Surfaceome profiling enables isolation of cancer-specific exosomal cargo in liquid biopsies from pancreatic cancer patients.

Background: Detection of circulating tumor DNA can be limited due to their relative scarcity in circulation, particularly while patients are actively undergoing therapy. Exosomes provide a vehicle through which cancer-specific material can be enriched from the compendium of circulating non-neoplastic tissue-derived nucleic acids. We carried out a comprehensive profiling of the pancreatic ductal adenocarcinoma (PDAC) exosomal 'surfaceome' in order to identify surface proteins that will render liquid biopsies amenable to cancer-derived exosome enrichment for downstream molecular profiling. Patients and methods: Surface exosomal proteins were profiled in 13 human PDAC and 2 non-neoplastic cell lines by liquid chromatography-mass spectrometry. A total of 173 prospectively collected blood samples from 103 PDAC patients underwent exosome isolation. Droplet digital PCR was used on 74 patients (136 total exosome samples) to determine baseline KRAS mutation call rates while patients were on therapy. PDAC-specific exosome capture was then carried out on additional 29 patients (37 samples) using an antibody cocktail directed against selected proteins, followed by droplet digital PCR analysis. Exosomal DNA in a PDAC patient resistant to therapy were profiled using a molecular barcoded, targeted sequencing panel to determine the utility of enriched nucleic acid material for comprehensive molecular analysis. Results: Proteomic analysis of the exosome 'surfaceome' revealed multiple PDAC-specific biomarker candidates: CLDN4, EPCAM, CD151, LGALS3BP, HIST2H2BE, and HIST2H2BF. KRAS mutations in total exosomes were detected in 44.1% of patients undergoing active therapy compared with 73.0% following exosome capture using the selected biomarkers. Enrichment of exosomal cargo was amenable to molecular profiling, elucidating a putative mechanism of resistance to PARP inhibitor therapy in a patient harboring a BRCA2 mutation. Conclusion: Exosomes provide unique opportunities in the context of liquid biopsies for enrichment of tumor-specific material in circulation. We present a comprehensive surfaceome characterization of PDAC exosomes which allows for capture and molecular profiling of tumor-derived DNA.

High prevalence of mutant KRAS in circulating exosome-derived DNA from early-stage pancreatic cancer patients.

Background: Exosomes arise from viable cancer cells and may reflect a different biology than circulating cell-free DNA (cfDNA) shed from dying tissues. We compare exosome-derived DNA (exoDNA) to cfDNA in liquid biopsies of patients with pancreatic ductal adenocarcinoma (PDAC). Patients and methods: Patient samples were obtained between 2003 and 2010, with clinically annotated follow up to 2015. Droplet digital PCR was performed on exoDNA and cfDNA for sensitive detection of KRAS mutants at codons 12/13. A cumulative series of 263 individuals were studied, including a discovery cohort of 142 individuals: 68 PDAC patients of all stages; 20 PDAC patients initially staged with localized disease, with blood drawn after resection for curative intent; and 54 age-matched healthy controls. A validation cohort of 121 individuals (39 cancer patients and 82 healthy controls) was studied to validate KRAS detection rates in early-stage PDAC patients. Primary outcome was circulating KRAS status as detected by droplet digital PCR. Secondary outcomes were disease-free and overall survival. Results: KRAS mutations in exoDNA, were identified in 7.4%, 66.7%, 80%, and 85% of age-matched controls, localized, locally advanced, and metastatic PDAC patients, respectively. Comparatively, mutant KRAS cfDNA was detected in 14.8%, 45.5%, 30.8%, and 57.9% of these individuals. Higher exoKRAS MAFs were associated with decreased disease-free survival in patients with localized disease. In the validation cohort, mutant KRAS exoDNA was detected in 43.6% of early-stage PDAC patients and 20% of healthy controls. Conclusions: Exosomes are a distinct source of tumor DNA that may be complementary to other liquid biopsy DNA sources. A higher percentage of patients with localized PDAC exhibited detectable KRAS mutations in exoDNA than previously reported for cfDNA. A substantial minority of healthy samples demonstrated mutant KRAS in circulation, dictating careful consideration and application of liquid biopsy findings, which may limit its utility as a broad cancer-screening method.

