Search PubMed⌕ Search

Biomedical subjects

R K Jain

Publications and source records attributed to R K Jain.

At least 91 records · Page 5Linked to original sources
PubMed2000

Properties of the trihydroxytoluene oxygenase from Burkholderia cepacia R34: an extradiol dioxygenase from the 2,4-dinitrotoluene pathway.

Burkholderia cepacia R34 mineralizes 2,4-dinitrotoluene via an oxidative pathway. The initial steps in the degradative pathway lead to formation of 2,4,5-trihydroxytoluene, which serves as the substrate for the ring cleavage dioxygenase. The trihydroxylated substrate differs from the usual substituted catechols found in pathways for aromatic compound degradation. To determine whether the characteristics of the trihydroxytoluene oxygenase reflect the unusual ring cleavage substrate of the 2,4-dinitrotoluene pathway, the gene encoding trihydroxytoluene oxygenase (dntD) was cloned and sequenced, and ring cleavage activity determined from recombinant bacteria carrying the cloned gene. The findings were compared to the trihydroxytoluene oxygenase from Burkholderia sp. strain DNT and to other previously described ring cleavage dioxygenases. The comparison revealed that only 60% identity was shared by the two trihydroxytoluene oxygenases, but the amino acid residues involved in cofactor binding, catalysis, and protein folding were conserved in the DntD sequence. The enzyme catalyzed meta-fission of trihydroxytoluene as well as the substrate analogues 1,2,4-benzenetriol, catechol, 3-methylcatechol, 4-methylcatechol, 3-chlorocatechol, 4-chlorocatechol and 2,3-dihydroxybiphenyl. However, results from enzyme assays indicated a strong preference for trihydroxytoluene, implying that it was the native substrate for the enzyme. The apparent enzyme specificity, its similarity to the trihydroxytoluene oxygenase from Burkholderia sp. strain DNT, and the distant genetic relationship to other ring cleavage enzymes suggest that dntD evolved expressly to carry out trihydroxytoluene transformation.

Burkholderia cepacia↗
PubMed2000

Openings between defective endothelial cells explain tumor vessel leakiness.

Leakiness of blood vessels in tumors may contribute to disease progression and is key to certain forms of cancer therapy, but the structural basis of the leakiness is unclear. We sought to determine whether endothelial gaps or transcellular holes, similar to those found in leaky vessels in inflammation, could explain the leakiness of tumor vessels. Blood vessels in MCa-IV mouse mammary carcinomas, which are known to be unusually leaky (functional pore size 1.2-2 microm), were compared to vessels in three less leaky tumors and normal mammary glands. Vessels were identified by their binding of intravascularly injected fluorescent cationic liposomes and Lycopersicon esculentum lectin and by CD31 (PECAM) immunoreactivity. The luminal surface of vessels in all four tumors had a defective endothelial monolayer as revealed by scanning electron microscopy. In MCa-IV tumors, 14% of the vessel surface was lined by poorly connected, overlapping cells. The most superficial lining cells, like endothelial cells, had CD31 immunoreactivity and fenestrae with diaphragms, but they had a branched phenotype with cytoplasmic projections as long as 50 microm. Some branched cells were separated by intercellular openings (mean diameter 1.7 microm; range, 0.3-4.7 microm). Transcellular holes (mean diameter 0.6 microm) were also present but were only 8% as numerous as intercellular openings. Some CD31-positive cells protruded into the vessel lumen; others sprouted into perivascular tumor tissue. Tumors in RIP-Tag2 mice had, in addition, tumor cell-lined lakes of extravasated erythrocytes. We conclude that some tumor vessels have a defective cellular lining composed of disorganized, loosely connected, branched, overlapping or sprouting endothelial cells. Openings between these cells contribute to tumor vessel leakiness and may permit access of macromolecular therapeutic agents to tumor cells.

Animals↗
PubMed2000

Degradation of 4-nitrocatechol by Burkholderia cepacia: a plasmid-encoded novel pathway.

