Expression of the human tumor suppressor p53 induces cell death in Pichia pastoris

The human tumor suppressor p53 is known as guardian of genome because of its involvement in many signals related to cell life or death. In this work, we report that human p53 induces cell death in the yeast Pichia pastoris. We showed a growth inhibition effect, which increased with the p53 protein expression level in recombinant Mut(s) (methanol utilization slow) strain of Pichia. However, no effect of p53 was observed in recombinant strain of Mut(+) (methanol utilization plus) phenotype. Interestingly, human p53 induces cell death in recombinant strains Mut(s) with characteristic markers of apoptosis such as DNA fragmentation, exposure of phosphatidylserine, and reactive oxygen species generation. Taken together, our results strongly suggest that human p53 is biologically active in this heterologous context. Thus, we propose that P.?pastoris could be a useful tool to better understand the biological function of human p53.     

Spatial fiber type distribution in normal human muscle: Histochemical and tensiomyographical evaluation

The variability of fiber type distribution in nine limb muscles was examined with histochemical and tensiomyographical (TMG) methods in two groups of 15 men aged between 17 and 40 years. The aim of this study was to determine the extent to which the relative occurrence of different fiber types and subtypes varies within human limb muscles in function to depth and to predict fiber type proportions with a non-invasive TMG method.

The distribution of different fiber types varied within the muscles, as a function of depth, with a predominance of type 2b fibers at the surface and type 1 fibers in deeper regions of the muscle. For all the analyzed muscles the contraction times measured at stimulus intensity 10% of supramaximal stimulus (10% MS) were significantly (p<0.05) shorter than the contraction times measured at 50% of supramaximal stimulus intensity (50% MS). The Pearson's correlation coefficient between percentage of type 1 muscle fibers measured at the surface of the muscle and contraction time at 10% MS, obtained by TMG was statistically significant (r=0.76,P<0.01). Also the Pearson's correlation coefficient between percentage of type 1 muscle fibers measured in the deep region of the muscle and contraction time at 50% MS obtained by TMG was also statistically significant (r=0.90,P<0.001).

These findings suggest that the contraction time obtained by TMG may be useful for non-invasive examining of muscle fiber types spatial distribution in humans.     

Antihypertensive and antioxidant potential of vanillic acid, a phenolic compound in L-NAME-induced hypertensive rats: a dose-dependence study

We investigated the antihypertensive and antioxidant potential of vanillic acid (VA) in N(ω)-Nitro-L-arginine methyl ester hydrochloride (L-NAME) - treated adult male albino Wistar rats. Treatment of rats with L-NAME (40 mg/kg Bw for 30 days) caused a sustained increase in systolic- (SBP) and diastolic blood pressure (DBP) and significantly decreased the concentration of nitrite/nitrate (NO(x)) in plasma as compared with that in the control. Rats treated with VA restored SBP and DBP to normal level and preserve the plasma NO metabolites concentration. Moreover, VA reduced lipid peroxidation products (thiobarbituric acid reactive substances, lipid hydroperoxides, conjugated dienes) and significantly restored enzymatic antioxidants (superoxide dismutase, catalase, and glutathione peroxidase), non-enzymatic antioxidants (vitamin C, vitamin E, and reduced glutathione) in the plasma. To assess the toxicity if any of VA treatment, hepatic and renal function markers were measured. Our results showed that the effect at a dose of 50 mg/kg Bw of VA was more pronounced than that of the other two doses, 25 and 100 mg/kg Bw. These results were supported by histopathology studies. We conclude that VA possesses an antihypertensive and antioxidant activity in L-NAME-induced hypertensive rats.     

Secreted recombinant P53 protein from Pichia pastoris is a useful antigen for detection of serum p53: autoantibody in patients with advanced colorectal adenocarcinoma

The detection of P53 alteration by serological method is easier to perform, does not require tumor tissues and is of interest for patients monitoring. In this study, we described the development of a home made ELISA test based on recombinant human P53 protein produced in Pichia pastoris and used as antigen for the detection of serum p53-Abs in colorectal carcinoma patients. The human P53 was secreted as a His-tagged protein by recombinant KM71 strain (Kα21) via the peptide signal α of the Saccharomyces cerevisiae mating type gene. The recombinant P53-His was able to detect p53-Abs in 23.4 % of patients. Serum p53-Abs correlated significantly with surgical treatment (P = 0.007), relapse during follow-up (P = 0.036), depth of invasion (P = 0.036) and the level of CA19-9 (P = 0.034). Survival analysis showed that patients negative for serum p53-Abs exhibited a prolonged disease free survival period (P log rank = 0.012). In conclusion, the secreted recombinant human P53-His produced in P. pastoris seems to be a useful antigen for detection of serum p53 Abs in patients with colorectal carcinoma.     

Cytomorphology of induced octoploid Chili pepper (Capsicum annuum L.)

Summary Octoploidy was induced in Chili pepper (Capsicum annuum cultivar cerasiformis) through the application of colchicine and the cytomorphological features of two octoploid plants were described. In general, the octoploids did not exhibit gigas characters when compared to the tetraploids; on the contrary they were less vigorous, suggesting that the optimum and desirable ploidy level for Capsicum is probably tetraploid. Chromosome associations such as octovalents and hexavalents, in addition to IVs, IIIs, IIs and Is, were recorded at diakinesis and metaphase I. Meiosis was highly irregular and the pollen and seed fertility was very low. Cytological features of octoploid Chili peppers are compared with octoploids of Physalis and Petunia.     

