Postprandial intradialytic dysglycaemia and diabetes in maintenance haemodialysis patients.

BACKGROUND: Although the risk of developing dysglycaemia has been investigated in different communities this incidence is poorly studied in patients on maintenance haemodialysis (MHD). MATERIALS AND METHODS: In a multicentre observational cohort study the occurrence of dysglycaemia was assessed in 239 primary normoglycaemic end stage renal disease (ERSD) patients on MHD. Dysglycaemia (fasting blood glucose > 110 mg dL(-1), > 140 mg dL(-1) 2 h after food intake) or diabetes (fasting blood glucose > 126 mg dL(-1) or > 200 mg dL(-1) at any time) were defined according to WHO criteria and cases were compared with age matched controls within the cohort. RESULTS: Dysglycaemia was found in 82 primary normoglycaemic ESRD patients (34%) within 31 months after initiation of MHD. In 31 of these patients type 2 diabetes was diagnosed. When compared with matched control MHD patients differences in body mass index (BMI), HbA1c and postprandial blood glucose were detectable (P < 0.05). Increments in 0.1% of HbA1c were related with 11% higher odds for dysglycaemia (P = 0.002). In a subgroup of 36 primary normoglycaemic MHD patients who developed dysglycaemia event-free survival was 64%, 53%, 31%, 17% and 11% after 1, 2, 3, 4 and 5 years of haemodialysis treatment. CONCLUSION: Onset of dysglycaemia or diabetes is frequent in ESRD patients after onset of chronic haemodialysis. Routine measurement of blood glucose before and after haemodialysis should be implemented as a standard of care during MHD.

Connective tissue changes in a mouse model of vein graft disease.

AIM: The extracellular matrix plays an important physiological role in the architecture of the vascular wall. In arterialized vein grafts severe early changes, such as thrombosis and neointimal hyperplasia occur. Paclitaxel is in clinical use as antiproliferative coating of coronary stents. We aimed to investigate the early connective tissue changes in arterialized vein grafts and the influence of perivascular paclitaxel treatment in an in vivo model. METHODS: C57 black mice underwent interposition of the vena cava into the carotid artery. Neointimal hyperplasia, thrombosis, acid mucopolysaccharides (Alcian), collagen fibers (trichrome Masson), elastic fibers, and apoptosis rate (TUNEL) were quantified in paclitaxel treated veins and controls. RESULTS: In both, controls and paclitaxel treated vein grafts acid mucopolysaccharides and elastic fibers were found predominantly in the neointima, whereas collagen fibers were found mainly in the media and adventitia. At 4 weeks postoperatively the neointimal thickness in controls was 52 (13-130) microm, whereas in 0.6 mg/mL l paclitaxel treated veins it was 103 (43-318) microm (P=0.094). At 8 weeks postoperatively paclitaxel treated veins showed a significantly increased neointimal thickness of 136 (87-199) microm compared with 79 (62-146) microm in controls (P=0.032). There was no difference in apoptosis rate between the two groups (P=NS). Even with the lowest concentration of 0.008 mg/mL paclitaxel veins showed a neointimal thickness of 67 (46-205) microm at 4 weeks postoperatively (P=NS vs controls). CONCLUSION: Early vein graft disease is characterised by an accumulation of acid mucopolysaccharides and elastic fibers in the thickened neointima. Paclitaxel treatment increases the neointimal hyperplasia in mouse vein grafts in vivo.

Expression and prognostic significance of Tetranectin in invasive and non-invasive bladder cancer.

