Significant improvement of a nevus spilus-type congenital melanocytic nevus with oral selumetinib.

Giant congenital melanocytic nevi (GCMN) can be cosmetically significant and can lead to melanoma. There is no standard pharmacologic treatment for GCMN. We present the case of an 8-year-old female with kaposiform lymphangiomatosis caused by an NRAS mutation whose nevus spilus-type GCMN improved on oral selumetinib.

Reduced tissue osmolarity increases TRPV4 expression and pro-inflammatory cytokines in intervertebral disc cells.

The mechanical behaviour and cellular metabolism of intervertebral discs (IVDs) and articular cartilage are strongly influenced by their proteoglycan content and associated osmotic properties. This osmotic environment is a biophysical signal that changes with disease and may contribute to the elevated matrix breakdown and altered biologic response to loading observed in IVD degeneration and osteoarthritis. This study tested the hypothesis that changes in osmo-sensation by the transient receptor potential vallinoid-4 (TRPV4) ion channel occur with disease and contribute to the inflammatory environment found during degeneration. Immunohistochemistry on bovine IVDs from an inflammatory organ culture model were used to investigate if TRPV4 is expressed in the IVD and how expression changes with degeneration. Western blot, live-cell calcium imaging, and qRT-PCR were used to investigate whether osmolarity changes or tumour necrosis factor α (TNFα) regulate TRPV4 expression, and how altered TRPV4 expression influences calcium signalling and pro-inflammatory cytokine expression. TRPV4 expression correlated with TNFα expression, and was increased when cultured in reduced medium osmolarity and unaltered with TNFα-stimulation. Increased TRPV4 expression increased the calcium flux following TRPV4 activation and increased interleukin-1ß (IL-1ß) and IL-6 gene expression in IVD cells. TRPV4 expression was qualitatively elevated in regions of aggrecan depletion in degenerated human IVDs. Collectively, results suggest that reduced tissue osmolarity, likely following proteoglycan degradation, can increase TRPV4 signalling and enhance pro-inflammatory cytokine production, suggesting changes in TRPV4 mediated osmo-sensation may contribute to the progressive matrix breakdown in disease.

Wafer-scale surface roughening for enhanced light extraction of high power AlGaInP-based light-emitting diodes.

A new approach to surface roughening was established and optimized in this paper for enhancing the light extraction of high power AlGaInP-based LEDs, by combining ultraviolet (UV) assisted imprinting with dry etching techniques. In this approach, hexagonal arrays of cone-shaped etch pits are fabricated on the surface of LEDs, forming gradient effective-refractive-index that can mitigate the emission loss due to total internal reflection and therefore increase the light extraction efficiency. For comparison, wafer-scale FLAT-LEDs without any surface roughening, WET-LEDs with surface roughened by wet etching, and DRY-LEDs with surface roughened by varying the dry etching time of the AlGaInP layer, were fabricated and characterized. The average output power for wafer-scale FLAT-LEDs, WET-LEDs, and DRY3-LEDs (optimal) at 350 mA was found to be 102, 140, and 172 mW, respectively, and there was no noticeable electrical degradation with the WET-LEDs and DRY-LEDs. The light output was increased by 37.3% with wet etching, and 68.6% with dry etching surface roughening, respectively, without compromising the electrical performance of LEDs. A total number of 1600 LED chips were tested for each type of LEDs. The yield of chips with an optical output power of 120 mW and above was 0.3% (4 chips), 42.8% (684 chips), and 90.1% (1441 chips) for FLAT-LEDs, WET-LEDs, and DRY3-LEDs, respectively. The dry etching surface roughening approach developed here is potentially useful for the industrial mass production of wafer-scale high power LEDs.

Anal sphincter fibrillation: is this a new finding that identifies resistant chronic anal fissures that respond to botulinum toxin?

