De novo organogenesis and plant regeneration in <Emphasis Type="Italic">Pongamia pinnata</Emphasis>, oil producing tree legume

Role of Thidiazuron (TDZ) in inducing adventitious organogenesis in Pongamia was studied. TDZ at different concentrations (0, 0.45, 2.27, 4.54, 6.71, 9.08, 11.35, 13.12 and 22.71 μM) were used for induction of caulogenic bud formation in deembryonated cotyledon explants. Each cotyledon was cut into three segments and identified as proximal, middle and distal. Duration of TDZ exposure, influence of the segment and orientation of the explant were studied. TDZ at 11.35 μM concentration was optimum for the induction of shoots and rapid elongation. Shoots induced at higher concentration elongated after several passages in growth regulator free medium, thereby extending the period of differentiation. Exposure of the explant for 20 days yielded more number of buds than 10 days. Proximal segment of the cotyledon was more responsive. Contact of abaxial surface in the medium was more effective and generated more buds than the adaxial side. Buds differentiated and elongated on transfer to MS basal medium for 8–12 passages of 15 days each. Rooting and elongation of shoots was achieved in charcoal supplemented half-strength MS medium. Rooted plantlets survived on transfer to sand soil mixture. The plants were hardened and transferred to green house. This is the first report on in vitro regeneration of Pongamia pinnata via adventitious organogenesis using TDZ. This protocol may find application in studies in genetic transformation, isolation of somaclonal variants and in induction of mutants. It also provides a system to study the inhibitory role of TDZ on shoot differentiation.     

The Phylogenetic Relationships of the Members of the DROSOPHILA ROBUSTA Group

The phylogenetic relationships among the species of the D. robusta group were investigated by the analysis of chromosomal differences. Six of the ten known members of the D. robusta group were available for the study: D. colorata and D. robusta from the United States, and D. sordidula, D. pseudosordidula, D. lacertosa, and D. moriwakii from Japan. Analysis of the metaphase chromosomes from larval ganglion cells suggests that D. moriwakii and D. colorata, with rod-shaped X-chromosomes, are the more ancestral species, while D. sordidula, D. pseudosordidula, D. robusta, and D. lacertosa, with V-shaped X-chromosomes, are derived. The ancestral position of D. colorata and D. moriwakii is further strengthened by the fact that these are the two species in the D. robusta group that are cytologically closest to D. nigromelanica of the related D. melanica group. Of the four derived species, D. sordidula was found to be the closest to the ancestral species. The phylogeny based on the analysis of the gene sequences in the homologous chromosomes agreed with that indicated by the metaphase chromosomes. Since all attempts to obtain hybrids were unsuccessful except for the cross involving D. moriwakii females and D. colorata males, photographic maps of the salivary chromosomes were used to determine homology between the chromosomes of the different species. Evidence is presented to indicate that the D. robusta group originated in Asia (Japan), and that there were two migrations to the New World, the first leading to D. robusta, and the second to D. colorata. It is suggested that the route of migrations was across the Bering Land Bridge, and further, that the migrations occurred during the period from late Oligocene to middle Miocene, 20-25 million years ago.     

Effects of endogenous calcium transport inhibitor from heart muscle on the active calcium uptake and passive calcium release properties of sarcoplasmic reticulum

In the present study, the effects of the cytosolic Ca2+ transport inhibitor on ATP-dependent Ca2+ uptake by, and unidirectional passive Ca2+ release from, sarcoplasmic reticulum enriched membrane vesicles were examined in parallel experiments to determine whether inhibitor-mediated enhancement in Ca2+ efflux contributes to inhibition of net Ca2+ uptake. When assays were performed at pH 6.8 in the presence of oxalate, low concentrations (less than 100 micrograms/mL) of the inhibitor caused substantial inhibition of Ca2+ uptake by SR (28-50%). At this pH, low concentrations of the inhibitor did not cause enhancement of passive Ca2+ release from actively Ca2+-loaded sarcoplasmic reticulum. Under these conditions, high concentrations (greater than 100 micrograms/mL) of the inhibitor caused stimulation of passive Ca2+ release but to a much lesser extent when compared with the extent of inhibition of active Ca2+ uptake (i.e., twofold greater inhibition of Ca2+ uptake than stimulation of Ca2+ release). When Ca2+ uptake and release assays were carried out at pH 7.4, the Ca2+ release promoting action of the inhibitor became more pronounced, such that the magnitude of enhancement in Ca2+ release at varying concentrations of the inhibitor (20-200 micrograms/mL) was not markedly different from the magnitude of inhibition of Ca2+ uptake. In the absence of oxalate in the assay medium, inhibition of Ca2+ uptake was observed at alkaline but not acidic pH.(ABSTRACT TRUNCATED AT 250 WORDS)     

The critical relationship between free radicals and degrees of ischemia: evidence for tissue intolerance of marginal perfusion

