Surfactin inhibits the growth of Propionibacterium acnes by destroying the cell wall and membrane.

Propionibacterium acnes plays a major role in acne vulgaris. In the pre-experiment, the growth of P. acnes was inhibited effectively using surfactin; however, the antibacterial mechanism has not been described. Therefore, the aim of this study was to evaluate antibacterial activity and analyse the mechanism of surfactin against P. acnes. Minimum inhibitory concentration, time-killing kinetics and scanning electron microscopy were used to evaluate the activity of surfactin against P. acnes, which showed that 128 µg ml-1 effectively inhibited growth. Cell wall permeability was evaluated by detecting the extracellular alkaline phosphatase activity, which increased to 1·83- and 2·32-fold after incubating with 128 and 256 µg ml-1 of surfactin for 10 h, respectively. Propidium iodide fluorescence, leakage of nucleic acid, protein, K+ , and Ca2+ , membrane potential and the leakage of calcein from small unilamellar vesicles all increased after incubation with surfactin, indicating that its strong biological activities act mainly by altering membrane integrity. In a mouse model of acne, surfactin significantly reduced P. acnes-induced epidermal swelling and erythema. These results indicate that surfactin effectively inhibited the growth of P. acnes by destroying the cell wall and membrane, and is a potential candidate for acne treatment.

The effect of three-dimensional conformal radiotherapy on locally recurrent nasopharyngeal carcinoma and on the expression of succinate dehydrogenase B.

OBJECTIVE: Investigate the effect of three-dimensional conformal radiotherapy (3DCRT) on locally recurrent nasopharyngeal carcinoma (NPC) on the expression of succinate dehydrogenase B (SDHB). PATIENTS AND METHODS: Eighty-six patients diagnosed with locally recurrent NPC in our hospital were selected and divided into the control group (43 cases) and observation group (43 cases). Conventional two-dimensional radiotherapy was applied in the control group, and 3DCRT was adopted in the observation group. The curative effect of both groups was compared. RESULTS: The effective rate and the degree of alleviation of the observation group were higher than those of the control group, and the differences were statistically significant (p<0.05). There were no differences in the occurrence rate of complications from radiotherapy between the two groups (p>0.05). The survival rate and median survival time of the observation group were significantly higher than those of the control group (p<0.05). The positive expression rate of SDHB in the observation group after radiotherapy was significantly higher than that of the control group (p<0.05), and the median survival time of patients with positive expression of SDHB was significantly higher than patients with negative expression (p<0.05). CONCLUSIONS: 3DCRT applied for treatment of locally recurrent NPC was safe and effective. It also improved the positive expression rate of SDHB, which was associated with increased survival time.

Production of gamma-aminobutyric acid by Streptococcus salivarius subsp. thermophilus Y2 under submerged fermentation.

Gamma-aminobutyric acid (GABA), a major inhibitory neurotransmitter in the central nervous system, has several well-known physiological functions and has been applied to the production of many drugs and functional foods. The technology of GABA production via submerged fermentation by Streptococcus salivarius subsp. thermophilus Y2 was investigated in this paper. It indicated that the GABA production was related to the biochemical characteristics of glutamate decarboxylase (GAD) of S. salivarius subsp. thermophilus Y2. After 24 h of fermentation at 37 degrees C, which is the suitable culture conditions for GAD-production, then the culture condition were adjusted to the optimal temperature (40 degrees C) and pH (4.5) for the GAD reaction activity in biotransformation of cells and pyridoxal 5'-phosphate (0.02 mmol/l) were added to the broth at the 48 h, the GABA production was increased up to 1.76-fold, reaching 7984.75 +/- 293.33 mg/l. The strain shows great potential use as a starter for GABA-containing yoghurt, cheese and other functional fermented food productions.

Biodegradation of nicotine from tobacco waste extract by Ochrobactrum intermedium DN2.

Ochrobactrum intermedium DN2 was used to degrade nicotine in tobacco waste extracts. The optimal temperature and pH of nicotine degradation by strain DN2 was 30-37 degrees C and 7.0, respectively. Under these optimal conditions, the average degradation rate of nicotine in a 30L fed-batch culture was 140.5 mg 1(-1) h(-1). The results of this study indicate that strain DN2 may be useful for reducing the nicotine content of reconstituted tobacco.

