Tissue-specific control of midbody microtubule stability by Citron kinase through modulation of TUBB3 phosphorylation.

Cytokinesis, the physical separation of daughter cells at the end of cell cycle, is commonly considered a highly stereotyped phenomenon. However, in some specialized cells this process may involve specific molecular events that are still largely unknown. In mammals, loss of Citron-kinase (CIT-K) leads to massive cytokinesis failure and apoptosis only in neuronal progenitors and in male germ cells, resulting in severe microcephaly and testicular hypoplasia, but the reasons for this specificity are unknown. In this report we show that CIT-K modulates the stability of midbody microtubules and that the expression of tubulin ß-III (TUBB3) is crucial for this phenotype. We observed that TUBB3 is expressed in proliferating CNS progenitors, with a pattern correlating with the susceptibility to CIT-K loss. More importantly, depletion of TUBB3 in CIT-K-dependent cells makes them resistant to CIT-K loss, whereas TUBB3 overexpression increases their sensitivity to CIT-K knockdown. The loss of CIT-K leads to a strong decrease in the phosphorylation of S444 on TUBB3, a post-translational modification associated with microtubule stabilization. CIT-K may promote this event by interacting with TUBB3 and by recruiting at the midbody casein kinase-2α (CK2α) that has previously been reported to phosphorylate the S444 residue. Indeed, CK2α is lost from the midbody in CIT-K-depleted cells. Moreover, expression of the nonphosphorylatable TUBB3 mutant S444A induces cytokinesis failure, whereas expression of the phospho-mimetic mutant S444D rescues the cytokinesis failure induced by both CIT-K and CK2α loss. Altogether, our findings reveal that expression of relatively low levels of TUBB3 in mitotic cells can be detrimental for their cytokinesis and underscore the importance of CIT-K in counteracting this event.

Heparin protection against Fe2+ -and Cu2+ -mediated oxidation of liposomes.

Heparin (HE) exhibited a protective effect on liposome peroxidation induced by Fe2+ and Cu2+, decreasing the formation of both conjugated dienes and thiobarbituric acid reactive substances (TBARS) in a dose-dependent manner. The antioxidant activity was more relevant in the oxidizing system employing Fe2+ and H2O2 and generating the highly reactive OH radical. The analysis of liposome size distribution by quasielastic laser light scattering showed that: (1) the native structure of the particles was completely lost after exposure to Fenton reagent; (2) the presence of HE in the reaction mixture completely prevented the peroxidative damage on liposomes. Thus, HE acts as an antioxidant factor on membrane lipid bilayer. This suggests that HE, released from mast-cell granules during inflammatory processes, might locally protect the cell membrane from the oxidative injuries.

The effect of heparin on Cu(2+)-mediated oxidation of human low-density lipoproteins.

The effect of heparin (HE) on the susceptibility of human low-density lipoprotein (LDL) to Cu(2+)-induced oxidation was investigated by monitoring conjugated diene formation. HE did not modify the maximum formation of conjugated diene, but increased markedly the lag phase. The plot of change in oxidation rate vs. time showed that the absolute value of Vmax was dependent on Cu2+ concentration and that HE increased the time necessary to reach Vmax. The value of constant K (the Cu2+ concentration producing a tlag of twice the minimum value) increased in the presence of HE, whereas the value of tmin (the time theoretically required for LDL oxidation at an infinite Cu2+ concentration) was not substantially affected. These results indicate that HE might play a protective antioxidant effect on LDL, probably affecting both the structural properties of the particle and the amount of Cu2+ available for the oxidation.

Water soluble proteoglycans from bovine duodenal mucosa.

Glycosaminoglycan-protein complexes were extracted from bovine duodenal mucosa with distilled water, resulting in solubilization of a fraction of the total proteoglycan of the tissue. The extracted material was purified by anion exchange chromatography on DEAE-Sephadex A-25, and then characterized by chemical analysis and by fractionation on Dowex 1. By using these procedures, two major fractions were identified, which were eluted from Dowex with 1.0-1.25 M NaC1 and with 1.5-1.75 M NaC1 respectively. Analyses showed that both fractions were mainly composed of glucosamine-containing, hyaluronidase-resistant polysaccharides, which were identified by their N-sulphate: D-glucosamine and total sulphate: D-glucosamine ratios as heparan-sulphate in the less acidic fraction, and as heparin in the more acidic fraction. Dermatan sulphate molecules were also present in both preparations, with an approximate ratio 1:3 to the glucosamine-containing polysaccharides. Solubility behaviour of the complexes formed by the isolated polyanionic molecules with cetylpyridinium chloride was strongly modified by papain digestion of the duodenal material. This reduction of molecular size of papain treatment suggests that the molecules extracted with water from duodenal mucosa are complex proteoglycans, perhaps in the native state.

