Study of X-ray topography using the super-Borrmann effect.

X-ray topography exerting the super-Borrmann effect has been performed using synchrotron radiation to display dislocation images with a high-speed and high-resolution CMOS camera. Forward-transmitted X-rays are positively employed instead of reflected X-rays to reveal dislocations in relatively thick crystals by simultaneously exciting a pair of adjacent {111} planes owing to the super-Borrmann effect. Before the experiment, minimum values of the attenuation coefficients AminP for σ and π polarizations of the incident X-rays in the three-beam case are calculated. Results demonstrate that AminP for both polarizations are almost 20 times larger than those in the two-beam (usual Borrmann effect) case. The transmitted X-rays can be used to confirm the efficacy of taking topographs under the super-Borrmann conditions, as well as under multiple-diffraction conditions. Furthermore, super-Borrmann topographs can be considered for relatively thick crystals, where a conventional Lang X-ray topography technique is difficult to apply.

Expression of Heparin-Binding EGF-Like Growth Factor (HB-EGF) in Bovine Endometrium: Effects of HB-EGF and Interferon-τ on Prostaglandin Production.

Heparin-binding EGF-like growth factor (HB-EGF) regulates several cell functions by binding to its membrane receptor (ErbB1 and ErbB4). Experimental evidences suggest that HB-EGF, prostaglandins (PGs) and interferon-τ (IFN-τ) regulate uterine function for pregnancy establishment in ruminants. In this study, the mRNA expressions of HB-EGF, ErbB1 and ErbB4 in bovine endometrium and the effects of HB-EGF and IFN-τ on PGE2 and PGF2-α production by endometrial cells were investigated. RT-PCR analysis revealed that HB-EGF mRNA was greater at the mid-luteal stage than at the early and regressed luteal stages (p < 0.05). ErbB1 mRNA expression was greater at the mid- and late luteal stages than at the other luteal stages (p < 0.05). IFN-τ increased the expression of HB-EGF, ErbB1 and ErbB4 mRNA in epithelial cells (p < 0.05). HB-EGF did not affect PGF2-α or PGE2 production by bovine endometrial epithelial cells, but increased PGF2-α and PGE2 production by bovine endometrial stromal cells (p < 0.05). IFN-τ significantly decreased HB-EGF-stimulated PGF2-α (p < 0.05), but not PGE2 (p > 0.05) production by stromal cells. These results indicate that HB-EGF and its receptors expression changed in bovine endometrium throughout the oestrous cycle. IFN-τ increased their expression in cultured endometrial cells. HB-EGF and IFN-τ have the ability to regulate PGs production by stromal cells and therefore may play a role in the local regulation of uterine function at the time of implantation in cattle.

Adipose tissue hypoxia induces inflammatory M1 polarity of macrophages in an HIF-1α-dependent and HIF-1α-independent manner in obese mice.

AIMS/HYPOTHESIS: As obesity progresses, adipose tissue exhibits a hypoxic and inflammatory phenotype characterised by the infiltration of adipose tissue macrophages (ATMs). In this study, we examined how adipose tissue hypoxia is involved in the induction of the inflammatory M1 and anti-inflammatory M2 polarities of ATMs. METHODS: The hypoxic characteristics of ATMs were evaluated using flow cytometry after the injection of pimonidazole, a hypoxia probe, in normal-chow-fed or high-fat-fed mice. The expression of hypoxia-related and inflammation-related genes was then examined in M1/M2 ATMs and cultured macrophages. RESULTS: Pimonidazole uptake was greater in M1 ATMs than in M2 ATMs. This uptake was paralleled by the levels of inflammatory cytokines, such as TNF-α, IL-6 and IL-1ß. The expression level of hypoxia-related genes, as well as inflammation-related genes, was also higher in M1 ATMs than in M2 ATMs. The expression of Il6, Il1ß and Nos2 in cultured macrophages was increased by exposure to hypoxia in vitro but was markedly decreased by the gene deletion of Hif1a. In contrast, the expression of Tnf, another inflammatory cytokine gene, was neither increased by exposure to hypoxia nor affected by Hif1a deficiency. These results suggest that hypoxia induces the inflammatory phenotypes of macrophages via Hif1a-dependent and -independent mechanisms. On the other hand, the expression of inflammatory genes in cultured M2 macrophages treated with IL-4 responded poorly to hypoxia. CONCLUSIONS/INTERPRETATION: Adipose tissue hypoxia induces an inflammatory phenotype via Hif1a-dependent and Hif1a-independent mechanisms in M1 ATMs but not in M2 ATMs.