Oral mucosal immunity and HIV/SIV infection.

Human Immunodeficiency Virus (HIV) transmission through genital and rectal mucosa has led to intensive study of mucosal immune responses to HIV and to the development of a vaccine administered locally. However, HIV transmission through the oral mucosa is a rare event. The oral mucosa represents a physical barrier and contains immunological elements to prevent the invasion of pathogenic organisms. This particular defense differs between micro-compartments represented by the salivary glands, oral mucosa, and palatine tonsils. Secretory immunity of the salivary glands, unique features of cellular structure in the oral mucosa and palatine tonsils, the high rate of oral blood flow, and innate factors in saliva may all contribute to the resistance to HIV/Simian Immunodeficiency Virus (SIV) oral mucosal infection. In the early stage of HIV infection, humoral and cellular immunity and innate immune functions in oral mucosa are maintained. However, these particular immune responses may all be impaired as a result of chronic HIV infection. A better understanding of oral mucosal immune mechanisms should lead to improved prevention of viral and bacterial infections, particularly in immunocompromised persons with Acquired Immune Deficiency Syndrome (AIDS), and to the development of a novel strategy for a mucosal AIDS vaccine, as well as vaccines to combat other oral diseases, such as dental caries and periodontal diseases.

Optimization of a medium for enhancing nicotine biodegradation by Ochrobactrum intermedium DN2.

AIMS: To optimize a medium for nicotine degradation by Ochrobactrum intermedium DN2 in presence of yeast extract, glucose and Tween 80 using response surface methodology (RSM). METHODS AND RESULTS: In this study, the effects of yeast extract, glucose and Tween 80 on nicotine degradation were investigated in flasks using a novel nicotine-degrading bacterium, O. intermedium DN2. A full factorial central composite design was applied in the design of experiments and in the analysis of the experimental data. The results showed that the most significant variable influencing nicotine degradation was yeast extract, followed by glucose, and then Tween 80. Moreover these three factors interacted with each other and combined to produce positive effects on nicotine degradation. The experimental data also allowed the development of an empirical model (P < 0.0001) describing the inter-relationship between independent and dependent variables. By solving the regression equation, the optimal values of the variables were determined as: yeast extracts 0.094%, glucose 0.101% and Tween 80 0.080%. Using the medium obtained, about 1,220 mg l(-1) of nicotine was degraded (95.55%) within 10 h at the specific biodegradation of 116.59 mg l(-1) h(-1) in 30-l bioreactor containing 25-l tobacco extract. CONCLUSIONS: An optimal medium of nicotine degradation by the strain DN2 was obtained. SIGNIFICANCE AND IMPACT OF THE STUDY: RSM proved to be reliable in developing the model, optimizing factors and analysing interaction effects. The results provide better understanding on the interactions between yeast extract, glucose and Tween 80 for nicotine biodegradation.

The strength of B cell immunity in female rhesus macaques is controlled by CD8+ T cells under the influence of ovarian steroid hormones.

To understand more clearly how mucosal and systemic immunity is regulated by ovarian steroid hormones during the menstrual cycle, we evaluated the frequency of immunoglobulin- and antibody-secreting cells (ISC, AbSC) in genital tract and systemic lymphoid tissues of normal cycling female rhesus macaques. The frequency of ISC and AbSC was significantly higher in tissues collected from animals in the periovulatory period of the menstrual cycle than in tissues collected from animals at other stages of the cycle. The observed changes were not due to changes in the relative frequency of lymphocyte subsets and B cells in tissues, as these did not change during the menstrual cycle. In vitro, progesterone had a dose-dependent inhibitory effect, and oestrogen had a dose-dependent stimulatory effect on the frequency of ISC in peripheral blood mononuclear cell (PBMC) cultures. The in vitro effect of progesterone and oestrogen on ISC frequency could not be produced by incubating enriched B cells alone with hormone, but required the presence of CD8+ T cells. Following oestrogen stimulation, a CD8+ enriched cell population expressed high levels of IFN-gamma and IL-12. The changes in B cell Ig secretory activity that we document in the tissues of female rhesus macaques during the menstrual cycle is due apparently to the action of ovarian steroid hormones on CD8+ T cells. Thus, CD8+ T cells control B cell secretory activity in both mucosal and systemic immune compartments. Understanding, and eventually manipulating, the CD8+ regulatory cell-B cell interactions in females may produce novel therapeutic approaches for autoimmune diseases and new vaccine strategies to prevent sexually transmitted diseases.