AIM: Anal fissures can be resistant to treatment and some patients may undergo several trials of medical therapy before definitive surgery. It would be useful to identify predictors of poor response to medical therapy. This study assesses the role of anorectal physiological criteria to identify patients with anal fissure predicted to fail botulinum toxin (BT) treatment. METHOD: A retrospective analysis of anorectal physiological data collected for patients with resistant chronic anal fissures, referred to one consultant surgeon between 2007 and 2011, was undertaken. These were correlated with treatment plans and healing rates. RESULTS: Twenty-five patients with idiopathic chronic anal fissures underwent anorectal physiology studies and were subsequently treated with BT injection. Eleven had a characteristic high-frequency low-amplitude 'saw tooth' waveform or anal sphincter fibrillation (ASF) and higher anal sphincter pressures. Nine (82%) of these patients had resolution of their anal fissure symptoms following treatment with BT. Of 14 patients with no evidence of ASF and a greater range of anal sphincter pressures, only one (7%) had resolution following BT. CONCLUSION: ASF appears to be an anorectal physiological criterion that helps predict response of anal fissures to BT injection. This could help streamline fissure management.

Fgf8 is required for outgrowth and patterning of the limbs.

The expression pattern and activity of fibroblast growth factor-8 (FGF8) in experimental assays indicate that it has important roles in limb development, but early embryonic lethality resulting from mutation of Fgf8 in the germ line of mice has prevented direct assessment of these roles. Here we report that conditional disruption of Fgf8 in the forelimb of developing mice bypasses embryonic lethality and reveals a requirement for Fgf8 in the formation of the stylopod, anterior zeugopod and autopod. Lack of Fgf8 in the apical ectodermal ridge (AER) alters expression of other Fgf genes, Shh and Bmp2.

Analysis of ultrasonographic images using a deep learning-based model as ancillary diagnostic tool for diagnosing gallbladder polyps.

BACKGROUND: Accurately diagnosing gallbladder polyps (GBPs) is important to avoid misdiagnosis and overtreatment. AIMS: To evaluate the efficacy of a deep learning model and the accuracy of a computer-aided diagnosis by physicians for diagnosing GBPs. METHODS: This retrospective cohort study was conducted from January 2006 to September 2021, and 3,754 images from 263 patients were analyzed. The outcome of this study was the efficacy of the developed deep learning model in discriminating neoplastic GBPs (NGBPs) from non-NGBPs and to evaluate the accuracy of a computer-aided diagnosis with that made by physicians. RESULTS: The efficacy of discriminating NGBPs from non- NGBPs using deep learning was 0.944 (accuracy, 0.858; sensitivity, 0.856; specificity, 0.861). The accuracy of an unassisted diagnosis of GBP was 0.634, and that of a computer-aided diagnosis was 0.785 (p<0.001). There were no significant differences in the accuracy of a computer-aided diagnosis between experienced (0.835) and inexperienced (0.772) physicians (p = 0.251). A computer-aided diagnosis significantly assisted inexperienced physicians (0.772 vs. 0.614; p < 0.001) but not experienced physicians. CONCLUSIONS: Deep learning-based models discriminate NGBPs from non- NGBPs with excellent accuracy. As ancillary diagnostic tools, they may assist inexperienced physicians in improving their diagnostic accuracy.

Ara h 2: crystal structure and IgE binding distinguish two subpopulations of peanut allergic patients by epitope diversity.

BACKGROUND: Peanut allergy affects 1% of the population and causes the most fatal food-related anaphylactic reactions. The protein Ara h 2 is the most potent peanut allergen recognized by 80-90% of peanut allergic patients. METHODS: The crystal structure of the major peanut allergen Ara h 2 was determined for the first time at 2.7 Å resolution using a customized maltose-binding protein (MBP)-fusion system. IgE antibody binding to the MBP fusion construct vs the natural allergen was compared by ELISA using sera from peanut allergic patients. RESULTS: The structure of Ara h 2 is a five-helix bundle held together by four disulfide bonds and related to the prolamin protein superfamily. The fold is most similar to other amylase and trypsin inhibitors. The MBP--Ara h 2 fusion construct was positively recognized by IgE from 76% of allergic patients (25/33). Two populations of patients could be identified. Subpopulation 1 (n = 14) showed an excellent correlation of IgE antibody binding to natural vs recombinant Ara h 2. Subpopulation 2 (n = 15) showed significantly reduced IgE binding to the MBP fusion protein. Interestingly, about 20% of the IgE binding in subpopulation 2 could be recovered by increasing the distance between MBP and Ara h 2 in a second construct. DISCUSSION: The reduced IgE binding to the MBP--Ara h 2 of subpopulation 2 indicates that the MBP molecule protects an immunodominant epitope region near the first helix of Ara h 2. Residues involved in the epitope(s) are suggested by the crystal structure. The MBP--Ara h 2 fusion constructs will be useful to further elucidate the relevance of certain epitopes to peanut allergy.

