Candida albicans morphologies revealed by scanning electron microscopy analysis

Scanning electron microscope (SEM) observations were used to analyze particular morphologies of Candida albicans clinical isolate (strain 82) and mutants defective in hyphae-promoting genes EFG1 (strain HLC52) and/ or CPH1 (strains HLC54 and Can16). Transcription factors Efg1 and Cph1 play role in regulating filamentation and adhesion of C. albicans' morphologies. Comparative analysis of such mutants and clinical isolate showed that Efg1 is required for human serum-induced cell growth and morphological switching. In the study, distinct differences between ultrastructural patterns of clinical strain's and null mutants' morphologies were observed (spherical vs tube-like blastoconidia, or solid and fragile constricted septa vs only the latter observed in strains with EFG1 deleted). In addition, wild type strain displayed smooth colonies of cells in comparison to mutants which exhibited wrinkled phenotype. It was observed that blastoconidia of clinical strain exhibited either polarly or randomly located budding. Contrariwise, morphotypes of mutants showed either multiple polar budding or a centrally located single bud scar (mother-daughter cell junction) distinguishing tube-like yeast/ pseudohyphal growth (the length-to-width ratios larger than 1.5). In their planktonic form of growth, blastoconidia of clinical bloodstream isolate formed constitutively true hyphae under undiluted human serum inducing conditions. It was found that true hyphae are essential elements for developing structural integrity of conglomerate, as mutants displaying defects in their flocculation and conglomerate-forming abilities in serum. While filamentation is an important virulence trait in C. albicans the true hyphae are the morphologies which may be expected to play a role in bloodstream infections.

Candida albicans morphologies revealed by scanning electron microscopy analysis.

Scanning electron microscope (SEM) observations were used to analyze particular morphologies of Candida albicans clinical isolate (strain 82) and mutants defective in hyphae-promoting genes EFG1 (strain HLC52) and/or CPH1 (strains HLC54 and Can16). Transcription factors Efg1 and Cph1 play role in regulating filamentation and adhesion of C. albicans' morphologies. Comparative analysis of such mutants and clinical isolate showed that Efg1 is required for human serum-induced cell growth and morphological switching. In the study, distinct differences between ultrastructural patterns of clinical strain's and null mutants' morphologies were observed (spherical vs tube-like blastoconidia, or solid and fragile constricted septa vs only the latter observed in strains with EFG1 deleted). In addition, wild type strain displayed smooth colonies of cells in comparison to mutants which exhibited wrinkled phenotype. It was observed that blastoconidia of clinical strain exhibited either polarly or randomly located budding. Contrariwise, morphotypes of mutants showed either multiple polar budding or a centrally located single bud scar (mother-daughter cell junction) distinguishing tube-like yeast/pseudohyphal growth (the length-to-width ratios larger than 1.5). In their planktonic form of growth, blastoconidia of clinical bloodstream isolate formed constitutively true hyphae under undiluted human serum inducing conditions. It was found that true hyphae are essential elements for developing structural integrity of conglomerate, as mutants displaying defects in their flocculation and conglomerate-forming abilities in serum. While filamentation is an important virulence trait in C. albicans the true hyphae are the morphologies which may be expected to play a role in bloodstream infections.

Discrimination between the enzymatic activities of Candida albicans pleomorphic forms determined using the api® ZYM test.

Enzymatic activity profiles for two morphotypes of 37 Candida albicans clinical isolates were compared. Yeast and hyphal forms were grown using yeast extract-peptone-glucose broth or undiluted human serum, respectively. Both morphotypes were documented under scanning electron microscopy. The api(®) ZYM (BioMérieux, France) test was used to evaluate the enzymatic activity profiles for particular pleomorphic forms. None of the examined enzymatic activities showed good agreement (kappa, κ > 0.80) for the two morphotypes of the tested strains. Only leucine arylamidase activity in blastoconidia and hyphae of 35 out of 37 strains appeared to be in significant agreement (κ = 0.770). This phenomenon should be explored further for clinical benefits. For morphotypes of all tested strains, activity profiles of 11 hydrolytic enzymes demonstrated weak agreement (κ = 0.044-0.197). Moreover, satisfactory (κ = 0.218-0.348) and moderate agreement (κ = 0.413-0.479) were noted for enzymatic activity values of five and two enzymes, respectively. The distinct differences in activity profiles of hydrolytic enzymes between hyphae and blastoconidia is suggested to be related to the specific roles of these two morphotypes in particular steps of pathogenesis. Moreover, both morphotypes should be examined by strain biotyping methods. Beta-N-hexosaminidase (HexNAcase) activity assessed by the api(®) ZYM test and on CHROMagar Candida(®) medium (Becton Dickinson, USA) is also discussed.

