Mode of action of Fusarium moniliforme endopolygalacturonase towards acetylated pectin

Endopolygalacturonase from Fusarium moniliforme was used to degrade acetylated homogalacturonan previously prepared from sugar beet pulp. The initial velocity and the final percentage of hydrolysis decreased very rapidly with increasing degree of acetylation, showing that acetyl substitution markedly affected the enzymatic activity. MALDI-TOF mass spectrometry was used to analyse the reaction products and to show acetyl groups on the oligogalacturonates. The results demonstrated that the enzyme was able to accommodate acetyl groups in its active site cleft. The influence of acetyl groups on the mode of action of the enzyme was discussed and compared to the influence of methyl groups.     

The inducible and cytochrome P450-containing dehydroepiandrosterone 7α-hydroxylating enzyme system of Fusarium moniliforme

7α-Hydroxylation of DHEA by Fusarium moniliforme was investigated with regard to inducibility and characterization of the responsible enzyme system. Using GC/MS, the 7-hydroxylated metabolites of DHEA produced after biotransformation by Fusarium moniliforme mycelia were identified. The strain of Fusarium moniliforme hydroxylated DHEA predominantly at the 7α-position, with minor hydroxylation occurring at the 7β-position. Constitutive 7α-hydroxylation activity was low, but DHEA induced the enzyme complex responsible for 7α-hydroxylation via an increase in protein synthesis. DHEA 7α-hydroxylase was found to be mainly microsomal, and the best production yields of 7α-hydroxy-DHEA (28.5 ± 3.51 pmol/min/mg protein) were obtained with microsomes prepared from 18-h-induced mycelia. Kinetic parameters (K_M=1.18 ± 0.035 μM and V_max=909 ± 27 pmol/min/mg protein) were determined. Carbon monoxide inhibited 7α-hydroxylation of DHEA by microsomes of Fusarium moniliforme. Also, exposure of mycelia to DHEA increased microsomal P450 content. These results demonstrated that: (i) DHEA is 7α-hydroxylated by microsomes of Fusarium moniliforme; (ii) DHEA induces Fusarium moniliforme 7α-hydroxylase; (iii) this enzyme complex contains a cytochrome P450.     

Phosphorylations of αA- and αB-crystallin

In addition to being refractive proteins in the vertebrate lens, the two α-crystallin polypeptides (αA and αB) are also molecular chaperones that can protect proteins from thermal aggregation. The αB-crystallin polypeptide, a functional member of the small heat shock family, is expressed in many tissues in a developmentally regulated fashion, is stress-inducible, and is overexpressed in many degenerative diseases and some tumors indicating that it plays multiple roles. One possible clue to α-crystallin functions is the fact that both polypeptides are phosphorylated on serine residues by cAMP-dependent and cAMP-independent mechanisms. The cAMP-independent pathway is an autophosphorylation that has been demonstrated in vitro, depends on magnesium and requires cleavage of ATP. Disaggregation of αA-, but not αB-crystallin into tetramers results in an appreciable increase in autophosphorylation activity, reminiscent of other heat shock proteins, and suggests the possibility that changes in the aggregation state of αA-crystallin are involved in yet undiscovered signal transduction pathways. The α-crystallin polypeptides differ with respect to their abilities to undergo cAMP-dependent phosphorylation, with preference given to the αB-crystallin chain. These differences and complexities in α-crystallin phosphorylations, coupled with the differences in expression patterns of the two α-crystallin polypeptides, are consistent with the idea that each polypeptide has distinctive structural and metabolic roles.     

α3β1 integrin promotes cell survival via multiple interactions between 14-3-3 isoforms and proapoptotic proteins

Laminin-5 and α3β1 integrin promote keratinocyte survival; however, the downstream signaling pathways for laminin-5/α3β1 integrin-mediated cell survival had not been fully established. We report the unexpected finding of multiple interactions between 14-3-3 isoforms and proapoptotic proteins in the survival signaling pathway. Ln5-P4 motif within human laminin-5 α3 chain promotes cell survival and anti-apoptosis by inactivating Bad and YAP. This effect is achieved through the formation of 14-3-3ζ/p-Bad and 14-3-3σ/p-YAP complexes, which is initiated by α3β1 integrin and FAK/PI3K/Akt signaling. These complexes result in cytoplasmic sequestration of Bad and YAP and their subsequent inactivation. An increase in Akt1 activity in cells induces 14-3-3ζ and σ, p-Bad, and p-YAP, promoting cell survival, whereas decreasing Akt activity suppresses the same proteins and inhibits cell survival. Suppression of 14-3-3ζ with RNA-interference inhibits cell viability and promotes apoptosis. These results reveal a new mechanism of cell survival whereby the formation of 14-3-3ζ/p-Bad and 14-3-3σ/p-YAP complexes is initiated by laminin-5 stimulation via the α3β1 integrin and FAK/PI3K/Akt signaling pathways, thereby resulting in cell survival and anti-apoptosis.     