Ultrastructure of the yeast actin cytoskeleton and its association with the plasma membrane.

We characterized the yeast actin cytoskeleton at the ultrastructural level using immunoelectron microscopy. Anti-actin antibodies primarily labeled dense, patchlike cortical structures and cytoplasmic cables. This localization recapitulates results obtained with immunofluorescence light microscopy, but at much higher resolution. Immuno-EM double-labeling experiments were conducted with antibodies to actin together with antibodies to the actin binding proteins Abp1p and cofilin. As expected from immunofluorescence experiments, Abp1p, cofilin, and actin colocalized in immuno-EM to the dense patchlike structures but not to the cables. In this way, we can unambiguously identify the patches as the cortical actin cytoskeleton. The cortical actin patches were observed to be associated with the cell surface via an invagination of plasma membrane. This novel cortical cytoskeleton-plasma membrane interface appears to consist of a fingerlike invagination of plasma membrane around which actin filaments and actin binding proteins are organized. We propose a possible role for this unique cortical structure in wall growth and osmotic regulation.

Cofilin is an essential component of the yeast cortical cytoskeleton.

We have biochemically identified the Saccharomyces cerevisiae homologue of the mammalian actin binding protein cofilin. Cofilin and related proteins isolated from diverse organisms are low molecular weight proteins (15-20 kD) that possess several activities in vitro. All bind to monomeric actin and sever filaments, and some can stably associate with filaments. In this study, we demonstrate using viscosity, sedimentation, and actin assembly rate assays that yeast cofilin (16 kD) possesses all of these properties. Cloning and sequencing of the S. cerevisiae cofilin gene (COF1) revealed that yeast cofilin is 41% identical in amino acid sequence to mammalian cofilin and, surprisingly, has homology to a protein outside the family of cofilin-like proteins. The NH2-terminal 16kD of Abp1p, a 65-kD yeast protein identified by its ability to bind to actin filaments, is 23% identical to yeast cofilin. Immunofluorescence experiments showed that, like Abp1p, cofilin is associated with the membrane actin cytoskeleton. A complete disruption of the COF1 gene was created in diploid cells. Sporulation and tetrad analysis revealed that yeast cofilin has an essential function in vivo. Although Abp1p shares sequence similarity with cofilin and has the same distribution as cofilin in the cell, multiple copies of the ABP1 gene cannot compensate for the loss of cofilin. Thus, cofilin and Abp1p are structurally related but functionally distinct components of the yeast membrane cytoskeleton.

NF-kappaB inhibition enhances caspase-3 degradation of Akt1 and apoptosis in response to camptothecin.

DNA damaging agents, such as camptothecin, and ionizing radiation (IR), can induce both NF-kappaB activation and apoptosis, however, the mechanism of their inter-regulation is not yet clear. In the present study, we discovered that Akt1 is degraded when cells deficient in Ataxia Telangiectasia mutated (ATM) were treated to CPT for apoptosis induction. While CPT-induced NF-kappaB activation could not be detected in ATM-deficient AT5BIVA cells, caspase-3 activation occurred and was even further enhanced by pretreatment with proteasome inhibitor-1 (Pro1), a NF-kappaB inhibitor. In contrast, activation of NF-kappaB but not of caspase-3 by CPT could be found in normal MRC5CV1 cells. NF-kappaB inhibition by Pro1, dominant negative mutant IkappaBalpha (S32/36) or p65 (N250), however, induced the caspase-3 activation in the normal cells, indicating the role of ATM-mediated NF-kappaB activation against cell apoptosis. On the other hand, interestingly, CPT significantly reduced the level of Akt1, this effect further enhanced by Pro1 pretreatment in AT5BIVA cells. In MRC5CV1 cells, however, Akt1 level could be reduced only when CPT and NF-kappaB inhibitors were co-treated to the cells, and this reversed by DEVD-cho treatment, demonstrating the caspase-3-mediated Akt1 degradation. Moreover, although MRC5CV1 cells were much more resistant to CPT compared with AT5BIVA, wortmannin and LY294002 significantly increased the chemosensitivity of MRC5CV1 cells to CPT. Given the accumulating evidences demonstrating Akt as a promising anticancer therapeutic target, all these results suggest that DNA damage induced apoptosis could be regulated by ATM-mediated NF-kappaB activation, and that Akt1 degradation be necessarily required for this apoptotic process.