Tetranectin (TN) is a plasminogen kringle-4 binding protein that can be detected in the plasma and the extracellular matrix. In malignancies, TN is thought to enhance proteolytic processes enabling tumor cells to invade and metastasize. So far, TN expression has not been described in transitional cell bladder cancer (TCC), and there is no information on the prognostic significance of its in situ expression. TN expression was studied in 99 TCC patients diagnosed between 1994 and 1997. Immunohistochemistry was performed on a tissue microarray using a monoclonal antibody against TN (clone 11F1). Within the mean follow-up period of 60 months, pTa and pT2-4 TCC patients with stainable TN in the tumor stroma had a significant shorter recurrence-free survival and higher risk of recurrence compared to those without stainable TN (p = 0.0002 for both). TN expression in the tumor cells did not influence recurrence-free survival. In conclusion, TN is expressed in a subgroup of bladder cancer patients with a higher risk of recurrence who may take benefit from a closer follow-up.

Component-resolved diagnosis (CRD) of type I allergy with recombinant grass and tree pollen allergens by skin testing.

The diagnosis of Type I allergy is based on the measurement of allergen-specific IgE antibodies and on provocation with allergens, most frequently conducted by skin testing. Both forms of diagnosis are currently performed with allergen extracts that are difficult to standardize regarding their allergen contents, and which contain additional undefined nonallergenic components. We report the expression in Escherichia coli and purification of some of the most relevant timothy grass- and birch pollen allergens. Recombinant timothy grass- (rPhl p 1, rPhl p 2, rPhl p 5) and birch pollen (rBet v 1, rBet v 2) allergens were purified and used for the measurement of allergen-specific IgE and IgG subclass responses as well as for skin prick testing in 55 pollen allergic patients and 10 nonatopic individuals. Results obtained showed that the recombinant allergens allowed in vivo allergy diagnosis in 52 of 54 of the grass pollen and in 35 of 36 of the birch pollen allergic patients. Positive skin reactions were observed almost exclusively in patients containing detectable allergen-specific IgE antibodies but not in the nonatopic group; however, sensitivity to a given allergen as measured by skin reactivity was weakly correlated with the levels of allergen-specific IgE. Our results demonstrate that recombinant allergens can be used for component-resolved skin test diagnosis (CRD) of the patients' allergen sensitization profile, whereas allergen extracts at best allow to identify allergen-containing sources. CRD may thus represent the basis for novel forms of patient-tailored immunotherapy.

Cloning and analysis of a human 86-kDa heat-shock-protein-encoding gene.

An 86-kDa heat-shock-protein-encoding (hsp86) cDNA probe permitted to identify, in whole genomic human DNA, two EcoRI fragments of 2.6 and 5.3 kb. These two fragments, as well as an homologous phage lambda VIII1 harboring about 19 kb of human DNA, were isolated from genomic libraries. Sequence analysis revealed that three different genomic hsp86 sequences had been cloned, one of them being the 5' half of a functional gene. This gene contains several introns, as compared to the entire Hsp86-encoding sequence found in lambda VIII1, which represents a processed pseudogene. Cloned hsp86 promoter, with its TATA-box and a heat-shock element upstream at nt positions -25 and -75, respectively, was functional, as verified by fusion to the bacterial chloramphenicol acetyltransferase-encoding gene and its transient expression in vivo. The typical hsp86-type heat-shock regulation was observed, i.e., significant basal activity associated with an inducibility at elevated temperatures. Furthermore, accurate and efficient in vitro transcription was initiated at this hsp86 promoter, resulting in expression of the hsp86 gene, as well as the unrelated sequences.

Expression of Zm13, a pollen specific maize protein, in Escherichia coli reveals IgE-binding capacity and allergenic potential.

Plant proteins belong to the most frequent elicitors of type I allergic symptoms in industrialized countries. Several relevant plant allergens have been found to be either specifically expressed or highly upregulated in mature pollen. The cDNA coding for a pollen specific maize protein, Zm13, shows significant sequence homology with a number of pollen or anther specific proteins from monocot and dicot plants as well as with recently described allergens from olive and rye grass. To test whether the Zm13 protein might possess IgE-binding capacity, Zm13 was expressed in E. coli. The coding region of Zm13 was PCR amplified from a genomic clone and expressed as a glutathione-S-transferase fusion protein. The recombinant Zm13 fusion protein bound a Zm13 specific rabbit antiserum and reacted with serum IgE from grass pollen allergic patients indicating that Zm13 and homologous proteins represent a family of conserved plant allergens.

