Secretion of tumor necrosis factor by human leukocytes stimulated with shark cartilage

To assess the role of shark cartilage as an immune modulator, acid, salt-soluble, and phosphate-buffered saline extracts were prepared from three different commercial sources (SL, TL, FDC) of cartilage and used to stimulate human leukocytes in vitro. Duplicate leukocyte cultures were set up, each containing 50 $\mu$l of endotoxin-free extract, 200 $\mu$l of cell suspension (2.4-2.5 $\times$ 10$\sp5$ cells) and 100 $\mu$l of medium and incubated at 37$\sp\circ$C. Cultures stimulated with LPS (5 $\mu$g/ml) or medium served as the positive and negative controls, respectively. Culture supernatants were assayed for TNF$\alpha$ by ELISA. Cartilage extracts stimulated cells to release significant levels of TNF$\alpha$ (p $<$.005); the highest response was obtained with the acid extract of SL cartilage. In comparison, response to corresponding extracts of bovine cartilage was lower (p $<$.05). The stimulatory activity was reduced (85%) following proteolytic digestion, and lost when extract was heated (60$\sp\circ$C, 20 min) or treated with urea (6M), suggesting that the active component(s) is a protein. ^

Effects of invasive Africanized honey bees (Apis mellifera scutellata) on native stingless bee populations (Meliponinae) and traditional Mayan beekeeping in central Quintana Roo, Mexico

The Maya of the Yucatan region have a long history of keeping the native stingless bees (subfamily Meliponinae). However, market forces in the last few decades have driven the Maya to favor the use of invasive Africanized honey bees (Apis mellifera scutellata) for producing large quantities of high quality honey that has an international market. Furthermore, the native bees traditionally used by the Maya are now disappearing, along with the practice of keeping them. ^ An interdisciplinary approach was taken in order to determine the social factors behind the decrease in stingless beekeeping and the ecological driving forces behind their disappearance from the wild. Social research methods included participant observation with stingless beekeepers, Apis beekeepers, and marketing intermediaries. Ecological research methods included point observations of commonly known melliferous and polliniferous plants along transects in three communities with different degrees of human induced ecosystem disturbance. ^ The stingless bee species most important to the Maya, Melipona beecheii, has become extremely rare, and this has caused a breakdown of stingless beekeeping tradition, compounded with the pressure of the market economy, which fuels Apis beekeeping and has lessened the influence of traditional practices. The community with the heaviest amount of human induced ecosystem disturbance also had the highest degree of dominance of Apis mellifera, while the area with the most intact ecosystem had the highest diversity of stingless bees, though Apis mellifera was still the dominant species. Aggressive competitive behavior involving physical attacks by Apis mellifera against stingless bees was observed on several occasions, and this is a new observation previously unreported by science. ^

Agrobacterium-mediated transformation of indica rice cultivars using binary and superbinary vectors

The Agrobacterium-mediated transformation system was extended to two indica cultivars: a widely cultivated breeding line IR-64 and an elite basmati cultivar Karnal Local. Root tips and shoot tips of seedlings, and scutellar-calli derived from mature seeds showed high-efficiency Agrobacterium tumefaciens infection and stable transformation. In addition to the superbinary vector pTOK233 in Agrobacterium strain LBA4404, almost equally high levels of transformation were achieved with a relatively much smaller (13.1 kb) binary vector (pCAMBIA1301) in a supervirulent host strain AGL1. In both cases, as well as in both cultivars, while 60–90% of the infected explants produced calli resistant to the selectable agent hygromycin, 59–75% of such calli tested positive for GUS. A high level (400 μM) of acetosyringone in the preinduction medium for Agrobacterium and a higher level (500 μM) in the cocultivation medium was necessary for an enhancement in transformation frequency of the binary vector to levels comparable to a superbinary. Hygromycin-resistant calli could be produced from all the explants used. Transformants could be regenerated for both cultivars using the superbinary and binary vector, but only for calli of scutellar origin. In addition to the molecular confirmation of hpt and gus gene transfer and transcription, absence of gene sequences outside the transferred DNA (T-DNA) region confirmed absence of any long T-DNA transfer.

