Cloning, Expression, and Sequencing of a Cell Surface Antigen Containing a Leucine-Rich Repeat Motif from Bacteroides forsythus ATCC 43037

Bacteroides forsythus is a recently recognized human periodontopathogen associated with advanced, as well as recurrent, periodontitis. However, very little is known about the mechanism of pathogenesis of this organism. The present study was undertaken to identify the surface molecules of this bacterium that may play roles in its adherence to oral tissues or triggering of a host immune response(s). The gene (bspA) encoding a cell surface-associated protein of B. forsythus with an apparent molecular mass of 98 kDa was isolated by immunoscreening of a B. forsythus gene library constructed in a lambda ZAP II vector. The encoded 98-kDa protein (BspA) contains 14 complete repeats of 23 amino acid residues that show partial homology to leucine-rich repeat motifs. A recombinant protein containing the repeat region was expressed in Escherichia coli, purified, and utilized for antibody production, as well as in vitro binding studies. The purified recombinant protein bound strongly to fibronectin and fibrinogen in a dose-dependent manner and further inhibited the binding of B. forsythus cells to these extracellular matrix (ECM) components. In addition, adult patients with B. forsythus-associated periodontitis expressed specific antibodies against the BspA protein. We report here the cloning and expression of an immunogenic cell surface-associated protein (BspA) of B. forsythus and speculate that it mediates the binding of bacteria to ECM components and clotting factors (fibronectin and fibrinogen, respectively), which may be important in the colonization of the oral cavity by this bacterium and is also a target for the host immune response.     

(ATT) trinucleotide repeats in the antithrombin gene and their use in determining the origin of repeated mutations

Two (ATT) trinucleotide repeat polymorphisms have been identified in the tails of Alu repeat elements in intron 5 of the antithrombin gene. The frequency and distribution of allele sizes for the Alu 5 and Alu 8 tail polymorphisms have been defined in a sample Caucasian population. The Alu 5 polymorphism has two alleles while that of Alu 8 has 10 alleles with a heterozygosity of 0.83. These polymorphisms have been used in combination with four previously described polymorphisms within the antithrombin gene to construct antithrombin gene haplotypes in the sample Caucasian population. Twenty-two different haplotypes were observed, with the Alu 8 polymorphism being particularly useful in subdividing the core haplotype based on the previously identified polymorphisms. The haplotype data were used to investigate the origin of repeat mutations within the antithrombin locus. We compared the haplotypes associated the mutant antithrombin genes in five families with the mutation 2759C→T (L99F) and five families with the mutation 5381C→T (R129Stop). The mutation 2759C→T (L99F), which occurs within a non-CpG dinucleotide, was carried on a gene associated with an identical haplotype in each of the five families. The mutation 5381C→T (R129Stop), a single base substitution within a CpG dinucleotide, was associated with at least two different haplotypes. The findings suggest a founder effect in the five families sharing the 2759C→T (L99F) and at least two independent origins for the CpG dinucleotide mutation 5381C→T (Rl29Stop). © 1994 Wiley-Liss, Inc.     

The developmentally regulated neural crest-associated glycotope HNK-1 predicts metastasis in cutaneous malignant melanoma

