Citral dehydrogenase involved in geraniol oxidation pathway: purification,characterization and kinetic studies from <Emphasis Type="Italic">Persicaria minor</Emphasis> (<Emphasis Type="Italic">Polygonum minus</Emphasis> Huds.)

Plants emit semiochemicals as alarm signals upon attack by herbivores or insect pests. Complex insect-plant interaction through alarm pheromones can be manipulated to improve crop protection. Geraniol, citral and geranic acid are monoterpenoid compounds from plants and they play a role as semiochemical alarm pheromones. In plants, the oxidation of geraniol into geranic acid is catalyzed by two oxidoreductases, geraniol dehydrogenase and citral dehydrogenase. In this study, citral dehydrogenase isoenzymes from Persicaria minor (Polygonum minus) leaves were purified to homogeneity and characterized. Enzyme purification through Toyopearl GigaCap Q-650 M column chromatography at pH 7.5 produced two activity peaks, suggesting the existence of two citral dehydrogenase isoenzymes. Both isoenzymes were different in isoelectric point and kinetic parameters but similar in pH and optimal temperature as well as in substrate specificity. Findings from this study will provide a basic understanding for the development of recombinant production of these particular enzymes. Further studies on molecular structure involved could be exploited in transgenic plant as an integrated pest management strategy.     

Acetylation of C-terminal lysines modulates protein turnover and stability of Connexin-32

Background

The gap junction protein, Connexin32 (Cx32), is expressed in various tissues including liver, exocrine pancreas, gastrointestinal epithelium, and the glia of the central and peripheral nervous system. Gap junction-mediated cell-cell communication and channel-independent processes of Cx32 contribute to the regulation of physiological and cellular activities such as glial differentiation, survival, and proliferation; maintenance of the hepatic epithelium; and axonal myelination. Mutations in Cx32 cause X-linked Charcot–Marie–Tooth disease (CMT1X), an inherited peripheral neuropathy. Several CMT1X causing mutations are found in the cytoplasmic domains of Cx32, a region implicated in the regulation of gap junction assembly, turnover and function. Here we investigate the roles of acetylation and ubiquitination in the C-terminus on Cx32 protein function. Cx32 protein turnover, ubiquitination, and response to deacetylase inhibitors were determined for wild-type and C-terminus lysine mutants using transiently transfected Neuro2A (N2a) cells.

Results

We report here that Cx32 is acetylated in transfected N2a cells and that inhibition of the histone deacetylase, HDAC6, results in an accumulation of Cx32. We identified five lysine acetylation targets in the C-terminus. Mutational analysis demonstrates that these lysines are involved in the regulation of Cx32 ubiquitination and turnover. While these lysines are not required for functional Cx32 mediated cell-cell communication, BrdU incorporation studies demonstrate that their relative acetylation state plays a channel-independent role in Cx32-mediated control of cell proliferation.

Conclusion

Taken together these results highlight the role of post translational modifications and lysines in the C-terminal tail of Cx32 in the fine-tuning of Cx32 protein stability and channel-independent functions.

     

A novel angiogenesis inhibitor suppresses rat adjuvant arthritis

Rat adjuvant arthritis (AA) is an animal model of rheumatoid arthritis in which pannus formation and destruction of joints occur after immunization with complete Freund's adjuvant. Neovascularization is present within the synovium and may be critical for pannus growth. In this study the effects of a novel angiogenesis inhibitor, AGM-1470, on AA were evaluated. Lewis rats were immunized with CFA to induce arthritis. AGM-1470 treatment was initiated prior to arthritis onset (preventative protocol) or administered to rats with established disease (suppressive protocol). The severity of synovitis and the immunologic status of all rats were then evaluated. Using clinical and radiographic criteria, AGM-1470 significantly reduced arthritis incidence (preventative protocol) (P < 0.01) and disease severity (both protocols, P < 0.001, compared to controls) without affecting T cell function in vitro or phenotype in vivo. Additionally, histologic sections from control rats revealed marked pannus formation, destruction of bone/cartilage, and neovascularization. These findings were absent in AGM-1470-treated rats. AGM-1470 may offer a new treatment option for rheumatoid arthritis and other angiogenesis-dependent diseases.     

