Dipeptidyl aminopeptidase yspI mutants of Schizosaccharomyces pombe: Genetic mapping of dpa1+ on chromosome III

Abstract A mutant strain of Schizosaccharomyces pombe lacking dipeptidyl aminopeptidase yspI was isolated from a strain already defective in aminopeptidase activity by means of a staining technique with the chromogenic substrate ala-pro-4-methoxy-β-naphthylamide to screen colonies for the absence of the enzyme. The defect segregated 2⁺ :2⁻ in meiotic tetrads, indicating a single chromosomal gene mutation, which was shown to be recessive. Gene dosage experiments indicated that the mutation resides in the structural gene of dipeptidyl aminopeptidase yspI, dpa 1⁺. The dpa 1⁺ gene was located on chromosome III by using l m- fluorophen-ylalanine-induced haploidization and mitotic analysis. dpa1 mutants did not show any obvious phenotype under a variety of conditions tested.     

Characterisation of Trypanosoma cruzi populations by dna polymorphism of the cruzipain gene detected by single-stranded dna conformation polymorphism (SSCP) and direct sequencing

Fifty fresh isolates of Trypanosoma cruzi from Triatoma dimidiata vectors and 31 from patients with Chagas disease were analysed for DNA polymorphisms within the 432-bp core region of the cruzipain gene which encodes the active site of cathepsin L-like cystein proteinase. The cruzipain gene showed signs of polymorphism consisting of four different DNA sequences in Central and South American isolates of T. cruzi. The PCR fragments of Guatemalan isolates could be divided into three groups, Groups 1, 2 and 3, based on different patterns of single-stranded DNA conformation polymorphism. All of the strains isolated from Brazil, Chile, and Paraguay, except for the CL strain, showed a Group 4 pattern. Two to four isolates from each group were analysed by cloning and sequencing. A silent mutation occurred between Groups 1 and 2, and five nucleotides and two aa substitutions were detected between Groups 1 and 3. The DNA sequence of Group 4 contained five nucleotides and one aa substitution from Group 1. All of the DNA sequences corresponded well with the single-stranded DNA conformation polymorphism. The Group 1 isolates, the majority in the Guatemalan population (70/81, 86.4%), were isolated from both triatomines and humans, but Group 3 were isolated only from humans. Moreover, the Group 2 isolates were detected only in triatomine vectors (9/50; 18%), but never in humans (0/32, P<0.05) suggesting that this group has an independent life-cycle in sylvatic animals and is maintained by reservoir hosts other than humans.     

Insulin activation of phosphatidylinositol 3-kinase in human skeletal muscle in vivo

Hickey, Matthew S., Charles J. Tanner, D. Sean O'Neill,Lydia J. Morgan, G. Lynis Dohm, and Joseph A. Houmard. Insulin activation of phosphatidylinositol 3-kinase in human skeletal muscle invivo. J. Appl. Physiol. 83(3):718-722, 1997.The purpose of this investigation was to determinewhether insulin-stimulated phosphatidylinositol 3-kinase (PI3-kinase)activity is detectable in needle biopsies of human skeletal muscle.Sixteen healthy nonobese males matched for age, percent fat, fastinginsulin, and fasting glucose participated in one of two experimentalprotocols. During an intravenous glucose tolerance test (IVGTT)protocol, insulin-stimulated PI3-kinase activity was determined frompercutaneous needle biopsies at 2, 5, and 15 min post-insulinadministration (0.025 U/kg). In the second group, a 2-h, 100 mU · m² · min¹euglycemic hyperinsulinemic clamp was performed, and biopsies wereobtained at 15, 60, and 120 min after insulin infusion was begun.Insulin stimulated PI3-kinase activity by 1.6 ± 0.2-, 2.2 ± 0.3-, and 2.2 ± 0.4-fold at 2, 5, and 15 min, respectively, duringthe IVGTT. During the clamp protocol, PI3-kinase was elevated by 5.3 ± 1.3-, 8.0 ± 2.6-, and 2.7 ± 1.4-fold abovebasal at 15, 60, and 120 min, respectively. Insulin-stimulatedPI3-kinase activity at 15 min post-insulin administration wassignificantly greater during the clamp protocol vs. the IVGTT(P < 0.05). These observations suggest that insulin-stimulated PI3-kinase activity is detectable inneedle biopsies of human skeletal muscle, and furthermore, that theeuglycemic, hyperinsulinemic clamp protocol may be a useful tool toassess insulin signaling in vivo.

