Characterization of Nine Novel Mutations in the CD40 Ligand Gene in Patients with X-Linked Hyper IgM Syndrome of Various Ancestry

X-linked immunodeficiency with hyper-IgM (HIGMX-1) is a rare disorder caused by defective expression of the CD40 ligand (CD40L) by activated T lymphocytes, resulting in inefficient T-B cell cooperation and failure of B cells to undergo immunoglobulin isotype switch. In the present work, we describe nine patients of various ancestry who bear different mutations in the X chromosome–specific CD40L gene. Two of the mutations were nonsense mutations, one each resulting in premature stop codons at amino acid residues 39 and 140. Three patients had single point missense mutations, one each at codons 126, 140, and 144. Another patient had a 4-bp genomic deletion in exon 2, resulting in a frameshift and premature termination. Three patients showed insertions, one each of 1, 2, and 4 nt, probably because of polymerase slippage, resulting in frameshift mutation and premature termination. Overall, these observations confirm the heterogeneity of mutations in HIGMX-1. However, the identification of two patients whose mutation involves codon 140 (previously shown to be altered in two other unrelated subjects) suggests that this may be a hotspot of mutation in HIGMX-1. In two additional patients with clinical and immunological features indistinguishable from canonical HIGMX-1, no mutation was detected in the coding sequence, in the 5' flanking region, or in the 3' UTR.     

Chromatographic resolution of some cyclic sulfoximides derived from prochiral and chiral sulfoxides

Three endocyclic sulfoximides of the 1-aryl- and 1-alkyl-3-oxo-benzo[d]-isothia (IV)-azole 1-oxide type (1-substituent = 2′-carboxyphenyl, 2′-carbethoxyphenyl, and octyl, respectively) were found to be well resolved on a chiral phase derived from bovine serum albumin (BSA). Selectivities (α) of 1.74, 1.12, and 1.44, respectively, were obtained. The retention behaviour of 1-octyl-3-oxo-benzo[d]isothia(IV)-azole 1-oxide was further investigated in some detail as a function of the mobile phase composition and the elution order was established from optically active material obtained from the enantiopure sulfoxide precursor. An enantiomeric excess of 85.4% was obtained in the cyclocondensation reaction of the octyl-substituted sulfoxide precursor with hydrazoic acid to the corresponding endocyclic sulfoximide. © 1995 Wiley-Liss, Inc.     

The PiGMaP consortium linkage map of the pig (Sus scrofa)

A linkage map of the porcine genome has been developed by segregation analysis of 239 genetic markers. Eighty-one of these markers correspond to known genes. Linkage groups have been assigned to all 18 autosomes plus the X Chromosome (Chr). As 69 of the markers on the linkage map have also been mapped physically (by others), there is significant integration of linkage and physical map data. Six informative markers failed to show linkage to these maps. As in other species, the genetic map of the heterogametic sex (male) was significantly shorter (16.5 Morgans) than the genetic map of the homogametic sex (female) (21.5 Morgans). The sex-averaged genetic map of the pig was estimated to be 18 Morgans in length. Mapping information for 61 Type I loci (genes) enhances the contribution of the pig gene map to comparative gene mapping. Because the linkage map incorporates both highly polymorphic Type II loci, predominantly microsatellites, and Type I loci, it will be useful both for large experiments to map quantitative trait loci and for the subsequent isolation of trait genes following a comparative and candidate gene approach.     

Purification and characterization of a vanadate-sensitive nucleotide tri- and diphosphatase with acid pH optimum from rat bone

Rat bone was extracted with KCl and Triton X-100, and a tartrate-resistant acid phosphatase activity was purified by protamine sulfate precipitation, ion-exchange chromatography (CM-cellulose), and gel filtration on Sephadex G-200 according to previously described procedures. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and silver staining demonstrated a major band with an apparent monomer molecular size of approximately 14,000 Da. The enzyme is active with p-nitrophenylphosphate (p-NPP) but exhibits a 5- to 10-fold higher affinity towards several nucleotides of which ATP and ADP are the most readily hydrolyzed substrates based on kinetic studies. Based on sensitivity towards proteolytic treatment and detergent removal, as well as pH-optimum studies, a single enzyme was found to be responsible for activity towards nucleotide phosphates as well as p-NPP. This nucleotide tri- and diphosphatase constitutes around 15% of the total acid phosphatase activity in rat bone. The activity with ATP as substrate in contrast to that with p-NPP was inhibited in a noncompetitive fashion by MgCl2, sodium metavanadate, and p-chloromercuribenzoate. Enzyme activity with p-NPP and ATP is dependent on the presence of KCl and detergent and is activated by Fe3+ and ascorbate. The reported characteristics of the enzyme suggest that it functions as a unique membrane acid ATPase.     

