Aerobic 2-ketogluconate metabolism of Klebsiella pneumoniae NCTC 418 grown in chemostat culture.

Klebsiella pneumoniae NCTC 418 is able to convert 2-ketogluconate intracellularly to 6-phosphogluconate by the combined action of an NADPH-dependent 2-ketogluconate reductase and gluconate kinase. Synthesis of the former enzyme was maximal under 2-ketogluconate-limited growth conditions. An instantaneous transition to a 2-ketogluconate-excess condition resulted in an acceleration of catabolism of this carbon source, accompanied by complete inhibition of biosynthesis. It is suggested that the cause of this inhibition resides in depletion of the NADPH pool due to the high rate at which NADPH is oxidized by 2-ketogluconate reductase.     

Changes in cell-surface carbohydrates of Trypanosoma cruzi during metacyclogenesis under chemically defined conditions.

Highly purified lectins with specificities for receptor molecules containing sialic acid, N-acetylglucosamine (D-GlcNAc), N-acetylgalactosamine (D-GalNAc), galactose (D-Gal), mannose-like residues (D-Man) or L-fucose (L-Fuc), were used to determine changes in cell-surface carbohydrates of the protozoal parasite Trypanosoma cruzi during metacyclogenesis under chemically defined conditions. Of the D-GalNAc-binding lectins, BS-I selectively agglutinated metacyclic trypomastigotes, MPL was selective for replicating epimastigotes, whereas SBA strongly agglutinated all developmental stages of T. cruzi. WGA (sialic acid and/or D-GlcNAc specific) was also reactive with differentiating epimastigotes and metacyclic trypomastigotes but displayed a higher reactivity with replicating epimastigote forms. A progressive decrease in agglutinating activity was observed for jacaline (specific for D-Gal) during the metacyclogenesis process; conversely, a progressive increase in affinity was observed for RCA-I (D-Gal-specific), although the reactivity of other D-Gal-specific lectins (PNA and AxP) was strong at all developmental stages. All developmental stages of T. cruzi were agglutinated by Con A and Lens culinaris lectins (specific for D-Man-like residues); however, they were unreactive with the L-fucose-binding lectins from Lotus tetragonolobos and Ulex europaeus. These agglutination assays were further confirmed by binding studies using 125I-labelled lectins. Neuraminidase activity was detected in supernatants of cell-free differentiation medium using the PNA hemagglutination test with human A erythrocytes. The most pronounced differences in lectin agglutination activity were observed between replicating and differentiating epimastigotes, suggesting that changes in the composition of accessible cell-surface carbohydrates precede the morphological transformation of epimastigotes into metacyclic trypomastigotes.     

The role of the IGF1 receptor in the regulation of cdc2 mRNA levels in fibroblasts

The levels of cdc2 mRNA increase when quiescent cells are stimulated by growth factors. In BALB/c 3T3, both platelet-derived growth factor and insulin-like growth factor 1 (IGF-1) are required to increase cdc2 mRNA levels. In p6 cells, which constitutively overexpress the IGF-1 receptor, IGF-1 is sufficient. The importance of the IGF-1/IGF-1 receptor interaction in regulating the levels of cdc2 mRNA was further confirmed by showing that an antisense oligodeoxynucleotide to the IGF-1 receptor RNA inhibited the IGF-1-mediated increase.     

Interaction between flocculent and nonflocculent cells of Saccharomyces cerevisiae.

Interaction between nonflocculent and flocculent cells of Saccharomyces cerevisiae was studied. Adhesion experiments were done using three types of nonflocculent cells and a flocculent one. Two types of nonflocculent cells were obtained from the flocculent strain by changing environmental growth conditions. The integration of nonflocculent cells in the flocs was observed by two different methods: measurement of the sedimentation capacity of mixtures and microscopic observation of stained nonflocculent cells blended with flocculent cells. It was possible to verify that cell-cell interaction corresponds to a true stable binding and not to a simple entrapment inside the floc matrix.     

