Treatment of Chinese hamster ovary (CHO) cells by the aldehyde containing calpain inhibitor I resulted in the induction of a 35-kDa protein that was partially sequenced and shown to be a member of the aldo-keto reductase superfamily (Inoue, S., Sharma, R. C., Schimke, R. T., and Simoni, R. D. (1993) J. Biol. Chem. 268, 5894 -5898). Using rapid amplification of cDNA ends polymerase chain reaction, we have sequenced the cDNA for this protein (CHO reductase). This enzyme is a new member of the aldo-keto reductase superfamily and shows greatest amino acid sequence identity to mouse fibroblast growth factor-regulated protein and mouse vas deferens protein (92 and 80% sequence identity, respectively). The enzyme exhibits about 70% sequence identity with the aldose reductases (ALR2; EC 1.1.1.21) and about 47% with the aldehyde reductases (ALR1; EC 1.1.1.2). Northern analysis showed that it is induced in preference to either ALR1 or ALR2 and RNase protection assays showed gene expression in bladder, testis, jejunum, and ovary in descending order of expression. The cDNA for this inducible reductase was cloned into the pET16b vector and expressed in BL21(DE3) cells. Expressed CHO reductase showed kinetic properties distinct from either ALR1 or ALR2 including the ability to metabolize ketones. This protein joins a growing number of inducible aldo-keto reductases that may play a role in cellular regulation and protection.To characterize the calpain inhibitor I-sensitive protease(s) involved in the degradation of 3-hydroxy-3-methylglutaryl-CoA reductase, Simoni's group (1) attempted to isolate Chinese hamster ovary (CHO) 1 cells resistant to this peptide aldehyde (N-acetyl-leucyl-leucyl-norleucinal (ALLN)). Instead of inducing a protease, a 35-kDa protein was overexpressed which gave tryptic peptide fragments with a high degree of sequence identity to members of the aldo-keto reductase superfamily. 2 This superfamily is a rapidly growing group of monomeric oxidoreductases containing at least 40 members at present (2). This group includes the aldehyde and aldose reductases, a number of hydroxysteroid dehydrogenases, Shaker channels, and plant chalcone reductases. They are characterized by a TIM-barrel structure (3) and the preferential use of NADPH over NADH. Substrates include aliphatic and aromatic aldehydes, monosaccharides, steroids, prostaglandins, polycyclic aromatic hydrocarbons, and isoflavinoid phytoalexins.Several members of this family have been shown to be induced in response to hormonal or chemical factors. These include fibroblast growth factor-regulated protein (FR-1) (4), mouse vas deferens protein (MVDP) (5), aldose reductase (ALR2) (6 -8), and dihydrodiol dehydrogenase (9). To determine the relationship of this new reductase to the other members of the family we have used RACE PCR to isolate and sequence its mRNA from CHO cells. It shows highest sequence identity to FR-1 and MVDP at 92 and 80%, respectively. FR-1 and MVDP have not been extensively characterized kinetically but CHO reductase showed a greater ...