Cytotoxic cell granule-mediated apoptosis. Characterization of the macromolecular complex of granzyme B with serglycin

We have recently shown that the physiological mediator of granule-mediated apoptosis is a macromolecular complex of granzymes and perforin complexed with the chondroitin-sulfate proteoglycan, serglycin (Metkar, S. S., Wang, B., Aguilar-Santelises, M., Raja, S. M., Uhlin-Hansen, L., Podack, E., Trapani, J. A., and Froelich, C. J. (2002) Immunity 16, 417-428). We now report our biophysical studies establishing the nature of granzyme B-serglycin (GrB.SG) complex. Dynamic laser light scattering studies establish that SG has a hydrodynamic radius of approximately 140 +/- 23 nm, comparable to some viral particles. Agarose mobility shift gels and surface plasmon resonance (SPR), show that SG binds tightly to GrB and has the capacity to hold 30-60 GrB molecules. SPR studies also indicate equivalent binding affinities (K(d) approximately 0.8 microm), under acidic (granule pH) and neutral isotonic conditions (extra-cytoplasmic pH), for GrB.SG interaction. Finally, characterization of GrB.SG interactions within granules revealed complexes of two distinct molecular sizes, one held approximately 4-8 molecules of GrB, whereas the other contained as many as 32 molecules of GrB or other granule proteins. These studies provide a firm biophysical basis for our earlier reported observations that the proapoptotic granzyme is exocytosed predominantly as a macromolecular complex with SG.     

Haplotype analysis of p53 polymorphisms: Arg72Pro,Ins16bp and G13964C in Tunisian patients with familial or sporadic breast cancer

Background: The p53 polymorphisms have been extensively studied as putative breast cancer susceptibility variants. The present study was undertaken to investigate the association of p53 Arg72Pro, Ins16bp and G13964C polymorphisms and their haplotypes with breast cancer risk in Tunisian women. Methods: Genotyping was performed by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) on 159 patients and 132 controls. Results: The G13964C intronic variant was significantly associated with familial breast cancer risk (p = 0.0018) while the genotypic distribution was similar for p53 Arg72Pro and Ins16bp in patients and controls. Moreover, the (NoIns-C), (Arg-C) and (NoIns-Arg-C) haplotypes were significantly associated with familial breast cancer risk (p = 0.0021, p = 0.0096 and p = 0.0084, respectively) while there was a trend of association between the (Ins-Arg) and (Ins-Arg-G) haplotypes and the risk of sporadic breast cancer. Only the G/C genotype as well as the (NoIns-C) haplotype remained significant after correction for multiple testing. Conclusion: Our data revealed an association between the G/C genotype and the (NoIns-C) haplotype and the risk of familial breast cancer in Tunisian women. However, these observations need to be confirmed due to the limited statistical power of our study and the small number of cases.     

A minority of 46,XX true hermaphrodites are positive for the Y-DNA sequence including SRY

Summary A total of 30 cases of 46,XX true hermaphroditism was analysed for Y-DNA sequences including the recently cloned gene for male testis-determination SRY. In 3 cases, a portion of the Y chromosome including SRY was present and, in 2 cases, was localised, to Xp22 by in situ hybridisation. Since previous studies have shown that the majority of XX males are generated by an X-Y chromosomal interchange, the Xp22 position of the Yp material suggests that certain cases of hermaphroditism can arise by the same meiotic event. The phenotype in the 3 SRY-positive cases may be caused by X-inactivation resulting in somatic mosaicism of testis-determining factor expression giving rise to both testicular and ovarian tissues. Autosomal or X-linked mutation(s) elsewhere in the sex-determining pathway may explain the phenotype observed in the remaining 27 SRY-negative cases.     

Mechanistic investigation of peptidylglycine alpha-hydroxylating monooxygenase via intrinsic tryptophan fluorescence and mutagenesis

The biosynthesis of the majority of biologically active peptides ends with an obligatory alpha-amidation step that is catalyzed only by peptidylglycine alpha-hydroxylating monooxygenase (PHM). The utility of two mechanisms proposed for this copper- and ascorbate-dependent monooxygenase was examined using site-directed mutagenesis and intrinsic tryptophan fluorescence. Retention of full activity by PHMccGln(170)Ala and -Asn eliminates a critical role for Gln(170) in a substrate-mediated electron transfer pathway. The 20-fold reduction in V(max) observed for PHMccGln(170)Glu and -Leu is consistent with a key role for conformational changes in this region. Mutation of Tyr(79), situated near Cu(A), to Trp reduced V(max) 200-fold. Measurement of changes in intrinsic fluorescence allowed determination of a K(d) for copper (0.06 microM) and for a peptidylglycine substrate, Phe-Gly-Phe-Gly (0.8 microM). Although the peptidylglycine substrate bound more tightly at pH 7.0 than at pH 5.5, V(max) decreased 25-fold at neutral pH. Total quenching of the signal from Trp(79) in apoPHMccTyr(79)Trp along with its greatly reduced V(max) defines a critical role for Cu(A) in the rate-limiting step of the reaction. Taking into account our data and the results of kinetic, spectroscopic, and crystallographic studies, we propose a mechanism in which substrate-mediated activation of molecular oxygen binding at Cu(A) completes a pathway for electron transfer from Cu(B).