A spectrophotometric microassay for sulfated glycosaminoglycans using a laser densitometer

The absorption spectrum of the dye 1,9-dimethylmethylene blue shifts if complexed with sulfated glycosaminoglycans. The present method uses the decrease in A633 rather than the increase in A535, described in a recent method, to measure the sulfated glycosaminoglycan content of biological samples. A conventional spectrophotometer was used to estimate the levels of sulfated glycosaminoglycan in papain extracts from intestinal wall tissue, by measuring both the A535 and the A633 and comparing them with a chondroitin sulfate standard: a highly significant correlation (r = 0.974, n = 17) was obtained. Also, interference by substances like RNA, DNA, and hyaluronic acid was similar for both methods. These results allowed us to employ a laser densitometer with a helium/neon laser emitting at 633 nm to improve the sensitivity and the capacity of the assay. The combination of a small reaction volume and a high-intensity light source allows the detection of less than 0.1 microgram chondroitin sulfate, a 40-fold improvement in sensitivity as compared with the original method. A very significant correlation (r = 0.885, n = 17) existed between results obtained with the macroassay, using a spectrophotometer, and those found by employing the microassay, using the laser densitometer. The use of microtiter plates and the screening potential of the densitometer yields an assay which is fast, very sensitive, and suitable for processing large numbers of samples.     

études sur les phénomenes électrocapillaires

Sans résuméI. Analyse électrocapillaire des matières colorantes. Rev. gén. Mat. Col. 1926 Vol. 30 pp 34–45II. Phénomènes électrocapillaires et le problème du cancer. Arch. Med. Exper. 1926 Vol. I p 381III. Phénomènes électrocapillaires et l'antagonismes microbiens. Bol. Istituto Sier. Milano 1927 Vol. VI p 313.     

The evolutionary position of the rhodophytePorphyra umbilicalis and the basidiomyceteLeucosporidium scottii among other eukaryotes as deduced from complete sequences of small ribosomal subunit RNA

Summary The complete small ribosomal subunit RNA (srRNA) sequence was determined for the red algaPorphyra umbilicalis and the basidiomyceteLeucosporidium scottii, representing two taxa for which no srRNA sequences were hitherto known. These sequences were aligned with other published complete srRNA sequences of 58 eukaryotes. Evolutionary trees were reconstructed by a matrix optimization method from a dissimilarity matrix based on sections of the alignment that correspond to structurally conservative areas of the molecule that can be aligned unambiguously. The overall topology of the eukaryotic tree thus constructed is as follows: first there is a succession of early diverging branches, leading to a diplomonad, a microsporidian, a euglenoid plus kinetoplastids, an amoeba, and slime molds. Later, a nearly simultaneous radiation seems to occur into a number of taxa comprising the metazoa, the red alga, the sporozoa, the higher fungi, the ciliates, the green plants, plus some other less numerous groups. Because the red alga diverges late in the evolutionary tree, it does not seem to represent a very primitive organism as proposed on the basis of morphological and 5S rRNA sequence data. Asco- and basidiomycetes do not share a common ancestor in our tree as is generally accepted on the basis of conventional criteria. In contrast, when all alignment positions, rather than the more conservative ones, are used to construct the evolutionary tree, higher fungi do form a monophyletic cluster. The hypothesis that higher fungi and red algae might have shared a common origin has been put forward. Although the red alga and fungi seem to diverge at nearly the same time, no such relationship can be detected. The newly determined sequences can be fitted into a secondary structure model for srRNA, which is now relatively well established with the exception of uncertainties in a number of eukaryote-specific expansion areas. A specific structural model featuring a pseudoknot is proposed for one of these areas.     

Differential methylation at the 5′ and the 3′ CCGG sites flanking the X chromosomal hypervariable DXS255 locus

Summary The degree of methylation at the 5 and 3 CCGG sequences flanking the variable number of tandem repeat (VNTR) region of the DXS255 locus at Xp11.22 was analysed separately in several haematopoietic cell lineages. The 5 CCGG site on active chromosomes was found to be completely methylated in B and T lymphocytes and granulocytes. Methylation of the 5 site on inactive X chromosomes differed between females (0%–60%), but was consistent in different cell lineages obtained from individual females. In contrast, methylation at the 3 CCGG site on active chromosomes was found to vary in B lymphocytes (40%–100%), whereas complete methylation was found in T lymphocytes and granulocytes. The extent of methylation on inactive X chromosomes was found to differ significantly between B lymphocytes (17%), T lymphocytes (54%) and granulocytes (82%). Thus, methylation at the 5 CCGG site seems to be primarily related to the status of X chromosome inactivation, whereas methylation at the 3 CCGG site is mainly subject to cell-lineage-specific influences.     

