Antimicrobial and antitumor activity and diversity of endophytic fungi from traditional Chinese medicinal plant Cephalotaxus hainanensis Li.

Endophytes from Cephalotaxus hainanensis Li, an important source of anti-leukemia drugs, have not been widely explored. In this study, 265 endophytic fungal isolates from C. hainanensis Li were screened for antimicrobial activities against tilapia, banana, rice, and rape and for antitumor activities against human leukemia cell lines (K562, NB4, and HL-60). Diversity was also analyzed. The results showed that 17.7% of the endophytic fungi had antimicrobial activities against at least three different test microbes, and activity against Fusarium oxysporum RKY102 was the highest at 15.8%. Cytotoxicity against at least one tumor cell line tested was observed in 18.5% of the endophytic fungi; with the highest value of 10.6% against K562. The endophytic fungal strains also showed relatively high activities against K562, NB4, and HL-60 while relatively fewer strains were cytotoxic against the human hepatic Hep-G2 and colon LoVo cancer cell lines. Thirty endophytic fungal strains showed both high antimicrobial and antitumor activities. Moreover, the analyses of the diversity of the 30 highly active strains showed they belonged to 20 species from 14 genera, and this is the first report of endophytic fungi Albonectria rigidiuscula, Colletotrichum magnisporum, and Nemania diffusa being isolated from Cephalotaxus plants. These findings suggest that natural antibacterial products for humans and tilapia; antifungal compounds for rice, rape, and banana; and antitumor compounds for leukemia therapy could be isolated from fungal strains derived from C. hainanensis Li.

Secondhand cigarette smoke exposure causes upregulation of cerebrovascular 5-HT(1) (B) receptors via the Raf/ERK/MAPK pathway in rats.

AIM: Cigarette smoke exposure increases the risk of stroke. Upregulation of 5-hydroxytryptamine 1B (5-HT(1) (B) ) receptors is associated with the pathogenesis of cerebral ischaemia. This study examined the hypothesis that the expression of 5-HT(1) (B) receptors is altered in brain vessels after secondhand smoke (SHS) exposure. METHODS: Rats were exposed to SHS in vivo for 200 min daily for 8 weeks. The contractile responses of isolated cerebral arteries were studies by a sensitive myograph. The mRNA and protein expression for 5-HT(1) (B) receptors were examined by real-time PCR, Western blot and immunofluorescence respectively. In addition, the phosphorylation of Raf/extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinases (MAPK) pathway was evaluated. RESULTS: The results showed that SHS exposure shifted the 5-HT(1) (B) receptor-mediated concentration-contraction curve towards the left with a markedly increased maximal contraction. Furthermore, there were significant elevations in mRNA level and protein expression of 5-HT(1) (B) receptors in SHS-exposed rats. Immunostaining revealed that the 5-HT(1) (B) receptors were localized to the smooth muscle cells of cerebral arteries. SHS was also found to induce the phosphorylation of Raf-1 and ERK1/2 proteins. The administration of a Raf-1 inhibitor GW5074 attenuated the 5-HT(1) (B) receptor upregulation. CONCLUSION: Secondhand smoke exposure upregulates cerebrovascular 5-HT(1) (B) receptors in rats. The receptor upregulation is associated with Raf/ERK/MAPK activation.

Protection against myocardial ischaemia/reperfusion injury by PPAR-alpha activation is related to production of nitric oxide and endothelin-1.

