Bladder cancer biomarker array to detect aberrant levels of proteins in urine.

Bladder cancer (BCa) is a serious malignancy of the urinary tract worldwide and also prominent for its high rate of recurrence incorporating 50% of all treated patients. To reduce relapse of BCa, lifelong surveillance of patients is essential leading to high treatment costs. The gold standard for the diagnosis of bladder cancer is cystoscopy. It is very sensitive, but due to high costs and its invasive nature this method for routine diagnosis of bladder cancer remains questionable. Because of this and the required surveillance of patients suffering from bladder cancer, urine based markers represent a new potential field of investigation. Literature at the National Center of Biological Information (NCBI) was retrieved for a potential marker panel offering specific protein signatures and used to develop a sensitive and accurate chip assay to monitor BCa. Discovery of possible bladder cancer protein markers is compiled by extensive literature search including 1077 recently (15.01.2008-20.03.2014) published research articles. Validation of this literature is done by selection based on prior defined inclusion and exclusion criteria. A set of six putative biomarkers (VEGF, IL-8, MMP-9, MMP-7, survivin and Cyfra 21.1) was identified and a non-invasive microarray developed to be used for further clinical validation. Investigation regarding optimized urine preparation and assay development, to enhance assay sensitivity for the marker panel, was carried out. This protein based BCa chip enables the fast (within 5 h), simultaneous, easy to operate, cheap, early and non-invasive determination of BCa and is ready for clinical evaluation.

High glucose-induced transforming growth factor beta1 production is mediated by the hexosamine pathway in porcine glomerular mesangial cells.

Previous studies revealed that exposure of mesangial cells to high glucose concentration induces the production of matrix proteins mediated by TGF-beta1. We tested if structural analogues of D-glucose may mimic the high glucose effect and found that D-glucosamine was strikingly more potent than D-glucose itself in enhancing the production of TGF-beta protein and subsequent production of the matrix components heparan sulfate proteoglycan and fibronectin in a time- and dose-dependent manner. D-Glucosamine also promoted conversion of latent TGF-beta to the active form. Therefore, we suggested that the hexosamine biosynthetic pathway (the key enzyme of which is glutamine:fructose-6-phosphate amidotransferase [GFAT]) contributes to the high glucose-induced TGF-beta1 production. Inhibition of GFAT by the substrate analogue azaserine or by inhibition of GFAT protein synthesis with antisense oligonucleotide prevented the high glucose-induced increase in cellular glucosamine metabolites and TGF-beta1 expression and bioactivity and subsequent effects on mesangial cell proliferation and matrix production. Overall, our study indicates that the flux of glucose metabolism through the GFAT catalyzed hexosamine biosynthetic pathway is involved in the glucose-induced mesangial production of TGF-beta leading to increased matrix production.

Cementless acetabular reconstruction and structural bone-grafting in dysplastic hips. Surgical technique.

BACKGROUND: Studies of acetabular reconstruction with use of cement and bulk bone graft have demonstrated increasing rates of cup failure in patients with dysplastic hips seven years after total hip arthroplasty. Comparable data on the long-term results of bulk bone-grafting done in conjunction with cementless implants are limited. The aim of this study was to review the clinical and radiographic results of autologous bulk bone-grafting in conjunction with a cementless cup. METHODS: From 1987 to 1992, forty-seven patients (forty women and seven men, with an average age of 50.4 years) who had developmental dysplasia of the hip underwent fifty-six total hip arthroplasties and received a structural graft in combination with a cementless Harris-Galante type-I cup. All patients were followed prospectively. In fifty-five hips, implant migration was measured with single-image radiographic analysis. RESULTS: After an average duration (and standard deviation) of 10.2 +/- 2.9 years, three patients (four hips) had died. In the surviving patients, four implants had been revised and two had radiographic evidence of loosening. With use of revision and loosening as end points, the eleven-year survival rates were 91.6% and 88.9%, respectively. Of the fifty implants that had no loosening, fourteen had measurable cup migration, thirty-five had no migration, and one implant could not be measured. All migrations but one were progressive. With loosening used as the end point, the survival rate at eleven years was 100% for the implants with no migration; however, the survival rate for the cups that had migrated was 69.3% (p = 0.0012). CONCLUSIONS: The eleven-year survival rate for the spherical press-fit cups in combination with bulk bone-grafting is satisfactory, given the complexity of these reconstructions. However, the difference between the survival of the implants that had migrated and those that had not was significant. We expect that the thirteen implants with progressive acetabular migration at the time of the latest follow-up are at risk for loosening, which will increase the revision rate for this series in the coming years.

