Cancer targeting Gene-Viro-Therapy of liver carcinoma by dual-regulated oncolytic adenovirus armed with TRAIL gene.

Liver cancer is a common and aggressive malignancy, but available treatment approaches remain suboptimal. Cancer targeting Gene-Viro-Therapy (CTGVT) has shown excellent anti-tumor effects in a preclinical study. CTGVT takes advantage of both gene therapy and virotherapy by incorporating an anti-tumor gene into an oncolytic virus vector. Potent anti-tumor activity is achieved by virus replication and exogenous expression of the anti-tumor gene. A dual-regulated oncolytic adenoviral vector designated Ad·AFP·E1A·E1B (Δ55) (Ad·AFP·D55 for short thereafter) was constructed by replacing the native viral E1A promoter with the simian virus 40 enhancer/α-fetoprotein (AFP) composite promoter (AFPep) based on an E1B-55K-deleted construct, ZD55. Ad·AFP·D55 showed specific replication and cytotoxicity in AFP-positive hepatoma cells. It also showed enhanced safety in normal cells when compared with the mono-regulated vector ZD55. To improve the anti-hepatoma activities of the virus, the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) gene was introduced into Ad·AFP·D55. Ad·AFP·D55-TRAIL exhibited remarkable anti-tumor activities in vitro and in vivo. Treatment with Ad·AFP·D55-TRAIL can induce both autophagy owing to the Ad·AFP·D55 vector and caspase-dependent apoptosis owing to the TRAIL protein. Therefore, Ad·AFP·D55-TRAIL could be a potential anti-hepatoma agent with anti-tumor activities due to AFP-specific replication and TRAIL-induced apoptosis.

A meta-analysis of the efficacy and safety of recombinant activated factor VII for patients with acute intracerebral hemorrhage without hemophilia.

Hematoma growth is common in intracerebral hemorrhage (ICH) and is associated with a poor outcome for patients. To evaluate the efficacy and safety of recombinant activated factor VII (rFVIIa) used as a hemostatic agent in patients with ICH without hemophilia, we searched Medline, Scopus, the Cochrane Library, Clinicaltrials.gov and the Stroke Trials Directory. Five randomized controlled trials were selected for analysis. Although rFVIIa can reduce the change in ICH volume, there was no significant difference in mortality, modified Rankin Scale (mRS) score or extended Glasgow Outcome Scale (GOS-E) score in patients treated with rFVIIa or placebo. There was a significant increase in arterial thromboembolic adverse events (TAE) in patients treated with rFVIIa. There was an increase in deep vein thrombosis in patients with spontaneous ICH and traumatic ICH. In conclusion, the use of rFVIIa reduces the growth of the hematoma but does not improve patient survival or functional outcome after ICH; in addition, rFVIIa increases the incidence of arterial TAE.

Changes and effects of plasma arginine vasopressin in traumatic brain injury.

BACKGROUND: Traumatic brain injury (TBI) is a common disease accompanied by chronic morbidity and mortality. The pathological mechanism and effective pharmacological treatments of TBI remain undetermined. It is suggested that AVP is involved in TBI. It is thus interesting to investigate the changes and effects of plasma AVP in clinical trials. METHODS: The serum concentrations of AVP, serum electrolytes, and serum osmolarity in a total of 23 TBI patients were dynamically monitored (on admission, Day 1, Day 3, and Day 5). Relationship between AVP and severity of brain injury and functional outcome were evaluated, respectively. RESULTS: The mean AVP serum concentrations in the TBI group were significantly higher than those recorded in the control (CTRL) group on intensive care unit (ICU) admission and Day 1 (p<0.05). On Day 3 and Day 5, the differences between those groups were not significant (p>0.05). The negative correlations were found between sodium and AVP (r=-0.35; p<0.05) and between osmolarity and AVP (r=-0.42; p<0.05). In poor outcome group, the mean AVP serum concentrations were significantly higher than in good outcome group and CTRL group (p<0.05). A statistically significant correlation was also found between AVP on ICU admission and the initial Glasgow Coma Scale (r=0.47; p<0.05). CONCLUSION: We suggest that AVP is involved in the pathophysiology process of secondary brain damage after TBI. It seems that AVP antagonist is a promising target for the treatment of TBI, while further studies should be carried out.