BACKGROUND: Ligands of peroxisome proliferator-activated receptor alpha (PPAR-alpha) have been shown to reduce ischaemia/reperfusion injury. The mechanisms behind this effect are not well known. We hypothesized that activation of PPAR-alpha exerts cardioprotection via a mechanism related to nitric oxide (NO) and endothelin-1 (ET-1). METHODS: Five groups of anaesthetized open-chest Sprague-Dawley rats were given the PPAR-alpha agonist WY 14643 1 mg/kg (WY; n = 7), dimethyl sulfoxide (DMSO, vehicle for WY; n = 6), the combination of WY and the NO synthase inhibitor N-nitro-L-arginine (L-NNA, 2 mg/kg) (n = 7), L-NNA only (n = 8) or 0.9% sodium chloride (NaCl, vehicle for DMSO and L-NNA; n = 8) i.v. before a 30 min period of coronary artery occlusion followed by 2 h of reperfusion. Infarct size (IS), eNOS and iNOS protein and ET-1 mRNA expression were determined. RESULTS: There were no haemodynamic differences between the groups during the experiment. The IS was 78 +/- 3% of the area at risk in the DMSO group and 77 +/- 2% in the NaCl group (P = NS). WY reduced IS to 56 +/- 3% (P < 0.001 vs. DMSO group). When WY was administered in combination with L-NNA the cardioprotective effect was abolished (IS 73 +/- 3%, P < 0.01 vs. WY 14643). L-NNA did not affect IS per se (78 +/- 2%, P = NS). The expression of eNOS but not iNOS protein in ischaemic myocardium from rats was increased in the group given WY (P < 0.05). ET-1 mRNA levels were lower in the ischaemic myocardium following WY administration. CONCLUSION: The results suggest that the PPAR-alpha activation protects the rat myocardium against ischaemia/ reperfusion injury via a mechanism related to production of NO, and possibly ET-1.

Endocardial expression and functional characterization of endothelin-1.

Endothelin-1 (ET-1), a 21 amino acid peptide exerts a wide range of biological activities including vasoconstriction, mitogenesis and inotropic effects on the heart. In this study, we examined whether endocardial endothelial cells express ET-1 and evaluated its functional properties. Using immunofluorescence localization method, we demonstrated cytoplasmic staining of ET-1 in the human endocardial endothelial cells from the right atrium and left ventricle. Employing reverse transcriptase polymerase chain reaction (RT-PCR) expression of ET-1 mRNA and its receptors ET(A) and ET(B) mRNAs were found in human myocardial as well as in endocardial endothelial cells. Biological activity of endocardial endothelial cells derived ET-1 was established as the conditioned media obtained from cultured porcine endocardial endothelial cells induced a slowly developing, strong and long-lasting contraction of circular rat aortic segments, with similar characteristics to that obtained with exogenous ET-1. Furthermore, the selective endothelin-A receptor antagonist, FR 139317, blocked the conditioned media induced contractions. Our results suggest that endocardial endothelial cells express and release biologically active ET-1 which could play a pivotal role in the regulation of myocardial contractility as well as a circulatory peptide may further act in other peripheral target organs.

[Effect of aloe-emodin on c-myc gene expression of cultured vascular smooth muscle cells of injured artery].

OBJECTIVE: To study the molecular mechanism of aloe-emodin (AE) in inhibiting smooth muscle cells' (SMC) proliferation. METHODS: Deendothelialization was performed in abdominal aorta of Japanese white ear rabbits by using 3F Fogarty arterial embolectomy catheter. The media of abdominal aorta was isolated 48 hrs later for performing primary SMC culture. Cells were synchronized with G0/G1 phase by serum starvation, the AE was then added to the culture medium of experimental group, at a concentration of 20 micrograms/ml, and to the control group, equal volume of culture solution was added instead. The c-myc mRNA and protein expressions were examined 3 hrs later by using techniques of Northern blotting, Western blotting and immunocytochemistry respectively. RESULTS: In comparing with the control group, neither the expression of c-myc mRNA nor the expression of c-myc protein was changed after addition of AE. CONCLUSION: The inhibition of AE on SMC might not be due to influencing c-myc expression, but via other pathway.

[Cloning and expression of ScFv gene against alpha-toxin of Clostridium perfringens type A].

The VH and VL genes from a hybridoma cell line producing mouse McAb against alpha-toxin of Clostridium perfringens type A were amplified by RT-PCR. The VH and VL genes were connected thought a flexible linker (Gly4Ser)3 and the VH-linker-VL (ScFv) gene was cloned into a vector pGEM-T. The ScFv gene consists of 726 bp encoding 242 amino acid residues. Both VH and VL genes were confirmed as functionally rearranged mouse immunoglobulin variable region. According to kabat classed method, the VH and VL gene segments belong to mouse Ig heavy chain subgroup II (B) and kappa light chain subgroup III respectively. The ScFv gene was amplified inserted the expression vector pHOG21 and transformed into E coli XL1-BLUE. The ScFv protein was highly expressed in recombinant strain XL1-BLUE (pHOG-2E3) and the expression level of the ScFv was about 25% of total bacteria protein by SDS-PAGE. The neutralization assay showed that the expressed ScFv protein could neutralize the phospholipase C activities of alpha-toxin.