     

The Nup2 meiotic-autonomous region relieves inhibition of Nup60 to promote progression of meiosis and sporulation in Saccharomyces cerevisiae

During meiosis, chromosomes undergo dramatic changes in structural organization, nuclear positioning, and motion. Although the nuclear pore complex has been shown to affect genome organization and function in vegetative cells, its role in meiotic chromosome dynamics has remained largely unexplored. Recent work in the budding yeast Saccharomyces cerevisiae demonstrated that the mobile nucleoporin Nup2 is required for normal progression through meiosis I prophase and sporulation in strains where telomere-led chromosome movement has been compromised. The meiotic-autonomous region, a short fragment of Nup2 responsible for its role in meiosis, was shown to localize to the nuclear envelope via Nup60 and to bind to meiotic chromosomes. To understand the relative contribution these 2 activities have on meiotic-autonomous region function, we first carried out a screen for meiotic-autonomous region mutants defective in sporulation and found that all the mutations disrupt interaction with both Nup60 and meiotic chromosomes. Moreover, nup60 mutants phenocopy nup2 mutants, exhibiting similar nuclear division kinetics, sporulation efficiencies, and genetic interactions with mutations that affect the telomere bouquet. Although full-length Nup60 requires Nup2 for function, removal of Nup60’s C-terminus allows Nup60 to bind meiotic chromosomes and promotes sporulation without Nup2. In contrast, binding of the meiotic-autonomous region to meiotic chromosomes is completely dependent on Nup60. Our findings uncover an inhibitory function for the Nup60 C-terminus and suggest that Nup60 mediates recruitment of meiotic chromosomes to the nuclear envelope, while Nup2 plays a secondary role counteracting the inhibitory function in Nup60’s C-terminus.     

The Aspergillus fumigatus Protein GliK Protects against Oxidative Stress and Is Essential for Gliotoxin Biosynthesis

The function of a number of genes in the gliotoxin biosynthetic cluster (gli) in Aspergillus fumigatus remains unknown. Here, we demonstrate that gliK deletion from two strains of A. fumigatus completely abolished gliotoxin biosynthesis. Furthermore, exogenous H₂O₂ (1 mM), but not gliotoxin, significantly induced A. fumigatus gliK expression (P = 0.0101). While both mutants exhibited significant sensitivity to both exogenous gliotoxin (P < 0.001) and H₂O₂ (P < 0.01), unexpectedly, exogenous gliotoxin relieved H₂O₂-induced growth inhibition in a dose-dependent manner (0 to 10 μg/ml). Gliotoxin-containing organic extracts derived from A. fumigatus ATCC 26933 significantly inhibited (P < 0.05) the growth of the ΔgliK²⁶⁹³³ deletion mutant. The A. fumigatus ΔgliK²⁶⁹³³ mutant secreted metabolites, devoid of disulfide linkages or free thiols, that were detectable by reverse-phase high-performance liquid chromatography and liquid chromatography-mass spectrometry with m/z 394 to 396. These metabolites (m/z 394 to 396) were present at significantly higher levels in the culture supernatants of the A. fumigatus ΔgliK²⁶⁹³³ mutant than in those of the wild type (P = 0.0024 [fold difference, 24] and P = 0.0003 [fold difference, 9.6], respectively) and were absent from A. fumigatus ΔgliG. Significantly elevated levels of ergothioneine were present in aqueous mycelial extracts of the A. fumigatus ΔgliK²⁶⁹³³ mutant compared to the wild type (P < 0.001). Determination of the gliotoxin uptake rate revealed a significant difference (P = 0.0045) between that of A. fumigatus ATCC 46645 (9.3 pg/mg mycelium/min) and the ΔgliK⁴⁶⁶⁴⁵ mutant (31.4 pg/mg mycelium/min), strongly suggesting that gliK absence and the presence of elevated ergothioneine levels impede exogenously added gliotoxin efflux. Our results confirm a role for gliK in gliotoxin biosynthesis and reveal new insights into gliotoxin functionality in A. fumigatus.     

Developmental pattern of Δ5 3β-hydroxysteroid dehydrogenase activity in isolated rat Leydig cells

A direct method for determination of Δ⁵ 3β-hydroxysteroid dehydrogenase (3β-HSD) activity was employed in isolated Leydig cells (LC) derived from rats on fetal day 19 (F₁₉) and postnatal (N) days 1,12,24, 34 and 45 and adults. The activity of 3β-HSD in the adult LC was 1.15 ± 0.02 (μmole/μg DNA/hr, mean ± SEM, n = 73). Activities in the other groups, expressed as a percentage of the respective adult control, were: F₁₉-38%; N₁-39%; N₁₂-8%; N₂₄-89%; N₃₄-166%; and N₄₅-118%. A good correlation was found between histochemical staining for 3β-HSD and the quantitive method employed. Using (³H)-DHA as a substrate, LC isolated from F₁₉, n₁ and N₁₂ produced testosterone in appreciable amounts (41%, 55% and 20% of the toal products respectively) whereas at advanced stages of development (N₂₄ to adulthood) the major product was androstenedione (93 ± 1%). These findings may be explained by the observed decrease in 17β-hydroxysteroid dehydrogenase (17β-HSD) activity, due to an insufficient supply of NADPH, in the older vs. earlier stages of development. This study indicates the presence of steroidogenic enzymatic activity in LC throughout development in the rat. It also provides a relatively simple in vitro model for studies of testicular regulation during development.