Microbial reduction of cyclohexanones

A Brazilian strain of the bacteria Serratia rubidaea CCT 5742 quantitatively reduced 4-tert-butylcyclohexanone and 4-methylcyclohexanone to the less thermodynamically stable diastereoisomeric alcohols cis-4-tert-butylcyclohexanol and cis-4-methylcyclohexanol with a diastereoisomeric excesses (de) of 96% and 44%, respectively. 2-Methylcyclohexanone was also totally reduced to the corresponding alcohols affording the trans-(+)-(1S, 2S)-2-methylcyclohexanol with 78% of de and an enantiomeric excess (ee) of 80%. The cis-(+)-(1S, 2R)-2-methylcyclohexanol was obtained in 98% ee.     

Differential expression of the pr1A gene in Metarhizium anisopliae and Metarhizium acridum across different culture conditions and during pathogenesis

The entomopathogenic fungi of the genus Metarhizium have several subtilisin-like proteases that are involved in pathogenesis and these have been used to investigate genes that are differentially expressed in response to different growth conditions. The identification and characterization of these proteases can provide insight into how the fungus is capable of infecting a wide variety of insects and adapt to different substrates. In addition, the pr1A gene has been used for the genetic improvement of strains used in pest control. In this study we used quantitative RT-PCR to assess the relative expression levels of the pr1A gene in M. anisopliae and M. acridum during growth in different culture conditions and during infection of the sugar cane borer, Diatraea saccharalis Fabricius. We also carried out a pathogenicity test to assess the virulence of both species against D. saccharalis and correlated the results with the pattern of pr1A gene expression. This analysis revealed that, in both species, the pr1A gene was differentially expressed under the growth conditions studied and during the pathogenic process. M. anisopliae showed higher expression of pr1A in all conditions examined, when compared to M. acridum. Furthermore, M. anisopliae showed a greater potential to control D. saccharalis. Taken together, our results suggest that these species have developed different strategies to adapt to different growing conditions.     

Low prevalence of hepatitis C virus infection in Amerindians from Western Venezuela

Previous studies have not found hepatitis C virus (HCV) infection in Amerindians from Western Venezuela. A survey of 254 Bari and Yukpa natives aged 10-60 years (mean +/- SD age = 35 +/- 5.4 years) from four communities, two Bari and two Yukpa, in this area were studied to assess the prevalence of antibodies to HCV (anti-HCV) and HCV RNA among these indigenous populations. Serum samples were examined initially for anti-HCV by a four generation enzyme-linked immunosorbent assay (ELISA). Reactive samples were then tested using a third generation recombinant immunoblot assay (RIBA-3). Viral RNA was investigated in all immunoblot-reactive samples by a nested polymerase chain reaction (PCR) method. Six (2.3%) of 254 natives were positive by ELISA, one (2.2%) of these reactive samples were positive by RIBA, and four (1.5%) were indeterminate. Only two (0.8%) were positive by PCR, corresponding to 1 (2.1%) of 47 inhabitants of a Yukpa community and to 1 (2.2%) of 45 subjects of a Bari community. Iatrogenic is thought to play a role in acquisition of the infection. The findings indicate a HCV focus of low endemicity and are compatible with a low degree of exposures of the natives to the virus. Studies are necessary to assess the risk factors for infection in these Amerindians.     

Cutting edge: Complement (C3d)-linked antigens break B cell anergy

C3dg adducts of Ag can coligate complement receptor type 2 (CR2; CD21) and the B cell Ag receptor. This interaction significantly amplifies BCR-mediated signals in Ag-naive wild-type mice, lowering the threshold for B cell activation and the generation of humoral immune responses as much as 1000-fold. In this study we demonstrate that CR2-mediated complementation of BCR signals can also overcome B cell anergy. Unlike Ag alone, BCR/CR2 costimulation (Ars-CCG/C3dg complexes) of anergic Ars/A1 B cells led to Ca(2+) mobilization in vitro and the production of autoantibodies in vivo. Interestingly, the in vivo immune response of anergic cells occurs without the formation of germinal centers. These results suggest that the Ag unresponsiveness of anergic B cells can be overcome by cross-reactive (self-mimicking) Ags that have been complement-opsonized. This mechanism may place individuals exposed to complement-fixing bacteria at risk for autoimmunity.     