Metabolic fluxes in riboflavin-producing Bacillus subtilis.

The pentose phosphate pathway and the pyruvate shunt were identified as major pathways of glucose catabolism in a recombinant, riboflavin-producing Bacillus subtilis strain. Reactions connecting the tricarboxylic acid cycle and glycolysis, catalyzed by the malic enzyme and phosphoenolpyruvate carboxykinase, consume up to 23% of the metabolized glucose. These are examples of important fluxes that can be accessed explicitly using a novel analysis based on synergistic application of flux balancing and recently introduced techniques of fractional 13C-labeling and two-dimensional nuclear magnetic resonance spectroscopy. The overall flux distribution also suggests that B. subtilis metabolism has an unusually high capacity for the reoxidation of NADPH. Under the conditions investigated, riboflavin formation in B. subtilis is limited by the fluxes through the biosynthetic rather than the central carbon pathways, which suggests a focus for future metabolic engineering of this system.

Cementless acetabular reconstruction and structural bone-grafting in dysplastic hips.

BACKGROUND: Studies of acetabular reconstruction with use of cement and bulk bone graft have demonstrated increasing rates of cup failure in patients with dysplastic hips seven years after total hip arthroplasty. Comparable data on the long-term results of bulk bone-grafting done in conjunction with cementless implants are limited. The aim of this study was to review the clinical and radiographic results of autologous bulk bone-grafting in conjunction with a cementless cup. METHODS: From 1987 to 1992, forty-seven patients (forty women and seven men, with an average age of 50.4 years) who had developmental dysplasia of the hip underwent fifty-six total hip arthroplasties and received a structural graft in combination with a cementless Harris-Galante type-I cup. All patients were followed prospectively. In fifty-five hips, implant migration was measured with single-image radiographic analysis. RESULTS: After an average duration (and standard deviation) of 10.2 +/- 2.9 years, three patients (four hips) had died. In the surviving patients, four implants had been revised and two had radiographic evidence of loosening. With use of revision and loosening as end points, the eleven-year survival rates were 91.6% and 88.9%, respectively. Of the fifty implants that had no loosening, fourteen had measurable cup migration, thirty-five had no migration, and one implant could not be measured. All migrations but one were progressive. With loosening used as the end point, the survival rate at eleven years was 100% for the implants with no migration; however, the survival rate for the cups that had migrated was 69.3% (p = 0.0012). CONCLUSIONS: The eleven-year survival rate for the spherical press-fit cups in combination with bulk bone-grafting is satisfactory, given the complexity of these reconstructions. However, the difference between the survival of the implants that had migrated and those that had not was significant. We expect that the thirteen implants with progressive acetabular migration at the time of the latest follow-up are at risk for loosening, which will increase the revision rate for this series in the coming years.

Sigma factor and sporulation genes in Clostridium.

