Refining the application of direct embryogenesis in sugarcane: Effect of the developmental phase of leaf disc explants and the timing of DNA transfer on transformation efficiency.

A rapid in vitro protocol using direct somatic embryogenesis and microprojectile bombardment was investigated to establish the developmental phases most suitable for efficient sugarcane transformation. Immature leaf roll disc explants with and without pre-emergent inflorescence tissue were compared. It was shown that for effective transformation to occur, explants should be cultured for several days to allow initiation of embryo development prior to bombardment. Leaf roll discs with pre-emergent inflorescences showed a higher degree of embryogenic competence than non-flowering explants, and transformation efficiency was higher when explants containing floral initials were bombarded. Despite the occurrence of high numbers of phenotypically negative plants, combining the use of inflorescent leaf roll discs with direct embryogenic regeneration has the potential to improve the speed and efficiency of transgenesis in sugarcane.

The detection of occult renal tumors in children by elevated hematocrit on routine complete blood count: a report of two cases.

Wilms' tumor is a renal neoplasm usually found in young children and is rarely seen in teenagers. The production of erythropoietin by these tumors may result in secondary erythrocytosis, which should be reflected in complete blood counts (CBC). A search of the literature for reports of occult Wilms' tumors initially suspected on the basis of erythrocytosis was unrewarding. Two teenagers are reported in whom unexplained erythrocytosis was the initial indication of a Wilms' tumor. In both cases, a previous CBC showed elevations in hemoglobin and hematocrit that might have led to an earlier diagnosis. We conclude that unexplained erythrocytosis should sound the alert for further diagnostic studies to evaluate the possibility of occult renal neoplasia in older children.

Granular cell tumor of the breast.

Granular cell tumors are rare tumors occasionally located in the breast. They are generally benign, but can mimic breast carcinoma thus complicating diagnosis and treatment. The etiology of these tumors remains obscure, but may be neurogenic in origin. We report a case of granular cell tumor of the breast and present a review of the topic.

Nitrogen catabolite repression of asparaginase II in Saccharomyces cerevisiae.

The biosynthesis of asparaginase II in Saccharomyces cerevisiae is subject to strong catabolite repression by a variety of nitrogen compounds. In the present study, asparaginase II synthesis was examined in a wild-type yeast strain and in strains carrying gdhA, gdhCR, or gdhCS mutations. The following effects were observed: (i) In the wild-type strain, the biosynthesis of asparaginase II was strongly repressed when either 10 mM ammonium sulfate or various amino acids (10 mM) served as the source of nitrogen. (ii) In a yeast strain carrying the gdhA mutation, asparaginase II was synthesized at fully derepressed levels when 10 mM ammonium sulfate was the source of nitrogen. When amino acids (10 mM) served as the nitrogen source, asparaginase II synthesis was strongly repressed. (iii) In a strain carrying the gdhCR mutation, the synthesis of asparaginase II was partially (30 to 40%) derepressed when either 10 mM ammonium sulfate or amino acids were present in the medium. (iv) In a yeast strain containing both gdhA and gdhCR mutations, asparaginase II synthesis was fully derepressed when 10 mM ammonium sulfate was the nitrogen source and partially derepressed when 10 mM amino acids were present. (v) Yeast strains carrying the gdhCS mutation were indistinguishable from the wild-type strain with respect to asparaginase II synthesis.

Reactions of asparaginase II of Saccharomyces cerevisiae. A mechanistic analysis of hydrolysis and hydroxylaminolysis.

Detailed kinetic analysis was performed on asparaginase II, a cell wall glycoprotein from Saccharomyces cerevisiae. The enzyme was highly active in the hydrolysis and hydroxylaminolysis reactions with D- and L-asparagine and with a variety of N-substituted analogues. The data from studies involving pH dependencey, substrate saturation, and product inhibition support the hypotheses that (a) the yeast asparaginase mechanism proceeds via an acyl enzyme intermediate; (b) an ionizable group on the enzyme, pK approximately 6.0, is involved in the acylation and deacylation reactions; and (c) yeast asparaginase II is a peptidoasparaginase.

Characterization of two forms of asparaginase in Saccharomyces cerevisiae.

Saccharomyces cerevisiae X2180-1A synthesizes two forms of asparaginase: L-asparaginase I, an internal constitutive enzyme, and asparaginase II, an external enzyme which is secreted in response to nitrogen starvation. The two enzymes are biochemically and genetically distinct. The structural gene for asparaginase I (asp 1) is closely linked to the trp 4 gene on chromosome IV. The gene controlling the synthesis of asparaginase II is not linked to either the trp 4 or asp 1 genes. The rate of biosynthesis of asparaginase II is unaltered in yeast strains carrying the structural gene mutation for asparaginase I. Asparaginase II has been purified approximately 300-fold from crude extracts of Saccharomyces by heat and pH treatment, ethanol fractionation, ammonium sulfate fractionation followed by Sephadex G-25 chromatography, and DEAE-cellulose chromatography. Multiple activity peaks were obtained which, upon gas chromatographic analysis, exhibit varying mannose to protein ratios. Asparaginase I has been purified approximately 100-fold from crude extracts of Saccharomyces by protamine sulfate treatment, ammonium sulfate fractionation, gel permeation chromatography, and DEAE-cellulose chromatography. No carbohydrate component was observed upon gas chromatographic analysis. Comparative kinetic and analytic studies show the two enzymes have little in common except their ability to hydrolyze L-asparagine to L-aspartic acid and ammonia.