A novel cobiotic-based preventive approach against high-fat diet-induced adiposity, nonalcoholic fatty liver and gut derangement in mice.

BACKGROUND: High-fat diets (HFDs) induce systemic inflammation, gut microbial derangements and disturb metabolic homeostasis, resulting in weight gain, insulin resistance and nonalcoholic fatty liver (NAFL). Numerous antioxidants and prebiotic/probiotics per se may prevent HFD-associated comorbidities, but there are no reports related to their combination. OBJECTIVE: In the present study, we aim to evaluate a cobiotic combination of lycopene (antioxidant) and isomalto-oligosaccharides (IMOs, a prebiotic) for prevention of HFD-induced alterations. DESIGN: Male Swiss albino mice were fed either normal pellet diet (NPD) or HFD and lycopene (5 and 10 mg kg(-1)), IMOs (0.5 and 1 g kg(-1)) or their combination for 12 weeks. Systemic adiposity, glucose tolerance, insulin sensitivity, feeding regulators in hypothalamus, hepatosteatosis and liver inflammation, cecal short chain fatty acids (SCFAs), serum inflammatory cytokines, gut morphology and alterations in selected gut microbes were studied. RESULTS: Lycopene, IMOs and their combination prevented weight gain, adiposity, improved adipose tissue fat mobilization and reduced insulin resistance. Hypothalamic orexigenic and anorectic genes have also been modulated by these treatments. Dietary interventions prevented NAFL-like symptoms and improved glucose homeostasis. Improvement in selected gut microbial abundance and SCFA concentration along with reduced systemic inflammation, metabolic endotoxemia and improved ileal and colonic health were observed in mice supplemented with lycopene, IMOs and their combination. Interestingly, cobiotic combination synergistically improved many of the HFD-induced alterations. CONCLUSION: The present work provide evidence that new approach based on cobiotic combination (antioxidant plus prebiotic) can be employed to develop novel class of functional foods for their application against HFD-associated pathological complications.

Immunoprotective responses of T helper type 1 stimulatory protein-S-adenosyl-L-homocysteine hydrolase against experimental visceral leishmaniasis.

It is well known that a patient in clinical remission of visceral leishmaniasis (VL) remains immune to reinfection, which provides a rationale for the feasibility of a vaccine against this deadly disease. In earlier studies, observation of significant cellular responses in treated Leishmania patients as well as in hamsters against leishmanial antigens from different fractions led to its further proteomic characterization, wherein S-adenosyl-L-homocysteine hydrolase (AdoHcy) was identified as a helper type 1 (Th1) stimulatory protein. The present study includes immunological characterization of this protein, its cellular responses [lymphoproliferation, nitric oxide (NO) production and cytokine responses] in treated Leishmania-infected hamsters and patients as well as prophylactic efficacy against Leishmania challenge in hamsters and the immune responses generated thereof. Significantly higher cellular responses were noticed against recombinant L. donovani S-adenosyl-L-homocysteine hydrolase (rLdAdoHcy) compared to soluble L. donovani antigen in treated samples. Moreover, stimulation of peripheral blood mononuclear cells with rLdAdoHcy up-regulated the levels of interferon (IFN)-γ, interleukin (IL)-12 and down-regulated IL-10. Furthermore, vaccination with rLdAdoHcy generated perceptible delayed-type hypersensitivity response and exerted considerably good prophylactic efficacy (∼70% inhibition) against L. donovani challenge. The efficacy was confirmed by the increased expression levels of inducible NO synthase and Th1-type cytokines, IFN-γ and IL-12 and down-regulation of IL-4, IL-10 and transforming growth factor (TGF)-ß. The results indicate the potentiality of rLdAdoHcy protein as a suitable vaccine candidate against VL.

Immunostimulatory potential and proteome profiling of Leishmania donovani soluble exogenous antigens.