Minimally invasive genomic and transcriptomic profiling of visceral cancers by next-generation sequencing of circulating exosomes.

BACKGROUND: The ability to perform comprehensive profiling of cancers at high resolution is essential for precision medicine. Liquid biopsies using shed exosomes provide high-quality nucleic acids to obtain molecular characterization, which may be especially useful for visceral cancers that are not amenable to routine biopsies. PATIENTS AND METHODS: We isolated shed exosomes in biofluids from three patients with pancreaticobiliary cancers (two pancreatic, one ampullary). We performed comprehensive profiling of exoDNA and exoRNA by whole genome, exome and transcriptome sequencing using the Illumina HiSeq 2500 sequencer. We assessed the feasibility of calling copy number events, detecting mutational signatures and identifying potentially actionable mutations in exoDNA sequencing data, as well as expressed point mutations and gene fusions in exoRNA sequencing data. RESULTS: Whole-exome sequencing resulted in 95%-99% of the target regions covered at a mean depth of 133-490×. Genome-wide copy number profiles, and high estimates of tumor fractions (ranging from 56% to 82%), suggest robust representation of the tumor DNA within the shed exosomal compartment. Multiple actionable mutations, including alterations in NOTCH1 and BRCA2, were found in patient exoDNA samples. Further, RNA sequencing of shed exosomes identified the presence of expressed fusion genes, representing an avenue for elucidation of tumor neoantigens. CONCLUSIONS: We have demonstrated high-resolution profiling of the genomic and transcriptomic landscapes of visceral cancers. A wide range of cancer-derived biomarkers could be detected within the nucleic acid cargo of shed exosomes, including copy number profiles, point mutations, insertions, deletions, gene fusions and mutational signatures. Liquid biopsies using shed exosomes has the potential to be used as a clinical tool for cancer diagnosis, therapeutic stratification and treatment monitoring, precluding the need for direct tumor sampling.

Effects of guar gum ingestion on postprandial blood pressure in older adults.

OBJECTIVES: The aim of this study was to investigate the effects of guar gum on postprandial blood pressure in older people. DESIGN: A randomized, double-blind, placebo-controlled, cross-over design. SETTING: Community senior centers in B city, South Korea. PARTICIPANTS: Twenty-two older female adults aged 67 to 88 with postprandial hypotension. INTERVENTION: The participants were randomly assigned to guar gum (semi-fluid food with 9 gram) or placebo intervention during the first treatment phase. After a washout period of 1 week, the two interventions were switched to the other in the second treatment phase. MEASUREMENTS: Blood pressure was measured during both phases before having a meal and every 15 minutes during 120 minutes after a meal with automated sphygmomanometer. RESULTS: Change in systolic blood pressure (SBP) over time was significantly different between guar gum and placebo groups (F=4.07, p=0.001). Compared with placebo group, guar gum group had significantly low prevalence of postprandial hypotension (PPH) (guar gum group=18.2% vs. placebo group=72.7%; χ² =13.20, p<0.001). It also had significant difference in change of diastolic blood pressure (DBP) over time between guar gum and placebo groups (F=2.49, p=0.027). CONCLUSION: This findings show that guar gum could be effective on postprandial drops in blood pressure in older female adults.

Diffusion-weighted MRI for differentiation of benign from malignant lesions in the gallbladder.