Characterization of heme as activator of Toll-like receptor 4

Heme is an ancient and ubiquitous molecule present in organisms of all kingdoms, composed of an atom of iron linked to four ligand groups of porphyrin. A high amount of free heme, a potential amplifier of the inflammatory response, is a characteristic feature of diseases with increased hemolysis or extensive cell damage. Here we demonstrate that heme, but not its analogs/precursors, induced tumor necrosis factor-alpha (TNF-alpha) secretion by macrophages dependently on MyD88, TLR4, and CD14. The activation of TLR4 by heme is exquisitely strict, requiring its coordinated iron and the vinyl groups of the porphyrin ring. Signaling of heme through TLR4 depended on an interaction distinct from the one established between TLR4 and lipopolysaccharide (LPS) since anti-TLR4/MD2 antibody or a lipid A antagonist inhibited LPS-induced TNF-alpha secretion but not heme activity. Conversely, protoporphyrin IX antagonized heme without affecting LPS-induced activation. Moreover, heme induced TNF-alpha and keratinocyte chemokine but was ineffective to induce interleukin-6, interleukin-12, and interferon-inducible protein-10 secretion or co-stimulatory molecule expression. These findings support the concept that the broad ligand specificity of TLR4 and the different activation profiles might in part reside in its ability to recognize different ligands in different binding sites. Finally, heme induced oxidative burst, neutrophil recruitment, and heme oxygenase-1 expression independently of TLR4. Thus, our results presented here reveal a previous unrecognized role of heme as an extracellular signaling molecule that affects the innate immune response through a receptor-mediated mechanism.     

Isolation of Brazilian marine fungi capable of growing on DDD pesticide

The fungi Aspergillus sydowii Ce15, Aspergillus sydowii Ce19, Aspergillus sydowii Gc12, Bionectria sp. Ce5, Penicillium miczynskii Gc5, Penicillium raistrickii Ce16 and Trichoderma sp. Gc1, isolated from marine sponges Geodia corticostylifera and Chelonaplysylla erecta, were evaluated for their ability to grow in the presence of DDD pesticide. Increasing concentrations of DDD pesticide, i.e., 5.0 mg (1.56 × 10⁻¹² mmol), 10.0 mg (3.12 × 10⁻² mmol) and 15.0 mg (4.68 × 10⁻² mmol) in solid and liquid culture media were tested. The fungi Trichoderma sp. Gc1 and Penicillium miczynskii Gc5 were able to grow in the presence of up to 15.0 mg of DDD, suggesting their potential for biodegradation. A 100% degradation of DDD was attained in liquid culture medium when Trichoderma sp. Gc1 was previously cultivated for 5 days and supplemented with 5.0 mg of DDD in the presence of hydrogen peroxide. However, the quantitative analysis showed that DDD was accumulated on mycelium and biodegradation level reached a maximum value of 58% after 14 days.     

Production and characterization of a collagenolytic serine proteinase by <Emphasis Type="Italic">Penicillium aurantiogriseum</Emphasis> URM 4622: A factorial study