Polynuclear Aromatic Hydrocarbons (PAHs) in fish from the Red Sea Coast of Yemen

A detailed analytical study using combined normal phase high pressure liquid chromatography (HPLC), gas chromatography (GC) and gas chromatography/mass spectrometry (GC/MS) of Polynuclear Aromatic Hydrocarbons (PAHs) in fish from the Red Sea was undertaken. This investigation involves a preliminary assessment of the sixteen parent compounds issued by the U.S. Environmental Protection Agency(EPA). The study revealed measurable levels of Σ PAHs (the sum of three to five or six ring parent compounds) (49.2 ng g⁻¹ dry weight) and total PAHs (all PAH detected) (422.1 ng g⁻¹ dry weight) in edible muscle of fishes collected from the Red Sea. These concentrations are within the range of values reported for other comparable regions of the world. Mean concentrations for individual parent PAH in fish muscles were; naphthalene 19.5, biphenyl 4.6, acenaphthylene 1.0, acenaphthene 1.2, fluorene 5.5, phenanthrene 14.0, anthracene 0.8, fluoranthene 1.5, pyrene 1.8, benz(a)anthracene 0.4, chrysene 1.9, benzo(b)fluoranthene 0.5, benzo(k)fluoranthene 0.5, benzo(e)pyrene 0.9, benzo(a)pyrene 0.5, perylene 0.2, and indeno(1,2,3-cd)pyrene 0.1 ng g⁻¹ dry weight respectively. The Red Sea fish extracts exhibit the low molecular weight aromatics as well as the discernible alkyl-substituted species of naphthalene, fluorene, phenanthrene and dibenzothiophene. Thus, it was suggested that the most probable source of PAHs is oil contamination originating from spillages and/or heavy ship traffic. It was concluded that the presence of PAHs in the fish muscles is not responsible for the reported fish kill phenomenon. However, the high concentrations of carcinogenic chrysene encountered in these fishes should be considered seriously as it is hazardous to human health. Based on fish consumption by Yemeni‘s population it was calculated that the daily intake of total carcinogens were 0.15 μg/person/day. This revised version was published online in August 2006 with corrections to the Cover Date.     

Structure and behavior of contractile vacuoles inChlamydomonas reinhardtii

Summary The contractile vacuole (CV) cycle ofChlamydomonas reinhardtii has been investigated by videomicroscopy and electron microscopy. Correlation of the two kinds of observation indicates that the total cycle (15 s under the hypo-osmotic conditions used for videomicroscopy) can be divided into early, middle, and late stages. In the early stage (early diastole, about 3 s long) numerous small vesicles about 70–120 nm in diameter are present. In the middle stage (mid-diastole, about 6 s long), the vesicles appear to fuse with one another to form the contractile vacuole proper. In the late stage (late diastole, also about 6 s long), the CV increases in diameter by the continued fusion of small vesicles with the vacuole, and makes contact with the plasma membrane. The CV then rapidly decreases in size (systole, about 0.2 s). In isosmotic media, CVs do not appear to be functioning; under these conditions, the CV regions contain numerous small vesicles typical of the earliest stage of diastole. Fine structure observations have provided no evidence for a two-component CV system such as has been observed in some other cell types. Electron microscopy of cryofixed and freeze-substituted cells suggests that the irregularity of the profiles of larger vesicles and vacuoles and some other morphological details seen in conventionally fixed cells may be shrinkage artefacts. This study thus defines some of the membrane events in the normal contractile vacuole cycle ofChlamydomonas, and provides a morphological and temporal basis for the study of membrane fusion and fluid transport across membranes in a cell favorable for genetic analysis.Abbrevations CV contractile vacuole - PM plasma membrane     

Biocontrol of Fusarium Root Rot in the Common Bean (Phaseolus vulgaris L.) by using Symbiotic Glomus mosseae and Rhizobium leguminosarum

Abstract The interaction between VA mycorrhiza Glomus mosseae (Gm), root rodulating symbiont Rhizobium leguminosarum (Rl), and root rot pathogen Fusarium solani (Fs) on the common bean (Phaseolus vulgaris) in relation to plant growth, nutrient uptake, disease severity, rhizosphere microbial biomass, and nutrient availability was investigated. Mycorrhizal plants yielded significantly greater plant biomass and mobilized more N and P uptake as compared to nonmycorrhizal plants or those infected with Fs. However, the mycorrhizal root colonizing ability, in presence of Fs, was reduced by 27%, whereas Rl enhanced it by 37%. The inoculation of Gm, besides decreasing propagule number of Fs in the rhizosphere, decreased pathogenic root rot by 34 to 77%. However, in the presence of Rl, Gm-inoculated plants were more tolerant of the fungal root pathogen. The Gm + Rl inoculated plants not only had maximum plant biomass and root nodulation, but also exhibited higher microbial biomass, alkaline phosphatase activity, and available phosphorus in their rhizosphere. Rl, alone or in association with Gm, caused the maximum increase in mineral nitrogen (NH₄ ⁺ and NO₃ ⁻) content in soil. These results indicate that Gm has a vital role in inhibiting the root pathogen from invasion, more so in the presence of R. leguminosarum. Received: 26 February 1996; Revised: 12 July 1996     

