In vitro propagation of the medicinal plant Plumbago zeylanica L. Through nodal explants

Summary A protocol for rapid in vitro propagation using nodal explants obtained from 2-yr-old, field-grown medicinal plants of Plumbago zeylanica L. belonging to the family Plumbaginaceae is described. High frequency bud break and fast development of shoots were induced on Murashige and Skoog's basal medium supplemented with 27.2 μM adenine sulfate +2.46 μM indole-3-butyric acid (IBA). Induction of rooting was achieved by transferring the shoots to the same basal medium containing 4.92 μM IBA. Using our protocol from one twig of P. zeylanica (eight responsive nodes per explant shoot) within a period of 5 mo., eight plantlets could be raised. After a hardening period of 4 wk, there was a 90% transplantation success in the field compared to the 60–65% survival of plantlets recorded in the experiments of previous workers. The plantlets derived through in vitro propagation mimic the growth and morphological characteristics of the donor plants.     

Deglycosylation studies on tracheal mucin glycoproteins

Following several model experiments, conditions were developed for optimal deglycosylation of tracheal mucin glycoproteins. Exposure of rigorously dried material to trifluoromethanesulfonic acid at 0 degree C for up to 8 h results in cleavage of essentially all fucose, galactose, and N-acetylglucosamine, about 80% of the N-acetylneuraminic acid (NeuNAc), and a variable amount of N-acetylgalactosamine (GalNAc), the sugar involved in linkage to protein. Residual N-acetylneuraminic acid is sialidase susceptible and apparently in disaccharide units, presumably NeuNAc2----GalNAc. The remaining N-acetylgalactosamine is mostly present as monosaccharides, and a few Gal beta 1----3GalNAc alpha units are also present; both are cleaved by appropriate enzymatic treatment. The saccharide-free proteins obtained from either human or canine mucin glycoproteins have molecular weights of about 100,000 and require chaotropic agents or detergents for effective solubilization.     

Short course chemotherapy for tuberculous lymphadenitis in children.

OBJECTIVE--To assess the efficacy of a short course chemotherapy regimen for treating tuberculosis of the lymph nodes in children. DESIGN--Open, collaborative, outpatient clinical trial. SETTING--Outpatient department of the Tuberculosis Research Centre, paediatric surgery departments of the Institute of Child Health and Hospital for Children and the Government Stanley Hospital, Madras, South India. PATIENTS--Children aged 1-12 years with extensive, multiple site, superficial tuberculous lymphadenitis confirmed by biopsy (histopathology or culture). INTERVENTIONS--Patients were treated with a fully supervised intermittent chemotherapy regimen consisting of streptomycin, rifampicin, isoniazid, and pyrazinamide three times a week for two months followed by streptomycin and isoniazid twice a week for four months on an outpatient basis. Surgery was limited to biopsy of nodes for diagnosis and assessment. MAIN OUTCOME MEASURES--Response to chemotherapy was assessed by regression of lymph nodes and healing of sinuses and abscesses during treatment and follow up. Compliance with treatment and frequency of adverse reactions were also estimated. RESULTS--197 Patients were admitted to the study and 168 into the analysis. The regimen was well tolerated and compliance was good with 101 (60%) patients receiving the prescribed chemotherapy within 15 days of the stipulated period of six months. Those whose chemotherapy extended beyond that period received the same total number of doses. Clinical response was favourable in most patients at the end of treatment. Sinuses and abscesses healed rapidly. Residual lymphadenopathy (exceeding 10 mm diameter) was present in 50 (30%) patients at the end of treatment; these nodes were biopsied. Fresh nodes, increase in size of nodes, and sinuses and abscesses occurred both during treatment and follow up. After 36 months of follow up after treatment only 5 (3%) patients required retreatment for tuberculosis. CONCLUSION--Tuberculous lymphadenitis in children can be successfully treated with a short course chemotherapy regimen of six months.     

