An acid phosphatase from Manihot glaziovii as an alternative to alkaline Phosphatase for molecular cloning experiments.

An acid phosphatase, free of deoxyribonuclease activity, was isolated from Manihot glaziovii leaves. It had a Mr of 78 kDa and was optimally active at pH 4.3 and 52 degrees C. It was inactivated at 65 degrees C over 15 min. It had a broad substrate specificity with strongest activity towards p-nitrophenyl phosphate. The enzyme dephosphorylated linearized pUC18 DNA and preventing self-ligation under the same conditions used for calf intestine alkaline phosphatase.

Assessing cyanogen content in cassava-based food using the enzyme-dipstick method.

The cyanogen content of 35 cassava-based foods was determined using the enzyme-dipstick method. The analyses showed presence of residual cyanogens in these products, and they ranged from 2 to 88 mg HCN equivalent/kg. Foodstuff prepared from grated cassava roots exhibited a lower level of cyanogen content (2-31 mg HCN equivalent/kg) compared with those prepared as cassava root slices or from cassava flour (28-88 mg HCN equivalent/kg). These results showed that current commercial processing methods did not remove all the cyanogen present from cassava roots and it might be worthwhile re-examining these processing practices. In this respect, the report showed that an overnight treatment of cassava root slices with linamarase and cellulase might facilitate the removal of cyanogens. This study also demonstrated the usefulness of enzyme-dipsticks for assessing cyanogen content in cassava food products.

Somatic embryogenesis, plant regeneration and cyanogenesis in Manihot glaziovii Muell. Arg. (ceara rubber).

The report describes a system for somatic embryogenesis and direct plant regeneration from the embryos of Manihot glaziovii. Somatic embryos were obtained by culturing young leaf lobes (3-6 mm long) adjacent to the apex in Murashige and Skoog medium containing 18 µM 2,4-dichlorophenoxy acetic acid for 20 days and then transferring them to a maturation medium with 0.5 µM 6-benzylaminopurine. Secondary embryogenesis was induced from cotyledonary segments of somatic embryos by using the same protocol as that for primary embryogenesis. For regeneration, somatic embryos were cultured in medium supplemented with 10-4 M kinetin and 53.4% of them developed into plantlets. Linamarin and linamarase were not detected in calli or in somatic embryos. Linamarin content was found to be highest in leaves of regenerated plantlets, followed by stem and root tissues. Levels of linamarase activity were almost the same in leaves and stem tissues and very low in roots.

Customisation of AFLP analysis for cassava varietal identification.

Amplified fragment length polymorphism (AFLP) markers were used in the characterization of eight cassava varieties. This nonradioactive AFLP system was customized in terms of the choice of restriction enzymes used and the selection of nucleotides added to the 3' end of primers. EcoRI/MseI and HindIII/MseI fragments generally gave monomorphic profiles while ApaI/TaqI fragments produced polymorphic profiles suggesting a genome with high G + C content. It was possible to identify the eight cassava varieties used in this study using CTG as selective bases at the TaqI primer. For cassava, the AFLP system provided a higher number of loci detected per run when compared to RAPD. The reliability accompanying AFLP analysis would thus make it suitable for the characterization of cassava varieties.

Benefits of a multidisciplinary predialysis program in maintaining employment among patients on home dialysis.

We evaluated the benefits of a multidisciplinary predialysis intervention to help home dialysis patients maintain employment. This program was shown to be successful with in-center hemodialysis patients in a case-controlled study published in the American Journal of Kidney Disease in September, 1993. We looked at 30 patients on home dialysis (28 on continuous ambulatory peritoneal dialysis and 2 on home hemodialysis) as of March, 1995. The age of the patients ranged from 28 to 63 years, with a mean of 46.8 years. Forty-three percent of the patients were diabetic. The patients went through a multidisciplinary predialysis program which consisted of: (1) psychosocial assessment, (2) education about dialysis and choice of modalities, (3) orientation to dialysis unit, and (4) counseling sessions with patient, significant family members, and others. During this time, patients were referred to the program approximately six months before beginning dialysis. There was continued collaboration among members of the multidisciplinary team. Of the 30 patients, 11 were already disabled when beginning dialysis. Of the 19 who were working once dialysis began, 14 maintained employment (3 diabetic), with 1 retiring and 4 becoming disabled. Therefore, 74% of patients maintained employment. In conclusion, employment was maintained by this predialysis multidisciplinary program.

