Hepatocellular carcinoma and focal nodular hyperplasia in patients with Fontan-associated liver disease: characterisation using dynamic gadolinium ethoxybenzyl diethylenetriamine pentaacetic acid-enhanced MRI.

AIM: To identify the characteristic diagnostic features of hepatocellular carcinoma (HCC) and focal nodular hyperplasia (FNH) in Fontan-associated liver disease (FALD) patients using dynamic gadolinium ethoxybenzyl diethylenetriamine pentaacetic acid (Gd-EOB-DTPA)-enhanced magnetic resonance imaging (MRI). MATERIALS AND METHODS: Thirty-one FALD patients (mean age, 28.3 ± 7.2 years) with liver nodules who underwent dynamic Gd-EOB-DTPA-enhanced MRI were enrolled prospectively. Twenty-five patients (mean age, 72.8 ± 11.4 years) with hepatitis C virus (HCV)-related HCC constituted the control group. The tumour-to-liver signal intensity (SI) ratio was measured at 30, 60, 100, 180 seconds and 15 minutes, and the SI ratio was compared among FALD-HCC, FALD-FNH, and HCV-HCC. RESULTS: FALD-HCC exhibited weak early enhancement with mild washout in late phases. FALD-FNH exhibited marked early enhancement that continued until the late phases. The SI ratio was significantly lower for FALD-HCC than for FALD-FNH in all phases. The SI ratio was significantly lower for FALD-HCC than for HCV-HCC only at 30 seconds (p<0.05), whereas poorer washout was seen in FALD-HCC than HCV-HCC in other phases. In 15 minutes, FALD-HCC had a significantly lower SI ratio compared to FALD-FNH (p<0.001). CONCLUSIONS: The time course of Gd-EOB-DTPA-enhanced MRI signal intensity in FALD-HCC was different from that in FALD-FNH or HCV-HCC. This imaging finding may be useful adjunctive information to distinguish FALD-HCC from FALD-FNH.

Bendamustine-120 plus rituximab therapy for relapsed or refractory follicular lymphoma: a multicenter phase II study.

The optimal dose, schedule, and other aspects of bendamustine plus rituximab treatment remain unclear for patients with relapsed or refractory follicular lymphoma (FL). Herein, we analyzed the efficacy of bendamustine combined with rituximab (RB-120) treatment for Japanese patients with relapsed or refractory FL. This phase II clinical trial included patients with relapsed or refractory FL who received 375 mg/m2 rituximab on day 1 and 120 mg/m2 bendamustine on days 2 and 3 every 28 days for up to 6 cycles. The primary endpoint was the overall response rate (ORR), and the secondary endpoints included the complete response (CR) rate, progression-free survival (PFS), overall survival (OS), and safety. Thirty-seven patients were enrolled in the trial (median age 62 years, range 42-75 years). All patients were previously treated with rituximab-containing chemotherapy, and 83.8% were previously treated with the R-CHOP regimen. A median of 5 cycles (range 1-6) and 48.6% of patients completed 6 cycles. The ORR was 91.9% (95% confidence interval [CI] 78.1-98.3%), with a CR rate of 86.5% (95% CI 71.2-95.5%). The 3-year PFS and OS were 70.9% (95% CI 52.3-83.3%) and 88.9% (95% CI 73.1-95.7%), respectively, with the median 39.5 months follow-up duration. The most-frequently observed grade 3/4 adverse events were hematologic: lymphopenia (95%) and neutropenia (70%). No treatment-related deaths were observed. RB-120 showed a good efficacy with equivalent toxicities, compared with the bendamustine 120 mg/m2 monotherapy. However, the problem of high drop-out incidences cannot be ignored.

Efficacy and safety of multitarget therapy with cyclophosphamide and tacrolimus for lupus nephritis: a prospective, single-arm, single-centre, open label pilot study in Japan.