The genus Clostridium, represented by Gram-positive, anaerobic, spore-forming bacteria, is well known for its clinical importance and considerable biotechnological potential. Recently, evidence for a functional role of the transcription factors sigma A, sigma E, sigma G, and sigma K in this genus was provided by cloning and sequencing these genes from C. acetobutylicum. In C. kluyveri, a partially sequenced open reading frame was found to encode the N terminus of the putative sigma factor L with significant similarity to members of the sigma 54 family. The identification of sequences with high similarity to the Bacillus sigma F (C. acetobutylicum), sigma H (several clostridial species), and sigma D (C. thermocellum)-controlled consensus promoters renders the existence of these transcription factors in clostridia very likely. These data are in agreement with information obtained by RNA transcript mapping (sigma A, sigma H), heterologous DNA hybridization (sigma D, sigma H), and immuno characterization of purified proteins (sigma A) from various clostridial species. Thus, the picture emerges that a fundamental similarity exists at the genetic level between the regulation of various cellular responses, in particular sporulation, in the genera Bacillus and Clostridium. The different induction patterns of sporulation in Bacillus spp. (nutrient starvation) and many clostridial species (cessation of growth or exposure to oxygen in the presence of excess nutrients) are most interestingly not reflected in the general regulatory features of this developmental process.

Solventogenic enzymes of Clostridium acetobutylicum: catalytic properties, genetic organization, and transcriptional regulation.

The enzymes acetoacetate decarboxylase and coenzyme A transferase catalyse acetone production from acetoacetyl-CoA in Clostridium acetobutylicum. The adc gene encoding the former enzyme is organized in a monocistronic operon, while the ctf genes form a common transcription unit with the gene (adhE) encoding a probable polyfunctional aldehyde/alcohol dehydrogenase. This genetic arrangement could reflect physiological requirements at the onset of solventogenesis. In addition to AdhE, two butanol dehydrogenase isozymes and a thiolase are involved in butanol synthesis. RNA analyses showed a sequential order of induction for the different butanol dehydrogenase genes, indicating an in vivo function of BdhI in low level butanol formation. The physiological roles of AdhE and BdhII most likely involve high level butanol formation, with AdhE being responsible for the onset of solventogenesis and BdhII ensuring continued butanol production. Addition of methyl viologen results in artificially induced butanol synthesis which seems to be mediated by a still unknown set of enzymes. Although the signal that triggers the shift to solventogenesis has not yet been elucidated, recent investigations suggest a possible function of DNA supercoiling as a transcriptional sensor of the respective environmental stimuli.

Myocardial blood flow and flow reserve after coronary reimplantation in patients after arterial switch and ross operation.

BACKGROUND: Coronary reimplantation is used in therapy for congenital heart disease, such as in the arterial switch (ASO) and Ross operations. The adequacy of myocardial perfusion may remain a matter of concern. The aim of the present study was to stratify the effect of coronary reimplantation on myocardial perfusion and to highlight the clinical relevance of any attenuation in myocardial perfusion. METHODS AND RESULTS: A total of 21 children with transposition of the great arteries at a mean interval of 11.2+/-2.9 years after ASO and 9 adolescents at a mean interval of 4.2+/-2.1 years after the Ross procedure were investigated. All patients were asymptomatic and had a normal exercise capacity. On stress echocardiography, 2 of the ASO patients had dyskinetic areas within the left ventricular myocardium, and 5 had adenosine-induced perfusion defects on positron emission tomography. No coronary obstruction was detected on coronary angiography in any patient, but a common finding was right coronary dominance and a small caliber of the distal part of the left anterior descending artery. Coronary flow reserve (CFR) was significantly reduced in all patients after ASO when compared with 10 normal healthy volunteers (age, 25.6+/-5.3 years). CFR was normal in the 9 patients who had the Ross operation (age, 19.2+/-7.6 years); exercise-induced perfusion defects were not detected in the Ross patients. CONCLUSIONS: Children after ASO are asymptomatic, without clinical signs of coronary dysfunction. In contrast to patients who had the Ross operation, stress-induced perfusion defects and an attenuated CFR were documented. The prognostic implications of these findings and the clinical consequences are unclear; nevertheless, close clinical follow-up of ASO patients is mandatory.