The measurement of solute diffusion coefficients in dilute liquid alloys: the influence of unit gravity and g-jitter on buoyancy convection.

Liquid diffusion experiments conducted on the MIR space station using the Canadian Space Agency QUELD II processing facility and the microgravity isolation mount (MIM) showed that g-jitter significantly increased the measured solute diffusion coefficients. In some experiments, milli-g forced vibration was superimposed on the sample when isolated from the ambient g-jitter; this resulted in markedly increased solute transport. To further explore the effects arising in these long capillary diffusion couples from the absence of unit-gravity and the presence of the forced g-jitter, the effects of a 1 milli-g forcing vibration on the mass transport in a 1.5 mm diameter long capillary diffusion couple have been simulated. In addition, to increase understanding of the role of unit gravity in determining the extent to which gravity can influence measured diffusion coefficient values, comparative experiments involving gold, silver, and antimony diffusing in liquid lead have been carried out using a similar QUELD II facility to that employed in the QUELD II/MIM/MIR campaign but under terrestrial conditions. It was found that buoyancy-driven convection may still persist in the liquid even when conditions are arranged for a continuously decreasing density gradient up the axis of a vertical long capillary diffusion couple due to the presence of small radial temperature gradients.

Strong magnetic field effect on the dissolution process of tetragonal lysozyme crystals.

Either a homogeneous or inhomogeneous magnetic field has been known to dampen the protein crystal growth. To date the mechanism is not clear. However, it was generally proposed that the magnetic field may dampen the convection in the solution, resulting in a reduced crystal growth rate and possibly a good crystal quality, similar to the case of protein crystal growth in space. To understand the mechanism of the magnetic field effect on protein crystal growth, further explorations on the magnetic field effect on protein solution, on the processes of crystal growth and dissolution, and on different crystallization (solution) systems, should be valuable. In this paper we present our recent efforts to study magnetic field effects on the dissolution processes of tetragonal lysozyme crystals under a strong magnetic field. A layer of oriented tetragonal lysozyme crystals was prepared under a temperature gradient and magnetic field, after that the crystals were dissolved by increasing the temperature of the solution. The lysozyme molecules will diffuse upwards due to the steep concentration gradient at the lower side of the cell caused by the dissolution. The evolution of the concentration in the solution was measured in-situ using a Mach-Zehnder interferometer. The results confirmed that the dissolution process of the crystals was slowed by the magnetic field. Judging from the concentration evolution versus time at different positions in the solution, we concluded that the apparent diffusion coefficient of lysozyme molecules was decreased by the magnetic field. The results were discussed using a suspended crystal model in the initial dissolution stage.

An investigation of magnetic field effects on the dissolution of lysozyme crystal and related phenomena.

It is now widely known that a magnetic field, either homogeneous or inhomogeneous, depresses the growth process of protein crystals. In this report, the dissolution process of tetragonal lysozyme crystals is also confirmed to be depressed by a homogeneous magnetic field (inhomogeneity <1.5%). The dissolution process was monitored using a Mach-Zehnder interferometer. The results showed that the concentration change during the dissolution process was slowed in a magnetic field compared with that in the absence of a magnetic field. It was concluded that the diffusion coefficient of the lysozyme molecules in the solution was decreased by the magnetic field. The decrease in the diffusion coefficient may contribute to the slowed growth process. The changes in the spatial concentration distribution under a vertical temperature gradient before crystallization in the absence of a magnetic field was also studied. The concentration in the lower, colder part of the cell increased, while it decreased in the upper, hotter part, a similar phenomenon to that discovered by previous investigators in an isothermal supersaturated solution system. Aggregated domain formation is proposed to explain the concentration redistribution before crystal growth and a suspended crystal model is proposed to explain the decrease of diffusivity in a magnetic field.

[Determination of salicin in extract of willow bark by high performance liquid chromatography].

