Biomedical subjects

P A Rajakumar

Publications and source records attributed to P A Rajakumar.

10 recordsLinked to original sources

PubMed2005

Extra-placental expression of vascular endothelial growth factor receptor-1, (Flt-1) and soluble Flt-1 (sFlt-1), by peripheral blood mononuclear cells (PBMCs) in normotensive and preeclamptic pregnant women.

The soluble VEGF receptor, sFlt-1 (otherwise referred to as sVEGFR-1), has been implicated in the pathogenesis of preeclampsia. The preeclamptic placenta has been previously demonstrated to produce high levels of the soluble VEGF receptor. Here we tested the hypothesis that peripheral blood mononuclear cells (PBMCs) may also represent an additional source for circulating sFlt-1 during normal and preeclamptic pregnancies. We first demonstrate that preeclamptic placentae show five-fold increased Flt-1 and sFlt-1 mRNA levels. We also show that the Flt-1 and sFlt-1 levels are eight-fold higher in preeclamptic placentae if we collect biopsies without rinsing them in saline to remove excess blood. Cultured villous explants from women with preeclampsia failed to show the increased amount of Flt-1 and sFlt-1 mRNA that was observed in the placental biopsies of normal pregnancy and preeclampsia. Under normoxic conditions the Flt-1 and sFlt-1 mRNA levels in the explants were 3.11+/-0.6 fold in normal pregnancy and 3.6+/-0.4 fold in women with preeclampsia (p = NS by ANOVA). However, the same villous explants showed hypoxic induction of Flt-1 mRNA (NP 3.96+/-0.4 fold, p = NS and PE 5.24+/-0.6 fold, p < 0.05 by ANOVA). We analyzed Flt-1 and sFlt-1 protein levels in the peripheral blood mononuclear cells (PBMCs) to analyze the possibility of an extra-placental sFlt-1 source. Our results indicate that PBMCs of pregnant women are capable of expressing variable amounts of Flt-1 proteins. PBMCs from pregnant women exposed to hypoxia show up-regulation of HIF-1alpha and Flt-1 proteins. PBMCs obtained from women with preeclampsia (n = 9) produced significantly higher amounts of sFlt-1 under normal tissue culture conditions (104.6+/-14.3 pg/ml vs. 46.23+/-5.03 pg/ml, p < 0.05 by ANOVA) and much higher concentrations under hypoxia (196.74+/-26.3pg/ml vs. 83.3+/-13.6pg/ml, p < 0.05 by ANOVA) than PBMCs from normal pregnant women (n = 11). Moreover, analysis of PBMCs from a different group of women with a history of preeclampsia showed persistent abnormality of Flt-1 women one year post-partum. The present study indicates that Flt-1 dysregulation in PBMCs of pregnant women resulting in over-expression of sFlt-1 could be an additional (extra-placental) source of sFlt-1 that contributes to the pathogenesis of preeclampsia.

Adult↗

PubMed2002

Species-specific variation in SIV disease progression between Chinese and Indian subspecies of rhesus macaque.

The shortage of rhesus macaques of Indian origin for acquired immune deficiency syndrome (AIDS) research has prompted a search for an alternate species. As rhesus macaques of Chinese origin are more readily obtainable, we have defined the parameters of infection in seven members of this subspecies with the primary virulent isolate, SIV/delta B670. Viremic peaks and set points as determined by real time polymerase chain reaction were, in general, lower than that observed in Indian origin rhesus macaques. As expected, these values were associated with maintenance of CD4+ lymphocytes and significantly longer survival, with six of seven Chinese origin animals living significantly longer than Indian origin rhesus macaques. Interestingly, these findings were associated with a selective amplification of one of two major phylogenetic groups found within the inoculum. This observation is in contrast to Indian origin animals where both phylogenetic groups are commonly identified. Together, these data suggest prudence in the design of experimental protocols using rhesus macaques of Chinese origin where survival and rapid loss of CD4+ lymphocytes are desired endpoints.

Animals↗

PubMed1999

Effect of development and hypoxic-ischemia upon rabbit brain glucose transporter expression.