Properties of Saccharopolyspora erythraea strains after protoplast regeneration.

Protoplast formation, stabilization and regeneration was improved for 4 strains (erythromycin producers) of Saccharopolyspora erythraea. A modified medium was developed for protoplast regeneration. Parental and protoplast-regenerated strains exhibited changes in morphology, ultrastructure, and antibiotic production.

In situ localization of manganese peroxidase production in mycelial pellets of Phanerochaete chrysosporium.

The ultrastructure of Phanerochaete chrysosporium hyphae from pellets in submerged liquid cultures was investigated in order to learn more about the interrelation between fungal architecture and manganese peroxidase (MnP) production. At day 2 of cultivation, some subapical regions of hyphae in the outer and middle zones of the pellet initiated differentiation into intercalary thick-walled chlamydospore-like cells of about 10 micro m diameter. At the periphery of the cytoplasm of these cells, a large number of mitochondria and Golgi-like vesicles were observed. The sites of MnP production were localized at different stages of cultivation by an immunolabelling procedure. The immunomarker of MnP was mainly concentrated in the chlamydospore-like cells and principally distributed in Golgi-like vesicles located at the periphery of the cytoplasm. The apices of hyphae in the outer layer of the pellets were apparently minor sites of MnP production. Maximal MnP release into the culture supernatant coincided with apparent autolysis of the chlamydospore-like cells. Production of extracellular autolytic chitinase and protease coincided with the disappearance of these structures from the pellets. The chlamydospore-like cells observed in the mycelial pellets of P. chrysosporium could be metabolically active entities operating as an enzyme reservoir, delivering their content into the surrounding medium possibly by an enzyme-mediated autolytic process.

[Efficacy of amnioinfusion in anhydramnios at term before induction of labor and newborn outcome]. / Wplyw amnioinfuzji przedporodowej na przebieg indukcji porodu i stan noworodka w ciazy z malowodziem.

Oligohydramnios complicates about 0.5-5% of pregnancies and is associated with an increased rate of perinatal morbidity and mortality. Our purpose was determine the effectiveness of amnioinfusion before induction of labour in reducing the incidence of fetal distress in pregnancies with anhydramnios at term. The rate of cesarean sections performed for fetal distress was higher in the control group (57.1%) than in the amnioinfused group (20.3%). Anhydramnios was associated with an increased abnormalities fetal heart rate. Amnioinfusion is effective option for the prevention of fetal distress in anhydramnios at term.

Screening and search for novel inhibitors of DD-peptidase 64-575.

The aim of the work was search for inhibitors of PBPs (penicillin binding proteins, DD-carboxypeptidase/transpeptidase, DD-peptidase). Between 141 tested Streptomyces strains, 25% showed activity of inhibitors production. The inhibitors were produced by selected Streptomyces strains (NIH Culture Collection). The culture supernatants of Streptomyces rimosus B PZH (inhibitor B) and Streptomyces rimosus NRRL 2234 (inhibitor 2234) exhibited the highest inhibition activity. Both inhibitors were purified by the use of: anion exchange chromatography and reversed phase chromatography (HPLC). Inhibitor B is a gamma-lactam compound and inhibitor 2234 is a beta-lactam.

Overexpression of the Saccharomyces cerevisiae mannosylphosphodolichol synthase-encoding gene in Trichoderma reesei results in an increased level of protein secretion and abnormal cell ultrastructure.

Production of extracellular proteins plays an important role in the physiology of Trichoderma reesei and has potential industrial application. To improve the efficiency of protein secretion, we overexpressed in T. reesei the DPM1 gene of Saccharomyces cerevisiae, encoding mannosylphosphodolichol (MPD) synthase, under homologous, constitutively acting expression signals. Four stable transformants, each with different copy numbers of tandemly integrated DPM1, exhibited roughly double the activity of MPD synthase in the respective endoplasmic reticulum membrane fraction. On a dry-weight basis, they secreted up to sevenfold-higher concentrations of extracellular proteins during growth on lactose, a carbon source promoting formation of cellulases. Northern blot analysis showed that the relative level of the transcript of cbh1, which encodes the major cellulase (cellobiohydrolase I [CBH I]), did not increase in the transformants. On the other hand, the amount of secreted CBH I and, in all but one of the transformants, intracellular CBH I was elevated. Our results suggest that posttranscriptional processes are responsible for the increase in CBH I production. The carbohydrate contents of the extracellular proteins were comparable in the wild type and in the transformants, and no hyperglycosylation was detected. Electron microscopy of the DPM1-amplified strains revealed amorphous structure of the cell wall and over three times as many mitochondria as in the control. Our data indicate that molecular manipulation of glycan biosynthesis in Trichoderma can result in improved protein secretion.