Isolation of the soluble exogenous antigens (SEAgs), its immune response study and proteome profiling is an essential prerequisite for understanding the molecular pathogenesis of Leishmania donovani. The immunostimulatory potential of L. donovani SEAgs, purified from culture of L. donovani clinical isolate, was evaluated for their ability to induce cellular responses in treated/cured hamsters. SEAgs induced significant proliferative responses in lymphocytes (SI 5.6 ± 2.3; P < 0.01) isolated from cured hamster. In addition, significant NO production in response to SEAgs was also noticed in macrophages of hamsters, mouse and human cell lines (J774A-1 and THP1). Western blot analyses with antibodies against proteophosphoglycan (PPG; surface-expressed and secreted molecule) of L. donovani revealed that PPG molecules are also present in L. donovani SEAgs. Mass spectrometry (MS)-based proteome analysis of 12 protein bands of SEAgs through MALDI-TOF/TOF endorsed the identification of some Th1-stimulatory immunogenic proteins. These immunogenic proteins may offer increased hope for the discovery of new promising vaccine candidates against visceral leishmaniasis (VL). The overall results suggest that immunostimulatory molecules are present in the SEAgs, which may be further exploited, for developing a subunit vaccine against VL a fatal human disease.

Simulation of vertical concentration gradient of influenza viruses in dust resuspended by walking.

UNLABELLED: Particles are resuspended from the floor by walking and are subject to turbulent transport in the human aerodynamic wake. These processes may generate a vertical concentration gradient of particles. To estimate the magnitude of turbulence generated by walking, we measured the velocity field in the wake from floor to ceiling at 10-cm intervals with a sonic anemometer. The resulting eddy diffusion coefficients varied between 0.06 and 0.20 m(2) /s and were maximal at ~0.75-1 m above the floor, approximately the height of the swinging hand. We applied the eddy diffusion coefficients in an atmospheric transport model to predict concentrations of resuspended influenza virus as a function of the carrier particle size, height in the room, and relative humidity, which affects the resuspension rate coefficient and virus viability. Results indicated that the concentration of resuspended viruses at 1 m above the floor was up to 40% higher than at 2 m, depending on particle size. For exposure to total resuspended viruses, the difference at 1 vs. 2 m was 11-14%. It is possible that shorter people are exposed to higher concentrations of resuspended dust, including pathogens, although experimental evidence is needed to verify this proposition. PRACTICAL IMPLICATIONS: Forces generated by walking can resuspend particles from the floor and create higher concentrations close to the floor and lower concentrations above it. These particles may include pathogens, such as the influenza virus, that were previously emitted into the air by an infected individual and that settled to the ground. Due to particle resuspension and turbulent transport, it is possible that shorter people are exposed to higher concentrations of particles, including certain pathogens, than are taller people. This work could be used in support of epidemiological investigations into the incidence of influenza as a function of a person's height and to guide the design of more effective control strategies to reduce transmission of influenza.

Validity of heart rate based nomogram fors estimation of maximum oxygen uptake in Indian population.

Maximal oxygen uptake (VO2max) during a graded maximal exercise test is the objective method to assess cardiorespiratory fitness. Maximal oxygen uptake testing is limited to only a few laboratories as it requires trained personnel and strenuous effort by the subject. At the population level, submaximal tests have been developed to derive VO2max indirectly based on heart rate based nomograms or it can be calculated using anthropometric measures. These heart rate based predicted standards have been developed for western population and are used routinely to predict VO2max in Indian population. In the present study VO2max was directly measured by maximal exercise test using a bicycle ergometer and was compared with VO2max derived by recovery heart rate in Queen's College step test (QCST) (PVO2max I) and with VO2max derived from Wasserman equation based on anthropometric parameters and age (PVO2max II) in a well defined age group of healthy male adults from New Delhi. The values of directly measured VO2max showed no significant correlation either with the estimated VO2max with QCST or with VO2max predicted by Wasserman equation. Bland and Altman method of approach for limit of agreement between VO2max and PVO2max I or PVO2max II revealed that the limits of agreement between directly measured VO2max and PVO2max I or PVO2max II was large indicating inapplicability of prediction equations of western population in the population under study. Thus it is evident that there is an urgent need to develop nomogram for Indian population, may be even for different ethnic sub-population in the country.

In-vitro shoot regeneration via caulogenesis in fern, Pteris vittata L.

