Biofilm formation by hyperpiliated mutants of Pseudomonas aeruginosa

Under static growth conditions, hyperpiliated, nontwitching pilT and pilU mutants of Pseudomonas aeruginosa formed dense biofilms, showing that adhesion, not twitching motility, is necessary for biofilm initiation. Under flow conditions, the pilT mutant formed mushroom-like structures larger than those of the wild type but the pilU mutant was defective in biofilm formation. Therefore, twitching motility affects the development of biofilm structure, possibly through modulation of detachment.     

Interprotein Disulfide Bonds Formed During Isolation Process Tighten the Structure of the Postsynaptic Density

Postsynaptic densities (PSDs) isolated by biochemical means consist of a complex mixture of proteins that tightly bond to each other. The purpose of this report is to study whether the numerous interprotein disulfides found in the isolated PSDs contribute to the tight structure of the PSDs and whether these interprotein disulfides exist in vivo. PSDs were isolated from pig cerebral cortex by conventional methods except that iodoacetic acid (IAA) was added to all solutions to curtail the formation of disulfides during the isolation process. The PSDs thus isolated were fragmented easily by treatment with chaotropic reagents or ionic detergents, whereas the PSDs isolated in IAA-free solutions were resistant to these treatments. Electron microscopy revealed that the PSDs isolated in IAA-containing solutions were more fragmented than those isolated in IAA-free solutions. Furthermore, the PSD sample isolated in IAA-free solutions contained very large disulfide-linked aggregates that were virtually absent from the PSDs isolated in IAA-containing solutions. Our results suggest that the exceptionally tight structure of the PSDs isolated by conventional methods is due largely to the new disulfides formed during the isolation process and that the PSD proteins under in vivo conditions are held together primarily by noncovalent interactions.     

Population structure of <Emphasis Type="Italic">Nouelia insignis</Emphasis> (Asteraceae), an endangered species in southwestern China,based on chloroplast DNA sequences: recent demographic shrinking

Nouelia insignis, an endangered species, is distributed in the Jinsha and Nanpan drainage areas in southwestern China. In this study, we examined the genetic diversity and population structure based on the sequences of the cpDNA rpL 16 intron. Low levels of genetic variation were detected within all populations of the endemic species. A gene genealogy of 11 haplotypes recovered two major lineages I and II, with haplotypes H1 and H6 nested as interior nodes, respectively. Haplotype H1 was widespread in all populations, while haplotype H6 was restricted to populations southern of the Jinsha River. Low levels of genetic differentiation were detected, as most F _st values between populations were zero. This result, however, contradicts previous studies based on allozymes and fingerprinting. Genetic analyses suggested that coancestry due to low evolutionary rates resulted in the lack of geographical subdivision. Molecular dating estimated that the two lineages split about 3.224 MYA (95% CI 1.070–6.089 MYA). Maintenance of ancestral polymorphisms was possibly attributable to a long-standing large effective population size until recently. Postglacial demographic expansion was supported by a unimodal mismatch distribution and star-like phylogenies.     

Inhibition of soil-borne plant pathogens by the treatment of sinigrin and myrosinases released from reconstructed Escherichia coli and Pichia pastoris

Myrosinases (thioglucoside glucohydrolase, EC 3.2.3.1) are able to hydrolyse glucosinolates in natural plant products. In Arabidopsis thaliana three different genes with different tissue-specific expressions and distribution patterns encode myrosinases. cDNAs of myrosinase genes (TGG1 and TGG2) were isolated from A. thaliana and expressed in Escherichia coli and Pichia pastoris. The enzyme activities of myrosinase TGG1 and TGG2 genes expressed in P. pastoris were higher than those expressed in E. coli. Among six glucosinolates tested for specificity to myrosinases TGG1 and TGG2, the suitable substrates for these two genes expressed in P. pastoris and E. coli were sinigrin, gluconapin, glucobrassicanapin and glucoraphanin. Treatment of sinigrin with myrosinases excreted from reconstructed E. coli and P. pastoris with TGG1 and TGG2 genes showed strong fungicidal effects on mycelial growth of Rhizoctonia solani AG-4, Sclerotium rolfsii, and Pythium aphanidermatum. This study suggests that the combination of glucosinolate with myrosinases excreted from the reconstructed microbes may be of potential for control of soil-borne diseases.     

