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.     

Inactivation of chloroplast photosynthetic electron-transport activity by Ni

Ni²⁺ inhibits electron-transport activity of isolated barley chloroplasts and this inhibition of electron transport by Ni²⁺ is distinctly different from other heavy metal ion (e.g., Pb²⁺, Cd²⁺, Zn²⁺)-induced inhibition of chloroplast function. Ni²⁺ inactivates Photosystem II (PS II) activity at a lower concentration than that required for the same extent of inhibition of Photosystem I (PS I)-mediated electron flow. Ni²⁺ induces changes in chlorophyll a (Chl a) emission characteristics and brings about a lowering of the Chl a fluorescence yield, and this lowering of Chl a fluorescence intensity is not relieved by the exogenously supplied electron donor NH₂OH which donates electrons very close to the PS II reaction centres. Immobilization of the chloroplast membrane structure with glutaraldehyde fails to arrest the Ni²⁺-induced loss of PS II activity. Also, Ni²⁺-treated chloroplasts do not regain the ability to photoreduce 2,6-dichlorophenolindophenol even after washing of chloroplasts with buffer. These results indicate that unlike Zn²⁺ or Pb²⁺, Ni²⁺ induces alterations in the chloroplast photosynthetic apparatus resulting in an irreversible loss of electron-transport activity.     

Regulation of protoporphyrin IX biosynthesis by intraplastidic compartmentalization and adenosine triphosphate

Subplastidic preparations from cotyledons of cucumber (Cucumis sativus L.) were tested for their ability to synthesize protoporphyrin IX from the substrate 5-aminolevulinic acid. Envelope or thylakoid membranes failed to synthesize protoporphyrin IX from the substrate 5-aminolevulinic acid. Stromal preparations synthesized a very low amount of protoporphyrin IX. In a reconstitution experiment using stroma + envelope membranes, protoporphyrin IX synthesis from 5-aminolevulinic acid was enhanced by 660% over that of stroma alone. However, when thylakoids were added to the stroma + envelope mixture, protoporphyrin IX synthesis from 5-aminolevulinic acid was completely inhibited. In the reconstituted stroma + envelope membrane mixture, the reducing agent dithiothreitol enhanced the protoporphyrin IX-synthesizing ability and completely abolished the inhibition of protoporphyrin IX synthesis by thylakoids. This suggested that the oxidizing agents usually associated with the thylakoid membranes inhibited protoporphyrin IX biosynthesis and the inhibition was alleviated by the reducing power of dithiothreitol. This study exposes the weakness of in vitro reconstitution experiments in mimicking the in vivo-conditions. Addition of ATP stimulated protoporphyrin IX synthesis by 50% in the supernatant fraction of chloroplast lysate. This ATP-induced stimulation of protoporphyrin IX synthesis was due to the enhancement of the activities of uroporphyrinogen decarboxylase and protoporphyrinogen oxidase, involved in tetrapyrrole biosynthesis. The ATP-induced stimulation of porphyrinogen oxidase activity was an energy-dependent reaction. Received: 21 March 2000 / Accepted: 9 May 2000     

Deciphering the Probiotic Potential of Bacillus amyloliquefaciens COFCAU_P1 Isolated from the Intestine of Labeo rohita Through In Vitro and Genetic Assessment

Probiotics and Antimicrobial Proteins - In this study, a bacterial strain COFCAU_P1, isolated from the digestive tract of a freshwater teleost rohu (Labeo rohita), was identified as Bacillus...     

HCMV Activates the IL-6-JAK-STAT3 Axis in HepG2 Cells and Primary Human Hepatocytes

Objectives

There has been increased interest in the possible role of human cytomegalovirus (HCMV) in carcinogenesis during the last decade. HCMV seroprevalence was enhanced in patients with hepatocellular carcinoma (HCC) but a possible relationship between HCC and HCMV infection remained to be assessed. The aim of this work was to investigate the pro-tumor influence of HCMV on primary human hepatocytes (PHH) and HepG2 cells.