An attempt was made to standardize the protocol for the shoot regeneration via caulogenesis in Pteris vittata L. employing leaf primordium explants. Calli were induced on Murashige and Skoog's (MS) and Parkers and Thompson's (P and T) media supplemented with different combinations of 2, 4-dichlorophenoxyaceticacid (2, 4-D), 6-benzylaminopurine (BAP), alpha-naphthalene acetic acid (NAA) and Indole-3-acetic acid (IAA). A combination of full strength MS medium with 2, 4-D (2.26 microM) and BAP (2.22 microM) was found to be ideal for profuse callusing (80%) against other combinations. More shoot differentiation (2.8 +/- 0.06) was achieved from the calli on one-fourth strength of P and T media fortified with BAP (4.44 microM) and NAA (2.68 microM) when compared to other combinations but statistically not significant.

Antimicrobial activity of some important Adiantum species used traditionally in indigenous systems of medicine.

Adiantum Linn. of Adiantaceae family is one of the most common and widely distributed species. Ethnomedicinally, the genus is important and popularly known as "Hansraj" in Ayurvedic System of Medicine. It has been used in cold, tumors of spleen, liver and other viscera, skin diseases, bronchitis and inflammatory diseases. It is also considered as tonic and diuretic. In the present study its four important species, i.e. Adiantum capillus-veneris, Adiantum peruvianum, Adiantum venustum and Adiantum caudatum were collected and extracted with methanol. These extracts were tested for their antimicrobial agents against five gram positive, six gram negative (including multiresistant bacteria Staphylococcus aureus) and eight fungal strains using standard microdilution assay. The maximum activity was exhibited by the methanolic extract of Adiantum venustum followed by Adiantum capillus-veneris, Adiantum peruvianum and Adiantum caudatum. The methanolic extract of Adiantum capillus-veneris had very low MIC value (0.48 microg/ml) against Escherichia coli whereas, Adiantum venustum extract against Aspergillus terreus with MIC of 0.97 microg/ml. Total phenolic constituents of Adiantum species viz. Adiantum venustum, Adiantum capillus-veneris, Adiantum peruvianum and Adiantum caudatum were 0.81% (w/w), 0.83% (w/w), 0.71% (w/w) and 0.52% (w/w), respectively (as gallic acid equivalent); implying that the observed activity could be related to the amount of phenolics.

Transient receptor potential (TRP) channels: a metabolic TR(i)P to obesity prevention and therapy.

Cellular transport of ions, especially by ion channels, regulates physiological function. The transient receptor potential (TRP) channels, with 30 identified so far, are cation channels with high calcium permeability. These ion channels are present in metabolically active tissues including adipose tissue, liver, gastrointestinal tract, brain (hypothalamus), pancreas and skeletal muscle, which suggests a potential role in metabolic disorders including obesity. TRP channels have potentially important roles in adipogenesis, obesity development and its prevention and therapy because of their physiological properties including calcium permeability, thermosensation and taste perception, involvement in cell metabolic signalling and hormone release. This wide range of actions means that organ-specific actions are unlikely, thus increasing the possibility of adverse effects. Delineation of responses to TRP channels has been limited by the poor selectivity of available agonists and antagonists. Food constituents that can modulate TRP channels are of interest in controlling metabolic status. TRP vanilloid 1 channels modulated by capsaicin have been the most studied, suggesting that this may be the first target for effective pharmacological modulation in obesity. This review shows that most of the TRP channels are potential targets to reduce metabolic disorders through a range of mechanisms.

Specifically targeted killing of carcinoembryonic antigen (CEA)-expressing cells by a retroviral vector displaying single-chain variable fragmented antibody to CEA and carrying the gene for inducible nitric oxide synthase.

The generation of retroviral vectors that infect specific cell types through recognition of cell surface antigens is a promising and effective approach to targeted gene therapy of cancer. Carcinoembryonic antigen (CEA), a highly characterized, cell surface glycoprotein overexpressed by various tumor cells, provides a specific tool for tumor tissue-specific targeting by retroviral vectors. The conventional suicidal gene delivery systems need additional drugs other than their gene products. The inducible nitric oxide synthase (iNOS) gene product yields nitric oxide (NO), which directly induces autocytotoxicity and cytolysis of bystander cells. In the present study, we have developed a novel bifunctional Moloney murine leukemia virus-based recombinant retroviral vector that displays a chimeric envelope protein containing a single-chain variable fragmented (scFv) antibody to CEA and carries the iNOS gene in the genome. The resultant bifunctional retroviral vector showed a specific delivery of the iNOS gene to human CEA-expressing carcinoma cells, resulting in the direct and efficient killing of CEA-expressing carcinoma cells by induction of apoptosis. This is the first report of successful killing of CEA-expressing cells by specific targeting of the iNOS gene. This approach may offer a one-step procedure for effective gene therapy of CEA-expressing tumors.