Mixed-lineage-leukemia (MLL) fusion protein collaborates with Ras to induce acute leukemia through aberrant Hox expression and Raf activation.

Mixed-lineage-leukemia (MLL) fusion oncogenes are closely involved in infant acute leukemia, which is frequently accompanied by mutations or overexpression of FMS-like receptor tyrosine kinase 3 (FLT3). Earlier studies have shown that MLL fusion proteins induced acute leukemia together with another mutation, such as an FLT3 mutant, in mouse models. However, little has hitherto been elucidated regarding the molecular mechanism of the cooperativity in leukemogenesis. Using murine model systems of the MLL-fusion-mediated leukemogenesis leading to oncogenic transformation in vitro and acute leukemia in vivo, this study characterized the molecular network in the cooperative leukemogenesis. This research revealed that MLL fusion proteins cooperated with activation of Ras in vivo, which was substitutable for Raf in vitro, synergistically, but not with activation of signal transducer and activator of transcription 5 (STAT5), to induce acute leukemia in vivo as well as oncogenic transformation in vitro. Furthermore, Hoxa9, one of the MLL-targeted critical molecules, and activation of Ras in vivo, which was replaceable with Raf in vitro, were identified as fundamental components sufficient for mimicking MLL-fusion-mediated leukemogenesis. These findings suggest that the molecular crosstalk between aberrant expression of Hox molecule(s) and activated Raf may have a key role in the MLL-fusion-mediated-leukemogenesis, and may thus help develop the novel molecularly targeted therapy against MLL-related leukemia.

Usefulness of endoscopic ultrasonography (EUS) in diagnosing esophageal infiltration of thyroid cancer.

Pre-operative evaluation of esophageal infiltration is sometimes difficult in patients with advanced thyroid cancer even with recent imaging modalities. We evaluated the accuracy of endoscopic ultrasonography (EUS) in diagnosing esophageal infiltration of thyroid cancer. Twenty-nine patients with advanced thyroid cancer underwent EUS and other imaging examinations before surgery. The diagnostic accuracy of EUS was compared with that of magnetic resonance imaging (MRI) and esophagography based on pathologic findings in 27 of the 29 cases. EUS clearly demonstrated the 5-layer structure of the esophageal wall. EUS detected cancer invasion into the muscularis propria of the esophagus correctly in 8 of 10 patients diagnosed pathologically with muscular infiltration. EUS was significantly more accurate than MRI and esophagography (88.9% vs 63.0% and 66.7%, respectively). The specificity of EUS was also significantly better than the specificities of MRI or esophagography (94.1% vs 58.8% and 64.7%, respectively). The sensitivity, positive predictive value and negative predictive value of EUS tended to be better than those of MRI and esophagography. EUS is useful in evaluating the esophageal infiltration of thyroid cancer. This method has the further advantage of detecting the exact depth of cancer invasion into the esophageal wall.

Essential role of Stat5 for IL-5-dependent IgH switch recombination in mouse B cells.

