[Screening and preliminary validation of biomarkers in sputum-negative pulmonary tuberculosis based on positron emission tomography/computed tomography and transcriptomics].

Objective: To screen and perform preliminary clinical validation of biomarkers of activity based on positron emission tomography/computed tomography (PET-CT) and transcriptomics in sputum-negative pulmonary tuberculosis lesion tissue. Methods: Nine patients with sputum-negative pulmonary tuberculosis treated surgically at the Shanghai Public Health Clinical Center for Thoracic Surgery from January 1, 2017 to December 31, 2019 were retrospectively collected as the discovery group, including four males and five females, aged 20-57 years (mean 36 years). All of the patients underwent PET-CT scanning before surgery, and the resected specimens were postoperatively classified according to preoperative PET-CT. The resected specimens were divided into areas with increased fluorodeoxyglucose (FDG) metabolism (SUVmax>3) and areas with normal FDG metabolism (SUVmax ≤ 3) according to the preoperative PET-CT performance. After sample processing, total RNA was extracted from the tissues of different regions, and then whole gene transcriptome sequencing was performed. Bioinformatics analysis of the two sets of data was performed to discover the expression profiles of the differences in whole gene transcriptome data between the two regions and to screen for candidate biomarkers. Eighty patients with sputum-negative pulmonary tuberculosis admitted to Shanghai Public Health Clinical Center from January 1, 2019 to January 1, 2021 were retrospectively collected as the validation group, including 37 males and 43 females, aged 20-62 years, with an average age of 39 years. The validation group was divided into a group with increased SUV (n=40) and a group without lesions on CT imaging (n=40). Enzyme-linked immunosorbent assay (ELISA) was used to determine the protein levels of candidate biomarkers in the peripheral plasma of patients. The effect of biomarkers was assessed using subject operating characteristic (ROC) curves. Student's t-test was used to determine whether the difference in protein levels between the two groups was statistically significant. Results: Bioinformatics analysis revealed that the expression levels of C1QB, CCL19, CCL5 and HLA-DMB correlated with the metabolic activity of sputum-negative tuberculosis lesion tissue. Further screening and validation by the validation group confirmed that the difference in C1QB protein levels in the peripheral plasma of patients was statistically significant between the group with increased SUV and the group without lesions on CT imaging [(3.55±0.34) mg/L vs. (2.75±0.21) mg/L, t=4.12, P<0.001]. And the ROC curve showed that the area under the curve for C1QB protein levels was 0.731, which had potential clinical value. Conclusion: The C1QB protein level can be used to assess the activity of lesions in patients with sputum-negative tuberculosis and is a potential biomarker.

Carcinogenesis of Male Oral Submucous Fibrosis Alters Salivary Microbiomes.

Most oral squamous cell carcinoma (OSCC) tumors arise from oral premalignant lesions. Oral submucous fibrosis (OSF), usually occurring in male chewers of betel quid, is a premalignant stromal disease characterized by a high malignant transformation rate and high prevalence. Although a relationship between the inhabited microbiome and carcinogenesis has been proposed, no detailed information regarding the oral microbiome of patients with OSF exists; the changes of the salivary microbiome during cancer formation remain unclear. This study compared the salivary microbiomes of male patients with OSCC and a predisposing OSF background (OSCC-OSF group) and those with OSF only (OSF group). The results of high-throughput sequencing of the bacterial 16S rRNA gene indicated that OSF-related carcinogenesis and smoking status significantly contributed to phylogenetic composition variations in the salivary microbiome, leading to considerable reductions in species richness and phylogenetic diversity. The microbiome profile of OSF-related malignancy was associated with increased microbial stochastic fluctuation, which dominated the salivary microbiome assembly and caused species co-occurrence network collapse. Artificial intelligence selection algorithms consistently identified 5 key species in the OSCC-OSF group: Porphyromonas catoniae, Prevotella multisaccharivorax, Prevotella sp. HMT-300, Mitsuokella sp. HMT-131, and Treponema sp. HMT-927. Robust accuracy in predicting oral carcinogenesis was obtained with our exploratory and validation data sets. In functional analysis, the microbiome of the OSCC-OSF group had greater potential for S-adenosyl-l-methionine and norspermidine synthesis but lower potential for l-ornithine and pyrimidine deoxyribonucleotide synthesis and formaldehyde metabolism. These findings indicated that the salivary microbiome plays important roles in modulating microbial metabolites during oral carcinogenesis. In conclusion, our results provided new insights into salivary microbiome alterations during the malignant transformation of OSF.

