CD44s adhesive function spontaneous and PMA-inducible CD44 cleavage are regulated at post-translational level in cells of melanocytic lineage.

Adhesion between the CD44s receptor and hyaluronic acid plays an important role in cell migration, tumour growth and progression. Although the alternative splicing of CD44 variant exons represents the principal regulatory mechanism of CD44-mediated functions, CD44v spliced variants are scantily expressed in melanoma cells. For this reason, we have investigated the possibility that post-translational modifications of the CD44 standard receptor could play a pivotal role in regulating CD44-mediated functions in melanoma. Using metabolic inhibitors of N- and O-glycosylation, as well as melanoma transfectants expressing CD44s O-glycosylation site-specific mutants, we performed structural and functional analysis of N- and O-deglycosylated CD44s molecules expressed in melanoma cells. We discovered that complete N- and O-glycosylation is not required by CD44s to be correctly expressed on the melanoma cell surface. Indeed, variably glycosylated and functionally different CD44s molecules were constitutively expressed in primary and metastatic lesions. Furthermore, we observed that changes in N- and O-glycosylation of CD44s could modulate its cleavage. In fact, spontaneous CD44s shedding was dependent on the presence of partial or complete O-glycosylation of four serine-glycine motifs localized in the membrane-proximal CD44 ectodomain. Mutation of these serine residues, as well as an extensive metabolic O-deglycosylation, strongly impaired spontaneous CD44 shedding. Furthermore, an O-glycosylation-independent mechanism of CD44 cleavage has been identified. This alternative mechanism of receptor cleavage is phorbol 12-myristate-13-acetate (PMA) inducible, mediated by metalloproteinase and requires the presence of N-linked sugar residues. Our findings demonstrate that the post-translational modification of CD44s represents the principal regulatory mechanism of CD44s-mediated functions in melanoma.

Galectin-3 and CD44v6 isoforms in the preoperative evaluation of thyroid nodules.

PURPOSE: Thyroid cancer is the most frequently occurring endocrine malignancy; however, preoperative diagnosis of some lesions, in particular those with follicular histology, is difficult, and a consistent number of not otherwise-specified "follicular nodules" are surgically resected more for diagnosis than therapeutic purposes. In this study, we investigated whether the lectin-related molecules CD44v6 and galectin-3, the expression of which is altered during deregulated cell growth and malignant transformation, could be potential markers for improving the diagnostic accuracy of conventional cytology. MATERIALS AND METHODS: A comparative immuno-chemical and molecular analysis was performed on 157 thyroid specimens representative of normal, benign, and malignant tissues, and on 36 cytologic samples obtained preoperatively by fine-needle aspiration biopsy from nonselected patients with palpable thyroid nodules. RESULTS: Normal thyrocytes did not express galectin-3 nor CD44v6. Although the expression of CD44v6 isnegligible in thyroiditis, these molecules are variably detected in benign and malignant proliferative lesions. Interestingly, galectin-3 is never expressed in benign lesions, but it is invariably detected in cancers. A comparative evaluation of CD44v6 and galectin-3 expression in thyroid malignancies demonstrated that these molecules are coexpressed at the messenger RNA and protein level in almost all lesions. CONCLUSION: Our findings suggest that CD44v6 and galectin-3 could be potential markers to preoperatively identify malignant transformed thyrocytes. Immunodetection of these molecules on cytologic specimens obtained by fine-needle aspiration biopsy is an accurate and improved method for selecting, on a molecular basis, those nodular lesions of the thyroid gland that need to be surgically resected.

Structural variability of CD44v molecules and reliability of immunodetection of CD44 isoforms using mAbs specific for CD44 variant exon products.

CD44 can be considered structurally and functionally one of the most variable surface molecules. Alternative splicing of variant exons as well as posttranslational modifications of the molecule (differences in glycosylation) generate a rich repertoire of CD44 isoforms (CD44v), some of which seem to play a key role in tumor growth and progression. Immunodetection of CD44 isoforms in vivo, using mAbs specific for CD44 variant exon products, is largely used to identify those CD44 molecules involved in tumor growth and progression and to interfere with CD44-mediated processes. In the present work we demonstrate that the immunoreactivity of some mAbs directed to CD44 exon-specific epitopes can be impaired by the structural variability of the molecule. Our findings demonstrate that (1) specific exon assortment and/or posttranslational modifications of CD44v molecules can mask CD44 exon-specific epitopes; (2) glycosaminoglycan side chains, carried by some CD44v isoforms of high molecular weight, may play a critical role in determining the exact conformation of the molecule, which is necessary for the detection of CD44 variant epitopes by specific mAbs; and (3) in a panel of stable transfectants expressing CD44 N-glycosylation site-specific mutants, generated in the constant region of CD44 extracellular domain, asparagine-isoleucine substitution is sufficient per se to impair the immunoreactivity of several mAbs to pan-CD44. Thus, conformational changes due to the alternative splicing of CD44 variant exons and/or posttranslational modifications of the molecule (different degree of glycosylation), which are cell type-specific, are likely to generate CD44 variants that elude immunodetection. These findings strongly suggest that immunohistochemical analysis of CD44 expression in vitro and in vivo, using mAbs specific for CD44 variant exon epitopes, can potentially be impaired by a large number of false negative results.

Sequence of the GLT1 gene from Saccharomyces cerevisiae reveals the domain structure of yeast glutamate synthase.

Glutamate synthase (GOGAT) and glutamine synthetase play a crucial role in ammonium assimilation and glutamate biosynthesis in the yeast Saccharomyces cerevisiae. The GOGAT enzyme has been purified and the GOGAT structural gene (GLT1) has been cloned, showing that this enzyme is a homotrimeric protein with a monomeric size of 199 kDa. We report the GLT1 nucleotide sequence and the amino acid sequence of its deduced protein product. Our results show that there is a high conservation with the corresponding genes of Escherichia coli, Medicago sativa (alfalfa) and Zea mais (maize). Binding domains for glutamine, cofactors (FMN and NADH) and the cysteine clusters (which comprise the iron-sulfur centres) were tentatively identified on the basis of sequence comparison with GOGAT sequences from E. coli, alfalfa and maize.

Immune function and renal transplantation in Fabry's disease.

A deficient leucocyte immunological function could cause the reported high rate of lethal infections following renal transplantation in patients affected by Fabry's disease. We have studied humoral immunity, peripheral lymphocyte subsets, mitogenic lymphocyte response in vitro and granulocyte function in three patients with Fabry's disease. The immunological state appears to be quite similar to that of the uraemic population in general, not showing any specific impairment.

[Blood folates in chronic uremic patients in dialysis treatment]. / La folatemia negli uremici cronici in trattamento dialitico.

The authors, after having reviewed folic acid and folates chemical and biochemical characteristics and their absorption and excretion modalities, determine serum folate levels by radioassay method on 30 patients with renal failure in chronic dialysis and on 24 normal controls. Low serum folate levels concerned 14 uremic patients (46.6% of th cases): 10 in hemodialysis and 4 in peritoneal dialysis. The mean serum folate values was 3.31 ng/ml (+/- 1.93) for the uremic patients' group and 4.29 ng/ml (+/- 1.21) for the control group. The difference between the mean level of these groups was statistically significant (p less than 0.05). No significant difference was observed among the mean serum folate levels of the uremic patients in peritoneal dialysis and of those in hemodialysis. Significantly low serum folate levels were finally found for the uremic HBsAg-positive patients, but in the same subjects the dialytic treatment period had been very prolonged. The authors conclude emphasizing the usefulness of folic acid treatment in uremic patients on dialysis, also without having an evident hematologic picture of megaloblastic anemia.