Natural twilight phase-response curves for the cave-dwelling bat, Hipposideros speoris.

Phase-response curves (PRCs) for the circadian rhythm of flight activity of the microchiropteran bat (Hipposideros speoris) were determined in a cave, employing discrete natural dawn and dusk twilight pulses. These PRCs are reported for the first time for any circadian system and they are unlike other PRCs constructed for nocturnal mammals. Dawn and dusk twilight pulses evoked advance and delay phase shifts, respectively. Advance phase shifts were followed by 3 to 4 advancing transients and a subsequent shortening of free-running period (tau); whereas, the delay phase shifts were instantaneous without any transients but with a subsequent lengthening of tau.

Altitudinal variation in the circadian rhythm of oviposition in Drosophila ananassae.

The effect of altitude on four basic properties of the pacemaker controlling the circadian rhythm of oviposition in two strains of Drosophila ananassae was determined. The high altitude (HA) strain from Badrinath (5123 m above sea level) had a low amplitude peak in the forenoon while the low altitude (LA) strain from Firozpur (179 m a.s.l.) had a high amplitude peak after the lights-off of LD 12:12 cycles. Free running periods in continuous darkness were about 22.6 and 27.4 h in the HA and LA strains, respectively. The light pulse phase response curve (PRC) for the HA strain showed a low amplitude and a dead zone of 8h; the ratio for the advance to delay region (A/D) was less than 1, while the PRC for the LA strain had a high amplitude, which was devoid of a dead zone and showed a ratio of A/D > 1. The magnitude of the delay phase shifts at CT 18 evoked by light pulses of 1 h duration, but varying light intensity was significantly different in the HA and LA strain, which suggests that the photic sensitivity of the clock photoreceptors mediating the phase shifts had been affected by the altitude.

Specific targeting strategies of cancer gene therapy using a single-chain variable fragment (scFv) with a high affinity for CEA.

Two specific targeting strategies of cancer gene therapy using carcinoembryonic antigen (CEA) as a target are briefly reviewed here. One method is the specific targeting of suicide genes to CEA-expressing tumor cells by a retrovector displaying anti-CEA single-chain variable fragment (scFv). We reconstructed a recombinant retroviral vector that displays both anti-CEA scFv-expressing chimeric and normal envelope proteins and carries the inducible nitric oxide synthase (iNOS) gene. This recombinant retrovirus specifically bound, infected and killed only CEA-expressing tumor cells, indicating the cell specific retroviral vector delivery of the iNOS gene. Another novel method is the specific redirecting of cytotoxic T-cells to CEA-expressing tumor cells through chimeric receptors. We also reconstructed a chimeric receptor gene which encoded an anti-CEA scFv antibody and the zeta-chain of human TCR/CD3 complex and expressed it on T-cell surface. When incubated with CEA-expressing tumor cells in vitro, the transduced T-cells tended to make a rosette-like formation around the tumor cells, suggesting the cell specific targeting of T-cells.

Carcinoma-associated antigens MK-1 and CEA in urological cancers.

BACKGROUND: The MK-1 antigen, recognized by monoclonal antibody FU-MK-1, is widely associated with human carcinomas. However, the expression and distribution of MK-1 in urological cancers is not well known. MATERIALS AND METHODS: We examined the expression of MK-1 in 10 urological tumor cell lines using flow cytometry and reverse transcription-polymerase chain reaction (RT-PCR) and in 15 cancer tissue specimens by immunohistochemical staining, and then compared it with that of carcinoembryonic antigen (CEA). RESULTS: When analyzed by flow cytometry, MK-1 was positive in 2 out of 3 bladder, 3 out of 3 prostate and one out of 4 renal tumor cell lines, whereas CEA was negative in all the 10 tumor cell lines. RT-PCR confirmed the presence of MK-1 mRNA in all the six MK-1-positive tumor cell lines. An immunohistochemical study demonstrated that MK-1 was positive in 2 out of 5 bladder, 2 out of 5 prostate and one out of 5 renal cancer tissues. Again, however, CEA was negative in all the 15 urological cancer tissues tested. CONCLUSION: These findings suggest that MK-1 seems to be a useful biological marker for malignant urological tumors, especially in cases of bladder and prostate cancer.