Cardiovascular actions of prostaglandins F2α; 15-me-F2α; F1α; F2β and F1β in the cat

Prostaglandin F_2α (5μg/kg, i.v.) causes an increase in pulmonary arterial pressure, decrease in systemic arterial pressure, and reflex bradycardia in the anesthetized cat. The same dose of the 15-methyl analogue of PGF_2α produces the same triad of effects but of greater magnitude and duration. Although prostaglandins F_1α, F_2β and F_1β also cause the same cardiovascular effects as F_2α, there is a decrease in potency for all parameters measured, with PGF_2α>PGF_1α>PGF_2β>PGF_1β. When compared to the actions of PGF_2α in producing an increase in pulmonary arterial pressure, PGs F_1α, F_2β and F_1β were less potent by approximately 10, 100, and 1000 fold respectively.     

Antinoceptive effect of triterpenoid α,β-amyrin in rats on orofacial pain induced by formalin and capsaicin

The effects of α,β-amyrin, a pentacyclic triterpene isolated from Protium heptaphylum was investigated on rat model of orofacial pain induced by formalin or capsaicin. Rats were pretreated with α,β-amyrin (10, 30, and 100 mg/kg, i.p.), morphine (5 mg/kg, s.c.) or vehicle (3% Tween 80), before formalin (20 μl, 1.5%) or capsaicin (20 μl, 1.5 μg) injection into the right vibrissa. In vehicle-treated controls, formalin induced a biphasic nociceptive face-rubbing behavioral response with an early first phase (0–5 min) and a late second phase (10–20 min) appearance, whereas capsaicin produced an immediate face-rubbing (grooming) behavior that was maximal at 10–20 min. Treatment with α,β-amyrin or morphine significantly inhibited the face-rubbing response in both test models. While morphine produced significant antinociception in both phases of formalin test, α,β-amyrin inhibited only the second phase response, more prominently at 30 mg/kg, in a naloxone-sensitive manner. In contrast, α,β-amyrin produced much greater antinociceptive effect at 100 mg/kg in the capsaicin test, which was also naloxone-sensitive. These results provide first time evidence to show that α,β-amyrin attenuates orofacial pain atleast, in part, through a peripheral opioid mechanism but warrants further detailed study for its utility in painful orofacial pathologies.     

η-Allyl-cobalt (II) complexes

(η³-Cyclooctenyl)Co(bisphosphine) compounds react with HBF₄ in the presence of alkenes with oxidation of the metal to give the novel, paramagnetic organocobalt(II) species [(η³-cyclooctenyl)Co(bisphosphine)]⁺BF₄⁻, (η³-2-RC₃H₄)Co(bisphosphine) complexes react similarly. The Co(II) compounds form adducts with CO and NO (the latter being diamagnetic) and undergo facile chemical and electrochemical reduction.     

Bis(η-allyl) (η-pentalene) zirconium: preparation, structure and structural dynamics

The preparation of a novel mononuclear complex of zirconium having an η⁸-bonded pentalene ligand and two η³-allyl groups is described. Its structure has been determined by ¹H and ¹³C NMR spectroscopy. At room temperature some of the NMR signals are broadened, revealing that the compound is structurally dynamic. It is shown that the compound has C₂ symmetry with the enantiomeric forms undergoing racemisation.     