Influence of putative exopolysaccharide genes on Pseudomonas putida KT2440 biofilm stability

We report a study of the role of putative exopolysaccharide gene clusters in the formation and stability of Pseudomonas putida KT2440 biofilm. Two novel putative exopolysaccharide gene clusters, pea and peb, were identified, and evidence is provided that they encode products that stabilize P. putida KT2440 biofilm. The gene clusters alg and bcs, which code for proteins mediating alginate and cellulose biosynthesis, were found to play minor roles in P. putida KT2440 biofilm formation and stability under the conditions tested. A P. putida KT2440 derivative devoid of any identifiable exopolysaccharide genes was found to form biofilm with a structure similar to wild-type biofilm, but with a stability lower than that of wild-type biofilm. Based on our data, we suggest that the formation of structured P. putida KT2440 biofilm can occur in the absence of exopolysaccharides; however, exopolysaccharides play a role as structural stabilizers.     

Effects of epinephrine and progesterone on the ocular hypertensive response to intravenous infusion of prostaglandin A2

Intravenous infusion of prostaglandin A₂ (0.24 μg/min) increased intraocular pressure and decreased mean arterial blood pressure in rabbits. This ocular hypertensive response was antagonized by a single topical pretreatment with 0.1 ml of a 1% 1-epinephrine and by pretreatment with intramuscular injections of progesterone (25 mg/kg, twice daily for a total of five injections). These results and others suggested that plasma prostaglandin A or E or both might be involved in the pathogenesis of ocular hypertension in glaucoma.     

Opposite and selective effects of epidermal growth factor and human platelet transforming growth factor-beta on the production of secreted proteins by murine 3T3 cells and human fibroblasts

Growth regulators such as epidermal growth factor (EGF) and type beta transforming growth factor (TGF-beta) regulate the synthesis and secretion of certain proteins by cells in culture. The secretion pattern of each cell line and the effect of growth regulators on the secretion pattern are unique. EGF increased the secreted and intracellular levels of mitogen-regulated protein (MRP) and major excreted protein (MEP) by Swiss 3T3 cells. MRP is related by sequence to prolactin. MEP is a thiol protease located intracellularly in the lysosomes. EGF also selectively induced a 52,000-dalton mitogen-induced protein (MIP 52) secreted by human fibroblasts. Two types of TGF-betas were tested for their effects on the expression of secreted proteins in mouse and human fibroblasts: TGF-beta from human platelets and a growth inhibitor (GI/TGF-beta) secreted by BSC-1 cells. Each selectively decreased the levels of the two secreted proteins induced by growth factors in mouse embryo 3T3 cells and one secreted protein induced by growth factors in human fibroblasts. Platelet TGF-beta and GI/TGF-beta also induced one 48,000-dalton protein secreted by human fibroblasts. Synthesis of DNA and the incorporation of [35S]methionine into total protein in Swiss 3T3 cells were not affected by platelet TGF-beta or GI/TGF-beta. Thus, the inhibitory effect of platelet TGF-beta on the synthesis and secretion of these three proteins is due to a specific effect of platelet TGF-beta on the regulation of MRP and MEP that does not interfere with the ability of EGF to stimulate DNA or protein synthesis.     

Regulation of cholesterol 7 alpha-hydroxylase in the liver. Cloning, sequencing, and regulation of cholesterol 7 alpha-hydroxylase mRNA.

