Analysis of genes involved in methyl halide degradation in Aminobacter lissarensis CC495

Aminobacter lissarensis CC495 is an aerobic facultative methylotroph capable of growth on glucose, glycerol, pyruvate and methylamine as well as the methyl halides methyl chloride and methyl bromide. Previously, cells grown on methyl chloride have been shown to express two polypeptides with apparent molecular masses of 67 and 29 kDa. The 67 kDa protein was purified and identified as a halomethane:bisulfide/halide ion methyltransferase. This study describes a single gene cluster in A. lissarensis CC495 containing the methyl halide utilisation genes cmuB, cmuA, cmuC, orf 188, paaE and hutI. The genes correspond to the same order and have a high similarity to a gene cluster found in Aminobacter ciceronei IMB-1 and Hyphomicrobium chloromethanicum strain CM2 indicating that genes encoding methyl halide degradation are highly conserved in these strains.     

Methyl jasmonate disrupts shoot formation in tobacco thin cell layers by over-inducing mitotic activity and cell expansion

The aim of the present study was to determine early cyto-histological events associated with the reduced number of shoots formed at the end of culture in tobacco (Nicotiana tabacum L.) thin cell layers treated with methyl jasmonate (MJ) [S. Biondi et al. (2001) J Exp Bot 52:1–12]. The results show that 0.1–10 M MJ strongly stimulated mitotic activity early in culture relative to untreated controls. Treatment with MJ also induced anomalous mitoses. Enhanced proliferative growth was confirmed by northern analysis and in situ hybridisation using cDNA probes of the G1/S phase-specific genes ubiquitin carboxyl-extension protein (ubi-CEP), topoisomerase 1 (top1) and ribonucleotide reductase (RNR). The formation of meristematic cell clusters on day 5 was also enhanced by 1 M MJ, but subsequent development of these cell clusters into meristemoids and shoot primordia was reduced by all MJ concentrations in a dose-dependent manner. Cell expansion was stimulated by MJ concentrations ranging from 0.001 to 10 M; expanded cells frequently occurred around and within meristemoids and shoot primordia, and displayed thickened and suberised cell walls; cell wall thickness increased with increasing MJ concentration. These cytological events caused alterations in the tunica and stem differentiation of the shoot dome. The apparently paradoxical role of MJ, which deregulates shoot formation through a stimulation of growth events, i.e., mitotic activity and cell expansion, is discussed.     

Methyl farnesoate controls adult male morphogenesis in the crayfish, Procambarus clarkii

Adult male crayfish Procambarus clarkii exist in two morphotypes. They continue to molt as adults, switching between Form Is and Form IIs. Form Is are primary reproductive types, with large chelae and spines on the ischiopodites of the third and fourth pair of walking legs. Form IIs are non-reproductive types with smaller chelae and no spines on the ischiopodites. We investigated the hormonal control of these transitions in two ways, by eyestalk ablation and by methyl farnesoate (MF) treatments. Eyestalk ablation accelerates molting and increases MF levels in the blood. MF is a hormone that regulates both reproduction and morphogenesis. MF concentrations were determined in two ways. The hemolymph samples were extracted first, then purified, using normal phase HPLC. The fractions containing MF were collected and analyzed for MF concentration, utilizing both internal and external standards by GC/MS. The other hemolymph samples were analyzed from individual animals by HPLC. The concentrations of ecdysteroids were determined by radioimmunoassay. In the control animals, 4 out of 4 untreated Form I males molted into Form II, while 6 out of 7 Form IIs molted into Form Is. Eight of 8 ablated Form Is molted into Form IIs as expected, while 5 of 5 ablated Form IIs molted into Form IIs, instead of Form Is. MF treatment of intact animals resulted in 6 of 7 Form Is becoming Form IIs and 5 of 6 Form IIs becoming Form IIs. These results were highly significant in comparison of Form I and IIs in each treatment (eyestalk intact, eyestalk ablated and eyestalk intact with MF) by a chi square analysis, P = 0.006, P < 0.0005, and P = 0.013, respectively. MF premolt blood levels suggested that Form IIs were produced in the presence of 1.3 ng/ml MF, while Form Is result from MF levels less than 0.5 ng/ml. Since both eyestalk ablation and MF treatment resulted in the failure of Form IIs becoming Form Is, it was concluded that the control of morphogenesis of primary reproductives (Form Is) depends on a low level of MF prior to the molt, while Form IIs are formed in the presence of increased levels of MF.     

1,5-Disubstituted imidazoles inhibit juvenile hormone biosynthesis by the corpora allata of the mosquito Aedes aegypti

We investigated the effect of fifteen 1,5-disubstituted imidazoles (1,5-dis) on juvenile hormone III (JH III) and methyl farnesoate (MF) biosynthesis by the corpora allata (CA) of the mosquito Aedes aegypti in vitro. Four compounds (TH-35, TH-83, TH-62 and TH-28) significantly decreased JH biosynthesis in the CA dissected from 3-day old sugar-fed females. The decrease of JH synthesis was not always associated with increased MF. TH-30 and TH-83 increased MF levels, while TH-85 and TH-61 significantly decreased MF levels. Five compounds (TH-26, TH-60, TH-83, TH-35 and TH-30) significantly inhibited JH biosynthesis in the CA dissected from females 15 h after a blood meal. Four 1,5-dis (TH-30, TH-26, TH-28 and TH-66) caused MF increases in CA from blood-fed females. 1,5-Disubstituted imidazoles had higher inhibitory activity on JH synthesis when substituted at position 5 by a 3-benzyloxyphenyl group and at position 1 by a benzyl group (such as TH-35). Inhibition of JH and MF biosynthesis by TH-35 was age-dependent and influenced by nutritional status; inhibition differed when evaluated in the CA dissected from sugar-fed females at different days after emergence and in the CA dissected from females at different hours after a blood meal. Inhibition was always higher when the CA was more active. The addition of TH-35 significantly reduced the stimulatory effect of Aedes-allatotropin and farnesoic acid on JH synthesis. This is the first report of an inhibitory effect of 1,5-disubstituted imidazoles on JH synthesis in Diptera.     