Deconstructing the electron transfer chain in a complex molybdoenzyme: Assimilatory nitrate reductase from Neurospora crassa

Nitrate reductase (NR) from the fungus Neurospora crassa is a complex homodimeric metallo-flavoenzyme, where each protomer contains three distinct domains; the catalytically active terminal molybdopterin cofactor, a central heme-containing domain, and an FAD domain which binds with the natural electron donor NADPH. Here, we demonstrate the catalytic voltammetry of variants of N. crassa NRs on a modified Au electrode with the electrochemically reduced forms of benzyl viologen (BV²⁺) and anthraquinone sulfonate (AQS^?) acting as artificial electron donors. The biopolymer chitosan used to entrap NR on the electrode non-covalently and the enzyme film was both stable and highly active. Electrochemistry was conducted on two distinct forms; one lacking the FAD cofactor and the other lacking both the FAD and heme cofactors. While both enzymes showed catalytic nitrate reductase activity, removal of the heme cofactor resulted in a more significant effect on the rate of nitrate reduction. Electrochemical simulation was carried out to enable kinetic characterisation of both the NR:nitrate and NR:mediator reactions.     

A Novel NAD<Superscript>+</Superscript>-dependent aldehyde dehydrogenase encoded by the <Emphasis Type="Italic">puuC</Emphasis> gene of <Emphasis Type="Italic">Klebsiella pneumoniae</Emphasis> DSM 2026 that utilizes 3-hydroxypropionaldehyde as a substrate

3-Hydroxypropionic acid (3-HP), a versatile and valuable platform chemical, has diverse industrial applications; but its biological production from glycerol is often limited by the capability of the enzyme aldehyde dehydrogenase (ALDH) to convert an intermediary compound, 3-hydroxypropionaldehyde (3-HPA), to 3-HP. In this study, we report a new ALDH, PuuC, from Klebsiella pneumoniae DSM 2026, that efficiently converts 3-HPA to 3-HP. The identified gene puuC was cloned, expressed in Escherichia coli, purified, and characterized for its properties. The recombinant enzyme with a molecular weight of 53.8 kDa exhibited broad substrate specificity for various aliphatic aldehydes, especially C₂–C₅ aldehydes. NAD⁺ was the preferred coenzyme for the oxidation of most aliphatic and aromatic aldehydes tested. The optimum pH and temperature for PuuC activity were pH 8.0 and 45°C. The K _m values for 3-HPA and NAD⁺ were 0.48 and 0.09 mM, respectively. The activity of PuuC was enhanced in the presence of reducing agents such as 2-mercaptoethanol or dithiothreitol, while several metal ions, particularly Hg²⁺, Ag⁺, and Cu²⁺ inhibited its activity. The predicted structure of PuuC indicated the presence of K191 and E194 in close proximity to the glycine motif, suggesting that PuuC belongs to class 2 ALDHs.     

3-hydroxyisobutyrate dehydrogenase-I from Pseudomonas denitrificans ATCC 13867 degrades 3-hydroxypropionic acid

