Contribution to the knowledge of the enzymatic activity of yeasts of clinical interest

The determination of the enzymatic activity of the yeasts has been applied to the identification of species, specially that ofCandida albicans. In order to know its usefulness in species of clinical interest, we have tested the commercial system API ZYM (Bio Mérieux) on 500 isolated strains of different organic samples, belonging to eight genera and twenty species. All the strains showed positivity to Phosphatase alcaline, Esterase (C4), Esterase lipase (C8), Leucine arylamidase and Phosphatase acid, and negativity to Lipase (C14), Trypsin, Chymotrypsin, -galactosidase, -glucoronidase, -manosidase and -fucosidase. Fourteen enzymatic activity patterns were obtained considering the substrates with variable results for the whole of the strains: Valine arylamidase, Cystine arylamidase, Naphthol-AS-BI-phosphohydrolase, -galactosidase, -glucosidase, -glucosidase and N-acetyl--glucosaminidase. In the majority of the species, the enzymatic profile did not have very specific results since it is usually shared by more than one species.C. albicans is that which presents the greatest number of enzymatic variations, some of these are similar to those of other common clinical species, such asCandida krusei, Candida parapsilosis andCandida tropicalis. This system is proposed as a rapid method for identification and as an epidemiological marker of medically important yeasts.Abbreviations AGL -glucosidase - BGA -galactosidase - BGL -glucosidase - CAA Cystine arylamidase - NAG N.Acetyl--glucosaminidase - PHO Naphthol-AS-BI-phosphohydrolase - VAA Valine arylamidase     

Assignment of human ferritin genes to chromosomes 11 and 19q13.3→19qter

Summary Extracts of hamster-human and mouse-human hybrids, some with translocations involving chromosome 19, have been assayed for both human spleen ferritin (rich in L subunits) and human heart ferritin (rich in H subunits). Hybrid lines retaining part of the long arm of chromosome 19 including the region 19q13.319qter produced human L type ferritin. This confirms the previous assignment of the ferritin gene to chromosome 19 (Caskey et al. 1983). However, lines retaining chromosome 11 were found to contain human H type ferritin suggesting that the gene for the H subunit is on this chromosome. The presence of chromosome 6 was not necessary for the expression of either H or L type human ferritin. It thus seems unlikely that the gene for idiopathic haemochromatosis is a ferritin gene.     

Mass spectrometric analysis of genetic and post-translational heterogeneity in the lectins Jacalin andMaclura pomifera agglutinin

Jacalin andM. pomifera agglutinin are T-antigen specific lectins with ₄₄ structures that show far greater microheterogeneity than plant lectins from other families, due to multiple genetic isoforms and post-translational processing. Electrospray mass spectrometry and combined liquid chromatography-electrospray mass spectrometry were used to characterize the various forms. For both lectins, the mass data were consistent with previous protein sequencing of the major -chain species of 133 residues and three -chain species of 20 or 21 residues. In addition, for jacalin the mass of one minor -chain species was consistent with a second of the four reported gene sequences. However, the glycopeptide -chain form and one -chain form did not match any of the genes, suggesting a fifth gene remains to be found. ForM. pomifera agglutinin, three more -chain forms were found, but all six could arise from only two genes, with additional post-translational proteolysis and post-translational substitution with an unidentified component of 106 Da creating the set of six forms. Only two -chain forms were found also, with no glycosylation.     

Inactivation of voltage-dependent calcium current in an insulinoma cell line

We have studied the mechanism of Ca current inactivation in the -cell line HIT-T15 by conventional and perforated patch recording techniques, using two pulse voltage protocols and a combination of current and tail current measurements. In 5 mM Ca, from a holding potential of - 80 mV, the maximum current showed a complex time course of inactivation: a relatively fast, double exponential inactivation (_h1 12 ms and _h2 60 ms) and a very slowly inactivating component ( > 1 s). The faster component (_h1) was due to the voltage-dependent inactivation of a low-threshold-activated (LVA), T-type current, which deactivates more slowly ( 3–5 ms) than the other components ( 0.2–0.3 ms). The intermediate component (_h2) was due to the Ca-dependent inactivation of a portion of the high-threshold-activated (HVA) current. A saturating dose of the dihydropyridine (DHP) nifedipine (10 M) did not affect the LVA current, but inhibited by 68 ± 5% the transient, Ca-sensitive portion of the HVA current and by 33 ± 12% the long lasting component. We suggest that three components of the calcium current can be resolved in HIT cells and the main target of DHPs is a HVA current, which inactivates faster than the DHP-resistant HVA component and does so primarily through calcium influx. Correspondence to: C. Marchetti     