Unfractionated heparin and low molecular weight heparin do not inhibit the growth of proliferating human arterial smooth muscle cells in culture.

OBJECTIVES: To clarify the effects of unfractionated heparin (UH) and low molecular weight heparin (LMWH) on proliferating human smooth muscle cells (SMC) compared to growth arrested SMC. DESIGN: A cell culture study where proliferating SMC were exposed to different concentrations of UH and LMWH and the effect on proliferation and collagen secretion was studied. Growth arrested SMC were stimulated with serum and the effect of UH on proliferation was measured. SETTING: Sections of Medical Angiology and Vascular Surgery, Malmö General Hospital, Sweden. MATERIALS: Human SMC were established from arterial tissue obtained at vascular surgery or at organ donation. CHIEF OUTCOME MEASURES: Effects of UH and LMWH on total cellular DNA, 3H-thymidine incorporation and collagen secretion using proliferating and growth arrested human SMC in culture. MAIN RESULTS: In proliferating SMC that had not been growth arrested, 1 and 10 IU/ml UH and LMWH significantly increased total cellular DNA compared to controls while DNA synthesis was not influenced. The higher cellular DNA was probably not a consequence of increased proliferation as DNA synthesis was not affected by UH or LMWH. The increased total cellular DNA could instead be due to reduced cell death. Higher concentrations (10 IU/ml) of UH and LMWH also increased collagen secretion. In control experiments with UH DNA, synthesis was decreased in stimulated human SMC that had been growth arrested previously to heparin exposure. CONCLUSIONS: The effects of UH and LMWH on SMC proliferation will depend on the proliferative state of the SMC. The results might be of relevance for the understanding of the atherosclerotic process and for pharmacologic interventions to prevent restenosis after angioplasty or surgery.

Interactions between cultured bovine arterial endothelial and smooth muscle cells; effects of modulated low density lipoproteins on cell proliferation and prostacyclin release.

We exposed bovine aortic endothelial cells in culture to native LDL (n-LDL) and LDL modulated by dimethylsulfoxide (DMSO-LDL), dimethylsulfoxide-soluble particles from cigarette smoke (DSP-LDL) or Cu2+ (Cu(2+)-LDL) to explore the hypothesis that these LDL-forms might influence interactions between endothelial and smooth muscle cells. It was shown that 3H-thymidine incorporation into endothelial cells was decreased by DMSO-LDL, DSP-LDL and Cu(2+)-LDL compared to n-LDL, while it was higher by DSP-LDL compared to its control i.e. DMSO-LDL. These effects could be transferred by conditioned medium to smooth muscle cell cultures. DSP-LDL or Cu(2+)-LDL decreased total cellular protein of endothelial cells. Initial (15 min) prostacyclin release from endothelial cells was increased by all LDL preparations compared to medium without LDL, most pronounced for Cu(2+)-LDL. If n-LDL was control, only Cu(2+)-LDL significantly increased the release of prostacyclin during 15 min and during 24 h. The release of prostacyclin assayed after 24 h was depressed by DSP-LDL compared to DMSO-LDL. This study demonstrated that interactions between endothelial and smooth muscle cells could be influenced by LDL treated by dimethylsulfoxide-soluble particles from cigarette smoke or by Cu2+, and their effects were not similar. DSP-LDL, in contrast to Cu(2+)-LDL, significantly decreased the release of prostacyclin by endothelial cells after 24 h incubations and via endothelial cell conditioned medium stimulated smooth muscle cell proliferation judged by increased 3H-thymidine incorporation. The results might be of relevance for atherogenesis.

Percutaneous transluminal excimer laser coronary angioplasty. Clinical report of six cases.

