Volume 23, Issue 3
September 1998, pages 163-283
pp 163-164 September 1998 Clipboard
pp 164-167 September 1998 Clipboard
pp 169-176 September 1998 Perspectives
pp 177-184 September 1998
We showed previously that ifDictyostelium discoideum cells are sucked up into a small glass capillary with air at one end and plugged with mineral oil at the other, a sharp band of fast moving cells with prestalk characteristics formed within a minute at the air end of the cell mass. We now demonstrate that oxygen inside the capillary is responsible for the initiation and positioning of the sharp division line between prestalk-like and prespore-like cells, and that the length of the prestalk zone is regulated by the oxygen concentration. Our results are compared to a quantitative theory, showing good agreement with the experiments. We also discuss the relevance of these observations to the differentiation of prestalk and prespore zones in normal slugs and the origins of polarity in this organism.
pp 185-191 September 1998
Vitamin A has manifold effects on the development, growth and pattern formation of several amphibians. At the same time it causes severe embryonic malformations. The histological changes brought about by vitamin A in the tail tissues of anurans are quite amazing. A common morphological change brought about by vitamin A in tail-amputated tadpoles ofBufo melanostictus include the formation of a large bulbular mass at the distal end of the tail following tail regeneration. Histology revealed that the bulbular mass consisted of notochordal cells only. Other histological changes are: a thickening of the epidermis and the basement membrane, enlargement of the notochord and the nerve cord, thickening of the sheath covering the notochord and the myelin sheath covering the nerve cord and the disorganization of muscle bundles. The significance of such changes is discussed.
pp 193-200 September 1998
Improved methods are described for the detection of G1P-binding proteins (G-proteins) in the protonema of mossFunaria hygrometrica and coleoptiles of corn(Zea mays) and sorghum(Sorghum vulgare). We optimized conditions for the transfer of proteins to nitrocellulose, production of high titer polyclonal anti-Gα (common) antibodies and finally the detection of G-proteins by amplification. In addition to the α-subunit of heterotrimeric G-proteins (Mr 41–43 kDa), a small molecular weight class (< 30 kDa) was also detected by anti-Gα (common) antibodies. An easy, reliable and efficient filter assay is also described to quantify the toxin catalyzed ADP-ribosylation. The apparentKm of the NAD has been determined to be approximately 1.5μM for the microsomal fraction of moss. Inclusion of G1P stimulated ADP-ribosylation by 2–27-fold. One to three polypeptides representing the α-subunit of heterotrimeric G-proteins of (Mr 37–43 kDa) were ADP-ribosylated in all three plants. The anti-Gβ (C-terminus) antibody cross-reacted strongly with 39 and 34 kDa polypeptide in moss and corn respectively. By employing improved methods two classes of G-proteins have been shown to be present in three plant species.
pp 201-207 September 1998
The role of divalent cations like magnesium (Mg2+) and calcium (Ca2+) was irrvestigated on energy distribution process ofHydrilla verticillata thylakoids. Effect of these cations was tested on relative quantum yield of photosystem (PS) II catalyzed electron transport activity, room and liquid nitrogen temperature fluorescence emission properties and thylakoid light scattering characteristics. The electron transport activity was found to be stimulated in the presence of these cations in a light intensity independent manner. The concentration of cation required for maximum stimulation was nearly 10–12 mM. Comparatively, Ca2+ was more effective than Mg2+. Cation induced stimulation in electron transport activity was not accompanied by increase in chlorophylla fluorescence intensity either at room (25°C) or liquid nitrogen (77°K) temperatures. Furthermore, 540 nm absorption and 90° light scattering properties of thylakoids remained insensitive towards divalent cations. These facts together suggest that divalent cations inHydrilla thylakoids are not effective in supporting the excitation distribution between the interacting photosystem complexes.