Immunocytochemical identification of neuroendocrine markers in human cardiac paraganglion — like structures

Summary Paraganglion-like structures (PLS) containing chromaffin-positive cells have been reported to be present in the adult human heart. The present work was initiated in order to evaluate the densitity of these structures in the interatrial septum and to study the presence of immunoreactivity of their cells to NSE and PGP 9,5 antibodies, two neuroendocrine markers. Six hundred 6-m paraffin serial sections were obtained from the upper third of the interatrial septum from six adult human hearts. From 2 to 12 paraganglia were found in each case, and their principal cells stained positively with NSE and PGP 9,5 antibodies. Depending on how these PLS related to other cardiac structures, four different types were identified: Type I — True paraganglia (located adjacent to ganglia or nerve fibers); Type II — Free paraganglia (immersed in the interatrial adipose tissue, without evident connection to other structures); Type III — Intraganglionic paraganglia (located within the nervous ganglia); Type IV — Intramyocardic paraganglia (small nests of immunoreactive cells closely related to myocardiocyte bundles). These cardiac paraganglia, which probably belong to the visceral-autonomic group, may have a role in the regulation of the cardiac function and in the adaptive mechanisms of the heart. Its is also possible that they originate functioning and non-functioning tumours.Work supported by grants from FINEP and CNPq (Brazil)     

The active centers of adenylylsulfate reductase from Desulfovibrio gigas. Characterization and spectroscopic studies

In order to utilize sulfate as the terminal electron acceptor, sulfate-reducing bacteria are equipped with a complex enzymatic system in which adenylylsulfate (AdoPSO4) reductase plays one of the major roles, reducing AdoPSO4 (the activated form of sulfate) to sulfite, with release of AMP. The enzyme has been purified to homogeneity from the anaerobic sulfate reducer Desulfovibrio gigas. The protein is composed of two non-identical subunits (70 kDa and 23 kDa) and is isolated in a multimeric form (approximately 400 kDa). It is an iron-sulfur, flavin-containing protein, with one FAD moiety, eight iron atoms and a minimum molecular mass of 93 kDa. Low-temperature EPR studies were performed to characterize its redox centers. In the native state, the enzyme showed an almost isotropic signal centered at g = 2.02 and only detectable below 20 K. This signal represented a minor species (0.10-0.25 spins/mol) and showed line broadening in the enzyme isolated from 57Fe-grown cells. Addition of sulfite had a minor effect on the EPR spectrum, but caused a major decrease in the visible region of the optical spectrum (around 392 nm). Further addition of AMP induced only a minor change in the visible spectrum whereas major changes were seen in the EPR spectrum; the appearance of a rhombic signal at g values 2.096, 1.940 and 1.890 (reduced Fe-S center I) observable below 30 K and a concomitant decrease in intensity of the g = 2.02 signal were detected. Effects of chemical reductants (ascorbate, H2/hydrogenase-reduced methyl viologen and dithionite) were also studied. A short time reduction with dithionite (15 s) or reduction with methyl viologen gave rise to the full reduction of center I (with slightly modified g values at 2.079, 1.939 and 1.897), and the complete disappearance of the g = 2.02 signal. Further reduction with dithionite produces a very complex EPR spectrum of a spin-spin-coupled nature (observable below 20 K), indicating the presence of at least two iron-sulfur centers, (centers I and II). M?ssbauer studies on 57Fe-enriched D. gigas AdoPSO4 reductase demonstrated unambiguously the presence of two 4Fe clusters. Center II has a redox potential less than or equal to 400 mV and exhibits spectroscopic properties that are characteristic of a ferredoxin-type [4Fe-4S] cluster. Center I exhibits spectra with atypical M?ssbauer parameters in its reduced state and has a midpoint potential around 0 mV, which is distinct from that of a ferredoxin-type [4Fe-4S] cluster, suggesting a different structure and/or a distinct cluster-ligand environment.     

Changes in RNA polymerase II in a cell cycle-specific temperature-sensitive mutant of hamster cells

tsAF8 cells are a temperature-sensitive mutant of BHK cells that arrest at the nonpermissive temperature in the G₁ phase of the cell cycle. The activity of solubilized RNA polymerase II and its ability to bind [³H]-γ-amanitin decrease in tsAF8 cells at 40.6°, with a half-life of ～ 10 hr. No appreciable changes occur in these two parameters in tsAF8 cells at 34° or in BHK cells at either 34° or 40.6°. Protein synthesis is not appreciably affected for at least 24 hr after tsAF8 cells are shifted to 40.6°. These results indicate that in tsAF8 cells at the nonpermissive temperature, there is a defect in either the synthesis, the assembly, or the stability of RNA polymerase II, and that the loss of RNA polymerase II molecules is not due to widespread cellular damage.