AIM: To investigate the value of diffusion-weighted imaging (DWI) for differentiating benign from malignant gallbladder lesions. MATERIALS AND METHODS: One hundred and twenty-six patients who had undergone magnetic resonance imaging (MRI) with DWI, in whom the histopathological diagnosis of their gallbladder lesions was confirmed by biopsy or surgery were retrospectively analysed. Thirty-six malignant and 90 benign lesions were included. Two radiologists categorized gallbladder lesions into seven types on two imaging sets [T2-weighted imaging (WI) alone and combined T2WI and DWI (b = 800 s/mm(2))] according to the presence of wall thickening, layered patterns, morphology of the mass, and diffusion restriction. Disagreements were resolved in consensus. The sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of each imaging set for diagnosing gallbladder carcinoma were calculated. The diagnostic performance of each imaging set was calculated using receiver operating characteristic (ROC) curve analysis. Additionally, ADC values of malignant and benign gallbladder lesions were compared separately for 1.5 and 3 T MRI. RESULTS: The sensitivity, specificity, PPV, and NPV of diagnosis at T2WI were 97.2%, 86.7%, 74.5%, and 98.7%, respectively. The sensitivity, specificity, PPV, and NPV using combined T2WI and DWI were 97.2%, 92.2%, 83.3%, and 98.8%, respectively. Diagnostic accuracy for gallbladder carcinoma slightly improved after adding DWI, from 0.92 to 0.95 (p < 0.05). ADC values for gallbladder carcinoma were significantly lower than those for benign lesions. Mean ADC values of malignant and benign lesions were 0.97 ± 0.25 × 10(-3) and 1.72 ± 0.56 × 10(-3) mm(2)/s, respectively, at 1.5 T (p < 0.001), and 1.04 ± 0.38 × 10(-3) and 2.2 ± 0.72 × 10(-3) mm(2)/s, respectively, at 3 T (p < 0.001). CONCLUSION: DWI can improve diagnostic accuracy for differentiating benign from malignant gallbladder lesions.

Cbl-independent degradation of Met: ways to avoid agonism of bivalent Met-targeting antibody.

The Met receptor tyrosine kinase, found to be constitutively activated in many tumors, has become a leading target for cancer therapy. Disruptions in Met downregulation have been associated with aggressive tumor progression with several therapeutic strategies addressing this aspect of Met biology. Castias B-lineage lymphoma (Cbl) E3 ligase-mediated degradation, which attenuates Met signaling via ligand-dependent Met internalization, is a major negative regulator of Met expression. It is believed that one of the mechanisms by which the therapeutic anti-Met antibodies induce cancer cell death in Met overexpressing tumors is via internalization and subsequent degradation of Met from the cell surface. However, a previously reported Met-targeting antibody demonstrated intrinsic agonistic activity while being capable of inducing Cbl-mediated degradation of Met, suggesting that Cbl-mediated degradation requires receptor activation and impedes therapeutic application. We have developed a potent and selective bivalent Met-targeting antibody (SAIT301) that invokes Met degradation using an alternative regulator LRIG1. In this report, we demonstrate that LRIG1 mediates degradation of Met by SAIT301 and this degradation does not require Met activation. Furthermore, SAIT301 was able to downregulate Met and dramatically inhibit growth of tumors with low or no Cbl expression, as well as tumors with Met exon 14 deletion that prevents Met binding to Cbl. In summary, we demonstrate the enhanced therapeutic potential of a novel tumor-inhibiting anti-Met antibody, SAIT301, which utilizes a Cbl-independent, LRIG1-mediated Met degradation pathway and thereby avoids the agonism that limits the effectiveness of previously reported anti-Met antibodies.

Splenic FDG uptake predicts poor prognosis in patients with unresectable cholangiocarcinoma.

AIM: Diffuse splenic 18F-Fluorodeoxyglucose (FDG) uptake has shown to be associated with concurrent inflammation. We evaluated the prognostic value of diffuse splenic FDG uptake for predicting prognosis in cholangiocarcinoma patients. PATIENTS, METHODS: Sixty-four patients with unresectable cholangiocarcinoma performed Positron emission tomography/computed tomography (PET/CT) using FDG between July 2009 and April 2012. Patients were divided into two groups according to splenic FDG uptake relative to hepatic FDG uptake. Eleven patients showing splenic FDG uptake exceeding hepatic uptake were included in group A, while 53 patients with hepatic FDG uptake exceeding splenic uptake were included in group B. Prognostic factors for overall survival were evaluated using log-rank test. Variables with a probability of less than or equal to 0.1 on univariate analysis were considered as possible independent factors. Cox-proportional hazards model was used to analyze univariate and multivariate analysis. RESULTS: Mean standardized uptake value of the liver (Liver SUVmean)/Spleen SUVmean (L/S) ratio <1 (p = 0.0034), WBC > 10 000 (p = 0.1155) and CEA >30 (p = 0.0946) were predictors of overall survival on univariate analysis. In a subsequent multivariate analysis, L/S ratio <1 remained a significant independent predictor of poor prognosis (HR 6.0153, 95% CI, 1.7193-21.0460, p = 0.0052). CONCLUSION: Our study has shown that splenic FDG uptake could be a predictor of overall survival of unresectable cholangiocarcinoma patients.