Magnetic resonance angiography of mesenteric arteries. A review.

There has been continued development of MRI techniques for evaluating mesenteric vascular disease. Contrast-enhanced magnetic resonance angiography (MRA) can provide reproducible high resolution, high contrast images of the arterial and venous mesenteric vasculature and may allow detection of segmental ischemia by detection of segmental delayed mesenteric or bowel wall enhancement. Cine phase-contrast MRA can provide additional information about the rate and volume of flow within the major mesenteric arteries and veins. Real-time MRI can provide interactive visualization of the mesenteric vessels in any plane, and with suitable bowel contrast, it can be used to monitor global and segmental small bowel motility. With in vivo MR oximetry, flow independent measurements of the T2 relaxation of blood allow the oxygen saturation of the mesenteric circulation to be determined. These MR techniques can be combined for evaluating both anatomic and functional aspects of the mesenteric circulation.

Preparation of mouse monoclonal antibodies and evidence for a host immune response to the preacetabular gland proteinase of Schistosoma mansoni cercariae.

Forty-six monoclonal antibodies were produced against the preacetabular gland secretions of Schistosoma mansoni cercariae by two different immunization protocols. These antibodies were of both the IgG and the IgM classes. One IgM monoclonal antibody (Ia4D6) was further characterized. It was specific to the cercarial stage by ELISA and showed specific binding to the 30,000 Mr proteinase in crude cercarial secretions by Western blot analysis. Preincubation of this antibody with purified cercarial proteinase resulted in inhibition of proteolytic activity, and it mediated complement-dependent cytotoxicity to cercariae in vitro. Immunoperoxidase staining of cercariae localized this antibody to vesicles visible within the preacetabular glands and their secretory ducts, and to secreted material. ELISA and Western blot analysis also showed that sera from infected mice and patients with schistosomiasis reacted with the cercarial proteinase. These studies demonstrate that a proteinase secreted into the host by invading cercariae is immunogenic and provide a monoclonal antibody probe for further characterization of its structure and function.

Construction of a Chlamydomonas reinhardtii mutant with an intronless psbA gene.

Efficient chloroplast transformation systems now available allow the manipulation of the evolutionarily highly conserved psbA gene in the eucaryotic organism Chlamydomonas reinhardtii. Two copies of this gene in the inverted repeat region of the chloroplast genome contain four large group I introns. To analyse possible functions of these introns and to generate a mutant for simplified psbA gene manipulations, a psbA cDNA fragment was introduced into a psbA deletion mutant using the biolistic transformation method. A transformant with no introns in the psbA gene has been obtained and represents the first example of the removal of a complete set of introns from a chloroplast gene. The newly generated strain is photosynthetically competent and contains no detectable recipient genome copies. The loss of all four introns appears to be phenotypically silent.

Analysis of a herbicide resistant mutant obtained by transformation of the Chlamydomonas chloroplast.

A herbicide resistant Chlamydomonas double mutant (I219A264) has been obtained by transforming the psbA deletion mutant FuD7 with a cloned psbA gene fragment containing mutations in codons 219 and 264. Copies from both the recipient (FuD7) genome and the genome carrying the mutated psbA gene persist in the transformant. This stable heteroplasmic state appears to be required for photoautotrophic growth. Comparison of resistance profiles for classical and phenol-type inhibitors of the double mutant and the corresponding single mutants demonstrates independent, additive contributions of both amino acids to herbicide binding. The approach chosen here to modify the psbA gene should be useful in those cases where consequences of psbA gene manipulations are not predictable with respect to inhibitor resistance.