γ -Ray-Induced DNA Damage and Repair in Methanosarcina barkeri

The capacity of the mesophilic archaeon, Methanosarcina barkeri (DSM 804) for DNA double strand break repair following⁶⁰Co- γ irradiation was investigated. The genome (1.9 Mb) of Methanosarcina barkeri was largely fragmented and was found to be repaired on incubation in medium under anaerobic conditions at 37°C for 4 h. To get an insight into its repair process a set of inhibitors were used. The methanogenesis inhibitor, bromoethanesulfonate showed partial inhibition of repair in Methanosarcina barkeri but not in Escherichia coli or human peripheral blood mononuclear cells. The Methanosarcina barkeri cells could also partially repair the DNA damage in a non-nutrient medium. Arabinosine-CTP, a nucleoside analogue and a polymerase inhibitor, completely inhibited repair in this archaeon. Arabinosine-CTP did not affect DSB (double-strand break) repair in human peripheral blood mononuclear cells but completely inhibited repair in Escherichia coli (a bacterium). The involvement of polymerase indicates recombination to be the underlying mechanism in DSB repair of Methanosarcina barkeri. 3-Aminobenzamide, a poly (ADP-ribose) polymerase inhibitor, completely inhibited repair in this archaeon as well as in eukarya but not in Escherichia coli showing the involvement of poly (ADP-ribose) polymerase in the DSB repair of Methanosarcina barkeri.     

Suppression of maize root diseases caused by Macrophomina phaseolina, Fusarium moniliforme and Fusarium graminearum by plant growth promoting rhizobacteria

A plant growth-promoting isolate of a fluorescent Pseudomonas sp. EM85 and two bacilli isolates MR-11(2) and MRF, isolated from maize rhizosphere, were found strongly antagonistic to Fusarium moniliforme, Fusarium graminearum and Macrophomina phaseolina, causal agents of foot rots and wilting, collar rots/stalk rots and root rots and wilting, and charcoal rots of maize, respectively. Pseudomonas sp. EM85 produced antifungal antibiotics (Afa⁺), siderophore (Sid⁺), HCN (HCN⁺) and fluorescent pigments (Flu⁺) besides exhibiting plant growth promoting traits like nitrogen fixation, phosphate solubilization, and production of organic acids and IAA. While MR-11(2) produced siderophore (Sid⁺), antibiotics (Afa⁺) and antifungal volatiles (Afv⁺), MRF exhibited the production of antifungal antibiotics (Afa⁺) and siderophores (Sid⁺). Bacillus spp. MRF was also found to produce organic acids and IAA, solubilized tri-calcium phosphate and fixed nitrogen from the atmosphere. All three isolates suppressed the diseases caused by Fusarium moniliforme, Fusarium graminearum and Macrophomina phaseolina in vitro. A Tn5:: lac Z induced isogenic mutant of the fluorescent Pseudomonas EM85, M23, along with the two bacilli were evaluated for in situ disease suppression of maize. Results indicated that combined application of the two bacilli significantly (P = 0.05) reduced the Macrophomina-induced charcoal rots of maize by 56.04%. Treatments with the MRF isolate of Bacillus spp. and Tn5:: lac Z mutant (M23) of fluorescent Pseudomonas sp. EM85 significantly reduced collar rots, root and foot rots, and wilting of maize caused by Fusarium moniliforme and F. graminearum (P = 0.05) compared to all other treatments. All these isolates were found very efficient in colonizing the rhizotic zones of maize after inoculation. Evaluation of the population dynamics of the fluorescent Pseudomonas sp. EM85 using the Tn5:: lac Z marker and of the Bacillus spp. MRF and MR-11(2) using an antibiotic resistance marker revealed that all the three isolates could proliferate successfully in the rhizosphere, rhizoplane and endorhizosphere of maize, both at 30 and 60 days after seeding. Four antifungal compounds from fluorescent Pseudomonas sp. EM85, one from Bacillus sp. MR-11(2) and three from Bacillus sp. MRF were isolated, purified and tested in vitro and in thin layer chromatography bioassays. All these compounds inhibited R. solani, M. phaseolina, F. moniliforme, F. graminearum and F. solani strongly. Results indicated that antifungal antibiotics and/or fluorescent pigment of fluorescent Pseudomonas sp. EM85, and antifungal antibiotics of the bacilli along with the successful colonization of all the isolates might be involved in the biological suppression of the maize root diseases.     

Transformation of 3-hydroxy-steroids by Fusarium moniliforme 7alpha-hydroxylase.