[Clinical analysis of 71 cases of anti-N-methyl-D-aspartate receptor encephalitis in children].

Objective: To investigate the clinical features, treatment strategies and long term outcomes of children with anti-N-methyl-D-aspartate receptor (NMDAR) encephalitis. Methods: The data of clinical features, auxiliary examinations, treatments and prognosis in children with anti-NMDAR encephalitis in Xiangya Hospital of Central South University from March 2014 to October 2017 were collected and retrospectively analyzed. A total of 71 patients were enrolled, including 33 males and 38 females. The youngest age of onset was 4 months old, and the age of onset was (9±4) years. The first-line immunotherapy treatment for anti-NMDAR encephalitis was short course corticosteroid (high-dose impulse therapy and oral maintenance therapy for 1 month in acute period) and (or) immunoglobulin. The clinical evaluation was performed 2 weeks after first-line immunotherapy treatment. The second-line immunotherapy treatment, including rituximab and (or) cyclophosphamide, would be started if the symptoms did not improve significantly and the modified Rankin scale (mRS) score ≥3. All patients were followed up and evaluated for prognosis. T-test, Mann-Whitney U, Chi square test and Fisher's exact probability method were used for comparison between good outcome group and poor outcome group, first-line immunotherapy group and first-line immunotherapy combined with second-line immunotherapy group. Results: The more common clinical manifestations were psychiatric symptoms (n=61, 86%), dyskinesia (n=55, 77%) and convulsions (n=51, 72%). Two cases (3%) had tumors. Electroencephalogram (EEG), cerebro-spinal fluid (CSF) and brain magnetic resonance imaging (MRI) studies were abnormal in 83% (59/71), 39% (27/69) and 38% (27/71) patients, respectively. For the treatment regimens, all the 71 patients underwent first-line immunotherapy, resulting in improvement within 14 days in 40 cases (56%), and 1 case (1%) died. The rest 30 cases (42%) received second-line immunotherapy. The patients were followed up for 5.0-41.8 months, with a median of 19.3 months. At the last follow-up, 49 cases (69%) recovered completely, 15 cases (21%) had mild disability, 6 cases (8%) had severe disability, 1 case (1%) died and 3 cases (4%) had relapse. There were significant differences between the groups with good prognosis and poor prognosis on admission to pediatric intensive care unit (PICU) and consciousness disorder (10/64 vs. 5/7, 39/64 vs. 7/7, P=0.047, 0.004). There were significant differences between first-line immunotherapy group and the first-line combined second-line immunotherapy group on admission to PICU, consciousness disorder, sleep disorder and first mRS score (12% (5/41) vs. 33% (10/30), 44% (18/41) vs. 93% (28/30), 56% (23/41) vs. 90% (27/30), 3 (1-5) vs. 4 (3-5), respectively; χ(2)=4.645, 18.555, 9.560, Z=5.184, P=0.031, <0.01, 0.002, <0.01, respectively). Conclusions: Anti-NMDAR encephalitis can occur in all ages of children. The most common clinical manifestations are psychotic symptoms, dyskinesia and convulsions. Paraneoplastic cases are less common in children. Immunotherapy is effective. The second-line immunotherapy should be given after the failure of first-line therapy (mRS score≥3).

MiR-99a exerts anti-metastasis through inhibiting myotubularin-related protein 3 expression in oral cancer.