Allospecific T cell recognition of HLA-A2 antigens: Evidence for group-specific and subgroup-specific epitopes

Interleukin 2-dependent alloreactive cytotoxic T cell lines, with activity predominantly directed against the HLA-A2 antigen, have been generated in vitro by stimulating blood mononuclear cells from donors nonimmune to the Epstein-Barr (EB) virus with appropriate numbers of EB virus-transformed B cells from A2-homozygous individuals. Such effector cells were tested against a panel of EB virus-transformed target cell lines all expressing the serologically defined A2 antigen but typed into common A2 and variant A2 subgroups on the basis of their recognition by A2-restricted EB virus-specific cytotoxic T cells. Variant A2 responder cells cocultivated with common A2-bearing stimulators gave rise to effector T cell lines which recognized only the common A2-bearing subgroup of targets. By contrast, responder cells from A2-negative donors stimulated with common A2-bearing cells produced effector T cell lines in which the strong lysis of common A2-bearing targets was accompanied by a lower, but still significant, lysis directed against all targets within the variant A2 subgroup. In both cases, lysis of the target cells was blocked equally well by the anti-A2-specific monoclonal antibody MA2.1 as by the monoclonal antibody W6/32 specific for HLA-A, -B, and -C determinants. This suggests that HLA-A2 molecules possess at least two distinct sets of epitopes capable of inducing alloreactive T cell cytotoxicity: first, epitopes probably associated with T cell-restricting sites, which generate subgroup-specific responses, and second, epitopes shared by all A2 molecules, and perhaps associated with serologically defined sites, which generate pan A2 group-specific responses.Abbreviations used in this paper EB Epstein-Barr - IL-2 Interleukin 2 - UM unfractionated mononuclear - AET aminoethylisothiouroniumbromide hydrobromide     

Immobilized Lotus tetragonolobus agglutinin binds oligosaccharides containing the Lex determinant

A defined set of oligosaccharides and glycopeptides containing -linked fucose were used to examine the specificity of the immobilized fucose-binding lectin Lotus tetragonolobus agglutinin (LTA1), also known as lotus lectin. Glycans containing the Lewis x determinant (Lex) Gal1-4[Fuc1-3]GlcNAc1-3-R were significantly retarded in elution from high density LTA-Emphaze columns. The lectin also bound the fucosylated lacdiNAc trisaccharide GalNAc1-4[Fuc1-3]GlcNAc. The lectin did not bind glycans containing either sialylLex or VIM-2 determinants, nor did it bind the isomeric Lea, Gal1-3[Fuc1-4]GlcNAc-R. Although 2-fucosyllactose Fuc1-2Gal1-4Glc) was retarded in elution from the columns, larger glycans containing the H-antigen Fuc1-2Gal1-3(4)GlcNAc-R interacted poorly with immobilized LTA. Our results demonstrate that immobilized LTA is effective in isolating glycans containing the Lex antigen and is useful in analyzing specific fucosylation of glycoconjugates. Abbreviations: LTA, Lotus tetragonolobus agglutinin; UEA-1, Ulex europaeus agglutinin-I; LNT, AAL, Aleuria aurantia agglutinin; Gal1-3GlcNAc1-3Gal1-3Glc; LNnT, Gal1-4GlcNAc1-3Gal1-3Glc; Lex, Lewis x antigen; Lea, Lewis a antigen; GDPFuc, guanosine 5-diphosphate--L-fucose     

Die kerngrössenverhältnisse in der larvenentwicklung verschiedener kasten bei formiciden