Transformation of physiologically important 3-hydroxy-steroids by the DHEA-induced 7alpha-hydroxylase of F. moniliforme was investigated. Whereas DHEA was almost totally 7alpha-hydroxylated, PREG, EPIA and ESTR were only partially converted into their 7alpha-hydroxylated derivatives because hydroxylation at other undetermined positions as well as reduction of ketone at C17 or C20 into hydroxyl also occurred. Cholesterol was not transformed by the enzyme. Kinetic parameters of the 7alpha-hydroxylation for these substrates were determined and confirmed that DHEA was the best substrate of the 7alpha-hydroxylase. Inhibition studies of DHEA 7alpha-hydroxylation by the other 3-hydroxy-steroids were also carried out and proved that DHEA, PREG, EPIA and ESTR shared the same active site of the enzyme. Induction effects of these steroids were compared, and DHEA appeared to be the best inducer of the 7alpha-hydroxylase of F. moniliforme.     

Bioactive and osteoblast cell attachment studies of novel α- and β-chitin membranes for tissue-engineering applications

Chitin is a novel biopolymer and has excellent biological properties such as biodegradation in the human body and biocompatible, bioabsorable, antibacterial and wound healing activities. In this work, α- and β-chitin membranes were prepared using α- and β-chitin hydrogel. The bioactivity studies were carried out using these chitin membranes with the simulated body fluid solution (SBF) for 7, 14 and 21 days. After 7, 14 and 21 days the membranes were characterized using SEM, EDS and FT-IR. The SEM, EDS and FT-IR studies confirmed the formation of calcium phosphate layer on the surface of the both chitin membranes. These results indicate that the prepared chitin membranes were bioactive. Cell adhesion studies were also carried out using MG-63 osteoblast-like cells. The cells were adhered and spread over the membrane after 24 h of incubation. These results indicated that the chitin membranes could be used for tissue-engineering applications.     

Reversible β-hydride elimination of amine ligands forming stable cis-η-iminium hydride Os complexes

Octahedral tetraammineosmium(II) species are generated from their Os^III precursors containing an amine ligand cis to a labile alcohol or triflate. These compounds undergo reversible β-hydride eliminations resulting in the formation of cis-η²-iminium hydride complexes. Judging from NMR data, the η²-iminium group in these products lies parallel to the osmium-hydride bond with the iminium carbon eclipsing the hydride. Attempts to form η²-arene complexes of an Os^II ammine system bearing a stereogenic carbon are also described.     

Homologous long-chain δ-lactones in leaf cuticular waxes of Cerinthe minor

A homologous series of eleven δ-lactones (1,5-alkanolides) was identified in cuticular waxes from leaves of Cerinthe minor L., six of them representing novel compounds. They accounted for 79% of the total coverage of 41 μg wax per cm² leaf area. Various chemical transformations with product identification by GC-mass spectrometry and GC-FTIR were employed to assign the structures. The chain-lengths of the δ-lactones ranged from C₂₂ to C₃₂ and even-numbered homologues were prevalent. Additionally, aldehydes (C₂₆–C₃₀), alkanes (C₂₃–C₂₉), primary alcohols (C₂₆–C₃₂), alkanoic acids (C₂₀–C₃₂), wax esters (C₄₀–C₅₆) and lupeol were detected.     

Glucose/Citrate Cometabolism in Lactococcus lactis subsp. lactis biovar diacetylactis with Impaired α-Acetolactate Decarboxylase

The pyruvate metabolism of a Lactococcus lactis subsp. lactis biovar diacetylactis mutant deficient in α-acetolactate decarboxylase and its wild-type strain was studied during batch cultivations. A chemically defined medium was used containing glucose as carbon- and energy-source. The α-acetolactate decarboxylase deficiency had no effect on the specific growth rate. Addition of citrate was found to increase the specific growth rate of both strains under aerobic and anaerobic conditions. The product formation was monitored throughout the cultivations. The carbon- and redox-balances were within the accuracy of the experimental data. When citrate was added, α-acetolactate, diacetyl, and acetoin were formed, and aeration was shown to have a positive effect on the formation of these metabolites. By omitting lipoic acid (required for a functional pyruvate dehydrogenase complex) from the growth medium, a similar stimulatory effect on α-acetolactate, diacetyl, and acetoin formation was observed under aerobic conditions. The strain with impaired α-acetolactate decarboxylase activity accumulated α-acetolactate which resulted in an increased diacetyl formation compared to the wild-type strain, under aerobic and anaerobic conditions.     