Influence of flavonoid-copper complexes on cross linking in elastin.

A protective and curative effect of some flavonoids on collagen maturation in lathyrism has been previously demonstrated. This action appeared to involve cross linking of collagen. This paper deals with the effect in vitro of flavonoids and flavonoid-copper complexes on the oxidative deamination of lysine epsilon-amino groups in [4,5-3H]-lysine-labelled elastin. Flavonoids alone do not affect the reaction but it is evident that some flavonoid copper complexes are strongly effective: the aldehyde groups that are quickly formed then enable cross linking to occur. The lysine epsilon-amino groups of elastin are specifically concerned: in fact no effect was observed on free [4,5-3H]-lysine or on [4,5-3H]-lysine-labelled proteins obtained from mouse liver. The lysyl oxidase seems to interfere with the flavonoid-copper complees to regulate the oxidative deamination of lysine epsilon-amino groups.

Biosynthesis of glycosaminoglycan precursors: evidences for different tissue specific forms of UDP-glucose dehydrogenase.

UDP-glucose dehydrogenase (UDPGDH) was extracted and partially purified from different rat tissues and the kinetic parameters and some properties of the enzyme were determined and compared. The pH optimum ranged between 8.6 and 9.4 for liver and kidney UDPGDH and between 8.4 and 8.6 for skin and lung UDPGDH. Liver and kidney enzymes showed a similar affinity for both UDPG and NAD. Lung and skin enzymes also showed similar affinity for both substrates, which differed however from that of liver and kidney UDPGDH. Both liver and kidney enzymes had a higher heat stability and a different electrophoretic mobility compared to skin and lung UDPGDH. These data suggest the existence of different tissue specific forms of the enzyme.

Collagenase-extractable proteoglycans from lesion-free areas of human aorta.

Different proteoglycan (PG) populations were isolated from normal human aorta by extraction of minced tissue with 4M GuHCl and by further digestion of the residue with collagenase. Dissociative extraction induced a complete disappearance of Alcian Blue positive material, which was demonstrable by transmission electron microscopy before the treatment around collagen fibrils and in pericellular areas. However, 4M GuHCl extraction solubilized only an average of 60% of aorta total hexuronate content. Collagenase treatment of the residue resulted in a complete loss of collagen fibril organization, which was coupled with a further hexuronate recovery, accounting for about one third of total tissue content. The bulk of PGs obtained in collagenase digest was retained by Sepharose CL-4B column. Their sulphated glycosaminoglycan (GAG) composition differed from PGs extracted with 4M GuHCl, containing only chondroitin sulphate (CS) and heparan sulphate (HS), without detectable traces of dermatan sulphate (DS). Moreover, they contained hyaluronic acid. The results obtained by agarose polyacrylamide gel electrophoresis (APGE) and Octyl-Sepharose chromatography, followed by further APGE and Sepharose CL-4B gel-filtration, carried out before and after treatment with Chondroitinase ABC and AC and Heparinase I and III, suggested that collagenase digest contained different PG populations, carrying mainly either CS or HS chains. Moreover, HS containing PGs showed higher hydrodynamic size and stronger properties of hydrophobic interactions than CS containing PGs.

Electrophoretic and lectin-binding properties of glycopeptides released from the membrane during "in vitro" aging of human erythrocytes.

During in vitro aging of human erythrocytes sialopeptides are lost from the membrane in a process which appears to act on glycophorins. This glycopeptide material can be purified by affinity chromatography on Wheat germ agglutinin-Sepharose, as glycophorin does. The electrophoretic behaviour of the purified material suggests that the glycopeptide comes from the breakdown of the domain of glycophorin exposed on the surface of the membrane. The binding properties toward Phaseolus vulgaris E lectin indicate that the only N-linked sugar chain of glycophorin is present in the sialopeptide released from the membrane; therefore we can argue that the glycophorin breakdown during in vitro aging of red cell takes place beyond the 26th residue of the sequence, and probably quite near the lipid bilayer.

Membrane processes during 'in vivo' aging of human erythrocytes.

Membrane modifications occurring during in vitro and in vivo aging in human erythrocytes have been investigated. The structural damages are the consequence of proteolytic and oxidative processes and involve both glycoprotein and protein component of the membrane. The target of these processes seems to be the same during in vitro and in vivo aging.

Identification of a sialoglycopeptide released by self-digestion from human erythrocyte membranes.

Membranes from human O Rhesus-positive erythrocyte 'ghosts' were tested in vitro for their ability to digest their own glycoproteins. 'Ghost' membranes incubated in Tris/HCl buffer, pH 7.4, release a sialoglycopeptide, which contains glucosamine, galactosamine, galactose and mainly polar amino acids. Chemical composition, molecular size and aggregation properties suggest that this glycopeptide may be a fragment of glycophorin.

