Somatic mosaicism in two unrelated patients with X-linked chronic granulomatous disease characterized by the presence of a small population of normal cells

X-linked chronic granulomatous disease (X-CGD) is a primary immunodeficiency disease of phagocytes caused by mutations in the cytochrome b(558)β (CYBB) gene. We, for the first time, detected somatic mosaicism in two unrelated male patients with X-CGD caused by de novo nonsense mutations (p.Gly223X and p.Glu462X) in the CYBB gene. In each patient, a small subset of granulocytes was normal in terms of respiratory burst (ROB) activity, gp91(phox) expression, and CYBB sequences. Cells with wild-type CYBB sequence were also detected in buccal swab specimens and in peripheral blood mononuclear cells. The normal cells were shown to be of the patient origin by fluorescent in situ hybridization analysis of X/Y chromosomes, and by HLA DNA typing. Two possible mechanisms for this somatic mosaicism were considered. The first is that the de novo disease-causing mutations in CYBB occurred at an early multicellular stage of embryogenesis with subsequent expansion of the mutated cells, leaving some unmutated cells surviving. The second possibility is that the de novo mutations occurred in oocytes which was followed by reversion of the mutations in a small subset of cells in early embryogenesis.     

Suitability of lipid materials for culture ofMalassezia as evaluated from its cellular fatty acid composition

Malassezia is a facultative or obligatory lipophilic yeast. We devised new lipid-supplemented media suitable for the culture ofMalassezia. Malassezia furfur andM. pachydermatis grew well on both solid and liquid media supplemented with creaming powder preparations which are commercially available at moderate prices. Striking differences were found between the cellular fatty acid compositions ofM. furfur grown on media supplemented with creaming powder and that grown on media with conventional olive oil.Malassezia furfur grown on media with olive oil had nearly the same fatty acid composition as olive oil, with C18:1 amounting to 80%, while that grown on media supplemented with creaming powder had C16, C18:1 and C18:2 as the principal components. The use of these supplementary lipids appeared not to inhibit the normal synthesis of fatty acid inM. furfur. For the culture ofM. pachydermatis, media supplemented with creaming powder were also found more suitable than lipid-free media. The media devised are considered excellent, because they appear to provide a more natural growth environment forMalassezia.     

The binding of VIP36 and alpha-amylase in the secretory vesicles via high-mannose type glycans

Vesicular integral protein of 36 kDa (VIP36) is an intracellular lectin recognizing high-mannose type glycans and is highly expressed in salivary glands, especially the parotid gland, which secretes alpha-amylase in large quantities. Here immunoelectron microscopy demonstrated that VIP36 was primarily localized to secretory vesicles in the glandula parotis of the rat, where alpha-amylase also resided. A secretory vesicle fraction, prepared by Percoll density gradient centrifugation, contained both VIP36 and alpha-amylase. Moreover, alpha-amylase that was localized to these secretory vesicles contained high-mannose type glycans. In addition, VIP36 coprecipitated with alpha-amylase in an endo H treatment-sensitive manner. These results suggest that VIP36 is involved in the secretion of alpha-amylase in the rat parotid gland.     

Unique sensitivity to alpha-galactosylceramide of NKT cells in the uterus

It was previously reported that NKT cells, which are mainly present in the liver of mice, are also present in the uterus and that these uterine NKT cells are associated with abortion under overactivated conditions. In this study, we further examined their phenotypic and functional properties. In parallel with the progression of pregnancy, the number of uterine lymphocytes increased. This increase accompanied an increase in the number of TCRalphabeta(+) T cells and NKT cells in the uterus, although the number of NKT cells decreased at late pregnancy. Approximately one-third of the TCRalphabeta(+) cells were NKT cells at the early pregnant stage, while this ratio decreased toward late pregnancy. These uterine NKT cells were found to respond to alpha-galactosylceramide (alpha-GalCer) differently in comparison with liver NKT cells. In contrast to the apoptotic response of liver NKT cells on day 1 after alpha-GalCer injection, uterine NKT cells expanded prominently without such apoptosis. The majority of liver NKT cells were CD4(+). In contrast, almost all of the uterine NKT cells were double negative CD4(-)8(-). However, similar to liver NKT cells, uterine NKT cells used an invariant chain of Valpha14Jalpha281 gene for TCRalpha. The resistance against apoptosis might be due to the high expression of bcl-2 on uterine NKT cells after alpha-GalCer activation. Other evidence was that macrophages which existed in the pregnant uterus carried an activation marker, CD69, and could potentially produce many cytokines by their activation. The present results suggest that uterine NKT cells and surrounding macrophages are quite different in function from those in the liver.     

Dynamic simulation of an in vitro multi-enzyme system

Parameters often are tuned with metabolite concentration time series data to build a dynamic model of metabolism. However, such tuning may reduce the extrapolation ability (generalization capability) of the model. In this study, we determined detailed kinetic parameters of three purified Escherichia coli glycolytic enzymes using the initial velocity method for individual enzymes; i.e., the parameters were determined independently from metabolite concentration time series data. The metabolite concentration time series calculated by the model using the parameters matched the experimental data obtained in an actual multi-enzyme system consisting of the three purified E. coli glycolytic enzymes. Thus, the results indicate that kinetic parameters can be determined without using an undesirable tuning process.     

Identification of amino acid substitutions that render the Arabidopsis cytokinin receptor histidine kinase AHK4 constitutively active

In Arabidopsis, three genes (AHK2, AHK3 and AHK4/CRE1) encode histidine kinases (His-kinases), which serve as cytokinin receptors. To understand how the external cytokinin signal activates the His-kinase across the cell membrane, we exploited the power of microbial genetics to isolate several AHK4 mutants that function independently of cytokinin in both prokaryotic and eukaryotic assay systems. In each mutant, a single amino acid substitution within the second membrane-spanning segment, or within the region around the phosphorylation His site, renders the His-kinase constitutively active. These mutant receptors appear to have a 'locked-on' conformation, even in the absence of stimulus. We discuss the implications of these data for the structure and function of the cytokinin receptor His-kinases in plants.     

Brassinolide promotes adventitious shoot regeneration from cauliflower hypocotyl segments

When hypocotyl segments of cauliflower (Brassica oleracea var. boturytis L.) were cultured on MS medium containing brassinolide (BR) in the light, a significant stimulation of adventitious shoot regeneration was observed. Cytokinins (zeatin and iso-pentenylaminopurine) also promoted shoot regeneration. When BR was added together with these cytokinins, the maximal regeneration was strongly improved and the dose–response curve of cytokinin was shifted to the left. Regeneration was much lower in the dark. This was not due to a possible increased ethylene synthesis in the dark.