Immunoreactivity of a surface wall fraction produced by spherules of Coccidioides immitis.

The membranous spherule outer wall (SOW) isolated from liquid cultures of Coccidioides immitis has been shown to elicit reactivity with human anti-Coccidioides antibody by immunofluorescence and the immunodiffusion-tube precipitin assay. The serologically reactive components were extracted from SOW with the nonionic detergent N-octyl-beta-D-glucopyranoside (OG). The OG-soluble fraction of SOW was shown to be reactive with immunoglobulin G in 25 serum samples from coccidioidomycosis patients by an enzyme-linked immunosorbent assay. The isolated SOW and OG-soluble fraction of SOW were also demonstrated to be capable of eliciting lymphocyte blastogenesis. The antigenic and protein compositions of the OG-soluble fraction were examined by two-dimensional immunoelectrophoresis and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), respectively. Two antigens which were extracted from SOW were identified as antigens 2 and CS on the basis of the coccidioidin-anticoccidioidin reference system. The latter was isolated earlier and shown to correspond to a molecular mass (Mr) of 19 X 10(3) by SDS-PAGE under reducing conditions. This same electrophoresis band was shown to be reactive with sera from coccidioidomycosis patients by immunoblot analysis. One other SDS-PAGE component of the OG-soluble fraction of SOW with an Mr of 66 X 10(3) was shown to be reactive with sera from patients by immunoblot analysis. The SOW of C. immitis represents an important reservoir of immunoreactive wall components which has not previously been reported.     

Histone deposition protein Asf1 maintains DNA replisome integrity and interacts with replication factor C

Chromatin assembly and DNA replication are temporally coupled, and DNA replication in the absence of histone synthesis causes inviability. Here we demonstrate that chromatin assembly factor Asf1 also affects DNA replication. In budding yeast cells lacking Asf1, the amounts of several DNA replication proteins, including replication factor C (RFC), proliferating cell nuclear antigen (PCNA), and DNA polymerase epsilon (Pol epsilon), are reduced at stalled replication forks. In contrast, DNA polymerase alpha (Pol alpha) accumulates to higher than normal levels at stalled forks in asf1Delta cells. Using purified, recombinant proteins, we demonstrate that RFC directly binds Asf1 and can recruit Asf1 to DNA molecules in vitro. We conclude that histone chaperone protein Asf1 maintains a subset of replication elongation factors at stalled replication forks and directly interacts with the replication machinery.     

Genetic instability in superficial bladder cancer and adjacent mucosa: an interphase cytogenetic study

A systematic analysis of both tumors and the surrounding urothelium to help identify what lies behind the mechanism of multifocal tumor development has not yet been performed. In this study we investigated chromosome 1, 7, 9, and 17 aneusomy in 25 superficial papillary carcinomas and in 51 tissue samples taken from sites of macroscopically uninvolved urothelium surrounding the tumors, using the fluorescence in situ hybridization method. Our data demonstrated a close genetic relationship between all examined tumors and normal-appearing mucosa. Numeric aberrations of chromosomes 1, 7, 9, and 17 were found to exhibit similar patterns in all analyzed specimens, although with different frequencies.     

Erratum: Neurofibromatosis type 1 (NF1): Identification of eight unreported mutations in NF1 gene in Italian patients

In the original version of this article, the title was incorrect. Please find the correct title given here. The publisher deeply regrets this error. The original article to which this Erratum refers was published in Human Mutation 22:179–180 Human Mutation(2003) 22(2) 179–180     

Mechanisms of the ATP potentiation of hyposmotic taurine release in Swiss 3T3 fibroblasts

