The Use of Nonaqueous Fractionation to Assess the Ionic Composition of the Apoplast during Fruit Ripening

We have examined the possibility that pectin solubilization and cell separation in fruit may be due to organic acids disrupting calcium bridges between pectic polysaccharides. With fruit from a wild tomato (Lycopersicon pimpinellifolium [Dunal]) we demonstrated the validity of a nonaqueous fractionation method to obtain reliable estimates of the ionic content of the apoplast. In unripe fruit no organic acids were associated with the cell wall, which contained 67% of the total calcium and 47% of the magnesium. In ripe fruit 4% of the malate, 10% of the citrate, and 15% of the oxalate were estimated to be in the cell wall, together with 84% of the calcium and 52% of the magnesium. In contrast to the cultivated tomato, we did not find a consistent decrease in the degree of methyl esterification between unripe and ripe fruit, and an overall average of 75% was observed. In the cell walls of ripe fruit the ratio of calcium:magnesium:organic acid:unesterified uronic acid, on the basis of charge, was 15:4:4:16. The use of a computer program to predict the proportions of different ionic species in complex mixtures suggested that in ripe fruit 70% of the unesterified uronic acid would be complexed with calcium. Our results show that organic acids do not accumulate in the cell wall sufficiently to disrupt calcium cross-linking, nor is the calcium removed from the wall into the cell. We therefore conclude that organic acids do not contribute to cell separation during the ripening of tomato fruit.     

Modulation of IL-4 production in murine spleen cells by prostaglandins

Recently, it has been reported that IL-4 production by murine Th2 cell lines is insensitive to inhibition by E-type prostaglandins. In the present study, IL-4 production in vitro by freshly isolated concanavalin A (Con A)-stimulated murine spleen cells was readily suppressed by PGE₂ with an I₅₀ of 2 nM. Comparable suppression by PGE₂ was seen after priming by anti-CD3? antibody instead of Con A or with other changes in the culture conditions. PGE₂ was an effective inhibitor after elimination of Ly2.2⁺ T cells, consistent with a direct effect on Th2 cells. In the absence of added prostaglandins, IL-4 production was enhanced 1.5- to 7.0-fold by 0.2–2.0 μM indomethacin, indicating that endogenous arachidonate metabolites such as PGE₂ and PGI₂ regulate IL-4 production in our usual culture system. The inhibition of Th2 cell secretion by PGE₂in vitro may have physiologic and pharmacologic implications for the regulation of Th2 cell function and IgE production in vivo.     

Characterisation of repeated sequences from microdissected B chromosomes of Crepis capillaris

The B chromosome of Crepis capillaris was isolated from the standard chromosomes by microdissection, and the chromosomal DNA amplified using the degenerate oligonucleotide-primed polymerase chain reaction (DOP-PCR). The PCR product was cloned and a Bspecific library created and characterised. Southern and in situ hybridisation analyses of the DOP-PCR product from microdissected B chromosomes confirmed that the B chromosome is composed mainly of sequences also present in the A chromosomes but lacks the main repeated DNA families located in the A-chromosomal heterochromatin. From 100 clones analysed, 12% of the generated B-chromosomal library was shown to be composed of dispersed repeats located in both the A and B chromosomes. No B-specific repeated sequence was detected. One of the most abundant repeated DNAs within the library, the family B134, was further characterised. Repeating units show a sequence similarity range from 69% to 90% and are characterised by their richness in (CA)n repeats. In situ hybridisation revealed that members of this family are dispersed throughout the A and B chromosomes but are more concentrated in the pericentromeric heterochromatin of the B, indicating that the molecular organization of B heterochromatin is different from that of the A chromosomes. Compared with the A chromosomes, the Bs contain about 20,000 copies per micron more of the B134 sequence. This indicates that B134 was amplified on the B chromosome after its origin. The B134 sequences in the B chromosomes have also diverged from those on the A chromosomes. Although the DNA composition of A and B chromosomes is similar, Bs are evolving separately from A chromosomes at the molecular level.     

Taxonomy and function of C1 protein kinase C homology domains.

C1 domains are compact alpha/beta structural units of about 50 amino acids which tightly bind two zinc ions. These domains were first discovered as the loci of phorbol ester and diacylglycerol binding to conventional protein kinase C isozymes, which contain 2 C1 domains (C1A and C1B) in their N-terminal regulatory regions. We present a comprehensive list of 54 C1 domains occurring singly or doubly in 34 different proteins. Many C1 domains and C1 domain-containing proteins bind phorbol esters, but many others do not. By combining analysis of 54 C1 domain sequences with information from previously reported solution and crystal structure determinations and site-directed mutagenesis, profiles are derived and used to classify C1 domains. Twenty-six C1 domains fit the profile for phorbol-ester binding and are termed "typical." Twenty-eight other domains fit the profile for the overall C1 domain fold but do not fit the profile for phorbol ester binding, and are termed "atypical." Proteins containing typical C1 domains are predicted to be regulated by diacylglycerol, whereas those containing only atypical domains are not.     