Regulation of CapZ, an actin capping protein of chicken muscle, by anionic phospholipids.

Chicken muscle CapZ, a member of the capping protein family of actin-binding proteins, binds to the barbed end of actin filaments and nucleates actin polymerization. No regulation of the capping protein family has been described. We report that micelles of phosphatidylinositol 4,5-bisphosphate (PIP2) bind to CapZ and completely inhibit its ability to affect actin polymerization as measured by several independent assays. Higher concentrations of other anionic phospholipids also completely inhibit the activity of CapZ. Neutral phospholipids have no effect. Mixed vesicles of PIP2 with phosphatidylcholine or phosphatidylethanolamine also inhibit CapZ, but addition of Triton X-100 both prevents and reverses PIP2's inhibition of CapZ.

Contrast-enhanced three-dimensional fast spoiled gradient-echo renal MR imaging: evaluation of vascular and nonvascular disease.

Breath-hold contrast material enhanced three-dimensional (3D) fast spoiled gradient-echo (FSPGR) sequences are valuable techniques for evaluation of renal arteries and veins and diagnosis of significant renal arterial stenosis at magnetic resonance (MR) imaging. The excellent spatial and contrast resolution with these techniques, combined with the ability to perform studies in multiple vascular phases, also make them attractive for the diagnosis of a wide range of nonvascular processes that affect the kidneys, including renal infections, renal parenchymal diseases, and renal trauma. Particularly when combined with T1- and T2-weighted MR imaging, the contrast-enhanced techniques are highly effective for characterization of renal masses owing to the ability to portray dynamic contrast enhancement. The ability to display venous structures with contrast-enhanced 3D FSPGR techniques helps staging of renal cell carcinoma. This article presents examples of the wide range of vascular and nonvascular renal diseases that may be effectively imaged with contrast material enhanced 3D FSPGR techniques and illustrates the usefulness of the techniques for renal MR imaging.

Site-specific mutagenesis of the D1 subunit of photosystem II in wild-type Chlamydomonas.

The structure and functional mode of photosystem II reaction center protein D1 can be studied by analyzing the effects of amino acid substitutions within the binding niche for QB, the second stable electron acceptor of photosystem II, on herbicide binding. Here we report on site-directed mutagenesis of the psbA gene coding for the D1 protein in the unicellular alga Chlamydomonas reinhardtii. The chloroplasts of wild-type cells were transformed using the particle gun. The plasmids introduced carried an in vitro mutated fragment of the psbA gene. We obtained a double mutant with replacements of amino acids 264 and 266 and a triple mutant having an additional substitution in position 259. The sensitivities of both mutants toward several types of herbicides are given and compared with those of a mutant having only a substitution at position 264.

Extracellular protease production by Drosophila imaginal discs.

We are investigating the role of extracellular proteases in imaginal disc eversion to understand the mechanism that controls cell rearrangements within epithelia. We have identified three cation-dependent neutral proteases released by Drosophila leg discs everting in culture. Serine protease inhibitors block disc eversion and inhibit activity of disc proteases. The pattern of extracellular proteases changes when eversion is blocked with added protease inhibitors. Changes in protease activity occur when released disc proteases are treated with trypsin. Trypsin treatment of intact imaginal discs releases protease and inhibitor activities to the medium, indicating their presence on the cell surface before release. Our results suggest that extracellular proteases are required for imaginal disc morphogenesis and are regulated by more than one mechanism.

A comprehensive approach using MR imaging to diagnose acute segmental mesenteric ischemia in a porcine model.