Management of gastric epithelial neoplasia in patients requiring esophagectomy for esophageal cancer.

Esophageal squamous cell carcinoma is occasionally associated with malignancies located in other regions of the alimentary tract, as well as in the head, neck, and upper respiratory tract. The stomach is most commonly used for reconstruction of the alimentary tract after esophagectomy for esophageal cancer. When synchronous tumors are located in the stomach, it is often unsuitable for use in esophageal reconstruction. In such cases, an invasive procedure involving anastomosis between the esophagus and the colon must be performed. However, this procedure is associated with a high incidence of mortality and morbidity. Seven patients with synchronous esophageal cancer and gastric epithelial neoplasia were encountered. First, endoscopic submucosal dissection (ESD) was performed for the gastric epithelial neoplasia. Then, following successful ESD, Ivor-Lewis esophagectomy for esophageal cancer was planned 1 to 2 weeks later. A total of 11 gastric epithelial lesions were found in seven patients. En bloc resection by ESD was possible in all 11 lesions and histologically complete resection was achieved in all 11 lesions. Follow-up endoscopy was done 1-2 weeks after ESD; six patients with well-healing ulcers underwent esophagectomy the next day (8 or 15 days after ESD). In one patient with a poorly healed ulcer, a second follow-up endoscopy was done 1 week later and then esophagectomy was performed the next day (22 days after ESD). Post-surgical complications related to ESD, such as bleeding or mediastinal leak, were not seen in any of the seven patients. In patients with synchronous esophageal cancer and gastric epithelial neoplasia, ESD for gastric epithelial neoplasia followed by Ivor-Lewis esophagectomy 1 to 2 weeks later is an effective choice of treatment.

Randomized clinical trial for apogeotropic horizontal canal benign paroxysmal positional vertigo.

OBJECTIVE: To determine the immediate and long-term therapeutic efficacies of Gufoni and head-shaking maneuvers in apogeotropic type of benign paroxysmal positional vertigo involving the horizontal semicircular canal (HC-BPPV), a randomized, prospective, sham-controlled study was conducted. METHODS: In 10 nationwide dizziness clinics in Korea, 157 consecutive patients (95 women, age range: 18-89 years, mean age ± SD = 59.9 ± 13.6) with apogeotropic HC-BPPV were randomized to Gufoni (n = 52), head-shaking (n = 54), or sham maneuver (n = 51). For Gufoni maneuver, patients underwent ipsilesional side-lying and upward head-turn for migration of the debris toward the vestibule. Immediate responses were determined within 1 hour after a maximum of 2 trials of each maneuver and in the following day. The patients also had weekly follow-ups for 1 month after the initial maneuver. RESULTS: After a maximum of 2 maneuvers on the initial visit day, Gufoni (38/52, 73.1%) and head-shaking (33/53, 62.3%) maneuvers showed better responses than the sham maneuver (17/49, 34.7%). The cumulative therapeutic effects were also better with Gufoni (p < 0.001) and head-shaking (p = 0.026) maneuvers compared with the sham maneuver. However, therapeutic efficacies did not differ between the Gufoni and head-shaking groups in terms of both immediate (p = 0.129) and long-term (p = 0.239) outcomes. CONCLUSION: Using a prospective randomized trial, we demonstrated that the Gufoni and head-shaking maneuvers are effective in treating apogeotropic HC-BPPV. CLASSIFICATION OF EVIDENCE: This study provides Class II evidence that Gufoni and head-shaking maneuvers are effective in treating apogeotropic horizontal BPPV up to 1 month after initial treatment. CLINICAL TRIAL REGISTRATION: NCT00810641.

Guidelines for the diagnosis of antibody-mediated rejection in pancreas allografts-updated Banff grading schema.