This study examined the role and physiological relevance of 3-hydroxyisobutyrate dehydrogenase-I (3HIBDHI) of Pseudomonas denitrificans ATCC 13867 in the degradation of 3-hydroxypropionic acid (3-HP) during 3-HP production. The gene encoding 3HIBDH-I of P. denitrificans ATCC 13867 was cloned and expressed in Escherichia coli BL21 (DE3). The recombinant 3HIBDH-I was then purified on a Ni-NTA-HP column and characterized for its choice of substrates, cofactors, metals, reductants, and the optimal temperature and pH. The recombinant 3HIBDH-I showed a high catalytic constant (k _cat/K _m) of 604.1 ± 71.1 mM/S on (S)-3-hydroxyisobutyrate, but no detectable activity on (R)-3-hydroxyisobutyrate. 3HIBDH-I preferred NAD⁺ over NADP⁺ as a cofactor for its catalytic activity. The k _cat/K _m determined for 3-HP was 15.40 ± 1.43 mM/S in the presence of NAD⁺ at 37°C and pH 9.0. In addition to (S)-3-hydroxyisobutyrate and 3-HP, 3HIBDH-I utilized l-serine, methyl-d,l-serine, and methyl-(S)-(+)-3-hydroxy-2-methylpropionate; on the other hand, the k _cat/K _m values determined for these substrates were less than 5.0mM/S. Ethylenediaminetetraacetic acid, 2-mercaptoethanol, dithiothreitol and Mn²⁺ increased the activity of 3HIBDHI significantly, whereas the presence of Fe²⁺, Hg²⁺ and Ag⁺ in the reaction mixture at 1.0 mM completely inhibited its activity. This study revealed the characteristics of 3HIBDH-I and its significance in 3-HP degradation.     

A novel inhibitor protein of N-myristoyltransferase from Escherichia coli

Myristoyl-CoA:protein N-myristoyltransferase (NMT) catalyzes the covalent attachment of myristate to the N-terminal of the glycine residue of various eukaryotic and viral proteins of diverse functions. Earlier, we have demonstrated that NMT activity is elevated in colon and gall bladder cancer. Attenuation of NMT activity may prove a novel therapeutic protocol for cancer. We report here a novel inhibitor protein of NMT being expressed in Escherichia coli cells containing the human NMT gene on increasing the incubation period from 5 to 24h. The inhibitor protein was purified by SP-Sepharose column chromatography, heat treatment, ammonium sulfate precipitation, and Superose 12 HR/30 FPLC column chromatography. The inhibitor protein had an apparent molecular mass of 10kDa by gel filtration. It inhibited human NMT in a concentration-dependent manner with 50% inhibition at 640+/-4.68nM. The inhibitor protein showed no direct interaction with myristoyl-CoA and demonstrated no demyristoylase or protease activity. Therefore, we conclude that the inhibitor protein acts directly on NMT.     

Dietary Administration of Probiotic Aeromonas veronii V03 on the Modulation of Innate Immunity,Expression of Immune-Related Genes and Disease Resistance Against Aeromonas hydrophila Infection in Common Carp (Cyprinus carpio)

This study investigates the effects of dietary Aeromonas veronii V03 supplementation on growth performances, innate immunity, and expression of immune-related genes in lymphoid organs of Cyprinus carpio and resistance to Aeromonas hydrophila infection. Fish were fed for 4 weeks with basal diet (BD; without probiotic), and experiment diet containing different doses of A. veronii V03 at 3.2 × 10⁷ (DI) and 3.5 × 10⁹ (DII) CFU g⁻¹ of diet. At the end of the probiotic feeding trial, fish were challenged with A. hydrophila, and the percentage of survival rates was recorded over 7 days. Results revealed that fish fed with A. veronii V03 demonstrated a significant improvement in growth and enhancement of innate immunity, including respiratory burst, myeloperoxidase, and lysozyme activities, and total immunoglobulin level compared with BD fed to fish. Relatively, expression of cytokines (MyD88, IL-1β1, IL-8, and IL-10) and c- and g-type lysozymes were significantly up- and downregulated in lymphoid organs of fish. Moreover, dietary supplementation of A. veronii V03 exhibited significantly (p < 0.001) higher survival rates of DI (90%) and DII (96.66%) compared with BD (53.33%) fed fish against A. hydrophila infection. These findings help to understand the effects of probiotic A. veronii V03 administrated feed influences on growth and ailment resistance to A. hydrophila infection by regulating innate and systemic immunity in common carp fish.

     

Micro‐dose placement of phosphorus induces deep rooting of upland rice

Plant and Soil - Upland rice production is often constrained by phosphorus deficiency (P) and drought events. Methods are needed to maximize P use efficiency, while promoting deep root development...