OBJECTIVE: We aimed at studying the role of the most deregulated miR-99a, identifying its downstream targets, and exploring the clinical potential of miR-99a and its target(s) in oral cancer. SUBJECTS AND METHODS: Following confirmation of miR-99a deregulation in nine oral lines and 26 pairwise clinical specimens, miR-99a-manipulated oral cancer cells were subjected to cell proliferation, migration, invasion, and in vivo murine metastasis assays. We characterized putative miR-99a target(s) using luciferase reporter assays and genetic manipulation. The inverse relation of miR-99a and its target(s) was examined in clinical specimens using real-time PCR and Western blot analysis. RESULTS: MiR-99a down-regulation was confirmed both in tested oral cancer cell lines and clinical specimens. Ectopic miR-99a expression inhibited oral cancer cell migration and invasion. Anti-miR-99a, silencing miR-99a functions, had the opposite effect. Myotubularin-related protein 3 (MTMR3) with one evolutionarily conserved seed region in the 3'-untranslated region was a novel miR-99a target. Depleting MTMR3 expression significantly reduced cell proliferation, migration, or invasion. There was an inverse expression of miR-99a and MTMR3 protein in oral cancer lines and clinical specimens. CONCLUSION: miR-99a repressed oral cancer cell migration and invasion partly through decreasing MTMR3 expression. MTMR3 may serve as a therapeutic target for oral cancer treatment.

Lack of modulatory function of coding nucleotide polymorphism S100A2_185G>A in oral squamous cell carcinoma.

OBJECTIVE: S100A2, a Ca(2+) -binding protein with two EF-hands, is a tumor suppressor in oral cancer. Helix III flanking the C-terminal EF-hand is implicated to participate in the interaction of S100A2 and its target(s). The aim of this study was to examine if the coding sequence polymorphism S100A2_185G>A, leading to the peptide 62 substitution of asparagine (AAC, A allele) for serine (AGC, G allele) in helix III, had modulation effects on S100A-mediated tumor suppression. SUBJECTS AND METHODS: We sequenced the coding sequence of S100A2 gene in normal oral keratinocytes (NOKs), dysplastic oral keratinocytes (DOKs), eight oral cancer lines, and 54 pairwise oral cancer specimens. We also compared the in vitro anti-tumor effect of wildtype (G allele) and variant (A allele) S100A2 expression using cell proliferation, migration, invasion, and colony formation assays. RESULTS: With the exception of CAL27 and SCC-15 cancer lines being heterozygotes of A and G alleles, the remaining oral cells were homozygotic in G alleles. No alterations of anti-growth, anti-migration, anti-invasion, and anti-colony formation were observed between variant and wildtype cells. Moreover, no minor S100A2_185A allele was detected in 54-pairwise clinical specimens. CONCLUSION: The coding sequence polymorphism S100A2_185G>A had no regulatory role in S100A2-mediated tumor suppression in oral cancer.

Angiostatin K1-3 induces E-selectin via AP1 and Ets1: a mediator for anti-angiogenic action of K1-3.

BACKGROUND: Angiostatin, a circulating angiogenic inhibitor, is an internal fragment of plasminogen and consists of several isoforms, K1-3 included. We previously showed that K1-3 was the most potent angiostatin to induce E-selectin mRNA expression. The purpose of this study was to identify the mechanism responsible for K1-3-induced E-selectin expression and investigate the role of E-selectin in the anti-angiogenic action of K1-3. METHODS AND RESULTS: Quantitative real time RT-PCR and Western blotting analyses confirmed a time-dependent increase of E-selectin mRNA and protein induced by K1-3. Subcellular fractionation and immunofluorescence microscopy showed the co-localization of K1-3-induced E-selectin with caveolin 1 (Cav1) in lipid rafts in which E-selectin may behave as a signaling receptor. Promoter-driven reporter assays and site-directed mutagenesis showed that K1-3 induced E-selectin expression via promoter activation and AP1 and Ets-1 binding sites in the proximal E-selectin promoter were required for E-selectin induction. The in vivo binding of both protein complexes to the proximal promoter was confirmed by chromatin immunoprecipitation (ChIP). Although K1-3 induced the activation of ERK1/2 and JNK, only repression of JNK activation attenuated the induction of E-selectin by K1-3. A modulatory role of E-selectin in the anti-angiogenic action of K1-3 was manifested by both overexpression and knockdown of E-selectin followed by cell proliferation assay. CONCLUSIONS: We show that K1-3 induced E-selectin expression via AP1 and Ets-1 binding to the proximal E-selectin promoter (-356/+1), which was positively mediated by JNK activation. Our findings also demonstrate E-selectin as a novel target for the anti-angiogenic therapy.

