Biomedical subjects

Subburaman Mohan

Publications and source records attributed to Subburaman Mohan.

63 records · Page 4Linked to original sources

PubMed2002

Differential regulation of pregnancy associated plasma protein (PAPP)-A during pregnancy in human and mouse.

UNLABELLED: Serum IGFBP-4 proteolytic activity increases dramatically during human pregnancy and is mainly attributed to the pregnancy-associated plasma protein-A (PAPP-A). To understand the regulation and actions of PAPP-A in vivo, we evaluated the utility of a mouse model system. Serum from day-9 and day-17 pregnant mice and age-matched controls was tested for IGFBP-4 proteolytic activity using recombinant mouse IGFBP-4 as the substrate. Surprisingly, IGFBP-4 proteolytic activity in mouse pregnancy serum (mPS) was not significantly different from that of non-pregnancy serum (mNPS). Addition of IGF-II to mPS or mNPS at a dose sufficient to increase IGFBP-4 proteolysis by human PS failed to enhance IGFBP-4 proteolysis. PAPP-A neutralization antibody did not inhibit IGFBP-4 proteolysis by mPS or mNPS, but completely blocked IGFBP-4 proteolytic activity in human PS (hPS, mouse osteoblast conditioned medium, and mouse amniotic fluid). To determine whether the lack of PAPP-A activity in mPS was due to low expression of PAPP-A in the placenta, we cloned a mouse genomic DNA, which contained 1 kb of the entire exon 2 coding sequence and 2.5 kb of the flanking intron sequences. The exon 2-coded mouse and human PAPP-A shared 86% amino acid sequence identity. RT-PCR analysis revealed that the PAPP-A mRNA level in mouse placenta was lower compared to that in human placenta by at least two orders of magnitude. PAPP-A expression was also lower in mouse placenta compared to those in mouse kidney, osteoblasts, and bone marrow stromal cells. CONCLUSIONS: (1) Serum IGFBP-4 proteolytic activity is differentially regulated by pregnancy in human and mouse. (2) The lack of an increase in serum IGFBP-4 proteolytic activity during mouse pregnancy is due to the low level of PAPP-A expression in the placenta.

Amino Acid Sequence↗

PubMed2002

DNA microarrays: their use and misuse.

DNA microarray represents one of the major advances in functional genomics. Its ability to study expression of several thousands of genes or even all genes in the entire genome in a single experiment has changed the way in which we address basic biomedical questions. Numerous publications have shown its utility in drug discovery, disease diagnosis, novel gene identification, and understanding complex biological systems. However, there are substantive technical issues associated with the use of this technology that limit the interpretation of microarray data. In this review, we first give an overview of DNA microarray technology and then focus on uncertainty areas of microarray technology that include making microarrays, isolation of RNA and labeling, hybridization and scanning, and data analysis. The center theme of this review is to improve microarray reproducibility by addressing common technical problems. Finally, we briefly summarize microarray's applications in biomedical research.

Algorithms↗

PubMed2002

Effects of liver-derived insulin-like growth factor I on bone metabolism in mice.

Insulin-like growth factor (IGF) I is an important regulator of both skeletal growth and adult bone metabolism. To better understand the relative importance of systemic IGF-I versus locally expressed IGF-I we have developed a transgenic mouse model with inducible specific IGF-I gene inactivation in the liver (LI-IGF-I-/-). These mice are growing normally up to 12 weeks of age but have a disturbed carbohydrate and lipid metabolism. In this study, the long-term effects of liver-specific IGF-I inactivation on skeletal growth and adult bone metabolism were investigated. The adult (week 8-55) axial skeletal growth was decreased by 24% in the LI-IGF-I-/- mice whereas no major reduction of the adult appendicular skeletal growth was seen. The cortical cross-sectional bone area, as measured in the middiaphyseal region of the long bones, was decreased in old LI-IGF-I-/- mice. This reduction in the amount of cortical bone was caused mainly by decreased periosteal circumference and was associated with a weaker bone determined by a decrease in ultimate load. In contrast, the amount of trabecular bone was not decreased in the LI-IGF-I-/- mice. DNA microarray analysis of 30-week-old LI-IGF-I-/- and control mice indicated that only four genes were regulated in bone whereas approximately 40 genes were regulated in the liver, supporting the hypothesis that liver-derived IGF-I is of minor importance for adult bone metabolism. In summary, liver-derived IGF-I exerts a small but significant effect on cortical periosteal bone growth and on adult axial skeletal growth while it is not required for the maintenance of the trabecular bone in adult mice.