Targeting of LAK activity to CEA-expressing tumor cells with an anti-CEA scFv/IL-2 fusion protein.

BACKGROUND: Fusion of tumor-specific monoclonal antibody (MAb) and cytokines has proved to be an efficient way to target cytokines to tumor cells and hence focuses the killing activity of effector cells to the target cells. We previously produced a high affinity MAb, F11-39, against carcinoembryonic antigen (CEA), which is often overexpressed on the surface of various tumor cells. MATERIALS AND METHODS: To target the cytotoxicity of effector cells to CEA-expressing tumor cells, we employed recombinant DNA techniques to fuse recombinant human interleukin-2 (rhIL-2) to a single chain variable fragment (scFv) antibody derived from F11-39. The resulting fusion protein, designated F39scFv/IL-2, was expressed in the Sp2/0-Ag14 mouse hybridoma cells, purified by CEA-affinity chromatography and characterized for the CEA-binding specificity and the IL-2 biological activity. RESULTS: F39scFv/IL-2 protein effectively targeted rhIL-2 onto the surface of CEA-expressing tumor cells and consequently introduced a specific cytotoxicity of lymphokine-activated killer cells to the tumor cells. CONCLUSIONS: This approach may be used for in vivo administration to localize IL-2 to tumor tissues, maximizing the immune response to CEA-expressing tumors while keeping systemic side effects to a minimum.

Temperature dependent eclosion rhythmicity in the high altitude Himalayan strains of Drosophila ananassae.

The circadian pacemaker controlling the eclosion rhythm of the high altitude Himalayan strains of Drosophila ananassae captured at Badrinath (5123 m) required ambient temperature at 21 degrees C for the entrainment and free-running processes. At this temperature, their eclosion rhythms entrained to 12h light, 12h dark (LD 12:12) cycles and free-ran when transferred from constant light (LL) to constant darkness (DD) or upon transfer to constant temperature at 21 degrees C following entrainment to temperature cycles in DD. These strains, however, were arrhythmic at 13 or 17 degrees C under identical experimental conditions. Eclosion medians always occurred in the thermophase of temperature cycles whether they were imposed in LL or DD; or whether the thermophase coincided with the photophase or scotophase of the concurrent LD 12:12 cycles. The temperature dependent rhythmicity in the Himalayan strains of D. ananassae is a rare phenotypic plasticity that might have been acquired through natural selection by accentuating the coupling sensing mechanism of the pacemaker to temperature, while simultaneously suppressing the effects of light on the pacemaker.

Effects of temperature, photoperiod, and light intensity on the eclosion rhythm of the high-altitude Himalayan strain of Drosophila ananassae.

Eclosion rhythm of the high-altitude Himalayan strain of Drosophila ananassae from Badrinath (altitude 5123 m) was temperature-dependent and at 21 degrees C, it was entrained by cycles of 12h light: 12h darkness (LD 12:12) and free-ran in constant darkness, however, it was arrhythmic at 13 degrees C or 17 degrees C under identical experimental conditions (Khare, P. V., Barnabas, R. J., Kanojiya, M., Kulkarni, A. D., Joshi, D. S. (2002). Temperature dependent eclosion rhythmicity in the high altitude Himalayan strains of Drosophila ananassae. Chronobiol. Int. 19:1041-1052). The present studies were designed to see whether or not these strains could be entrained at 13 degrees C, 17 degrees C, and 21 degrees C by two types of LD cycles in which the photoperiod at 100 lux intensity varied from 6h to 18h, and the light intensity of LD 14:10 cycles varied from 0.001 lux to 1000 lux. All LD cycles entrained this strain at 21 degrees C but not at 13 degrees C or 17 degrees C. These results demonstrate that the entrainment of eclosion rhythm depends on the ambient temperature and not on the photoperiod or light intensity of LD cycles. Thus the temperature has taken precedence over the light in the entrainment process of eclosion rhythm of the high altitude Himalayan strain of D. ananassae. This may be the result of natural selection in response to the environmental temperature at Badrinath that resembles that of the sub-Arctic region but the photoperiod or light intensity are of the subtropical region.