Factors affecting 39K NMR detectability in rat tissue

In this study we have found that NMR detectability of 39K in rat thigh muscle may be substantially higher (up to 100% oftotal tissue potassium) than values previously reported of around 40%. The signal was found to consist of two superimposed components, one broad and one narrow, of approximately equal area. Investigations involving improvements in spectral parameters such as signal-to-noise ratio and baseline roll, together with computer simulations of spectra, show that the quality of the spectra has a major effect on the amount of signal detected, which is largely due to the loss of detectability of the broad signal component. In particular, lower-field spectrometers using conventional probes and detection methods generally have poorer signal-to-noise and worse baseline roll artifacts, which make detection of a broad component of the muscle signal difficult.

Effect of magnesium depletion and potassium and chlorothiazide on intracellular pH in the rat, studied by 31P NMR

1. Both dietary magnesium depletion and potassium depletion (confirmed by tissue analysis) were induced in rats which were then compared with rats treated with chlorothiazide (250 mg/kg diet) and rats on a control synthetic diet. 2. Brain and muscle intracellular pH was measured by using a surface coil and [31P]-NMR to measure the chemical shift of inorganic phosphate. pH was also measured in isolated perfused hearts from control and magnesium-deficient rats. Intracellular magnesium status was assessed by measuring the chemical shift of β-ATP in brain. 3. There was no evidence for magnesium deficiency in the chlorothiazide-treated rats on tissue analysis or on chemical shift of β-ATP in brain. Both magnesium and potassium deficiency, but not chlorothiazide treatment, were associated with an extracellular alkalosis. 4. Magnesium deficiency led to an intracellular alkalosis in brain, muscle and heart. Chlorothiazide treatment led to an alkalosis in brain. Potassium deficiency was associated with a normal intracellular pH in brain and muscle. 5. Magnesium depletion and chlorothiazide treatment produce intracellular alkalosis by unknown mechanism(s).

Aromatic l-amino acid decarboxylase : histochemical localization in rat kidney and lack of effect of dietary potassium or sodium loading on enzyme distribution

Utilizing a mono-specific antiserum produced in rabbits to hog kidney aromatic L-amino acid decarboxylase (AADC), the enzyme was localized in rat kidney by immunoperoxidase staining. AADC was located predominantly in the proximal convoluted tubules; there was also weak staining in the distal convoluted tubules and collecting ducts. An increase in dietary potassium or sodium intake produced no change in density or distribution of AADC staining in kidney. An assay of AADC enzyme activity showed no difference in cortex or medulla with chronic potassium loading. A change in distribution or activity of renal AADC does not explain the postulated dopaminergic modulation of renal function that occurs with potassium or sodium loading.

The role of enoyl reductase genes in phloridzin biosynthesis in apple

Phloridzin is the predominant polyphenol in apple (Malus× domestica Borkh.) where it accumulates to high concentrations in many tissues including the leaves, bark, roots and fruit. Despite its relative abundance in apple the biosynthesis of phloridzin and other related dihydrochalcones remains only partially understood. The key unidentified enzyme in phloridzin biosynthesis is a putative carbon double bond reductase which is thought to act on p-coumaroyl-CoA to produce the dihydro p-coumaroyl-CoA precursor. A functional screen of six apple enoyl reductase-like (ENRL) genes was carried out using transient infiltration into tobacco and gene silencing by RNA interference (RNAi) in order to determine carbon double bond reductase activity and contribution to foliar phloridzin concentrations. The ENRL-3 gene caused a significant increase in phloridzin concentration when infiltrated into tobacco leaves whilst a second protein ENRL-5, with over 98% amino acid sequence similarity to ENRL-3, showed p-coumaroyl-CoA reductase activity in enzyme assays. Finally, an RNAi study showed that reducing the transcript levels of ENRL-3 in transgenic 'Royal Gala' led to a 66% decrease in the concentration of dihydrochalcones in the leaves in the one available silenced line. Overall these results suggest that ENRL-3, and its close homolog ENRL-5, may contribute to the biosynthesis of phloridzin in apple.