OBJECTIVE: Acute mesenteric ischemia is a lethal disease that lacks a noninvasive diagnostic test. We evaluated the abilities of contrast-enhanced MR angiography, MR oximetry, and real-time interactive MR imaging to diagnose segmental mesenteric ischemia in a porcine model. MATERIALS AND METHODS: Segmental mesenteric ischemia was created by subselective Gelfoam embolization of the mesenteric circulation in eight pigs. Conventional digital subtraction angiography (DSA), MR oximetry, and real-time interactive MR imaging of the small bowel were performed before and after embolization. Changes in the perfusion pattern seen on DSA established the regions of true ischemia. Postembolization DSA and MR angiography were compared with this gold standard. RESULTS: Both MR angiography and DSA had high sensitivity (91% and 100%, respectively) for detecting ischemic regions. The difference was not statistically significant (p > .2). MR angiography yielded lower specificity than DSA (80% and 90%, respectively; p < .01). After embolization, the oxygen saturation in the superior mesenteric vein (SMV) dropped significantly (p < .005). After embolization, the SMV also showed oxygen saturation significantly lower than that in the inferior vena cava (p < .005). In two of the animals, segmental hypomotility of the small bowel was observed. CONCLUSION: MR oximetry is capable of detecting oxygen desaturation caused by segmental ischemia. A loss of oxygen saturation in the SMV relative to that in the inferior vena cava provides a convenient marker of mesenteric ischemia. Contrast-enhanced MR angiography has sensitivity and specificity approaching those of DSA. Both MR techniques hold promise for the detection of acute mesenteric ischemia.

Cloning sulfur assimilation genes of Brassica juncea L.: cadmium differentially affects the expression of a putative low-affinity sulfate transporter and isoforms of ATP sulfurylase and APS reductase.

The heavy-metal accumulator Brassica juncea L. is a high-biomass crop able to extract heavy-metal ions from the soil, a substantial part being translocated from root to shoot. Previous work has shown that Cd accumulation is accompanied by massive formation of phytochelatins (PCs). Rapid de novo synthesis of PCs in roots and leaves requires an increased synthesis of the tripeptide glutathione (GSH), which in turn depends on increased sulfur assimilation. Therefore. we have cloned cDNAs for three enzymes involved in sulfur assimilation, i.e. a putative low-affinity sulfate transporter (LAST) and two isoforms each for ATP sulfurylase (ATPS) and APS reductase (APSR). As degradation of glucosinolates might provide an additional sulfur source under stress, we also cloned a myrosinase (MYR). RNA blot analysis of transcript amounts indicated that upon Cd exposure (25 microM) the expression of ATPS and APSR in roots and leaves of 6-week-old Brassica juncea plants was strongly increased, whereas the expression of MYR was unaffected. LAST transcripts were significantly reduced in the root but remained unchanged in the leaves. Concomitant with Cd induction of ATPS and APSR mRNAs, cysteine concentrations in roots and leaves increased by 81% and 25%, respectively, whereas GSH concentrations decreased in roots and leaves by 39% and 48%, respectively. In agreement with our previous report on Cd induction of gamma-glutamylcysteine synthetase in B. juncea, the results indicate coordinate changes of expression for several sulfur assimilation enzymes in response to an increased demand for cysteine during PC synthesis.

Effects of CapZ, an actin capping protein of muscle, on the polymerization of actin.

We have studied the interaction of CapZ, a barbed-end actin capping protein from the Z line of skeletal muscle, with actin. CapZ blocks actin polymerization and depolymerization (i.e., it "caps") at the barbed end with a Kd of approximately 0.5-1 nM or less, measured by three different assays. CapZ inhibits the polymerization of ATP-actin onto filament ends with ATP subunits slightly less than onto ends with ADP subunits, and onto ends with ADP-BeF3- subunits about as much as ends with ADP subunits. No effect of CapZ is seen at the pointed end by measurements either of polymerization from acrosomal processes or of the critical concentration for polymerization at steady state. CapZ has no measureable ability to sever actin filaments in a filament dilution assay. CapZ nucleates actin polymerization at a rate proportional to the first power of the CapZ concentration and the 2.5 power of the actin concentration. No significant binding is observed between CapZ and rhodamine-labeled actin monomers by fluorescence photobleaching recovery. These new experiments are consistent with but do not distinguish between three models for nucleation proposed previously (Cooper & Pollard, 1985). As a prelude to the functional studies, the purification protocol for CapZ was refined to yield 2 mg/kg of chicken breast muscle in 1 week. The activity is stable in solution and can be lyophilized. The native molecular weight is 59,600 +/- 2000 by equilibrium ultracentrifugation, and the extinction coefficient is 1.25 mL mg-1 cm-1 by interference optics. Polymorphism of the alpha and beta subunits has been detected by isoelectric focusing and reverse-phase chromatography. CapZ contains no phosphate (less than 0.1 mol/mol).