An HPLC method for the determination of salicin in extract of willow bark is described. Chromatographic analysis was carried out on a Kromasil C18, 5 microns column(4.6 mm i.d. x 250 mm) with methanol-0.01 mol/L KH2PO4 buffer (pH 4.01) (15:85, volume ratio) as mobile phase. The detection wavelength was 265 nm. Salicin was extracted from samples with methanol-water(50:50, volume ratio), and centrifuged. Ten microL of supernatant were injected. The average recoveries were from 96.1% to 101.2% (n = 5), and the relative standard deviation (RSD) was 1.43%. The method is simple, rapid and accurate.

[Laser-induced fluorescence detection in micro-column separation].

Laser-induced fluorescence detection (LIFD) has been used extensively in micro-column separation due to its high sensitivity and selectivity. A review is presented on the present status and the trends of development of LIF detector with 52 references cited. The detection cells of the LIFD are mainly discussed.

[Expression and purification of recombinant human interleukin-11 in Pichia pastoris].

OBJECTIVE: To express recombinant human interleukin-11 (rhIL-11) in methylotropic yeast Pichia pastoris. METHODS: By designing and synthesizing an artificial gene for IL-11, the expression vector pPICZ alpha-A-IL-11 was constructed and introduced into Pichia pastoris by linearized electroporation. The rhIL-11 protein was identified by ELISA and SDS-PAGE analysis. The bioactivity was analyzed by B9-11 cell line. A combination of liquid chromatography was developed to purify the rhIL-11 from ferment supernatant. RESULTS: The nucleotide sequence analysis indicated that the sequence of cloned artificial IL-11 gene accorded with that of designed; the secreted yield of rhIL-11 by yeast Pichia pastoris KM71-2424 in flask reached 60 mg/L. The biological activity of IL-11 in yeast supernatant and E. coli standard determined by B9-11 was 5.5 x 10(7) U/mg and 2.2 x 10(7) U/mg respectively. The rhIL-11 was purified to electrophoretic purity by a combination of liquid chromatography. CONCLUSION: The human IL-11 artificial gene was obtained and successfully expressed in the Pichia pastoris(KM71-2424). The biological activity of IL-11 in yeast supernatant was significantly higher than that of E. coli standard. The rhIL-11 was purified to electrophoretic purity.

[Study on the characters of hydrogen bonds in protein and nucleic acid of the breast cancer tissues].

Some remarkable spectral differences are observed between the normal breast tissues and breast cancer tissues, including those of association pattern and degree of the hydrogen bonds in the protein and nucleic acid biomacromolecules. These differences are found in the relative intensity, absorption position and shape of the characteristic bands: (1) amide I band, stretching vibration bands of N-H group and C-O(H) in the residues of some amino-acids, and (2) the relative intensity of asymmetric stretching vibration of the phosphodiester group in nucleic acid. The hydrogen bond is the major force which maintain and promote the formation of the high structure of protein and nucleic acid. It is possible to diagnose the breast cancer and forecast the possibility of canceration using above spectral characters of the hydrogen bond with other spectral characters.

[Effect of organic solvents on DNA conformation by fluorescent probe method].

In this paper, the conformation of calf thymus DNA(DNA) is investigated in some common solvents by spectrofluorimetry. The result of fluorescence spectra and scatchard plot indicate that the binding constant between DNA and ethidium bromide (EB) will decrease with the increasing concentration of ethanol, and the number of binding sites will increase. The change of binding constant elucidate that the interaction between DNA and EB will become poor, and the change of binding sites shows that the conformation of DNA has transformed from B-form to A-form. In the course of this change, the distance between both neighboring base plan will be decreased. When the EB molecule just fit into the interval, the fluorescence intensity will be maximum. When DNA shrink further, the EB molecules will be excluded from DNA, then the fluorescence intensity will decrease. Although the conformation of DNA will be changed in organic solvents, it will not denaturate or dehelix, because the absorption of DNA will not increase greatly with the increasing concentration of ethanol. The melting temperature of DNA decreases continually as the weight fraction of ethanol concentration is increased. It shows that the DNA will be more distabilized in ethanol, so it helps to explain some phenomena of life and physiology. Meanwhile, the results offer an opportunity to further study the interaction DNA and insoluble drugs.