We have cloned and sequenced a full length rabbit GLUT 1 and partial rabbit GLUT 3 cDNAs. The derived rabbit GLUT 3 peptide revealed 84% homology to the mouse, 82% to the rat, human, dog, and sheep, and 69% to the chicken GLUT 3 peptides. Using Northern blot analysis, we investigated the tissue and brain cellular distribution of GLUT 1 and GLUT 3 expression. In addition, we examined the effect of development and hypoxic-ischemia upon brain GLUT 1 and GLUT 3 mRNA levels. While GLUT 1 mRNA was observed in most tissues, GLUT 3 was expressed predominantly in the brain, placenta, stomach, and lung with minor amounts in the heart, kidney and skeletal muscle. In the brain, both GLUT 1 and GLUT 3 were noted in neuron- and glial-enriched cultures. Both GLUT 1 and GLUT 3 mRNA levels demonstrated a similar developmental progression (p<0.05) secondary to post-transcriptional mechanisms. Further, while hypoxic-ischemia did not significantly affect brain GLUT 1 mRNA and protein, it altered GLUT 3 mRNA levels in a region-specific manner, with a three-fold increase in the cerebral cortex, a two-fold increase in the hippocampus, and a 50% increase in the caudate nucleus (p<0.05). We conclude, that the rabbit GLUT 3 peptide sequence exhibits 82-84% homology to that of other species in the coding region with a 62-89% sequence identity in the 3'-untranslated region. The tissue-specific expression of rabbit GLUT 3 mimics that of the human closely. Postnatal development and hypoxic-ischemia with reperfusion injury cause an increase in brain GLUT 3 expression, as a response to synaptogenesis and substrate deprivation, respectively.

Aging↗

PubMed1998

Neuropeptide Y gene expression in immortalized rat hippocampal and pheochromocytoma-12 cell lines.

Employing clonal cell lines derived from rat embryonic hippocampal cells, we detected neuropeptide Y (NPY) mRNA in three progenitor subcloned cell lines. These cell lines upon differentiation express markers indicative of commitment to either neuronal (H19-7; NF +, GFAP -), glial (H19-5; GFAP +, NF -), or bipotential (H583-5; NF +, GFAP + ) lineages. Induction of differentiation was associated with the persistence of the NPY mRNA, however, in the differentiated H19-7 cells a 20-fold increase in NPY mRNA levels was observed (P<0.05). NPY immunoreactivity was observed only in cells with a differentiated neuronal phenotype. The cellular radioimmunoassayable NPY peptide levels increased twelve-fold without a change in extracellular NPY peptide levels by multi-factorially induced neuronal or glial cell differentiation. The differentiated H19-5 cells expressed lower levels of NPY that could not be immunocytochemically detected. The peripheral sympathetic PC-12 neuronal cells examined in the undifferentiated and nerve growth factor-driven differentiated states expressed NPY only upon differentiation. We conclude that NPY is expressed by the cultured undifferentiated and differentiated rat hippocampal clonal cell lines, while the peripheral sympathetic PC-12 neuronal cell line only expresses the NPY gene upon differentiation. These immortalized embryonic neural cell line(s) will provide a hippocampal cell line(s) to conduct future in-vitro investigations targeted at determining the cellular and molecular mechanisms governing NPY gene expression.

Animals↗

PubMed1998

Effect of uteroplacental insufficiency upon brain neuropeptide Y and corticotropin-releasing factor gene expression and concentrations.

