Polysialic acid, a unique glycan that is developmentally regulated by two polysialyltransferases, PST and STX, in the central nervous system: From biosynthesis to function

Polysialic acid is a developmentally regulated carbohydrate composed of a linear homopolymer of a-2,a-linked sialic acid residues. This unique glycan is mainly attached to the neural cell adhesion molecule (N-CAM) and implicated in many morphogenic events of the neural cells by modulating the adhesive property of N-CAM. Recently, the cDNA that encodes polysialyltransferase, which is responsible for the polysialylation of N-CAM, was successfully cloned from three mammalian species. This review focuses on the molecular cloning of human polysialyltransferase, designated PST. it then describes the number of enzymes actually required for the polysialylation of N-CAM using an in vitro polysialyltransferase assay. Comparisons between PST and another polysialyltransferase, sialyltransferase X (STX), are made and it Is demonstrated that both enzymes can independently form polysiatic acid In vitro , but that during neural development they coordinately but distinctly synthesize polysialic acid on N-CAM. The role of polysialic acid in the central nervous system is also discussed. Finally, evidence that the two polysialyltransferases, PST and STX, apparently have distinct roles in the development of neural cells is provided by using a neurite outgrowth assay.     

Glutaraldehyde-treated bioprosthetic substitute for rabbit Achilles tendon

The biomechanical properties and histocompatibility of a glutaraldehyde-treated xenograft were examined after implanting it in 27 rabbits. This xenograft was used as an Achilles tendon substitute placed in vivo for 2 to 48 wk. The specimens were then subjected to mechanical strength testing using a specially constructed tensioning device. The contralateral Achilles tendon was used as the reference baseline. Mechanical testing showed that the strength of the implanted graft increased to 33.5% of the normal side at week 10 and to 79.5% at week 14. The host generated a fibrous cord around the xenograft, linking the two ends of the Achilles tendon. Light and electron microscopic examination showed fibroblastic infiltration of varying magnitude in all specimens but no statistical correlation between degree of infiltration and duration of implantation was found. Also, there was no histological evidence of immunological rejection within the 48 wk of study.     

Evaluation of screened blood donations for human immunodeficiency virus type 1 infection by culture and DNA amplification of pooled cells

BACKGROUND. Reports of transmission of the human immunodeficiency virus type 1 (HIV-1) from transfusions of screened blood and reports of silent, antibody-negative HIV-1 infections in persons at high risk continue to foster concern about the safety of the blood supply. Previous estimates of the risk of HIV-1 range from 1 in 38,000 to 1 in 300,000 per unit of blood but are based on either epidemiologic models or the demonstration of seroconversion in recipients. METHODS. We isolated peripheral-blood mononuclear cells from blood that was fully screened and found to be seronegative, combined them into pools of cells from 50 donors, and tested them for HIV-1 by viral culture and the polymerase chain reaction, using protocols specifically adapted for this analysis. RESULTS. The 1530 pools of mononuclear cells were prepared from 76,500 blood donations made in San Francisco between November 1987 and December 1989. Of these pools, 1436 (representing 71,800 donations) were cultured successfully; 873 (43,650 donations) were evaluated by the polymerase chain reaction. Only one pool was confirmed as HIV-1--infected by both methods. After adjustment for sample-based estimates of the sensitivity of the detection systems using culture and the polymerase chain reaction, the probability that a screened donor will be positive for HIV-1 was estimated as 1 in 61,171 (95 percent upper confidence bound, 1 in 10,695). CONCLUSIONS. Silent HIV-1 infections are exceedingly rare among screened blood donors, so the current risk of HIV-1 transmission from blood transfusions, even in high-prevalence metropolitan areas, is extremely low.     

