Thymidine phosphorylase inhibits the expression of proapoptotic protein BNIP3

An angiogenic factor, thymidine phosphorylase (TP), confers resistance to apoptosis induced by hypoxia. We investigated the molecular basis for the suppressive effect of TP on hypoxia-induced apoptosis using Jurkat cells transfected with TP cDNA, Jurkat/TP, and a mock transfectant, Jurkat/CV. TP and 2-deoxy-d-ribose, a degradation product of thymidine generated by TP enzymatic activity, suppressed hypoxia-induced apoptosis. They also inhibited the upregulation of hypoxia-inducible factor (HIF) 1α and the proapoptotic factor, BNIP3, and caspase 3 activation induced by hypoxia. Introduction of siRNA against BNIP3 in Jurkat cells decreased the proportion of apoptotic cells under hypoxic condition. These findings suggest that the suppression of BNIP3 expression by TP prevents, at least in part, hypoxia-induced apoptosis. Expression levels of TP are elevated in many malignant solid tumors and thus 2-deoxy-d-ribose generated by TP in these tumors might play an important role in tumor progression by preventing hypoxia-induced apoptosis.     

The Unilateral Urogenital Anomalies (UUA) Rat: A New Mutant Strain Associated with Unilateral Renal Agenesis, Cryptorchidism, and Malformations of Reproductive Organs Restricted to the Left Side

We established an inbred rat strain with unilateral urogenital anomalies from an incidentally identified male rat with unilateral renal agenesis and an undescended left testis. These rats were characterized by unilateral renal agenesis in both sexes, undescended testes with agenesis and hypoplasia of the accessory sex organs in male rats, and complete and partial agenesis of the uterine horn in female rats. All of these urogenital anomalies were unilateral and restricted to the left side; we named this phenotype unilateral urogenital anomalies (UUA). Breeding tests showed that these abnormalities were inherited as polygenic traits. The weight of right kidneys of affected rats was 1.7-fold higher than that of normal rats; histologically, glomerulosclerosis, tubular dilations, and tubular casts were detected at 30 wk of age. These alterations may have resulted from compensatory renal adaptation to the lack of 1 kidney. The cryptorchid left testes of affected male rats showed atrophy of seminiferous tubules and degeneration of spermatocytes and spermatids. These results indicate that the UUA rat may be a good model to study the etiology of unilateral renal agenesis accompanied by agenesis of the reproductive tract and to study compensatory alterations resulting from the congenital loss of 1 kidney.Abbreviations: A, affected; F, female; FUBI, failure of ureteric bud invasion; M, male; N, normal; UUA, unilateral urogenital anomaliesUnilateral renal agenesis (URA) is a common malformation of the kidney,¹⁵ occurring in 1:1000 to 1:500 persons.²⁴ This disorder often is associated with ipsilateral absence of the deferent duct in men and hypoplasia of the uterine horn in women.²⁴ Although some cases of familial renal agenesis have been reported,^2,11 the genetic etiology of this condition is almost completely unknown. Unilateral renal agenesis also occurs in other species, including dogs,¹² cats,¹⁴ and guinea pigs.¹⁶ Although spontaneously mutant strains of renal agenesis in mice⁹ and rats^6,18 have been reported, the genes responsible for renal agenesis have not yet been identified.We identified a male rat with renal agenesis and an undescended left testis in a group of rats obtained from a laboratory animal supplier. Using this rat as a founder, we started brother–sister breeding to establish an inbred rat strain that could be used as an animal model in the study of unilateral renal agenesis. We named the rat phenotype unilateral urogenital anomalies (UUA). In the present study, we show that UUA is a congenitally genetic disorder characterized by unilateral renal agenesis with agenesis and hypoplasia of the reproductive organs restricted to the left side.     

Manipulation of major membrane lipid synthesis and its effects on sporulation in Saccharomyces cerevisiae

During the sporulation process of Saccharomyces cerevisiae, meiotic progression is accompanied by de novo formation of the prospore membrane inside the cell. However, it remains to be determined whether certain species of lipids are required for spore formation in yeast. In this study, we analyzed the requirement of the synthesis of phosphatidylethanolamine (PE), phosphatidylcholine (PC), and ergosterol for spore formation using strains in which the synthesis of these lipids can be controlled. When synthesis of PE and PC was repressed, sporulation efficiency decreased. This suggests that synthesis of these phospholipids is vital to proper sporulation. In addition, sporulation was also impaired in cells with a lowered sterol content, raising the possibility that sterol content is also important for spore formation.     

Pentose Metabolism in Candida utilis

In attempts to obtain GMP producing strains, Brevibacterium ammoniagenes was treated with UV, N.T.G. or D.E.S. as a mutagen. Adenine-guanine requiring mutants were obtained from an adenine-requiring mutant of Brev. ammoniagenes, KY 3482–9 and two of them, presumably adenine-xanthine requiring mutants, were then reverted to mutants which required only adenine for their growth.

Although these revertants were not able to accumulate a copious amount of GMP, most of them and of adenine-guanine requiring mutants produced larger amounts of IMP than the parent adenine-requiring strain.

Effects of Mn²⁺ and purine bases in the medium on IMP production by these mutants were examined and IMP productivities of these mutants were compared with the parent strain under optimal conditions.

These mutagenic treatments were thus proved to be effective for the increase of de novo IMP production by Brev. ammoniagenes mutants.

Brevibacterium ammoniagenes ATCC 6872 accumulates 5′-GDP and -GTP, or 5′-ADP and -ATP together with GMP or AMP in nucleotide fermentation by salvage synthesis.

With cell free extract of this strain, transphosphorylating reactions of AMP or GMP were investigated.

ATP-AMP transphosphorylating enzyme(s) was partially purified to 21.7 fold with acid treatment, salting-out and column chromatography.

