Uptake of hematite nanoparticles in maize and their role in cell cycle dynamics, PCNA expression and mitigation of cadmium stress.

Cadmium toxicity is considered a major threat to several crops worldwide. Hematite nanoparticles (NPs), due to their small size and large specific surface area, could be applied as an adsorbent for toxic heavy metals in soil. Also, they serve as an efficient nano-fertilizer, promoting Fe availability and biomass production in plants, thus enabling Cd2+ -induced stress tolerance. The phytotoxicity of five different concentrations of hematite NPs, ranging from 500 to 8,000 mg·kg-1 , and Cd2+ concentrations (110 or 130 mg·kg-1 Cd2+ ) alone or combined with 500 mg·kg-1 NPs was evaluated in maize. The changes in fresh weight, element analysis, cell cycle regulation, DNA banding patterns and proliferating cell nuclear antigen (PCNA) expression were used as biomarkers. The results revealed that increased fresh weight and fewest polymorphic DNA bands were detectable after treatment with 500 mg·kg-1 NPs. However, at 8,000 mg·kg-1 NPs, PCNA expression increased significantly, which resulted in cell cycle arrest at the G1/S checkpoint in roots. Significant reductions in fresh weight, altered nutrient profiles and cell cycle perturbations are considered symptoms of Cd2+ toxicity in maize. Conversely, amending 500 mg·kg-1 NPs with 130 mg·kg-1 Cd2+ increased fresh weight, Fe concentration and genomic template stability, while reducing Cd2+ uptake and PCNA1 expression. Overall, 8,000 mg·kg-1 hematite NPs interfered with the cellular homeostatic balance of maize, resulting in a cascade of genotoxic events, leading to growth inhibition. Although 500 mg·kg-1 hematite NPs alleviated Cd2+ -induced DNA damage to a certain extent, their impact on cell cycle progression requires further verification.

Fetal renal biopsies in obstructive uropathy: feasibility and clinical correlations--preliminary results.

Final assessment on the outcome of fetal obstructive uropathy is a challenging matter. Ultrasonography, fetal urine electrolytes, and beta 2 microglobulin are postulated as being useful in many cases. For cases in which renal function remains unclear, ultrasound-guided fetal kidney biopsy may be used in order to detect histologic features distinctive of renal dysplasia. We present preliminary results aimed at studying the feasibility and possible risks. Biopsies were initially performed in 11 severely malformed fetuses, three of them with associated renal abnormalities. The success rate in obtaining renal material was 63.6 per cent with no maternal complications. In the next phase of this study, ten biopsies and urine collections were performed in fetuses with bilateral obstructive uropathy. The success rate was 50 per cent with no complications. Normal fetal renal histology was seen in 80 per cent of cases. In one case, although electrolytes were normal, a histologic diagnosis of renal dysplasia was made, showing a good correlation with outcome. In conclusion, fetal kidney biopsies for obstructive uropathy are feasible and further studies are needed to show their clinical relevance and risks.

Secretion, purification and characterization of a soluble form of the yeast KEX1-encoded protein from insect-cell cultures.

The Saccharomyces cerevisiae KEX1 gene encodes a carboxypeptidase involved in the C-terminal processing of the lysine and arginine residues from the precursors of K1 and K2 killer toxins and alpha-factor (mating pheromone). In order to produce large quantities of this unique carboxypeptidase for structural studies, a functional soluble form was obtained by deleting 224 amino acids from the C-terminus of the KEX1-encoded protein which includes a putative membrane-spanning domain. The resulting truncated KEX1 gene (KEX1 delta) has been expressed in the baculovirus/insect cell system. The protein (Kex1 delta p) is efficiently secreted into the culture medium and was purified to apparent homogeneity with a yield of approximately 4 mg/l culture. Kex1 delta p is a glycoprotein with a molecular mass of 56 kDa, its N-terminal sequence is identical to that of the full-length membrane-associated form of the enzyme [Latchinian-Sadek, L. & Thomas, D. Y. (1993) J. Biol. Chem. 268, 534-540], and like the full-length enzyme it is not made as a proenzyme. For the soluble enzyme form, the optimum pH for activity was 5.5-6.0, and the apparent pI value of the protein determined by isoelectric focusing was 4.2. The enzyme cleaves arginine from the C-terminus of the synthetic peptide benzoyl-Phe-Ala-Arg with Km 335 microM and Vmax 282 mumol.min-1 x mg protein-1. Insect-cell-derived Kex1 delta p processes alpha-factor-Lys-Arg, a known natural substrate, to mature active alpha-factor in a manner similar to the membrane-associated full-length enzyme. This secreted form of the enzyme is a convenient source for the isolation of substantial quantities of the pure enzyme for detailed kinetic and structural studies.

Expression, purification, and characterization of the yeast KEX1 gene product, a polypeptide precursor processing carboxypeptidase.