Microvilli in sea urchin eggs : Differences in their formation and type

The elongation of microvilli, which normally occurs upon fertilization in sea urchin eggs, was also observed in unfertilized eggs treated with the enzyme, papain. Cortical granule exocytosis, which is thought to be the source of membrane used in microvillar elongation, does not occur in the papain-treated eggs. It appears, therefore, that there is more than one way in which the egg plasma membrane can increase very quickly and to a great extent. In addition, the kinds of microvilli formed in the two instances appear to be different. Previous work with reaggregating sea urchin cells is also cited to support the suggestion that microvilli can form in different ways and are of different types.     

Selective radioactive labeling of cell surface sialoglycoproteins by periodate-tritiated borohydride.

Low concentrations of sodium metaperiodate induce specific oxidative cleavage of sialic acids between carbon 7 and carbon 8 or carbon 8 and carbon 9. The aldehydes formed can easily be reduced with NaB3H4 to tritiated 5-acetamido-3,5-dideoxy-L-arabino-2-heptulosonic acid or 5-acetamido-3,5-dideoxy-L-arabino-2-octulosonic acid. At 0 degrees, the periodate anion penetrates the cell plasma membrane very slowly and only externally exposed sialic acids are oxidized. This was shown by (a) limited labeling of the sialoglycoproteins in a preparation of inside-out erythrocyte vesicles; (b) trapping 14C-labeled fetuin within resealed erythrocyte ghosts; fetuin was then poorly labeled, whereas the erythrocyte sialoglycoproteins were highly labeled; (c) comparison of labeled glycoproteins of mouse lymphoid cells before and after treatment with neuraminidase. This simple method of specifically introducing a radioactive label into cell surface sialic acids is useful in the study of cell surface sialic acid-containing glycoproteins.     

Improvement of anther culture technique: Activated charcoal bound in agar medium in combination with liquid medium and elevated CO2 concentration

Anthers of different species of the genera Anemone, Clematis, Papaver and Nicotiana were cultured by floating on a liquid medium which overlay an agarified charcoal medium . This technique proved to be superior to conventional methods i.e. culture on either solid or liquid media. Cold treatment of Anemone anthers for 7 days after inoculation on the double layer medium gave about the same frequency of embryos per anther as corresponding cultures cold treated before inoculation. An elevation of the CO₂ concentration to 2% stimulated embryogenesis in anther cultures of Anemone canadensis, Anemone vitifolia, Papaver setigerum and Papaver radicatum . Cold treatment of cultures of Anemone canadensis inhibited embryogenesis if the ensuing culture was performed in 2% CO_2. On the other hand, cold treatment was stimulating, with an optimum of about 20 days, if the cultures were maintained in normal air. Chemical analysis of untreated anthers of Anemone canadensis showed the presence of abscisic acid (2.2 × 10⁻⁶ g/g anthers). Cold treatment reduced the concentration of abscisic acid to 0.6 × 10⁻⁶ g/g anthers. By use of assays with Lemna gibba as test organism, activated charcoal was shown to adsorb abscisic acid that was added to the medium. Medium treated with charcoal before inoculation of anthers of Anemone canadensis provided to inhibit embryo production.     

The effects of post-treatments with caffeine during S and G2 on the frequencies of chromosomal aberrations induced by thiotepa in root tips of Vicia faba and in human lymphocytes in vitro

The effects of post-treatments with caffeine on the frequencies of chromosomal aberrations induced by the trifunctional alkylating agent thiotepa were studied in human lymphocytes and in root tips of Vicia faba. In lymphocytes the frequency of aberrations induced in G0 or G1 was most strongly increased when the caffeine post-treatments were given during G2. In Vicia faba, on the other hand, the frequency of aberrations induced in early interphase was unaffected by post-treatments with caffeine during G2, but strongly increased when the root tips were exposed to caffeine during the S phase.     

Chemical modification of enzymes: Critical evaluation of the graphical correlation between residual enzyme activity and number of groups modified

In the study of chemical modification of enzymes and other biologically active proteins, plots of fractional residual activity as a function of number of groups modified per enzyme molecule are often used to establish a correlation between the chemical modification and enzyme inactivation reactions and to determine the stoichiometry of the modification reaction. This paper presents a critical examination of the underlying theoretical framework of such graphs. Whereas these plots are usually presented as linear functions, it is shown here that the general equation describing the relationship between inactivation and modification contains an exponential term; therefore, in the general case, the plot is actually a curve. It is suggested that caution be exercised in the interpretation of such plots and that equations such as those derived in the text be used to fit theoretical curves to the data, in order to maximize the information gained from chemical modification experiments.