Background Pulsed cyclophosphamide or mycophenolate mofetil for lupus nephritis has limited efficacy. We previously reported a case of mixed-class IV + V lupus nephritis successfully treated with cyclophosphamide and tacrolimus. This study assessed the efficacy and safety of multitarget therapy with cyclophosphamide and tacrolimus for the treatment of lupus nephritis. Methods In a prospective, single-arm, open label pilot study, we recruited 15 patients aged 18-64 years with active lupus nephritis who met the American College of Rheumatology criteria for a diagnosis of systemic lupus erythematosus (1997). The treatment protocol was a starting dose of prednisolone of 0.6-1.0 mg/kg/day for 2 weeks and then tapered to a maintenance dose, intravenous cyclophosphamide (500 mg biweekly for 3 months) and tacrolimus (3.0 mg/day). Tacrolimus was continued as maintenance therapy. Complete remission was defined as a spot urine protein/creatinine ratio of < 0.5 g/gCr with no active urine casts and a serum creatinine level that was either normal or within 30% of a previously abnormal baseline level. We retrospectively compared results for the study patients with those of 18 historical controls conventionally treated with cyclophosphamide and prednisolone. Results At baseline, the mean patient age was 41.5 ± 14.6 years (male:female ratio 2:13), urine protein/creatinine ratio 3.9 ± 2.3 g/gCr and serum creatinine 84.6 ± 34.6 µmol/L. Lupus nephritis classifications included classes IV ( n = 8), III + V ( n = 1), IV + V ( n = 5) and unclassified ( n = 1). Eleven patients completed the treatment protocol and four withdrew. At 6 months, 12 of 15 (80.0%) had achieved complete remission using intention-to-treat analysis, significantly more than historical controls (seven of 18 patients, 38.9%). A transient increase in serum creatinine and gastric symptoms occurred in three cases. One patient withdrew due to cytomegalovirus antigenemia and severe diabetes, and one patient died of thrombotic microangiopathy. Conclusions Multitarget therapy with cyclophosphamide and tacrolimus can be a therapeutic option for lupus nephritis. Clinical trials registration Combination therapy of tacrolimus and intravenous cyclophosphamide for remission induction of lupus nephritis, UMIN: 000004893, URL: https://upload.umin.ac.jp/cgi-open-bin/ctr/ctr.cgi?function=brows&action=brows&type=summary&recptno=R000005830&language=E . Date of registration: 18 January 2011.

Catastrophic gastric rupture in a horse secondary to psyllium pharmacobezoars.

A mare was euthanized because of gastric rupture secondary to complete duodenal obstruction by 2 bezoars located in the pylorus and proximal duodenum. Infrared spectroscopy showed that the bezoars were composed of psyllium. The mare had been receiving treatment with a pelleted psyllium product at 4 times the recommended dosage. Veterinarians should be aware that treatment of colic in horses with pelleted psyllium products could be associated with gastric impaction.

Rupture gastrique catastrophique secondaire à des pharmacobézoards de psyllium en boulettes chez un cheval. Une jument a été euthanasiée en raison d'une rupture gastrique secondaire à une obstruction duodénale complète par deux bézoards situés dans le pylore et le duodénum proximal. La spectroscopie infrarouge a montré que les bézoards se composaient de psyllium. La jument avait reçu un traitement composé d'un produit de psyllium en boulettes à quatre fois la dose recommandée. Les vétérinaires devraient être au courant que le traitement des coliques chez les chevaux avec des produits de psyllium en boulettes pourrait être associé à une impaction gastrique.(Traduit par Isabelle Vallières).

An Effective New Cryopreservation Procedure for Pancreatic Islets Using Hollow Fiber Vitrification.

The present study aimed at establishing a new cryopreservation method for mouse pancreatic islets by vitrification using hollow fibers as a container. A unique feature of the hollow fiber vitrification (HFV) method is that this method achieves stable vitrification using a minimum volume of cryoprotectant (CPA) solution, thereby ensuring high viability of the islets. The cytotoxicity, optimum composition, and concentration of the CPAs for vitrifying islets were examined. The viability, functional-integrity of vitrified islets were evaluated in comparison with those vitrified by conventional methods. Insulin secretion was measured in vitro by a static incubation assay and the metabolic functions was tested after transplantation into Streptozotocin-induced diabetic mice. The combination of 15% dimethyl sulfoxide+15% ethylene glycol resulted in the best CPA solution for the HFV of islets. HFV showed the highest viability in comparison to 2 vitrification methods, open pulled straws and vitrification with EDT324 solution. The vitrified islets stably expressed ß-cells markers NeuroD, Pancreatic and duodenal homeobox-1, and MafA. Transplantation of the vitrified islets achieved euglycemia of the host diabetic mice and response to an intraperitoneal glucose tolerance test to a similar extent as non-vitrified transplanted islets. The HFV method allows for efficient long-term cryopreservation of islets.