[Affinity of exocellular DD-carboxypeptidase/transpeptidase from Saccharopolyspora erythraea PZH TZ-575 to beta-lactam compounds]. / Powinowactwo zewnatrzkomórkowej DD-karboksypeptydazy/transpeptydazy szczepu Saccharopolyspora erythraea PZH TZ 64-575 do zwiazków beta-laktamowych.

The DD-carboxypeptidase/transpeptidases (DD-peptidases) involved in bacterial cell wall metabolism, catalyse the attack of C-terminal D-alanyl-D-alanine peptide bond of the peptydoglycan precursor. These enzymes are inactivated by beta-lactam antibiotics. DD-peptidase from Saccharopolyspora erythraea PZH TZ 64-575 was purified by the use of DEAE-cellulose, Sephadex G-100, Q-Sepharose resins and FPLC (Mono Q). After each step the effluent was concentrated by Amicon ultrafiltration. The purified enzyme showed DD-carboxypeptidase specific activity of 50.9 U/mg. The enzyme exhibited high affinity to beta-lactam compounds e.g. cefamandole, cefapirin, cefradin 1.5-2.6 x 10(-8) M. It was used to screen strains from the Culture Collection of the National Institute of Hygiene in Warsaw for the production of DD-peptidase inhibitors.

Glucose-induced secretion of Trichoderma reesei xylanases.

To produce two xylanases with Trichoderma reesei grown on glucose, recombinant strains which carry either the xyn1 or the xyn2 (xylanase I and II [XYN I and XYN II]-encoding) structural genes under the expression signals of the homologous pki1 (pyruvate kinase-encoding) gene were constructed. The two types of transformants secreted XYN I or II, respectively, during growth on glucose, as demonstrated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunostaining. The corresponding specific xylanase activities of the best transformants on glucose were 76 and 145 U/mg of protein for XYN I and XYN II, respectively, as opposed to that obtained by the parent strain (26 U/mg of protein). When related to the amount of biomass formed, however, they produced only about 4 to 5 U/g, in contrast to much higher activities (10 to 12 U/g) during growth on xylan. The ultrastructural location of XYN II in the transformant strain producing the highest constitutive XYN II formation (ATX2-12) was investigated by immunoelectron microscopy and compared with that in the wild-type strain growing on xylan. Cell extracts from both types of transformants grown on glucose exhibited a higher intracellular xylanase activity than did the parent strain grown on xylan. By using electron microscopy and immunogold labelling, XYN II was detected in the endoplasmic reticulum, Golgi-like vesicles, secretory vesicles, vacuoles, and cell walls. The immunolabel in the vacuoles was detected preferentially in subapical cells. When a recombinant strain which expressed xyn2 from the pki1 promoter was compared with the parent strain during growth on xylan, the former exhibited a less proliferated endoplasmic reticulum and a smaller number of secretory vesicles; however, a higher density of labelling was observed. The relationship of these findings to the efficacy of protein secretion during growth on glucose is discussed.

[Localization of penicillin G chemoreceptors in cells of Streptomyces sp. R61]. / Lokalizacja chemoreceptorów penicyliny G w komórkach szczepu Streptomyces sp. R61.

The purpose of the present work was localization of penicillin-binding proteins (PBPs) in the cells of Streptomyces sp. R61 using immunological and autoradiographic methods and electron microscopy. The cells were treated with H3-penicillin G. PBPs of protoplasts were marked with peroxidase labelled IgG to DD-carboxypeptidase of the strain. The results indicate areas privileged in PBPs content at the cytoplasmic membrane and in vesicles located in the periplasmic space. PBPs were visualized in bulges of the protoplasts membrane. In the cells PBPs are present at hyphal tips and in centers at the periphery of cells, which are places of cross wall biosynthesis or branching of hyphae and overproduction of cell wall material.

Reversion of L-lysine inhibition of penicillin G biosynthesis by 6-oxopiperidine-2-carboxylic acid in Penicillium chrysogenum PQ-96.

6-Oxopiperidine-2-carboxylic acid (OCA; cyclic alpha-aminoadipic acid) reverses the L-lysine inhibition of penicillin G production by Penicillium chrysogenum PQ-96. The reaction probably depends on the recovery of L-alpha-aminoadipic acid for penicillin G production from OCA.