Zusammenfassung Zur Klärung des Problems der Kastendetermination bei Formiciden konnte durch die Untersuchung der endomitotischen Polyploidisierung im Verlauf der Larvenentwicklung beigetragen werden. Endomitosen können hierbei nicht direkt beobachtet werden, die Polyploidisierung ist nur aus dem Wachstum der Kerne zu erschließen.Die Polyploidisierung sieben verschiedener Gewebe von Myrmica- wurde untersucht. Alle Tiere wachsen unter ständiger Polyploidisierung bis zum Puppenstadium heran. Während der Metamorphose werden alle hochpolyploiden Gewebe abgebaut. Besonders hohe Polyploidiegrade erreichen Gewebe der Stoffwechselorgane, wie Mitteldarm und Malpighische Gefäße. Oenocyten zeigen sehr unübersichtliche Verhältnisse. Die Spinndrüse wird im Zusammenhang mit dem Sekretionszyklus hochpolyploid. Fettzellen, Epidermis und Ganglien zeigen dagegen nur geringe Polyploidiegrade.Die Unterschiede in den verschiedenen Kasten werden festgestellt. Es zeigte sich, daß a anfänglich haploid sind and Geschlechtstiere einen Endomitoseschritt mehr ausführen als .Die Polyploidisierung entsprechender Gewebe von Lasius niger zeigt die gleiche Entwicklungstendenz. Futter- ud Temperatureinflüsse konnten festgestellt werden. Zwerg- zeigten Polyploidiegrade, die von denen der Normal- abweichen und dadurch auf blastogene Determination schließen lassen.-Brut gibt bei Ausschluß der Nestbegattung stets , die sick in ihren Kerngrößen nicht von den aus weiselrichtigen Nestern unterscheiden.Alle untersuchten Formicidenarten weisen die gleiche Entwicklungstendenz auf.Beobachtungen über Entwicklungsdauer, Eiablage und -Brut-Entwicklung werden angefügt.Auf Grund der Ergebnisse wurde zu Fragen der endomitotischen Polyploidisierung Stellung genommen. Die Gründe, die zur Annahme eines Polyploidisierungsvorganges in der Larvenentwicklung der Formiciden führen, werden diskutiert. Polyploidie wird in Beziehung gesetzt zur Körpergröße der Tiere, zur phylogenetischen Entwicklungshöhe und zur Gewebsfunktion (Deutung als Sparsamkeitsmaßnahme). Hypothesen zur Kastendetermination werden durch die Ergebnisse unterstützt.     

On age morphological changes of males of Chydoridae (Cladocera)

Summary Young and adult males of 11 species of Chydoridae are studied, their figures being published here (fig. 1–15). The necessity is stressed to distinguish young forms of males and gynandromorphic individuals.Pleuroxus balatonicus is considered to be described from the population ofPleuroxus unicatus having under Balaton Lake conditions retarded transformation of young males into adult form, and accordingly having unusually numerous young males. \qO\qs\qn\qo\qv\qn\qy\ye \qr\ye\qz\qu\ql\Qj\qt\qa\qt\qy 11 (. 1–15). . , Pleuroxus uncinatus , Pleuroxus balatonicus.     

Die Feinstruktur der Zirbeldrüse normaler,trächtiger und experimentell beeinflußter Meerschweinchen

Zusammenfassung In der Meerschweinchenzirbeldrüse lassen sich elektronenmikroskopisch helle und dunkle Pinealzellen sowie einzelne Gliazellen nachweisen. In den bei weitem überwiegenden hellen Pinealzellen zeichnet sich ein Teil der vesicle-crowned rodlets (VCR) durch lokale Auftreibungen aus. Von VCR deutlich abzugrenzen sind die vesicle-crowned balls (VCB). Erstmalig beschrieben wird das Vorkommen von sog. Zylindern, die als Vorstufen von VCB aufgefaßt werden. In den relativ seltenen dunklen Pinealzellen, die sich durch chromatinreiche Kerne und elektronendichtes Zytoplasma auszeichnen, sind Vesikel, VCR, VCB und Zylinder seltener als in hellen Pinealzellen. Die reichlich vorhandenen marklosen Nervenfasern finden sich vor allem in perivasculären Räumen, seltener im Parenchym. Synapsen zwischen Nerven und Pinealzellen wurden nicht beobachtet. In den Zirbeldrüsen trächtiger Meerschweinchen zeichnen sich in der 2. Hälfte der Tragzeit die hellen Pinealzellen durch stärkere Lappung der Kerne, gehäuftes Auftreten von laktiven Zonen, Vermehrung von Mitochondrien, glattem ER, agranulären Vesikeln, VCR, VCB und Zylindern aus. Die dunklen Pinealzellen nehmen während der Tragzeit an Zahl zu. Post partum bilden sich diese Veränderungen innerhalb einer Woche zurück. Längerer Aufenthalt der Tiere in Dunkelheit führt zu einer Aktivierung der hellen Pinealzellen mit auffallender Vermehrung der VCR und zu einer Zunahme der dunklen Zellen. Unter Dauerbelichtung kommt es in den hellen Zellen zu einer Abnahme fast aller Zellorganellen und zu einer starken Vermehrung der VCR, die nach 70 Tagen auch Formveränderungen aufweisen. Nach Reserpinbehandlung beobachtet man eine Verminderung und degenerative Veränderungen der VCR. Es wird diskutiert, daß die VCR als prae- bzw. postsynaptische Strukturen der Erregungsübertragung von Nerven zu Pinealzellen bzw. von Pinealzellen untereinander dienen könnten.

