Fermentation of galacturonic acid and pectin-rich materials to ethanol by genetically modified strains of Erwinia

Evaluation of the four ethanologenic constructs of bacteria in the genus Erwinia indicates that two strains E. chrysanthemi EC16 and E. carotovora SR38 show promise for development of direct hydrolysis and fermentation of pectin-rich substrates to mixtures of ethanol and acetate. Both strains fermented glucose to ethanol in nearly theoretical yields, but produced mainly acetate and ethanol by fermentation of D-galacturonic acid. Both strains depolymerized citrus pectin, polygalacturonic acid and polysaccharides in citrus peel and converted resulting sugars to carbon dioxide, acetate, ethanol and lesser amounts of formate and succinate.     

A theory of preattentive texture discrimination based on first-order statistics of textons

The many indistinguishable texture pairs having identical second-, but different third- and higher-order statistics, led to the conjecture thatglobally the preattentive texture discrimination system cannot process statistical parameters of third- or higher-order. Thus in cases when iso-second-order textures yield discrimination this must be based onlocal conspicuous features calledtextons (Julesz, 1980). Here it is shown that globally even second-order statistical parameters, such as autocorrelation, cannot be processed by the textural system, and texture discrimination is solely the result of first-order statistics (density) of textons. It is also shown that the perceivable distance of statistical constraints (coherence distance) in densely packed stochastic textures is very short, four dots or less. As of now, only three texton classes were found: color, elongated blobs (line segments) of given width, orientation, and length, and the terminators (end-points) of these elongated blobs. The strength of these textons is demonstrated by several examples.     

Vasoconstrictor mechanisms in the cerebral circulation are unaffected by insulin resistance

Insulin-resistance (IR) impairs agonist-induced relaxation in cerebral arteries, but little is known about its effect on constrictor mechanisms. We examined the vascular responses of the basilar artery (BA) and its side branches in anesthetized Zucker lean (ZL) and IR Zucker obese (ZO) rats using a cranial window technique. Endothelin-1 (ET-1) constricted the BAs in both the ZL and ZO rats, but there was no significant difference between the two groups (ZL: 36 +/- 8%; ZO: 33 +/- 3% at 10(-8) M). Inhibition of the ET(A) receptors by BQ-123 slightly increased the diameters of the BAs, with no difference shown between the ZL (6 +/- 1%) and ZO (5 +/- 3%) rats. Expressions of the ET(A) receptors and ET-1 mRNA examined by immunoblot analysis and RT-PCR, respectively, were also similar in the ZL and ZO groups. Phorbol 12,13-dibutyrate (PDBu), an activator of protein kinase C (PKC), and the thromboxane A(2) (TxA(2)) mimetic U-46619 constricted the BAs, but similarly to ET-1, there was no significant difference between the ZL and ZO groups (10(-6) M PDBu: ZL: 33 +/- 2%; ZO: 32 +/- 4%; and 10(-7) M U-46619: ZL: 23 +/- 1%; ZO: 19 +/- 2%). Inhibition of Rho-kinase with Y-27632 induced dilation of the BAs, and these responses were also comparable in the ZL and ZO rats (ZL: 39 +/- 4%; ZO: 38 +/- 2% at 10(-5) M). In contrast, nitric oxide-dependent relaxation to bradykinin was significantly reduced in the ZO rats (10(-6) M: 10 +/- 3%) compared with ZLs (29 +/- 7%, P < 0.01). These findings indicate that vasoconstrictor responses of the BA mediated by ET-1, TxA(2), PKC, and Rho-kinase are not affected by IR.     

Accumulation of mutant alpha1-antitrypsin Z in the endoplasmic reticulum activates caspases-4 and -12, NFkappaB, and BAP31 but not the unfolded protein response

In alpha(1)-antitrypsin (alpha1AT) deficiency, a polymerogenic mutant form of the secretory glycoprotein alpha1AT, alpha1ATZ, is retained in the endoplasmic reticulum (ER) of liver cells. It is not yet known how this results in liver injury in a subgroup of deficient individuals and how the remainder of deficient individuals escapes liver disease. One possible explanation is that the "susceptible" subgroup is unable to mount the appropriate protective cellular responses. Here we examined the effect of mutant alpha1ATZ on several potential protective signaling pathways by using cell lines with inducible expression of mutant alpha1AT as well as liver from transgenic mice with liver-specific inducible expression of mutant alpha1AT. The results show that ER retention of polymerogenic mutant alpha1ATZ does not result in an unfolded protein response (UPR). The UPR can be induced in the presence of alpha1ATZ by tunicamycin excluding the possibility that the pathway has been disabled. In striking contrast, ER retention of nonpolymerogenic alpha1AT mutants does induce the UPR. These results indicate that the machinery responsible for activation of the UPR can distinguish the physical characteristics of proteins that accumulate in the ER in such a way that it can respond to misfolded but not relatively ordered polymeric structures. Accumulation of mutant alpha1ATZ does activate specific signaling pathways, including caspase-12 in mouse, caspase-4 in human, NFkappaB, and BAP31, a profile that was distinct from that activated by nonpolymerogenic alpha1AT mutants.     