[Physico-chemical studies of aluminum hydroxide in biological preparations preserved in different conditions]. / Ocena fizyko-chemicznego stanu wodorotlenku glinu w biopreparatach przechowywanych w róznych warunkach.

An observation of physico-chemical properties of 0.5% Al(OH)3 and vaccines adsorbed to it (Di, Te, Di-Te, Di-Te-Per) stored for 3-24 months in various pH (5, 7, 8) and temperatures -18 degrees, +37 degrees, +45 degrees and +65 degrees was carried out. For this purpose an analysis of sedimentation rate, microscopic observation and ++roentgenographic analysis were performed. It was found that a decrease of storage temperature of Al(OH)3 gel and vaccines down to -18 degrees C resulted in some changes in structure and morphology of this absorbent (big precipitates) and it led to a significant increase of sedimentation rate.

[Serum cathepsin A activity in pregnant, parturient and puerperal patients]. / Die Aktivität des Kathepsins A im Blutserum Schwangerer, Gebärender und Wöchnerinnen.

Cathepsin A activity has been determinal in sera of nonpregnant, pregnant (1st, 2nd and 3rd trimesto), puerperal (first, third and fifth day post partum) and parturient women in second stage (retroplacental blood and blood from umbilical cord) by means of N-Cbz-L-glutamyl-L-tyrosin after incubation with 37 degrees C and pH 5.5. There was an increasing cathepsin A activity with the duration of pregnancy, during labour and in the first puerperal days. Maximum of activity of cathepsin A has been determined in serum of retroplacental blood.

[The activities of cathepsin A in the placenta, fetal membranes and amniotic fluid in physiologic pregnancy and pregnancy complicated by EPH gestosis]. / Die Aktivitäten des Kathepsins A in der Plazenta, den Eihäuten und der Amnionflüssigkeit in der physiologischen und durch EPH-Gestose komplizierten Schwangerschaft.

Activity of cathepsin A was determined in placenta, fetal membranes and amniotic fluid as well in normal pregnancy as in complicated pregnancy by EPH-gestosis. Measurement of activity was done by N-CbZ-L-glutamyl-tyrosine with pH 5.5. Compared with normal pregnancy activity of cathepsin A was lower in the three materials of EPH-gestosis.

Biosynthesis of benzylpenicillin by Penicillium chrysogenum and its Golgi apparatus.

The fine structure of high and low-yield mutants of Penicillium chrysogenum producing 10,000 and 100 units of benzylpenicillin was compared. The cells of both mutants showed typical eukaryotic ultrastructure. The Golgi vesicles, present in largest number in cells of high-yield mutant, fuse with the cell membrane and play an important role in the transport of benzylpenicillin from the cytoplasm to the cell environment. Benzylpenicillin was localized in cells of the high-yield mutant by means of enzymatical and immunological methods. The results indicate that benzylpenicillin is stored in the vesicles of the Golgi apparatus. The Golgi vesicles isolated from the protoplasts of high-yield mutant showed activities of enzymes of the pathway of benzylpenicillin biosynthesis i.e., delta-/L-alpha-aminoadipyl/-L-cysteinyl-D-valine synthetase, isopenicillin N synthetase, phenylacetyl: coenzyme A ligase, and acyl-exchange activity. Cell-free biosynthesis of antibiotic by the native Golgi vesicles was investigated in a well-defined reaction mixture. The native Golgi vesicles produced antibiotic in amount corresponding to 320 nmol.mg protein-1.h-1. The activity yield of the calcium alginate immobilized Golgi vesicles was 44%. Moreover, a hypothetical scheme for localization of the enzymes of pathway of benzylpenicillin biosynthesis in the cells of high-yield mutant is presented.

Bacteriophages of Bacillus polymyxa.

Virulent bacteriophages of colistin--producing Bacillus polymyxa strains were studied. The phages were found to differ in lytic spectrum and were active only against strains of B. polymyxa. They did not attack other strains of the genus Bacillus. The virulent bacteriophages belong to two morphological groups differing in size. The size of the DNA of the bacteriophages of both groups is similar and ranges from 74.9 X 10(6) to 87.8 X 10(6) daltons. The cells of different B. polymyxa strains were also found to carry various defective phages which could be shown after mitomycin C induction of cell cultures. The antibacterial activity of mitomycin C induced cell lysates was not detected. Strains of B. polymyxa most probably devoid of defective bacteriophages (delysogenized) were isolated.