Ki-67 is a strong predictor of central nervous system relapse in patients with mantle cell lymphoma (MCL).

BACKGROUND: Central nervous system (CNS) relapse is an uncommon but challenging complication in patients with mantle cell lymphoma (MCL). Survival after CNS relapse is extremely poor. Identification of high-risk populations is therefore critical in determining patients who might be candidates for a prophylactic approach. PATIENTS AND METHODS: A total of 608 patients (median age, 67 years; range 22-92) with MCL newly diagnosed between 1994 and 2012 were evaluated. Pretreatment characteristics and treatment regimens were evaluated for their association with CNS relapse by competing risk regression analysis. RESULTS: None of the patients received intrathecal prophylaxis. Overall, 33 patients (5.4%) experienced CNS relapse during a median follow-up of 42.7 months. Median time from diagnosis to CNS relapse was 20.3 months (range: 2.2-141.3 months). Three-year cumulative incidence of CNS relapse was 5.6% [95% confidence interval (95% CI) 3.7% to 8.0%]. Univariate analysis revealed several risk factors including blastoid variant, leukemic presentation, high-risk MCL International Prognostic Index and high Ki-67 (proliferation marker). Multivariate analyses revealed that Ki-67 ≥ 30 was the only significant risk factor for CNS relapse (hazard ratio: 6.0, 95% CI 1.9-19.4, P = 0.003). Two-year cumulative incidence of CNS relapse in patients with Ki-67 ≥ 30 was 25.4% (95% CI 13.5-39.1), while that in the patients with Ki-67 < 30 was 1.6% (95% CI 0.4-4.2). None of the treatment modalities, including rituximab, high-dose cytarabine, high-dose methotrexate or consolidative autologous stem-cell transplant, were associated with a lower incidence of CNS relapse. Survival after CNS relapse was poor, with median survival time of 8.3 months. There was no significant difference in the survival by the site of CNS involvement.

Cardiovascular anomaly, impaired actin bundling and resistance to Src-induced transformation in mice lacking p130Cas.

p130Cas (Cas), the protein encoded by the Crkas gene (also known as Cas), is an adaptor molecule with a unique structure that contains a Src homology (SH)-3 domain followed by multiple YXXP motifs and a proline-rich region. Cas was originally cloned as a highly tyrosine-phosphorylated protein in cells transformed by v-Src (refs 2,3) or v-Crk (ref. 4) and has subsequently been implicated in a variety of biological processes including cell adhesion, cell migration, growth factor stimulation, cytokine receptor engagement and bacterial infection. To determine its role in vivo, we generated mice lacking Cas. Cas-deficient embryos died in utero showing marked systemic congestion and growth retardation. Histologically, the heart was poorly developed and blood vessels were prominently dilated. Electron microscopic analysis of the heart revealed disorganization of myofibrils and disruption of Z-disks. In addition, actin stress fiber formation was severely impaired in Cas-deficient primary fibroblasts. Moreover, expression of activated Src in Cas-deficient primary fibroblasts did not induce a fully transformed phenotype, possibly owing to insufficient accumulation of actin cytoskeleton in podosomes. These findings have defined Cas function in cardiovascular development, actin filament assembly and Src-induced transformation.

Chronic stress, metabolism, and metabolic syndrome.

The prevalence of obesity has rapidly escalated and now represents a major public health concern. Although genetic associations with obesity and related metabolic disorders such as diabetes and cardiovascular disease have been identified, together they account for a small proportion of the incidence of disease. Environmental influences such as chronic stress, behavioral and metabolic disturbances, dietary deficiency, and infection have now emerged as contributors to the development of metabolic disease. Although epidemiological data suggest strong associations between chronic stress exposure and metabolic disease, the etiological mechanisms responsible remain unclear. Mechanistic studies of the influence of chronic social stress are now being conducted in both rodent and nonhuman primate models, and phenotypic results are consistent with those in humans. The advantage of these models is that potential neural mechanisms may be examined and interventions to treat or prevent disease may be developed and tested. Further, circadian disruption and metabolic conditions such as diabetes mellitus could increase susceptibility to other stressors or serve as a stressor itself. Here, we review data from leading investigators discussing the interrelationship between chronic stress and development of metabolic disorders.