C-reactive protein binds to integrin α2 and Fcγ receptor I, leading to breast cell adhesion and breast cancer progression.

C-reactive protein (CRP) is an acute phase protein synthesized upon the inflammatory responses, associated with breast cancer. The process of tumor cell invasion and metastasis involves the adherence of cells to the extracellular matrix via integrin as a receptor for matrix molecules. The present study investigated the role of CRP in the adhesive phenotype of breast cells and the underlying mechanisms. Here, we first showed that CRP induces adhesion of MCF10A human breast epithelial cells through the activation of integrin α2 signaling. Expression of integrin α2 was induced by CRP in which transcription factors c-fos and SP1 may be involved. Binding of CRP with integrin α2 leads to the activation of focal adhesion kinase (FAK), paxillin and ERKs. CRP also binds to an Fcγ receptor Fcγ receptor I (FcγRI), and induces activation of paxillin, FAK and ERKs. Integrin α2 and FAK have crucial roles in the adhesive and invasive phenotypes as well as MMP-9 upregulation induced by CRP in MCF10A cells. Treatment with an inflammatory lipid sphingosine-1-phosphate induced CRP, which may be secreted and exert an autocrine effect by binding to FcγRI and integrin α2. Involvement of CRP in adhesion, invasion, anchorage-independent growth and upregulation of integrin α2, paxillin and FAK was observed in MDA-MB-231 triple-negative human breast cancer (TNBC) cells. Using an in vivo invasion model and an orthotopic mouse tumor model with MDA-MB-231 cells, we showed that CRP has an important role in intravasation and tumor growth in vivo, demonstrating the in vivo relevance of our in vitro results. The present study elucidates a critical molecular basis between CRP, integrin α2 and FcγRI pathways in MCF10A breast cells and MDA-MB-231 TNBC cells, thereby providing useful information on CRP-induced aggressiveness of breast cells in the inflammatory microenvironment.

Complementation analysis of fatty acid oxidation disorders.

We assayed [9,10(n)-3H]palmitate oxidation by fibroblast monolayers from patients with fatty acid oxidation disorders. Activities in the different disorders were (percent control): short-chain acyl-coenzyme A (CoA) dehydrogenase deficiency (115%), medium chain acyl-CoA dehydrogenase deficiency (18%), long-chain acyl-CoA dehydrogenase deficiency (28%), multiple acyl-CoA dehydrogenation disorder, mild and severe variants (49% and 7%), and palmityl-carnitine transferase deficiency (4%). Multiple acyl-CoA dehydrogenation disorder, medium chain acyl-CoA dehydrogenase-deficient lines, and long-chain acyl-CoA dehydrogenase-deficient lines all complemented one another after polyethylene glycol fusion, with average activity increases of 31-83%. We detected two complementation groups in the severe multiple acyl-CoA dehydrogenation disorder lines, consistent with deficiencies of either electron transfer flavoprotein or electron transfer flavoprotein:ubiquinone oxidoreductase. The metabolic block in the latter cell lines is threefold more severe than in the former (P less than 0.001). No intragenic complementation was observed within either group. We assigned two patients with previously unreported severe multiple acyl-CoA dehydrogenation disorder to the electron transfer flavoprotein:ubiquinone oxido-reductase-deficient group.