Crystal structure of a cohesin module from Clostridium cellulolyticum: implications for dockerin recognition

In the assembly of the Clostridium cellulolyticum cellulosome, the multiple cohesin modules of the scaffolding protein CipC serve as receptors for cellulolytic enzymes which bear a dockerin module. The X-ray structure of a type I C. cellulolyticum cohesin module (Cc-cohesin) has been solved using molecular replacement, and refined at 2.0 A resolution. Despite a rather low sequence identity of 32 %, this module has a fold close to those of the two Clostridium thermocellum cohesin (Ct-cohesin) modules whose 3D structures have been determined previously. Cc-cohesin forms a dimer in the crystal, as do the two Ct-cohesins. We show here that the dimer exists in solution and that addition of dockerin-containing proteins dissociates the dimer. This suggests that the dimerization interface and the cohesin/dockerin interface may overlap. The nature of the overall surface and of the dimer interface of Cc-cohesin differ notably from those of the Ct-cohesin modules, being much less polar, and this may explain the species specificity observed in the cohesin/dockerin interaction of C. cellulolyticum and C. thermocellum. We have produced a topology model of a C. cellulolyticum dockerin and of a Cc-cohesin/dockerin complex using homology modeling and available biochemical data. Our model suggests that a special residue pair, already identified in dockerin sequences, is located at the center of the cohesin surface putatively interacting with the dockerin.     

Vaccinia virus infection and gene transduction in cultured neurons

The study of neurons in culture would benefit from the development of a gene transduction system capable of delivering foreign genes at high efficiency, as transduction of primary neurons with existing systems is inefficient. The efficacy of lytic vaccinia virus (VV) infection of primary retinal cultures and PC12 cells (a model of neuronal differentiation) was examined in order to determine the efficiency of gene transduction using VV in neuronal primary culture. VV was able to infect retinal cells and PC12 cells and express transgenes of Escherichia coli beta-galactosidase (lacZ) and epithelial fatty acid binding protein (E-FABP) in a virus dose-dependent manner. Most (50-100%) of the retinal cells were positive for transgene protein at multiplicities of infection (MOI) between 10 and 100 plaque-forming units (PFU), while over 50% of VV-infected PC12 cells expressed the virus encoded gene at an MOI = 10. The production of foreign mRNA and protein by VV following infection was verified by PCR and Western blot. Because VV is a lytic virus, cytopathic effects were examined. Retinal cultures maintained for 0.5 days in vitro showed greater than 90% survival at 24 h post-infection, while 14-day cultures were equally viable for 48 h. Retinal ganglion cells and differentiated PC12 cells appear to be more protected against lytic VV infection than proliferating glial and undifferentiated PC12 cells. These data suggest that VV may be a useful vector for delivering foreign genes to neuronal cells with an efficient transient transgene expression.     

Importance of UVA photoprotection as shown by genotoxic related endpoints: DNA damage and p53 status

In order to demonstrate the importance of photoprotection in the UVA range (320-400 nm), an in vitro approach where sun formulations are spread on a quartz slide, and placed over human keratinocytes in culture is proposed as a convenient test for photoprotection assessment at the DNA level. Using the comet assay, DNA strand breaks, oxidative DNA damage or drug-induced DNA breaks were assessed. Accumulation of p53 protein was also studied as a marker for UV-induced genotoxic stress. Such a method was used to compare two formulations with different photostability. Spectroradiometry showed that a photounstable formulation lost its effectiveness in UVA screening when pre-irradiated by simulated sunlight (UVB+UVA). As a consequence, it was also shown that this formulation was not as protective as the photostable one at the genomic level. These data demonstrate that the loss of absorbing efficiency within UVA wavelengths due to photounstability may have detrimental consequences leading to impairments implicated in genotoxic events.     

