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Biomedical subjects

K Souza

Publications and source records attributed to K Souza.

13 recordsLinked to original sources
PubMed2001

Study of developmental changes on hexoses metabolism in rat cerebral cortex.

We have studied the developmental changes of glucose, mannose, fructose and galactose metabolism in rat cerebral cortex. As the animals aged, glucose, mannose and fructose oxidation to CO2 increased, whereas galactose oxidation decreased. Lipid synthesis from glucose and fructose also increased with age, that from mannose decreased and galactose did not change. Cytochalasin B, a potent non-competitive inhibitor of sodium-independent glucose transport, significantly impaired glucose, mannose and galactose metabolism, but had no effect on fructose metabolism. Both galactose or fructose did not change, whereas mannose declined the glucose metabolism. Glucose decreased fructose, galactose and mannose metabolism. Our results show that besides glucose, the metabolism of mannose, galactose and fructose present developmental changes from fetal to adult age, and reinforce the literature data indicating that mannose and galactose are transported by glucose carriers, while fructose is not.

Aging↗
PubMed2001

Cytokine-induced p38 activation feedback regulates the prolonged activation of AKT cell survival pathway initiated by reactive oxygen species in response to UV irradiation in human keratinocytes.

A previous study has shown that UV activates the PI3K/AKT cell survival pathway while inducing cell death in human skin in vivo and cultured human keratinocytes in vitro, and yet the upstream pathway leading to the activation of AKT has not been thoroughly investigated. In this study we found that UV-induced phosphorylation of p38 and AKT in a time-dependent manner. The phosphorylation of p38 started at 5 min post UV irradiation, peaked at about 30 min, and remained elevated up to 2 h. The phosphorylation of AKT started at 15 min post UV treatment, peaked at about 1 h, and remained elevated up to 2 h. We also found that H2O2 induced phosphorylation of p38 and AKT in a time- dependent manner. Pretreatment with NAC abolished UV-induced AKT phosphorylation, suggesting the involvement of reactive oxygen species in AKT activation. Interestingly, SB203085, a known p38 inhibitor, had partially inhibited UV-induced AKT phosphorylation. Further studies showed that cytokines such as TNF-alpha and IL-1beta induced AKT phosphorylation in a time-dependent manner. Pretreatment with SB203085 inhibited IL-1beta-induced p38 and AKT phosphorylation. Collectively, our data suggest that UV activation of PI 3-kinase/AKT pathway is initiated by ROS and prolonged by feedback activation of p38 induced by released cytokines in response to UV irradiation in cultured human keratinocytes.

Apoptosis↗
PubMed2001

Arsenite activation of P13K/AKT cell survival pathway is mediated by p38 in cultured human keratinocytes.

BACKGROUND: Arsenic has been considered as a carcinogen. Recently the issue of arsenic in drinking water raised an unprecedented social concern on human health, and yet the molecular mechanisms through which arsenic induces cancer remain unknown. Activation of cell survival pathway leading to the activation of eNOS has been associated with various types of cancer. The objective of this study was to investigate the pathway leading to the activation of eNOS in response to arsenite using human keratinocytes. MATERIALS AND METHODS: Cultured keratinocytes (HaCat cells) were exposed to arsenite with or without pretreatment of various inhibitors. Western blot analysis was performed to determine the activation of p38, AKT, eNOS. EGFR tyrosine phosphorylation was detected by immunoprecipitation and Western blot analysis. pNPP assay was used to measure phosphatase activity in cell lysate. FACS analysis was performed for the determination of generation of reactive oxygen species. RESULTS: Arsenite induced the activation of AKT at both Ser473 and Thr308, and its downstream effector eNOS in cultured human keratinocytes. Arsenite also induced phosphorylation of p38. PI-3-kinase inhibitors, Wortmannin and LY294002 inhibited arsenite-induced phosphorylation of AKT and eNOS but had no effect on phosphorylation of p38. Interestingly, however, SB203580, a known p38 inhibitor, completely inhibited arsenite-induced phosphorylation of AKT and eNOS. Arsenite induced generation of reactive oxygen species and inactivated phosphatase activity, but did not activate EGF receptor tyrosine phosphorylation. CONCLUSIONS: Collectively, our data indicate that arsenite induces activation of AKT and eNOS, via PI-3-kinase and p38 pathway, likely bypassing the activation of EGF receptor in cultured human keratinocytes.