Six patients with 95% to 100% occluded atherosclerotic lesions underwent percutaneous transluminal excimer laser coronary angioplasty (PTELCA). Among them, 5 were male and 1 was female; their age ranged from 28 to 66 years. Four patients had LAD stenosis and 2 LCX lesions. Acute angiographic and clinical success was achieved in all patients but one, with a success rate of 83.3%. It was demonstrated that PTELCA is a safe and effective therapy for selected patients with coronary artery disease.

Glutathione transferase activity in human vessels and in cultured arterial smooth muscle cells.

Glutathione transferases play an important role in the detoxification of many different endogeneous and exogenous compounds such as metabolites of polycyclic aromatic hydrocarbons (PAH) of cigarette tar. There is evidence that PAH may be atherogenic. The glutathione transferase activity towards trans-stilbene oxide (GST-tSBO) can be separated in blood in GST-positive and GST-negative phenotypes. We have previously suggested that the GST-negative phenotype may be associated with a higher morbidity in intermittent claudication among middle aged smokers. In the present study, GST-tSBO could easily be measured in human, rabbit and bovine arterial smooth muscle cells (SMC) in culture. The level of GST-tSBO was higher in rabbit than in bovine SMC. It was stable in bovine SMC during 5 cell passages and it could be induced twofold by long-time incubation with dimethylsulfoxide-soluble particulate matter from cigarette smoke or 3,4-benzo(a)pyrene. There was a positive correlation between the level of GST-tSBO in blood and in "healthy" arterial and venous tissue from individuals operated with coronary bypass. The enzyme levels in arterial tissue were lower than in venous tissue. GST-tSBO in atherosclerotic segments of human arteries was lower than in "healthy" segments from the same artery. These findings suggest that the arterial wall may have a low defense against toxic compounds that may decrease further as atherosclerosis proceeds. It is concluded that SMC are suitable for the study of the effects of PAH in relation to GST-tSBO and that the enzyme activity in blood will reflect the individual GST-tSBO phenotype also in vascular tissues.

Interactions between cultured bovine arterial endothelial and smooth muscle cells: studies on uptake and degradation of low density lipoproteins by smooth muscle cells.

This study was designed to investigate the effects of substances released from non-injured and injured bovine arterial endothelial cells on 125I-low density lipoprotein uptake and degradation by smooth muscle cells in culture. It was demonstrated that endothelial cell-released non-dialysable (molecular weight cut off 12-14000) substances significantly stimulated 125I-low density lipoprotein uptake and degradation by smooth muscle cells. Endothelial cell-released dialysable substances and endothelin-1 did not cause this stimulation. The increase in 125I-low density lipoprotein uptake and degradation by smooth muscle cells could be dissociated from cell proliferation. However, in endothelial cell-smooth muscle cell co-culture 125I-low density lipoprotein uptake and degradation by smooth muscle cells were not stimulated. Injury to endothelial cells by lipid-soluble smoke particles or ultraviolet light, which reduced total cellular protein by 15-25%, enhanced the endothelial cell release of the substances stimulating 125I-low density lipoprotein uptake. The results are discussed in relation to atherogenesis.

[Percutaneous transluminal peripheral excimer-laser angioplasty: two successful cases report].

The excimer-laser (XeC1) operating at 308 nm, ablates by means of photochemical mechanisms with minimal thermal injury. Two male patients, 53 years and 67 years, with symptomatic peripheral vascular disease underwent percutaneous transluminal excimer-laser angioplasty (PTPELA). Both of them experienced claudication at a short walking distance and had superficial femoral or initial segmental femoral occlusions from 4 cm to 7 cm in length. The laser emitted 120ns pulses at 20 Hz with delivering 16-18 mJ by 7F and 9F catheter, energy density was about 4mJ/mm. The irradiation time was 157 seconds and 185 seconds respectively. Successful recanalization occurred in our two cases, the residual stenosis were less than 20%. The acute outcome was good without any complications.

Schistosoma mansoni 28-kDa glutathione S-transferase and immunity against parasite fecundity and egg viability. Role of the amino- and carboxyl-terminal domains.

