Cleavage of chromogranin A N-terminal domain by plasmin provides a new mechanism for regulating cell adhesion

It has been proposed that chromogranin A (CgA), a protein secreted by many normal and neoplastic neuroendocrine cells, can play a role as a positive or a negative modulator of cell adhesion. The mechanisms that regulate these extracellular functions of CgA are unknown. We show here that plasmin can regulate the anti/pro-adhesive activity of CgA by proteolytic cleavage of the N-terminal domain. Limited proteolytic processing decreased its anti-adhesive activity and induced pro-adhesive effects in fibronectin or serum-dependent fibroblast adhesion assays. Cleavage of Lys(77)-Lys(78) dibasic site in CgA(1-115) was relatively rapid and associated with an increase of pro-adhesive effect. In contrast, antibodies against the region 53-90 enhanced the anti-adhesive activity of CgA and CgA(1-115). Structure-activity relationship studies showed that the conserved region 47-64 (RILSILRHQNLLKELQDL) is critical for both pro- and anti-adhesive activity. These findings suggest that CgA might work on one hand as a negative modulator of cell adhesion and on the other hand as a precursor of positive modulators, the latter requiring proteolytic processing for activation. Given the importance of plasminogen activation in tissue invasion and remodeling, the interplay between CgA and plasmin could provide a novel mechanism for regulating fibroblast adhesion and function in neuroendocrine tumors.     

Efficient development of dinucleotide microsatellite markers in Norway spruce ( Picea abies Karst.) through dot-blot selection

The development of microsatellite markers can be a time-consuming process, especially in species such as conifers where many microsatellites have been shown to be associated with the repetitive fraction of the genome and to produce complex banding patterns following electrophoresis. Therefore, procedures to eliminate this fraction from further processing are sought. In this paper, we report on the development of 53 dinucleotide SSR markers in Norway spruce, 35 of which (66%) produce simple, polymorphic patterns. This high efficiency is obtained by introducing a dot-blot selection against high copy number sequences, performed on the microsatellite-containing clones. The resulting markers turned out to be polymorphic and useful for population genetic studies and for linkage mapping. Seven additional markers that were not subject to the dot-blot selection are also presented.     

Polygalacturonase inhibiting protein in plant cell walls

Polygalacturonase inhibiting protein (PGIP) is localized in plant cell walls and plays an important role both in pectic substance metabolism and in prevention of the penetration of phytopathogenic microorganisms. Apparently, PGIP is responsible for the specificity of cell--cell interactions during pollination or inoculation by fungi nonpathogenic for the particular plant. PGIPs from different plants share a basic common structure. They are rather thermostable glycoproteins enriched with leucine and contain about 20% carbohydrates; the molecular weight varies between 37-54 kD. The synthesis of PGIP is encoded by one gene, and its expression is stimulated by injury and fungal infection. The resistance of plant tissues to infection frequently correlates with PGIP expression and with inhibiting action on fungal PG. Thus, PGIP is believed to be useful for gene engineering to obtain transgenic plants resistant to fungal infection or retaining commercial value during storage.     

Loss of heterozygosity and K-ras gene mutations in gastric cancer

In order to identify relevant genetic lesions in gastric carcinoma, we searched for tumor suppressor gene inactivation and K-ras gene mutations by analyzing tumor and control DNAs from 34 patients. These were from an epidemiologically defined area of Italy characterized by one of the world's highest incidences of stomach cancer. Allele losses were investigated by the Southern blotting procedure at 16 polymorphic loci on 11 different chromosomes. Our data demonstrate that chromosomal regions 5q, 11p, 17p and 18q are frequently deleted, and that 7q and 13q chromosome arms are also involved, although at a lower frequency. Loss of heterozygosity (LOH) at region 11p was not found during other surveys carried out on patients of different geographic origins. No specific combination of allelic losses could be recognized in the samples analyzed, the only exception being that tumors with 17p allelic loss also showed LOH on the 18q region. When matching frequent LOH events and the stage of progression of the tumors, we observed a trend of association between advanced stages and allelic losses on 17p and 18q chromosome arms. The analysis of K-ras, carried out by the polymerase chain reaction and denaturing gradient gel electrophoresis, demonstrated transforming mutations in only 3 out of 32 cases. Colorectal tumorigenesis proceeds by the accumulation of genetic alterations, including K-ras mutations and inactivation of tumor suppressor genes on the 5q, 17p and 18q regions. Our data indicate that, although gastric and colorectal neoplasias share common genetic alterations, they probably progress through different pathways.     

Evidence for an inhibitory effect exerted by yeast NMN adenylyltransferase on poly(ADP-ribose) polymerase activity

We have previously reported for the first time the purification to homogeneity of the enzyme NMN adenylyltransferase (EC 2.7.7.1) from yeast and its major molecular and catalytic properties. The homogeneous enzyme was found to be a glycoprotein containing 2% carbohydrate and 1 mol of adenine residue and 2 mol of phosphate covalently bound per mole of protein. Such a stoichiometry, apparently consistent with that of ADP-ribose, prompted us to further investigate the possibility that NMN adenylyltransferase could be subjected to poly(ADP-ribosylation) in vitro in a reconstituted system. Poly(ADP-ribose) polymerase was purified to homogeneity from bull testis by means of a rapid procedure involving two batchwise steps on DNA-agarose and Reactive Blue 2 cross-linked agarose and a column affinity chromatography step on 3-aminobenzamide-Sepharose; the optimal conditions for the poly(ADP-ribosylation) of exogenous substrates were determined. When pure NMN adenylyltransferase was incubated in the presence of the homogeneous poly(ADP-ribose) polymerase, a marked inhibition of the polymerase was observed, both in the presence and in the absence of histones, while the activity of NMN adenylyltransferase was not affected. The inhibition could not be prevented by increasing the concentrations of either DNA or NAD. Mg2+ did not affect the activity or the inhibition. The significance of such a phenomenon is at present unknown, but it may be of biological relevance in view of the close topological and metabolic relationship between the two enzymes.     

