Electron Microscope Study of Lens Fibers

Comparative electron microscope investigations on sections of the lens cortex of the normal, mature rat, rabbit, monkey, and the normal calf reveal similar patterns of intracellular organization. The superficial lens fiber contains a nucleus, mitochondria, endoplasmic reticulum, dense granules, Golgi complex, and a quantity of small structures of low opacity which appear as filamentous and spherical configurations. Variations in number, distribution, and spatial arrangement of cytoplasmic elements in lens fibers are described. These changes in the pattern of cytoplasmic organization are concomitant with development of fibers and their displacement towards the center of the lens. Structural details of the various zones of the lens epithelium and the lens fibers are compared.     

Genetic analysis of agronomic traits in a cross between sugarcane (Saccharum officinarum L.) and its presumed progenitor (S. robustum Brandes & Jesw. ex Grassl)

Saccharum robustum Brandes & Jesw. ex Grassl has been suggested as the immediate progenitor species of cultivated sugarcane (S. officinarum L.) [4]. Chromosome pairing and assortment in these two species were previously studied by genetic analysis of single-dose DNA markers in parents in and 44 F₁ progeny of a cross between euploid, meiotically regular 2n=80S. officinarum LA Purple andS. robustum Mol 5829 [2]. This same population was subsequently clonally propagated and evaluated in replicated trials for quantitative traits important to sugarcane breeders. Numbers of stalks, tasseled stalks, and stalks with smut, and the average diameter of two stalks were determined one day prior to harvest. At harvest, plant material from each plot was weighed and evaluated for pol (sucrose content) and fiber percentages. Clones were significantly different (P<0.01) for all traits analyzed. Associations of 83 single-dose arbitrarily primed PCR genetic markers with quantitative trait loci (QTL) of recorded traits was determined by single-factor ANOVA, and multiple regression. QTL analysis revealed markers significantly (P<0.05) associated with the expression of each trait analyzed. Markers associated with QTL after multiple regression were tested for digenic linear × linear epistatic interactions. The various multilocus models explained between 23% and 58% of the total phenotypic variation and 32% and 76% of the genotypic variation for the various traits. Digenic interactions were uncommon. Implications for marker-assisted selection in sugarcane and sugarcane domestication are discussed.     

Molecular cloning and analysis of CDC28 and cyclin homologues from the human fungal pathogen Candida albicans

In the budding yeast Saccharomyces cerevisiae, progress of the cell cycle beyond the major control point in G1 phase, termed START, requires activation of the evolutionarily conserved Cdc28 protein kinase by direct association with GI cyclins. We have used a conditional lethal mutation in CDC28 of S. cerevisiae to clone a functional homologue from the human fungal pathogen Candida albicans. The protein sequence, deduced from the nucleotide sequence, is 79% identical to that of S. cerevisiae Cdc28 and as such is the most closely related protein yet identified. We have also isolated from C. albicans two genes encoding putative G1 cyclins, by their ability to rescue a conditional GI cyclin defect in S. cerevisiae; one of these genes encodes a protein of 697 amino acids and is identical to the product of the previously described CCN1 gene. The second gene codes for a protein of 465 residues, which has significant homology to S. cerevisiae Cln3. These data suggest that the events and regulatory mechanisms operating at START are highly conserved between these two organisms.     

Kiwifruit β-galactosidase: Isolation and activity against specific fruit cell-wall polysaccharides

A -galactosidase (EC 3.2.1.23) capable of degrading a number of fruit cell-wall polysaccharides in vitro, was isolated from ripening kiwifruit (Actinidia deliciosa [A. Chev.] C.F. Liang et A.R. Ferguson cv. Hayward). The enzyme has a molecular weight of approximately 60 kDa by gel permeation and consists of several basic isoforms. Several polypeptides were enriched during purification, with 33-, 46- and 67-kDa bands being predominant after sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The optimum activity of the enzyme against p-nitrophenyl--d-galactopyranoside was at pH 3.2, but against a galactan purified from kiwifruit cell walls, it was at pH 4.9. The enzyme was specific for galactosyl residues in the -configuration, releasing galactose from a variety of kiwifruit cell-wall polysaccharide fractions including cell wall material, Na₂CO₃-soluble pectin, high-molecular-weight galactan, xyloglucan, and galactoglucomannan. A galactosylated glucuronomannan found throughout the kiwifruit plant was also a substrate for the enzyme. The results indicate that the enzyme attacks the non-reducing end of galactose side chains, cleaving single galactose residues which may be attached to the 2, 3, 4, or 6 position of the aglycone. Activity of the enzyme in-vitro was too low to account for the total loss of galactose from the cell walls during ripening. If the -galactosidase of this study is solely responsible for the removal of galactose from the cell wall during ripening then its in-vivo activity must be much greater than that observed in-vitro.Abbreviations CWM cell wall material - SDS-PAGE sodium dodecyl sulphate-polyacrylamide gel electrophoresis We thank Bronwyn Culling and Teresa Wegrzyn for assistance and acknowledge a contribution towards the cost of the research from the New Zealand Kiwifruit Marketing Board.     

