Morphogenetic movements during axolotl neural tube formation tracked by digital imaging

During neurulation in vertebrate embryos, epithelial cells of the neural plate undergo complex morphogenetic movements that culminate in rolling of the plate into a tube. Resolution of the determinants of this process requires an understanding of the precise movements of cells within the epithelial sheet. A computer algorithm that allows automated tracking of epithelial cells visible in digitized video images is presented. It is used to quantify the displacement field associated with morphogenetic movements in the axolotl (Ambystoma mexicanum) neural plate during normal neural tube formation. Movements from lateral to medial, axial elongations and area changes are calculated from the displacement field data and plotted as functions of time. Regional and temporal differences are identified. The approach presented is suitable for analyzing a wide variety of morphogenetic movements.     

Stimulation of natural killer (NK) and antibody-dependent cellular cytotoxicity (ADCC) activities in murine leukocytes by bombesin-related peptides requires the presence of adherent cells

Bombesin and the two mammalian bombesin-related peptides, gastrin-releasing peptide (GRP) and neuromedin C, at physiological concentrations have been previously shown to stimulate significantly in vitro the antibody-dependent cellular cytotoxicity (ADCC) and natural killer (NK) activities in BALB/c mouse leukocytes from axillary nodes, spleen and thymus. In the present work we have shown that adherent cells are required in leukocyte samples for stimulation of cytotoxicity by the neuropeptides, which suggests that this effect may be mediated by those cells. Here we demonstrate the specificity of the effects by reversing them in the presence of the bombesin-antagonist (Leu¹³-ΨCH₂NH-Leu¹⁴)-BN, and by detecting specific receptors for GRP on macrophages of high and low affinity. Using the same binding technics, no receptors for this neuropeptide were found in non-adherent leukocytes.     

Mutant Escherichia coli penicillin acylase with enhanced stability at alkaline pH

Increased stability at alkaline pH should be a valuable attribute for the utilization of penicillin acylase in bioreactors employed to convert penicillins into 6-aminopenicillanic acid, a precursor of semisynthetic penicillins. In these systems, base is added for pH control, which results in local alkaline conditions that promote enzyme inactivation. Hydrolysis and synthesis reactions are also pH dependent. Here, we report work in which the gene coding for Escherichia coli penicillin acylase was subjected to oligonucleotide-directed random mutagenesis at regions coding for amino acids predicted to be at the surface of the enzyme. The resulting mutant library, cloned in E. coli, was screened by a filter paper assay of the colonies for the presence of penicillin acylase activity with enhanced stability at alkaline pH. Characterization of one of the selected clones revealed the presence of a mutation, Trp431-Arg, which would presumably alter the surface charge of the protein. In vitro experiments demonstrated a near twofold increase in the half-life of the mutant enzyme when stored at pH 8.5 as compared with the wild-type enzyme, with a comparable specific activity at several pH values. In general, the mutant displayed increased stability toward the basic side in the pH-stability profile. (c) 1995 John Wiley & Sons, Inc.     

Exon and intron sequences, respectively, repress and activate splicing of a fibroblast growth factor receptor 2 alternative exon.

Two alternative exons, BEK and K-SAM, code for part of the ligand binding site of fibroblast growth factor receptor 2. Splicing of these exons is mutually exclusive, and the choice between them is made in a tissue-specific manner. We identify here pre-mRNA sequences involved in controlling splicing of the K-SAM exon. The short K-SAM exon sequence 5'-TAGGGCAGGC-3' inhibits splicing of the exon. This inhibition can be overcome by mutating either the exon's 5' or 3' splice site to make it correspond more closely to the relevant consensus sequence. Two separate sequence elements in the intron immediately downstream of the K-SAM exon, one of which is a sequence rich in pyrimidines, are both needed for efficient K-SAM exon splicing. This is no longer the case if either the exon's 5' or 3' splice site is reinforced. Furthermore, if the exon inhibitory sequence is removed, the intron sequences are not required for splicing of the K-SAM exon in a cell line which normally splices this exon. At least three elements are thus involved in controlling splicing of the K-SAM exon: suboptimal 5' and 3' splice sites, an exon inhibitory sequence, and intron activating sequences.     

