A transformation system for the yeast Candida utilis: use of a modified endogenous ribosomal protein gene as a drug-resistant marker and ribosomal DNA as an integration target for vector DNA.

We have developed a transformation system for the yeast Candida utilis. A novel strategy was applied to construct the transformation system, since auxotrophic mutants which could be used as hosts for transformation are not available. A gene encoding the ribosomal protein L41 was cloned from C. utilis, which is sensitive to cycloheximide, and used as a marker gene conferring cycloheximide resistance after modification of its amino acid sequence. The marker gene was constructed by substitution of the proline codon at position 56 with the glutamine codon by in vitro mutagenesis, as it had been reported previously that the 56th amino acid residue of L41 is responsible for the cycloheximide sensitivity of various organisms (S. Kawai, S. Murao, M. Mochizuki, I. Shibuya, K. Yano, and M. Takagi, J. Bacteriol. 174:254-262 1992). The ribosomal DNA (i.e., DNA coding for rRNA) of C. utilis was also cloned and used as a multiple-copy target for the integration of vector DNA into the genome, which resulted in a high transformation efficiency. Transformants were obtained by electroporation with a maximum efficiency of approximately 1,400 transformants per 1 microgram of linearized DNA carrying the gene for cycloheximide resistance and part of the ribosomal DNA. No transformants were obtained with intact plasmids. Multiple copies of the linearized plasmid were integrated into the host chromosome by homologous recombination. Southern analysis of the transformants in which vector DNA was integrated at the L41 gene locus indicated that there are two copies of gene for the L41 protein per cell, suggesting that C. utilis is diploid. Transformants were obtained from a variety of C. utilis strains, indicating that this method is applicable to the transformation of other C. utilis strains, even though there is significant heterogeneity in chromosomal karyotypes among these strains.     

Acute nonlymphocytic leukemia (M2) with chromosome abnormality trisomy 4 developing eight years after radiation therapy for breast cancer

We report here the development, 8 years after radiation therapy for breast cancer, of acute nonlymphocytic leukemia (ANLL), type M2 of the FAB classification, in which trisomy 4 was detected as the only chromosomal abnormality. Simultaneous observation of cytologic and cytogenetic features of individual colonies derived from leukemic progenitor (L-CFU) and early progenitor (CFU mix) cultures in this patient revealed that all colonies examined had a normal karyotype, although the clone with trisomy 4 was predominant in the direct bone-marrow culture. These findings suggest that progenitor cells with trisomy 4 were less predominant in colony growth when stimulated by colony-stimulating factors (CSFs) than were stem cells with a normal karyotype.     

Polymorphism of pentanucleotide repeats in the 5′ flanking region of glutathione S-transferase (GST) π gene

The upstream sequence of the glutathione S-transferase gene contains pentanucleotide (ATAAA) repeats. Analysis of the region using polymerase chain reaction indicated that the repeat sequence was polymorphic and segregation of the polymorphic alleles was codominant heredity. Heterozygosity of the new VNTR was 0.818 in healthy Japanese and 0.794 in American whites. Allelic frequencies among healthy controls and alcoholics as well as other diseases were not significantly different.     

Role of T‐bet,the master regulator of Th1 cells,in the cytotoxicity of murine CD4+ T cells

Although CD4⁺ T cells are generally regarded as helper T cells, some activated CD4⁺ T cells have cytotoxic properties. Given that CD4⁺ cytotoxic T lymphocytes (CTLs) often secrete IFN‐γ, CTL activity among CD4⁺ T cells may be attributable to Th1 cells, where a T‐box family molecule, T‐bet serves as the “master regulator”. However, although the essential contribution of T‐bet to expression of IFN‐γ has been well‐documented, it remains unclear whether T‐bet is involved in CD4⁺ T cell‐mediated cytotoxicity. In this study, to investigate the ability of T‐bet to confer cytolytic activity on CD4⁺ T cells, the T‐bet gene (Tbx21) was introduced into non‐cytocidal CD4⁺ T cell lines and their cytolytic function analyzed. Up‐regulation of FasL (CD178), which provided the transfectant with cytotoxicity, was observed in Tbx21transfected CD4⁺ T cells but not in untransfected parental cells. In one cell line, T‐bet transduction also induced perforin gene (Prf1) expression and Tbx21 transfectants efficiently killed Fas^? target cells. Although T‐bet was found to repress up‐regulation of CD40L (CD154), which controls FasL‐mediated cytolysis, the extent of CD40L up‐regulation on in vitro‐differentiated Th1 cells was similar to that on Th2 cells, suggesting the existence of a compensatory mechanism. These results collectively indicate that T‐bet may be involved in the expression of genes, such as FasL and Prf1, which confer cytotoxicity on Th1 cells.

     

Improved method for the determination of trimethadione and its demethylated metabolite, dimethadione, in human serum by gas chromatography

An improved gas chromatographic method, involving the use of a wide-bore capillary column, for the determination of trimethadione and its only demethylated metabolite, dimethadione, in human serum is described. The results indicate that both substances and the internal standard (maleinimide) were well separated with no tailing peak. The detection limit was 10 ng/ml for trimethadione and 50 ng/ml for dimethadione. This improved method is reliable in terms of sensitivity, selectivity and reproducibility for the simultaneous determination of both compounds in human serum.     