Expression of glutamine:fructose-6-phosphate amidotransferase in human tissues: evidence for high variability and distinct regulation in diabetes.

Recent in vitro and in vivo studies suggested that the increased flux of glucose through the hexosamine biosynthetic pathway may contribute to glucose-induced insulin resistance and to the induction of the synthesis of growth factors. Because glutamine:fructose-6-phosphate amidotransferase (GFAT) catalyzes the first and rate-limiting step in the formation of hexosamine products, this enzyme is the key regulator in this pathway and is therefore possibly also involved in the alterations occurring in preclinical or manifest diabetic patients. To study the expression of GFAT in human tissues, we produced and characterized a peptic antiserum specifically recognizing GFAT protein and a riboprobe for the detection of GFAT mRNA. Immunohistochemical and nonradioactive in situ hybridization analysis revealed high levels of expression of GFAT protein and mRNA in adipocytes and skeletal muscle. Furthermore, a marked GFAT expression was found in vascular smooth muscle cells with unexpectedly high variability and lower levels in other cells, e.g., peripheral nerve sheath cells or endocrine-active cells, including the pancreatic islet cell. GFAT protein expression was below detection level in endothelium, osteocytes, lymphocytes, granulocytes, and in most quiescent fibroblasts. In renal tissue, GFAT was expressed in tubular epithelial cells, while glomerular cells remained essentially unstained. Renal sections obtained from patients with diabetic nephropathy showed significant GFAT expression in some glomerular epithelial and mesangial cells, indicating that GFAT expression may be induced by manifest diabetes. Our data indicate that GFAT is expressed in most tissues involved in the development of diabetic late complications. Furthermore, the results suggest that GFAT gene expression is highly regulated.

Myocardial blood flow and coronary flow reserve late after anatomical correction of transposition of the great arteries.

OBJECTIVES: Myocardial blood flow (MBF) in children late after arterial switch operation (ASO) was investigated quantitatively by positron emission tomography (PET). BACKGROUND: In children with transposition of the great arteries (TGA), ASO is widely accepted as the management of choice. The long-term patency of coronary arteries after surgical transfer to the neo-aorta, however, remains a concern. METHODS: Twenty-two normally developed, symptom-free children were investigated by PET with nitrogen-13 ammonia at rest and during adenosine vasodilation 10+/-1 years after ASO. A subgroup of 15 children (9+/-1 years; group A) had simple TGA and underwent ASO within 20 days after birth while 7 (13+/-3 years; group B) had complex TGA and underwent ASO and correction of associated anomalies later after birth. Ten young, healthy adults (26+/-6 years) served as the control group. RESULTS: Resting MBF was not different between groups. After correction for the rate-pressure product as an index of cardiac work, younger children of group A had significantly higher MBF at rest compared to healthy adults (102+/-29 vs. 77+/-6 ml/100 g/min; p = 0.012) while flow in group B was not different from the other groups (85+/-22 ml/100 g/min; p = NS). Hyperemic blood flows were significantly lower in both groups after ASO compared to normals (290+/-42 ml/100 g/min for group A, 240+/-28 for group B, 340+/-57 for normals; p < 0.01); thus, coronary flow reserve was significantly lower in both groups after ASO compared to healthy adults (3.0+/-0.6 for group A, 2.9+/-0.6 for group B, 4.6+/-0.9 for normals; p < 0.01). CONCLUSIONS: Blood flow measurements suggest decreased coronary reserve in the absence of ischemic symptoms in children late after arterial switch repair of TGA. The global impairment of stress flow dynamics may indicate altered vasoreactivity; however, the prognostic significance of these findings needs to be determined.

Cumulative site-directed charge-change replacements in bacteriophage T4 lysozyme suggest that long-range electrostatic interactions contribute little to protein stability.