Lymphoid aggregates in gonads of embryos, hatchlings, and young of turtles with temperature-dependent sex determination

Cellular infiltrations forming lymphoid-like aggregates were previously observed in gonads of two turtle species exhibiting temperature-dependent sex determination (TSD): at hatching in Chelydra serpentina; at and after hatching in Emys orbicularis. We show here that such aggregates are also present in gonads of Testudo graeca by the end of embryonic development, suggesting that their occurrence is general in turtles. Since in C. serpentina, infiltrations were observed mainly in testes exhibiting remnants of the germinal epithelium, it was assumed that their occurrence was an expression of maleness leading to rejection of this epithelium. The generality of this hypothesis was tested in E. orbicularis by looking for lymphoid-like aggregates in three types of gonads (testes, ovotestes, and ovaries) and for the stages at which they occur. Gonads were from embryos, hatchlings, and young incubated at various temperatures. Ovotestes obtained by treatment with an aromatase inhibitor of eggs incubated at female-producing temperature were also examined. In these gonads, the differentiation of Sertoli cells in testicular cords/tubes was ascertained by expression of SOX9. Moreover, the cell composition of aggregates was determined on electron micrographs. Aggregates appear in ovaries and ovotestes by the end of embryonic development and are present in the majority of these gonads at hatching, and at least up to one year after hatching. They are composed mainly of lymphocytes and fibroblasts. Aggregates are not present in typical testes. Since they occur in most ovaries, they cannot be seen as an expression of maleness. Rather, lymphocytic infiltration and formation of lymphoid aggregates in turtle gonads can be seen as components of the immune system, and can be under the control of gonadal endogenous sex steroids.     

Expression of Sorsby's fundus dystrophy mutations in human retinal pigment epithelial cells reduces matrix metalloproteinase inhibition and may promote angiogenesis

Sorsby's fundus dystrophy (SFD) is an autosomal dominant degenerative disease of the macula caused by mutations in the tissue inhibitor of metalloproteinase-3 (TIMP-3) gene. Choroidal neovascularization is a hallmark of this disease, which closely resembles the exudative form of age-related macular degeneration. However, the mechanism by which TIMP-3 mutations induce the disease phenotype in SFD remains unknown. To address this question we established human retinal pigment epithelial cell lines expressing wild type or S156C (Ser(156) changed to cysteine) mutant TIMP-3. S156C TIMP-3 had reduced matrix metalloproteinase (MMP) inhibitory activity in retinal pigment epithelial cells and resulted in increased secretion and activation of gelatinase A and B. The conditioned medium from these cells induced angiogenesis in "in vivo" chick chorioallantoic membrane assays that could be reversed with recombinant wild type TIMP-3. Our data indicate that the choroidal neovascularization in SFD may be a result of increased MMP activity, which could lead to the stimulation of angiogenesis. These results also suggest the potential therapeutic use of TIMP-3 or synthetic MMP inhibitors in this disease.     

Slow Onset Inhibition of Bacterial ��-Ketoacyl-acyl Carrier Protein Synthases by Thiolactomycin

Thiolactomycin (TLM), a natural product thiolactone antibiotic produced by species of Nocardia and Streptomyces, is an inhibitor of the β-ketoacyl-acyl carrier protein synthase (KAS) enzymes in the bacterial fatty acid synthase pathway. Using enzyme kinetics and direct binding studies, TLM has been shown to bind preferentially to the acyl-enzyme intermediates of the KASI and KASII enzymes from Mycobacterium tuberculosis and Escherichia coli. These studies, which utilized acyl-enzyme mimics in which the active site cysteine was replaced by a glutamine, also revealed that TLM is a slow onset inhibitor of the KASI enzymes KasA and ecFabB but not of the KASII enzymes KasB and ecFabF. The differential affinity of TLM for the acyl-KAS enzymes is proposed to result from structural change involving the movement of helices α5 and α6 that prepare the enzyme to bind malonyl-AcpM or TLM and that is initiated by formation of hydrogen bonds between the acyl-enzyme thioester and the oxyanion hole. The finding that TLM is a slow onset inhibitor of ecFabB supports the proposal that the long residence time of TLM on the ecFabB homologues in Serratia marcescens and Klebsiella pneumonia is an important factor for the in vivo antibacterial activity of TLM against these two organisms despite the fact that the in vitro MIC values are only 100–200 μg/ml. The mechanistic data on the interaction of TLM with KasA will provide an important foundation for the rational development of high affinity KasA inhibitors based on the thiolactone skeleton.