Analysis of Potential Radiosensitizing Materials for X-Ray-Induced Photodynamic Therapy

For a development of deep tumor treatment in photodynamic therapy, a feasibility of novel radiosensitizers induced by x-ray was investigated. The sensitizers are designed to generate reactive oxygen species (ROS) inside or outside the cell, possibly leading to damage exclusively on tumor cells and reservation of normal cells along the x-ray path. Taking note of the similarity in energy transfer mechanism in photocatalysts, scintillators, and particulate semiconductors, we chose TiO₂, ZnS:Ag, CeF₃, and quantum dots (CdTe and CdSe) in particulate form, which contain heavy atoms for efficient absorption of x-rays. A parameter study for x-ray operating conditions showed that in a typical scenario, photons with 20 to 170 keV energy are attenuated by 90% through the region of particle dispersed aqueous solution at varying concentration between 0.01 and 100 wt%. The amount of ROS generation under the exposure of polychromatic x-ray was measured using dihydroethidium reagent which detects an integrated amount of several species. Proportional increase in ROS generation to x-ray dose was observed for varying concentrations of TiO₂, ZnS:Ag, CeF₃, and CdSe quantum dot dispersions. Then, HeLa cells were mixed with aqueous solutions dispersed with sensitizing materials at a concentration of 3.0 mg/ml and were exposed to x-ray. Their survival fraction obtained by a cell proliferation reagent WST-1 immediately after the irradiation showed insignificant effects of sensitizing materials except at large doses. To enhance the sensitization effect, bio-conjugated CdSe quantum dots were internalized in the cytoplasm up to a concentration of 1.0 ng/ml. The cells were irradiated by x-ray up to 5 Gy, and their survival fraction was measured by the colony forming ability 9 days after irradiation. Survival fraction of the cells treated with quantum dots were less than those without quantum dots for all doses, suggesting that the colony forming ability is impaired by the internalized quantum dots.     

Production of plant virus inhibitor by Phytolacca americana suspension culture.

The inhibitory activity of tobacco mosaic virus (TMV) infection was assayed with the extracts of various callus tissues derived from the intact plants. Phytolacca americana callus was selected as a producer of the virus inhibitor and its cultural conditions in suspension were examined for cell growth and the inhibitor production. A modified liquid medium containing twofold concentrations of all components in that of Murashige and Skoog plus2,4-D (1.0 mg/liter) and sucrose (6%), but without any vitamins and glycine was chosen for production of higher levels of the inhibitor. TMV infections in tobacco, bean, and tomato plants were markedly inhibited by the introduction of the disrupted whole broth of suspension cultured P. americana.     

Differential effects of undernourishment on the differentiation and maturation of rat enteric neurons

The colocalization, number, and size of various classes of enteric neurons immunoreactive (IR) for the purinergic P2X₂ and P2X₇ receptors (P2X₂R, P2X₇R) were analyzed in the myenteric and submucosal plexuses of control, undernourished, and re-fed rats. Pregnant rats were exposed to undernourishment (protein-deprivation) or fed a control diet, and their offspring comprised the following experimental groups: rats exposed to a normal diet throughout gestation until postnatal day (P)42, rats protein-deprived throughout gestation and until P42, and rats protein-deprived throughout gestation until P21 and then given a normal diet until P42. Immunohistochemistry was performed on the myenteric and submucosal plexuses to evaluate immunoreactivity for P2X₂R, P2X₇R, nitric oxide synthase (NOS), choline acetyltransferase (ChAT), calbindin, and calretinin. Double-immunohistochemistry of the myenteric and submucosal plexuses demonstrated that 100% of NOS-IR, calbindin-IR, calretinin-IR, and ChAT-IR neurons in all groups also expressed P2X₂R and P2X₇R. Neuronal density increased in the myenteric and submucosal plexuses of undernourished rats compared with controls. The average size (profile area) of some types of neurons in the myenteric and submucosal plexuses was smaller in the undernourished than in the control animals. These changes appeared to be reversible, as animals initially undernourished but then fed a normal diet at P21 (re-feeding) were similar to controls. Thus, P2X₂R and P2X₇R are present in NOS-positive inhibitory neurons, calbindin- and calretinin-positive intrinsic primary afferent neurons, cholinergic secretomotor neurons, and vasomotor neurons in rats. Alterations in these neurons during undernourishment are reversible following re-feeding.     

Production of Nucleic Acid-Related Substances by Fermentative Processes

The presence of psicofuranine in the fermentation medium caused the accumulation of a copious amount of 5′–XMP by Brevibacterium ammoniagenes. The accumulation of 5′–XMP in the medium was considered to be due to the inhibition of converting 5′–XMP to 5′–GMP by psicofuranine, which is known as a specific inhibitor of XMP aminase.

It was previously reported that in 5′–IMP fermentation with Br. ammoniagenes pantothenate and thiamine, in addition to biotin which was required for the growth of the microorganism, were exclusively required. This requirement for both vitamins was also observed in 5′–XMP production induced by the antibiotic.

The addition of manganese in excess to the fermentation medium promoted the bacterial growth greatly and inhibited IMP production, whereas XMP production induced by piscofuranine was not affected by the addition of excess manganese.

The accumulation of XMP induced by the antibiotic was completely suppressed by the presence of purine derivatives such as guanine, and xanthine derivatives, and partially by hypoxanthine.

5′–XMP was identified by chemical and enzymatic analyses and by UV absorption spectrum.