Linamarin sensors: interference-based sensing of linamarin using linamarase and peroxidase.

An interference-based linamarin sensor is developed. Horseradish peroxidase (HRP) is adsorbed on a pyrolytic graphite (PG) electrode, and then linamarase from cassava is cross-linked with glutaraldehyde on the electrode surface. The prepared bienzyme electrode is poised at -300 mV vs Ag/AgCl for 40 s to reduce dissolved O(2) to H(2)O(2) at the PG surface. The potential is then stepped to 0 mV, at which point the accumulated H(2)O(2) is reduced, though the O(2) reduction does not proceed. Since the H(2)O(2) reduction is catalyzed by HRP, the transient cathodic current is inhibited by cyanide, which is liberated from linamarin by linamarase. Therefore, the transient current is a function of the linamarin concentration. This sensor responds to 1 × 10(-5)-5 × 10(-3) M linamarin and can estimate a linamarin concentration of a cassava extract.

Sources of fungal linamarases.

Forty-four strains of Aspergillus, Penicillium, Fusarium, Trichoderma and Rhizopus were grown on a liquid medium containing glucose and cassava-root extract. All of the Aspergillus and Fusarium strains, eight out of 10 Penicillium strains and three of seven Trichoderma strains showed linamarase activity. No such activity was detected in any Rhizopus strain. The crude enzyme preparation from F. oxysporum had the highest affinity for linamarin whereas that from A. nidulans was the most heat-stable.

Increased risk of renal cell cancer among women using diuretics in the United States.

Use of prescription diuretics and incidence of renal cell cancer have increased in the United States in the past 25 years. Recent interview-based epidemiologic studies have reported an association between diuretic use and renal cell cancer risk. Our study evaluated this hypothesis using, for the first time, medical records as the source of the information on prescription diuretic use. Using medical records of women from a prepaid health plan, we identified 191 cases and 191 controls matched on age, membership duration, and membership at diagnosis. Diuretics use and history of potential confounding factors were ascertained by a standardized review of the medical records of each subject, without reference to case or control status. There was a strong and statistically significant association between renal cell cancer and prescription diuretics (odds ratio [OR] adjusted for hypertension, smoking, and obesity = 2.9, 95 percent confidence interval [CI] = 1.7-4.7). Risk tended to increase with dose, measured by number of prescriptions. Since renal cancer can induce hypertension, which is treated by diuretics, and thereby confound the association with diuretics, we examined diuretic use 10 or more years prior to diagnosis when secondary hypertension would be unlikely. The OR for prescriptions 10 or more years before diagnosis was 3.5 (CI = 1.7-7.4). Our results support earlier reports of an excess risk of renal cell cancer among users of prescription diuretics and indicate need for further study to evaluate this relationship, especially due to the extensive use of diuretics and the increasing incidence of this cancer.

Isolation and characterization of ß-glucosidases from Aspergillus nidulans mutant USDB 1183.

Two extracellular ß-glucosidases (cellobiase, EC 3.2.1.21), I and II, from Aspergillus nidulans USDB 1183 were purified to homogeneity with molecular weights of 240,000 and 78,000, respectively. Both hydrolysed laminaribiose, ß-gentiobiose, cellobiose, p-nitrophenyl-ß-L-glucoside, phenyl-ß-L-glucoside, o-nitrophenyl-ß-L-glucoside, salicin and methyl-ß-L-glucoside but not α-linked disaccharides. Both were competitively inhibited by glucose and non-competitively (mixed) inhibited by glucono-1,5-lactone. ß-Glucosidase I was more susceptible to inhibition by Ag(+) and less inhibited by Fe(2+) and Fe(3+) than ß-glucosidase II.

Protoplast fusion of beta-glucosidase-producing Aspergillus niger strains.