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The fine structure of the pineal gland of normal, pregnant and experimentally affected guinea-pigs

Summary By means of electron microscopy light and dark pinealocytes can be distinguished in the guinea-pig pineal gland. Glial cells are rare. In the light pinealocyte. the most frequent cell type, some vesicle-crowned rodlets (VCR) show circumscribed thickenings. From these structures vesicle-crowned balls (VCB) have to be clearly distinguished. Furthermore cylinders occur, which, it is suggested, are precursors of VCB. Dark pinealocytes characterized by chromatin-rich nuclei and electron-dense cytoplasm are rare and contain fewer vesicles, VCR, VCB and cylinders than light pinealocytes. Numerous non-myelinated nerve fibres are situated within perivascular spaces, a few also in the parenchyma. Synapses between nerve fibres and pinealocytes were not observed. In the pineal gland of pregnant guinea-pigs the following changes can be observed in the second half of gestation. The light cells show many nuclear indentations and an increase of active zones, mitochondria, smooth ER, agranular vesicles, VCR, VCB, and cylinders respectively. The dark cells increase in number. After birth these changes reverse to normal within one week. Constant darkness leads to an activation of the light cells accompanied by an increase of the VCR and to an increase in number of the dark cells. Under constant illumination the light cells show a decrease of their organelles and a strong increase of the VCR. After 70 days the VCR also show a change in shape. Following reserpine treatment the VCR decrease in number and show signs of degeneration. It is discussed that the VCR function as pre- or postsynaptic structures and that they are involved either in transmitting impulses from nerve fibres to pinealocytes or from one pinealocyte to the other.

Untersuchung unter Leitung von Univ.-Doz. Dr. L. Vollrath.     

Conservation status of parrot populations in an Atlantic rainforest area of southeastern Brazil

A census of four species of syntopic parrots was carried out using distance sampling methods on São Sebastião island, SE Brazil. Most of the 33593 ha island is covered by mature and secondary Atlantic rainforest. Almost 80% of these forests are within the Ilhabela Park. Although the species counted have marked differences in size and weight, density (individuals/km²) and estimated population size in 23500 ha of well-preserved forests were similar: Amazona farinosa (13.82±5.94; 3247±1395), Pionus maximiliani (15.79±7.04; 3712±1654), Brotogeris tirica (15.05±4.87; 3537±1143) and Pyrrhura frontalis (13.06±5.53; 3068±1298). Encounter rates of Forpus crassirostris and Pionopsitta pileata were very low, which suggests that there is only a small population of these species on the island. The São Sebastião forests still support healthy populations of parrots. Although woodpecker population estimates on the island are large enough to provide nesting sites for parrots, competition for holes with other secondary cavity nesters such as toucans, flycatchers and tytiras, and the selective cutting of dead trees for canoe construction, which is a common practice on the island, may limit hole availability for parrots.     