Aberrant glycosylation is a common feature of metastatic sub-clones of malignant tumours and in uveal melanoma in particular, the HNK-1 glycotope has been positively correlated with poor prognosis. So far, no such correlation has been investigated in cutaneous melanoma. In order to do so, HNK-1 expression was evaluated immunohistochemically in 100 primary cutaneous melanomas and correlated with metastasis after up to 10-years' follow-up. Furthermore, HNK-1 expression was analysed in metastatic deposits (19 distant cutaneous metastases and six sentinel lymph node metastases), as well as in benign nevi. Kaplan-Meier analysis revealed a positive association between HNK-1 expression and metastasis (p < 0.005) and multivariate Cox regression analysis adjusted for the standard prognostic markers ulceration and vertical tumour thickness confirmed HNK-1 expression as an independent prognostic marker. HNK-1 expression was preserved in 42% of the distant cutaneous metastases, but metastatic cells in lymph nodes were devoid of HNK-1 immunoreactivity. None of the benign pigmented lesions exhibited HNK-1 immunoreactivity. Expression of the HNK-1 glycotope in cutaneous malignant melanoma is an independent prognostic marker of metastasis. Differential HNK-1 expression at the metastatic sites implies that its expression is modulated by the surrounding environment. As HNK-1 is also transiently expressed during migration of melanocyte precursor cells derived from the neural crest, recapitulation of this transient expression might occur during metastatic spread of cutaneous malignant melanoma.     

Die Bedeutung des Natriums für den renal-tubulären Calcium-Transport bei Rana ridibunda

Zusammenfassung In Anlehnung an frühere Experimente zur Na⁺-Abhängigkeit der renal-tubulären Transporte von Glucose, PAH und Urea wurden Versuche über die Bedeutung des Na⁺ für die Ca⁺⁺-Reabsorption an der isolierten künstlich perfundierten Niere von Rana ridibunda durchgeführt.Erniedrigung des Na⁺-Angebotes von 76,5 auf 20,0 mMol/l vermindert sowohl die Menge reabsorbierten Natriums als auch die des reabsorbierten Calciums, letztere allerdings weniger als erstere. Der Quotient reabsorbiertes Na⁺/reabsorbiertes Ca⁺⁺ verschiebt sich zugunsten des Ca⁺⁺.Fehlendes Na⁺-Angebot auf der Lumen- oder der Blutseite der Tubulusepithelien hat keinen sicheren Einfluß auf die Menge reabsorbierten Calciums, dagegen sinkt die Reabsorptionsrate von Ca⁺⁺ auf weniger als 10% des filtrierten, wenn Na⁺ weder auf der Lumen- noch auf der Blutseite angeboten wird.Die Ca⁺⁺-Reabsorptionsrate ist gesteigert bei Angebot von Ca⁺⁺ ausschließlich auf der Lumenseite, vermutlich wegen des unter physiologischen Bedingungen nicht existenten Konzentrationsgradienten, der blutwärts gerichtet ist. Bei Angebot von Ca⁺⁺ nur von der Blutseite erscheint kein Ca⁺⁺ im Harn: die Tubulusepithelien sind für Ca⁺⁺ im Richtungssinne der Sekretion undurchlässig.Durch die Hemmstoffe der Na⁺-Reabsorption Convallatoxin und Furosemid wird auch die Reabsorption von Ca⁺⁺ — sogar relativ stärker als die von Na⁺ — gehemmt. Da in den Versuchen mit Hemmstoffen das Na⁺-Angebot (extracelluläre Konzentration) konstant bleibt, wird geschlossen, daß die Ca⁺⁺-Reabsorption mit dem Na⁺ über dessen Transport gekoppelt ist.Im Prinzip folgt die Verknüpfung der Ca⁺⁺-Reabsorption mit dem Na⁺ den gleichen Gesetzmäßigkeiten wie die Verknüpfung des Na⁺ mit den Transporten von Glucose, PAH und Harnstoff. Damit ist ein weiteres Indiz für die Vermutung gewonnen, daß die Na⁺-Abhängigkeit ein zellphysiologisches Prinzip von weiter Gültigkeit ist.