pp 209-212 September 1998
Callus cultures ofCapsicum frutescens capable of producing a maximum of 53 μg capsaicin/g FW were exposed to various levels of p-fluorophenyialanine (PFP) at 100, 400, 1000 and 2000 μM to develop a resistant cell line that over produces capsaicin. After 15 days of culturing on media lacking PFP, cell lines resistant to 100, 400 and 1000 μM registered 18%, 34.5% and 45% increase in capsaicin content over normal cell line (cells not exposed to PFP). Capsaicin accumulation was inhibited in 2000 μM PFP resistant cell line. The profile of phenylalanine ammonia lyase (PAL), the key enzyme in pheny1propanoid pathway in resistant cell cultures was studied and compared with normal cell cultures to understand its role in capsaicin formation. Importantly increased production of capsaicin was obtained using PFP resistant cell lines. The activity profile of PAL had no correlation with capsaicin content in both control and PFP resistant cells.
pp 213-223 September 1998
A cDNA library was constructed in λZAPII vector from poly(A)+ RNA isolated from developing seeds of chickpea (Cicer arietinum L.). Two cDNA clones encoding legumin protein were obtained by screening the library by plaque hybridization using a heterologous pea legumin cDNA probe. The pBluescript plasmids were excised from the phage clones and two clones designated pCSSK4 and pCSSK5 with inserts of 1.45 kb and 1.82 kb respectively were mapped by restriction and partially sequenced. The partial nucleotide sequence showed that these two cDNAs are not identical and showed sequence homology with the storage protein cDNA clones of other legumes.
pp 225-233 September 1998
The interaction of Cibacron blue F3GA with ribosome inactivating proteins, ricin, ricin A-chain and momordin has been investigated using difference absorption spectroscopy. Ricin was found to bind the dye with a 20- and 2-fold lower affinity than ricin A-chain and momordin, respectively. A time dependent increase in the amplitude of Cibacron blue difference spectrum in the presence of ricin was observed on addition of β-mercaptoethanol. Analysis of the kinetic profile of this increase showed a biphasic phenomenon and the observed rates were found to be independent of the concentration of β-mercaptoethanol. Kinetics of reduction of the intersubunit disulphide bond in ricin by β-mercaptoethanol showed that reductionper se is a second order reaction. Therefore, the observed changes in the difference spectra of Cibacron blue probably indicate a slow change in the conformation of ricin, triggered by reduction of the intersubunit disulphide bond.
pp 235-246 September 1998
Carbonic anhydrase I (CAI) is one out of ten CA isoenzymes that have been identified in humans. X-ray crystallographic and inhibitor complex studies of human carbonic anhydrase I (HCAI) and related studies in other CA isoenzymes identified several residues, in particular Thr199, GlulO6, Tyr7, Glull7, His l07, with likely involvement in the catalytic activity of HCAI. To further study the role of these residues, we undertook, site-directed mutagenesis of HCAI. Using a polymerase chain reaction based strategy and altered oligonucleotide primers, we modified a cloned wild type hCAI gene so as to produce mutant genes encoding proteins with single amino acid substitutions. Thrl99Val, Thrl99Cys, Thr199Ser, GlulO6Ile, Glul06Gln, Tyr7Trp, Glu.117Gln, and His 107Val mutations were thus generated and the activity of each measured by ester hydrolysis. Overproduction of the Glu117Gln and HisI07Val mutant proteins inEscherichia coli resulted in a large proportion of the enzyme forming aggregates probably due to folding defect. The mutations Thr199Val, GlulO6Ile and GlulO6Gln gave soluble protein with drastically reduced enzyme activity, while the Tyr7Trp mutation had only marginal effect on the activity, thus s.uggesting important roles for Thr199 and Glu lO6 but not for Tyr7 in the catalytic function of HCAI.
pp 247-254 September 1998
The genome ofLeishmania donovani AG83, a virulent strain causing kala-azar, was resolved into 29 chromosomal bands by pulsed field gel electrophoresis (pFGE) under standardized conditions. Comparison of the karyotype with those of other strains and species revealed variations. By Southern hybridization, specific genes were localized to individual chromosomes. Twenty-two copies ofβ-tubulin genes are located on band 27 (1.63 Mb); minor copies are present in band 16 (850 kb) and band 9 (650 kb). Aβ-tubulin related nontranscribed locus was isolated from a genomic library and shown to contain repetitive sequences hybridizing throughout the genome. Single chromosomes contain multicopy clusters of gp63 and rnini-exon-derived RNA genes, but interspecific variations were observed in each case. The results emphasize the importance of using a standard reference strain ofLeishmania donovani for coordinated genome mapping of this clinically important organism.