Variation of glucose 6-phosphate dehydrogenase in the HeLa and Detroit 98 cell lines and their clonal derivatives

Contrary to other reports, we have found that the A type G6PD found in two permanent cell lines—HeLa (Gey), with its single cell clonal derivative HeLa S3, and Detroit 98, with its four clonal derivative lines—is not a single variant but rather at least three different isozymes. One is heat stable with normal specific activity and normal A type electrophoretic migration, another is heat labile with normal specific activity and normal A type electrophoretic migration, and the third is heat labile with reduced specific activity and slightly slow A type electrophoretic migration. We also found that in a mosaic cell population with respect to G6PD phenotype, the predominant G6PD phenotype varied randomly over a 5-month period, that the G6PD phenotype might be mutable in permanent cell lines, and that spontaneous human cell lines might not be HeLa cell contaminants as has been suggested.Aided by the National Institutes of Health General Research Support Grant # 5 S01 FR05507.     

Protein engineering in the α-amylase family: catalytic mechanism,substrate specificity,and stability

Most starch hydrolases and related enzymes belong to the -amylase family which contains a characteristic catalytic (/)₈-barrel domain. Currently known primary structures that have sequence similarities represent 18 different specificities, including starch branching enzyme. Crystal structures have been reported in three of these enzyme classes: the -amylases, the cyclodextrin glucanotransferases, and the oligo-1,6-glucosidases. Throughout the -amylase family, only eight amino acid residues are invariant, seven at the active site and a glycine in a short turn. However, comparison of three-dimensional models with a multiple sequence alignment suggests that the diversity in specificity arises by variation in substrate binding at the loops. Designed mutations thus have enhanced transferase activity and altered the oligosaccharide product patterns of -amylases, changed the distribution of -, - and -cyclodextrin production by cyclodextrin glucanotransferases, and shifted the relative -1,4:-1,6 dual-bond specificity of neopullulanase. Barley -amylase isozyme hybrids and Bacillus -amylases demonstrate the impact of a small domain B protruding from the (/)₈-scaffold on the function and stability. Prospects for rational engineering in this family include important members of plant origin, such as -amylase, starch branching and debranching enzymes, and amylomaltase.Abbreviations CGTase cyclodextrin glucanotransferase - SBD starch binding domain - TAA taka-amylase A - TIM triose-phosphate isomerase. The mutations are described with the one-letter code, i.e. D164A is a mutant in which A in the mutant is substituted for D in the wild-type.     

Lipid profile of rat myelin subfractions

Myelin from adult rat brains was separated on a discontinuous sucrose gradient into three subfractions. Analysis of light, heavy and membrane fraction lipid classes was performed by HPTLC and densitometry while fatty acid composition was determinated by GLC. The more interesting results observed are: i) the membrane fraction resembles in its lipid and fatty acid composition other cell membranes (particulary oligodentrocytes); ii) light and heavy myelin are quite similar between them but the former has a higher content of sphingomyelin, a lower hydroxy/nohhydroxy cerebrosides ratio and a lower content of monoenoic fatty acids than the heavy subfraction. The results obtained could explain the different structures observed in each myelin subfraction since fatty acid composition, hydroxy fatty acids, sphingomyelin and cholesterol play a key role in the stability and structure of membranes.     

Über das Verhalten von Champignonstämmen in Mischkultur

Zusammenfassung Mischungen aus dem Mycel sweier weißer oder zweier brauner Stämme des Kulturchampignons hatten keinen Einfluß auf den Fruchtkörperetrag.Bei Mischungen aus einem weißen und einem braunen Stamm was dagegen der Ertrag immer niedriger als erwartet und das Mycel verschwand verhältnismäßig schnell. Der Ertragsausfall beruht in der Regel auf einer Unterdrückung des braunen Stammes.Herrn Prof. von Sengbusch zum 65. Geburtstag in Dankbarkeit und Verehrung gewidment.     

Effect of Intercalators on the Properties of Liquid-Crystalline Dispersions of Nucleic Acid–Chitosan Complex

Molecules of deoxyribonucleic acid and synthetic polydeoxyribonucleotides (NA) in the particles of liquid-crystalline dispersions resulting from interaction with chitosan are accessible to interaction with intercalators. The intercalation is accompanied by alteration in the direction of spatial twist of cholesterics of NA–chitosan complexes. This effect is absent in the case of classical cholesterics produced from NA molecules via phase exclusion, i.e., the cholesteric structure of NA–chitosan complex is very labile as distinct from classical cholesteric NA.     