Application of mass spectrometry to determine the activity and specificity of pectin lyase A

Electrospray ionization (ESI) with quadrupole ion-trap mass spectrometry was used to assess the activity and specificity of the enzyme pectin lyase A (PLA) (EC 4.2.2.10) on model pectins with varying degrees and patterns of methyl esterification. PLA is a pectinase which cleaves alpha-(1-->4)-glycosidic linkages in pectin by a trans-elimination process. Using pectins with different degrees and patterns of methyl esterification, there was a significant variation in the activity rate of PLA. The enzymatic products generated at various time intervals were structurally analyzed by mass spectrometry to determine the specificity of PLA. Although the preferred substrate for PLA is fully methyl esterified polygalacturonate, cleavage was also observed with a non-methyl esterified galacturonic acid residue on either the non-reducing end or the reducing end. The current study shows that although PLA prefers fully methyl esterified substrates it can also accept partially esterified ones. It also demonstrates the suitability of ESI ion-trap mass spectrometry in determining enzyme specificities.     

Methyl mercury uptake and associations with the induction of chromosomal aberrations in Chinese hamster ovary (CHO) cells

In order to evaluate possible health effects of environmental exposure of humans towards methyl mercury species, relevant exposure experiments using methyl mercury chloride in aqueous solution and Chinese hamster ovary (CHO) cells were performed. The solution was monitored for the presence of monomethyl, dimethyl and elemental mercury by several analytical techniques including chromatographic as well as atomic absorption and mass spectrometric methods. Methyl mercury induces structural chromosomal aberrations (CA) and sister chromatid exchanges (SCE) in CHO cells. At a concentration of methyl mercury in the culture medium of 1.0 x 10(-6) M where the frequencies of CA and SCE are significantly elevated, the intracellular concentration was 1.99 x 10(-16) mol/cell. Possible biochemical processes leading to the cytogenetic effects are discussed together with toxicological consequences, when humans (e.g. workers at waste deposits) are exposed to environmental concentrations of methyl mercury.     

Synthesis, crystal structure, and high-resolution NMR spectroscopy of methyl 3-azido-2,3-dideoxy-4,6-di-O-p-tolylsulfonyl-alpha-D-xylo-hexopyranoside

Synthesis of methyl 3-azido-2,3-dideoxy-4,6-di-O-p-tolylsulfonyl- and -6-O-p-tolylsulfonyl-alpha-D-xylo-hexopyranosides is presented. High-resolution 1H and 13C NMR spectral data for both compounds and their precursors, and the single-crystal X-ray diffraction analysis for methyl 3-azido-2,3-dideoxy-4,6-di-O-p-tolylsulfonyl-alpha-D-xylo-hexopyranoside are reported. The influence of the O-protective group on the chemical shift of adjacent atoms in the 1H and 13C NMR spectra is discussed.     

Methyl jasmonate treatments reduce chilling injury and activate the defense response of guava fruits

Tropical fruits cannot be stored at low temperatures due to the chilling injury phenomena. With the goal of reducing the chilling injury, we tested 10(-4) and 10(-5) M of methyl jasmonate (MJ) treatment before the storage of red and white cultivars of guava fruits at 5 degrees C for up to 15 days plus two days at 20 degrees C. Every five days, we evaluated chilling injury index, ion leakage percentage, vitamin C, sugars, total phenols, and the activity of the enzymes lipoxygenase (LOX) and phenylalanine-ammonia lyase (PAL). We found that methyl jasmonate treatments reduce the chilling injury index and the ion leakage percentage. Furthermore, MJ did not affect vitamin C, chlorophyll, and total phenols. MJ increased sugar content, PAL, and LOX activities. We concluded that MJ reduces chilling injury and activates the fruit defense response as indicated by the behavior of total phenols and the increase in sugar content, PAL, and LOX activities.     

Essential oils from New Zealand manuka: triketone and other chemotypes of Leptospermum scoparium

The triketone chemotype of manuka, Leptospermum scoparium (Myrtaceae), is commercially important because of its antimicrobial activity. Oils from 36 individual plants on the East Cape of New Zealand all showed similar high triketone contents (>20% total triketones) with little seasonal variation. Analyses of oils from 261 individual manuka plants collected from 87 sites throughout New Zealand showed that the high triketone chemotype was localised on the East Cape, although oils with triketone levels up to 20% were found in the Marlborough Sounds area of the South Island. Cluster analysis revealed other chemotypes localised on other areas. Ten further chemotypes are described: alpha-pinene; sesquiterpene-rich with high myrcene; sesquiterpene-rich with elevated caryophyllene and humulene; sesquiterpene-rich with an unidentified sesquiterpene hydrocarbon; high geranyl acetate; sesquiterpene-rich with high gamma-ylangene + alpha-copaene and elevated triketones; sesquiterpene-rich with no distinctive components; sesquiterpene-rich with high trans-methyl cinnamate; high linalol; and sesquiterpene-rich with elevated elemene and selinene. Some of the chemotypes contained aroma compounds at relatively high levels, with a geranyl acetate-rich oil being most notable. Possible origins for this complex array of chemotypes are proposed.