Kiwifruit β-galactosidase: Isolation and activity against specific fruit cell-wall polysaccharides

A -galactosidase (EC 3.2.1.23) capable of degrading a number of fruit cell-wall polysaccharides in vitro, was isolated from ripening kiwifruit (Actinidia deliciosa [A. Chev.] C.F. Liang et A.R. Ferguson cv. Hayward). The enzyme has a molecular weight of approximately 60 kDa by gel permeation and consists of several basic isoforms. Several polypeptides were enriched during purification, with 33-, 46- and 67-kDa bands being predominant after sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The optimum activity of the enzyme against p-nitrophenyl--d-galactopyranoside was at pH 3.2, but against a galactan purified from kiwifruit cell walls, it was at pH 4.9. The enzyme was specific for galactosyl residues in the -configuration, releasing galactose from a variety of kiwifruit cell-wall polysaccharide fractions including cell wall material, Na₂CO₃-soluble pectin, high-molecular-weight galactan, xyloglucan, and galactoglucomannan. A galactosylated glucuronomannan found throughout the kiwifruit plant was also a substrate for the enzyme. The results indicate that the enzyme attacks the non-reducing end of galactose side chains, cleaving single galactose residues which may be attached to the 2, 3, 4, or 6 position of the aglycone. Activity of the enzyme in-vitro was too low to account for the total loss of galactose from the cell walls during ripening. If the -galactosidase of this study is solely responsible for the removal of galactose from the cell wall during ripening then its in-vivo activity must be much greater than that observed in-vitro.Abbreviations CWM cell wall material - SDS-PAGE sodium dodecyl sulphate-polyacrylamide gel electrophoresis We thank Bronwyn Culling and Teresa Wegrzyn for assistance and acknowledge a contribution towards the cost of the research from the New Zealand Kiwifruit Marketing Board.     

Internodal elongation and orientation of cellulose microfibrils and microtubules in deepwater rice

Excised stem sections of deepwater rice (Oryza sativa L.) containing the highest internode were used to study the induction of rapid internodal elongation by gibberellin (GA). It has been shown before that this growth response is based on enhanced cell division in the intercalary meristem and on increased cell elongation. In both GA-treated and control stem sections, the basal 5-mm region of the highest internode grows at the fastest rate. During 24 h of GA treatment, the internodal elongation zone expands from 15 to 35 mm. Gibberellin does not promote elongation of internodes from which the intercalary meristem has been excised. The orientation of cellulose microfibrils (CMFs) is a determining factor in cell growth. Elongation is favored when CMFs are oriented transversely to the direction of growth while elongation is limited when CMFs are oriented in the oblique or longitudinal direction. The orientation of CMFs in parenchymal cells of GA-treated and control internodes is transverse throughout the internode, indicating that CMFs do not restrict elongation of these cells. Changes in CMF orientation were observed in epidermal cells, however. In the basal 5-mm zone of the internode, which includes the intercalary meristem, CMFs of the epidermal cell walls are transversely oriented in both GA-treated and control stem sections. In slowly growing control internodes, CMF orientation changes to the oblique as cells are displaced from this basal 5-mm zone to the region above it. In GA-treated rapidly growing internodes, the reorientation of CMFs from the transverse to the oblique is more gradual and extends over the 35-mm length of the elongation zone. The CMFs of older epidermal cells are obliquely oriented in control and GA-treated internodes. The orientation of the CMFs parallels that of the cortical microtubules. This is consistent with the hypothesis that cortical microtubules determine the direction of CMF deposition. We conclude that GA acts on cells that have transversely oriented CMFs but does not promote growth of cells whose CMFs are already obliquely oriented at the start of GA treatment.     