Bacteriophage T4 lysozyme is a basic molecule with an isoelectric point above 9.0, and an excess of nine positive charges at neutral pH. It might be expected that it would be energetically costly to bring these out-of-balance charges from the extended, unfolded, form of the protein into the compact folded state. To determine the contribution of such long-range electrostatic interactions to the stability of the protein, five positively charged surface residues, Lys16, Arg119, Lys135, Lys147 and Arg154, were individually replaced with glutamic acid. Eight selected double, triple and quadruple mutants were also constructed so as to sequentially reduce the out-of-balance formal charge on the molecule from +9 to +1 units. Each of the five single variant proteins was crystallized and high-resolution X-ray analysis confirmed that each mutant structure was, in general, very similar to the wild-type. In the case of R154E, however, the Arg154 to Glu replacement caused a rearrangement in which Asp127 replaced Glu128 as the capping residue of a nearby alpha-helix. The thermal stabilities of all 13 variant proteins were found to be fairly similar, ranging from 0.5 kcal/mol more stable than wild-type to 1.7 kcal/mol less stable than wild-type. In the case of the five single charge-change variants, for which the structures were determined, the changes in stability can be rationalized in terms of changes in local interactions at the site of the replacement. There is no evidence that the reduction in the out-of-balance charge on the molecule increases the stability of the folded relative to the unfolded form, either at pH 2.8 or at pH 5.3. This indicates that long-range electrostatic interactions between the substituted amino acid residues and other charged groups on the surface of the molecule are weak or non-existent. Furthermore, the relative stabilities of the multiple charge replacement mutant proteins were found to be almost exactly equal to the sums of the relative stabilities of the constituent single mutant proteins. This also clearly indicates that the electrostatic interactions between the replaced charges are negligibly small. The activities of the charge-change mutant lysozymes, as measured by the rate of hydrolysis of cell wall suspensions, are essentially equal to that of the wild-type lysozyme, but on a lysoplate assay the mutant enzymes appear to have higher activity.(ABSTRACT TRUNCATED AT 400 WORDS)

Optimizing processing parameters for signal enhancement of oligonucleotide and protein arrays on ARChip Epoxy.

Signal enhancement of oligonucleotide and protein arrays on ARChip Epoxy was achieved by optimizing chip processing parameters. The parameters investigated were fabrication, blocking and guide dot concentration, probe concentration and modification, print buffer, humidity during arraying, slide agitation, spot volume and spotter compatibility. The optimum oligonucleotide concentration was 20 microM, while the optimum protein concentration was 0.05 mg/ml. Amino-modified oligonucleotides were best able to be bound to the resin's epoxy groups at pH 8, whereas thiol-modified oligonucleotides displayed an optimum coupling value of pH 7. So as to avoid background (BG) contamination of probes around bright guide dots, the concentration of fluorescent guide dots was set to 1 muM. The most suitable print buffers for oligonucleotide arrays using both piezo- and contact-printing systems proved to be 3 x SSC/1.5 M betaine and commercial ArrayLink. When 0.01% monochlortriazinyl-beta-cyclodextrin sodium salt (MCT) was added, the hybridization signal doubled in strength as compared to plain buffer. The optimum print buffer for proteins was 0.1 N phosphate buffer, pH 8/10% glycerine. The optimum humidity for arraying oligonucleotides was 60% and for proteins 40%. Initially agitating slides for 15 min was found just as effective as agitating slides over the total hybridization period (2.5 h), and this resulted in a three times stronger signal.

Cloning and expression of the Zymomonas mobilis pyruvate kinase gene in Escherichia coli.