Pseudomonas cepacia RKJ200 (now described as Burkholderia cepacia) has been shown to utilize p-nitrophenol (PNP) as sole carbon and energy source. The present work demonstrates that RKJ200 utilizes 4-nitrocatechol (NC) as the sole source of carbon, nitrogen and energy, and is degraded with concomitant release of nitrite ions. Several lines of evidence, including thin layer chromatography, gas chromatography, 1H-nuclear magnetic resonance, gas chromatography-mass spectrometry, spectral analyses and quantification of intermediates by high performance liquid chromatography, have shown that NC is degraded via 1,2, 4-benzenetriol (BT) and hydroquinone (HQ) formation. Studies carried out on a PNP- derivative and a PNP+ transconjugant also demonstrate that the genes for the NC degradative pathway reside on the plasmid present in RKJ200; the same plasmid had earlier been shown to encode genes for PNP degradation, which is also degraded via HQ formation. It is likely, therefore, that the same sets of genes encode the further metabolism of HQ in NC and PNP degradation.

Burkholderia cepacia↗
PubMed2000

Indigo production by naphthalene-degrading bacteria.

A wild-type naphthalene-degrading strain Pseudomonas putida RKJ1 and two recombinant strains each of Ps. putida and Escherichia coli carrying the genes for naphthalene degradation on a recombinant plasmid pRKJ3, produced indigo and indirubin pigments from indole. Naphthalene, salicylate and IPTG induced cells of naphthalene-degrading recombinant bacteria produced up to two times higher indigo compared with the uninduced cells. The maximum rates of indigo formation by Ps. putida RKJ1, Ps. putida RKJ5/pRKJ3, Ps. putida KT2442/pRKJ3, E. coli TB1/pRKJ3 and E. coli AB1157/pRKJ3 were 0.60, 0.80, 0.60, 1.20 and 1.50 nmol min-1 mg dry biomass-1, respectively, using indole as the substrate. The apparent Km values of indigo formation by these same bacteria were 0.22, 0.15, 0.10, 0. 21 and 0.20 mmol l-1, respectively, again using indole as the substrate. The present study revealed that E. coli AB1157 was the most efficient of the hosts tested for the expression of the plasmid encoded genes (pRKJ3) from the wild-type strain Ps. putida RKJ1. In addition, both recombinant E. coli strains were capable of producing indigo directly from nutrient medium.

Coloring Agents↗
PubMed2000

Vascular permeability in a human tumour xenograft: molecular charge dependence.

Molecular charge is one of the main determinants of transvascular transport. There are, however, no data available on the effect of molecular charge on microvascular permeability of macromolecules in solid tumours. To this end, we measured tumour microvascular permeability to different proteins having similar size but different charge. Measurements were performed in the human colon adenocarcinoma LS174T transplanted in transparent dorsal skinfold chambers in severe combined immunodeficient (SCID) mice. Bovine serum albumin (BSA) and IgG were fluorescently labelled and were either cationized by conjugation with hexamethylenediamine or anionized by succinylation. The molecules were injected i.v. and the fluorescence in tumour tissue was quantified by intravital fluorescence microscopy. The fluorescence intensity and pharmacokinetic data were used to calculate the microvascular permeability. We found that tumour vascular permeability of cationized BSA (pI-range: 8.6-9.1) and IgG (pI: 8.6-9.3) was more than two-fold higher (4.25 and 4.65x10(-7) cm s(-1)) than that of the anionized BSA (pI approximately 2.0) and IgG (pI: 3.0-3.9; 1.11 and 1.93x10(-7) cm s(-1), respectively). Our results indicate that positively charged molecules extravasate faster in solid tumours compared to the similar-sized compounds with neutral or negative charges. However, the plasma clearance of cationic molecules was approximately 2x faster than that of anionic ones, indicating that the modification of proteins enhances drug delivery to normal organs as well. Therefore, caution should be exercised when such a strategy is used to improve drug and gene delivery to solid tumours.

Animals↗
PubMed2000

Differential dynamics of CD4(+) and CD8(+) T-lymphocyte proliferation and activation in acute simian immunodeficiency virus infection.