Skin-flap ischemia has been associated with the presence of free radicals. In this study, two enzyme systems involved in free-radical metabolism were used to compare a distal skin flap to a skin graft. Forty-two rats were divided into several test groups. A 10 X 3 cm dorsal rat flap was used, and tissue biopsies for xanthine oxidase and malonyldialdehyde (MDA) were obtained 2.5, 5.5, and 8.5 cm from the base of the flap at the hours given. In group I (control), the flap was outlined but not elevated, and biopsies were obtained. In group II, the flap was elevated, and biopsies were obtained at 6 hours. In group III, the flap was elevated, the distal 4 X 3 cm was amputated and replaced as a full-thickness skin graft, and biopsies were obtained at 6 hours. In group IV, the flap was elevated, and biopsies were obtained at 12 hours. In group V, the flap was treated as in group III, and biopsies were obtained at 12 hours. In group VI, the flap was elevated, and biopsies were obtained at 24 hours. In group VII, the flap was treated as in group III, and biopsies were obtained at 24 hours. Results: Xanthine oxidase was significantly higher in all distal biopsies compared to proximal biopsies. Xanthine oxidase also increased with time. Malonyldialdehyde increased over time as well as with distance from the flap base. Distal flap biopsies at 24 hours had greatly increased levels of malonyldialdehyde compared to skin grafts (p less than 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)     

Reaction products of a new anti-cancer agent, Pt(IV)(cyclohexyldiamine)Cl4, with guanosine and 9-methylguanine: first crystal structures of Pt(cyclohexyldiamine) complexes with a nucleobase and a nucleoside

Pt(IV)(dach)Cl4 (dach = cyclohexyldiamine) was reacted with guanosine and 9-methylguanine and their reaction products were analyzed by single-crystal x-ray diffraction. In both cases the resulting complexes, [Pt(dach)(guanosine)2]2+ and [Pt(dach)(9-methylguanine)2]2+ respectively, corresponded to an unanticipated reduction of the octahedral Pt(IV) starting material to a square planar Pt(II) species. The nature of the reducing agent is presently unknown.     

In vivo expression of a nonselected gene transferred into murine hematopoietic stem cells by electroporation

Mouse bone marrow cells were subjected to electroporation in the presence of RSVCAT and SV2NEO plasmids. CAT activity was detected in the G-418 resistant granulocyte-macrophage colonies. RSVCAT electroporated into primary bone marrow cells, repopulated lethally irradiated mice as demonstrated by the persistence of CAT activity in the hematopoietic tissues showing that electroporation can offer a powerful mode of gene transfer into bone marrow cells.     

Inhibition of sarcoplasmic reticulum calcium pump by cytosolic protein(s) endogenous to heart and slow skeletal muscle but not fast skeletal muscle

Cytosol from rabbit heart and slow and fast skeletal muscles was fractionated using (NH₄)₂SO₄ to yield three cytosolic protein fractions, viz., CPF-I (protein precipitated at 30% saturation), CPF-II (protein precipitated between 30 and 60% saturation), and cytosol supernatant (protein soluble at 60% saturation). The protein fractions were dialysed and tested for their effects on ATP-dependent, oxalate-supported Ca²⁺ uptake by sarcoplasmic reticulum from heart and slow and fast skeletal muscles. CPF-I from heart and slow muscle, but not from fast muscle, caused marked inhibition (up to 95%) of Ca²⁺ uptake by sarcoplasmic reticulum from heart and from slow and fast muscles. Neither unfractionated cytosol nor CPF-II or cytosol supernatant from any of the muscles altered the Ca²⁺ uptake activity of sarcoplasmic reticulum. Studies on the characteristics of inhibition of sarcoplasmic reticulum Ca²⁺ uptake by CPF-I (from heart and slow muscle) revealed the following: (a) Inhibition was concentration- and temperature-dependent (50% inhibition with approx. 80 to 100 μg CPF-I; seen only at temperatures above 20°C). (b) The inhibitor reduced the velocity of Ca²⁺ uptake without appreciably influencing the apparent affinity of the transport system for Ca²⁺. (c) Inhibition was uncompetitive with respect to ATP. (d) Sarcoplasmic reticulum washed following exposure to CPF-I showed reduced rates of Ca²⁺ uptake, indicating that inhibition results from an interaction of the inhibitor with the sarcoplasmic reticulum membrane. (e) Concomitant with the inhibition of Ca²⁺ uptake, CPF-I also inhibited the Ca²⁺-ATPase activity of sarcoplasmic reticulum. (f) Heat-treatment of CPF-I led to loss of inhibitor activity, whereas exposure to trypsin appeared to enhance its inhibitory effect. (g) Addition of CPF-I to Ca²⁺-preloaded sarcoplasmic reticulum vesicles did not promote Ca²⁺ release from the vesicles. These results demonstrate the presence of a soluble protein inhibitor of sarcoplasmic reticulum Ca²⁺ pump in heart and slow skeletal muscle but not in fast skeletal muscle. The characteristics of the inhibitor and its apparently selective distribution suggest a potentially important role for it in the in vivo regulation of sarcoplasmic reticulum Ca²⁺ pump, and therefore in determining the duration of Ca²⁺ signal in slow-contracting muscle fibers.