Evolution of the mitochondrial DNA control region in the mbuna (Cichlidae) species flock of lake Malawi, East Africa

Considerable controversy has surrounded the application of mitochondrial DNA data to reconstruction of evolutionary relationships among the endemic cichlids of Lake Malawi. Central to this debate has been the issue of whether lineage sorting is complete, and thus whether these data actually reflect species phylogeny, or simply gene genealogy. Review of all mtDNA control region sequences available for members of one monophyletic subset of this species flock, the Malawi rockfishes, or mbuna, strongly indicates that lineage sorting is incomplete: Character-based analyses of these sequences reconstruct gene, not species, interrelationships. Analysis of the pattern of nucleotide substitutions differentiating these mtDNA alleles suggests that pyrimidine residues undergo transition substitutions more often than do purines. Estimation of the magnitude of derived sequence differentiation in light of the reconstructed gene genealogy suggests that the mbuna may be of considerably more recent vintage than previous molecular characterizations have indicated. Received: 6 April 1996 / Accepted: 3 March 1997     

Decompression comparison of helium and hydrogen in rats

Lillo, R. S., E. C. Parker, and W. R. Porter.Decompression comparison of helium and hydrogen in rats.J. Appl. Physiol. 82(3): 892-901, 1997.The hypothesis that there are differences in decompression riskbetween He and H₂ wasexamined in 1,607 unanesthetized male albino rats subjected to dives on2% O₂-balance He or 2%O₂-balanceH₂ (depths  50 ATA, bottom times  60 min). The animals were decompressed to 10.8 ATA with profilesvarying from rapid to slow, with up to four decompression stops of up to 60 min each. Maximum likelihood analysis was used to estimate therelative decompression risk on a per unit pressure basis (termed "potency") and the rate of gas uptake and elimination, bothfactors affecting the decompression sickness risk, from a specific dive profile. H₂ potency for causingdecompression sickness was found to be up to 35% greater than that forHe. Uptake rates were unresolvable between the two gases with the timeconstant (TC) estimated at ~2-3 min, leading to saturation inboth cases in <15 min. Washout of both gases was significantly slowerthan uptake, with He washout (TC ~1.5-3 h) substantially slowerthan H₂ washout (TC ~0.5 h). Itis unknown whether the decompression advantage of the faster washout ofH₂ or the disadvantage of itsincreased potency, observed in the rat, would be important for humandiving.

     

Optical measurement of isolated canine lung filtration coefficients at normal hematocrits

Klaesner, Joseph W., N. Adrienne Pou, Richard E. Parker,Charlene Finney, and Robert J. Roselli. Optical measurement ofisolated canine lung filtration coefficients at normal hematocrits. J. Appl. Physiol. 83(6):1976-1985, 1997.In this study, lung filtration coefficient(K_fc) valueswere measured in eight isolated canine lung preparations at normalhematocrit values using three methods: gravimetric, blood-correctedgravimetric, and optical. The lungs were kept in zone 3 conditions andsubjected to an average venous pressure increase of 10.24 ± 0.27 (SE) cmH₂O. The resulting K_fc(ml · min¹ · cmH₂O¹ · 100 g dry lung wt¹) measuredwith the gravimetric technique was 0.420 ± 0.017, which wasstatistically different from theK_fc measured bythe blood-corrected gravimetric method (0.273 ± 0.018) or theproduct of the reflection coefficient(_f) andK_fc measuredoptically (0.272 ± 0.018). The optical method involved the use of aCellco filter cartridge to separate red blood cells from plasma, whichallowed measurement of the concentration of the tracer in plasma atnormal hematocrits (34 ± 1.5). The permeability-surface areaproduct was measured using radioactive multiple indicator-dilutionmethods before, during, and after venous pressure elevations. Resultsshowed that the surface area of the lung did not change significantlyduring the measurement ofK_fc. Thesestudies suggest that_fK_fccan be measured optically at normal hematocrits, that this measurement is not influenced by blood volume changes that occur during the measurement, and that the optical_fK_fcagrees with theK_fc obtained viathe blood-corrected gravimetric method.

     

Estimation of sample zooplankton abundance from Folsom splitter sub-samples

Procedures are described for estimating the abundance of eachzooplanklon species in a sample after counting at least twosub-samples from a Folsom splitter. A multinomiai model is assumedfor the splitting process. The method may be summarized as follows:two sub-samples, balanced with respect to the left — rightsplits, are counted. The counts for each species are testedfor homogeneity using a x² test. If the counts are homogeneous,an estimator is given which permits the estimation of the numberof animals in the sample, and the variance of this estimate.If the subsample counts are heterogeneous, it is assumed thatclumping has occurred. A procedure is described in which additionalsub-samples are counted to locate the clump, and estimatorsof abundance (and variance estimators) are derived. If the splitterbias is <5%, bias of the abundance estimator is negligiblecompared to the binomial sampling error. Comparisons of speciesabundance between plankton samples is made more rigorous usingthe estimates of mean abundance, and the variance of this mean,provided by the methods described here. ^*Present address: Dunoon Road, Dorroughby, NSW 2480, Australia.