Efficacy Assessment Of Trichogramma Evanescens And T. Embryophagum ( Bdhym.: Trichogrammatidae), For Control Of Stored Products Moth Pests In Bulk Wheat

The potential ofTrichogramma evanescens Westwood andT. embryophagum Quednau to controlEphestia kuehniella andE. elutella was evaluated under laboratory conditions. BothTrichogramma species parasitised eggs of the twoEphestia spp. in bulk wheat at 1, 2 and 5 cm depth. The release ofT. evanescens was more effective than that ofT. embryophagum resulting in higher rates of parasitism, predation and parasitoid induced mortality. At 17?C and 26?C, the mortality ofEphestia due to the release ofT. evanescens was 67, 78% and ofT. embryophagum 27 and 38%, respectively. The difference in host finding efficiency between the two species was large, at 5 cm depth and 26?C,T. evanescens parasitised about four times more host eggs compared to T.embryophagum.     

The use of confocal microscopy in the investigation of cell structure and function in the heart,vascular endothelium and smooth muscle cells

In recent years, fluorescence microscopy imaging has become an important tool for studying cell structure and function. This non invasive technique permits characterization, localisation and qualitative quantification of free ions, messengers, pH, voltage and a pleiad of other molecules constituting living cells. In this paper, we present results using various commercially available fluorescent probes as well as some developed in our laboratory and discuss the advantages and limitations of these probes in confocal microscopy studies of the cardiovascular system.     

Metabolizable energy requirements for maintenance and growth of Awassi lambs

Twenty four Awassi lambs (mean BW 24.4 kg) were used in a two periods experiment to measure maintenance (zero growth) and growth requirements of metabolizable energy (ME). During the pre-treatment period (period 1, 3 weeks) all lambs were maintained at a constant level of feed intake (2% BW). During the treatment period (period 2, 10 weeks) the lambs were divided into four equal groups and fed different amounts (2, 3, 4 and 5% of BW). Six additional lambs of similar BW and age were killed at the beginning of the experiment to compare final empty body weight (FEBW) with initial body weight (IBW). Twelve lambs, divided into four equal groups were fed the experimental diet in the same amounts as in the growth experiment (period 2) to determine the digestibility of energy.

Energy requirement for maintenance (EM) was measured by both constant weight technique, using data collected during period 1, and regression technique by regressing ME intake on body weight gain (BWG) and empty body weight gain (EBWG). EM measured during the constant weight period (pre-treatment) and overall experimental period (pre-treatment plus treatment periods) were 0.482 and 0.466 MJ of ME per kg M^0.75 per day. Predicted energy requirements for growth (Eg) calculated from equations derived during the treatment period were 0.433, 0.623, 0.782, 0.910 and 1.007 (MJ per kg M^0.75 per day) for the growth rates 50, 100, 150, 200, and 250 (g per day) respectively. It is concluded to be more appropriate to use the values derived during the overall experimental period for maintenance and the overall treatment period for growth as they are most applicable to production situations.     

Selective quantification of free 3-nitrotyrosine in exhaled breath condensate in asthma using gas chromatography/tandem mass spectrometry.

Reactive nitrogen species can cause oxidative modifications of certain amino acid residues in proteins, notably the modification of tyrosine to 3-nitrotyrosine (3-NT), which is a potentially useful marker of oxidative stress. Since lung diseases are associated with airway inflammation and oxidative stress, quantification of 3-NT in exhaled breath condensate (EBC) may provide a non-invasive means for monitoring ongoing inflammatory processes. 3-NT-like immunoreactivity has previously been detected in EBC, but no definitive evidence for the presence of 3-NT in EBC is available. Here, a method based on gas chromatography/negative ion chemical ionization/tandem mass spectrometry was established for the quantification of free 3-NT in EBC. The detection limit was 0.56 pM (corresponding to 3.0 amol microl(-1) sample injected) and the method was found to give linear results (r2 > 0.999) in the concentration range of 0-5.0 nM. The coefficient of variation (CV) for within-day and between-day precision were 11 and 12%, respectively. No artifactual nitration was observed during sample processing. The method was applied to study subjects with asthma (n = 8), and healthy subjects (n = 10), but only a slight non-significant increase in 3-NT levels was found in the former group (median [interquartile ranges]; 99 [50-547] amol s(-1) vs. 75 [35-147] amol s(-1)). No correlation with exhaled nitric oxide (NO), pulmonary function or EBC levels of total protein was observed. The 3-NT levels were much lower compared to previously reported levels, based on immunochemical measurements. The method does not allow the simultaneous quantification of tyrosine in samples.