Expression of matrix metalloproteinases and the tissue inhibitors of metalloproteinases and their local invasiveness and metastasis in Chinese human pancreatic cancer.

BACKGROUND AND OBJECTIVES: The objective was to evaluate the potent role of matrix metalloproteinases(MMPs) and the tissue inhibitors of metalloproteinases(TIMPs) in processes leading to metastasis and local invasiveness of Chinese human ductal adenocarcinomas of the pancreas. We also evaluated a possible biological association between the gene expression and clinical manifestations. METHODS: Northern blot and in situ hybridization have shown MMP and TIMP gene expression in the pancreas and alterations associated with neoplastic transformation. Fifteen cases of surgical pancreatic specimens were examined, using cDNA probes to MMP2, MMP9, and TIMP1. Findings were correlated with the size of tumor section, CA19-9, pathological classification, thrombosis, and infiltration of capsule and lymphonoids. RESULTS: Increased levels of the mRNA of MMP2, MMP9, and TIMP1 genes, MMP2 approximately MMP9

Clinical classification of chronic prostatitis: a preliminary investigation.

AIM: To propose a practical clinical classification for the chronic prostatitis (CP). METHODS: The clinical features and the findings in the expressed prostatic secretion (EPS) in 804 cases of CP patients were retrospectively analyzed. RESULTS: Four types of CP were identified based on the clinical manifestations and the amounts of white blood cells (WBC) and lecithin in EPS. They were the latent type (85 cases; 10.6%), the common type (423 cases; 52.6%), the persisting type (104 cases; 12.9%), and the active type (192 cases, 23.9%). The therapeutic efficacy for these 4 subtypes were 40.4%, 76.8%, 30.8% and 37%, respectively; a statistical difference was noticed between the common type and the persisting type (P < 0.01). CONCLUSION: The method of classification proposed by the authors may help clinicians in the diagnosis and predicting the prognosis of CP.

No-scalpel vasectomy outside China.

Since 1985, the no-scalpel vasectomy technique has been widely used outside China. The prevalence of this technique has helped to increase the acceptability of male sterilization in many parts of the world. More than 5000 physicians in twenty-five developing countries have been trained in the no-scalpel vasectomy technique. In the United States in 1995, nearly one third of vasectomies employed the no-scalpel technique, and in the whole Northern American region, a total of 1100 doctors have been made familiar with the technique. Doctors believe that there are several advantages of the no-scalpel technique, including no incision, no stitches, faster procedure, faster recovery, less chance of bleeding, less discomfort and high efficacy. The key steps of the technique include fixation of the vas and infiltration anaesthesia of the spermatic cord, as well as grasping, delivering and isolating the vas. No-scalpel technique provides a good approach to expose the vas, in conjunction with which, different vas-end occlusion methods may be used.

Prediction of dry weight through changes in blood volume and plasma cyclic 3',5'-guanosine monophosphate in patients under maintenance hemodialysis.

Dry weight evaluation is generally made from clinical observation of body weight (BW) changes, edema, blood pressure, and chest radiograph. In fact, 25-50% of patients on chronic hemodialysis had an incorrectly determined dry weight. To predict dry weight, twenty stable patients on regular hemodialysis were enrolled to investigate the correlation among dry weight, hematocrit, blood volume (BV), and vasoactive hormones including plasma renin activity (PRA), aldosterone (PA), and cyclic 3',5'-guanosine monophosphate (cGMP) values. BV was estimated by an infrared light reflection method. PRA, PA, and plasma cGMP were determined by commercial radioimmunoassay kits. The results showed significantly decreasing plasma cGMP values toward the end of hemodialysis compared with before hemodialysis (15.76 +/- 3.56 pmol/ml vs 33.57 +/- 3.98 pmol/ml; p < 0.05). A significant correlation exists between changes in plasma cGMP values and BV (p < 0.05). In addition, no significant correlation exists between changes in plasma cGMP and BW. A good correlation was found between changes in BV and hematocrit throughout dialysis (r = -0.774; p < 0.001). PRA and PA values predict neither BV nor BW changes. All patients were treated to attain a further ultrafiltration of 0.5 to 1.0 L after reaching dry weight, and we found that the critical point in blood pressure drop occurred when BV decreased by 8% or when plasma cGMP values decreased by 50% from their initial values. Continuous BV monitoring with infrared light reflection and detecting of cGMP throughout hemodialysis could help predict dry weight and avoid dialysis hypotension.