The role of ethylene and cold temperature in the regulation of the apple POLYGALACTURONASE1 gene and fruit softening

Fruit softening in apple (Malus 3 domestica) is associated with an increase in the ripening hormone ethylene. Here, we show that in cv Royal Gala apples that have the ethylene biosynthetic gene ACC OXIDASE1 suppressed, a cold treatment preconditions the apples to soften independently of added ethylene. When a cold treatment is followed by an ethylene treatment, a more rapid softening occurs than in apples that have not had a cold treatment. Apple fruit softening has been associated with the increase in the expression of cell wall hydrolase genes. One such gene, POLYGALACTURONASE1 (PG1), increases in expression both with ethylene and following a cold treatment. Transcriptional regulation of PG1 through the ethylene pathway is likely to be through an ETHYLENE-INSENSITIVE3-like transcription factor, which increases in expression during apple fruit development and transactivates the PG1 promoter in transient assays in the presence of ethylene. A coldrelated gene that resembles a COLD BINDING FACTOR (CBF) class of gene also transactivates the PG1 promoter. The transactivation by the CBF-like gene is greatly enhanced by the addition of exogenous ethylene. These observations give a possible molecular mechanism for the coldand ethylene-regulated control of fruit softening and suggest that either these two pathways act independently and synergistically with each other or cold enhances the ethylene response such that background levels of ethylene in the ethylene-suppressed apples is sufficient to induce fruit softening in apples.

Interaction between Arabidopsis Brca2 and its partners Rad51, Dmc1, and Dss1

The Arabidopsis (Arabidopsis thaliana) orthologs of Brca2, a protein whose mutations are involved in breast cancer in humans, were previously shown to be essential at meiosis. In an attempt to better understand the Brca2-interacting properties, we examined four partners of the two isoforms of Brca2 identified in Arabidopsis (AtRad51, AtDmc1, and two AtDss1 isoforms). The two Brca2 and the two Dss1 isoforms are named AtBrca2(IV), AtBrca2(V), AtDss1(I), and AtDss1(V) after their chromosomal localization. We first show that both AtBrca2 proteins can interact with either AtRad51 or AtDmc1 in vitro, and that the N-terminal region of AtBrca2 is responsible for these interactions. More specifically, the BRC motifs (so called because iterated in the Brca2 protein) in Brca2 are involved in these interactions: BRC motif number 2 (BRC2) alone can interact with AtDmc1, whereas BRC motif number 4 (BRC4) recognizes AtRad51. The human Rad51 and Dmc1 proteins themselves can interact with either the complete (HsRad51) or a shorter version of AtBrca2 (HsRad51 or HsDmc1) that comprises all four BRC motifs. We also identified two Arabidopsis isoforms of Dss1, another known partner of Brca2 in other organisms. Although all four Brca2 and Dss1 proteins are much conserved, AtBrca2(IV) interacts with only one of these AtDss1 proteins, whereas AtBrca2(V) interacts with both of them. Finally, we show for the first time that an AtBrca2 protein could bind two different partners at the same time: AtRad51 and AtDss1(I), or AtDmc1 and AtDss1(I).

Perturbed Lignification Impacts Tree Growth in Hybrid Poplar-A Function of Sink Strength, Vascular Integrity, and Photosynthetic Assimilation

The effects of reductions in cell wall lignin content, manifested by RNA interference suppression of coumaroyl 3'-hydroxylase, on plant growth, water transport, gas exchange, and photosynthesis were evaluated in hybrid poplar trees (Populus alba 3 grandidentata). The growth characteristics of the reduced lignin trees were significantly impaired, resulting in smaller stems and reduced root biomass when compared to wild-type trees, as well as altered leaf morphology and architecture. The severe inhibition of cell wall lignification produced trees with a collapsed xylem phenotype, resulting in compromised vascular integrity, and displayed reduced hydraulic conductivity and a greater susceptibility to wall failure and cavitation. In the reduced lignin trees, photosynthetic carbon assimilation and stomatal conductance were also greatly reduced, however, shoot xylem pressure potential and carbon isotope discrimination were higher and water-use efficiency was lower, inconsistent with water stress. Reductions in assimilation rate could not be ascribed to increased stomatal limitation. Starch and soluble sugars analysis of leaves revealed that photosynthate was accumulating to high levels, suggesting that the trees with substantially reduced cell wall lignin were not carbon limited and that reductions in sink strength were, instead, limiting photosynthesis.