Microarray profiling of gene expression patterns in bladder tumor cells treated with genistein.

Microarray technology was used to gain an insight into the molecular events of tumor cell growth inhibition mediated by the soy isoflavone genistein. For this, a susceptible bladder tumor line TCCSUP was treated with the inhibitory dose (50 microM) of genistein for various periods of time, followed by mRNA isolations, cDNA probe preparations, and hybridization individually to cDNA chips containing 884 sequence-verified known human genes. After analyzing the hybridization signals with a simple quantitative method developed by this study, we detected that egr-1, whose expression has been associated with proliferation and differentiation, was transiently induced and this expression pattern was later confirmed by RT-PCR. Thus, microarray technology is a reliable and powerful tool for profiling gene expression patterns in many biological systems related to cancer. We further detected many groups of genes with distinct expression profiles and most of them encode for proteins that regulate the signal transduction or the cell cycle pathways. These genes warrant further investigation as regards their roles in the susceptibility of the tumor cell line to the antitumor drug.

Distinct regulation of genes by bFGF and VEGF-A in endothelial cells.

A finely tuned balance of angiogenic inhibitors and inducers controls the activity of angiogenesis characterized by proliferation, migration and differentiation of endothelial cells. Among many angiogenic factors, basic fibroblast growth factor (bFGF) was first identified to be angiogenic whereas vascular endothelial growth factor A (VEGF-A) is an endothelial cell specific mitogen. In addition to being a specific mitogen, VEGF-A is also known as a vascular permeability factor. The majority of growth factors transduce their mitogenic signals from cell surface to nucleus where gene expression occurs. Whether these ligands utilize a distinct or a common molecular pathway to exert their biological effects on human endothelial cells remains elusive. We thus studied the expression profile of 884 human genes under the influence of either bFGF or VEGF-A alone in the context of human endothelial cells. A total of ninety-four genes were differentially regulated by more than two folds. The expression patterns of 32 genes are similar between the treatment of either factor alone whereas those of the remaining 62 genes are only regulated by one but not the other factor. Their function in the control of angiogenesis will be discussed and apoptotic signaling in the regulation of angiogenesis is also implicated.

Utilization of distinct signaling pathways by receptors for vascular endothelial cell growth factor and other mitogens in the induction of endothelial cell proliferation.

This study was initiated to identify signaling proteins used by the receptors for vascular endothelial cell growth factor KDR/Flk1, and Flt1. Two-hybrid cloning and immunoprecipitation from human umbilical vein endothelial cells (HUVEC) showed that KDR binds to and promotes the tyrosine phosphorylation of phospholipase Cgamma (PLCgamma). Neither placental growth factor, which activates Flt1, epidermal growth factor (EGF), or fibroblast growth factor (FGF) induced tyrosine phosphorylation of PLCgamma, indicating that KDR is uniquely important to PLCgamma activation in HUVEC. By signaling through KDR, VEGF promoted the tyrosine phosphorylation of focal adhesion kinase, induced activation of Akt, protein kinase Cepsilon (PKCepsilon), mitogen-activated protein kinase (MAPK), and promoted thymidine incorporation into DNA. VEGF activates PLCgamma, PKCepsilon, and phosphatidylinositol 3-kinase independently of one another. MEK, PLCgamma, and to a lesser extent PKC, are in the pathway through which KDR activates MAPK. PLCgamma or PKC inhibitors did not affect FGF- or EGF-mediated MAPK activation. MAPK/ERK kinase inhibition diminished VEGF-, FGF-, and EGF-promoted thymidine incorporation into DNA. However, blockade of PKC diminished thymidine incorporation into DNA induced by VEGF but not FGF or EGF. Signaling through KDR/Flk1 activates signaling pathways not utilized by other mitogens to induce proliferation of HUVEC.