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Summary With regard to earlier experiments about the influence of Na⁺ on the tubular transport of glucose, PAH and urea, the effect of Na⁺ on the reabsorption of Ca⁺⁺ in the isolated artificially perfused kidney of Rana ridibunda was studied.Lowering of the Na⁺-concentration from 76.5–20.0 mMol/l results in a decrease of the reabsorbed amounts of both Na⁺ and Ca⁺⁺, the decrease being less marked in the latter. The quotient Na⁺ reabsorbed/Ca⁺⁺ reabsorbed changes in favour of the calcium.Lack of Na⁺ on the luminal or the contraluminal side of the tubular wall does not significantly influence the amount of Ca⁺⁺ being reabsorbed. If, however, Na⁺ is missing on both sides the rate of reabsorption of Ca⁺⁺ falls below 10% of the filtered amount.The rate of Ca⁺⁺-reabsorption increases if Ca⁺⁺ is offered on the luminal side only. Presumably this increase is due to a concentration gradient for Ca⁺⁺ which does not exist under physiological conditions. If Ca⁺⁺ is offered on the contraluminal side only, no Ca⁺⁺ appears in the urine. Apparently the tubular epithelium is impermeable for Ca⁺⁺ from the blood to the tubular lumen.The decrease of the reabsorption of Ca⁺⁺ brought about by substances inhibiting the reabsorption of Na⁺ such as Convallatoxin and Furosemid is relatively more marked than the concomitant inhibition of the reabsorption of Na⁺.Since in the experiments with the above mentioned inhibitors the extracellular Na⁺-concentration remains constant, it is concluded that the Ca⁺⁺-reabsorption is coupled with the transport of Na⁺.In principle the Na⁺-dependence of the Ca⁺⁺-reabsorption follows the same pattern as the Na⁺-dependence of the transports of glucose, PAH and urea, thus supporting our contention that the Na⁺-dependence of active transports is a physiological mechanism of general importance.

     

Epidermal growth factor receptor expression correlates with poor survival in gastric adenocarcinoma from Mexican patients: a multivariate analysis using a standardized immunohistochemical detection system.

The aim of the study was to determine epidermal growth factor receptor (EGFR) expression in gastric adenocarcinoma by standardized immunohistochemistry and to correlate EGFR expression with clinical features and patient survival. EGFR expression was investigated in paraffin sections of resection specimens of 89 gastric carcinomas from Mexican Mestizo patients using standardized immunohistochemistry with antigen retrieval (Dako EGFRpharmDx assay detection system). Membrane staining of EGFR was evaluated in the neoplastic cells and graded using a semiquantitative score (0-3+). Of the 89 carcinomas examined, staining of neoplastic cells was weak in 17 (19.1%, score 1+), moderate in 16 (18.0%, score 2+), and strong in nine cases (10.1%, score 3+). EGFR reactivity was heterogeneous, frequently showing completely negative up to 3+ positive areas within an individual tumor. EGFR reactivity score correlated with distant metastases (P=0.002) and clinical stage (P=0.033). EGFR score 0/1+ was significantly associated with an increase in patient survival when compared to score 2+/3+ (P=0.0006). In a multivariate analysis, EGFR positive cells in muscularis or subserosa (P=0.004), distant metastases (P=0.016) and residual disease (P=0.039) were significantly correlated with decreased survival. The prognosis was associated with the EGFR reactivity score (P=0.003), distant metastases (P=0.0001) and residual disease (P=0.012) in a univariate analysis. EGFR reactivity in neoplastic cells is an independent prognostic factor in gastric adenocarcinoma. The relevance of the heterogeneity in EGFR expression with regard to tumor progression, metastasis and anti-EGFR therapy needs to be studied.     

Monitoring of basophil activation using CD63 and CCR3 in allergy to muscle relaxant drugs