Absorptiometry, Photon↗

PubMed2002

Two different pathways for the maintenance of trabecular bone in adult male mice.

Androgens may regulate the male skeleton either directly via activation of the androgen receptor (AR) or indirectly via aromatization of androgens into estrogen and, thereafter, via activation of estrogen receptors (ERs). There are two known estrogen receptors, ER-alpha and ER-beta. The aim of this study was to investigate the relative roles of ER-alpha, ER-beta, and AR in the maintenance of trabecular bone in male mice. Seven-month-old male mice, lacking ER-alpha (ERKO), ER-beta (BERKO), or both receptors (DERKO), were orchidectomized (orx) and treated for 3 weeks with 0.7 microg/mouse per day of 17beta-estradiol or vehicle. No reduction in trabecular bone mineral density (BMD) was seen in ERKO, BERKO, or DERKO mice before orx, showing that neither ER-a nor ER-beta is required for the maintenance of a normal trabecular BMD in male mice. After orx, there was a pronounced decrease in trabecular BMD, similar for all groups, resulting in equal levels of trabecular BMD in all genotypes. This reduction was reversed completely in wild-type (WT) and BERKO mice treated with estrogen, and no significant effect of estrogen was found in ERKO or DERKO mice. In summary, the trabecular bone is preserved both by a testicular factor, presumably testosterone acting via AR and by an estrogen-induced activation of ER-alpha. These results indicate that AR and ER-alpha are redundant in the maintenance of the trabecular bone in male mice. In contrast, ER-beta is of no importance for the regulation of trabecular bone in male mice.

Absorptiometry, Photon↗

PubMed2002

Human osteoblasts' proliferative responses to strain and 17beta-estradiol are mediated by the estrogen receptor and the receptor for insulin-like growth factor I.

The mechanism by which mechanical strain and estrogen stimulate bone cell proliferation was investigated using monolayer cultures of human osteoblastic TE85 cells and female human primary (first-passage) osteoblasts (fHOBs). Both cell types showed small but statistically significant dose-dependent increases in [3H]thymidine incorporation in response to 17beta-estradiol and to a single 10-minute period of uniaxial cyclic strain (1 Hz). In both cell types, the peak response to 17beta-estradiol occurred at 10(-8) - 10(-7) M and the peak response to strain occurred at 3500 microstrain ((mu)epsilon). Both strain-related and 17beta-estradiol-related increases in [3H]thymidine incorporation were abolished by the estrogen receptor (ER) modulator ICI 182,780 (10-8 M). Tamoxifen (10(-9) - 10(-8) M) increased [3H]thymidine incorporation in both cell types but had no effect on their response to strain. In TE85 cells, tamoxifen reduced the increase in [3H]thymidine incorporation associated with 17beta-estradiol to that of tamoxifen alone but had no such effect in fHOBs. In TE85 cells, strain increased medium concentrations of insulin-like growth factor (IGF) II but not IGF-I, whereas 17beta-estradiol increased medium concentrations of IGF-I but not IGF-II. Neutralizing monoclonal antibody (MNAb) to IGF-I (3 microg/ml) blocked the effects of 17beta-estradiol and exogenous truncated IGF-I (tIGF-I; 50 ng/ml) but not those of strain or tIGF-II (50 ng/ml). Neutralizing antibody to IGF-II (3 microg/ml) blocked the effects of strain and tIGF-II but not those of 17beta-estradiol or tIGF-I. MAb aIR-3 (100 ng/ml) to the IGF-I receptor blocked the effects on [3H]thymidine incorporation of strain, tIGF-II, 17beta-estradiol, and tIGF-I. HOBs and TE85 cells, act similarly to rat primary osteoblasts and ROS 17/2.8 cells in their dose-related proliferative responses to strain and 17beta-estradiol, both of which can be blocked by the ER modulator ICI 182,780. In TE85 cells (as in rat primaries and ROS 17/2.8 cells), the response to 17beta-estradiol is mediated by IGF-I, and the response to strain is mediated by IGF-II. Human cells differ from rat cells in that tamoxifen does not block their response to strain and reduces the response to 17beta-estradiol in TE85s but not primaries. In both human cell types (unlike rat cells) the effects of strain and IGF-II as well as estradiol and IGF-I can be blocked at the IGF-I receptor.