Although lymphocyte turnover in chronic human immunodeficiency virus and simian immunodeficiency virus (SIV) infection has been extensively studied, there is little information on turnover in acute infection. We carried out a prospective kinetic analysis of lymphocyte proliferation in 13 rhesus macaques inoculated with pathogenic SIV. A short-lived dramatic increase in circulating Ki-67(+) lymphocytes observed at 1 to 4 weeks was temporally related to the onset of SIV replication. A 5- to 10-fold increase in Ki-67(+) CD8(+) T lymphocytes and a 2- to 3-fold increase in Ki-67(+) CD3(-) CD8(+) natural killer cells accounted for >85% of proliferating lymphocytes at peak proliferation. In contrast, there was little change in the percentage of Ki-67(+) CD4(+) T lymphocytes during acute infection, although transient increases in Ki-67(-) and Ki-67(+) CD4(+) T lymphocytes expressing CD69, Fas, and HLA-DR were observed. A two- to fourfold decline in CD4(+) T lymphocytes expressing CD25 and CD69 was seen later in SIV infection. The majority of Ki-67(+) CD8(+) T lymphocytes were phenotypically CD45RA(-) CD49d(hi) Fas(hi) CD25(-) CD69(-) CD28(-) HLA-DR(-) and persisted at levels twofold above baseline 6 months after SIV infection. Increased CD8(+) T-lymphocyte proliferation was associated with cell expansion, paralleled the onset of SIV-specific cytotoxic T-lymphocyte activity, and had an oligoclonal component. Thus, divergent patterns of proliferation and activation are exhibited by CD4(+) and CD8(+) T lymphocytes in early SIV infection and may determine how these cells are differentially affected in AIDS.

Acute Disease↗
PubMed2000

Evidence for plasmid-mediated chemotaxis of Pseudomonas putida towards naphthalene and salicylate.

A naphthalene (Nap) and salicylate (Sal) degrading microorganism, Pseudomonas putida RKJ1, is chemotactic towards these compounds. This strain carries a 83 kb plasmid. A 25 kb EcoRI fragment of the plasmid contains the genes responsible for Nap degradation through Sal. RKJ5, the plasmid-cured derivative of RKJ1, is neither capable of degradation nor is chemotactic towards Nap or Sal. The recombinant plasmid pRKJ3, which contained a 25 kb EcoRI fragment, was transferred back into the plasmid-free wild-type strain RKJ5, and the transconjugant showed both degradation and chemotaxis. The recombinant plasmid pRKJ3 was also transferred into motile, plasmid-free P. putida KT2442. The resulting transconjugant (RKJ15) showed chemotaxis towards both Nap and Sal. Two mutant strains carrying deletions in pRKJ3 (in KT2442) with phenotypes Nap- Sal+ and Nap- Sal-, were also tested for chemotaxis. It was found that the Nap- Sal+ mutant strain showed chemotaxis towards Sal only, whereas the Nap- Sal- mutant strain is non-chemotactic towards both the compounds. These results suggest that the metabolism of Nap and Sal may be required for the chemotactic activity.

Chemotaxis↗
PubMed2000

Enhancement of seed oil content by expression of glycerol-3-phosphate acyltransferase genes.

Arabidopsis thaliana was transformed with a plastidial safflower glycerol-3-phosphate acyltransferase (GPAT) and an Escherichia coli GPAT. The genes were used directly and in modified forms with, as applicable, the plastidial targeting sequence removed, and with an endoplasmic reticulum targeting sequence added. Seeds of plants transformed using only the vector were indistinguishable in oil content from wild-type control plants. All other gene constructs increased seed oil content. The unmodified safflower gene (spgpat) produced oil increases ranging from 10 to 21%. On average, the greatest increase (+22%) was observed in seeds of transformants carrying the spgpat with the targeting peptide removed. The E. coli plsB gene increased seed oil content by an average of 15%.

Arabidopsis↗
PubMed2000

Intraoperative penile erection.

OBJECTIVE: Penile erection following epidural anesthesia is an infrequent occurrence of unclear etioogy. It is very troublesome to perform the procedure during penile erection and attempting it leads to various complications. Three such cases are reported herein. The literature on the pathophysiology and management of intraoperative penile erection is discussed. METHODS: Eighteen hundred patients who received epidural anesthesia for various transurethral procedures like cystoscopy, ureterorenoscopy, transurethral resection of prostate and bladder tumors form the clinical material. RESULTS: Out of 1800 cases, we have encountered penile erection only in three cases. Two cases were managed with local application of cold saline and cold sponging followed by terbutaline. The third case could be managed with local application of cold saline and cold sponging of scrotum and penis. Afterwards the procedure could be performed safely. CONCLUSION: The pathophysiology and management of intraoperative penile erection is poorly understood. With the onset of erection during an operative procedure, the anesthesiologist must quickly initiate the therapy, as duration of erection is the critical factor in the detumescence of the penis.

Adult↗
PubMed2000

Transgenic strategies for genetic improvement of Basmati rice.