The homotetrameric pyruvate kinases (PK) constitute a fine example of allosteric enzymes subjected to sophisticated regulatory mechanisms. We have cloned and sequenced the Zymomonas mobilis structural gene for the first prokaryotic dimeric PK, as an initial step toward understanding the peculiar properties of this enzyme. The deduced amino acid sequence of the pyk gene consists of 475 residues with a calculated molecular mass of 51.4kDa and exhibits up to 50% sequence identity with other PKs. Heterologous expression in Escherichia coli was not obtained from the native promoter, but only when the pyk gene was under the control of a strong inducible promoter when a ribosome-binding site was present upstream of the putative TTG start codon of the pyk gene. Kinetic characterization of PK in concentrated crude cell extracts showed that the enzyme is not activated by sugar phosphates or AMP but is slightly inhibited by ATP. Thus, PK of Z. mobilis is unique among the characterized prokaryotic PKs due to its high activity in the absence of any allosteric activator. Amino acid sequence alignments revealed that glutamate 381 may play a role in ineffective binding of the usual PK activator, fructose-1,6-bisphosphate.

Chloride binding by the AML1/Runx1 transcription factor studied by NMR.

It is known that the DNA binding Runt domain of the AML1/Runx1 transcription factor coordinates Cl(-) ions. In this paper we have determined Cl(-) binding affinities of AML1 by (35)Cl nuclear magnetic resonance (NMR) linewidth analysis. The Runt domain binds Cl(-) with a dissociation constant (K(d,Cl)) of 34 mM. If CBFbeta is added to form a 1:1 complex, the K(d,Cl) value increases to 56 mM. Homology modeling suggests that a high occupancy Cl(-) binding site overlaps with the DNA binding surface. NMR data show that DNA displaces this Cl(-) ion. Possible biological roles of Cl(-) binding are discussed based on these findings.

Crystallization and preliminary crystallographic studies of recombinant dimerization cofactor of transcription factor HNF1/pterin-4 alpha-carbinolamine dehydratase from liver.

The bi-functional protein dimerization cofactor of HNF1 (DCoH)/pterin-4 alpha-carbinolamine dehydratase (PCD) is found in liver cell nuclei bound to the transcription factor hepatocyte nuclear factor 1 (HNF1) as well as in the cytoplasm acting as an enzyme involved in the phenylalanine hydroxylation system. Deficiency of DCoH/PCD activity in liver causes an atypical hyperphenylalaninemia and deficiency in human epidermis is related to the depigmentation disorder vitiligo. DCoH/PCD from rat liver, which is identical to the human protein, was expressed in E. coli, purified to homogeneity and crystallized. The crystals belong to the trigonal space group P3(1)21 (or P3(2)21) with unit cell dimensions of a = b = 106.2 A, c = 197.1 A. Native crystals diffract to a resolution of 2.5 A.

Development of the pulmonary circulation in ventricular septal defect: a quantitative structural study.

The application of quantitative morphometric techniques to evaluation of the lungs of nine children who died with a ventricular septal defect between the ages of 3 months and 4 years showed that the presence of pulmonary hypertension interferes with the growth and development of the pulmonary circulation. In all cases the preacinar arteries were of normal size and not dilated, and arterial size and number within the acinus were reduced and similar to those seen in the normal child at birth. Arterial and venous muscularity was increased as judged by an increase in wall thickness and by the presence of muscle in smaller and more peripheral arteries than is normal. Elevation of resistance was associated with failure of the intraacinar pulmonary circulation to develop normally rather than to obliterative pulmonary vascular disease. In view of the rapidity with which impairment of growth and elevation of resistance can develop, closure of a large defect is recommended before age 2 years.

Compression of intrapulmonary bronchi by abnormally branching pulmonary arteries associated with absent pulmonary valves.

