Inhibition of protein and lipid synthesis in muscle by 2,4-dinitrofluorobenzene, an inhibitor of creatine phosphokinase

A photoaffinity label, 4-azidobenzoyltrimethionine has been synthesized. It competitively inhibits trimethionine uptake in the yeast C. albicans. Upon UV irradiation it irreversibly and specifically blocks oligopeptide uptake. These results give the first example of photoinhibition of peptide uptake in yeast.     

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.     

Starch-degrading enzymes of two species ofFusarium

Starch supported production of maximum α-amylases (dextrinizing and saccharifying) byFusarium oxysporum andF. scirpi. Addition of gibberellic acid resulted in an increased production of α-amylase. Presence of glucose depressed the enzyme production. pH 4.5 and 4.0 was found to be optimum for the dextrinizing enzyme secreted by both species. The temperature of 25 and 40 °C was optimum for the dextrinizing enzyme secreted byF. oxysporum andF. scirpi, respectively. Saccharifying enzymes of both species showed their optimum at pH 6.9. The optimum temperature for the activity of the saccharifying enzyme was 30 and 40 °C, respectively.     

Overproduction and rapid purification of the phosphoenolpyruvate:sugar phosphotransferase system proteins enzyme I, HPr, and Protein IIIGlc of Escherichia coli.

We present methods for the rapid, simple purification of Enzyme I, HPr, and Protein IIIGlc of the Escherichia coli phosphoenolpyruvate:sugar phosphotransferase system (PTS) using plasmids overproducing gene products. The gene for HPr (ptsH) was cloned into the expression vector pKC30. A simple procedure was devised for the purification to homogeneity of this protein from extracts of heat-induced cells containing pKC30/ptsH recombinant clone. The genes for Enzyme I (ptsI) and Protein IIIGlc (crr) were cloned separately into the expression vector pRE1. Rapid purification procedures were developed for the isolation of homogeneous preparations of these two proteins from extracts of heat-induced cells containing pRE1/ptsI and pRE1/crr recombinants. From about 6 g of cells, these procedures yielded 100, 86, and 50 mg of Enzyme I, HPr, and Protein IIIGlc, respectively. The activity of the proteins purified by these methods was comparable to that of the proteins isolated by previously published less efficient procedures.     

Pattern of protein profiles of reproductive organs after induced bilateral cryptorchidism in albino rats

Bilateral cryptorchidism was induced surgically in albino rats and pattern of protein profiles was studied in reproductive organs. Cryptorchidism activated tissue proteolysis leading to overall degradation in soluble and structural protein fractions and in amino acids leading to prevalence of negative nitrogen balance in the reproductive organs. The testicular hypoalbuminic and hypoglobulinic conditions seem to be responsible for oligo-astheno-spermia associated with cryptorchidism.     

Immobilization of amyloglucosidase on polystyrene anion exchangeresin II. Kinetics and stabilities

Summary Amyloglucosidase (exo-1, 4- -D-glucosidase, EC 3.2.1.3), was coupled to glutaraldehyde activated Indion 48-R (a cross-linked macroporous anion exchanger) by Schiff base reaction. Immobilization brought about a marginal increase in the apparent K_m. The bound enzyme exhibited increased stability towards urea and metal ions, but was less stable in the presence of guanidine hydrochloride. Immobilized amyloglucosidase could be stored at 4°C (in wet state) for 6–8 months without any apparent loss of activity.     

32P-analysis of DNA adducts in somatic and reproductive tissues of rats treated with the anticancer antibiotic, mitomycin C

Mitomycin C (MMC) is a clinically used drug with mutagenic and antitumor activities, presumably elicited through its covalent binding to DNA, however, little is known about MMC binding to DNA in vivo. A 32P-postlabeling method that does not require radiolabeled test compounds was employed here to study the formation of DNA adducts in somatic and reproductive tissues of rats 24 h after an i.p. dose of 9 mg/kg MMC. Among 14 tissues studied in female rats, MMC-DNA adduct levels were within a 2-fold range in 11 tissues, i.e. bladder, colon, esophagus, heart, kidney, liver, lung, ovary, pancreas, small intestine and stomach (minimum levels of 9.6-21.9 adducts per 10(7) N). Three other tissues, i.e. brain, spleen and thymus, exhibited lower adduct levels (0.2 5.4 and 1.4 adducts, respectively, per 10(7) N). Liver DNA adduct levels were 32% lower in male than in female rats. Testicular DNA contained 2.5 adducts per 10(7) N, i.e. 5.3 times less than ovarian DNA. 32P-labeled adduct patterns were qualitatively similar among the different tissues and consisted of 10 adducts, one of which comprised 71 (+/- 5)% of the total. All these adducts were chromatographically identical to adducts formed by the reaction of chemically reduced MMC with DNA in vitro, demonstrating that metabolic activation of MMC occurred via reduction. Using homopolydeoxyribonucleotides modified with MMC, in vivo adducts were shown to be mostly (greater than 90%) guanine derivatives and small amounts of adenine, cytosine and thymine products. Most of the adducts appeared to be monofunctional derivatives of DNA nucleotides. Dose-dependent MMC-DNA adduct formation was determined in rat liver over an 82-fold range of MMC administered (0.11-9.0 mg/kg). The lowest dose level studied was 4.5 times lower than the recommended single dose for human cancer chemotherapy (20 mg/m2). Thus, these results predict that 32P-postlabeling methodology is suitable to monitor and quantify DNA adducts in tissue biopsies of patients receiving MMC chemotherapy.     

Calcium-Regulated in Vivo Protein Phosphorylation in Zea mays L. Root Tips

Calcium dependent protein phosphorylation was studied in corn (Zea mays L.) root tips. Prior to in vivo protein phosphorylation experiments, the effect of calcium, ethyleneglycol-bis-(β-aminoethyl ether)-N-N′-tetraacetic acid (EGTA) and calcium ionophore (A-23187) on phosphorus uptake was studied. Calcium increased phosphorus uptake, whereas EGTA and A-23187 decreased it. Consequently, phosphorus concentration in the media was adjusted so as to attain similar uptake in different treatments. Phosphoproteins were analyzed by two-dimensional gel electrophoresis. Distinct changes in phosphorylation were observed following altered calcium levels. Calcium depletion in root tips with EGTA and A-23187 decreased protein phosphorylation. However, replenishment of calcium following EGTA and ionophore pretreatment enhanced phosphorylation of proteins. Preloading of the root tips with ³²P in the presence of EGTA and A-23187 followed by a ten minute calcium treatment, resulted in increased phosphorylation indicating the involvement of calcium, calcium and calmodulin-dependent protein kinases. Calmodulin antagonist W-7 was effective in inhibiting calcium-promoted phosphorylation. These studies suggest a physiological role for calcium-dependent phosphorylation in calcium-mediated processes in plants.