Biomedical subjects

H Martinetto

Publications and source records attributed to H Martinetto.

6 recordsLinked to original sources

PubMed1999

Partial characterization of guanylyl cyclase activity in calf thyroid.

The aim of the present study was to perform the partial characterization of the enzyme guanylyl cyclase (GC) in bovine thyroid. The results obtained showed the presence of two types of GC: one is soluble and comprises around 79% of total activity, while the other is particulate. Treatment with 1% Triton X-100 increased both activities. When the kinetics of the enzyme was analyzed, using the complex MnGTP as a substrate, the results showed a Michaelis type kinetics for the soluble enzyme, with a Km of 0.037 mM, whereas the particulate GC showed a positive allosteric behavior with a S0.5 of 0.214 mM and a Hill coefficient of 1.9, indicating that the enzyme has at least two binding sites for the substrate. When the influence of different Mn2+ concentrations was studied, a positive allosteric behavior for the soluble GC was found, with a S0.5 of 1.2 mM and a Hill coefficient of 2.2. The kinetics of the particulate enzyme under similar conditions was of Michaelis type, with a Km of 0.752 mM. Although the enzyme is highly dependent on Mn2+, it was of interest to investigate the possible effects of other divalent cations, such as Ca2+ and Mg2+. The replacement of Mn2+ for Mg2+ caused a complete disappearance of the particulate enzyme activity, while the soluble activity decreased by 85%. Addition of Ca2+ had no effect on either GC. However, with suboptimum. concentrations of Mn2+, high Ca2+ concentration caused an increase in soluble activity, but it comprised only 20% of maximum activity with optimum Mn2+ concentrations. With the particulate enzyme a slight but significant inhibition was observed.

Allosteric Regulation↗

PubMed1998

Multicellular stalk-like structures in Saccharomyces cerevisiae.

Stalk formation is a novel pattern of multicellular organization. Yeast cells which survive UV irradiation form colonies that grow vertically to form very long (0.5 to 3.0 cm) and thin (0.5 to 4 mm in diameter) multicellular structures. We describe the conditions required to obtain these stalk-like structures reproducibly in large numbers. Yeast mutants, mutated for control of cell polarity, developmental processes, UV response, and signal transduction cascades were tested and found capable of forming stalk-like structures. We suggest a model that explains the mechanism of stalk formation by mechanical environmental forces. We show that other microorganisms (Candida albicans, Schizosaccharomyces pombe, and Escherichia coli) also form stalks, suggesting that the ability to produce stalks may be a general property of microorganisms. Diploid yeast stalks sporulate at an elevated frequency, raising the possibility that the physiological role of stalks might be disseminating spores.

Candida albicans↗

PubMed1995

Identification of a dual specificity kinase that activates the Jun kinases and p38-Mpk2.

One Ras-dependent protein kinase cascade leading from growth factor receptors to the ERK (extracellular signal-regulated kinases) subgroup of mitogen-activated protein kinases (MAPKs) is dependent on the protein kinase Raf-1, which activates the MEK (MAPK or ERK kinase) dual specificity kinases. A second protein kinase cascade leading to activation of the Jun kinases (JNKs) is dependent on MEKK (MEK kinase). A dual-specificity kinase that activates JNK, named JNKK, was identified that functions between MEKK and JNK. JNKK activated the JNKs but did not activate the ERKs and was unresponsive to Raf-1 in transfected HeLa cells. JNKK also activated another MAPK, p38 (Mpk2; the mammalian homolog of HOG1 from yeast), whose activity is regulated similarly to that of the JNKs.

Amino Acid Sequence↗

PubMed1994

The UV response involving the Ras signaling pathway and AP-1 transcription factors is conserved between yeast and mammals.

UV irradiation of mammalian cells activates AP-1 through a Ras-dependent pathway, independently of DNA damage. We show that the yeast S. cerevisiae has a remarkably similar UV response involving the AP-1 factor Gcn4, which is distinct from the DNA damage response. Transcriptional activation of HIS3 and HIS4 by Gcn4 is triggered by UV irradiation in a Ras-dependent fashion. Moreover, resistance of yeast to UV irradiation is correlated with the level of Ras activity and Gcn4 function. Like mammalian cells in which activated Ras leads to increased c-Jun synthesis and phosphorylation, the effects in yeast involve increased translation of GCN4 mRNA and a posttranslational event. However, this effect on GCN4 translation is different from the response to amino acid or purine starvation. Therefore, a UV signaling pathway involving Ras and AP-1 is an ancient and universal mechanism involved in protection against damage to cellular components other than DNA.

Amitrole↗

PubMed1992

Characterization of a Gi-protein from Trypanosoma cruzi epimastigote membranes.

A guanosine 5'-[gamma-[35S]thio]triphosphate-binding activity was detergent-extracted from Trypanosoma cruzi membranes. This binding activity was co-eluted from gel-filtration columns with a factor which, in a heterologous reconstitution system, blocks glucagon stimulation of adenylate cyclase activity in liver membranes. ADP-ribosylation of these membranes by pertussis toxin eliminated this blocking capacity. Incubation of T. cruzi membranes with activated pertussis toxin and [adenylate-32P]NAD+ led to the incorporation of radioactivity into a labelled product with an apparent M(r) of approx. 43,000. Crude membranes were electrophoresed on SDS/polyacrylamide gels and analysed, by Western blotting, with GA/1 anti-alpha common, AS/7 anti-alpha t, anti-alpha i1 and anti-alpha i2 polyclonal antibodies. These procedures led to the identification of a specific polypeptide band of about 43 kDa. Another polypeptide reacting with the SW/1 anti-beta antibody, of about 30 kDa, was also detected in the membrane fraction.

Adenosine Diphosphate↗

PubMedDate not supplied

Factors from Trypanosoma cruzi interacting with AP-1 sequences.

Interaction between factors from Trypanosoma cruzi extracts and AP-1 sequences was studied by electrophoretic mobility shift assays. Using a double-stranded probe carrying the AP-1 sequence from the SV40 promoter, three specific complexes designated A, B, and C were detected. Complexes A and C were formed when using single-stranded probes. The relative amount of complex B, specific for double-stranded DNA, increased as a function of probe length. Complexes were stabilized by cross-linking with UVC irradiation and resolved on denaturing SDS-PAGE. Complex A generated bands of 60- and 39 kDa; complex B produced two bands of 46- and 43 kDa; and complex C generated one band of 43 kDa. The AP-1 binding activity was much higher in purified nuclear preparations than in soluble fractions, and was detected in crude extracts from the three forms of the parasite. The binding signal, however, was much stronger in amastigote and trypomastigote than in the epimastigote forms. Specific binding was increased by oxidative stress. Antibodies raised against peptides corresponding to conserved domains of mammalian c-Jun and c-Fos detected bands of 40- and 60 kDa, respectively, in a nuclear epimastigote preparation.

Animals↗