VRAP is an adaptor protein that binds KDR, a receptor for vascular endothelial cell growth factor.

A protein that binds the intracellular domain of KDR (KDR-IC), a receptor for vascular endothelial cell growth factor (VEGF), was identified by two-hybrid screening. Two-hybrid mapping showed that the VEGF receptor-associated protein (VRAP) interacted with tyrosine 951 in the kinase insert domain of KDR. Northern blot analysis identified multiple VRAP transcripts in peripheral leukocytes, spleen, thymus, heart, lung, and human umbilical vein endothelial cells (HUVEC). The predominant VRAP mRNA encodes a 389-amino acid protein that contains an SH2 domain and a C-terminal proline-rich motif. In HUVEC, VEGF promotes association of VRAP with KDR. Phospholipase C gamma and phosphatidylinositol 3-kinase, effector proteins that are downstream of KDR and important to VEGF-induced endothelial cell survival and proliferative responses, associate constitutively with VRAP. These observations identify VRAP as an adaptor that recruits cytoplasmic signaling proteins to KDR, which plays an important role in normal and pathological angiogenesis.

[Neurootologic study of acoustic neuroma].

OBJECTIVE: To summarize the outcome of neurootologic examination of acoustic neuroma and study of diagnostic significance of neurootologic examination on acoustic neuroma. METHOD: 13 patients were examined with pure tune test, acoustic immittance, electronystagmography, acoustic brainstem evoked response, electrocochleogram, otoacoustic emissions and facial electroneurography. RESULT: All patients had subjective hearing loss. One patient had normal pure tone audiogram, other 4 patients had normal pure tone test at the frequency < 2000 Hz. The outcome of acoustic immittance is various. 5 patients were normal in caloric test, but all were abnormal in visual-vestibular optokinetic response. ABR are positive in 90.9% of cases, but 2 patients had no response. 7 patients were abnormal in facial electroneurography, 3 (of 3) eletrocochleogram and 2 (of 2) otoacoustic emissions revealed retrocochlear pathology. CONCLUSION: Neurootologic examination played important role in filtering diagnosis of acoustic neuroma, results should be considered synthetically, acoustic neuroma should not be ruled out if any one test was negative.

Tumor necrosis factor employs a protein-tyrosine phosphatase to inhibit activation of KDR and vascular endothelial cell growth factor-induced endothelial cell proliferation.

Vascular endothelial cell growth factor (VEGF) binds to and promotes the activation of one of its receptors, KDR. Once activated, KDR induces the tyrosine phosphorylation of cytoplasmic signaling proteins that are important to endothelial cell proliferation. In human umbilical vein endothelial cells (HUVECs), tumor necrosis factor (TNF) inhibits the phosphorylation and activation of KDR. The ability of TNF to diminish VEGF-stimulated KDR activity was impaired by sodium orthovanadate, suggesting that the inhibitory activity of TNF was mediated by a protein-tyrosine phosphatase. KDR-initiated responses specifically associated with endothelial cell proliferation, mitogen-activated protein kinase activation and DNA synthesis, were also inhibited by TNF, and this was reversed by sodium orthovanadate. Stimulation of HUVECs with TNF induced association of the SHP-1 protein-tyrosine phosphatase with KDR, identifying this phosphatase as a candidate negative regulator of VEGF signal transduction. Heterologous receptor inactivation mediated by a protein-tyrosine phosphatase provides insight into how TNF may inhibit endothelial cell proliferative responses and modulate angiogenesis in pathological settings.

The proteasome regulates caspase-dependent and caspase-independent protease cascades during apoptosis of MO7e hematopoietic progenitor cells.