Influence of food on the absorption of albuterol Repetabs

A study was conducted in 12 healthy, nonsmoking male volunteers to examine the effect of food intake on the absorption profile of albuterol repeat-action tablets. This randomized crossover study consisted of two phases separated by a 1-week washout period. All subjects fasted 10 hours preceding drug administration. Each subject received two 4 mg albuterol repeat-action tablets with and without a high fat content breakfast. Plasma albuterol concentrations were determined by a gas chromatographic/mass spectrophotometric assay. Relative bioavailability was assessed by comparing areas under the plasma-albuterol concentration time curves as well as peak concentrations and time to peak concentration. No significant differences were noted between the two treatment phases in the area under the curve or peak plasma concentrations. The areas under the curve were 100 and 105 hr.ng/ml when the drug was administered with and without food, respectively. The corresponding peak plasma concentration values were 9.4 and 10.4 ng/ml, respectively. The only significant difference observed was in the maximum time to reach peak plasma concentrations, which was delayed by about 1 hour when the drug was administered with food. Therefore, food has minimal effect on the absorption of albuterol from repeat-action tablets.     

T-cell receptor V beta selectivity in T-cell clones alloreactive to HLA-Dw14.

The HLA-DR4 subtypes Dw14 and Dw4 are T-cell-defined allospecificities encoded by the DRB1*0404 and DRB1*0401 genes, respectively. Although these allelic subtypes differ in only two amino acids, allorecognition between Dw14 and Dw4-positive individuals is brisk. This provides an opportunity to analyze T-cell receptor (TCR) usage in a very limited and specifically targeted case, namely the Dw4 anti-Dw14 allogeneic T-cell response. The variable (V), diversity (D), and joining (J) region sequences of the TCR beta chain from two different Dw14-specific alloreactive T-cell clones derived from a Dw4 donor were examined. Clone EMO25 recognized the Dw14.1, Dw14.2, and Dw15 subtypes, which share a DRB1 polymorphism at codon 71 on a DR4 background, while clone EMO36 reacted with only the Dw14.1 subtype associated with polymorphisms at codons 71 and 86. TCR beta cDNA from each clone was amplified using an anchored polymerase chain reaction (PCR) and subsequently expanded with V beta- and C beta-specific primers for asymmetric PCR and direct DNA sequencing. Both clones were found to express the same TCR V beta 8.2 gene segment; however, they have several different residues within the V beta-D beta-J beta junctional regions. V beta 8 usage was also enriched in polyclonal cells obtained from mixed lymphocyte cultures performed between the Dw4 and Dw14 responder-stimulator combination from which EMO25 and EMO36 were derived.     

PCR-Based assay to quantify human immunodeficiency virus type 1 DNA in peripheral blood mononuclear cells

An assay that quantifies the amount of human immunodeficiency virus type 1 (HIV-1) DNA in peripheral blood mononuclear cells has been developed. PCR amplification of the HIV-1 DNA is performed in the presence of an internal quantitation standard, and colorimetric detection of the amplified product is performed with microwell plates. The copies of HIV-1 DNA are normalized to total genomic DNA input. The assay has an analytical sensitivity of 10 input copies per amplification reaction and a three-log detection range. In an analysis of sequential samples from patients on combination therapy, HIV-1 DNA was quantifiable for all individuals tested, including those with undetectable plasma HIV-1 RNA. In a separate study, a comparison of HIV-1 DNA levels was made with a group of long-term survivors and progressors. The mean HIV-1 DNA levels were lower in the long-term survivors than in the progressors (P, 0.04). The mean HIV-1 RNA levels were also lower, but the difference was not statistically significant (P, 0.164). A quantitative DNA assay will provide an additional tool to gain insight into the natural history of infection and the continued efficacy of potent antiretroviral therapies.     

Plasmodium falciparum sexual stage antigens: immunogenicity and cell-mediated responses.

Antibody and cell-mediated immune responses to the transmission-blocking target antigens of Plasmodium falciparum, Pfs 48/45, were determined in infected non-immune patients and in immune individuals from an endemic area. Characterization of the B cell epitopes with monoclonal antibodies showed that there were five regions identifiable but there could be interactions between them causing either competitive or enhancing effects. Sera from infected non-immune patients contained antibodies that would compete with one or more of the mAbs to the different epitopes. Immune responsiveness to purified Pfs 48/45 in P. falciparum-immune adults measured as lymphoproliferation, production of interferon-gamma, or as Pfs 48/45-specific antibody was very limited. This did not appear to be due to MHC class II restriction, to diversity in structure of the parasite antigens or to a failure of immunological memory. The antibody-response data were more consistent with down-regulation of immunity as a result of prolonged exposure to infection.     