Protection against simian immunodeficiency virus vaginal challenge by using Sabin poliovirus vectors.

Here we provide the first report of protection against a vaginal challenge with a highly virulent simian immunodeficiency virus (SIV) by using a vaccine vector. New poliovirus vectors based on Sabin 1 and 2 vaccine strain viruses were constructed, and these vectors were used to generate a series of new viruses containing SIV gag, pol, env, nef, and tat in overlapping fragments. Two cocktails of 20 transgenic polioviruses (SabRV1-SIV and SabRV2-SIV) were inoculated into seven cynomolgus macaques. All monkeys produced substantial anti-SIV serum and mucosal antibody responses. SIV-specific cytotoxic T-lymphocyte responses were detected in three of seven monkeys after vaccination. All 7 vaccinated macaques, as well as 12 control macaques, were challenged vaginally with pathogenic SIVmac251. Strikingly, four of the seven vaccinated animals exhibited substantial protection against the vaginal SIV challenge. All 12 control monkeys became SIV positive. In two of the seven SabRV-SIV-vaccinated monkeys we found no virological evidence of infection following challenge, indicating that these two monkeys were completely protected. Two additional SabRV-SIV-vaccinated monkeys exhibited a pronounced reduction in postacute viremia to <10(3) copies/ml, suggesting that the vaccine elicited an effective cellular immune response. Three of six control animals developed clinical AIDS by 48 weeks postchallenge. In contrast, all seven vaccinated monkeys remained healthy as judged by all clinical parameters. These results demonstrate the efficacy of SabRV as a potential human vaccine vector, and they show that the use of a vaccine vector cocktail expressing an array of defined antigenic sequences can be an effective vaccination strategy in an outbred population.

The number and distribution of immune cells in the cervicovaginal mucosa remain constant throughout the menstrual cycle of rhesus macaques.

A number of studies have shown that the ovarian hormone cycle affects genital tract immunoglobulin (Ig) levels and T cell function in both humans and rhesus monkeys. We hypothesized that shifts in immune cell populations occurring in response to hormone cycles are involved in the observed changes in genital tract immunity. To test this hypothesis, we characterized the type, number, and distribution of immune cells in the cervicovaginal mucosa at different stages of the menstrual cycle. Tissues from 18 normal female rhesus macaques were studied by immunohistochemistry and computerized morphometric analysis. The number and distribution of CD1a+ Langerhans' cells, CD2+, CD3+, CD4+, and CD8+ T cells, CD20+ B cells, and surface Ig+ plasma cells did not change in samples collected at the different stages of the cycle. However, in no relation to the stage of the menstrual cycle, the number of Langerhans' cells and other immune cell types was different in the various regions of the cervicovaginal mucosa examined. In addition, variation in thickness of the ectocervical and vaginal epithelium during a normal menstrual cycle of rhesus macaques is not accompanied by changes in intraepithelial immune cell populations. We conclude that steroid hormones do not influence genital mucosal immunity by changing the number or distribution of immune cells in the lower reproductive tract.

Route of simian immunodeficiency virus inoculation determines the complexity but not the identity of viral variant populations that infect rhesus macaques.