Methods

Following infection of PHH and HepG2 cells by two different strains of HCMV, we measured the production of IL-6 in culture supernatants by ELISA and the protein levels of STAT3, pSTAT3, JAK, cyclin D1, survivin, p53, p21, and Mdm2 by western Blotting in infected and uninfected cells. Cell proliferation and transformation were investigated using Ki67Ag expression measurement and soft-agar colony formation assay respectively.

Results

Infection of HepG2 cells and PHH by HCMV resulted in the production of IL-6 and the subsequent activation of the IL-6R-JAK-STAT3 pathway. HCMV increased the expression of cyclin D1 and survivin. Cell proliferation was enhanced in HepG2 and PHH infected with HCMV, despite a paradoxical overexpression of p53 and p21. More importantly, we observed the formation of colonies in soft agar seeded with PHH infected with HCMV and when we challenged the HepG2 cultures to form tumorspheres, we found that the HCMV-infected cultures formed 2.5-fold more tumorspheres than uninfected cultures.

Conclusion

HCMV activated the IL-6-JAK-STAT3 pathway in PHH and HepG2 cells, favored cellular proliferation, induced PHH transformation and enhanced HepG2 tumorsphere formation. Our observations raise the possibility that HCMV infection might be involved in the genesis of hepatocellular carcinoma.     

The N-acylethanolamine-mediated regulatory pathway in plants

While cannabinoids are secondary metabolites synthesized by just a few plant species, N-acylethanolamines (NAEs) are distributed widely in the plant kingdom, and are recovered in measurable, bioactive quantities in many plant-derived products. NAEs in higher plants are ethanolamides of fatty acids with acyl-chain lenghts of C12-C(18) and zero to three C=C bonds. Generally, the most-abundant NAEs found in plants and vertebrates are similar, including NAE 16 : 0, 18 : 1, 18 : 2, and 18 : 3. Like in animal systems, NAEs are formed in plants from N-acylphosphatidylethanolamines (NAPEs), and they are hydrolyzed by an amidase to yield ethanolamine and free fatty acids (FFA). Recently, a homologue of the mammalian fatty acid amide hydrolase (FAAH-1) was identified in Arabidopsis thaliana and several other plant species. Overexpression of Arabidopsis FAAH (AtFAAH) resulted in plants that grew faster, but were more sensitive to biotic and abiotic insults, suggesting that the metabolism of NAEs in plants resides at the balance between growth and responses to environmental stresses. Similar to animal systems, exogenously applied NAEs have potent and varied effects on plant cells. Recent pharmacological approaches combined with molecular-genetic experiments revealed that NAEs may act in certain plant tissues via specific membrane-associated proteins or by interacting with phospholipase D-alpha, although other, direct targets for NAE action in plants are likely to be discovered. Polyunsaturated NAEs can be oxidized via the lipoxygenase pathway in plants, producing an array of oxylipin products that have received little attention so far. Overall, the conservation of NAE occurrence and metabolic machinery in plants, coupled with the profound physiological effects of elevating NAE content or perturbing endogenous NAE metabolism, suggest that an NAE-mediated regulatory pathway, sharing similarities with the mammalian endocannabinoid pathway, indeed exists.     

Fungus mediated synthesis of gold nanoparticles and their conjugation with genomic DNA isolated from Escherichia coli and Staphylococcus aureus

Biological systems employing microorganisms have been used as an alternative to conventional chemical techniques for synthesizing gold nanoparticles. In the present study, gold nanoparticles have been synthesized from the supernatant broth (SB) and live cell filtrate (LCF) of the industrially important fungus Penicillium rugulosum. Additionally, potato dextrose broth (PDB) medium which is used for the growth of the fungus has also been able to synthesize gold nanoparticles. The size of the particles has been investigated by Bio-TEM before purification as well as after purification to find the difference in morphology pattern of the nanoparticles. Different characterization techniques like X-ray diffraction (XRD), infra-red (FTIR), X-ray photoelectron (XPS) and UV–vis spectroscopy have been used for analysis of the particles. SB of the fungus has yielded nanoparticles with better morphology and hence further optimization studies were conducted for controlling the size and shape of the above by altering pH and concentration of gold salt. A pH range of 4–6 has favored the synthesis process whereas increasing concentration of gold salt (beyond 2 mM) has resulted in the formation of bigger sized and aggregated nanoparticles. The optimized nanoparticles have been used to conjugate with isolated genomic DNA of bacteria Escherichia coli and Staphylococcus aureus. Visual observation of agarose gel electrophoresis images confirmed the binding of gold nanoparticles (4 μL and 6 μL) with isolated DNA (2 μL) fragments of both the organisms. The slight red shift of the surface plasmon (SP) band and minor aggregations noticed in Bio-TEM images for the DNA conjugated gold nanoparticles indicates that the genomic DNA could stabilize the particles against aggregation owing to negatively charged phosphate backbone.     