The first Banff proposal for the diagnosis of pancreas rejection (Am J Transplant 2008; 8: 237) dealt primarily with the diagnosis of acute T-cell-mediated rejection (ACMR), while only tentatively addressing issues pertaining to antibody-mediated rejection (AMR). This document presents comprehensive guidelines for the diagnosis of AMR, first proposed at the 10th Banff Conference on Allograft Pathology and refined by a broad-based multidisciplinary panel. Pancreatic AMR is best identified by a combination of serological and immunohistopathological findings consisting of (i) identification of circulating donor-specific antibodies, and histopathological data including (ii) morphological evidence of microvascular tissue injury and (iii) C4d staining in interacinar capillaries. Acute AMR is diagnosed conclusively if these three elements are present, whereas a diagnosis of suspicious for AMR is rendered if only two elements are identified. The identification of only one diagnostic element is not sufficient for the diagnosis of AMR but should prompt heightened clinical vigilance. AMR and ACMR may coexist, and should be recognized and graded independently. This proposal is based on our current knowledge of the pathogenesis of pancreas rejection and currently available tools for diagnosis. A systematized clinicopathological approach to AMR is essential for the development and assessment of much needed therapeutic interventions.

Formation of vitamin A lipid droplets in pancreatic stellate cells requires albumin.

OBJECTIVE: Quiescent pancreatic stellate cells (PSCs) store vitamin A as cytoplasmic lipid droplets, and, when activated by profibrogenic stimuli, they transform into myofibroblast-like cells characterised by the loss of vitamin A droplets. Activation of stellate cells is central to fibrogenesis, but the mechanism for the formation of vitamin A droplets and its relationship to stellate cell activation remain unclear. METHODS: With use of cultured PSCs, an attempt was made to characterise the function of albumin endogenously expressed in stellate cells. RESULTS: Albumin is endogenously expressed in quiescent PSCs, localised in cytoplasmic lipid droplets, and its levels are markedly reduced after stellate cell activation. Continuous albumin expression in stellate cells is sufficient to maintain their fat-storing phenotype even after cell passages and renders cells resistant to the activating effects of transforming growth factor beta (TGFbeta). Forced expression of albumin in PSCs after passage 2 (activated PSCs) induced the re-appearance of lipid droplets and phenotypic changes, which were previously reported with retinol treatment. Retinol increases albumin synthesis in activated PSCs and the suppression of albumin expression using small interfering RNA (siRNA) abolishes retinol-induced effects. CONCLUSIONS: The data demonstrate a novel role for albumin in the formation of cytoplasmic vitamin A lipid droplets in stellate cells, and suggest that albumin may have a direct influence on stellate cell activation.

Pleckstrin homology domain interacts with Rkp1/Cpc2, a RACK1 homolog, to modulate Pck2-mediated signaling process in Schizosaccharomyces pombe.

Rkp1/Cpc2, a fission yeast RACK1 homolog, interacts with Pck2, a PKC homolog, and is involved in the regulation of pck2-mediated signaling process. The N-terminal region of split pleckstrin homology domain (nPH) in human PLC-gamma1 bound to Rkp1/Cpc2 concomitantly with Pck2. nPH inhibited kinase activity of GST-Pck2 purified from Schizosaccharomyces pombe in vitro. The lethality induced by pck2(+) overexpression was suppressed by coexpression of either rkp1(+) or nPH domain. This result suggests that Rkp1/Cpc2 interacts with PH domain-containing protein and regulates the Pck2-mediated signaling process in S. pombe.

Isolation of a novel gene from Schizosaccharomyces pombe: stm1+ encoding a seven-transmembrane loop protein that may couple with the heterotrimeric Galpha 2 protein, Gpa2.

A putative seven transmembrane protein gene, stm1(+), which is required for proper recognition of nitrogen starvation signals, was isolated as a multicopy suppressor of a ras1 synthetic lethal mutant in Schizosaccharomyces pombe. Under nitrogen-deficient conditions, transcription of the stm1 gene was induced; deletion of stm1 was associated with early entry into G(1) arrest. Under nutritionally sufficient conditions, overexpression of Stm1 inhibited vegetative cell growth, resulted in decreased intracellular cAMP levels, increased the expression of the meiosis-specific genes ste11, mei2, and mam2, and facilitated sexual development in homothallic cells. However inhibition of vegetative cell growth and reduction of cAMP levels were not observed in a deletion mutant of the heterotrimeric G protein Galpha2 gene, gpa2, that is responsible for regulating intracellular cAMP levels, a key factor in determining the sexual development in S. pombe. Stm1 protein was shown to interact with Gpa2 through its C-terminal transmembrane domains 5-7. Mutation at Lys(199) in the C-terminal domain (stm1(K199A)) abolished the Stm1 overexpression effect on lowering cAMP levels. Induction of ste11, a meiosis-specific gene transcription factor, by Stm1 overexpression was enhanced in gpa2-deleted cells but was absent in a deletion mutant of sty1, a key protein kinase that links mitotic control with environmental signals and induces stress-responsive genes. Moreover, deletion of both stm1 and ras1 caused delayed entry into G(1) arrest in S. pombe when the cells were grown in a nitrogen-deficient medium. Thus we consider that the stm1 gene can function through Gpa2-dependent and/or -independent pathways and may play a role in providing the prerequisite state for entering the pheromone-dependent differentiation cycle in which heterotrimeric Galpha1 protein, Gpa1, and Ras1 play major roles. Stm1 could function as a sentinel molecule sensing the nutritional state of the cells, stopping the proliferative cell cycle, and preparing the cell to enter meiosis under nutritionally deficient conditions.