A better understanding of the host and viral factors associated with human immunodeficiency virus (HIV) transmission is essential to developing effective strategies to curb the global HIV epidemic. Here we used the rhesus macaque-simian immunodeficiency virus (SIV) animal model of HIV infection to study the range of viral genotypes that are transmitted by different routes of inoculation and by different types of viral inocula. Analysis of transmitted variants was undertaken in outbred rhesus macaques inoculated intravenously (IV) or intravaginally (IVAG) with a genetically heterogeneous SIVmac251 stock derived from a well-characterized rhesus macaque viral isolate. In addition, we performed serial IV and IVAG passage experiments using plasma from SIV-infected macaques as the inoculum. We analyzed the V1-V2 region of the SIV envelope gene from virion-associated RNA in plasma from infected animals by the heteroduplex mobility assay (HMA) and by DNA sequence analysis. We found that a more diverse population of SIV genetic variants was present in the earliest virus-positive plasma samples from all five IV SIVmac251-inoculated monkeys and from two of five IVAG SIVmac251-inoculated monkeys. In contrast, we found a relatively homogeneous population of SIV envelope variants in three of five monkeys inoculated IVAG with SIVmac251 stock and in two monkeys infected after IVAG inoculation with plasma from an SIV-infected animal. In some IVAG-inoculated animals, the transmitted SIV variant was the most common variant in the inoculum. However, a specific viral variant in the SIVmac251 stock was not consistently transmitted by IVAG inoculation. Thus, it is likely that host factors or stochastic processes determine the specific viral variants that infect an animal after IVAG SIV exposure. In addition, our results clearly demonstrate that the route of inoculation is associated with the extent and breadth of the genetic complexity of the viral variant population in the earliest stages of systemic infection.

Predominate HIV1-specific IgG activity in various mucosal compartments of HIV1-infected individuals.

Evaluating mucosal humoral immunity is important for understanding local immunity induced by HIV infection or vaccination and designing prophylactic strategies. To characterize the mucosal humoral immunity following HIV infection, the levels of immunoglobulins (Igs), antibodies (Abs), and HIV1-specific Ab activity were evaluated in cervicovaginal secretions (CVS), saliva, breast milk, and sera of HIV-infected individuals. HIV1-specific IgG activity was significantly higher than that of IgA in CVS, saliva, and breast milk. The highest HIV1-specific IgG activity was found in breast milk. The data suggest that anti-HIV1 Abs in CVS were most likely serum derived. However, HIV1-specific Abs in saliva and breast milk were mainly locally produced. The prevalence of HIV1-specific Abs in seropositive subjects was 97% for IgG and 95% for IgA in CVS, 100% for IgG and 80% for IgA in saliva, and 59% for IgG and 94% for IgA in breast milk. These data provide evidence for both a better understanding of the nature of humoral mucosal responses after HIV1 infection and the development of strategies to induce desirable functional mucosal immunity for preventing HIV transmission.

New method for quantifying anti-HIV1-gp160 antibodies in saliva, cervicovaginal secretions, and serum of infected women.

Antibody titers or units as measured by ELISA or similar assays represent only a semi-quantitative measurement of the antibody concentration. In the present study, a truly quantitative assay for evaluating antibody concentrations was developed. In the new assay, IgG and IgA antibody concentrations are expressed in absolute gravimetric units (weight/ml) rather than by more ambiguous terms such as titers or units. The procedure is based on the comparison of known concentrations of IgG or IgA bound to anti-F(ab)'(2) with the binding of an unknown concentration of antibodies to a specific antigen. We applied the new assay to the measurement of HIV1-gp160 specific IgG and IgA concentrations in cervicovaginal secretions (CVS), saliva, and sera of infected women. Measurement of antibody activity was obtained by comparing the amount of specific antibodies to the amount of total immunoglobulins found in the same compartment. Because of its quantitative and comparative nature, the assay was named comparative antibody gravimetric evaluation assay (CAGE).

Immunoglobulin concentrations and antigen-specific antibody levels in cervicovaginal lavages of rhesus macaques are influenced by the stage of the menstrual cycle.

The levels of antigen-specific antibodies (Abs) and immunoglobulins in the cervical mucus of women vary with the menstrual cycle; the highest levels occur during menses, and the lowest occur during the periovulatory period. The rhesus macaque is a widely used animal model of female genital tract immunity. We sought to determine whether rhesus macaques have a cyclical pattern of changing cervicovaginal Ab and immunoglobulin levels that is similar to that of the human female. This study examined the relationship of the stages of the menstrual cycle to genital mucosal and systemic immunoglobulin concentrations and Ab levels in rhesus macaques. In all seven rhesus macaques studied, the immunoglobulins G and A and some antibodies in cervicovaginal lavages varied with the stages of the menstrual cycle, and in many cases this variation reached the level of statistical significance. In a pattern similar to that of women, the highest levels of Abs and immunoglobulins occurred during menses, and the lowest levels occurred around the time of ovulation. However, the Ab and immunoglobulin levels in serum and rectal lavages did not change with the menstrual cycle stage. The results of this study are consistent with the hypothesis that the ovarian hormones that drive the menstrual cycle influence genital tract immunity in female primates.