Arsenites↗
PubMed1999

Meeting the challenge: a survey of capabilities planned to meet future space biology research needs.

NASA/Ames Research Center Life Sciences has supported a large number of experiments and observations directed at understanding how biological systems perform or change in the microgravity space environment. These campaigns have been accomplished on a wide range of space-based platforms beginning with the Bion/Cosmos unmanned satellites and including the Space Shuttle middeck, Spacelab, SpaceHab, the Russian Space Station Mir and in the near future, the International Space Station (ISS). To further build upon this past experience, and to continue to make contributions towards the goals of the Human Exploration and Development of Space (HEDS) enterprise, a number of experiment systems and infrastructure are in development in an attempt to provide a comprehensive set of opportunities and capabilities to enable research into biological systems in space. Life support systems, or habitats are geared towards the maintenance of a wide range of biological specimens onboard ISS. Augmented with a set of ancillary equipment and sufficient expendable resources and crew time, researchers will have a robust set of tools to continue biological and physiological experiments in space.

Animals↗
PubMed1998

Swimming kinematics and respiratory behaviour of xenopus laevis larvae raised in altered gravity

We examined the respiratory behaviours and swimming kinematics of Xenopus laevis tadpoles hatched in microgravity (Space Shuttle), simulated microgravity (clinostat) and hypergravity (3 g centrifuge). All observations were made in the normal 1 g environment. Previous research has shown that X. laevis raised in microgravity exhibit abnormalities in their lungs and vestibular system upon return to 1 g. The tadpoles raised in true microgravity exhibited a significantly lower tailbeat frequency than onboard 1 g centrifuge controls on the day of landing (day0), but this behaviour normalized within 9 days. The two groups did not differ significantly in buccal pumping rates. Altered buoyancy in the space-flight microgravity tadpoles was indicated by an increased swimming angle on the day after landing (day1). Tadpoles raised in simulated microgravity differed to a greater extent in swimming behaviours from their 1 g controls. The tadpoles raised in hypergravity showed no substantive effects on the development of swimming or respiratory behaviours, except swimming angle. Together, these results show that microgravity has a transient effect on the development of locomotion in X. laevis tadpoles, most notably on swimming angle, indicative of stunted lung development. On the basis of the behaviours we studied, there is no indication of neuromuscular retardation in amphibians associated with embryogenesis in microgravity.

Journal Article↗
PubMed1998

Swimming kinematics and respiratory behaviour of Xenopus laevis larvae raised in altered gravity.

We examined the respiratory behaviours and swimming kinematics of Xenopus laevis tadpoles hatched in microgravity (Space Shuttle), simulated microgravity (clinostat) and hypergravity (3 g centrifuge). All observations were made in the normal 1 g environment. Previous research has shown that X. laevis raised in microgravity exhibit abnormalities in their lungs and vestibular system upon return to 1 g. The tadpoles raised in true microgravity exhibited a significantly lower tailbeat frequency than onboard 1 g centrifuge controls on the day of landing (day0), but this behaviour normalized within 9 days. The two groups did not differ significantly in buccal pumping rates. Altered buoyancy in the space-flight microgravity tadpoles was indicated by an increased swimming angle on the day after landing (day1). Tadpoles raised in simulated microgravity differed to a greater extent in swimming behaviours from their 1 g controls. The tadpoles raised in hypergravity showed no substantive effects on the development of swimming or respiratory behaviours, except swimming angle. Together, these results show that microgravity has a transient effect on the development of locomotion in X. laevis tadpoles, most notably on swimming angle, indicative of stunted lung development. On the basis of the behaviours we studied, there is no indication of neuromuscular retardation in amphibians associated with embryogenesis in microgravity.