IL-5 stimulation of CD38-activated murine splenic B cells induces mu-gamma1 CSR at the DNA level leading to a high level of IgG1 production. Further addition of IL-4 in the system enhances IL-5-dependent mu-gamma1 CSR. Although some of the postreceptor signaling events initiated by IL-5 in activated B cells have been characterized, the involvement of Stat in IL-5 signaling has not been thoroughly evaluated. In this study, we examined the activation of Stat5 and activation-induced cytidine deaminase (AID) in CD38-activated murine splenic B cells by IL-5. The role of Stat5a and Stat5b in IL-5-induced mu-gamma1 CSR and also IgG1 and IgM production was documented, as IL-5 does not act on CD38-stimulated splenic B cells from Stat5a(-/-) and Stat5b(-/-) mice. Expression levels of CD38-induced germline gamma1 transcripts and AID in Stat5a(-/-) and Stat5b(-/-) B cells upon IL-5 stimulation were comparable to those of wild-type B cells. The impaired mu-gamma1 CSR by Stat5b(-/-) B cells, but not by Stat5a(-/-) B cells, was rescued in part by IL-4, as the addition of IL-4 to the culture of CD38- and IL-5-stimulated B cells induced mu-gamma1 CSR leading to IgG1 production. Analysis of cell division cycle number of wild-type B cells revealed that mu-gamma1 CSR was observed after five or six cell divisions. Stat5a(-/-) and Stat5b(-/-) B cells showed similar cell division cycles, but they did not undergo mu-gamma1 CSR. Our data support the notion that both Stat5a and Stat5b are essential for IL-5-dependent mu;-gamma1 CSR and Ig secretion; however, their major target may not be AID. Stat5a and Stat5b are not redundant, but rather are at least partially distinctive in their function.

Bronchoscopic diagnosis of thyroid cancer with laryngotracheal invasion.

HYPOTHESIS: Some controversy exists concerning the appropriate surgical management for patients with thyroid cancer invading the laryngotracheal wall. We have used shaving of the wall when cancer invasion was confined to the perichondrium, and extensive resection when it invaded further. Preoperative assessment of the depth and length of laryngotracheal invasion is important when choosing an appropriate surgical procedure. DESIGN: Prospective study. SETTING: A Japanese center for thyroid diseases, where about 1400 thyroid operations are performed each year. PATIENTS: Of 171 patients with thyroid cancer who were surgically treated between January 1, 2000, and July 30, 2000, 37 were suspected to have laryngotracheal invasion on preoperative magnetic resonance imaging or ultrasonography. INTERVENTION: We used bronchoscopy to examine the 37 patients suspected to have laryngotracheal invasion. MAIN OUTCOME MEASURE: Bronchoscopic findings (localized mucosal redness, telangiectasia, mucosal elevation, mucosal edema, and mucosal erosion) were compared with pathological results in the 30 patients who underwent curative resections. Seven patients were excluded because of palliative resections. RESULTS: Of the 18 patients without localized mucosal changes, we performed shaving of the laryngotracheal wall in 4 patients because we found laryngotracheal invasion during surgery. Shaving of the laryngotracheal wall was performed successfully in terms of obtaining a cancer-free margin. Twelve patients with localized mucosal redness required extensive resections. Other mucosal changes were found depending on the depth of cancer invasion. CONCLUSION: Surgeons should perform extensive resections when encountering localized mucosal redness on bronchoscopy.

Impaired vitamin A-mediated mucosal IgA response in IL-5 receptor-knockout mice.

To clarify actions of vitamin A on mucosal immunity associated with interleukin-5 (IL-5), we examined effects of vitamin A on mucosal IgA level in IL-5 receptor alpha-chain-knockout (IL-5Ralpha(-/-)) mice. Daily supplementation of retinyl acetate (1 mg/mouse) increased Th2 cytokine levels and a number of their positive cells in the small intestinal mucosa of IL-5Ralpha(-/-) mice, as observed in wild-type or IL-5Ralpha(+/-) mice. Wild-type and heterozygous mice increased the IgA level and a number of IgA-containing cells in the mucosa in response to the vitamin A treatment, but not in IL-5Ralpha(-/-) mice. Retinyl acetate increased anti-cholera toxin (CT) IgA level in the mucosa of wild-type mice, improving their survival rate after an exposure to 0.4 mg of CT. However, retinyl acetate failed to induce resistance to CT toxicity in IL-5Ralpha(-/-) mice. Our results suggest that IL-5 may play an important role in an action of vitamin A on mucosal IgA system.