Rkp1/Cpc2, a fission yeast RACK1 homolog, is involved in actin cytoskeleton organization through protein kinase C, Pck2, signaling.

The Rkp1/Cpc2, a fission yeast RACK1 homolog, interacted with Pck2, one of the known PKC homologs, in vivo and in vitro. The rkp1-deletion mutants (Deltarkp1) are elongated and the pck2-deletion mutant (Deltapck2) showed abnormal morphology. The double-deletion mutant (Deltarkp1Deltapck2) showed more aberrant cell shapes and was sensitive to high salt concentration. Both Deltarkp1 and Deltapck2 cells were sensitive to latrunculin B (Lat B) which inhibits actin polymerization. The cells expressing the human RACK1 homolog complemented the latrunculin B sensitivity of Deltarkp1 indicating that human RACK1 is a functional homolog of Rkp1/Cpc2. We propose that Rkp1/Cpc2 may function as a receptor for Pck2 in the regulation of actin cytoskeleton organization during cell wall synthesis and morphogenesis of Schizosaccharomyces pombe.

Enhancement of lysophosphatidic acid-induced ERK phosphorylation by phospholipase D1 via the formation of phosphatidic acid.

We made stable cell lines overexpressing PLD1 (GP-PLD1) from GP+envAm12 cell, a derivative of NIH 3T3 cell. PLD1 activity and extracellular signal-regulated kinase (ERK) phosphorylation were enhanced in GP-PLD1 cells by the treatment of lysophosphatidic acid (LPA). In contrast, these LPA-induced effects were attenuated with the pretreatment of pertussis toxin (PTX) or protein kinase C (PKC) inhibitor. Moreover, accumulation of phosphatidic acid (PA), a product of PLD action, potentiated the LPA-induced ERK activation in GP-PLD1 cells while blocking of PA production with the treatment of 1-butanol attenuated LPA-induced ERK phosphorylation. From these results, we suggest that LPA activate PLD1 through pertussis toxin-sensitive G protein and PKC-dependent pathways, then PA produced from PLD1 activation facilitate ERK phosphorylation.

Junctional epidermolysis bullosa associated with congenital localized absence of skin, and pyloric atresia in two newborn siblings.

Congenital localized absence of the skin has been observed in various subsets of inherited epidermolysis bullosa (EB). Pyloric atresia is a rare disorder that has been seen in association with EB. Ureterovesical junction obstruction is a condition unique to the association between pyloric atresia and EB. The authors describe 2 premature male siblings with pyloric atresia, congenital localized absence of the skin, urinary obstruction, and EB at birth. Electron microscopic study of the biopsy specimen from the first sibling revealed characteristic findings of EB simplex. However, prenatal diagnosis of the next sibling was made by integrin B4 mutations and the electron microscopic study of the biopsy specimen after delivery confirmed junctional EB (JEB). These cases emphasize this unusual combination of defects and limitations of electron microscopy.

Molecular mechanism of staurosporine-induced apoptosis in osteoblasts.