Animals↗
PubMed1997

Formulation development and primary degradation pathways for recombinant human nerve growth factor.

The chemical and physical stabilities of recombinant human nerve growth factor (NGF) in aqueous solution were investigated between 5 and 37 degrees C and at pH 4.2-5.8. NGF chemical stability decreased with a decrease in pH due to Asp60-Pro61 cleavage, with the stability being greater in acetate buffer than in succinate buffer at each pH investigated. Aggregation was a significant degradation pathway at 37 degrees C, with the aggregation rate being greatest in succinate buffer at pH 5.8. Quantitation of NGF degradation by cation-exchange chromatography was complicated by the rearrangement of the NGF monomer variants into various mixed dimers over time. Treatment with dilute acid brought the dimer distribution rapidly to equilibrium, allowing NGF degradation to be accurately quantitated. An acetate-buffered formulation at pH 5.5 was investigated in more detail. To assist in degradation product identification, NGF degradation was accelerated with base, hydrogen peroxide, and temperature. These degradation products were shown to coelute on RP-HPLC with the variants found when the protein was stored at -70, 5, and 25 degrees C. By electrospray mass spectrometry, peptide maps, and LC/MS, these degradation products were shown to be monooxidized (Met37) and dioxidized (Met37 and Met92) NGF, with Met37 being more labile, deamidated NGF (Asn45), and NGF with Asp93 isomerized to beta-Asp93. NGF can be stored in pH 5.5 acetate buffer at 5 degrees C for 1.5 years with less than 10% conversion to these degradation products, with Asp93 isomerization being the primary degradation pathway.

Amino Acid Sequence↗
PubMed1996

1H-NMR detectable fatty acyl chain unsaturation in excised leiomyosarcoma correlate with grade and mitotic activity.

We report on the use of 1H-NMR two-dimensional total correlated spectroscopy (2D TOCSY) at 600 MHz for an ex vivo analysis of fatty acyl chain lipid in normal smooth muscle and a series of primary retroperitoneal leiomyosarcomas. These TOCSY spectra were used to identify and quantitate the methylene protons situated between unsaturated site protons (D) to those bordered by only one unsaturated site proton (C). The D/C cross-peak volume ratios determined for oleic (18:1), linoleic (18:2), linolenic (18:3), and arachidonic (20:4) acids were 0.0, 1.3, 2.7, and 4.0, respectively, suggesting that this ratio can be a measure of the degree of unsaturation for fatty acyl chains of lipids. The D/C cross-peak volume ratio was found to be proportional to the mean mitotic activity (r = 0.94) in nine smooth muscle tissues. These results suggest, that for leiomyosarcoma, the degree of fatty acyl unsaturation may be an important determinant of the metastatic potential of these tumors. Furthermore, application of TOCSY for the ex vivo study of smooth muscle tumors would potentially serve as a pathologist-independent and quantitative method for assessment of leiomyosarcoma grade and mitotic activity thereby rendering a more accurate staging of patients.

Amino Acids↗
PubMed1996

Regulative development of Xenopus laevis in microgravity.

To test whether gravity is required for normal amphibian development, Xenopus laevis females were induced to ovulate aboard the orbiting Space Shuttle. Eggs were fertilized in vitro, and although early embryonic stages showed some abnormalities, the embryos were able to regulate and produce nearly normal larvae. These results demonstrate for the first time that a vertebrate can ovulate in the virtual absence of gravity, and that the eggs can develop to a free-living stage.

Animals↗
PubMed1995

Pyruvate utilization, phosphocholine and adenosine triphosphate (ATP) are markers of human breast tumor progression: a 31P- and 13C-nuclear magnetic resonance (NMR) spectroscopy study.