Sidney Farber Cancer I n s t i t u t e , Department o f P e d i a t r i c s , Harvard Medical School. Boston. MA.-, -----P a t i e n t s w j t h ~e o v e r l o a d oiten.demonstrate s a t u r a t e d Fe b i n d i n g c a p a c i t i e s and low w h i t e c e l l C l e v e l s when measured by s t a n dard techniques.It has been shown t h a t serum Fe i n excess o f t h e Fe b i n d i n g c a p a c i t y ( " f r e e Fe") can be measured i n p a t i e n t s w i t h Fe o v e r l o a d by EDTA b i n d i n g and u l t r a f i l t r a t i o n .We found " f r e e Feu o f 8.2i2.0 ug% i n 15 p r e v i o u s l y unchelated p a t i e n t s w i t h t h a l , compared t o O.liO.l ug% i n normal c o n t r o l s . Since " f r e e Feu m i g h t be a p a r t i c u l a r l y p o t e n t o x i d a n t and t h e r e f o r e r e s p o n s i b l e f o r sane o f t h e patholoqy i n t h a l , and s i n c e C i s thought t o increase t h e r a t e o f t r a n s f e r o f Fe from one pool t o another, we examined 5 o f these p a t i e n t s t o determine t h e e f f e c t o f C and DF on " f r e e Fe". A l l 5 t h a l s had low i n i t i a l w h i t e c e l C l e v e l s . Each r e c e i v e d a l t e r n a t i n g 12-hour continuous I . V . r e g imens o f C alone, s a l i n e , and C + DF w i t h samples f o r " f r e e Feu drawn every 4 hours. Results showed t h a t " f r e e Feu was 9.8i2.3 ~g % d u r i n g s a l i n e i n f u s i o n s and t h a t t h e l e v e l was unchanged hen C was added. When DF was present, " f r e e Feu was e l i m i n a t e d These r e s u l t s show t h a t t h a l s w i t h Fe o v e r l o a d have measurable = amounts o f " f r e e Feu which a r e n o t a f f e c t e d by C. Continuous c h e l a t i o n w i t h DF i s e f f e c t i v e i n e l i m i n a t i n g t h i s p o t e n t i a l l y t o x i c p o o l . Forty-seven of 417 evaluable patients (11%) developed hematuria. The hematuria was moderate or severe in 80% of cases. It occurred with equal frequency throughout treatment and occurred for the first time in some patients after therapy was stopped. Severe neutropenia (<500/mm3) was observed in 67% of patient in Regimen E and 72% of patients in Regimen F (Clinical Groups I11 and IV). Adriamycin cardiomyopathy has not been observed in this study.Since most early deaths occurred in patients with head and neck primaries, the use of less intensive inltial chemotherapeutic regimens may be advisable fc these patients. EHE IThe purpose of this study was to investigate the cause of granulocytopenia in children with acute lymphocytic leukemia (ALL). The double layer agar technique for myeloid colony formation was utilized in a1 studies. Various concentrations of sera from 16 chi1 dren with ALL at the time of diagnosis were added to bone marrow cells in culture. Only 2 sera were observed to produce inhibition of colony formation. Various concentrations of initial bone marrow cells from children with ALL were mixed with normal bone marrow cells and cultured. No inhibition of colony formation was observed in a...

The deqree of FAS positivity was determined by counting 200 hlast cells in the initial bone marrow specimen of 38 childrendm ALL. Each cell was scored on a 0 to 5 scale accordinq to the number of PAS-psitive qranules per cell (0-4). or the presence of 1 or more larqe blocks per cell (5). The patients were stratified into 3 groups accordinq to their mean PAS score. Fourteen children had a low score (mean 65, ranqe 3-150), 12 were intermediate (mea~. 315, range 153-4451. and 12 were high (mean 692, range 465-860). The mean white blood count (WBC) at diaanosis was higher in the low-score qroup compared to the hiqh-score qroup, but thedf* erence was not siqnificant (mO.05); 5/14 low-score patients had an initial WBC below 20,000/mn3, and 3/12 high-score patientsww above 20,000/mm3. There were no sir~nificant differences among tha groups with respect to aqe, h-lobin and platelet levels at* nosis or presence of a mediastinal or abdominal mass. However, 13 of 14 lw-score patients were male, compared to 4/12 and 6/12 in the intermediate and high groups (p<0.05). In addition, 7 of 13 low-score patients were in relapse by 6 months from diaqnosis (yl had an initial WBC below 20,000/mm3), compared to none of the k d z~ mediate and only 1 of the high patients (~(0.05). These results suggest that a low PAS score is related to a poor proqnosis in childhood ALL, regardless of the level of the initial WBC. Fenales tend to have higher PAS scores than males, and also seem to have a better prognosis.