pp 255-263 September 1998
Fully sequenced prokaryotic genomes ofEscherichia coli, Haemophilus influenzae andMethanococcus janaschii were subjected .to genome analysis for nucleotide interactions. The analysis was restricted to inter-nucleotide relations like two nucleotides in a dinucleotide, three nucleotides in a codon and two codons in a dicon. This relational analysis was carried out in C language and was compiled on a C++ compiler. The relational analysis showed a preferential dinucleotide frequency (the observed frequencies of AA and TT were higher than the expected frequency and the observed frequencies of CC and GG). From codon frequency distribution analysis, sub-codonic elements have been noticed, exerting that the first one or first two nucleotide may reasonably determine the next nucleotide(s) in a codon. The analysis further reveals the existence of short-range randomness or chaotic behaviour in prokaryotic genomes, which might be a forerunner for the origin of introns in eukaryotes, besides being involved in a regulatory role.
pp 265-269 September 1998
Using inverse polymerase chain reaction (PCR), we have cloned partial intronic sequences from human glutamic acid decarboxylase (GAD) gene. A small 153 bp core region was selected from the GAD cDNA sequence to design outward primers corresponding to its 3′ and 5′ ends. EcoRI digested human DNA which had been circularized by self-ligation and then linearized withSacII was used as a substrate to can.y out PCR. This gave a 900 bp long product which was cloned into pUC19. The sequence analysis of this fragment revealed the presence of introns in the region flanking the selected core DNA. In this work we used this technique to walk into the upsteam region of the GAD gene using sequence information from its cloned cDNA.
pp 271-277 September 1998
Ovarian follicular fluid peptide (OFFP) purified from sheep ovaries has been earlier shown to induce degeneration of ovarian follicles in mice. In the present study, whether the effect of OFFP on granulosa cells was similar to apoptosis was studied using three parameters. Immature mice injected with pregnant mare serum gonadotropin on day 0 were administered with 10 or 20 μg of OFFP on day 1 and autopsied on day 2. The granulosa cells were collected from the ovarian follicles. The presence of apoptotic bodies were observed by staining the cells with acridine orange. DNA profiles of DAPI-stained cells analysed by flow cytometry also revealed apoptotic response to OFFP. Furthermore, agarose gel electrophoresis of low molecular weight DNA fraction extracted from the cells of OFFP-treated animals confirmed ladder formation and induction of apoptosis and not necrosis in granulosa cells. In conclusion, all the three parameters indicated apoptotic changes in granulosa cells of ovarian follicles in .mice treated with OFFP. The effect of OFFP seems to be exerted directly on the granulosa cells showing its autocrine role in the process of follicular atresia. This is discussed in the light of other intra/extra ovarian factors.
pp 279-283 September 1998
Previous studies have shown that exposure to urea-supplemented food inhibited fecundity inDrosophila females, and that this inhibition was not expressed when females were given a choice between regular and urea-supplemented food as an oviposition substrate. We assayed fecundity, on both regular food and urea-supplemented food, at 5, 15 and 25 days post eclosion on females from ten laboratory populations ofDrosophila melanogaster. The females assayed came from one of two treatments; they were maintained as adults on either regular or urea-supplemented food. We found that exposure to urea-supplemented food inhibited fecundity, relative to the levels exhibited on regular food, regardless of whether the urea was present in the assay medium, or in the medium on which the flies were maintained over the course of the experiment, thereby suggesting that urea has both a long-term (possibly physiological) as well as a short-term (possibly behavioural) inhibitory effect on fecundity ofDrosophila females. We also tested and ruled out the hypothesis that prior yeasting could ameliorate the inhibitory effect of urea in the assay medium on fecundity, as this was a possible explanation of why flies given a choice between regular and urea-supplemented food did not exhibit a preference for regular food in a previous study.