Evidence for WT1 as a Wilms tumor (WT) gene: intragenic germinal deletion in bilateral WT.

The inactivation of two alleles at a locus on the short arm of chromosome 11 (band 11p13) has been suggested to be critical steps in the development of Wilms tumor (WT), a childhood kidney tumor. Two similar candidate WT cDNA clones (WT33 and LK15) have recently been identified on the basis of both their expression in fetal kidney and their location within the smallest region of overlap of somatic 11p13 deletions in some tumors. These homozygous deletions, however, are large and potentially affect more than one gene. Using a cDNA probe to the candidate gene, we have analyzed DNA from both normal and tumor tissue from WT patients, in an effort to detect rearrangements at this locus. We report here a patient with bilateral WT who is heterozygous for a small (less than 11 kb) germinal deletion within this candidate gene. DNA from both tumors is homozygous for this intragenic deletion allele, which, by RNA-PRC sequence analysis, is predicted to encode a protein truncated by 180 amino acids. These data support the identification of this locus as an 11p13 WT gene (WT1) and provide direct molecular data supporting the two-hit mutational model for WT.     

Anomeric preference of glucose utilization in human erythrocytes loaded with glucokinase

Human erythrocytes were loaded with homogeneous rat liver glucokinase by an encapsulation method based on hypotonic hemolysis and isotonic resealing. As assayed at 10 mM glucose, glucokinase and hexokinase activities in glucokinase-loaded erythrocytes were 218 and 384 nmol/min/gHb, respectively; whereas hexokinase activity in both intact and unloaded red cells, which contain no glucokinase activity, was about 400 nmol/min/gHb. No difference in the rate of lactate production from glucose anomers between intact and unloaded erythrocytes suggested that the encapsulation procedure itself did not affect glucose utilization in red cells. Alpha-anomeric preference in lactate production from glucose was observed in glucokinase-loaded erythrocytes, whereas the beta anomer of glucose was more rapidly utilized than the alpha anomer in intact and unloaded erythrocytes. The results indicate that the step of glucose phosphorylation determines the anomeric preference in glucose utilization by human erythrocytes, since glucokinase and hexokinase are alpha- and beta-preferential, respectively, in glucose phosphorylation.     

High-performance liquid chromatographic analyses of hydroxymonocarboxylic acids and dicarboxylic acids in urine as their 2-nitrophenylhydrazides

Both hydroxymonocarboxylic acids and dicarboxylic acids in urine were converted into their 2-nitrophenylhydrazides without lengthy and cumbersome sample workup and were separated from each other by two-step extraction with diethyl ether at different pH values. HPLC analysis of each acid group was achieved isocratically within 30 min. By the use of a visible-range detector (400 nm) the detection limits ranged from 1 to 2 pmol and from 2 to 5 pmol per injection for the hydroxymonocarboxylic acids and dicarboxylic acids, respectively. The analytical results showed good recovery and reproducibility. Analysis profiles of the two acid groups in normal and diabetic subjects could be performed with 200 microliters of urine. The present method is superior over previously published methods because of its great simplicity and its time-, cost-, and labor-saving nature.     

Determination of (-)-threo-chlorocitric acid in human plasma by gas chromatography—positive chemical-ionization mass spectrometry

A method is described for measuring (-)-threo-chlorocitric acid in human plasma. Plasma is acidified to pH 1 to minimize lactonization and a¹³C analogue of (-)-threo-chlorocitric acid is added as internal standard. The acidified plasma is then extracted with ethyl acetate containing 10% methanol. The ethyl acetate—methanol extract is back-extracted with acetate buffer (pH 5). This extract, following adjustment to pH 1, is reextracted with ethyl acetate. The residue after removal of the ethyl acetate is treated with ethereal diazomethane. The wet residue is reconstituted in ethyl acetate and a portion of this solution is analyzed by gas chromatography—chemical ionization mass spectrometry. The mass spectrometer is set to monitorm/z269 [MH⁺ of trimethylated (-)-threo-chlorocitric acid] andm/z270 [MH⁺ of trimethylated (-)-threo-[¹³C]chlorocitric acid] in the gas chromatographic effluent. Them/z/269 tom/z270 ion ratio in a sample containing an unknown amount of (-)-threo-chlorocitric acid is converted to an amount of compound using a calibration curve. The calibration curve is generated by analyzing control plasma spiked with various known amounts of (-)-threo-chlorocitric acid and a fixed amount of (-)-threo-[¹³C]chlorocitric acid. The limit of quantitation is 0.1–0.6 μg ml⁻¹, depending on the characteristics of the calibration curve generated with each set of samples. The precision (relative standard deviation) at a concentration of 2 μg ml⁻¹ is 3.3%.     