Measurements of major ion concentration in settled coarse particles and aerosols at a semiarid rural site in India.

Deposition rates and deposition velocities of water-soluble ions (F, Cl, NO3, SO4, NH4, Ca, Mg, Na and K) were measured at a rural site (Gopalpura, Agra). Dry deposition samples were collected throughout the year from December 1995 to August 1997, while the aerosol samples were collected only during the winter season of 1996. Surrogate technique was used to collect the dry deposition samples, while aerosol samples were collected on PTFE membrane filter. Deposition velocities (Vd) of SO4 and NO3 are < or = 0.01 m s(-1) while Ca, Mg, Na, K, NH4, F and Cl exhibit greater than 0.01 m s(-1) Equivalent concentration ratios of K/Na, Ca/Na and Mg/Na conform with the corresponding ratios of local soil, indicating the dominant contribution of local sources. Deposition rates are maximum in winter, followed by summer and monsoon. No significant differences are found in dry deposition rates of all ions or in atmospheric concentrations of soil-derived elements with respect to wind direction. However, in aerosols, concentrations of F, Cl, NO3 and SO4 are higher with winds from southwesterly and westerly directions corresponding to pollution sources located in these directions. Deposition data have been used to calculate the critical load of S and N for soil with respect to Triticum vulgaris. The critical load of actual acidity was found to be 622.4 eq ha(-1) year(-1) within the range of 500-1,000 eq ha(-1) year(-1) as assessed by the RAINS-Asia model for this region. The present load of S and N (77.4 and 86.4 eq ha(-1) year(-1)) was much lower than the critical load of S and N (622.4 and 2,000 eq ha(-1) year(-1)), indicating that at present there is no harmful effect on ecosystem structure and function.

Photocatalytic degradation of 2-chlorophenol: a study of kinetics, intermediates and biodegradability.

The kinetics of photocatalytic (TiO(2)/UV) degradation of 2-chlorophenol (2-CP), characterization of intermediates and induction of biodegradability in treated chlorophenol solutions is reported. Approximately 95% of the 2-CP is removed in approximately 2h at pH 5 and 0.2g TiO(2)l(-1) when the 2-CP concentration is < or =100mgl(-1); the pseudo-first-order rate constant (k) is estimated to be 0.0183 min(-1). GC-MS analyses detected phenol, catechol, hydroxyhydroquinone (HHQ), and chlorohydroquinone (CHQ) intermediates during the short irradiation time (<1h); however two other higher carbon intermediates 2-hydroxy-benzaldehyde (HB) and [1.1'-biphenyl]-2,2'-diol (BPD) are found as major intermediates over longer irradiation times. The biochemical oxygen demand (BOD) of treated 2-CP solutions improved substantially. A tentative mechanistic pathway to explain formation of higher carbon intermediates is presented.

Identification and characterization of receptor for dengue virus-induced macrophage cytotoxin.

The present study was undertaken to identify the receptor for dengue virus type 2 (DV) induced macrophage cytotoxin (CF2) on mouse peritoneal macrophages (MPhi). The binding of 125I-labelled CF2 to MPhi was saturable (15 nM), reversible, temperature, pH- and time-dependent. The saturation concentration was similar to that causing cell death. Scatchard analysis showed the presence of intermediate type of affinity receptor and the number of receptor sites was 1.1 x 10(6) per cell with dissociation constant of 14.28 nM.

Differential identification of Mycobacterium tuberculosis from various clinical specimens from Sassoon General Hospital, Pune.

A total of 619 clinical specimens from cases of pulmonary and extrapulmonary tuberculosis were processed by smear, culture and biochemical tests. Acid fast bacilli could be demonstrated in 93 samples (15.02%) by Z.N. staining method. Culture yielded positive growth in 95 samples (15.35%) M. tuberculosis human type was the most predominient pathogen obtained from 82 cultures (13.40%) M tuberculosis bovine type was isolated from 2 cases of ascitic fluids (0.32%). Atypical Mycobacteria were isolated from 11 samples (1.73%). 5 turned out to be M. scrofulacium, 4 were M. Kansasii, 1 was M. phlei and 1 was M. smegmatis.