Genealogy of the α-crystallin—small heat-shock protein superfamily

Sequences of 40 very diverse representatives of the α-crystallin–small heat-shock protein (α-Hsp) superfamily are compared. Their characteristic C-terminal ‘α-crystallin domain' of 80–100 residues contains short consensus sequences that are highly conserved from prokaryotes to eukaryotes. There are, in addition, some positions that clearly distinguish animal from non-animal α-Hsps. The α-crystallin domain is predicted to consist of two hydrophobic β-sheet motifs, separated by a hydrophilic region which is variable in length. Combination of a conserved α-crystallin domain with a variable N-terminal domain and C-terminal extension probably modulates the properties of the various α-Hsps as stress-protective and structural oligomeric proteins. Phylogeny reconstruction indicates that multiple α-Hsps were already present in the last common ancestor of pro- and eukaryotes. It is suggested that during eukaryote evolution, animal and non-animal α-Hsps originated from different ancestral gene copies. Repeated gene duplications gave rise to the multiple α-Hsps present in most organisms.     

Quaternary structure of bovine α-crystallin: influence of temperature

The tertiary and quaternary structure of α-crystallin is still a matter of controversy. We have characterized the native α-crystallin quaternary structure by isolating it at the in vivo temperature and solvent conditions. It can be represented by a distribution of expanded particles with a weight average molar mass of 550 000 g/mol. On decreasing (to 4°C) or increasing (up to 50°C) the temperature, the size distribution increases to larger particles. Only at lower temperatures (4°C), a stable population of particles is obtained with weight average molar mass of 700 000 g/mol. In all conditions, α-crystallin behaves as a very expanded particle with a maximum hydrodynamic volume of 3.15 ml/g. The transitions in quaternary structure are rather slow: it takes several hours to evolve from a population of aggregates, characteristic for given solvent conditions, to another distribution in size and quaternary structure on changing the environment. The quaternary structure of α-crystallin is an uncharacteristic parameter of the particle: a broad distribution of values can be obtained on changing the environment. Any realistic model should include this property. Our studies favor an open loose structure, where peptides can be added or removed without drastic changes of secondary and tertiary structure of the peptides.     

α-phenyl-tert-butyl-nitrone inhibits free radical release in brain concussion

Traumatic brain injury (TBI) is one of the important causes of mortality and morbidity. The pathogenesis of the underlying brain dysfunction is poorly understood. Recent data have suggested that oxygen free radicals play a key role in the primary and secondary processes of acute TBI. We report direct electron spin resonance (ESR) evidence of hydroxyl (·OH) radical generation in closed-head injury of rats. Moderate brain concussion was produced by controlled and reproducible mechanical, fixed, closed-head injury. A cortical cup was placed over one cerebral hemisphere within 20 min of the concussion, perfused with artificial cerebrospinal fluid (aCSF) containing the spin trap agent pyridyl-N-oxide-tert-butyl nitrone (POBN, 100 mM), and superfusate samples collected at 10 min intervals for a duration up to 130 min post brain trauma. In addition, POBN was administered systematically (50 mg/kg body wt.) 10 min pretrauma and 20 min posttrauma to improve our ability to detect free radicals. ESR analysis of the superfusate samples revealed six line spectra (α_N = 15.4 and α^β_H = 2.5 G) characteristic of POBN-OH radical adducts, the intensity of which peaked 40 min posttrauma. The signal was undetectable after 120 min. Administration of α-phenyl-tert-butyl-nitrone (PBN), a spin adduct forming agent systemically (100 mg/kg body wt. IP 10 min prior to concussion) alone or along with topical PBN (100 mM PBN in aCSF),6significantly (P< 0.001) attenuated the ESR signal, suggesting its possible role in the treatment of TBI.     

The possible role of α-crystallins in human senile cataractogenesis

α-Crystallins possess molecular chaperone properties and are one of the most abundant of the lenticular proteins. Posttranslational modifications of these proteins have been implicated as a possible etiology of human cataracts. This article will review current knowledge concerning the effects of known posttranslational modifications upon the molecular chaperone properties and aggregation behavior of α-A and α-B crystallin. Based upon these effects, experimental approaches will be discussed that may be useful in the development of reagents that may selectively inhibit the cataractogenic process in the aging human lens.