Pattern of Expression and Substrate Specificity of Chloroplast Ferredoxins from Chlamydomonas reinhardtii

Ferredoxin (Fd) is the major iron-containing protein in photosynthetic organisms and is central to reductive metabolism in the chloroplast. The Chlamydomonas reinhardtii genome encodes six plant type [Fe₂S₂] ferredoxins, products of PETF, FDX2–FDX6. We performed the functional analysis of these ferredoxins by localizing Fd, Fdx2, Fdx3, and Fdx6 to the chloroplast by using isoform-specific antibodies and monitoring the pattern of gene expression by iron and copper nutrition, nitrogen source, and hydrogen peroxide stress. In addition, we also measured the midpoint redox potentials of Fd and Fdx2 and determined the kinetic parameters of their reactions with several ferredoxin-interacting proteins, namely nitrite reductase, Fd:NADP⁺ oxidoreductase, and Fd:thioredoxin reductase. We found that each of the FDX genes is differently regulated in response to changes in nutrient supply. Moreover, we show that Fdx2 (E_m = −321 mV), whose expression is regulated by nitrate, is a more efficient electron donor to nitrite reductase relative to Fd. Overall, the results suggest that each ferredoxin isoform has substrate specificity and that the presence of multiple ferredoxin isoforms allows for the allocation of reducing power to specific metabolic pathways in the chloroplast under various growth conditions.Ferredoxins are small (∼11,000-kDa), soluble, iron-sulfur cluster-containing proteins with strongly negative redox potentials (−350 to −450 mV) that function as electron donors at reductive steps in various metabolic pathways (1–3). In photosynthetic organisms, the well studied ferredoxin (Fd⁴; the product of the PETF gene) is the most abundant iron-containing protein in the chloroplast and is central to the distribution of photosynthetically derived reductive power (4).The most well known Fd-dependent reaction is the transfer of electrons from photosystem I (PSI) to NADPH, catalyzed by Fd:NADP⁺ oxidoreductase (FNR). The NADPH produced by this reaction donates electrons to the only reductant-requiring step in the Calvin cycle and other steps in anabolic pathways that require NADPH as reductant. In addition, reduced Fd directly donates electrons to other metabolic pathways by interacting with various enzymes in the chloroplast. This includes Fd:thioredoxin reductase (FTR), which converts a light-driven electron signal into a thiol signal that is transmitted to thioredoxins (TRXs) present in the plastid as different types (or different isoforms). Once reduced, TRXs interact with specific disulfide bonds on target enzymes, modulating their activities (5). Other Fd targets include hydrogenase, which is responsible for hydrogen production in anaerobic conditions in green algae; glutamine-oxoglutarate amidotransferase in amino acid synthesis; nitrite and sulfite reductases in nitrate and sulfate assimilation, respectively; stearoyl-ACP Δ9-desaturase in fatty acid desaturation; and phycocyanobilin:Fd oxidoreductase in synthesis of phytochromobilin (6). Fd also functions in non-photosynthetic cells. Here, FNR catalyzes the reduction of Fd by NADPH produced in the oxidative pentose phosphate pathway, enabling Fd-dependent metabolism to occur in the dark (7, 8).The single-celled green alga, Chlamydomonas reinhardtii is an excellent reference organism for studying both metabolic adaptation to nutrient stress and photosynthesis (9–13). The Chlamydomonas genome encodes six highly related plant type ferredoxin genes (9). Until recently, only the major photosynthetic ferredoxin, Fd (encoded by PETF), which mediates electron transfer between PSI and FNR, had been characterized in detail (14).Many land plants are known to have multiple ferredoxins. Typically, they are differently localized on the basis of their function. Photosynthetic ferredoxins reduce NADP⁺ at a faster rate and are localized to the leaves, whereas non-photosynthetic ferredoxins are more efficiently reduced by NADPH and are localized to the roots. Arabidopsis thaliana has a total of six ferredoxin isoforms (15). Of these, two are photosynthetic and localized in the leaves. The most abundant, AtFd2, is involved in linear electron flow, and the less abundant (5% of the ferredoxin pool), AtFd1, has been implicated in cyclic electron flow (16). There is one non-photosynthetic ferredoxin located in the roots, AtFd3, which is nitrate-inducible. This protein has higher electron transfer activity with sulfite reductase in in vitro assays compared with other Arabidopsis ferredoxin isoforms, suggesting in vivo function of AtFd3 in nitrate and sulfate assimilation (15, 17). In addition, there is one evolutionarily distant ferredoxin, AtFd4, of unknown function with a more positive redox potential present in the leaves and two other proteins which are “ferredoxin-like” and uncharacterized (15). Zea mays has four ferredoxin isoforms, two photosynthetic and two non-photosynthetic (18). One of the non-photosynthetic isoforms is specifically induced by nitrite, suggestive of a role in nitrate metabolism (19). A cyanobacterium, Anabaena 7120, has two ferredoxins, vegetative and heterocyst type (by analogy to leaf and root types, respectively). The heterocyst type is present only in cells that have differentiated into nitrogen-fixing cells, indicating that this form may serve to transfer electrons to nitrogenase (20).We hypothesize that the presence of as many as six ferredoxin isoforms in a single-celled organism like C. reinhardtii allows for the differential regulation of each isoform and therefore the prioritization of reducing power toward certain metabolic pathways under changing environmental conditions. To test this hypothesis, expression of the genes (PETF and FDX2–FDX6) encoding the six ferredoxin isoforms in Chlamydomonas reinhardtii was monitored under various conditions in which well characterized ferredoxin-dependent enzymes are known to be expressed. In addition, we also analyzed the interaction of Fd and Fdx2 with several ferredoxin-interacting proteins, such as NiR, FNR, and FTR, and determined the kinetic parameters of the corresponding reactions.We found that each of the FDX genes is indeed differently regulated in response to changes in nutrient supply. In the case of FDX2 whose product is most similar to classical Fd, we suggest that it has specificity for nitrite reductase based on its pattern of expression and activity with nitrite reductase.     