Antibodies, Monoclonal↗

PubMed2002

Insulin-like growth factor I effect on the number of osteoblast progenitors is impaired in ovariectomized mice.

Because insulin-like growth factor (IGF) I is an important regulator of bone formation, we proposed the hypothesis that IGF-I could contribute in regulating the number of osteoblast progenitors (colony-forming unit fibroblast with ALP activity [CFU-F/ALP+]). To test ex vivo and in vivo effects of IGF-I on the number of CFU-F/ALP+, bone marrow cells (BMCs) derived from normal mice, growth hormone (GH)-deficient lit/lit mice, or ovariectomized (OVX) mice were cultured and the CFU-F/ALP+ number was counted. Ex vivo treatment of IGF-I increased the CFU-F/ALP+ number in a dose-dependent manner compared with vehicle-treated control cultures. The CFU-F/ALP+ number was decreased by 20% (p < 0.01; n = 7-9) in GH-deficient lit/lit mice compared with age-matched control mice. Four weeks after OVX or sham operation, IGF-I (2 microg/g body wt) or vehicle was administered twice on day 1, and 5 days later, BMCs were removed from the femur and cultured for 10 days (n = 9-10 per group). IGF-I administration increased the CFU-F/ALP+ number by 63% (p < 0.01) and 19% (NS), respectively, in sham-operated (sham) and OVX mice compared with the vehicle-treated control group. The serum IGF-I level was similar in OVX mice compared with sham mice; this finding is different from that found in rats in which OVX increases the serum IGF-I level. This study showed that IGF-I is an important regulator of osteoblast-progenitor number in the BMCs of mice both ex vivo and in vivo and that the IGF-I response to increase the number of osteoblast progenitors was impaired in OVX mice.

Alkaline Phosphatase↗

PubMed2002

IGF-I, IGF-II, 'free' IGF-I and IGF-binding proteins-2 to -6 during high-dose oestrogen treatment in constitutionally tall girls.

OBJECTIVE: To investigate the effect of high-dose oestrogen treatment on IGF-I, IGF-II, free-dissociable IGF-I and the IGF-binding proteins (IGFBP)-2 to -6 in girls with constitutional tall stature. METHODS: In patient cohort 1, blood samples were drawn before and after 3 months of daily oral treatment with 0.1 mg ethinyloestradiol. In cohort 2, samples were collected at the end of the treatment period and 3 to 6 months afterwards. IGFs and IGFBPs were analysed by specific immunoassays and by Western ligand blot. RESULTS: Total IGF-I decreased significantly on oestrogen treatment and increased again after oestrogen withdrawal. Ligand blot analysis showed a clear reduction in a 34 kDa band, corresponding to IGFBP-2, and a strong induction of a 24 kDa band, corresponding to the non-glycosylated form of IGFBP-4. These changes were confirmed by specific immunological methods. The serum levels of IGFBP-3, IGFBP-5 and IGFBP-6 remained unchanged during the first 3 months of treatment. In cohort 2, IGFBP-3 and IGFBP-6 increased after oestrogen withdrawal. Free-dissociable IGF-I fell to 35+/-4% during oestrogen therapy and rose again when the treatment was stopped. CONCLUSIONS: Oestrogens modulate the serum concentrations of several components of the IGF system. The fall in total IGF-I is not explained by a decrease in IGFBPs but probably results from a decreased synthesis.