Aggregation of human lymphoblastoid cells by tumor-promoting phorbol esters and dihydroteleocidin B

Human lymphoblastoid cells transformed by Epstein-Barr virus aggregated rapidly in the presence of tumor-promoting phorbol esters and dihydroteleocidin B. Cell aggregation was almost complete after incubation for 6 hours. In amounts of a few ng, they induced significant aggregation. Their abilities to aggregate cells could be measured quantitatively and correlated well with their effects in promoting skin tumors.     

Anomeric preferences ofd-glucose uptake and utilization by cerebral cortex slices of rats

On aerobic incubation of rat cerebral cortex slices with anomers ofd-glucose and with 2-deoxy-d-glucose (2DG) for 5 min, the disappearance of -d-glucose from the incubation mixture was greater than that of -d-glucose and both anomers had a greater rate of disappearance than that of 2DG. In addition, there were significantly greater consumption of oxygen and production of lactate with the -anomer than with the -anomer. In similar experiments with³H-labeledd-glucose anomers and [1-³H]-3-O-methyl-d-glucose (3MG), the accumulation of [1-³H]--d-glucose (up to 5 min) by rat cerebral cortex slices was greater than that of [1-³H]--d-glucose. Although initially lower than that of the anomers, the accumulation of [1-³H]-3MG increased at a greater rate and, by 5 min of incubation, was greater than that of both glucose anomers. This preferential accumulation was seen to disappear when the slices were preincubated with 2DG (hexokinase inhibitor) or when the temperature of incubation was reduced to 20°C. Under those conditions the data with the glucose anomers were similar to those obtained with 3MG. Our data then suggested that the greater accumulation of -d-glucose than of -d-glucose by the slices was probably not due to differences in transport through brain cell membranes but rather to the preferential metabolism of the -d-glucose.     

Characterization of Temperature-Sensitive, Fertilization-Defective Mutants of the Nematode CAENORHABDITIS ELEGANS

The isolation and characterization of three Caenorhabditis elegans temperature-sensitive mutants that are defective at fertilization are described. All three are alleles of the gene fer-1. At the restrictive temperature of 25 degrees, mutant hermaphrodites make sperm and oocytes in normal numbers. No oocytes are fertilized, although they pass through the spermatheca and uterus normally. The oocytes can be fertilized by sperm transferred by wild-type males, indicating that the mutant defect is in the sperm. The temperature-sensitive period for the mutants coincides with spermatogenesis. Sperm made by mutants at 25 degrees cannot be distinguished from wild-type sperm by light microscopy. The sperm do contact oocytes in mutant hermaphrodites, but do not fertilize. Mutant sperm appear to be nonmotile. Mutant males are also steril when grown at 25 degrees. They trnasfer normal numbers of sperm to hermaphrodites at mating, but these sperm fail to migrate to the spermatheca and are infertile. The phenotype of these mutants is consistent with a primary defect in sperm motility, but the cause of this defect is not known.     

Human erythrocyte glutathione reductase. I. Purification and properties.

1. Glutathione reductase (NAD(P)H:oxidized-glutathione oxidoreductase, EC. 1.6.4.2) from human erythrocytes was purified 49 000-fold with an overall yield of 15% and a 280/460 nm absorbance ratio of 6.03. The procedure used was the method of Worthington and Rosemeyer modified by addition of heating and recrystallization. 2. It was concluded from the results of purification, electrofocusing and inhibition studies that glutathione reductase is a single enzyme which used both NADPH and NADH as hydrogen donors. 3. Apoenzyme cross-reacts with the antibody to the holoenzyme but has a slightly reduced affinity to the antibody. Apoenzyme can be removed from the hemolysate by heating and centrifugation without loss of holoenzyme. 4. Indirect immunological assay of the specific activity of the erythrocyte glutathione reductase is possible in the enzyme saturated with FAD.     

Composition of uterine milk and its changes with gestational period in red stingrays (Hemitrygon akajei)

Uterine milk is secreted in the uterus for embryo nutrition in several elasmobranch species and may contribute to rapid embryonic growth, but the details of its composition and its functions are poorly understood. In this study, to explore the roles of uterine milk for embryos, its components throughout the gestational period were analysed in detail. Uterine milk was collected from pregnant red stingrays (Hemitrygon akajei) in the early, middle and late gestational periods, respectively (n= 3 for each period). The crude composition, constituent proteins and fatty acids in the milk were analysed. The uterine milk was rich in proteins throughout the gestational period, whereas lipids dramatically increased in the middle period and reduced slightly towards the late period. Some proteins potentially associated with nutrition, cartilage growth and embryonic immunity were found. Several enzymes related to central metabolism were also detected. The constituent fatty acids in the middle and late periods were similar to those in the egg yolks of elasmobranchs, except for C18:2, which was rich only in the uterine milk. The most abundant fatty acid in the milk was C16:1, which could function as a lipokine to promote lipid metabolism in the embryo. This study's data suggest that uterine milk may be secreted in addition to the egg yolk in elasmobranchs to support rapid and healthy embryonic growth.