Increased α-tocopherol metabolism in horses with equine neuroaxonal dystrophy.

BACKGROUND: Equine neuroaxonal dystrophy/equine degenerative myeloencephalopathy (eNAD/EDM) is an inherited neurodegenerative disorder associated with a vitamin E deficiency within the first year of life. Vitamin E consists of 8 isoforms metabolized by the CYP4F2 enzyme. No antemortem diagnostic test currently exists for eNAD/EDM. HYPOTHESIS/OBJECTIVES: Based on the association of α-tocopherol deficiency with the development of eNAD/EDM, we hypothesized that the rate of α-tocopherol, but not γ-tocopherol or tocotrienol metabolism, would be increased in eNAD/EDM-affected horses. ANIMALS: Vitamin E metabolism: Proof of concept (POC) study; eNAD/EDM-affected (n = 5) and control (n = 6) horses. Validation study: eNAD/EDM-affected Quarter Horses (QHs; n = 6), cervical vertebral compressive myelopathy affected (n = 6) horses and control (n = 29) horses. CYP4F2 expression and copy number: eNAD/EDM-affected (n = 12) and age- and sex-matched control (n = 11-12) horses. METHODS: The rates of α-tocopherol/tocotrienol and γ-tocopherol/tocotrienol metabolism were assessed in equine serum (POC and validation) and urine (POC only) using liquid chromatography tandem mass spectrometry (LC-MS/MS). Quantitative reverse-transcriptase PCR (qRT-PCR) and droplet digital (dd)-PCR were used to assay expression and genomic copy number of a CYP4F2 equine ortholog. RESULTS: Metabolic rate of α-tocopherol was increased in eNAD/EDM horses (POC,P < .0001; validation, P = .03), with no difference in the metabolic rate of γ-tocopherol. Horses with eNAD/EDM had increased expression of the CYP4F2 equine orthologue (P = .02) but no differences in copy number. CONCLUSIONS AND CLINICAL IMPORTANCE: Increased α-tocopherol metabolism in eNAD/EDM-affected QHs provides novel insight into alterations in vitamin E processing in eNAD/EDM and highlights the need for high-dose supplementation to prevent the clinical phenotype in genetically susceptible horses.

Linkage group-chromosome correlation in Culex tritaeniorhynchus.

Linkage groups of a culicine mosquito, Culex tritaeniorhynchus, have been assigned to their respective chromosomes by genetic and cytologic observations of radiation-induced aberrations. Linkage group I is assigned to the smallest chromosome, linkage group II to the submetacentric chromosome, and linkage group III to the metacentric chromosome.

Stable integration and expression of a bacterial gene in the mosquito Anopheles gambiae.

Foreign DNA was successfully introduced into the germline of the African mosquito vector of malaria Anopheles gambiae. Stable integration of genes into the germlines of insects had been achieved previously only in Drosophila melanogaster and related species and required the use of the P element transposon. In these experiments with Anopheles gambiae, the plasmid pUChsneo was used, which contains the selectable marker neo gene flanked by P element inverted repeats. Mosquitoes injected with this plasmid were screened for resistance to the neomycin analog G-418. A single event of plasmid insertion was recovered. Integration appears to be stable and, thus far, resistance to G-418 has been expressed for eight generations. The transformation event appears to be independent of P.

Genetic selection of a Plasmodium-refractory strain of the malaria vector Anopheles gambiae.

The anopheline mosquito is the target in most malaria control programs, primarily through the use of residual insecticides. A mosquito was studied that is refractory to most species of malaria through a genetically controlled mechanism. A strain of Anopheles gambiae, which was selected for complete refractoriness to the simian malaria parasite Plasmodium cynomolgi, also has varying degrees of refractoriness to most other malaria species examined, including the human parasites P. falciparum, P. ovale, and P. vivax for which this mosquito is the principal African vector. Furthermore, the refractoriness extends to other subhuman primate malarias, to rodent malaria, and to avian malaria. Refractoriness is manifested by encapsulation of the malaria ookinete after it completes its passage through the mosquito midgut, approximately 16 to 24 hours after ingestion of an infective blood meal. Fully encapsulated ookinetes show no abnormalities in parasite organelles, suggesting that refractoriness is due to an enhanced ability of the host to recognize the living parasite rather than to a passive encapsulation of a dead or dying parasite. Production of fully refractory and fully susceptible mosquito strains was achieved through a short series of selective breeding steps. This result indicates a relatively simple genetic basis for refractoriness. In addition to the value these strains may serve in general studies of insect immune mechanisms, this finding encourages consideration of genetic manipulation of natural vector populations as a malaria control strategy.