Transgenic approach offers an attractive alternative to conventional techniques for the genetic improvement of Basmati rice because they enable the introduction of one or more genes into a leading cultivar without affecting its genetic background. During the last ten years, a rapid progress has been made towards the development of transformation methods in rice. Several transformation methods including Agrobacterium, biolistic, and DNA uptake by protoplasts, have been employed to produce transgenic rice. An array of useful genes is now available and many of these have already been transferred in rice to improve the resistance against biotic and abiotic stresses. In Basmati rice, a beginning has already been made regarding the development of tissue culture protocols, transformation methods and production of useful transgenic plants. The application and future prospects of transformation technology to engineer the resistance against insect pests (stem borer, leaf folder, brown plant hopper, gall midge), fungal diseases (blast, bakanae/foot, rot), bacterial diseases (bacterial leaf blight, sheath blight), abiotic stresses (salinity and drought) and improved nutritional quality (accumulation of provitamin A and essential amino acids in endosperm) in Basmati rice, have been addressed.

Breeding↗
PubMed1999

Cellular membrane permeability of anthracyclines does not correlate with their delivery in a tissue-isolated tumor.

The clearance of anthracyclines from the vasculature was studied in perfused tissue-isolated tumors. Human tumor lines MCF-7, U87, and LS174T were implanted in the ovarian fat pad of immune-deficient mice and grown isolated from the surrounding tissue. The initial and continuous tissue uptakes of doxorubicin, daunorubicin, and idarubicin were measured. The clearance of these anthracyclines from the perfused vasculature of the tissue-isolated tumor was calculated using BSA as an intravascular marker. The measured clearances ranged from 50-200 microl/min/g tumor tissue, and the fractional clearances were between 0.30 and 0.70. On the basis of the in vitro cellular uptake rates of the anthracyclines, we expected a higher clearance of idarubicin than of doxorubicin. No significant differences were found among the clearances of the anthracyclines or among the tumor lines. The observed similarities in clearance of the anthracyclines was largely explained by differences in their protein binding and tissue diffusion gradients.

Animals↗
PubMed1999

Augmentation of transvascular transport of macromolecules and nanoparticles in tumors using vascular endothelial growth factor.

The goal of this investigation was to measure changes in vascular permeability, pore cutoff size, and number of transvascular transport pathways as a function of time and in response to vascular endothelial growth factor (VEGF), placenta growth factor (PIGF-1 and PIGF-2), or basic fibroblast growth factor (bFGF). Two human and two murine tumors were implanted in the dorsal skin chamber or cranial window. Vascular permeability to BSA (approximately 7 nm in diameter) and extravasation of polyethylene glycol-stabilized long-circulating liposomes (100-400 nm) and latex microspheres (approximately 800 nm) were determined by intravital microscopy. Vascular permeability was found to be temporally heterogeneous. VEGF superfusion (100 ng/ml) significantly increased vascular permeability to albumin in normal s.c. vessels, whereas a 30-fold higher dose of VEGF (3000 ng/ml) was required to increase permeability in pial vessels, suggesting that different tissues exhibit different dose thresholds for VEGF activity. Furthermore, VEGF superfusion (1000 ng/ml) increased vascular permeability to albumin in a hypopermeable human glioma xenograft in cranial window, whereas VEGF superfusion (10-1000 ng/ml) failed to increase permeability in a variety of hyperpermeable tumors grown in dorsal skin chamber. Interestingly, low-dose VEGF treatment (10 ng/ml) doubled the maximum pore size (from 400 to 800 nm) and significantly increased the frequency of large (400 nm) pores in human colon carcinoma xenografts. PIGF-1, PIGF-2, or bFGF did not show any significant effect on permeability or pore size in tumors. These findings suggest that exogenous VEGF may be useful for augmenting the transvascular delivery of larger antineoplastic agents such as gene targeting vectors and encapsulated drug carriers (typical range, 100-300 nm) into tumors.

Animals↗
PubMed1999

Taxane-induced apoptosis decompresses blood vessels and lowers interstitial fluid pressure in solid tumors: clinical implications.