We have previously shown that a mAb that inhibits the enzymatic activity of the Schistosoma mansoni 28-kDa glutathione S-transferase (Sm28 GST) also reduces female worm fecundity and egg viability in vivo and in vitro. By peptidic epitope mapping and an activity reconstitution assay, the carboxyl terminus (CT) amino acid residues 190-211 and to a lesser extent the truncated amino terminus (NT) residues 10-43 of the enzyme were identified as mAb recognition sites. Sera from rats immunized with the NT (10-43) and CT (190-211) peptides showed a partial inhibitory effect on Sm28 GST activity in a late phase (6 to 7 wk) but not in an early phase (2 to 4 wk) after immunization. Passive transfer of Sm28 GST-inhibiting anti-N- and C-terminal sera, but not of the noninhibitory sera, protected the infected mice by reducing tissue egg deposition and the ability of eggs to hatch. In active immunization experiments, the CT peptide significantly decreased the worm burden (37 to 40%) in mice as did the rSm28 GST (28 to 52%). In terms of tissue egg deposition and egg-hatching ability, immunization with both the NT and CT peptides reproduced the reduction observed after immunization with rSm28 GST. A constant reduction in egg numbers was noted in the small intestines and the livers of the immunized mice. A clear reduction in the ability of intestinal or hepatic eggs to hatch was observed. The results are discussed in terms of the conformational participation of the NT and CT of Sm28 in the expression of GST activity.

Interactions between cultured bovine arterial endothelial and smooth muscle cells; further studies on the effects of injury and modification of the consequences of injury.

The hypothesis that cells of the arterial wall might modify the consequences of arterial injury was tested. Bovine aortic endothelial cells (EC) or smooth muscle cells (SMC) were exposed to the two toxic stimuli 3,4-benzo(a)pyrene (BP) and dimethylsulfoxide-soluble particulate matter from cigarette smoke (DSP) or factors released from platelets. The modification of the injury caused by these substances on arterial cells was studied by using a conditioned medium from arterial cells or an EC-SMC co-culture model. Direct addition of BP or DSP to the EC or SMC cultures induced toxic effects on the cells. DSP caused a decreased release of prostacyclin by EC. Conditioned medium from EC and SMC modified these toxic effects, which resulted in a reduced cell death and a further decreased cell proliferation, while conditioned medium from SMC increased the release of prostacyclin by EC injured by DSP. In EC-SMC co-culture the same modifications were obtained. The modification of cell injury was not linked to cell proliferation but instead the results suggested that the effects were mediated by multiple substances released from arterial cells. It is concluded that interactions between different cells in the arterial wall, in the non-injured as well as in the injured state, could be modified by endogeneous substances. This might be of relevance for atherogenesis.

IgA antibodies to a protective antigen in human Schistosomiasis mansoni.

The specific IgA antibody responses to the protective recombinant Schistosoma mansoni 28-kDa glutathione-S-transferase (Sm28GST) Ag and to derived synthetic peptides have been evaluated before and 6 mo after chemotherapy in S. mansoni-infected patients from Kenya. These studies revealed a parallelism between the age-dependent evolution of IgA antibody levels to Sm28GST and to one synthetic peptide (115-131) and the acquisition of resistance to reinfection. Functional analysis revealed that IgA antibodies to Sm28GST displayed a potent neutralizing effect on the enzymatic properties of the molecule, and also markedly impaired schistosome fecundity, by limiting both the egg laying of mature worms and the hatching capacity of schistosome eggs into viable miracidia. These results suggest that, in addition to IgE, IgA antibodies might participate in the protective immune response against schistosomiasis.

Interactions between cultured bovine arterial endothelial and smooth muscle cells; studies on the release of prostacyclin by endothelial cells.

The release of prostacyclin by endothelial cells (EC) in culture was studied after exposure to two toxic stimuli (UV light or dimethylsulfoxide-soluble smoke particles (DSP)) or to medium conditioned by smooth muscle cells (SMC), basically or after injury to the SMC. An activity stimulating the release of prostacyclin was found together with growth inhibiting activity from arterial SMC, but dissociated from growth stimulating activity. The prostacyclin stimulating activity was increased when SMC were exposed to UV light, while DSP caused a decrease. EC directly exposed to UV light or DSP generally released more prostacyclin than controls. One exception was very low concentrations of DSP. UV light induced a burst of release in contrast to DSP where a continuous release after a two hours lag period was seen. It is concluded that EC will increase the release of prostacyclin in response to injury but the release pattern will depend on the kind and doses of the stimulus. SMC release prostacyclin stimulating activity for EC, which can be modified by exposure to toxic stimuli. The results might have applications for atherogenesis.