High prevalence of vitamin D deficiency in Japanese female patients with Graves' disease.

We reported previously that vitamin D deficiency is a causal mechanism of postoperative tetany in patients with Graves' disease. The aim of the present study was to determine the prevalence of vitamin D deficiency by reviewing serum 25(OH)D levels in 208 patients with Graves' disease (146 women, 62 men) during a 1 year period. Serum 25(OH)D levels were significantly lower (p < 0.001) in female Graves' patients (31.8 +/- 13.3 nmol/l) than in male patients (41.3 +/- 15.0 nmol/l). Vitamin D deficiency (defined as a serum 25(OH)D value below 25 nmol/l) was found in 40% of female patients and in 18% of male patients (p < 0.005). There was a significant seasonal variation in the 25(OH)D concentrations in female patients [amplitude 6.38 (95% CI, 5.42-7.56)], with values below 25 nmol/l found in 58% of female patients during the winter months. There were significant (p < 0.001) differences in serum 25(OH)D levels between age groups in the female patients. The concentrations were lowest in patients in their twenties (25.1 +/- 8.2 nmol/l) and highest in patients in their fifties and sixties (43.2 +/- 13.7 nmol/l). Serum 25(OH)D concentrations might be monitored in patients with Graves' disease during antithyroid drug therapy, and vitamin D and/or calcium supplements are recommended for patients with vitamin D deficiency.

Effect of genistein analogs on oxygen radical production in leukocytes stimulated by unopsonized zymosan.

Effects of genistein analogs on oxygen radical production have been analyzed in human neutrophils, human monocytes or murine macrophages Raw264.7 stimulated with unopsonized zymosan by lucigenin- and luminol-enhanced chemiluminescence assays. Genistein exhibited IC50 values of 10.7-11.5 microM on the oxygen radical production in human neutrophils, 10.9-11.0 microM in human monocytes, and 14.8-27.3 microM in Raw264.7 cells. Orobol, a genistein analog with an additional hydroxy group at the 3' position, exhibited IC50 values of 3.0-3.3 microM on the oxygen radical production in human neutrophils, 2.8-3.1 microM in human monocytes, and 1.5-3.9 microM in Raw264.7 cells. Genistin and sophoricoside are genistein glycosides with a glucose moiety at 7 or 4' position, respectively. The genistein glycosides exhibited 23-37% inhibitory effects at 100 microM on the oxygen radical production.

Bruton's tyrosine kinase is required for signaling the CD79b-mediated pro-B to pre-B cell transition.

Formation of the pre-BCR complex is a critical check point during B cell development and induces the transition of pro-B to pre-B cells. CD79b (Igbeta) is a signaling component in the pre-BCR complex, since differentiation to the pre-B phenotype is induced by cross-linking the CD79b expressed on developmentally arrested pro-B cells from recombination-activating gene (RAG)-2-deficient mice. Bruton's tyrosine kinase (BTK) plays important roles in B cell development. However, its molecular mechanisms in early B cell development are not fully understood. To examine whether BTK functions in CD79b-mediated signaling for the pro-B/pre-B transition, we utilized RAG2/BTK double-knockout (DKO) mice. Pro-B cells from RAG2/BTK-DKO mice did not differentiate into pre-B cells following CD79b cross-linking, although tyrosine phosphorylation of cellular proteins including Erk1/2 and phospholipase C-gamma2 was induced in the same manner as RAG2-KO mice. BTK is phosphorylated after cross-linking of CD79b on RAG2-deficient pro-B cells. These findings suggest that BTK-dependent pathways downstream of CD79b are critical for the pro-B/pre-B transition and BTK-independent signaling pathways are also activated via the pre-BCR complex.

Functional dissection of the cytoplasmic subregions of the interleukin-5 receptor alpha chain in growth and immunoglobulin G1 switch recombination of B cells.