Mucosal immunization of cynomolgus macaques with two serotypes of live poliovirus vectors expressing simian immunodeficiency virus antigens: stimulation of humoral, mucosal, and cellular immunity.

Poliovirus live virus vectors are a candidate recombinant vaccine system. Previous studies using this system showed that a live poliovirus vector expressing a foreign antigen between the structural and nonstructural proteins generates both antibody and cytotoxic T-lymphocyte responses in mice. Here we describe a novel in vitro method of cloning recombinant polioviruses involving a hybrid-PCR approach. We report the construction of recombinant vectors of two different serotypes of poliovirus-expressing simian immunodeficiency virus (SIV) antigens and the intranasal and intravenous inoculations of four adult cynomolgus macaques with these poliovirus vectors expressing the SIV proteins p17(gag) and gp41(env). All macaques generated a mucosal anti-SIV immunoglobulin A (IgA) response in rectal secretions. Two of the four macaques generated mucosal antibody responses detectable in vaginal lavages. Strong serum IgG responses lasting for at least 1 year were detected in two of the four monkeys. SIV-specific T-cell lymphoproliferative responses were detected in three of the four monkeys. SIV-specific cytotoxic T lymphocytes were detected in two of the four monkeys. This is the first report of poliovirus-elicited vaginal IgA or cytotoxic T lymphocytes in any naturally infectable primate, including humans. These findings support the concept that a live poliovirus vector is a potentially useful delivery system that elicits humoral, mucosal, and cellular immune responses against exogenous antigens.

Identification of DNA--cisplatin adducts in a blind trial of in situ scanning tunneling microscopy.

Scanning tunneling microscopy (STM) reveals nanometer scale details of hydrated DNA but the interpretation of the images is controversial because of substrate artifacts and the lack of a theory for image contrast. We demonstrate that we have overcome these problems by identifying five DNA samples by their STM images alone in a blinded trial. The samples were single-stranded and double-stranded DNA with and without covalent modification by the anti-tumor drug cisplatin. The cisplatin adducts were distinguished by substantial kinking at the drug binding site. The oligomers were 20 bases in length, which was too short to permit the kinking angle to be determined with precision. However, models with a 45 degree kink gave a better fit to the images of the duplex adducts than models with a 90 degrees kink. A variety of structures was observed for the single-stranded adducts.

Isolation, structural identification, and characterization of a mutagen from Fusarium moniliforme.

Strains of Fusarium moniliforme produce a variety of toxins, including several uncharacterized mutagens that act directly in the Ames assay using Salmonella typhimurium, strain TA 100. The Ames assay was used to monitor isolation of the direct-acting mutagens from the F. moniliforme culture extracts. Seven strains were tested, of which strains F07 and F84 contained the highest levels of direct-acting mutagens. Extracts of strain F84 were fractionated on a silica gel column, eluted with methanol-chloroform (1:9). This fraction was then separated on a reverse-phase, C-18 column with 50% methanol in water as eluant and further purified by TLC. One compound was isolated and given the trivial name fusarin X (FX). Its structure was determined from its UV (lambda max 357 nm), 500-MHz NMR, and mass spectra, and those of its diacetate, to be the 1-hydroxy analog of the previously characterized fusarin C. FX was present in culture extracts of strains F07 and F84 at 83 and 8 micrograms/g, respectively, which was proportional to their relative mutagenicities. It was not detected in the other strains tested. Since exposure of FX most likely occurs in cooked corn, its thermal stability was measured; it, like FC, decomposes at 100 degrees C, especially at high pH. Again, in common with FC, it was decomposed by GSH. The possible role of these Fusarium metabolites in the etiology of human cancers has still to be resolved.