The Drosophila trio plays an essential role in patterning of axons by regulating their directional extension.

We identified the Drosophila trio gene, which encodes a Dbl family protein carrying two Dbl homology (DH) domains, each of which potentially activates Rho family GTPases. Trio was distributed along axons in the central nervous system (CNS) of embryos and was strongly expressed in subsets of brain regions, including the mushroom body (MB). Loss-of-function trio mutations resulted in the misdirection or stall of axons in embryos and also caused malformation of the MB. The MB phenotypes were attributed to alteration in the intrinsic nature of neurites, as revealed by clonal analyses. Thus, Trio is essential in order for neurites to faithfully extend on the correct pathways. In addition, the localization of Trio in the adult brain suggests its postdevelopmental role in neurite terminals.

The mammalian ShcB and ShcC phosphotyrosine docking proteins function in the maturation of sensory and sympathetic neurons.

Shc proteins possess SH2 and PTB domains and serve a scaffolding function in signaling by a variety of receptor tyrosine kinases. There are three known mammalian Shc genes, of which ShcB and ShcC are primarily expressed in the nervous system. We have generated null mutations in ShcB and ShcC and have obtained mice lacking either ShcB or ShcC or both gene products. ShcB-deficient animals exhibit a loss of peptidergic and nonpeptidergic nociceptive sensory neurons, which is not enhanced by additional loss of ShcC. Mice lacking both ShcB and ShcC exhibit a significant loss of neurons within the superior cervical ganglia, which is not observed in either mutant alone. The results indicate that these Shc family members possess both unique and overlapping functions in regulating neural development and suggest physiological roles for ShcB/ShcC in TrkA signaling.

Effect of glucose and pyruvate on nuclear and cytoplasmic maturation of porcine oocytes in a chemically defined medium.

The objective was to examine potential roles of glucose and pyruvate in nuclear and cytoplasmic maturation of porcine oocytes. Oocyte-cumulus complexes (OCC), derived from 3 to 6mm follicles, were cultured in a chemically defined medium (pyruvate-free mNCSU37-PVA), with or without 5.55 mM glucose, during in vitro maturation (IVM); in the absence of glucose, germinal vesicle breakdown (GVBD) and nuclear maturation were prevented (P<0.05). Subsequently, OCC were cultured for IVM in glucose-containing mNCSU37-PVA supplemented with 6-amononicotinamide (6-AN) and diphenyleneiodonium (DPI), inhibitors of the pentose phosphate pathway (PPP); both compounds (>or=10 microM 6-AN and >or=10 nM DPI) inhibited resumption of meiosis (P<0.05). Supplementation of glucose-free maturation medium with increasing concentrations of pyruvate induced resumption of meiosis and increased the incidence of oocytes reaching metaphase-II in a concentration-dependent manner (P<0.05). More mature oocytes were obtained in the presence of pyruvate+glucose (P<0.05). After culture to allow maturation, glutathione content was higher in oocytes cultured in the presence of pyruvate alone than in those cultured in glucose alone; inclusion of 6-AN abolished responses to pyruvate (P<0.05). In conclusion, both glucose and pyruvate played a critical role in the release of porcine oocytes from arrest at the GV-I stage, probably through the PPP, whereas supplementation with pyruvate improved cytoplasmic maturation, as determined by oocyte glutathione content.

Use of a locking compression plate for equine proximal interphalangeal joint arthrodesis: 29 cases (2008-2014).