The interleukin-5 receptor alpha chain (IL-5Ralpha) is known to regulate the development and function of B cells and eosinophils. Although the functions of IL-5Ralpha cytoplasmic domain subregions have been studied extensively using cultured cell lines, this approach has limitations when studying the functions of distinct primary B-cell subpopulations and their responsiveness to IL-5. In the present study, we generated mice on an IL-5Ralpha null background, each expressing a mutant form of an IL-5Ralpha transgene ligated to a mu enhancer and VH promoter, either lacking the cytoplasmic DC3 region or substituting two proline residues for alanine (ApvA) in the membrane-proximal ppvp motif of the cytoplasmic domain. The ppvp motif, which mediates activation of JAK2/STAT5 and Btk, also contributes to c-fos, c-jun and c-myc expression. IL-5Ralpha null mutant mice showed impaired B-1-cell development, reduced serum immunoglobulin G3 (IgG3) and IgM, no IL-5-induced enhancement of B-cell proliferation and IL-5-induced switch recombination from the mu gene to gamma1 gene; these were not recovered following the expression of the ApvA mutant. In contrast, absence of the DC3 region affected the IL-5-induced switch recombination from the mu to the gamma1 gene and B-1-cell development, while IL-5-induced proliferation and IgM production were at levels similar to those of B cells expressing wild-type IL-5Ralpha transgene. The results clearly indicated that the ppvp motif and the DC3 region of IL-5Ralpha played distinct roles in B-cell proliferation and differentiation. Thus, this present approach offers new insights into the functions of the cytoplasmic subregions of IL-5Ralpha, in particular its carboxy-terminal region.

Identification and characterization of a transcriptional regulator for the lck proximal promoter.

The lck gene encodes a protein-tyrosine kinase that plays a key role in signaling mediated through T cell receptor (TCR) and pre-TCR complexes. Transcription of the lck gene is regulated by two independent promoter elements: the proximal and distal promoters. Previous studies employing transgenic mice demonstrated that the sequence between -584 and -240 from the transcription start site in the mouse lck proximal promoter is required for its tissue-specific expression in the thymus. In this study, we demonstrate that a Krüppel-like zinc finger protein, mtbeta (BFCOL1, BERF-1, ZBP-89, ZNF148), previously cloned as a protein that binds to the CD3delta gene enhancer, binds to the -365 to -328 region of the lck proximal promoter. mtbeta is ubiquitously expressed in various cell lines and mouse tissues. Overexpressed mtbeta is more active in T-lineage cells than B-lineage cells for transactivating an artificial promoter consisting of the mtbeta binding site and a TATA box. Activity of the lck proximal promoter was significantly impaired by mutating the mtbeta binding site or by reducing mtbeta protein expression level by using antisense mRNA. Our results indicate that mtbeta activity is regulated in a tissue-specific manner and that mtbeta is a critical transactivator for the lck proximal promoter.

Reduction of atherosclerosis despite hypercholesterolemia in lyn-deficient mice fed a high-fat diet.

Oxidation and other modifications of serum low-density lipoprotein (LDL) are associated with the development of atherosclerosis, and a scavenger receptor and CD40 signalling are also known to play important roles in the process. We previously showed that the Src family protein-tyrosine kinase Lyn is physically and/or functionally associated with macrophage type-I and type-II class-A scavenger receptors (MSR-A) and CD40. In this study, we addressed whether Lyn is involved in the build-up of serum lipid levels and in atherosclerotic changes. When fed a normal diet, lyn-deficient mice had serum lipid levels that were no different from those of wild-type mice. By contrast, lyn-deficient mice fed a high-fat diet showed serum lipid levels that were much higher than those seen in wild-type mice. Curiously, however, the lyn-deficient mice fed either diet showed no increase in incidence of atherosclerotic lesions compared with wild-type mice. This may be partly explained by our data showing suppression of proliferation of peritoneal macrophages in response to oxidized LDL in the absence of Lyn, and failure of stimulation of the CD40 pathway in lyn-deficient macrophages to induce expression of monocytic chemoattractant protein-1 (MCP-1), which is related to atherosclerosis. These results suggest that Lyn plays an important role in the metabolism of serum lipids and in the development of atherosclerotic lesions on high-fat diets.