Various hypothalamic functions such as feeding behavior, energy expenditure, body weight gain, level of anxiety, and sexual maturation are mediated by a balance between the concentrations of neuropeptide Y (NPY) and corticotropin-releasing factor (CRF). To test the hypothesis that maternal uteroplacental insufficiency alters the offspring's brain NPY and/or CRF levels, we examined the effect of maternal uterine artery ligation with intrauterine growth restriction (IUGR) (p < 0.05) upon fetal (20 d) and postnatal (4, 14, and 21 d) brain NPY and CRF synthesis, concentrations, and regional distribution. An age-related increase in NPY (0.8 kb) and CRF (1.4 kb) mRNA levels with peak amounts at the 14-d postnatal age (p < 0.05) was observed. IUGR was associated with a 75% increase in fetal brain NPY mRNA levels (p < 0.05) with no change in NPY peptide, CRF mRNA and peptide amounts. Although the increase in NPY mRNA levels persisted postnatally (p < 0.05) at d 4 and 21, CRF mRNA amounts were 2.5-fold higher only in the 4-d IUGR (p < 0.05). Paralleling the mRNA changes, an age-related increase in RIA of NPY and CRF peptide concentrations was noted (p < 0.05). IUGR caused postnatal brain NPY and CRF peptide changes similar to corresponding mRNA levels (p < 0.05), despite normal postnatal circulating glucose, insulin, corticosterone, and leptin concentrations. The age-specific intergroup differences in the NPY and CRF peptide immunoreactivity appeared predominantly in the hypothalamic region. We conclude that maternal uteroplacental insufficiency causing IUGR leads to a pretranslational imbalance in the immediate (4 d) postnatal brain NPY and CRF peptide concentrations, thereby altering the developmental pattern. This alteration in NPY and CRF peptide concentrations, despite normalization of the metabolic milieu was associated with a persistent diminution in body weight. The IUGR-associated pretranslational increase in NPY and not CRF peptide levels at d 21, may herald changes in feeding behavior during the postsuckling phase.

Animals↗

PubMed1997

Developmental changes in ob gene expression and circulating leptin peptide concentrations.

We examined the developmental changes in murine white and brown adipose tissue leptin and circulating immunoreactive total leptin concentrations. The approximately 4.4 kb leptin mRNA levels were higher at 2 and 7d postnatal ages, but declined to adult levels by the 14d stage and remained so until 160d. Paralleling the mRNA concentrations, leptin peptide levels also were higher at 2d, 7d, and 14d, declining to adult values by the 21d weaning stage. No difference in mRNA levels was observed between brown-enriched and white adipose tissue. No sexual dimorphism was observed in the leptin mRNA or peptide levels between 14 and 160d; however, at 2 and 7d, while no sex related differences were observed in the peptide levels, adipose mRNA concentrations were mildly higher in males than in the females. We conclude that leptin mRNA and peptide levels are higher during consumption of a high fat milk diet. High levels of leptin with increasing food intake and body weight gain signify hypothalamic leptin receptor resistance during the immediate postnatal period.

Adipose Tissue↗

PubMed1997

Insulin gene expression in immortalized rat hippocampal and pheochromocytoma-12 cell lines.

Employing reverse transcription-polymerase chain reaction and clonal cell lines derived by retroviral transduction of the temperature sensitive simian virus 40 large T-antigen into dispersed rat embryonic hippocampal cells, we detected the ancestral gene-insulin II mRNA in three progenitor subcloned cell lines. These cell lines upon differentiation are known to express markers indicative of commitment to either neuronal (H19-7; NF + , GFAP -), glial (H19-5; GFAP +, NF -), or bipotential (H583-5, NF +, GFAP + ) lineages. No duplicated, i.e., insulin I gene expression, was observed in any of the three cell lines. Induction of differentiation was associated with the persistence of insulin II mRNA and in the cells expressing a neuronal phenotype (H19-7; NF +, GFAP -) a relative doubling in insulin II mRNA level was present (P < 0.05). Minimal cellular insulin immunoreactivity was detected only in a subpopulation of cells with a differentiated neuronal phenotype. Radioimmunoassayable insulin peptide in the H19-7 cellular conditioned medium revealed a 5-fold increase in the differentiated state. In contrast, peripheral sympathetic PC-12 neuronal cells both in the undifferentiated and nerve growth factor-driven differentiated states, failed to express both insulin I and insulin II genes. We conclude that insulin II is expressed by cultured rat hippocampal clonal cell lines, and not by the peripheral sympathetic PC-12 neuronal cell line.

Animals↗

PubMed1997

Effect of streptozotocin-induced maternal diabetes on fetal rat brain glucose transporters.