OBJECTIVE To describe clinical use of a locking compression plate (LCP) for proximal interphalangeal joint (PIPJ) arthrodesis in horses and compare outcomes for horses that underwent the procedure as treatment for fracture of the middle phalanx (P2) versus other causes. DESIGN Retrospective case series. ANIMALS 29 client-owned horses. PROCEDURES Medical records of 2 veterinary teaching hospitals from 2008 through 2014 were reviewed to identify horses that underwent PIPJ arthrodesis of 1 limb. Signalment, surgical, and outcome-related variables were recorded. Owners were contacted from 1 to 6 years after surgery to determine rehabilitation time, current use of the horse, and overall owner satisfaction with the procedure. Success was determined on the basis of owner satisfaction and outcome for intended use. Variables of interest were compared statistically between horses that underwent surgery for P2 fracture versus other reasons. RESULTS 14 horses underwent surgery for treatment of P2 fracture, and 15 had surgery because of osteoarthritis, subluxation, or osteochondrosis. Median convalescent time after surgery (with no riding or unrestricted exercise) was 7 months. Four horses were euthanized; of 23 known alive at follow-up, 22 were not lame, and 18 had returned to their intended use (8 and 10 at higher and lower owner-reported levels of work, respectively). Horses undergoing arthrodesis for reasons other than fracture were significantly more likely to return to their previous level of work. Twenty-two of 24 owners contacted indicated satisfaction with the procedure. CONCLUSIONS AND CLINICAL RELEVANCE Surgical arthrodesis of the PIPJ was successful in most horses of the study population. Various nuances of the system for fracture repair need to be understood prior to its use.

Immunological Response of Pigs to Human Cells, Including Issues Such as the Production of Natural Antibodies in Newborns.

Pigs have recently become very popular for use not only in xenotransplantation field, but in regeneration studies as well, sometimes with pigs being used as the scaffold. We have already presented our findings related to the pig immune system against human cells, including the complement systems, natural antibodies (NAs), and NK cells. In this study, we investigated the pig innate immunological reaction against human cells further. Our investigations included issues such as the production of NAs in newborns, day 0 and day 1, and sow colostrum. The alternative pathway for pig complement reacted with human cells, and pig NK cells and macrophages directly injured human aortic endothelial cells. Pig serum clearly contains the natural antibodies IgG and IgM to human peripheral blood mononuclear cells (PBMCs). Pig plasma from day 1 newborns contained almost the same levels of these natural antibodies to human PBMCs as those of sow plasma. On the other hand, pig plasma from day 0 newborns did not contain IgG and IgM to human PBMCs. In addition, sow colostrum clearly contained both IgG and IgM to human PBMCs. As expected, the pig innate immunity system reacted to human cells, including natural antibodies. However, the NAs of pigs, both IgM and IgG, against human cells do not exist in pig serum at day 0, but at day 1 and in mother's milk, indicating that NAs in newborns did not come from the placenta but from sow colostrum.

Evidence that SH2 domains promote processive phosphorylation by protein-tyrosine kinases.

BACKGROUND: Non-receptor protein-tyrosine kinases often contain at least one Src homology 2 (SH2) domain, a protein module that binds with high affinity to tyrosine-phosphorylated peptides. Because SH2 domains would be predicted to bind with high affinity to proteins phosphorylated by the kinase, but not to the unphosphorylated substrate, their presence in tyrosine kinases has been puzzling. An important role for the SH2 domain of the Abl tyrosine kinase was suggested by work showing that Abl requires an intact SH2 domain in order to malignantly transform cells, and that replacement of the Abl SH2 domain with heterologous SH2 domains alters the spectrum of proteins phosphorylated detectably by Abl in vivo. RESULTS: We have used purified wild-type and mutant Abl kinases to examine the roles of the Abl's SH2 and catalytic domains in phosphorylation of p130CAS, a model substrate that has multiple potential phosphorylation sites. We find that an SH2 domain is required for efficient hyperphosphorylation of p130 in vitro. We use chimeric mutants with heterologous SH2 domains to demonstrate that the SH2 domain of the oncogenically transforming adaptor protein Crk, which is the SH2 domain predicted to bind with highest affinity (of those tested) to potential phosphorylation sites in p130, is best able to facilitate hyperphosphorylation. This is the case whether the catalytic domain of the kinase is derived from Abl or from its distant relative, Src. These studies also reveal a role for binding of Crk to Abl in mediating phosphorylation by the kinase. Using purified proteins, we demonstrate that association with Crk strikingly enhances the ability of Abl to hyperphosphorylate p130. There is an excellent correlation between the ability of mutant Crk proteins to promote hyperphosphorylation of p130 by Abl and their ability to transform rodent fibroblasts. CONCLUSION: Our data suggest that, ultimately, the substrate specificity of a non-receptor tyrosine kinase is dependent on the binding specificity of its associated SH2 domain. The SH2 domain binds tightly to a subset of proteins phosphorylated by the catalytic domain, leading to processive phosphorylation of those proteins. Substrate specificity can be broadened by an association between the kinase and proteins, such as Crk, that contain additional SH2 domains; this may play a role in malignant transformation by Crk.