The expression pattern of the chicken homeobox-containing gene GHox-7 in developing polydactylous limb buds suggests its involvement in apical ectodermal ridge-directed outgrowth of limb mesoderm and in programmed cell death

Abstract. The limb buds of the polydactylous mutant embryos, talpid ² and diplopodia -5, possess expanded distal apexes surmounted by prolongated thickened apical ectodermal ridges that promote the outgrowth and formation of digits from both the anterior and posterior mesoderm of the mutant limb buds. The chicken homeobox-containing gene GHox-7 exhibits an expanded domain of expression throughout the expanded subridge mesoderm of the mutant limb buds, providing support for the hypothesis that GHox-7 expression by subridge mesenchymal cells is involved in the outgrowth-promoting effect of the apical ectodermal ridge. During normal limb development GHox-7 is also expressed by the mesoderm in the proximal anterior nonchondrogenic periphery of the limb bud, which includes, but is not limited to the anterior necrotic zone. GHox-7 is also expressed in the posterior necrotic zone at the mid-proximal posterior edge of the limb bud. In contrast, GHox-7 is not expressed in either the proximal anterior or posterior peripheral mesoderm of talpid ² and diplopodia -5 limb buds which lack proximal anterior and posterior necrotic zones. Furthermore, retinoic acid-coated bead implants, which diminish cell death in the anterior necrotic zone, elicit a local inhibition of GHox-7 expression in the proximal anterior peripheral mesoderm. These results support the suggestion that GHox-7 may be involved in defining regions of programmed cell death during limb development. Furthermore, these studies indicate that the distal subridge and proximal anterior nonchondrogenic mesodermal domains of GHox-7 expression are independently regulated.     

Distribution of NADPH-diaphorase activity in rat paravertebral,prevertebral and pelvic sympathetic ganglia

Summary Paravertebral (superior cervical and stellate), prevertebral (coeliac-superior mesenteric, inferior mesenteric) and pelvic (hypogastric) sympathetic ganglia of the rat were investigated by enzyme histochemistry to ascertain the distribution of nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-diaphorase) activity. In the paravertebral ganglia the majority of the sympathetic neuronal perikarya contained lightly and homogeneously distributed formazan reaction product but there was a range of staining intensities amongst the neuron population. In contrast, in the prevertebral ganglia, intense NADPH-diaphorase staining was present in certain neurons. Firstly, a population of neurons of the coeliac-superior mesenteric ganglion complex were surrounded by densely NADPH-diaphorase-positive baskets of fibres and other stained fibres were seen in interstitial nerve bundles and in nerve trunks connected to the ganglion complex. Secondly, in both the inferior mesenteric ganglion and hypogastric ganglion there were many very intensely NADPH-diaphorase positive neurons. Stained dendritic and axonal processes emerged from these cell bodies. In both ganglia this population of neurons was smaller in size than the lightly stained ganglionic neurons and commonly had only one long (presumably axonal) process. The similarity of these highly NADPH-diaphorase-positive neurons with previously described postganglionic parasympathetic neurons in the hypogastric ganglion is discussed.     

Structural characterization and physiological function of component B from Methanosarcina thermophila

The electron donor (component B) to the methyl coenzyme M methylreductase system from Methanosarcina thermophila was isolated as the 7-methyl derivative and characterized. Gas chromatography-mass spectrometry and ¹H NMR analyses identified this derivative as 7-methylthioheptanoylthreonine phosphate (CH₃-S-HTP), indicating that the original component B had the same structure (HS-HTP) as previously determined for component B from Methanobacterium thermoautotrophicum. The heterodisulfide of HS-HTP and coenzyme M (HS-CoM, 2-mercaptoethanesulfonate) was enzymatically reduced in cell extracts using electrons supplied by either H₂ or CO, confirming that HS-HTP was a functional molecule in M. thermophila.     

