Isolation and1H-NMR spectroscopic identification of the glucose-containing lipid-linked precursor oligosaccharide ofN-linked carbohydrate chains

The lipid-linked precursor ofN-type glycoprotein oligosaccharides was isolated from porcine thyroid microsomes after in cubation with UDP[³H] Glucose. The carbohydrate was released from dolichol pyrophosphate by mild acid hydrolysis, purified by gel filtration and characterized by 500-MHz¹H-NMR spectroscopy in combination with enzymatic degradation. The parent oligosaccharide was found to be Glc₃Man₉Glc-NAc₂. The three glucose residues are present in the linear sequence Glcα1-2Glα1-3 Glc, the latter being α(1-3)-linked to one of the mannose residues. In order to establish the branch location of the triglucosyl unit, the parent compound was digested with jack-bean α-mannosidase. The oligosaccharide product was purified by gel filtration, and identified by¹H-NMR as Glc₃Man₅GlcNAc₂ lacking the mannose residues A, D₂, B and D₃. Therefore, the structure of the precursor oligosaccharide is as follows: $$\begin{gathered} c b a D_1 C 4 \hfill \\ Glc\alpha 1 - 2Glc\alpha 1 - 3Glc\alpha 1 - 3Man\alpha 1 - 2Man\alpha 1 - 2Man\alpha 1 \hfill \\ 3 \swarrow 3 2 1 \hfill \\ Man\alpha 1 - 2Man\alpha 1 Man\beta 1 - 4GlcNAc\beta 1 - 4GlcNAc \hfill \\ D_{2 } A 3 6 \hfill \\ Man\alpha 1 \hfill \\ 6 \hfill \\ Man\alpha 1 - 2Man\alpha 1 \nwarrow 4 \hfill \\ D_3 B \hfill \\ \end{gathered} $$      

A new biallelic DNA polymorphism of the human COL5A1 gene

A cDNA probe of the human COL5A1 gene detects a frequent biallelic PstI polymorphism. Allele A has a frequency of 54% whereas that of allele B is 46%. This restriction fragment length polymorphism provides a useful marker for linkage analysis in 9q34.3.     

Cytotoxic effect of adriamycin and agarose-coupled adriamycin on glomerular epithelial cells: Role of free radicals

Summary It has been suggested that the generation of toxic radicals plays an important role in toxicity by Adriamycin (ADR) on cancer cell lines and in vivo. We have examined the role of free radicals in determining toxicity and resistance to ADR of rat glomerular epithelial cells in culture; this method provides a good model for analyzing the mechanisms responsible for ADR experimental nephrosis in rats. Three points were established: a) the intra- or extracellular site of ADR toxicity; b) the role of the superoxide anion and of the hydroxyl radical in determining intra- and-extracellular cytotoxicity; and c) the implication of oxido-reduction cycling as a potential route for ADR semiquinone transformation. Free ADR was found to induce the same inhibition of [³H]thymidine incorporation into DNA as ADR bound to an agarose macroporous bed which prevents the intracellular incorporation of the drug. Specific scavenging of free radical activity by the enzymes catalase and superoxide dismutase, the hydroxyl radical inhibitors dimethyl sulfoxide and dimethylthiourea (DMTU) and by chelation of intracellular free iron with deferoxamine produced only a partial restoration of [³H]thymidine incorporation into DNA, which was maximal for DMTU (30% of normal incorporation). DMTU treatment was unsuccessful in preventing the extracellular cytostatic effect of ADR. Finally, glomerular epithelial cell killing (⁵¹Cr-release method) by 5-iminodaunorubicin, an ADR analogue with a modified quinone function that prohibits oxido-reduction cycling, was higher than unmodified ADR. These results indicate that ADR may exert its cytotoxic effects on glomerular epithelial cells by interaction at the cell surface, whereas the intracellular compartment, principally DNA, does not seem to be the target of ADR effects. They also suggest that the free radicals are in part responsible for ADR intracellular cytotoxicity, but other mechanisms should also be hypothesized. Finally, the participation of the ADR semiquinone radical in oxido-reduction cycling seems not important for the induction of the cellular damage.     