A Method for the Gross and Microscopic Investigation of Vascular Reperfusion in Ischemic Myocardium

Fluorescein-isothiocyanate dextran (FITC-dextran; MW ∼ 70,000) was used in isolated rat hearts to compare normal vascular perfusion of ventricular myocardium with the pattern and extent of reperfusion following 60 minutes of global ischemia. Its gross distribution in frozen transverse sections through the ventricles was similar to that of sodium fluorescein. However, unlike 0.1% sodium fluorescein, 6.7% FITC-dextran has a viscosity similar to that of blood, and its much higher molecular weight prevents its diffusion beyond the ischemically injured vessels. Furthermore, staining by the alcoholic periodic acid-Schiff technique enabled tracer distribution to be confirmed microscopically and distinguished competent from incompetent vessels in paraffin embedded material.     

6-Ethyl-5-hydroxy-2,7-dimethoxy-1,4-naphthoquinone from Hendersonula toruloidea: A biosynthetic study using C labels detected by nuclear magnetic resonance and C tracers

Production of 6-ethyl-5-hydroxy-2,7-dimethoxy-1,4-naphthoquinone was obtained by growth of Hendersonula toruloidea on Czapek-Dox broth supplemented with malt extract. Stationary cultures were grown at 28°C for 21–22 days yielding about 6 mg of metabolite per 700 ml of culture fluid. The best incorporations of isotopic tracers were obtained by addition at the 20th day of growth, followed by harvest 24–48 hr later. With [2-¹⁴C]acetate, incorporation values were in the range of 0.1–0.3% with dilution values from 2000 to 5900. With [1-¹⁴C]propionate, incorporations were much lower (0.04%) and dilutions much higher (120,000). Activity from [¹⁴CH₃]methionine was incorporated only into the OCH₃ groups (incorporation values, 0.5–0.7%). Nuclear magnetic resonance studies confirmed that propionate was not a precursor. Using [1,2-¹³C]acetate, substantial enrichments were obtained at all carbon atoms except those of the OCH₃ groups. The following pairs of carbon atoms were shown to be derived from acetate units: C-1 + 2, C-3 + 4, C-5 + 10, C-6 + 7, C-8 + 9, C-11 + 12. The biosynthetic pathway is clearly that of acetate plus polymalonate. Experiments with [2-¹³C²H₃]acetate suggested that the “starter” acetate unit was located at positions C-12 + 11.     

6-Ethyl-5-hydroxy-2,7-dimethoxy-1,4-naphthoquinone from Hendersonula toruloidea: A biosynthetic study using 13C labels detected by nuclear magnetic resonance and 14C tracers

Production of 6-ethyl-5-hydroxy-2,7-dimethoxy-1,4-naphthoquinone was obtained by growth of Hendersonula toruloidea on Czapek-Dox broth supplemented with malt extract. Stationary cultures were grown at 28°C for 21–22 days yielding about 6 mg of metabolite per 700 ml of culture fluid. The best incorporations of isotopic tracers were obtained by addition at the 20th day of growth, followed by harvest 24–48 hr later. With [2-¹⁴C]acetate, incorporation values were in the range of 0.1–0.3% with dilution values from 2000 to 5900. With [1-¹⁴C]propionate, incorporations were much lower (0.04%) and dilutions much higher (120,000). Activity from [${}^{14}\text{CH}{}_3$]methionine was incorporated only into the OCH₃ groups (incorporation values, 0.5–0.7%). Nuclear magnetic resonance studies confirmed that propionate was not a precursor. Using [1,2-¹³C]acetate, substantial enrichments were obtained at all carbon atoms except those of the OCH₃ groups. The following pairs of carbon atoms were shown to be derived from acetate units: C-1 + 2, C-3 + 4, C-5 + 10, C-6 + 7, C-8 + 9, C-11 + 12. The biosynthetic pathway is clearly that of acetate plus polymalonate. Experiments with [2-¹³C²H₃]acetate suggested that the “starter” acetate unit was located at positions C-12 + 11.