Short-chain acyl-coenzyme A dehydrogenase deficiency. Clinical and biochemical studies in two patients.

We describe two patients with short-chain acyl-coenzyme A (CoA) dehydrogenase (SCADH) deficiency. Neonate I excreted large amounts of ethylmalonate and methylsuccinate; ethylmalonate excretion increased after a medium-chain triglyceride load. Neonate II died postnatally and excreted ethylmalonate, butyrate, 3-hydroxybutyrate, adipate, and lactate. Both neonates' fibroblasts catabolized [1-14C]butyrate poorly (29-64% of control). Neonate I had moderately decreased [1-14C]octanoate catabolism (43-60% of control), while neonate II oxidized this substrate normally; both catabolized radiolabeled palmitate, succinate, and/or leucine normally. Cell sonicates from neonates I and II dehydrogenated [2,3-3H]butyryl-CoA poorly (41 and 53% of control) and [2,3-3H]octanoyl-CoA more effectively (59 and 95% of control). Mitochondrial acyl-CoA dehydrogenase (ADH) activities with butyryl- and octanoyl-CoAs were 37 and 56% of control in neonate I, and 47 and 81% of control in neonate II, respectively. Monospecific medium-chain ADH (MCADH) antisera inhibited MCADH activity towards both butyryl- and octanoyl-CoAs, revealing SCADH activities to be 1 and 11% of control for neonates I and II, respectively. Fibroblast SCADH and MCADH activities were normal in an adult female with muscular SCADH deficiency.

Metacyclic variant surface glycoprotein genes of Trypanosoma brucei subsp. rhodesiense are activated in situ, and their expression is transcriptionally regulated.

During the metacyclic stage in the life cycle of Trypanosoma brucei subsp. rhodesiense, the expression of variant surface glycoproteins (VSGs) is restricted to a small subset of antigenic types. Previously we identified cDNAs for the VSGs expressed in metacyclic variant antigen types (MVATs) 4 and 7 and found that these VSG genes do not rearrange when expressed at the metacyclic stage (M. J. Lenardo, A. C. Rice-Ficht, G. Kelly, K. Esser, and J. E. Donelson, Proc. Nathl. Acad Sci. USA 81:6642-6646, 1984). We now provide further evidence that these genes do not rearrange and demonstrate that their 5' upstream regions lack the 72 to 76-base-pair repeats which are considered the substrate for duplication and transposition events. Pulsed field gradient electrophoresis showed that the MVAT VSG genes were located on the largest chromosome-sized DNA molecules, and the lack of the MVAT 4 gene in one of two different serodemes suggested that one mechanism for the evolution of MVAT repertoires is gene deletion. When MVATs were inoculated into the bloodstream of a mammalian host by a bite from the insect vector, they rapidly switched into nonmetacyclic VSG types. We found that this switch was accomplished by a loss of MVAT RNA concomitant with the loss of metacyclic VSGs. Transcription studies with isolated metacyclic nuclei showed that the MVAT genes were expressed in situ from a single locus and were regulated at the level of transcription.

Transformation of hepatitis C antiviral treatment in a national healthcare system following the introduction of direct antiviral agents.

BACKGROUND: Highly effective direct antiviral agents (DAAs) for hepatitis C virus (HCV) were introduced recently. Their utilisation has been limited by high cost and low access to care. AIM: To describe the effect of DAAs on HCV treatment and cure rates in the United States Veterans Affairs (VA) national healthcare system. METHODS: We identified all HCV antiviral treatment regimens initiated from 1 January 1999 to 31 December 2015 (n = 105 369) in the VA national healthcare system, and determined if they resulted in sustained virological response (SVR). RESULTS: HCV antiviral treatment rates were low (1981-6679 treatments/year) in the interferon era (1999-2010). The introduction of simeprevir and sofosbuvir in 2013 and ledipasvir/sofosbuvir and paritaprevir/ombitasvir/ritonavir/dasabuvir in 2014 were followed by increases in annual treatment rates to 9180 in 2014 and 31 028 in 2015. The number of patients achieving SVR was 1313 in 2010, the last year of the interferon era, and increased 5.6-fold to 7377 in 2014 and 21-fold to 28 084 in 2015. The proportion of treated patients who achieved SVR increased from 19.2% in 1999 and 36.0% in 2010 to 90.5% in 2015. Within 2015, monthly treatment rates ranged from 727 in July to 6868 in September correlating with the availability of funds for DAAs. CONCLUSIONS: DAAs resulted in a 21-fold increase in the number of patients achieving HCV cure. Treatment rates in 2015 were limited primarily by the availability of funds. Further increases in funding and cost reductions of DAAs in 2016 suggest that the VA could cure the majority of HCV-infected Veterans in VA care within the next few years.