We have used 31P- and 13C-nuclear magnetic resonance spectroscopy to measure key metabolite levels and fluxes through enzymes regulating phospholipid and mitochondrial metabolism in normal human mammary epithelial cells. We have compared these values to those found in a progression series of breast cancer cell lines of varying metastatic potential established from a single patient. We find a 16-19-fold increase in phosphocholine content in two primary breast cancer cell lines (21PT and 21NT) and a 27-fold increase in phosphocholine content in the metastatic breast cancer cell line (21MT-2) compared with the normal breast epithelial cell strain 76N. Thus, phosphocholine may serve as a metabolic marker for the human breast cell progression state. A 30% decrease in ATP levels, a 83% decrease in phosphocreatine levels, along with a 2-fold increase in NAD(+) + NADH levels in 21PT, 21NT, and 21MT-2 cells compared to the normal breast cells further suggests impaired mitochondrial metabolism in the breast carcinoma cell lines. Consistent with this suggestion is our finding that the primary breast cancer cell lines (21PT and 21NT) and the metastatic breast cell line (21MT-2) showed a 50 and 89% relative reduction, respectively, in the flux of pyruvate utilized for mitochondrial energy generation compared to pyruvate utilized to replenish tricarboxylic acid cycle intermediates. These results demonstrate that diminished mitochondrial energy generation may be quantitatively related to the progression state of human breast cells.

Adenosine Triphosphate↗
PubMed1994

Ex vivo two-dimensional proton nuclear magnetic resonance spectroscopy of smooth muscle tumors: advantages of total correlated spectroscopy over homonuclear J-correlated spectroscopy.

Two-dimensional total correlated proton nuclear magnetic resonance spectroscopy (TOCSY) at 600 MHz was used for an ex vivo biochemical analysis of a benign human smooth muscle tumor (leiomyoma) and high grade malignant smooth muscle tumor (leiomyosarcoma). While there are a number of applications of proton nuclear magnetic resonance magnitude-mode two-dimensional correlated spectroscopy (COSY) to the ex vivo study of tissues and cells in the literature, to our knowledge this is the first application of TOCSY for the ex vivo study of biological tissue. Comparison of TOCSY and purged COSY data demonstrate the potential sensitivity advantages of the TOCSY method for the study of heterogeneous biological tissues. These TOCSY spectra were used to identify and quantitate a wide range of metabolites such as amino acids, peptides, triglycerides, phospholipid precursors and degradation products, bound fucose, and other saccharides. The leiomyosarcoma was found to have 5-fold higher levels of triglycerides and a 7-fold increase in the glycerophosphocholine:choline ratio compared to the leiomyoma. These metabolite changes may enhance membrane fluidity in the leiomyosarcoma compared to leiomyoma and thus may be of fundamental importance to cell motility, recognition, sarcoma tumorigenesis, and metastatic potential.

Amino Acids↗
PubMedDate not supplied

Correlation of lipid content and composition with liposarcoma histology and grade.

BACKGROUND: The determination of sarcoma grade, histologic type, and differentiation is often pathologist dependent and requires considerable expertise. METHODS: Lipid content and composition was analyzed in ex vivo fat, lipoma, and liposarcoma tissue samples using proton-decoupled 13C nuclear magnetic resonance (13C-NMR) spectroscopy and correlated with the histologic type and grade of liposarcoma. RESULTS: The well-differentiated liposarcomas were found to have threefold increases in fatty acyl chain content compared with benign lipomas. The fatty acyl chain content of the dedifferentiated and pleomorphic liposarcomas was 1% of that found in lipoma and < 0.2% of that found in well-differentiated liposarcoma. The 2.1- to 2.8-fold increase in the degree of polyunsaturation in the dedifferentiated and pleomorphic liposarcomas compared with well-differentiated liposarcoma could largely be accounted for by the 2.3-fold increase in the percentage of fatty acyl chains of lipid containing linoleic acid. The dedifferentiated and pleomorphic liposarcomas contained both free fatty acids and phospholipids that were not NMR detectable in normal fat, lipoma, and well-differentiated liposarcoma. CONCLUSION: Ex vivo 13C-NMR spectroscopy may be used to distinguish lipoma from well-differentiated, dedifferentiated, and pleomorphic liposarcoma based on changes in lipid and phospholipid metabolite profiles and may serve as adjunct to conventional light microscopy for the determination of liposarcoma histologic type and thus grade.

Biomarkers, Tumor↗