Protoplast fusion, induced by polyethylene glycol and Ca2+, was carried out between two auxotrophic strains of Aspergillus niger. The fusion frequency ranged from 6.2 x 10(-2) - 9.1 x 10(-2). After induced haploidization of a diploid, various segregants showing combinations of the parental genetic markers were isolated. Unlike diploids, haploid segregants exhibited greater variations in their morphology and beta-glucosidase activities. One segregant showed a 2.5-fold increase in beta-glucosidase activity over those of the parents. Thus, this method appears promising for creating new recombinant strains of A. niger with improved beta-glucosidase activities.

Estimation of DNA restriction fragment size using a PC.

A computer program developed by Duggleby et al. (R. G. Duggleby, H. Kinns and J. I. Rood, A computer program for determining the size of DNA restriction fragments. Analytical Biochemistry 110, 49 (1981)) for determining DNA restriction fragment size was rewritten for the IBM compatible PC. Accuracy of curve fit was examined for molecular size range 22 to 0.6 kb. It showed a better fit of the data involving molecular size range 22 to 2 kb, or 2 to 0.6 kb, rather than for 22 to 0.6 kb, irrespective of agarose gel concentrations and electrophoretic running times. Thus, greater accuracy of DNA fragment size could be estimated by using two calibration ranges, i.e. 22 to 2 kb, or 2 to 0.6 kb, depending on the fragment size.

Variations in Kinetic Properties of Ribulose-1,5-bisphosphate Carboxylases among Plants.

Studies of ribulose-1,5-bisphosphate (RuBP) carboxylase from taxonomically diverse plants show that the enzyme from C(3) and crassulacean acid metabolism pathway species exhibits lower K(m)(CO(2)) values (12-25 micromolar) than does that from C(4) species (28-34 micromolar). RuBP carboxylase from aquatic angiosperms, an aquatic bryophyte, fresh water and marine algae has yielded consistently high K(m)(CO(2)) values (30-70 micromolar), similar in range to that of the enzyme from C(4) terrestrial plants. This variation in K(m)(CO(2)) is discussed in relation to the correlation between the existence of CO(2)-concentrating mechanisms for photosynthesis and the affinity of the enzyme for CO(2). The K(m)(RuBP) of the enzyme from various sources ranges from 10 to 136 micromolar; mean +/- sd = 36 +/- 20 micromolar. This variation in K(m)(RuBP) does not correlate with different photosynthetic pathways, but shows taxonomic patterns. Among the dicotyledons, the enzyme from crassinucellate species exhibits lower K(m)(RuBP) (18 +/- 4 micromolar) than does that from tenuinucellate species (25 +/- 7 micromolar). Among the Poaceae, RuBP carboxylase from Triticeae, chloridoids, andropogonoids, Microlaena, and Tetrarrhena has yielded lower K(m)(RuBP) values (29 +/- 11 micromolar) than has that from other members of the grass family (46 +/- 10 micromolar).

Variations in K(m)(CO(2)) of Ribulose-1,5-bisphosphate Carboxylase among Grasses.

A survey of the K(m)(CO(2)) values of ribulose-1,5-bisphosphate carboxylase from 60 grass species shows that enzyme from C(3) grasses consistently exhibits lower K(m)(CO(2)) than does that from C(4) grasses. Systematically ordered variation in K(m)(CO(2)) of ribulose-1,5-bisphosphate carboxylases from C(3) and C(4) grasses is also apparent and, among C(4) grasses, this shows some correlation with C(4) types.

Effects of 4-pentenoic acid on renal phosphate and calcium excretion in the dog.

The effects of 4-pentenoic acid (4-PA) on renal excretion of phosphate and calcium were studied in anesthetized mongrel dogs. The major metabolic action of 4-PA is inhibition of long-chain fatty acid oxidation. In intact dogs undergoing modest saline diuresis, 4-PA caused significant decrease in the percentage of filtered phosphate reabsorbed. No statistically significant calciuria was observed. Similar results were observed in intact dogs undergoing more brisk saline diuresis as well as in acutely and chronically thyroparathyroidectomized dogs. However, when results were pooled, both phosphate and calcium excretions were significantly increased. The results are interpreted as demonstrating the importance of fatty acid oxidation in providing energy for the work of tubular phosphate reabsorption. Either the energy source for calcium reabsorption differs from fatty acid oxodation, or calcium reabsorption is less sensitive to changes in energy sources. The action of 4-PA is independent of the forces involved in saline diuresis and of parathyroid hormone.