Microheterogeneity of serum glycosyl albumin in diabetes mellitus.

Proteins purified by affinity chromatography on Blue-Sepharose CL-6B from serum of 10 normal controls and 19 Type I diabetic patients were studied by means of combined ultrathin isoelectric focusing and photochemical silver stain. While only a single band of protein (characterized as albumin by crossed immunoelectrophoresis) with a pI of 4.7 was found in serum of normal subjects, 10 out of the diabetic group showed some bands of proteins with a pI greater than 4.7 and a single one with a pI less than 4.7. Concanavalin A-Sepharose removed all these bands with altered pIs which were further characterized as albumin by fused rocket immunoelectrophoresis of the eluates from Concanavalin A-Sepharose, direct immunofixation after isoelectric focusing of proteins with high purified anti-albumin antibodies, SDS-polyacrylamide gradient pore electrophoresis, aminoacidic analysis. The gas-chromatographic analysis of carbohydrates released from both the albumin bound to Concanavalin A-Sepharose and that not bound, revealed in addition to two unidentified peaks, the presence of glucose, galactose and mannose whose contents were greatly increased in albumin with affinity for the lectin. Serum glycosyl albumin concentration was not statistically different in serum of diabetic patients displaying cationic glycosyl albumin in comparison to patients without these proteins (0.2261 +/- 0.0186 versus 0.1874 +/- 0.015 nmole HMF/nmole albumin), whereas the first group showed statistically higher urinary excretion rates of albumin (28.6 +/- 1.2 micrograms/min versus 4.6 +/- 0.2 micrograms/min).(ABSTRACT TRUNCATED AT 250 WORDS)

Protection of rat gastric mucosa against ethanol injury by the new synthetic prostaglandin MDL 646.

Oral administration to fasted rats of absolute ethanol produces extensive necrotic lesions of gastric mucosa as well as a massive leakage of proteins and mucus glycoproteins into gastric lumen. When the new synthetic prostaglandin MDL 646, belonging to the PGE1 series, is administered intragastrically (2 or 10 micrograms/kg) 30 min before ethanol administration, a significant protection of rat gastric mucosa against alcohol injury is observed.

Reversible and irreversible structural modifications of erythrocyte membrane.

Membrane glycoprotein structure regulates the fate in the circulation of mammalian erythrocytes: the mechanism of this phenomenon was observed in rabbit and human red cells by in vivo and in vitro experiments. Glycoprotein remodeling can occur as a reversible desialylation process, produced by different events, or as an irreversible process consisting in a loss of sialoglycopeptides. The effect of these two phenomena on the viability of the red cell in the circulation was the subject of our investigation.

Self-digestion of human erythrocyte membranes. Role of adenosine triphosphate and glutathione.

Intact human erythrocytes incubated at 37 degrees C, pH7.4, release a sialoglycopeptide similar in its chemical composition, immunological and aggregation properties to the glycopeptide released by isolated 'ghost' membranes. The presence of ATP or reduced glutathione at physiological concentrations in the incubation medium of 'ghost' membranes inhibits this self-digestion process.

Mimotopes and proteome analyses using human genomic and cDNA epitope phage display.

In the post-genomic era, validation of candidate gene targets frequently requires proteinbased strategies. Phage display is a powerful tool to define protein-protein interactions by generating peptide binders against target antigens. Epitope phage display libraries have the potential to enrich coding exon sequences from human genomic loci. We evaluated genomic and cDNA phage display strategies to identify genes in the 5q31 Interleukin gene cluster and to enrich cell surface receptor tyrosine kinase genes from a breast cancer cDNA library. A genomic display library containing 2 x 106 clones with exon-sized inserts was selected with antibodies specific for human Interleukin-4 (IL-4) and Interleukin-13. The library was enriched significantly after two selection rounds and DNA sequencing revealed unique clones. One clone matched a cognate IL-4 epitope; however, the majority of clone insert sequences corresponded to E. coli genomic DNA. These bacterial sequences act as 'mimotopes' (mimetic sequences of the true epitope), correspond to open reading frames, generate displayed peptides, and compete for binding during phage selection. The specificity of these mimotopes for IL-4 was confirmed by competition ELISA. Other E. coli mimotopes were generated using additional antibodies. Mimotopes for a receptor tyrosine kinase gene were also selected using a breast cancer SKBR-3 cDNA phage display library, screened against an anti-erbB2 monoclonal antibody. Identification of mimotopes in genomic and cDNA phage libraries is essential for phage display-based protein validation assays and two-hybrid phage approaches that examine protein-protein interactions. The predominance of E. coli mimotopes suggests that the E. coli genome may be useful to generate peptide diversity biased towards protein coding sequences.