Renal Cell Cancer in a European Regional Renal Transplant Population: Is There a Role for Immediate Native Renal Radiological Surveillance Before and After Transplantation?

BACKGROUND: The risk of the development of renal cell cancer (RCC) in renal transplant recipients is several times higher than the general population. There can often be a delay between initial radiological imaging and patients undergoing renal transplantation. We present and evaluate the prevalence and clinical characteristics of RCC in renal transplant recipients at a single UK transplant center, with particular focus on tumors diagnosed in the immediate post-operative period, that is, likely present before transplantation. METHODS: This is a retrospective cohort study examining all renal transplant recipients with the diagnosis of RCC of native and/or graft kidneys followed up in a single UK transplant center. RESULTS: Between January 2002 and April 2014, 1386 patients underwent renal transplantation. 19 of 1386 patients had development of RCC (1.4%): 17 native and 2 graft tumors. The mean interval between pre-operative native renal imaging and transplantation was 3.5 years in 13 of 19 patients (range, 1-10 years). Six patients had no documented renal imaging before their renal transplant. The median time from transplantation to diagnosis of RCC was 5 years (range, 1 month to 30 years). In 5 patients (26.3%), RSS developed within 6 months of undergoing renal transplantation. CONCLUSIONS: In our study, we identified several patients with RCC diagnosed shortly after surgery, which raised the possibility that this was present before transplantation. With transplant recipients at increased risk of development of RCC and early detection key in the management of RCC, there appears to be a role for native renal radiological screening for patients undergoing renal transplantation.

Gα12 gep oncogene deregulation of p53-responsive microRNAs promotes epithelial-mesenchymal transition of hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) has a poor prognosis owing to aggressive phenotype. Gα12 gep oncogene product couples to G-protein-coupled receptors, whose ligand levels are frequently increased in tumor microenvironments. Here, we report Gα12 overexpression in human HCC and the resultant induction of zinc-finger E-box-binding homeobox 1 (ZEB1) as mediated by microRNA deregulation. Gα12 expression was higher in HCC than surrounding non-tumorous tissue. Transfection of Huh7 cell with an activated mutant of Gα12 (Gα12QL) deregulated microRNA (miRNA or miR)-200b/a/429, -194-2/192 and -194-1/215 clusters in the miRNome. cDNA microarray analyses disclosed the targets affected by Gα12 gene knockout. An integrative network of miRNAs and mRNA changes enabled us to predict ZEB1 as a key molecule governed by Gα12. Decreases of miR-200a/b, -192 and -215 by Gα12 caused ZEB1 induction. The ability of Gα12 to decrease p53 levels, as a result of activating protein-1 (AP-1)/c-Jun-mediated mouse double minute 2 homolog induction, contributed to transcriptional deregulation of the miRNAs. Gα12QL induced ZEB1 and other epithelial-mesenchymal transition markers with fibroblastoid phenotype change. Consistently, transfection with miR-200b, -192 or -215 mimic prevented the ability of Gα12QL to increase tumor cell migration/invasion. In xenograft studies, sustained knockdown of Gα12 decreased the overall growth rate and average volume of tumors derived from SK-Hep1 cell (mesenchymal-typed). In HCC patients, miR-192, -215 and/or -200a were deregulated with microvascular invasion or growth advantage. In the HCC samples with higher Gα12 level, a correlation existed in the comparison of relative changes of Gα12 and ZEB1. In conclusion, Gα12 overexpressed in HCC causes ZEB1 induction by deregulating p53-responsive miRNAs, which may facilitate epithelial-mesenchymal transition and growth of liver tumor. These findings highlight the significance of Gα12 upregulation in liver tumor progression, implicating Gα12 as an attractive therapeutic target.