Reducing osmolarity by 35% increased ³H-taurine efflux from Swiss 3T3 fibroblasts from 0.5% to a peak of 5.7%. The presence of ATP (10–100 µM; EC₅₀ 1.5 µM) increased taurine efflux up to 10%, and decreased the set point for hyposmotically stimulated taurine release (HTR). ATP potentiation was mimicked by UTP, reduced by addition of suramin and pyridoxal phosphate-6-azophenyl-2,4-disulphonic acid (PPADS) and unaffected by ADP, ,-methylene-ATP (,-ATP) or 2-methylthio-ATP (Me-ATP), suggesting its mediation by purinergic P2Y₂ and P2Y₄ metabotropic receptors. Under isosmotic conditions ATP increased the cytosolic [Ca²⁺] ([Ca²⁺]_i) markedly, but did not increase taurine release. HTR was independent of external Ca²⁺ but was reduced (by 56–59%) by BAPTA-AM, thapsigargin-induced depletion of intracellular Ca²⁺ stores, or phospholipase C (PLC) inhibition. Blockade of calmodulin (CaM) or calmodulin kinase II (CaMKII) reduced HTR by 54% and 76%, respectively. The ATP-mediated potentiation was prevented fully by all these treatments. HTR was reduced by 30–50% by blockers of protein tyrosine kinases (AG18), phosphoinositide 3-kinase (PI3K) (wortmannin), p21rho (toxin B), p21rho-kinase (Y27632) and the stress-activated kinase p38 (PD169316). ATP-mediated potentiation was reduced similarly by these blockers. Simultaneous inhibition of PI3K and CaMKII abolished HTR. Altogether, these results suggest a modulatory effect of ATP, probably exerted by a potentiation of the Ca²⁺-dependent fraction of HTR. This fraction has as signalling elements a PLC-dependent [Ca²⁺]_i increase, resulting from Ca²⁺ released from thapsigargin-sensitive internal stores, followed by activation of CaM/CaMKII reactions. The Ca²⁺/ATP effect operates only when the Ca²⁺-independent, tyrosine kinase-mediated pathway is already activated. Suggested elements of cross-talk between the two pathways are PLC, PI3K and CaMKII.     

Large deletions of the MECP2 gene detected by gene dosage analysis in patients with Rett syndrome

The original article to which this Erratum refers was published in Human Mutation 23:234–244 Human Mutation(2004) 23(3) 234–244     

Merlin expression in secretory meningiomas: evidence of an NF2-independent pathogenesis? Immunohistochemical study.

One of the most common chromosomal regions implicated in the meningiomas tumorigenesis is 22q12 where the neurofibromatosis 2 (NF2) gene resides. The NF2 tumor-suppressor gene encodes for the merlin/schwannomin protein, which is responsible for the inherited disease neurofibromatosis 2. NF2 gene mutations predominantly occur in transitional and fibroblastic meningiomas, whereas the meningothelial variant is less affected. Secretory meningioma is an infrequent meningioma subtype. Its most typical morphologic feature is the presence of intracytoplasmic or extracytoplasmic round hyaline, eosinophilic, and periodic acid Shiff-positive bodies in a lesion frequently otherwise classifiable as meningothelial meningioma. This study reviews the immunohistochemical merlin expression in 14 consecutive secretory meningiomas. Our purpose was to investigate if secretory meningiomas, analogous to meningothelial meningiomas, follow a molecular route of pathogenesis independent of the neurorofibromatosis 2 gene-associated pathway. All meningiomas showed positive immunocoloration involving the majority of the hyaline inclusions and secretory cells; in 12 (86%) meningiomas, a positive immunoreaction was also documented in nonsecretory tumoral cells. Our results may indicate a molecular, besides morphologic, similarity between secretory and meningothelial meningiomas: the almost constant merlin immunohistochemical expression in our series gives evidence for a possible NF2 gene-independent pathogenesis in secretory meningiomas.     

Trisomy 8 in myelodysplasia and acute leukemia is constitutional in 15-20% of cases.

The trisomy 8 found in malignancies may derive from a constitutional trisomy 8 mosaicism (CT8M), and in these cases the trisomy itself may be regarded as the first mutation in a multistep carcinogenetic process. To assess the frequency of CT8M in hematological dysplastic and neoplastic disorders with trisomy 8, an informative sample of 14 patients was collected. The data ascertained included chromosome analyses of fibroblast cultures and of PHA-stimulated blood cultures in patients with normal blood differential count, as well as possible CT8M clinical signs. One patient showed trisomy 8 in all cell types analyzed and undoubtedly has a CT8M; a second patient consistently showed trisomy 8 in PHA-stimulated blood cultures when no immature myeloid cells were present in blood and should be considered as having CT8M; a third patient, with Philadelphia-positive chronic myelocytic leukemia, was more difficult to interpret, but the possibility that she had CT8M is likely. A few clinical signs of CT8M were also present in these three patients. Our data indicate that the frequency of CT8M in hematological dysplastic and neoplastic disorders with trisomy 8 is approximately 15-20%.