Withdrawal of trophic support from growth factor-dependent MO7e human myeloid progenitor cells induces apoptosis characterized by DNA fragmentation and degradation of the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs). Inhibitors of caspase (ICE) protease family members did not inhibit apoptosis or DNA fragmentation induced by factor withdrawal, but blocked degradation of DNA-PKcs. Thus, caspase activity accounts for only a component of the apoptotic program in MO7e hematopoietic cells. The protease inhibitor TPCK, but not other protease inhibitors, blocked DNA fragmentation, but not degradation of DNA-PKcs during apoptosis of MO7e cells. Thus, caspase-independent and caspase-dependent protease cascades mediate distinct features of MO7e cell apoptosis. The proteasome inhibitors calpain inhibitor I and lactacystin promoted DNA fragmentation, degradation of DNA-PKcs and apoptosis of MO7e cells. The ability of lactacystin to promote DNA fragmentation was abrogated by TPCK, but not by caspase inhibitors, whereas the ability of lactacystin to promote degradation of DNA-PKcs was blocked by caspase inhibitors, but not by TPCK. Thus, caspase-dependent and caspase-independent protease cascades are downstream of and regulated by the proteasome, which plays a central role in regulating the multiple protease cascades that induce apoptosis.

Two-hybrid cloning of a gene encoding TNF receptor-associated protein 2, a protein that interacts with the intracellular domain of the type 1 TNF receptor: identity with subunit 2 of the 26S protease.

A protein that binds the intracellular domain of the type 1 TNFR (TNFR-1IC) has been identified by two-hybrid cloning. The 97-kDa TNFR-associated protein, TRAP2, shows sequence identity with internal amino acid sequences from subunit 2 of the 26S protease. TRAP2 antiserum recognizes subunit 2 of the 26S protease, which is consistent with the identity of these proteins. TRAP2 antiserum interacted with the 97-kDa protein in HeLa cell lysates and cytosol, the latter observation showing that TRAP2 resides in the same cellular compartment as TNFR-1IC. A fusion of glutathione-S-transferase and TNFR-1IC (GST-TNFR-1IC) precipitated TRAP2 from a HeLa cell lysate; conversely, GST-TRAP2 precipitated TNFR-1 from such a lysate. These observations show that the proteins interact in the cellular milieu. After in vitro transcription/translation and 35S labeling, TRAP2 was precipitated from a cellfree system by GST-TNFR-1IC, showing that TNFR-1IC and TRAP2 interact directly. TRAP2 was also precipitated from the cellfree translation system by a GST fusion containing the N-terminal half of TNFR-1IC, but not by a GST fusion containing the C-terminal half of TNFR-1IC that contains a "death domain" that plays an obligatory role in signaling cytotoxicity. The ability of deletion mutants of TNFR-1IC to interact with TRAP2 was tested using the two-hybrid system. This also showed that the amino acid sequences that mediate binding reside outside of the death domain in TNFR-1IC. The demonstration that a subunit of the 26S protease binds TNFR-1 may identify a novel TNF-signaling pathway.

Fluoride at mitogenic doses induces a sustained activation of p44mapk, but not p42mapk, in human TE85 osteosarcoma cells.

Fluoride, at micromolar concentrations, stimulates bone cell proliferation in vitro. In this study, we sought to test whether fluoride at mitogenic doses increases the tyrosyl phosphorylation level and specific activity of a mitogen-activated protein kinase (MAPK) in human TE85 osteosarcoma cells. Analysis by immunoprecipitation with antiphosphotyrosine antibody followed by Western analysis using an anti-pan extracellular signal-regulated kinase antibody revealed that fluoride at the optimal mitogenic dose (i.e. 100 mumol/L) induced a time-dependent increase in the steady state tyrosyl phosphorylation level of p44mapk, but not p42mapk, with the maximal increase (4- to 13-fold) after 1-3 h fluoride treatment. The effect was sustained in that a 9-fold increase was seen after 12 h of the fluoride treatment. The sustained nature of the effect is consistent with an inhibition of dephosphorylation rather than a direct stimulation of phosphorylation. The fluoride effect on the tyrosyl phosphorylation level of p44mapk was dose dependent, with the optimal dose being 100 mumol/L fluoride. The mitogenic dose of fluoride also increased the specific activity and the in-gel kinase activity of p44mapk, but not that of p42mapk, in a time-dependent manner similar to the effect on the p44mapk tyrosyl phosphorylation level. Fluoride at the same micromolar doses did not increase cell proliferation, tyrosyl phosphorylation, or specific activity of any MAPK in human skin foreskin fibroblasts, which are fluoride-nonresponsive cells. Consistent with the interpretation that the effect of fluoride on the steady state tyrosyl phosphorylation level of p44mapk is a consequence of an inhibition of a phosphotyrosyl phosphatase (PTP), mitogenic doses of orthovanadate, a bone cell mitogen and a PTP inhibitor, also increased the steady state tyrosyl phosphorylation level of p44mapk, but not p42mapk, in a time-dependent sustained manner similar to that observed with fluoride. Together, these findings support the concept that inhibition of a PTP activity in bone cells could lead to an activation of MAPK activity.