Gene dosage-dependent functions for phosphotyrosine-Grb2 signaling during mammalian tissue morphogenesis.

BACKGROUND: The mammalian Grb2 adaptor protein binds pTyr-X-Asn motifs through its SH2 domain, and engages downstream targets such as Sos1 and Gab1 through its SH3 domains. Grb2 thereby couples receptor tyrosine kinases to the Ras-MAP kinase pathway, and potentially to phosphatidylinositol (PI) 3'-kinase. By creating a null (Delta) allele of mouse Grb2, we have shown that Grb2 is required for endoderm differentiation at embryonic day 4.0. RESULTS: Grb2 likely has multiple embryonic and postnatal functions. To address this issue, a hypomorphic mutation, first characterized in the Caenorhabditis elegans Grb2 ortholog Sem-5, was engineered into the mouse Grb2 gene. This mutation (E89K) reduces phosphotyrosine binding by the SH2 domain. Embryos that are compound heterozygous for the null and hypomorphic alleles exhibit defects in placental morphogenesis and in the survival of a subset of migrating neural crest cells required for branchial arch formation. Furthermore, animals homozygous for the hypomorphic mutation die perinatally because of clefting of the palate, a branchial arch-derived structure. Analysis of E89K/Delta Grb2 mutant fibroblasts revealed a marked defect in ERK/MAP kinase activation and Gab1 tyrosine phosphorylation following growth factor stimulation. CONCLUSIONS: We have created an allelic series within mouse Grb2, which has revealed distinct functions for phosphotyrosine-Grb2 signaling in tissue morphogenesis and cell viability necessary for mammalian development. The placental defects in E89K/Delta mutant embryos are reminiscent of those seen in receptor tyrosine kinase-, Sos1-, and Gab1-deficient embryos, consistent with the finding that endogenous Grb2 is required for efficient RTK signaling to the Ras-MAP kinase and Gab1 pathways.

1 alpha,25-dihydroxyvitamin D3 suppresses proliferation and immunoglobulin production by normal human peripheral blood mononuclear cells.

Activated B and T lymphocytes from normal human subjects are known to have the specific high-affinity receptor for 1,25-dihydroxyvitamin D3 (1,25-(OH)2-D3). In an attempt to determine a functional role for the sterol in such cells, we studied the effect of 1,25-(OH)2-D3 on DNA synthesis and Ig production by normal human peripheral blood mononuclear (PBM) cells activated in vitro by the polyclonal lymphocyte activators pokeweed mitogen and phytohemagglutinin, and the specific antigen dermatophyton O. A dose-dependent inhibition of [3H]thymidine incorporation was observed in cells incubated with 1,25-(OH)2-D3 in concentrations ranging from 10(-10) to 10(-7) M. Production of IgG and IgM, determined by enzyme-linked immunosorbent assay, was similarly inhibited by increasing concentrations of 1,25-(OH)2-D3. Half-maximal inhibition of DNA and Ig synthesis was found at 10(-10) to 10(-9) M 1,25-(OH)2-D3. This suppressive effect was specific for 1,25-(OH)2-D3; of the other vitamin D metabolites examined, only 10(-7) M 24R,25 dihydroxyvitamin D3 (24,25-(OH)2-D3) had a similar inhibitory effect. 1,25-(OH)2-D3 was not cytotoxic and did not affect unactivated PBMs. These data demonstrate that 1,25-(OH)2-D3 is a potent inhibitor of human PBM Ig production in vitro and suggest that this action is mediated through the hormone's antiproliferative effect on Ig-producing B cells and/or helper T cells.