Trehalose Toxicity in Cuscuta refexal Correlation With low Trehalase Activity

A toxic effect of a,a-trehalose in an angiospermic plant, Cuscuta reflexa (dodder), Is described. This disaccharide and Its analogs, 2-aminotrehalose and 4-aminotbhakose, induced a raid blackening of the terminal region of the vine which is Involved in elongation growth. From the results of in vitro growth of several angkiopermic plants and determination of trehalase activity in them, it is concluded that the toxic effect of trehalose in Cucaa is because of the very low trehalas activity In the vine. As a result, trehalose accumulates In the vine and interferes with some process closely associated with growth. The growth potential of Lemma (a duckweed) in a medium containing trehalose as the carbon source was ihreversibly lost upon addition of trealosamine, an Inhibitor of trehalase activity. It is concluded that, if allowed to accumulate within the tissue, trehalose may be potentiaMly toxic or inhibitory to higher plants in generaL The presence of trhalase actvity in plants, where Its substrate has not been found to occur, is envisged to relieve the plant from the toxic effects of trehalose which it may encounter in soil or during association with fungi or insects.

Modelling Leaf Production and Crop Development in Maize (Zea mays L.) After Tassel Initiation Under Diverse Conditions of Temperature and Photoperiod

Prediction of the initiation, appearance and emergence of leaves is critically important to the success of simulation models of crop canopy development and some aspects of crop ontogeny. Data on leaf number and crop ontogeny were collected on five cultivars of maize differing widely in maturity and genetic background grown under natural and extended photoperiods, and planted on seven sowing dates from October 1993 to March 1994 at Gatton, South-east Queensland. The same temperature coefficients were established for crop ontogeny before silking, and the rates of leaf initiation, leaf tip appearance and full leaf expansion, the base, optimum and maximum temperatures for each being 8, 34 and 40 degrees C. After silking, the base temperature for ontogeny was 0 degrees C, but the optimum and maximum temperatures remained unchanged. The rates of leaf initiation, appearance of leaf tips and full leaf expansion varied in a relatively narrow range across sowing times and photoperiod treatments, with average values of 0.040 leaves (degrees Cd)-1, 0.021 leaves (degrees Cd)-1, and 0.019 leaves (degrees Cd)-1, respectively. The relationships developed in this study provided satisfactory predictions of leaf number and crop ontogeny (tassel initiation to silking, emergence to silking and silking to physiological maturity) when assessed using independent data from Gatton (South eastern Queensland), Katherine and Douglas Daly (Northern Territory), Walkamin (North Queensland) and Kununurra (Western Australia).

The freezing characteristics of wheat at ear emergence

Wheat is occasionally exposed to freezing temperatures during ear emergence and can suffer severe frost damage. Few studies have attempted to understand the characteristics of freezing and frost damage to wheat during late development stages. It was clearly shown that wheat appears to have an inherent frost resistance to temperatures down to −5 °C but is extensively damaged below this temperature. Acclimation, whilst increasing the frost resistance of winter wheat in a vegetative state was incapable of increasing frost resistance of plants at ear emergence. It is proposed that the ability to upregulate frost resistance is lost once vernalisation requirement is fulfilled. Culms and ears of wheat were able to escape frost damage at temperatures below −5 °C by supercooling even to as low as −15 °C and evidence collected by infrared thermography suggested that individual culms on a plant froze as independent units during freezing with little or no cross ice-nucleation strategies to protect wheat from frost damage in the field appear to revolve around avoiding ice nucleation.