Chemopreventive allylthiopyridazine derivatives induce apoptosis in SK-Hep-1 hepatocarcinoma cells through a caspase-3-dependent mechanism.

Dietary organosulphur compounds including diallylsulphide, a component of garlic oil, were shown to inhibit the proliferation of tumour cells. Since hepatocellular carcinoma is one of the most lethal malignancies and there is no effective preventive measure to date, we wished to pursue the chemopreventive potential of the synthetic allylthiopyridazine derivatives (K compounds) on hepatocarcinoma cells. Here, we report that the K compounds efficiently inhibited SK-Hep-1 cell proliferation through induction of apoptosis. Increased chain length at the 3-position of allylthiopyridazine ring improved the potency of growth inhibition. K compounds downregulated Bcl-2, while Bax remained unchanged, reducing the ratio of Bcl-2 to Bax. We also provide evidence that the K compound-induced apoptosis involves cytochrome c release and caspase-3 activation. These results suggest that the allythiopyridazine derivatives, especially 3-propoxy-6-allylthiopyridazine, induce apoptosis in SK-Hep-1 cells through a caspase-3-dependent mechanism, which may contribute to the chemopreventive function for hepatocellular carcinoma.

Cloning and characterization of an abundant Plasmodium knowlesi antigen which cross reacts with Gambian sera.

A 110 kDa Plasmodium knowlesi antigen, termed PK110, has been identified on the basis of messenger RNA abundance in late schizonts. Most Plasmodium genes previously cloned have been identified by immune sera, which have selected immunodominant antigens composed of repeating epitopes. Although PK110 was not selected by immune sera, it also contains amino acid repeats, indicating that this structure may be a common feature of malarial proteins. Determination of 296 codons in the PK110 gene revealed the presence of thirteen tandem repeats of twelve amino acids whose consensus sequence is E E T Q K T V E P E Q T. A termination codon interrupts the fourteenth repeat, indicating that these repeats are at the C-terminus of the protein. Indirect immunofluorescence experiments with sera raised against the lambda gt11 fusion protein indicate that PK110 is present in intra-erythrocytic late schizonts. Cloned PK110 is recognized by Gambian sera, and shares epitopes with Plasmodium ovale. PK110 does not cross react immunologically or by DNA hybridization with Plasmodium falciparum.

Induction of CTL responses and identification of a novel epitope of hepatitis B virus surface antigens in C57BL/6 mice immunized with recombinant vaccinia viruses.

MHC class I-restricted cytotoxic T lymphocyte (CTL) response to hepatitis B virus (HBV) surface antigens (HBsAg) has been suggested to play essential roles in viral clearance and pathogenesis of HBV-induced hepatitis. In the present study, we analyzed CTL responses to endogenously synthesized or exogenously introduced HBsAg in C57BL/6 mice (H-2(b)). We show that the endogenously synthesized surface antigens of adr-type HBV encoded by recombinant vaccinia virus efficiently elicit CTL responses in C57BL/6 mice previously defined as non-responders to vaccinia-HBV immunization. We also show that two peptides, S(179-186) (FVQWFVGL) and S(208-216) (ILSPFLPLL), serve as effective motifs for CTL response in H-2(b) system after in vitro restimulation of the primed T cells with either of the two synthetic peptides. S(208-215) has recently been identified as a CTL epitope which could be produced by exogenous pathway only, in contrast to the current result, while S(179-186) appeared a novel epitope for CTL response. In addition, we show that soluble HBsAg also elicits CTL responses in H-2(b) mice upon in vitro restimulation with the two peptides, although less efficiently compared with the recombinant vaccinia viruses. These findings may provide an efficient experimental system for studying H-2(b)-restricted immune responses against endogenously synthesized and exogenously introduced HBsAg.