Simulation of a reactor for glucose isomerization to fructose by immobilized glucose isomerase with continuous enzyme renewal

Two different dispositions of laboratory-scaled columns have been tested to simulate the isomerization of glucose to fructose in a mobile bed reactor where exhausted immobilized glucose isomerase is continuously renewed. If the simulation columns working at 65°C are arranged in parallel and connected to a section for final enzyme exploitation at 75°C, a syrup with constant composition can be produced, at relatively constant total throughput, by feeding the individual columns at flow rate decreasing according to the enzyme decay profile and following a programmed disphased mode of operation.This revised version was published online in October 2005 with corrections to the Cover Date.     

Are antibiotics indicated as initial treatment for children with acute otitis media? A meta-analysis.

OBJECTIVE: To determine the effect of antibiotic treatment for acute otitis media in children. DESIGN: Systematic search of the medical literature to identify studies that used antibiotics in randomised controlled trials to treat acute otitis media. Studies were examined blind, and the results of those of satisfactory quality of methodology were pooled. SUBJECTS: Six studies of children aged 7 months to 15 years. MAIN OUTCOME MEASURES: Pain, deafness, and other symptoms related to acute otitis media or antibiotic treatment. RESULTS: 60% of placebo treated children were pain free within 24 hours of presentation, and antibiotics did not influence this. However, at 2-7 days after presentation, by which time only 14% of children in control groups still had pain, early use of antibiotics reduced the risk of pain by 41% (95% confidence interval 14% to 60%). Antibiotics reduced contralateral acute otitis media by 43% (9% to 64%). They seemed to have no influence on subsequent attacks of otitis media or deafness at one month, although there was a trend for improvement of deafness at three months. Antibiotics were associated with a near doubling of the risk of vomiting, diarrhoea, or rashes (odds ratio 1.97 (1.19 to 3.25)). CONCLUSIONS: Early use of antibiotics provides only modest benefit for acute otitis media: to prevent one child from experiencing pain by 2-7 days after presentation, 17 children must be treated with antibiotics early.     

Studies of AIDS vaccination using an ex vivo feline immunodeficiency virus model: protection conferred by a fixed-cell vaccine against cell-free and cell-associated challenge differs in duration and is not easily boosted.

Cats immunized with cells infected with a primary isolate of feline immunodeficiency virus (FIV) and fixed with paraformaldehyde were challenged with cell-free or cell-associated homologous virus obtained ex vivo. Complete protection was observed in animals challenged with cell-free virus 4 months after completion of vaccination (p.v.) or with cell-associated virus 12 months p.v. In contrast, no protection was observed in cats challenged with cell-free virus 12 or 28 months p.v. or with cell-associated virus 37.5 months p.v. Prior to the 28- and 37.5-month challenges, the animals had received a booster dose of vaccine that had elicited a robust anamnestic immune response. These results show that vaccine-induced protection against ex vivo FIV is achievable but is relatively short-lived and can be difficult to boost.     

Expression of platelet membrane glycoproteins and alpha-granule proteins by a human erythroleukemia cell line (HEL)

We demonstrate that HEL, a human erythroleukemic cell line, has numerous megakaryocytic markers which were markedly enhanced following the addition of the inducers dimethyl sulfoxide or 12-O-tetradecanoylphorbol-13-acetate to the culture medium. Ultrastructural and cytochemical studies showed: (i) the presence of organelles morphologically resembling the platelet alpha-granules; and (ii) a peroxidase activity with the same characteristics as that specifically found in platelets. The platelet alpha-granule proteins (von Willebrand factor, platelet factor-4 and beta-thromboglobulin) were immunologically detected in the HEL cell cytoplasm and their amounts increased after induction. Of particular interest was the presence of platelet membrane proteins. A monoclonal antibody specific for glycoprotein Ib bound to HEL cells. Platelet membrane glycoproteins IIb and IIIa were identified on intact cells using specific antibodies in a binding assay or in cell lysates using either crossed immunoelectrophoresis or an immunoblotting procedure following SDS-polyacrylamide gel electrophoresis. Most HEL cells also expressed the platelet alloantigen PIA1. All of the platelet membrane proteins were present in higher amounts after induction. Glycophorin A, specific for the erythroid lineage, was also detected on HEL cells. Thus, while confirming the presence of erythroid markers, our studies provide evidence that the HEL cell line also expresses platelet antigens. As such, HEL cells represent a unique system with which to study the biosynthesis of platelet-specific proteins and glycoproteins.