Gene structure and properties of TIGR, an olfactomedin-related glycoprotein cloned from glucocorticoid-induced trabecular meshwork cells.

Expression of the trabecular meshwork inducible glucocorticoid response (TIGR) gene progressively increases from barely detectable levels to greater than 2% of total cellular mRNA over 10 days exposure of trabecular meshwork (TM) cells to dexamethasone. Cycloheximide blocked most of the TIGR mRNA induction, suggesting a requirement for ongoing protein synthesis. The genomic structure of TIGR (approximately 20 kilobases) consists of 3 exons, and a 5-kilobase promoter region that contains 13 predicted hormone response elements, including several glucocorticoid regulatory elements, and other potentially important regulatory motifs. TIGR cDNA encodes an olfactomedin-related glycoprotein of 504 amino acids with motifs for N- and O-linked glycosylation, glycosaminoglycan initiation, hyaluronic acid binding, and leucine zippers. Recombinant TIGR (rTIGR) showed oligomerization and specific binding to TM cells. Anti-rTIGR antibody detected multiple translational/post-translational forms of TIGR produced by the cells (including secreted 66 kDa/55 kDa glycoproteins/proteins in the media and 55 kDa cellular proteins), whereas Northern blot showed a single mRNA species. The findings suggest potential mechanisms by which TIGR could obstruct the aqueous humor fluid flow and participate in the pathogenesis of glaucoma.

Potent growth inhibition of human tumor cells in culture by arginine deiminase purified from a culture medium of a Mycoplasma-infected cell line.

Two kinds of growth-inhibitory substances were found in culture of a Rous sarcoma virus-transformed rat liver cell line, RSV-BRL. The two substances were purified from the serum-free culture medium and identified as transforming growth factor beta 1 and Mycoplasma-derived arginine deiminase (EC 3.5.3.6), respectively. The arginine deiminase was an acid-labile but dithiothreitol-resistant protein with a molecular weight of 45,000 and pI 4.7. Its Km value for L-arginine was 0.3 mM, which is about 30 times lower than that of bovine liver arginase. It was stable and active under culture conditions. When added into cultures, the arginine deiminase inhibited the growth of various human cancer cell lines at a dose of 5 ng/ml or higher by depleting L-arginine in the culture media. This effective dose was about 1000 times lower than that of bovine liver arginase. These results suggested the possibility of chemotherapeutic use of arginine deiminase for human cancers.

Structure-function studies on the bradykinin potentiating peptide from Chinese snake venom (Agkistrodon halys Pallas).

A bradykinin potentiating peptide (BPP) was purified from the Chinese snake venom (Agkistrodon halys Pallas). The amino acid sequence of this BPP was determined to be pyroGlu-Gly-Arg-Pro-Pro-Gly-Pro-Pro-Ile-Pro-Pro. Removal of the N-terminal residue with pyroglutamate aminopeptidase enhanced two-fold the activity of BPP, the resulting despyroGlu-BPP gradually lost its activity on further Edman degradation. However, around 90% of the original activity was still present in the C-terminal tripeptide Ile-Pro-Pro. Some analogs of this tripeptide were synthesized by the conventional method, and investigated by two biological assays, i.e., potentiating response on bradykinin (BK) and inhibitory activity on angiotensin converting enzyme (ACE). It was shown that the two biological activities inherent in the synthetic analogs were not parallel to each other. In addition, the isolated guinea pig ileum strips treated with chelating agent to irreversibly inactivate kininase (the same enzyme ACE) still responded to BPP. Consequently the potentiating effect of BPP on BK in vitro bioassay might be due to its influence on the binding receptor for BK rather than the inhibitory effect on kininase.