Staurosporine, a microbial alkaloid, is a strong inhibitor of protein kinases. We induced apoptosis in murine osteoblast MC3T3E-1 cells by exposure to the staurosporine. Staurosporine transiently increased the phosphotransferase activity of c-Jun N-terminal kinase-1 (JNK1), which in turn may activate the transcriptional activity of activating protein-1 (AP-1). We then prepared extracts from staurosporine-treated MC3T3E-1 cells and monitored the cleavage of acetyl-YVAD-AMC and acetyl-DEVD-AMC, fluorogenic substrates of caspase-1-like and caspase-3-like proteases, respectively. Staurosporine caused a significant increase in the proteolytic activity of caspase-3-like proteases, but not in the activity of caspase-1-like proteases. Furthermore, staurosporine increased the transcriptional activity of nuclear factor- kappa B (NF- kappa B). These data suggest that staurosporine-induced apoptosis in osteoblasts may occur via activation of JNK1, caspase-3-like proteases, and transcriptional factors including AP-1 and NF- kappa B.

Epidermolysis bullosa: novel and de novo premature termination codon and deletion mutations in the plectin gene predict late-onset muscular dystrophy.

Epidermolysis bullosa (EB) with late-onset muscular dystrophy (EB-MD) is a hemidesmosomal variant of EB due to mutations in the plectin gene (PLEC1). The age of onset of muscle involvement has been noted to vary from infancy to the fourth decade of life. Immunofluorescence of the patients' skin and muscle biopsies is usually negative for staining with antibodies recognizing plectin, a large cytoskeleton-associated anchorage protein. In this study we report novel plectin mutations in two families with EB. In both families, the proband was a newborn with neonatal blistering with no evidence for muscle weakness as yet. Peripheral blood DNA was isolated and examined by heteroduplex scanning strategy, protein truncation test (PTT), and/or direct sequencing of the plectin gene. One of the probands was compound heterozygote for nonsense mutations E2005X/K4460X, and the proband in the second family was compound heterozygote for deletion mutations 5083delG/2745-9del21, the latter mutation extending from -9 to +12 at the intron 22/exon 23 border. The mutations K4460X and 5083delG were not present in either one of the parents, thus being de novo events. In both cases, nonpaternity was excluded by microsatellite marker analysis. The stop codon mutations are predicted to result in the synthesis of a truncated protein lacking the carboxy-terminal globular domain of the protein and possibly causing nonsense-mediated decay of the corresponding mRNA. The 2745-9del21 deletion mutation abolishes the splice site at the intron 22/exon 23 junction, predicting abnormal splicing events. Because plectin deficiency is associated with muscular dystrophy, molecular diagnostics of the plectin gene provides prognostic value in evaluation of these patients who appear to be at risk to develop muscular dystrophy.

Molecular cloning and functional expression of a phospholipase D from cabbage (Brassica oleracea var. capitata).

We cloned and expressed a full-length cDNA encoding a phospholipase D of type alpha (PLDalpha) from cabbage. Analysis of the cDNA predicted an 812-amino-acid protein of 92.0 kDa. The deduced amino acid sequence of cabbage PLD has 83% and 80% identity with Arabidopsis PLDalpha and castor bean PLD, respectively. Expression of this cDNA clone in E. coli shows a functional PLD activity similar to that of the natural PLD.

Molecular cloning of gaf1, a Schizosaccharomyces pombe GATA factor, which can function as a transcriptional activator.

As a first step to elucidate the functions of Schizosaccharomyces pombe (S. pombe) GATA factors, we have isolated the gaf1+ gene (GATA-factor like gene) in S. pombe. The predicted amino acid (aa) sequence of Gaf1 reveals a single zinc finger domain typical of fungal GATA factors, and the zinc finger exhibits 60% aa identity to that of human GATA-1. The open reading frame of Gaf1 predicts a protein of Mr 32 kDa consisting of 290 intronless amino acids. Disruption of this gene has no effect on cell viability and growth rate. The GST-Gaf1 fusion protein binds specifically to GATA motifs of its own promoter as well as DAL7 UAS, a canonical GATA motif of Saccharomyces cerevisiae (S. cerevisiae) The specific DNA-binding activity resides within the N-terminal half of Gaf1 (Gaf1N; aa 1-120) containing the zinc finger, whereas the C-terminal half (Gaf1C; aa 121-290) contains transactivation sequences that induce the expression of the lacZ reporter when fused to the GAL4 DNA binding domain. These results demonstrate that Gaf1 may function as a transcriptional activator consisting of DNA-binding and transactivation domains.