Development of a Western Blot Assay for Detection of Antibodies against Coronavirus Causing Severe Acute Respiratory Syndrome

To identify a major antigenic determinant for use in the development of a rapid serological diagnostic test for severe acute respiratory syndrome (SARS) coronavirus infection and to study the immune response during SARS coronavirus infection in humans, we cloned the full length and six truncated fragments of the nucleocapsid gene, expressed them, and purified them as glutathione S-transferase-tagged recombinant proteins. The reactivities of the recombinant proteins to a panel of antibodies containing 33 SARS coronavirus-positive sera and 66 negative sera and to antibodies against other animal coronaviruses were screened. A truncated 195-amino-acid fragment from the C terminus of the nucleocapsid protein (N195) was identified that had a strong ability to detect antibodies against SARS coronavirus. No cross-reaction was found between the N195 protein and antibodies against chicken, pig, and canine coronaviruses. The N195 protein was used to develop a Western blot assay to detect antibodies against SARS coronavirus in 274 clinically blinded samples. The specificity and sensitivity of this test were 98.3 and 90.9%, respectively. The correlation between our Western blotting assay and an immunofluorescence assay (IFA) was also analyzed. The results of our Western blot assay and IFA for the detection of SARS coronavirus-positive sera were the same. Thus, the N195 protein was identified as a suitable protein to be used as an antigen in Western blot and other possible assays for the detection of SARS coronavirus infection.     

Evaluation of a Selective Transport Medium for Gastric Biopsy Specimens To Be Cultured for Helicobacter pylori

Since the means of culturing Helicobacter pylori may not be available in some laboratories, prolonging the survival of this organism during transportation is a major concern in terms of improving detection rates. A selective transport medium was evaluated for the preservation of H. pylori from 254 gastric biopsy specimens collected from a rural area in China where culturing is not feasible. Gastric biopsy specimens were inoculated in sterile broth consisting of brain heart infusion (BHI) broth, horse serum, and yeast extract supplemented with vancomycin, amphotericin B, and nalidixic acid (VAN). Of the 254 biopsy specimens, 238 were identified by histology to have H. pylori infection. Total rates of recovery of H. pylori from the H. pylori-positive gastric biopsy specimens stored in the BHI-VAN broth ranged from 76 to 46% after storage of specimens for 5 to 9 days. In conclusion, the selective medium is useful for prolonging the survival of H. pylori in gastric biopsy specimens for which immediate culture is not feasible.     

In vivo stability and discriminatory power of methicillin-resistant Staphylococcus aureus typing by restriction endonuclease analysis of plasmid DNA compared with those of other molecular methods.

We evaluated test discriminatory power and DNA type alterations among methicillin-resistant Staphylococcus aureus strains by testing 199 sequential isolates from 39 patients collected over 30 to 228 days. Isolates were typed by one or three different methods (restriction endonuclease analysis of plasmid DNA [REAP] with or without pulsed-field gel electrophoresis of genomic DNA [PFGE] and immunoblotting [IB]). REAP was highly discriminatory compared with PFGE and IB. However, the initial isolates from 4 of the 39 patients lacked detectable plasmid DNA and could not be typed by REAP. Typing of individual patient isolates showed that a different REAP type was identified only once every 138 days. Among 25 comparisons, seven sequential isolate pairs demonstrating REAP differences were also different by PFGE and IB. This likely represented the presence of more than one strain. Eighteen other pairs with REAP differences were identical or related to one another by PFGE and IB typing, and 17 of these differences were likely caused by a single genetic alteration within the same strain or clone. The rate of PFGE differences explicable by single genetic alterations among sequential isolates identical by REAP was similar to the overall rate for REAP differences in the whole collection. We conclude that REAP and PFGE typing differences explicable by single genetic alterations are relatively infrequent but not rare. These isolates should be examined by alternative typing systems to further support or refute clonality.