Some Aspects of Melanin Formation of Melanocytes Cultured on Collagen-Coated Microcarrier Beads

Melanocytes cultured on collagen-coated Cytodex 3 microcarrier Sephadex beads caused remarkable pigmentation of the beads during the period of culture when optimal density was reached. Electron microscopy of melanocytes on the microcarriers revealed that the cells and their dendrites invaginate into the microcarrier surface layer. Removal of the cells by trypsinization showed that some pigment granules were left on the carrier surface and within the cavities present on the microcarrier surface. In order to investigate whether the pigmentation of the microcarriers could be a result of indole intermediates of melanogenesis present in the culture medium, extracts were studied by gas chromatography/mass spectrometry for the presence of these compounds. Two compounds (5,6-dihydroxyindole-2-carboxylic acid and 6-hydroxy-5-methoxyindole-2-carboxylic acid) so far have been identified in the medium extracts. Results indicate that microcarrier culture of melanocytes can serve as an interesting model for electron microscopy studies of melanocytes with regard to pigmentation and cell attachment.     

Biovolume as a tool in biomass determination of Oligochaeta and Chironomidae

• 1 We used biovolume as a simple non-destructive means for estimating biomass for several tubificid and chironomid species. Biovolumes were determined by treating the organisms as cylinders and measuring body lengths and diameters; estimates were verified by measuring the displacement of water by live organisms in a 5-ml burette. In addition, factors were determined for the conversion of biovolume (V) into wet weight (WW), dry weight (DW) and ash-free dry weight (AFDW).
• 2 For six tubificid species, regression functions between the diameter of segment XI (D₁₁) and the biovolume enabled the prediction of any biovolume merely by measuring D₁₁. This method, however, underestimated biovolumes of Branchiura sowerbyi by 38–47% compared with burette measurements. This difference is largely attributable to the mountant we used during identification.
• 3 For ten chironomid species a linear relation was found between body length and the diameter of abdominal segment 5. The biovolume of Chironomus muratensis was underestimated by an average of 12% compared with burette measurements.
• 4 Specific gravity (WW:V) amounted to 1.07 and 1.05, DW:WW ratios were 0.20 and 0.142 and AFDW:WW ratios were 0.182 and 0.129 for Oligochaeta and Chironomidae, respectively.
• 5 The biovolume method is not yet very accurate for Oligochaeta, but to our knowledge, it is the only practical method that can be used in natural polyspecific communities that need to be treated with elucidating media for identification. For those Chironomidae that need no elucidation for identification, this method is time saving and just as accurate as the direct body shape—biomass method.

     

Cellular Processes Limiting Leaf Growth in Plants under Hypoxic Root Stress

The effects of root hypoxia on leaf growth of a Populus trichocarpa? deltoides hybrid have been assessed. Clonal plants were subjectedto hypoxic root conditions in pot culture by flooding and insolution culture by gassing with nitrogen. The rate of leafexpansion declined within 8 h and was suppressed for the durationof the treatment. Final leaf size was reduced by 35% to 60%compared to aerated plants. Final epidermal cell size and numberdepended both on the developmental stage of the leaf at theonset of stress and on the duration of the treatment. No differencesin bulk leaf water potential were measured between the hypoxicand aerated plants. Cell wall extensibility was lower, leafsolute potential was more negative and turgor potential washigher in leaves of hypoxia-treated plants than of aerated plants.These data suggest that leaf growth of hypoxia-stressed plantsis limited by cell wall extensibility. The mechanism by whichthe root stress induces changes in leaf cell wall characteristicsis not known. Key words: Populus, flooding     

Root Hypoxia-lnduced Changes in the Pattern of Translatable mRNAs In Poplar Leaves

Shoot responses to root stress may be modulated by hormonestransported from root tissues. In this study, the pattern ofin vitro translatable mRNAs from leaves on plants with oxygen-deprived(hypoxic) roots was used as a molecular phenotype to test forthe action and specificity of putative root-to-shoot signalmolecules. Root hypoxia caused a decrease in the abundance ofa group of leaf mRNAs which translated to give low molecularweight (LMW) proteins (12 to 28 kDa). Root excision induceda similar reduction in a subset of the same LMW leaf mRNAs.This suggests that the effects of root hypoxia on leaves arephysiologically related to the effects of root excision. Feedingabscisic acid to leaves did not mimic the effects of root aerationor hypoxia. Applications of zeatin riboside (ZR) to leaves ofplants with hypoxic roots duplicated the effects of root aerationon the LMW translation products. The specificity of the leafresponse to ZR was tested by feeding KC1 and sucrose to leavesof root excised plants. Applications of either KC1 or sucrosealso increased the abundance of the LMW translation products.Although the results reported in this paper are consistent withthe concept that shoot responses to root stress may be modulatedby substances moving in the xylem, a thorough understandingof how hormones control cellular responses needs to be resolvedbefore the mechanism of root-shoot integration can be understood. Key words: ABA, cytokinins, hypoxia, poplar     

O-Methylation of L-dopa in Melanin Metabolism and the Presence of Catechol-O-Methyltransferase in Melanocytes

O-Methylation of L-dopa was investigated as a possible regulatory mechanism in melanin metabolism. The methylation product of L-dopa, 3-O-methoxytvrosine was detected in extracts of cultured human melanocytes. The enzyme catechol-O-methyltransferase is responsible for this O-methylation and that of the dihydroxyindolic intermediates of melanogenesis. The enzyme is present in melanocytes in its soluble and membrane-bound isoforms. Immuno-electron microscopy suggests the presence of the membrane-bound enzyme in the endoplasmic reticulum. This localization may indicate a role of catechol-O-methyltransferase in protecting the melanocyte against reactive dihydroxyphenolic intermediates of melanogenesis leaking from the melanogenic compartments. On the other hand, the O-methylation of L-dopa may serve as a regulatory point in melanogenesis during early stage of tyrosinase processing in the endoplasmic reticulum.     

The Influence of Leaf Water Status and ABA on Leaf Growth and Stomata of Phaseolus Seedlings with Hypoxic Roots

The objective of this study was to assess the relative rolesof leaf water status and root-sourced signals in mediating beanleaf responses to root hypoxia. To do so, the roots of beanplants under varied VPD (0.95 kPa to 0.25 KPa) were made hypoxic.Under all conditions, leaf growth rates and stomatal conductanceswere reduced. There was a transitory decline in leaf water potentialat high VPD which accounted for the initial reduction in leafgrowth rates and stomatal conductance. At low VPD, no waterdeficits were detected. Leaf growth inhibition and reduced stomatalconductance under low VPD treatments were unrelated to leafwater status and must be induced by some other factor. In vitrogrowth of leaf discs was reduced by xylem sap collected fromhypoxic roots. Exogenously applied ABA, at high concentrationsin KCl and sucrose, or at low concentrations diluted in xylemsap from aerated plants, inhibited in vitro growth of leaf discs.Applications of ABA in the transpiration stream reduced stomatalconductance.