Energetics and regulations of formate and hydrogen metabolism by Methanobacterium formicicum

Accumulation of formate to millimolar levels was observed during the growth of Methanobacterium formicicum species on H₂–CO₂. Hydrogen was also produced during formate metabolism by M. formicicum. The amount of formate accumulated in the medium or the amount H₂ released in gas phase was influenced by the bicarbonate concentration. The formate hydrogenlyase system was constitutive but regulated by formate. When methanogenesis was inhibited by addition of 2-bromoethane sulfonate, M. formicicum synthesized formate from H₂ plus HCO _inf3 ^sup- or produced H₂ from formate to a steady-state level at which point the Gibbs free energy (G) available for formate synthesis or H₂ production was approximately -2 to -3 kJ/reaction. Formate conversion to methane was inhibited in the presence of high H₂ pressure. The relative rates of conversion of formate and H₂ were apparently controlled by the G available for formate synthesis, hydrogen production, methane production from formate and methane production from H₂. Results from ¹⁴C-tracer tests indicated that a rapid isotopic exchange between HCOO^- and HCO _inf3 ^sup- occurred during the growth of M. formicicum on H₂–CO₂. Data from metabolism of ¹⁴C-labelled formate to methane suggested that formate was initially split to H₂ and HCO _inf3 ^sup- and then subsequently converted to methane. When molybdate was replaced with tungstate in the growth media, the growth of M. formicicum strain MF on H₂–CO₂ was inhibited although production of methane was not Formate synthesis from H₂ was also inhibited.     

Localization of the gene for human heart fatty acid binding protein to chromosome 1p32-1p33

Among 639 spontaneous abortions between the 8th and 14th week of gestation 342 (53.5%) revealed an abnormal karyotype. While the rate of trisomies distinctly increased with advancing maternal age, a decrease in the rate of 45,X conceptuses and polyploidies was observed among abortions from older women. The overall relation of XXXXXXYY among the tetraploidies was 1411 and that of XXXXXYXYY among the triploidies was 26 361. However, when the latter was related to maternal age, a reversal of the XXXXXY ratio of 12 in the younger to 21 in the older age groups became evident. Furthermore a decrease in the rate of paternally derived partial hydatidiform moles was found among the triploid abortion specimens from older women. From these observations we conclude that digyny plays a major role in the origin of triploidy in the increased maternal age groups, while diandry related to immaturity of oocytes and impairment of oocyte cortical function is more frequent in triploid abortions from younger women.     

Summer drought exposure,stand structure,and soil properties jointly control the growth of European beech along a steep precipitation gradient in northern Germany

Increasing exposure to climate warming-related drought and heat threatens forest vitality in many regions on earth, with the trees' vulnerability likely depending on local climatic aridity, recent climate trends, edaphic conditions, and the drought acclimatization and adaptation of populations. Studies exploring tree species' vulnerability to climate change often have a local focus or model the species' entire distribution range, which hampers the separation of climatic and edaphic drivers of drought and heat vulnerability. We compared recent radial growth trends and the sensitivity of growth to drought and heat in central populations of a widespread and naturally dominant tree species in Europe, European beech (Fagus sylvatica), at 30 forest sites across a steep precipitation gradient (500–850 mm year⁻¹) of short length to assess the species' adaptive potential. Size-standardized basal area increment remained more constant during the period of accelerated warming since the early 1980s in populations with >360 mm growing season precipitation (April–September), while growth trends were negative at sites with <360 mm. Climatic drought in June appeared as the most influential climatic factor affecting radial growth, with a stronger effect at drier sites. A decadal decrease in the climatic water balance of the summer was identified as the most important factor leading to growth decline, which is amplified by higher stem densities. Inter-annual growth variability has increased since the early 1980s, and variability is generally higher at drier and sandier sites. Similarly, within-population growth synchrony is higher at sandier sites and has increased with a decrease in the June climatic water balance. Our results caution against predicting the drought vulnerability of trees solely from climate projections, as soil properties emerged as an important modulating factor. We conclude that beech is facing recent growth decline at drier sites in the centre of its distribution range, driven by climate change-related climate aridification.