Oxidized albumin. The long way of a protein of uncertain function

Background

Proteins are extremely reactive to oxidants and should represent a potential target of instable reactive oxygen. This may represent a problem for plasma proteins since they may be directly modified in vivo in a compartment where antioxidant enzymatic systems are scarcely represented. On the other hand, it is possible that some plasma components have evolved over time to guarantee protection, in which case they can be considered as anti-oxidants.

Scope of review

To present and discuss main studies which addressed the role of albumin in plasma antioxidant activity mainly utilizing in vitro models of oxidation. To present some advances on structural features of oxidized albumin deriving from studies carried out on in vitro models as well as albumin purified in vivo from patients affected by clinical conditions characterized by oxidative stress.

Major conclusions

There are different interaction with HOCl and chloramines. In the former case, HOCl produces an extensive alteration of ²³⁸Trp and ¹⁶²Tyr, ⁴²⁵Tyr, ⁴⁷Tyr, while thiol groups are only partially involved. Chloramines are extremely reactive with the unique free SH group of albumin (³⁴Cys) with the formation of sulfenic and sulfinic acid as intermediates and sulfonic acid as end-product. Oxidized albumin has a modified electrical charge for the addition of an acidic residue and presents α-helix and random coil reorganization with subtle changes in domain orientation.

General significance

Albumin, is the major antioxidants in plasma with a concentration (0.8 mM) higher than other antioxidants by an exponential factor. Functional and protective roles in the presence of oxidative stress must be defined. This article is part of a Special Issue entitled Serum Albumin.     

N-(4-hydroxyphenyl) retinamide inhibits cystogenesis by polycystic epithelial cell lines in vitro.

Primary tubular epithelial cells develop spherical monolayered cysts when cultured in collagenI matrix, a model that has been used to study the mechanism of cystogenesis. In an attempt to block cystogenesis, we have evaluated the effect of N-(4-hydroxyphenyl) retinamide (HPR), a synthetic derivative of retinoic acid, on both formation and growth of cysts in a human model of polycystic kidney cells. Number, dimension and submicroscopical characteristics of cysts were evaluated after 2 and 4 weeks from treatment with HPR. A marked inhibitory effect of HPR on cystogenesis was found at concentration of 1 microM, while a complete block was observed at concentration between 5 and 10 microM. Furthermore, treatment with HPR of already formed cysts resulted in their disruption. HPR at 10 microM also induced apoptosis of several tubular epithelial cell models suggesting a correlation between the two phenomena. Taken together these observations demonstrate that HPR blocks cystogenesis by polycystic kidney cells "in vitro" and that it also reverts the fate of already formed cysts. Apoptosis may be the mechanism which mediates the inhibitory effect on cystogenesis in this model.     

Increased expression of costimulatory markers CD134 and CD80 on interleukin-17 producing T cells in patients with systemic lupus erythematosus

Introduction  

There is growing evidence that interleukin 17 (IL-17) producing T cells are involved in the pathogenesis of systemic lupus erythematosus (SLE). Previous studies showed that increased percentages of T-cell subsets expressing the costimulatory molecules CD80 and CD134 are associated with disease activity and renal involvement in SLE. The aim of this study was to investigate the distribution and phenotypical characteristics of IL-17 producing T-cells in SLE, in particular in patients with lupus nephritis, with emphasis on the expression of CD80 and CD134.     

Baseline predictors of response and discontinuation of tumor necrosis factor-alpha blocking therapy in ankylosing spondylitis: a prospective longitudinal observational cohort study

Introduction  

Identifying ankylosing spondylitis (AS) patients who are likely to benefit from tumor necrosis factor-alpha (TNF-α) blocking therapy is important, especially in view of the costs and potential side effects of these agents. Recently, the AS Disease Activity Score (ASDAS) has been developed to assess both subjective and objective aspects of AS disease activity. However, data about the predictive value of the ASDAS with respect to clinical response to TNF-α blocking therapy are lacking. The aim of the present study was to identify baseline predictors of response and discontinuation of TNF-α blocking therapy in AS patients in daily clinical practice.