Analysis of higher-primate phylogeny from transversion differences in nuclear and mitochondrial DNA by Lake's methods of evolutionary parsimony and operator metrics

In the companion paper (Holmquist et al. 1988), we concluded that there is no agreement on either the correct branching order or differential rates of evolution among the higher primates, and we examined in depth why this uncertainty in the evolutionary understanding of our closest living relatives persists. Recently, Lake developed two novel methods, based on group properties of transition and transversion operators, that (a) permit, in principle, objective resolution of problems of the above type and (b) attach a statistical significance level to the conclusions drawn. In the present paper, we develop formulas for using these two methods in tandem and apply them to study transversion differences in (1) nuclear DNA for a 7-kb segment of the psi eta-globin locus and a 3-kb intergenic region between the psi beta- and delta- globin loci and (2) mitochondrial DNA for the 896-bp fragment of Brown et al. Although each of these nucleotide sequence regions has its characteristic tempo and mode of evolution, the nuclear and mitochondrial data together, comprising a total of 10,939 base positions, support a Homo/Pan clade at the 97% confidence level. If we calibrate the divergence point for humans and chimpanzees at 5 Myr, consideration of the transversion branch lengths for the combined nuclear data indicates that the gorilla lineage branched off 600,000- 900,000 years prior to that, although the 2 sigma sampling errors do not preclude either a temporal trifurcation for the three species or a considerably more ancient branch point for the gorilla. To resolve the length of this central branch to a relative accuracy of 25% and 30% will require a factor of 16 and nine times more data, respectively-- i.e., in excess of 100,000 homologous nucleotides for each of the four primates. For the nuclear genes, heterogeneity in evolutionary rates between different parts of the genome is mostly restricted to the human lineage for these two segments. The lineage leading to chimpanzees has evolved 0.4 (3-kb fragment) to 3.5 (7-kb segment) times as rapidly as the lineage leading to humans, and that leading to the gorilla has evolved approximately one-fifth to one-half as rapidly as that leading to chimpanzees. Thus, even local molecular clocks can "tick" badly. As significant is the fact that virtually contiguous parts of the genome tick at markedly different rates.(ABSTRACT TRUNCATED AT 400 WORDS)      

Properties of a Leu-Phe-Cleaving Endopeptidase Activity Putatively Involved in β-Endorphin Metabolism in Rat Brain

Incubation of beta-endorphin with cytosolic and particulate fractions of rat brain resulted in the formation of several peptides, including gamma-endorphin [beta-endorphin-(1-17)] and beta-endorphin-(18-31), indicating the presence of enzyme activity cleaving the Leu17-Phe18 bond of beta-endorphin. An assay for this Leu-Phe cleaving activity, based on the cleavage of the 14C-labeled substrate acetyl-Val-Thr-Leu-Phe-[epsilon-([14C]CH3)2]Lys-NHCH3, was used to examine the properties of this enzyme activity. beta-Endorphin-(1-31) competitively inhibited the Leu-Phe-cleaving enzyme activity on the pentapeptide substrate. Over 90% of activity was recovered in the cytosolic fraction. Leu-Phe-cleaving activity behaved like a thiol endopeptidase because it was inhibited by low concentrations of N-ethylmaleimide, p-chloromercuribenzoate, p-chloromercuribenzoyl sulfate, and low concentrations of Hg2+. Low concentrations of sulfhydryl compounds stimulated Leu-Phe-cleaving activity. The activity was optimal between pH 8.5 and 9.0. The Km of Leu-Phe-cleaving activity in the cytosolic fraction was 35 microM and in the particulate fraction 88 microM with Vmax values of 193 and 15 nmol mg protein-1 h-1, respectively. The apparent molecular mass of the Leu-Phe-cleaving enzyme was estimated by gel filtration to be approximately 200 kilodaltons. These properties of Leu-Phe-cleaving activity indicate that the Leu-Phe-cleaving enzyme is distinct from any known brain endopeptidase.     

Primary and secondary structure of the 18 S ribosomal RNA of the insect species Tenebrio molitor

The sequence of the 18 S rRNA of Tenebrio molitor is reported. A detailed secondary structure model for eukaryotic small subunit rRNAs is proposed. The model comprises 48 universal helices that eukaryotic and prokaryotic small subunit rRNAs have in common, plus a number of helices in areas of variable secondary structure. For the central area of the model, an alternative structure is possible, applicable only to eukaryotic small subunit rRNAs. Possibly, small subunit rRNA switched to this alternative conformation after the eukaryotic branch had been established in evolution. Another possibility is that the two conformers represent a dynamic structural switch functioning during the translational activity of the eukaryotic ribosome.