Inhibition of tumor necrosis factor signal transduction in endothelial cells by dimethylaminopurine.

Tumor necrosis factor (TNF) promotes diverse responses in endothelial cells that are important to the host response to infections and malignancies; however, less is known of the postreceptor events important to TNF action in endothelial cells than in many other cell types. Since phosphorylation cascades are implicated in cytokine signaling, the effects of the protein kinase inhibitor dimethylaminopurine (DMAP) on TNF action in bovine aortic endothelial cells (BAEC) were investigated. In BAEC, TNF promotes phosphorylation of eukaryotic initiation factor 4E (eIF-4E), c-Jun N-terminal kinase (JNK) and ceramide-activated protein kinase activities, Jun-b expression, prostacyclin production, and, when protein synthesis is inhibited, cytotoxicity. DMAP abrogated or significantly attenuated each of these responses to TNF, without affecting the specific binding of TNF to its receptors. Histamine, another agent active in the endothelium, promotes phosphorylation of elongation factor-2 (EF-2) and prostacyclin production, but not phosphorylation of eIF-4E in BAEC. Histamine-stimulated EF-2 phosphorylation was not inhibited and prostacyclin production was unaffected by DMAP. These observations demonstrate that a distinct signal transduction cascade, which can be selectively inhibited by DMAP, promotes the response of BAEC to TNF. Thus, we have identified a reagent, DMAP, that may be useful for characterizing the TNF signal transduction pathway.

Development of cellular resistance to pp60v-src kinase-induced cell death.

The v-src gene of Rous sarcoma virus (RSV) encodes pp60v-src, a tyrosine kinase that can initiate cellular transformation. High levels of v-src gene expression can either be cytotoxic or the cause of altered expression of cellular genes. Examination of cytotoxic thresholds is difficult because cells expressing high levels of a cytotoxic oncogene will die. To evaluate quantitatively the cytotoxicity of pp60v-src on growth, we amplified two different v-src genes, under the control of the human hsp70B heat shock promoter to establish cell clones with varying copy numbers of the heat-inducible v-src gene. The viability of cell lines over a prolonged period of time depended on the particular src gene, the expression of v-src mRNA, synthesis of the pp60v-src protein and, most importantly, the tyrosine kinase activity of the pp60v-src protein. We found a relatively sharp threshold in v-src-encoded tyrosine kinase activity above which cell viability rapidly declines. However, over time, tyrosine kinase activity was exponentially suppressed at about a 10-fold higher rate than pp60v-src protein during passage. Our results indicate that homeostasis of tyrosine phosphorylation is important for cell viability, that perturbation of this balance results in cell mortality, and that cells can evolve to accommodate overexpression of oncogene by downregulating the level of tyrosine kinase activity.

[An preliminary study on hepato-protective action of seed oil of Hippophae rhamnoides L. (HR) and mechanism of the action].

The seed oil of Hippophae rhamnoides markedly inhibits the rise of MDA in liver of mice and rats induced by CCl4, AAP and ethyl alcohol, decreases significantly the activity of SGPT and SGOT, and markedly checks the depletion of GSH in liver of mice induced by AAP. The microscopic and electron-microscopic examination has shown that the seed oil can lighten liver injury.