Monospecific antibody against purified rat liver cholesterol 7 alpha-hydroxylase cytochrome P-450 was used to screen a lambda gt11 cDNA library constructed from immuno-enriched polysomal RNA of cholestyramine-treated female rat liver. Two types of cDNA clones differing in the length of the 3'-untranslated region were identified, and DNA sequences were determined. The full length clone contains 3561 base pairs plus a long poly(A) tail. The amino acid sequence deduced from the open reading frame revealed a unique P-450 protein containing 503 amino acid residues which belonged to a new gene family designated family VII or CYP7. Southern blot hybridization experiments indicated that the minimal size of P-450 VII gene was 11 kilobase pairs (kb), and there was probably only one gene in this new family. Northern blot hybridization using specific cDNA probes revealed at least two major mRNA species of about 4.0 kb and 2.1 kb, respectively. These two mRNA species may be derived from the use of different polyadenylation signals and reverse-transcribed to two types of cDNA clones. Cholesterol 7 alpha-hydroxylase mRNAs were induced 2- to 3-fold in rat liver by cholestyramine treatment. The mRNA level was rapidly reduced upon the removal of the inducer. Similarly, cholesterol feeding induced enzyme activity, protein, and mRNA levels in the rat by 2-fold, suggesting that cholesterol is an important regulator of cholesterol 7 alpha-hydroxylase in the liver. On the other hand, dexamethasone and pregnenolone-16 alpha-carbonitrile drastically reduced the activity, protein, and mRNA levels. These experiments suggest that the induction of cholesterol 7 alpha-hydroxylase activity by cholestyramine or cholesterol and inhibition of cholesterol 7 alpha-hydroxylase activity by bile acid feedback are results of the rapid turnover of cholesterol 7 alpha-hydroxylase enzyme and mRNA levels.     

Efficient Removal of Co2+ from Aqueous Solution by 3-Aminopropyltriethoxysilane Functionalized Montmorillonite with Enhanced Adsorption Capacity

To achieve a satisfactory removal efficiency of heavy metal ions from wastewater, silane-functionalized montmorillonite with abundant ligand-binding sites (-NH₂) was synthesized as an efficient adsorbent. Ca-montmorillonite (Ca-Mt) was functionalized with 3-aminopropyl triethoxysilane (APTES) to obtain the APTES-Mt products (APTES_1.0CEC-Mt, APTES_2.0CEC-Mt, APTES_3.0CEC-Mt, APTES_4.0CEC-Mt) with enhanced adsorption capacity for Co²⁺. The physico-chemical properties of the synthesized adsorbents were characterized by spectroscopic and microscopic methods, and the results demonstrated that APTES was successfully intercalated into the gallery of Ca-Mt or grafted onto the surface of Ca-Mt through Si-O bonds. The effect of solution pH, ionic strength, temperature, initial concentrations and contact time on adsorption of Co²⁺ by APTES-Mt was evaluated. The results indicated that adsorption of Co²⁺ onto Ca-Mt, APTES_1.0CEC-Mt and APTES_2.0CEC-Mt can be considered to be a pseudo-second-order process. In contrast, adsorption of Co²⁺ onto APTES_3.0CEC-Mt and APTES_4.0CEC-Mt fitted well with the pseudo-first-order kinetics. The adsorption isotherms were described by the Langmuir model, and the maximum adsorption capacities of APTES_1.0CEC-Mt, APTES_2.0CEC-Mt, APTES_3.0CEC-Mt and APTES_4.0CEC-Mt were 25.1, 33.8, 61.6, and 61.9 mg·g^-1, respectively. In addition, reaction temperature had no impact on the adsorption capacity, while both the pH and ionic strength significantly affected the adsorption process. A synergistic effect of ion exchange and coordination interactions on adsorption was observed, thereby leading to a significant enhancement of Co²⁺ adsorption by the composites. Thus, APTES-Mt could be a cost-effective and environmental-friendly adsorbent, with potential for treating Co²⁺-rich wastewater.