Chloroplast Biogenesis 60 : Conversion of Divinyl Protochlorophyllide to Monovinyl Protochlorophyllide in Green(ing) Barley, a Dark Monovinyl/Light Divinyl Plant Species

In higher plants, most of the chlorophyll a is formed via the divinyl and monovinyl chlorophyll monocarboxylic biosynthetic routes. These two routes are strongly interconnected prior to protochlorophyllide formation in barley (Hordeum vulgare L. cv Morex), a dark monovinyl-light divinyl plant species, but not in cucumber (Cucumis sativus L. cv Beit Alpha MR), a dark divinyl-light divinyl plant species (BC Tripathy, CA Rebeiz, 1986 J Biol Chem 261: 13556-13564). It is shown that in dark monovinyl-light divinyl plant species such as barley, the divinyl and monovinyl monocarboxylic routes become interconnected at the level of protochlorophyllide during transition from the divinyl to the monovinyl protochlorophyllide biosynthetic mode. In cucumber, a dark divinyl-light divinyl plant species, in which the monovinyl monocarboxylic biosynthetic route becomes preponderant only after an abnormally long sojourn in darkness, the conversion of divinyl to monovinyl protochlorophyllide does not take place on the barley time-scale of incubation.     

Execution of programmed cell death by singlet oxygen generated inside the chloroplasts of Arabidopsis thaliana

Protoplasma - Absorption of excess excitation energy induces overproduction of singlet oxygen (1O2) in plants. The major sources of singlet oxygen production are chlorophyll and its intermediates...