Involvement of complement and fibronectin in eosinophil-mediated damage to Nippostrongylus brasiliensis larvae.

By using IL-5 transgenic mice, it has been shown that eosinophils might play a key role in elimination of larval stages of nematode infections. The present study was carried out to clarify molecular mechanisms involved in the eosinophil-mediated killing of Nippostrongylus brasiliensis larvae. The larvicidal activity was observed in the presence of normal serum in vitro. Electron microscopic observations revealed firm attachment of eosinophils to the cuticular surface of larvae, which was damaged by electron-dense materials released from eosinophils. The larvicidal activity was abrogated by heat- or zymosan-treatment of the serum, whereas depletion of IgG or IgM from the serum did not interfere with eosinophil adhesion and killing. Moreover, pretreatment of eosinophils with monoclonal antibodies against CD11b or VLA-4 inhibited the eosinophil-mediated killing of larvae. Immunofluorescent staining demonstrated the deposition of C3c and plasma fibronectin on the cuticle of the larvae. These results indicate that interactions between CD11b and VLA-4 and their respective counter-ligands deposited on the cuticle are essential in eosinophil-mediated adhesion and damage to larvae of N. brasiliensis.

Comparison of clinicopathological features of patients with hepatocellular carcinoma seropositive for alpha-fetoprotein alone and those seropositive for des-gamma-carboxy prothrombin alone.

BACKGROUND: There has been no comparative study of the clinicopathological features of HCC patients who are seropositive for alpha-fetoprotein (AFP) alone and those who are seropositive for des-gamma-carboxy prothrombin (DCP) alone. The authors, thus, performed this comparative study. METHODS: The clinicopathological features of patients with solitary hepatocellular carcinoma (HCC), who underwent a hepatectomy were compared among the four below groups according to the seropositivity of AFP and DCP: group A, seronegative for both AFP below 20 ng/mL and DCP below 40 mAU/mL; group B, seropositive for AFP above 100 ng/mL and seronegative for DCP; group C, seronegative for AFP and seropositive for DCP above 100 mAU/mL; and group D, seropositive for both AFP and DCP. RESULTS: Group B patients showed a higher incidence of HCC with an indistinct margin, and a somewhat higher incidence of small HCC less than 2 cm in greatest dimension compared with group C patients. By contrast, group C patients had a higher frequency of HCC with a distinct margin compared with that of an indistinct margin, large tumors more than 3 cm compared with that of small tumors less than 2 cm, and a somewhat higher frequency of moderately to poorly differentiated HCC compared with that of well-differentiated HCC. Our HCC cases showed advanced clinicopathological features in the order of group C, group B and group A. Groups C and D patients showed similar characteristics. CONCLUSIONS: Hepatocellular carcinoma patients who were seropositive for AFP alone demonstrated clinicopathological features of less advanced HCC compared with those who were seropositive for DCP alone.

Control of B cell production by the adaptor protein lnk. Definition Of a conserved family of signal-modulating proteins.

Lnk is an SH2 domain-containing adaptor protein expressed preferentially in lymphocytes. To illuminate the importance of Lnk, we generated lnk(-/-) mice. Whereas T cell development was unaffected, pre-B and immature B cells accumulated in the spleens. In the bone marrow, B-lineage cells were proportionately increased, reflecting enhanced production of pro-B cells that resulted in part from hypersensitivity of precursors to SCF, the ligand for c-kit. Hence, Lnk ordinarily acts to regulate B cell production. Further characterization of lnk(-/-) mice also revealed that full-length Lnk is a 68 kDa protein containing a conserved proline-rich region and a PH domain. Lnk is a representative of a multigene adaptor protein family whose members act, by analogy with Lnk, to modulate intracellular signaling.

Differential inhibitory effects of sophoricoside analogs on bioactivity of several cytokines.

