in Ile-de-France]. / Infection par le HIV1 dans les centres de dialyse. Etude prospective dans quatre centres d'Ile-de-France.

A prospective multicenter study was undertaken between february 1985 and august 1986 in 4 haemodialysis centers in the Paris area (France) in order to assess the prevalence of HIV1 infection and the risk of transmission of the virus within the centers. Every four months a follow-up was carried out in 221 patients undergoing haemodialysis (HD) and in 40 staff members caring for the patients in 2 centers. 62 patients undergoing peritoneal dialysis (PD) and 126 haemodialysis patients who transited through a center (HDT) were screened once. A questionnaire exploring risk factors was completed for each patient and staff member. Sera were tested for HIV1 antibodies by ELISA (ELAVIA) and confirmed by Western Blot. Of the 347 HD + HDT patients, 4 were found to be positive. Of the 221 HD patients, 1 multi-transfused haemophiliac and 1 multitransfused Nigerian without other risk factors were positive in the first screening. Another patient seroconverted after blood transfusion during the study; no other risk factors existed and the donor has not yet been found. One of the 126 HDT patients had received infected plasma. No staff members or PD patients were positive. Thus there is no evidence within the centers of transmission, from patient-to-patient or patient-to-staff. Although HIV1 seems to be less infectious than HBV, precautions to prevent transmission of HIV1 in dialysis centers should be maintained.

[Retroviral infections by HIV-1, HIV-2 and AIDS-related complex in the Ivory Coast]. / Infections retrovirales par HIV-1, HIV-2 et SIDA associé en Côte-d'Ivoire.

The study of 500 blood donors, of 23 prostitutes, 22 confirmed AIDS cases and 19 ARC patients in Abidjan showed that HIV-2 prevalence in blood donors (3.4%) was somewhat higher than that of HIV-1 (2.4%). Furthermore HIV-2 alone was found associated with 14% of AIDS cases and 21% of ARC, while HIV-1 was associated with 32% of the cases. Antibodies to HIV-1 and HIV-2 were found in 54% of AIDS and ARC cases, as well as in 48% of the prostitutes sera. These preliminary results suggest that HIV-2 is pathogenic, being causally involved in some AIDS cases and does not protect from HIV-1 pathogenic potential.

Cholangitis in the acquired immunodeficiency syndrome: report of two cases and review of the literature.

We report the cases of one patient with the acquired immunodeficiency syndrome as a result of human immunodeficiency virus type 1/lymphadenopathy associated virus type 1/human T-cell lymphotrophic virus type III (HIV-1/LAV-1/HTLV-III) infection and of another patient with AIDS related complex caused by human immunodeficiency virus type 2/lymphadenopathy associated virus type 2 (HIV-2/LAV-2) infection, who were suffering from cholangitis. The manifestations and possible mechanisms for cholangitis in these patients and in 10 previously reported similar cases are reviewed.

Retrospective seroepidemiology of AIDS virus infection in Nairobi populations.

Among 446 sera from prostitutes in Nairobi, the prevalence of antibody to human immunodeficiency virus (HIV) rose from 4% in 1981 to 61% in 1985. None of 118 men with chancroid seen in 1980 had antibody to HIV compared with 15% of 107 such men in 1985. Among pregnant women, 2.0% were seropositive in 1985 versus none of 111 in 1981. Seropositive prostitutes and women with sexually transmitted diseases (STDs) tended to have more sex partners and had a higher prevalence of gonorrhoea, and in women with STDs, significantly more seropositive women practiced prostitution. Pregnant women and men with STDs who were born in the most-western region of Kenya were more likely to have antibody to HIV than were such groups from other geographic areas. Our results indicate that the AIDS virus was recently introduced into Kenya, that HIV can rapidly disseminate in a high-risk group of heterosexuals, and that prostitutes may have significantly contributed to the spread of the virus.