Allergic or pseudoallergic reactions that occur during anesthesia have been increasing for the last few years. To date, the diagnosis of allergy to muscle relaxants remains difficult. In this respect, we developed a flow cytometric method for the study of drug-induced basophil degranulation using CD63 and CCR3. Fifty patients who developed clinical features evocative of allergic reactions immediately after induction of anesthesia were included and classified into two groups. Group 1 (n = 39) comprised true allergic patients, who developed typical signs of shock associated to positive skin testing. Group 2 (n = 11) consisted of patients whose clinical history was not typical and skin testing was negative or nonconclusive. Seventeen control subjects were also studied in this report. We compared data from flow cytometry to skin tests, specific IgE, and histamine release results. Flow cytometry showed a sensitivity of 54%, while that of specific IgE was similar, at 62%. Interestingly, when considering the sensitivity of IgE + CD63 for diagnosis, we reached a sensitivity value of 80%. Of 15 negative results for specific IgE, we found 7 positive CD63 tests, while histamine release gave positive results in only 2 cases. Furthermore, the CD63 protocol showed good specificity (100%). We conclude that our flow cytometry protocol is a promising tool in allergy diagnosis since it is specific and complementary to specific IgE detection.     

Bone marrow mast cell reaction in preleukaemic myelodysplasia and in aplastic anaemia

Summary The relationship of bone marrow mast cell counts to prognosis was investigated in 48 patients with preleukaemic myelodysplasia, in 59 patients with aplastic anemia and in a DMBA induced myelodysplasia/leukaemia rat model. In patients with myelodysplasia terminating in overt leukaemia the number of mast cells per square millimeter was not correlated to duration of the preleukaemic course. Leukaemia development probabilities of patients at risk were not different for low and elevated mast cell counts. In aplastic anaemia, however, a lower bone marrow mast cell count was related to a higher survival probability and longer survival time. In the animal model no significant differences could be found between myelodysplastic, leukaemic, and control animals.     

Bam32 links the B cell receptor to ERK and JNK and mediates B cell proliferation but not survival

Bam32 is an adaptor protein recruited to the plasma membrane upon B cell receptor (BCR) crosslinking in a phosphoinositol 3-kinase (PI3K)-dependent manner; however, its physiologic function is unclear. To determine its physiologic function, we produced Bam32-deficient mice. Bam32(-/-) B cells develop normally but have impaired T-independent antibody responses in vivo and diminished responses to BCR crosslinking in vitro. Biochemical analysis revealed that Bam32 acts in a novel pathway leading from the BCR to MAPK/ERK Kinases (MEK1/2), MAPK/ERK Kinase Kinase-1 (MEKK1), extracellular signal-regulated kinase (ERK), and c-jun NH2-terminal kinase (JNK), but not p38 mitogen-activated protein kinase (p38). This pathway appears to be initiated by hematopoietic progenitor kinase-1 (HPK1), which interacts directly with Bam32, and differs from all previously characterized BCR signaling pathways in that it is required for normal BCR-mediated proliferation but not for B cell survival.     

Novel OCTN2 mutations: No genotype–phenotype correlations: Early carnitine therapy prevents cardiomyopathy

Primary systemic carnitine deficiency or carnitine uptake defect (OMIM 212140) is a potentially lethal, autosomal recessive disorder characterized by progressive infantile‐onset cardiomyopathy, weakness, and recurrent hypoglycemic hypoketotic encephalopathy, which is highly responsive to L ‐carnitine therapy. Molecular analysis of the SLC22A5 (OCTN2) gene, encoding the high‐affinity carnitine transporter, was done in 11 affected individuals by direct nucleotide sequencing of polymerase chain reaction products from all 10 exons. Carnitine uptake (at Km of 5 μM) in cultured skin fibroblasts ranged from 1% to 20% of normal controls. Eleven mutations (delF23, N32S, and one 11‐bp duplication in exon 1; R169W in exon 3; a donor splice mutation [IVS3+1 G > A] in intron 3; frameshift mutations in exons 5 and 6; Y401X in exon 7; T440M, T468R and S470F in exon 8) are described. There was no correlation between residual uptake and severity of clinical presentation, suggesting that the wide phenotypic variability is likely related to exogenous stressors exacerbating carnitine deficiency. Most importantly, strict compliance with carnitine from birth appears to prevent the phenotype. © 2002 Wiley‐Liss, Inc.