A 2⁴ full factorial design was used to identify the main effects and interactions of the initial medium pH, soybean flour concentration, temperature and orbital agitation speed on extracellular collagenase production by Penicillium aurantiogriseum URM4622. The most significant variables for collagenase production were soybean flour concentration and initial medium pH that had positive main effects, and temperature that had a negative one. Protein concentration in soybean flour revealed to be a significant factor for the production of a collagenase serine proteinase. The most favorable production conditions were found to be 0.75% soybean flour, pH 8.0, 200 rpm, and 28°C, which led to a collagenase activity of 164 U. The enzyme showed an optimum activity at 37°C and pH 9.0, was stable over wide ranges of pH and temperature (6.0 ∼ 10.0 and 25 ∼ 45°C, respectively) and was strongly inhibited by 10 mM phenylmethylsulphonylfluoride. The firstorder rate constants for collagenase inactivation in the crude extract, calculated from semi-log plots of the residual activity versus time, were used in Arrhenius and Eyring plots to estimate the main thermodynamic parameters of thermoinactivation (E* _d = 107.4 kJ/mol and ΔH* _d = 104.7 kJ/mol). The enzyme is probably an extracellular neutral serine collagenase effective on azocoll, gelatin and collagen decomposition.     

Induction of Cell Cycle Arrest and Apoptosis by Ruthenium Complex cis-(Dichloro)tetramineruthenium(III) Chloride in Human Lung Carcinoma Cells A549

Lung cancer is one of the leading causes of death in the world, and non-small cell lung carcinoma (NSCLC) accounts for approximately 75-85% of all lung cancers. In the present work, we studied the cytotoxic activity, cell cycle arrest and induction apoptosis of the compound cis-(dichloro)tetramineruthenium(III) chloride {cis-[RuCl(2)(NH(3))(4)]Cl} in human lung carcinoma tumor cell line A549. The results of MTT and trypan blue assays showed that cis-[RuCl(2)(NH(3))(4)]Cl causes reduction in the viability of A549 cells when treating with 95 and 383 μM of the compound for 48 and 72 h. Lower concentrations of the compound (19, 3.8 and 0.38 μM), however, only slightly affected cell viability. The IC(50) value for the compound was about 383 μM. Survival analysis of the A549 cells after treatment with ruthenium(III) compound using long term clonogenic assay showed that it reduced colony formation ability at concentrations of 0.38 and 3.8 μM, and at concentrations of 95 and 383 μM no colonies were observed. Cell cycle analysis showed that compound ruthenium led to an accumulation of A549 cells in S phase and increased in the sub-G1 peak. In addition, cis-(dichloro)tetramineruthenium(III) chloride treatment induced apoptosis, as observed by the increased numbers of annexin V-positive cells and increased messenger RNA expression of caspase-3.     

Androgen- and estrogen-dependent regulation of insulin-degrading enzyme in subcellular fractions of rat prostate and uterus

Innumerous data support the fact that insulin-degrading enzyme (IDE) is the primary enzymatic mechanism for initiating and controlling cellular insulin degradation. Nevertheless, insulin degradation is unlikely to be the only cellular function of IDE, because it appears that some cellular effects of insulin are mediated by IDE as a regulatory protein. Insulin-degrading enzyme shows a significant correlation with various cellular functions, such as cellular growth and differentiation, and the expression of IDE is developmentally regulated. Besides insulin, other substrates are also degraded by IDE, including various growth-promoting peptides. It has also been shown that IDE enhances the binding of androgen to DNA in the nuclear compartment. It is also known that the androgen hormones have a stimulatory effect on prostate growth, and that estradiol stimulates uterine growth. To establish whether IDE is regulated by a cellular prostate/uterine growth stimulus, the present study assessed whether IDE was modified in quantity and activity during proliferative conditions (castration + testosterone in the male rat, or castration + estradiol or the proestrus phase of the estrous cycle in the female rat) and autolysis (castration or the metestrus phase of the estrous cycle) using cytosolic and nuclear fractions of rat prostate and cytosolic fractions of rat uterus. The activity and amount of IDE decreased in the cytosolic fraction with castration and during metestrus, and increased with testosterone or estradiol treatment and during proestrus. In the nuclear fraction, the quantity of the IDE followed the same pattern observed in the cytosolic fraction, although without degradative activity. The data presented here suggest that IDE may participate in prostatic and uterine growth and that the testosterone or estradiol and/or prostate and uterus insulin-like growth factors may be important factors for the expression and regulation of IDE in the prostate and uterus.