In 3 patients with absent pulmonary valve syndrome and absent ductus arteriosus, the lungs were injected and analyzed postmortem using morphometric techniques. Two patients had tetralogy of Fallot and 1 had D-transposition of the great arteries, the latter being the first autopsy-proved case of absent pulmonary valve with transposition. In addition to the expected dilatation of the central pulmonary arteries and compression of the mainstem bronchi, postmortem pulmonary arteriography revealed a bizarre pattern of hilar branching. Instead of single segmental arteries, tufts of arteries arose which entwined and compressed the intrapulmonary bronchi. In all 3 patients the histologic structure of the pulmonary arteries was abnormal. The elastic lamina of the media of the right and left pulmonary arteries were increased in number outside the lung, but were decreased within the lung. At both sites, the elastic laminae were thickened and fragmented. In the 2 ventilator-dependent patients, there was slight medial hypertrophy and extension of muscle into normally nonmuscular arteries. In 1 of the 2 cases in which the number of bronchial generations was counted, they were decreased, and in the 1 case in which bronchial count was unknown, alveolar multiplication was severely impaired. Therefore, our data may explain why, in some patients with absent pulmonary valve syndrome, relief of compression of the mainstem bronchi alone does not appreciably alleviate or reverse severe respiratory disease.

Early activation and proliferation of T cells in simian immunodeficiency virus-infected rhesus monkeys.

To longitudinally determine T cell activation and turnover in early simian immunodeficiency virus (SIV) infection of macaques, immunological and virological parameters were monitored in 10 SIV-infected animals starting before infection until 40 weeks postinfection (wpi). Lymphocyte subsets in blood and lymph nodes (LNs) were characterized by three-color flow cytometry for expression of markers of activation, proliferation, and differentiation. As early as 1 wpi, CD69 expression was upregulated both on CD4+ and CD8+ T cells, indicative of an early activation of these cells. Whereas this activation led to increased proliferation, determined by expression of Ki-67, and absolute numbers of CD8+ T cells, CD4+ T cells showed a decreased expression of Ki-67 and reduced counts in blood at 2 wpi. Later, the percentage of Ki-67-expressing CD4+ T cells in blood and LNs increased again above preinfection levels in most animals but remained low in two monkeys progressing to AIDS. These findings suggest that T cells are activated after SIV infection, leading to increased T cell proliferation already in the early asymptomatic phase. In addition, we found a correlation between the capacity to regenerate CD4+ T cells by peripheral proliferation and the disease course. Moreover, our data indicate that the increased peripheral T cell proliferation during immunodeficiency virus infection is probably not caused by the effort of the immune system to maintain T cell homeostasis but may be a reflection of the ongoing immune response against the virus.

Autosomal recessive lateralization and midline defects: blastogenesis recessive 1.

In this report, we present 2 sibships in which midline and lateralization anomalies are demonstrated. Because midline and lateralization processes are early embryological events, we suggest calling this sequence Blastogenesis Recessive 1 (BGR1). Since connexin 43 gene mutations were demonstrated in some polyasplenia patients and according to connexin 43 temporospatial tissue expression, we hypothesize that this gene could bear mutations responsible for the anomalies reported in these two sibships.

Divergence in distribution and prognostic significance of major basement components in laryngeal carcinomas.

In previous studies, we demonstrated a loss of major basement membrane (BM) components in laryngeal squamous cell carcinomas and provided initial evidence that this was of potential prognostic significance. In our current study, we extended the panel of BM antibodies and enlarged our study group in order to perform a multivariate statistical analysis. We analyzed 26 laryngeal squamous cell carcinomas immunohistochemically for the distribution of the BM-components collagen IV, collagen VII, laminin-1, laminin-5, perlecan and fibronectin. The resulting data were correlated with clinical prognostic factors and statistical correlation coefficients were determined for independent uni- and multivariate analysis. All carcinomas analyzed revealed defects of the peritumoral BM with more extensive loss of collagen VII than collagen IV, laminin-1, perlecan and fibronectin. Laminin-5 in contrast was present even in poorly differentiated tumors showing an enhanced intracytoplasmatic staining in the tumor cells. Furthermore, our statistical analysis did not show independent prognostic significance of any of the BM-components. Our observations indicate a divergence between the loss of several major BM-components (collagens IV, VII, laminin-1, perlecan) and an enhanced deposition of laminin-5. This suggests a severely altered cell-matrix interaction, since laminin-5 links the collagen VII-containing anchoring fibrils to cell receptors of the integrin type.