Testis-specific β2 tubulins are identical in Drosophila melanogaster and D. hydei but differ from the ubiquitous β1 tubulin

In Drosophila as in many organisms tubulins are encoded by a gene family. We have determined the complete nucleotide sequences coding for the 1 and 2 tubulins of Drosophila melanogaster and the 2 tubulin of D. hydei, and found these insect tubulins to be highly conserved and like tubulins of other organisms. This is discussed with reference to the possible functional domains of these proteins. — The 1 tubulin gene of Drosophila is constitutively expressed, whereas the 2 tubulin is expressed specifically in the testes. In D. melanogaster the amino acid sequences of these proteins are 95% homologous, differing at only 25 positions. In the testes the 2 tubulin participates in different microtubules as shown by genetic analysis (Kemphues et al. 1982). Interestingly, all of the amino acids characteristic of the testis-specific 2 tubulin are also present in the corresponding gene of D. hydei. Of special interest is the high degree of conservation of the carboxy-terminal domain in these functionally equivalent tubulins.     

Corticosteroid-binding globulin synthesis and distribution in rat white adipose tissue

The lactone isolated from Fusarium termed L659,699 is a potent specific inhibitor of the enzyme 3hydroxi3methylglutaril coenzyme A (HMG-CoA) synthase. In cultures of smooth muscle cells (SMC) isolated from aortic-arch of control (CSMC) and 5% of cholesterol diet (Ch-SMC) treated chicks, the incorporation of (¹⁴C)acetate to lipids (cholesterol, triacylglycerides and cholesterol ester) were greater in ChSMC cultures than in CSMC and the presence of 0.05 M L659,699 for 2 h in the incubation medium decrease the synthesis of cholesterol however the triacylglycerides synthesis increase. The effect of inhibitor is stronger in young cultures (3–4 steps) than in the older ones (11–12 steps). In young CSMC and ChSMC cultures the inhibition of cholesterol and triacylglycerides synthesis by L659,699 was reversal.     

Identification of a complete set of isogenic wheat/rye D-genome substitution lines by means of Giemsa C-banding

Summary A complete set of isogenic wheat/rye D-genome substitutions were produced by crossing an inbred line of spring rye Secale cereale L. cv. Prolific to a tetraploid wheat, the A-and B-genomes of which had previously been extracted from hexaploid wheat, Triticum aestivum L. em Thell. cv. Thatcher. After chromosome doubling, the derived hexaploid triticale (x Triticosecale Wittmack) was backcrossed to 6x Thatcher and selection for wheat/rye substitution lines was carried out in BCF₃ to BCF₆ families by using Giemsa C-banding. Five fertile disomic wheat/rye D-genome substitution lines were obtained and their chromosomal constitution was determined to be 1D/1R, 2D/2R, 7D/4R, 6D/6R, 7D/7R. The two remaining 3R and 5R substitutions are at the moment in a monosomic condition. Another 1D/7R substitution was detected but this plant was very weak and sterile, indicating that only substitutions between homoeologous chromosomes result in fertile, vigorous plants. Furthermore, many rye telocentrics as well as rye-rye and rye-wheat translocations were selected. Since all lines selected in this program share the same genetic background of Thatcher wheat, genetic heterogeneity is excluded. The material is very useful, therefore, for analyzing the effects of different rye chromosomes or chromosome segments in an otherwise homozygous background.Contribution No. 797     

Characteristics of βA chromosome haplotypes in Japanese

DNA polymorphism patterns linked to the ^A-globin gene were analyzed in healthy Japanese using four different restriction endonucleases. The chromosomes with the ^A-globin gene were mapped through an evaluation of the presence of seven different restriction sites (HincII 5 to ; HindIII in ^G and ^A; HincII in, and 3 to, ₁; AvaII in ; Bam-HI 3 to ). Among 36 chromosomes analyzed, 20 chromosomes had a haplotype of [+–––––+]. Among 55 individuals examined, 7 possessed a homozygous haplotye of [+–––––+]. All Japanese with the ^AT-globin gene had a subhaplotype of [–++–+] 5 to the -globin gene. Their major haplotypes were [–++–+–+] and [–++–++–]. It was expected that the presence of the ^AT-globin gene in Japanese may be deduced from subhaplotypes 5 to the -globin gene.