In vitro transformation of rat esophageal epithelial cells by fusarin C.

Fusarin C (FC) is a mutagenic and carcinogenic mycotoxin which was isolated from Fusarium moniliforme culture extracts. The Fusarium moniliforme is one of most prevalent fungi found on corn in Linxian, a high risk area for esophageal cancer. This paper reports, for the first time, the malignant transformation of rat esophageal epithelial cells induced by FC. The transformed cells showed several characteristics of transformation. Colonies were formed after seeding these transformed cells either into selective medium free of epidermal growth factor (EGF) and serum, or on semi-solid agar; there was an increase in chromosome number; the expression of c-myc and v-erb-B oncogenes was enhanced in the cells; and squamous cell carcinomas arose after inoculating the cells into nude mice. The results demonstrated transforming effect of FC on rat esophageal epithelial cells, and indicate that the abnormal expression of some oncogenes could serve as a new property of transformed cells.

Affinity capture of [Arg8]vasopressin-receptor complex using immobilized antisense peptide.

Solubilized noncovalent complexes of [Arg8]-vasopressin (AVP) with receptor proteins from rat liver membranes were isolated by selective binding to silica-immobilized antisense (AS) peptide. The affinity chromatographic support was prepared with a chemically synthesized AS peptide whose sequence is encoded by the AS DNA corresponding to the 20 amino-terminal residues of the AVP bovine neurophysin II biosynthetic precursor [pro-AVP/BNPII-(20-1)], a region that includes the AVP sequence at residues 1-9. The AVP-related AS peptide previously was shown to bind selectively to AVP. The AS peptide-AVP interaction mechanism hypothesized, contact by hydropathic complementarily at multiple sites along the peptide chains, led to the prediction that AVP bound to its receptor would still have enough free surface to interact with immobilized AS peptide. To test this prediction of a three-way interaction, [3H]AVP-receptor was obtained as a solubilized, partially purified fraction from rat liver membrane. When this fraction was eluted through AS pro-AVP/BNPII-(20-1) silica, a complex containing [3H]AVP was bound and separated from the major, unretarded membrane protein fraction as well as from free AVP. Chemical crosslinking of [3H]AVP complex, SDS/PAGE of the products, and analysis of gel slices by scintillation counting led to detection of two major radiolabeled bands of 31 and 38 kDa. Covalent labeling was blocked when unlabeled AVP was added as a competitor before crosslinking. A third radiolabeled protein band of 15 kDa was found when the receptor complex was solubilized from rat liver membranes in the absence of the protease inhibitor phenylmethylsulfonyl fluoride. Covalently crosslinked [3H]AVP complex also was bound to the AS peptide column; binding was blocked by competition with unlabeled AVP in the elution buffer. Since the AVP-linked 31- and 38-kDa proteins have the same apparent molecular mass on SDS/PAGE as found previously by photo-affinity labeling, we conclude that the AS peptide column has affinity-captured AVP-receptor complexes. The 15-kDa protein appears to be an active AVP-receptor fragment of one or both of the larger proteins. It is generally concluded that immobilized AS peptides may be useful to isolate peptide and protein-receptor complexes in other systems as well.

[The metabolism and DNA binding of 3H-fusarin C in rats].

In this paper, the distribution and elimination of 3H-Fusarin C (3H-FC) in rats and DNA-binding of FC in cultured explants of rat esophagus are reported. The radioactivity distribution in tissues was altered dynamically, and the highest levels of radioactivity were found in the intestine, stomach and liver after giving 3H-FC into the stomachs of rats. Kidney, bladder, esophagus and spleen followed. The radioactivity levels in the lung and brain were low. Radioactivity in the blood reached a peak three hours after giving 3H-FC to rats, but about 50% of the radioactivity remained in the blood even after 24 h. The total urinary excretion of radioactivity in rats was found to be about 30.7% within 48 h, and about 27.8% was present in the feces. Only 5.4% unchanged FC was excreted in the urine, while other metabolites of FC accounted for 94.6% of total urinary radioactivity. DNA-binding of FC occurred in rat esophageal explants.