In ATP-AMP and ATP-GMP transphosphorylating reactins, optimal conditions were decided such as for concentrations of enzyme, of MgCl₂ and of phosphate donor, pH and cell age as the enzyme sources.

Specificities of phosphate donors and acceptors were examined with both the partially purified enzymes or the sonicate. AMP and GMP were phosphorylated by ATP rapidly, but IMP and XMP were not, therefore supporting our previous finding that Brev. ammoniagenes could not accumulated IDP, ITP, XDP and XTP in IMP and XMP fermentation, respectively.

Although ATP was the best donor for both AMP and GMP phosphorylations, other nucleoside triphosphates and PRPP were used as phosphate donors.

Furthermore, phosphorylation of ADP to ATP was investigated and possible mechanisms of nucleoside di- or triphosphates synthesis in the nucleotide fermentation were discussed.

From these results, it is suggested as a possible mechanism for nucleoside di- and triphosphate accumulation by Brev. Ammoniagenes, that a nucleoside monophosphate formed is phosphorylated to a nucleoside di-phosphate with ATP or other phosphate donors and then the nucleoside diphosphate is converted to a triphosphate with these phosphate donors.

Both AMP and GMP were transphosphorylated rapidly to the corresponding nucleoside-diphosphates and triphosphates by ATP and by other high energy phosphate compounds with cell free extracts of Brevibacterium ammoniagenes.

Some enzyme inhibitors, such as metals and PCMB were shown to inhibit the phosphorylations of AMP and GMP. Higher levels of ATP, ADP, GTP and GDP also inhibited the activity of the partially purified ATP-AMP transphosphorylating enzyme(s).

In guanine nucleotides fermentation by salvage synthesis with this strain, addition of these inhibitors to the medium increased the amounts of GMP and total guanine nucleotides accumulated.

On the contrary, supplement of xylene or of other organic solvents to the medium stimulated the accumulation of both GTP and total guanine compouuds in this fermentation. From enzymatic studies, these solvents are presumed to have the ability to change cell permeability.

Such findings give an effective method for controlling the amounts of nucleotides accumulated in these fermentations.     

Determination of the Role of Cold Acclimation-Induced Diverse Changes in Plant Cells from the Viewpoint of Avoidance of Freezing Injury

Received 4 January 1999/ Accepted in revised form 7 April 1999     

Microbody defective mutants of arabidopsis

In germinating fatty seedlings, microbodies are differentiated to leaf peroxisomes from glyoxysomes during greening, and then transformed to glyoxysomes from leaf peroxisomes during senescence. These transformations of microbodies are regulated at various level, such as gene expression, splicing of the mRNA and degradation of microbody proteins. In order to clarify the regulatory mechanisms underlying these transformations of microbodies, we tried to obtain glyoxysome-deficient mutants of Arabidopsis. We screened 2,4-dichlorophenoxybutyric acid (2,4-DB) mutants of Arabidopsis which have defects in glyoxysomal fatty acid β-oxidation. Four mutants can be classified as carrying alleles at three independent loci, which we designatedped1, ped2, andped3, respectively (whereped stands for peroxisome defective). The characteristics of theseped mutants are described. The extended abstract of a paper presented at the 13th International Symposium in Conjugation with Award of the International Prize for Biology “Frontier of Plant Biology”     

Relationship between Germination and PFR Level in Spores of the Fern Lygodium japonicum

The relationship between germination and P_FR level in sporesof the fern Lygodium japonicum was investigated. Percent P_FRestimated from direct spectrophotometric measurement of sporesincreased with the logarithm of total fluence of 660 nm-light.The transformation from P_R to P_FR was saturated by giving ca.200 Jm^–2 of 660 nm-light and half-saturated by ca. 55J^–2 of 660 nm-light. Clear positive correlation was observedbetween % P_FR levels and germination rates in spores irradiatedwith 660 nm and/or 730 nm-light, or with 686 or 700 nm-light.The P_FR percentage in spores was raised to 16–34% by blue(415 nm) light irradiation. This P_FR level was enough to causesome germination when produced by monochromatic light of redto far-red region, but blue light did not cause any germination. After 660 nm-light irradiation, the P_FR level decreased graduallyin darkness (25±1°C) and P_FR completely disappearedin 8 h, but 730 nm-light given even 16 h after 660 nm-lightirradiation inhibited germination. ⁴Present address: Tropical Botanic Garden and Research Institute,Navaranga Road, Trivandrum 695 011, India. (Received March 15, 1983; Accepted June 4, 1983)     

Difference between dorsal and ventral iris in lens producing potency in normal lens regeneration is maintained after dissociation and reaggregation of cells from the adult newt, Cynops pyrrhogaster

In Wolffian lens regeneration, lentectomized newt eye can produce a new lens from the dorsal marginal iris, but the ventral iris has never shown such capabilities. To investigate the difference of lens regenerating potency between dorsal and ventral iris epithelium at the cellular level, a transplantation system using cell reaggregates was developed. Two methods were devised for preparing the reaggregates from pigmented iris epithelial cells. One was rotating cells in an agar-coated multiplate on a gyratory shaker and the other was incubating cells in a microcentrifuge tube after slight centrifugation. Reaggregates made of dorsal iris cells that had been completely dissociated into single cells were phenotypically transformed into a lens when placed in the pupillary region of the lentectomized host eye. None of the ventral reaggregates produced a lens. Even dorsal reaggregates could not transdifferentiate into lens when they were placed away from the pupil. The produced lenses from the reaggregates were morphologically and immunohistochemically identified. To obtain evidence whether produced lenses really originated from singly dissociated cells, we labeled dissociated cells with a fluorescent dye (PKH26) before reaggregate formation and then traced it in the produced lens.