Degradation of isooctane by Mycobacterium austroafricanum IFP 2173: growth and catabolic pathway.

AIMS: Isooctane (2,2,4-trimethylpentane), a major component of gasoline formulations, is recalcitrant to biodegradation probably because of the quaternary carbon group it contains. Information on the biodegradability of this hydrocarbon is essential to evaluate its fate in the environment. For these reasons, the degradation kinetics and the catabolic pathway of isooctane were investigated in Mycobacterium austroafricanum IFP 2173, the only strain characterized to use it as sole carbon and energy source. METHODS AND RESULTS: The selected strain exhibited a rather moderate maximum growth rate (micromax = 0.053 h(-1)) but degraded isooctane up to 99% with a mineralization yield of 45%, indicating attack of the quaternary carbon group. The GC/MS identification of metabolites, 2,4,4-trimethylpentanoic and dimethylpropanoic (pivalic) acids, which transiently accumulated in the cultures indicated that degradation started from the isopropyl extremity of the molecule and subsequently proceeded by catabolism of the tert-butyl moiety. The degradation of putative metabolic intermediates was investigated. The initial isooctane oxidation system was tentatively characterized. CONCLUSIONS: The isooctane-degrading strain harboured two candidate systems for initial alkane oxidation. Although a cytochrome P450 was induced by isooctane degradation, the functional oxidation system was probably a nonheme alkane monooxygenase as indicated by PCR amplification and RT-PCR expression of an alkB gene. SIGNIFICANCE AND IMPACT OF THE STUDY: Isooctane is a recalcitrant branched alkane. A plausible pathway of its degradation by Myco. austroafricanum was put forward.

Schistosoma mansoni: degradation of host extracellular matrix by eggs and miracidia.

A radioactively labeled in vitro model of the extracellular matrix of the mammalian intestinal wall and of snail tissue was used to determine whether proteolytic enzymes released by eggs and miracidia of Schistosoma mansoni could degrade connective tissue macromolecules in the type of interactive framework found in vivo. Eggs were collected and miracidia hatched in the presence of antibiotics to eliminate bacterial contamination. Uninfected livers were used as controls to ensure that the tissue dissociation and egg collection procedures did not produce proteolytic activity. One thousand live eggs incubated with the extracellular matrix for 72 hr at 37 C degraded 31% of the glycoprotein in the matrix; there was no degradation of elastin or collagen. Medium conditioned by incubation with eggs degraded 60% as much of the matrix as the live eggs themselves. The proteolytic activity of the egg-conditioned medium was greater in the presence of dithiothreitol. Miracidia incubated with the extracellular matrix in tissue culture medium at 27 or 37 C rapidly transformed to living sporocysts. This transformation was accompanied by a release of proteolytic activity, resulting in the degradation of 49 to 58% of the glycoprotein in the extracellular matrix by 1000 miracidia. Again, no elastin or collagen was degraded. The time course of degradation by miracidia was rapid over 24 hr and thus similar to that previously reported for cercariae. Degradation by eggs occurred more slowly over 72 hr. These data confirm that both eggs and miracidia secrete proteinases which are capable of degrading at least the glycoprotein components of extracellular matrix to facilitate their migration through intestinal wall or penetration of snail tissue.