Effects of sophoricoside and its analogs on proinflammatory cytokines have been investigated. Sophoricoside, genistein and orobol exhibited inhibitory effects on IL-5, IL-3, GM-CSF and IL-6 bioactivities. Genistin showed inhibitory effects on IL-5 and IL-3 bioactivities, but did not inhibit GM-CSF and IL-6 bioactivities. None of the sophoricoside analogs showed inhibitory effects on both IL-1beta and TNF-alpha bioactivities. Among the compounds, sophoricoside exhibited the highest inhibitory effects on IL-5, IL-3 and IL-6 bioactivities with IC50 values of 1.9 microM, 6.9 microM and 6.0 microM, respectively and orobol did show on GM-CSF bioactivity with an IC50 value of 18.0 microM. The result would provide an additional mechanism by which the compounds exert immunosuppressive and anti-inflammatory effects.

Screening of the Men1 gene and discovery of germ-line and somatic mutations in apparently sporadic parathyroid tumors.

Hyperparathyroidism is the first manifestation in a majority of multiple endocrine neoplasia (MEN1) patients. To discriminate between sporadic and hereditary parathyroid tumors and characterize MEN1 somatic mutations, we examined MEN1 gene mutations in patients who had undergone surgery for sporadic parathyroid tumors. DNA was extracted from fresh frozen parathyroid tumor specimens from 112 patients as well as from peripheral blood leukocytes from 64 of the 112 patients. Sequence analysis was performed to examine exons 2-10 of the MEN1 gene for mutations. Loss of heterozygosity (LOH) was also examined by an analysis of codon 418 and 541, which lie within a polymorphic region of MEN1. Somatic MEN1 mutations were found in 25 of the 112 patients (22%). Two patients had two point mutations (508del33 and Y341X and 363insT and 1767delT, respectively). A total of 27 mutations were characterized, 20 of which have not been reported previously. There were 7 nonsense mutations, 10 frameshift mutations, 2 splice site deletions, 5 missense mutations, and 3 in-frame mutations. Nineteen mutations (70%) predicted truncation of the menin protein. Germ-line MEN1 mutations were found in 3 of 64 patients (5%) who had no family history of endocrine tumors associated with MEN1, and these patients were identified as MEN1 gene probands. LOH at the MEN1 locus was detected in three parathyroid tumors showing germ-line mutation. LOH was significantly frequent in parathyroid tumors with somatic MEN1 mutations (15 of 22 tumors, 68%) but not in those without germ-line or somatic MEN1 mutations (14 of 51 tumors, 28%; P = 0.0011). Our findings suggest that alterations of both alleles of the MEN1 gene may be associated not only with endocrine tumors of affected MEN1 patients but also with sporadic parathyroid tumors. Germ-line MEN1 gene analysis can distinguish heritable from nonheritable parathyroid tumors, and MEN1 gene evaluation of patients with apparently sporadic parathyroid tumor is recommended before parathyroid surgery.

Identification and characterization of a molecule, BAM11, that associates with the pleckstrin homology domain of mouse Btk.

Bruton's tyrosine kinase (Btk) is required for normal B cell development and signal transduction through cell surface molecules, and its defects lead to X-linked immune deficiency in mice and X-linked agammaglobulinemia in humans. In this report, we will describe the identification and characterization of a molecule, BAM11, which binds to the pleckstrin homology domain of Btk. A sequence homology search revealed that BAM11 has 89% homology, at the amino acid level, to human LTG19/ENL, that was originally identified as one of the fusion partners involved in chromosomal translocations of 11q23, MLL/ALL-1/HRX, in leukemia cells. Deletion mutants demonstrated that the region of BAM11 required for binding to Btk was localized between amino acid residues 240 and 256. Forced expression of a truncated form of BAM11 (amino acids 246-368) inhibited IL-5-induced proliferation by 50%, whereas forced expression of full-length BAM11 in Y16 cells did not affect the IL-5 responsiveness. We have also shown that BAM11 (amino acids 246-368) inhibited the kinase activity of Btk. These results suggest that the binding of BAM11 to Btk plays a regulatory role in the Btk signal transduction pathway. A cell fractionation study and analysis using EGFP-fused Btk protein demonstrated that a proportion of Btk is present within the nucleus.