Glucose, an essential substrate for brain oxidative metabolism, is transported across the blood-brain barrier and into neuronal and glial cells via Glut 1 and Glut 3 facilitative glucose transporter isoforms. To examine the effect of excessive circulating glucose on fetal brain glucose transporter expression, we investigated the effect of streptozotocin-induced maternal diabetes (SEVERE-D; n = 29) on the 20-d gestation fetal rat brain Glut 1 and Glut 3. We studied the effect of streptozotocin alone (STZ-ND; n = 12) in a nondiabetic state as well, along with vehicle injected controls (C; n = 24). In the presence of fetal hyperglycemia (12.63 +/- 0.82 nM-SEVERE-D versus 2.35 +/- 0.28-STZ-ND and 2.42 +/- 0.16-C; p < 0.001) and hypoinsulinemia (0.38 +/- 0.03 nM-SEVERE-D versus 0.50 +/- 0.07-STZ-ND and 0.55 +/- 0.06-C; p < 0.02), no detectable change in fetal brain Glut 1 and Glut 3 pretranslational expression (transcription/elongation rates and corresponding steady state mRNA levels) was noted when simultaneously compared with the STZ-ND and C groups. In contrast, a trend toward a decline in Glut 1 (approximately 25 to 30%, p = 0.05) and a substantive decrease in Glut 3 (approximately 35 to 50%, p = 0.0006) protein concentrations was present in both the STZ-ND and SEVERE-D groups when compared with the C group. These observations support a chemical effect of streptozotocin independent of maternal diabetes upon the translation or posttranslational processing of fetal brain glucose transporters. Maternal diabetes with fetal hyperglycemia, however, failed to substantively alter fetal brain glucose transporters independent of the streptozotocin effects upon neuroectodermally derived tissues. We conclude that maternal diabetes with associated overt fetal hyperglycemia does not significantly change fetal brain glucose transporter levels.

Analysis of Variance↗

PubMed1994

Insulin gene expression and insulin synthesis in mammalian neuronal cells.

To demonstrate the presence of de novo synthesis in central mammalian neurons, we cloned and sequenced a rabbit insulin cDNA from pancreas and used it to define sequences encoding insulin mRNA from postnatal rabbit brain. We observed transcription/elongation of nascent insulin transcripts, characterized the size of these transcripts, and localized them to specific neurons in certain catecholaminergic-rich areas of the central nervous system. RNase protection assays using a rabbit probe spanning a region from 14 bases 5' to the translation start site through all but 18 bases of the sequence encoding the A-chain of insulin showed two bands in rabbit brain RNA and only one band in pancreas. The larger band in brain was the same size as that in pancreatic RNA; the other was approximately 10 bases shorter. Because the sequence of a reverse transcription-polymerase chain reaction product from brain RNA was identical to pancreatic RNA sequence in the region corresponding to the 3' region of the probe, the smaller band in brain is most consistent with a sequence mismatch in some brain mRNA in the region corresponding to the 5'-end of the probe. In situ hybridization localized insulin mRNA to anatomical regions involved with olfaction and higher association of the limbic system. High performance liquid chromatography, radioimmunoassay, and [35S]cysteine metabolic labeling of cultured neuronal and glial cells indicated extracellular secretion of immunoprecipitable insulin by neurons only. Presence of insulin transcripts within specific neurons with extracellular secretion of the peptide suggests a specialized biological role.

Aging↗

PubMed1993

Insulin II gene expression in rat central nervous system.

Controversy persists concerning the origin of insulin in the central nervous system. While there has been convincing evidence in vitro to demonstrate the presence of neuronal insulin mRNA, conventional assays have failed to detect the same in whole brain preparations. Here we employed RNAse-protection and sensitive reverse transcription-polymerase chain reaction (RT-PCR) assays in attempts to detect insulin I and II mRNAs in rat brains obtained from different developmental stages. The RNAse-protection assay did not detect insulin I or insulin II transcripts in fetal (13 to 21 day gestation) or adult brains. RT-PCR, while detecting low amounts of insulin I transcripts in other extrapancreatic tissues such as the rat yolk sac and fetal liver previously shown to express insulin II, failed to detect insulin I in brain at any age examined. Insulin II mRNA was detected by RT-PCR in fetal, neonatal and adult rat brains, just as in yolk sac, fetal and adult livers. We conclude that while the duplicated insulin I gene is not expressed, the ancestral insulin II gene is expressed in fetal, neonatal and adult rat brains. Our observations support the concept of de novo brain insulin II synthesis beyond the pre-pancreatic stage of embryonic development.

Aging↗