The Saccharomyces cerevisiae KEX1 gene encodes a protease with carboxypeptidase B-like activity involved in K1 and K2 killer toxins and alpha-factor (mating pheromone) precursors processing. The gene has been expressed using the baculovirus/insect cell system, and the KEX1 encoded protein (Kex1p) was purified to apparent homogeneity from detergent-solubilized membrane preparations of insect cells infected with the recombinant virus. The specific activity of the enzyme was enriched 126-fold as compared with the cell lysate, with a recovery of 29%. The NH2-terminal sequence of the purified active enzyme was identical to the predicted sequence after the removal of the signal peptide. This provides evidence that Kex1p, at least in insect cells, is not made as a proenzyme. The optimum pH for activity was 6.0, and the apparent pI value of the protein was below pH 3.0. The enzyme cleaves arginine or lysine from the COOH terminus of synthetic peptides: benzoyl-Phe-Ala-Arg (Km = 284 microM), furylacryloyl (fa)-Ala-Arg (Km = 516 microM), and fa-Ala-Lys (Km = 962 microM). The kinetic data obtained reveals that Kex1p preferentially cleaves the COOH-terminal arginine of peptides over the COOH-terminal lysine. Insect-derived Kex1p processes alpha-factor-Lys-Arg, its known natural substrate, to mature active alpha-factor, and this maturation event takes place in a sequential manner. Furthermore, the enzyme expresses very high affinity for the 15-amino acid-long peptide, alpha-factor-Lys-Arg (Ki = 22 microM), and somewhat lower affinity for the heptapeptides [Leu]enkephalin-Arg-Arg,-Arg-Lys, and [Met]enkephalin-Lys-Lys (Ki = 45, 57, and 81 microM, respectively). The data demonstrate that processing at the COOH terminus of the peptides tested stops after the cleavage of the Arg and/or Lys residues. The specificity of the enzyme for COOH-terminal basic amino acid residues of the peptides used in this study and its high affinity for alpha-factor-Lys-Arg confirms the role that Kex1p plays in polypeptide precursor processing in yeast.

[Fetal atrioventricular block]. / Bloqueio atrioventricular fetal.

PURPOSE: To study the pathogenesis, evolution and prognosis of the complete heart block of the fetus. METHODS: Bidimensional echocardiography associated to M-mode and doppler was performed in 600 patients. All cases of congenital heart block were referred because the fetuses presented hydrops, bradycardia and/or cardiac malformation suspected by routine ultrasound. RESULTS: Isolated heart block was found in 6 fetuses (5 cases of complete type and 1 case of 2nd degree type 2:1). Heart block associated with complex cardiac disease and left atrial isomerism was found in 6 fetuses with no survivors (5 cases of complete type and 1 case of 2nd degree type 2:1). Heart block associated with atrioventricular discordante was found in 1 case. CONCLUSION: The findings of this study agree the literature about the relation between maternal anti-RO antibodies and isolated complete heart block. We also found a poor prognosis in the group with heart block and complex cardiac malformations.

Flavonol ring B-specific O-glucosyltransferases: purification, production of polyclonal antibodies, and immunolocalization.

UDP-glucose: flavonol 2'- and 5'-O-glucosyltransferases (E.C.2.4.1.-) from leaves of Chrysosplenium americanum were copurified to apparent homogeneity by successive chromatography on Sephacryl S-200, UDP-glucuronic acid-agarose, Mono P, Superose 12, and Mono Q columns. Both enzymes have similar properties except for their substrate specificity and stability (J. Chromatogr. 388, 235, 1987). The purified protein was used as the source of antigen to produce polyclonal antibodies in rabbits. In situ localization of the O-glucosyltransferases was studied by applying a postembedding immunogold labeling technique on ultrathin sections of Lowicryl K4M- and LR White-embedded tissues. Postfixation with osmium tetroxide followed by embedding in LR White resulted in good preservation of membrane ultrastructure, although protein antigenicity was greatly reduced. Leaf sections embedded in Lowicryl K4M had an extracted appearance; however, they retained a high degree of protein antigenicity revealing the deposition of gold particles in the periplasmic region of cells. Considering the compromise chosen in this study to retain antigenicity over preservation of membrane ultrastructure, the results suggest that the "easily solubilized" O-glucosyltransferases of C. americanum may actually be associated with vesicle-like structures and cytoplasmic membranes.

Prohormone processing by yeast proteases.

Investigations of the precursors of alpha-pheromone and killer toxin in the yeast Saccharomyces cerevisiae have defined the genes coding (KEX1 and KEX2) for the proteases which are responsible for their processing. In addition to processing at pairs of basic residues it is evident that yeast can also process at monobasic sites. We present data on the Kex1p and Kex2p enzymes, their cellular localization, and their post-translational modification. In addition initial characterisation of the monobasic specific protease and the isolation of mutants defective in this activity are presented. The use of the yeast system as a model for the processing of mammalian prohormones is discussed.

Spacing in the human deciduous dentition in relation to tooth size and dental arch size.

Dental casts of 243 preschool children, 129 males and 114 females, aged 2.5-5.5 years, were analysed for the presence of interdental spaces, mesiodistal crown diameters, intercanine width, intermolar width and arch length (circumference). The prevalence of primate spaces (diastemata mesial to the upper canines and distal to the lowers) varied from 65 to 90% by arch and sex; it was lowest in the lower arch especially in females, and highest in the upper. These prevalences did not increase with age; however, other spaces which usually occur between incisors may develop towards the end of the deciduous dentition, commonly in arches with primate spaces. Generally, spacing of the deciduous anterior teeth was significantly related to mesiodistal crown diameter and intercanine arch width. The crowns were significantly broader and the arches significantly narrower in cases with no spaces than in those where spaces existed. As the genetic programming for tooth size also normally affects arch size, greater discrepancies between mesiodistal crown diameters of the deciduous anterior teeth and their permanent successors are probably associated with more deciduous arch spacing.