Meiotic Diploid Progeny and Meiotic Nondisjunction in SACCHAROMYCES CEREVISIAE

Abnormalities in chromosome number that occurred during meiosis were evaluated with a specially-constructed diploid strain of Saccharomyces cerevisiae. The strain is heterozygous for six markers of the right arm of chromosome V and heterozygous for cyh2 (resistance to cycloheximide) on chromosome VII.-Selection of meiotic spores on a medium containing cycloheximide and required nutrilites-except those for the markers of the right arm of chromosome V-allows the growth of aberrant clones belonging only to two classes: a) diploid clones, caused by failure of the second meiotic division, with a frequency of 0.54 x 10(-4) per viable spore; and b) diplo V, aneuploids derived from nondisjunctions in meiosis I or meiosis II, with a total spontaneous frequency of 0.95 x 10(-4) per viable spore. About two-thirds of the aneuploids originated during meiosis I, the rest during meiosis II. An investigation of these events in control meioses and after treatment with MMS, Benomyl and Amphotericin B suggests that this assay system is suitable for screening environmental mutagens for their effects on meiotic segregation.     

Bakers' yeast protease a purification and enzymatic and molecular properties

It has been previously demonstrated in our laboratory that uridine nucleosidase (EC 3.2.2.3) is inactivated by yeast protease A (EC 3.4.23.8). A complete purification procedure for protease A from bakers' yeast, which lacks the acidic activation step used by other workers, and the major properties of the enzyme are shown. The enzyme is homogeneous as judged by disc gel electrophoresis. Its molecular weight, calculated from both sodium dodecyl sulfate-disc gel electrophoresis and gel filtration experiments, is around 45,000. The protein does not possess quaternary structure. The isoelectric point is 4.1. Carbohydrate content is around 8%. Amino acids analysis and sulfur analysis reveal the presence of 1-SH group and two disulfide bridges. The free-SH group does not seem to be involved in catalysis. Amino terminal analysis shows that isoleucine is at the amino terminal position. The pH optima are 2.4 for the hydrolysis of azocasein and casein, and 3.3 for the hydrolysis of hemoglobin. The K_m value for hemoglobin is 1.7 × 10^?5m. The inhibition exerted by pepstatin on the proteolytic activity of protease A is pH dependent. Among various yest enzyme substrates only uridine nucleosidase is inactivated by protease A.     

Improving hydration in elite male footballers during a national team training camp – an observational case study

[Purpose] The purpose of this study was to (i) assess hydration levels in elite male football players during a national team training camp before and during qualifying matches, (ii) evaluate the effect of coaching strategies for hydration based on feedback from hydration monitoring, and (iii) assess possible relationships between hydration status and training load or wellness markers.[Methods] Thirty-one male players (age 27±4 yrs; height 185±6 cm; weight 82.9±6.7 kg; body fat 10.4±2.3%) representing a national team from the Union of European Football Associations (UEFA) participated. The players were studied during three different national team training camps related to the UEFA Nations League tournament. Urine specific gravity (U_SG) was measured to assess hydration status. During all camps, the players were actively coached on improving strategies for hydration and given individual feedback on their test results. The training load was measured using GPS technology, and wellness questionnaires were completed.[Results] U_SG decreased progressively and significantly (p<0.005) during camp 1 and hydration status improved over the three camps, with fewer dehydrated and more well-hydrated players identified during the last part of camp 3. Significantly (p<0.05) higher U_SG values were observed 2 days prior to a match (MD-2) than on match day (MD); consequently, 52% of the players were dehydrated on MD-2 and only 6% on MD. No correlations were observed between hydration status and training load or wellness markers.[Conclusion] Dehydration is a challenge in elite male football, but continuous monitoring of hydration status and coaching on hydration strategies can lead to major improvements and reduce the degree of dehydration.     

Identification and characterization of YLR328W, the Saccharomyces cerevisiae structural gene encoding NMN adenylyltransferase. Expression and characterization of the recombinant enzyme.

The enzyme nicotinamide mononucleotide (NMN) adenylyltransferase (EC 2.7.7.1) catalyzes the transfer of the adenylyl moiety of ATP to NMN to form NAD. A new purification procedure for NMN adenylyltransferase from Saccharomyces cerevisiae provided sufficient amounts of enzyme for tryptic fragmentation. Through data-base search a full matching was found between the sequence of tryptic fragments and the sequence of a hypothetical protein encoded by the S. cerevisiae YLR328W open reading frame (GenBank accession number U20618). The YLR328W gene was isolated, cloned into a T7-based vector and successfully expressed in Escherichia coli BL21 cells, yielding a high level of NMN adenylyltransferase activity. The purification of recombinant protein, by a two-step chromatographic procedure, resulted in a single polypeptide of 48 kDa under SDS-PAGE, in agreement with the molecular mass of the hypothetical protein encoded by YLR328W ORF. The N-terminal sequence of the purified recombinant NMN adenylyltransferase exactly corresponds to the predicted sequence. Molecular and kinetic properties of recombinant NMN adenylyltransferase are reported and compared with those already known for the enzyme obtained from different sources.