Interactions between cultured bovine arterial endothelial and smooth muscle cells: effects of injury on the release of growth stimulating and growth inhibiting substances.

Dimethylsulfoxide-soluble particles (DSP) from cigarette smoke and ultraviolet light caused a low degree (cell death less than 30%) and high degree (cell death 60-90%) injury to bovine arterial endothelial cells and smooth muscle cells in culture. Conditioned medium from low degree injured endothelial cells and smooth muscle cells generally inhibited DNA synthesis in new smooth muscle cells or endothelial cells while high degree injury increased DNA synthesis in new cells. Specifically, the growth stimulating activity from endothelial cells was decreased after low degree injury but increased after high degree. UV light released more growth stimulating substances from smooth muscle cells after both low and high degree injury. The release of growth inhibiting substances was dependent on both cell kind and degree of injury. In co-culture low and high degree DSP injury to endothelial cells inhibited smooth muscle cell proliferation, which was in contrast to the effect of conditioned medium from high degree injured endothelial cells. Conditioned medium from endothelial cells treated with LDL and glucose inhibited DNA synthesis in smooth muscle cells. It is concluded that injury to endothelial cells or smooth muscle cells will modify the release of growth inhibiting and growth stimulating activity and that this release will depend on cell kind as well as degree and kind of the injurious stimulus.

A monoclonal antibody blocking the Schistosoma mansoni 28-kDa glutathione S-transferase activity reduces female worm fecundity and egg viability.

The protective effects of two different monoclonal antibodies (mAb) raised against the Schistosoma mansoni 28-kDa glutathione S-transferase (Sm 28 GST) were investigated. Two mAb of the same isotype (IgM) have been selected according to the blocking effect on Sm 28 GST enzymatic activity (S13) or the lack of blockade (H12). When passively transferred into Fischer rats, both S13 and H12 significantly reduced the worm burden. In BALB/c mice clear effects on female worm fecundity and egg viability were observed when the S13 mAb was transferred; these effects included significantly reduced loads of intestinal eggs, reduced egg hatching rates and an increased proportion of non-living eggs. No effect on egg production and egg hatching was observed in H12-treated mice. In addition, worm pairs recovered from S13-but not H12-treated mice laid significantly fewer eggs in vitro, and normal worm pairs incubated in vitro with the S13 mAb produced significantly fewer eggs than those incubated with H12 mAb. The impairment of egg hatching ability was also reproduced in vitro by the S13 mAb. These data suggest the existence of two different effector mechanisms induced by immunization with Sm 28 GST. The effect on the schistosome worm burden appears to be independent of GST activity whereas the effect on S. mansoni female fecundity and egg viability seems to be significantly linked to the inactivation of the enzymatic site.

Increased smooth muscle cell proliferation by dimethylbenzanthracene is correlated to variations in activity of ornithine decarboxylase but not arylhydrocarbonhydroxylase.

Polycyclic aromatic hydrocarbons of cigarette smoke have been suggested to be involved in atherogenesis. After being converted to epoxides by monooxidases in the arterial wall the hydrocarbons may exert toxic or mutagenic effects on the smooth muscle cells (SMC). In a previous study we found that dimethylbenzanthracene (DMBA), an inducer of arylhydrocarbonhydroxylase (AHH), increased SMC proliferation and viability. In the present work we intended to study whether these effects were mediated by AHH. Alpha-naphtoflavone (ANF), a non specific AHH inhibitor, decreased SMC proliferation. The effects of ANF were totally counteracted by serum, partially by albumin and not at all by platelet derived growth factor. AHH activity was not detectable nor basally nor after induction in SMC, and this made us conclude that the effects of DMBA and ANF on SMC proliferation were not mediated by AHH. On the other hand the activity of ornithine decarboxylase was influenced by DMBA and ANF in parallel to proliferation, suggesting the involvement of this enzyme in the described DMBA effects on SMC proliferation. This mechanism might be of relevance for the pathogenesis of atherosclerosis especially in relation to cigarette smoking.