Elevated tumor interstitial fluid pressure (IFP) is partly responsible for the poor penetration and distribution of therapeutic agents in solid tumors. The etiology of tumor interstitial hypertension is poorly understood. We have postulated that the solid stress generated by tumor cells growing in a confined space compresses blood vessels and increases tumor microvascular pressure and IFP. To test the hypothesis that neoplastic cell loss would decompress blood vessels and lower IFP, we induced apoptosis in tumors with paclitaxel and docetaxel. Taxanes inhibited the growth of the murine mammary carcinoma (MCa-IV) and of the human soft tissue sarcoma (HSTS-26T). Taxanes induced apoptosis and reduced the density of intact neoplastic cells in both MCa-IV and HSTS-26T. To determine whether neoplastic cell loss decompressed blood vessels, we measured the diameter of tumor vessels in HSTS-26T tumors implanted in transparent dorsal skin fold chambers. At 48 and 96 h after paclitaxel, the diameter of tumor vessels was significantly increased. The increase in vascular diameters was associated with reductions in microvascular pressure and IFP. The changes in neoplastic cell density and IFP were also correlated. In the human glioblastoma U87, which is resistant to paclitaxel, the IFP and cellular density were not modified by paclitaxel treatment. Collectively, these results support the hypothesis that solid stress generated by neoplastic cell proliferation increases vascular resistance and IFP. The increase in vessel diameter induced by paclitaxel and docetaxel suggests that taxanes could improve tumor response by increasing the vascular surface area for delivery of therapeutic agents.

Animals↗
PubMed1999

Angiogenesis in the huPBL-SCID model of human transplant rejection.

BACKGROUND: Angiogenesis is characteristic of chronic inflammatory reactions. The process of angiogenesis is reported to be proinflammatory in part due to enhanced adhesion events and in part due to increased perfusion and permeability to sites of inflammation. However, little is known about the association between angiogenesis and rejection. METHODS: Severe combined immune deficient mice are permissive for the growth of human skin allografts and human peripheral blood mononuclear cells (PBMC). Human PBMC were injected into mice by intravenous or intraperitoneal injection. The infiltration of cells and the associated angiogenesis reactions in the skin allografts were analyzed temporally by videomicroscopy and spatially by immunohistochemistry. RESULTS: Human alloreactive mononuclear cells migrated to human skin but not mouse skin within hours after the intravenous infusion of PBMC. Within 3 days, areas of angiogenesis were observed in the skin grafts at the sites of infiltrates. The vessel densities in skin grafts were 24+/-6 vessels per calibrated grid at baseline on the day of the infusion and increased to 55+/-16 vessels per calibrated field by day 10. Skin grafts harvested from humanized severe combined immune deficient mice 7-14 days after the intraperitoneal infusion of human PBMC showed a similar increased density of vessels that were spatially associated with mononuclear cell infiltrates. CONCLUSIONS: A significant angiogenesis response was associated with the cell infiltrates in the human skin allografts. The onset of angiogenesis appeared after the initial development of localized infiltrates and preceded the development of microvascular destruction. These findings suggest that alloreactive T cells and/or monocytes mediate the angiogenesis response in skin allografts.

Adoptive Transfer↗
PubMed1999

Enhancement of fluid filtration across tumor vessels: implication for delivery of macromolecules.

Cancer therapies using genes and other macromolecules might realize their full clinical potential if they could be delivered to tumor tissue in optimal quantities. Unfortunately, the compromised circulation within tumors poses a formidable resistance to adequate and uniform penetration of these agents. Previously, we have proposed elevated interstitial fluid pressure (IFP) as a major physiological barrier to delivery of macromolecules. Here we postulate that modulation of tumor microvascular pressure (MVP) and associated changes in IFP would enhance macromolecular delivery into a solid tumor. To test our hypothesis, we altered tumor MVP by either periodic injection or continuous infusion of angiotensin II (AII) and measured the resulting changes in IFP and uptake of macromolecules. We used the nicotinyl hydrazine derivative of human polyclonal IgG (HYNIC-IgG) as a nonspecific macromolecule and CC49 antibody as a specific macromolecule. We found that both chronic and periodic modulation of tumor MVP enhances transvascular fluid filtration, leading to a 40% increase in total uptake of the specific antibody within 4 hr of its administration. Conversely, neither continuous nor periodic infusion of AII induced any increase in uptake of nonspecific antibodies. Strategies to improve delivery of macromolecules and limitations of this approach are identified.

Animals↗
PubMed1999

Effect of local anti-VEGF antibody treatment on tumor microvessel permeability.

Recent studies have shown that systemic injection of anti-VEGF antibody into tumor-bearing mice results in decreases in tumor vascular permeability, vessel diameters, and tumor regression. Using a similar animal model, we have applied anti-VEGF antibody directly to the tumor tissue growing in transparent window chambers in SCID mice. Similar to the results obtained with systemic injection, vascular permeability was greatly reduced, but the response was reached at much lower concentrations with local application. Implications of these findings on local control of tumors are discussed.

Animals↗