Adolescent↗

PubMed2002

Effect of low-dose of recombinant human growth hormone on bone metabolism in elderly women with osteoporosis.

BACKGROUND: There has been increasing evidence that the growth hormone (GH)-IGF-I axis plays an important part in the maintenance of bone mass. However, controversy still exists as to the effect of GH treatment on bone mineral density (BMD) in elderly patients with osteoporosis. OBJECTIVE: To investigate the effect of low-dose GH treatment on markers of body composition and bone turnover, serum concentrations of IGF-I and IGF-binding proteins (IGFBPs), and BMD at the radius and lumbar spine in eight elderly Japanese women with osteoporosis. METHODS: Participants were treated with GH as a single daily subcutaneous injection (0.125 IU/kg per week; 0.00595 mg/kg per day) for 48 weeks. RESULTS: Markers of bone formation and bone resorption were both increased up to 24 weeks of GH treatment. The bone formation markers remained increased during GH treatment, whereas the bone resorption markers returned to baseline values after 24 weeks of GH treatment. GH treatment caused a rapid (within 2 weeks) and sustained increase in serum IGF-I concentration. As for IGFBPs, serum concentrations of IGFBPs-2, -3 and -4 did not change significantly during GH treatment. In contrast, GH treatment caused a gradual increase in serum IGFBP-5 concentration, with a significant increase seen 48 weeks after the start of GH treatment. Radial BMD seemed to be increased during the late period of GH treatment, although the change was not significant. Lumbar BMD did not change during GH treatment. GH treatment caused a significant increase in hand grip strength. None of the GH-treated participants had new fractures and side effects such as edema and joint pain. Radial BMD was significantly increased after discontinuation of GH treatment for another 48 weeks and a similar tendency was observed at the lumbar spine (7.1+/-2.3% above pretreatment values for the radius and 3.6+/-2.0% for the lumbar spine). CONCLUSIONS: Low-dose GH treatment attenuated the decrease in muscle strength and bone mass in elderly women without side effects, although changes in nutrition and exercise might affect BMD. The present findings provide useful information regarding the use of low-dose GH treatment in elderly women with osteoporosis.

Aged↗

PubMed2001

Intact IGF-binding protein-4 and -5 and their respective fragments isolated from chronic renal failure serum differentially modulate IGF-I actions in cultured growth plate chondrocytes.

Impairment of longitudinal growth among children with chronic renal failure (CRF) may be partly attributable to the inhibition of insulin-like growth factor (IGF) activity by an excess amount of high-affinity IGF-binding proteins (IGFBP). Elevated levels of immunoreactive IGFBP-4 in CRF serum are inversely correlated with the standardized heights of these children, whereas levels of IGFBP-5, which circulates mainly as proteolyzed fragments, are positively correlated with growth parameters. To delineate the respective effects of these IGFBP on growth cartilage, the biologic effects of intact and fragmented forms of IGFBP-4 and IGFBP-5 on rat growth plate chondrocytes in primary cultures were characterized. Intact IGFBP-4 and IGFBP-5 and the amino-terminal fragment IGFBP-5(1-169) were recombinant proteins; the carboxy-terminal fragments IGFBP-5(144-252) and IGFBP-4(136-237) and the amino-terminal fragment IGFBP-4(1-122) were purified to homogeneity from CRF hemofiltrates. Intact IGFBP-4 and, to a lesser extent, IGFBP-4(1-122) inhibited IGF-I-induced cell proliferation. In contrast, intact IGFBP-5 was stimulatory in the absence or presence of exogenous IGF-I, whereas the amino-terminal fragment IGFBP-5(1-169) was inhibitory. Studies on the mechanism by which IGFBP-4 and IGFBP-5 exert opposite effects on chondrocyte proliferation demonstrated that intact IGFBP-4 prevented the binding of (125)I-IGF-I to chondrocytes, whereas intact IGFBP-5 enhanced ligand binding and was able to bind specifically to the cell membrane. These data suggest that intact IGFBP-4 and, to a lesser extent, IGFBP-4(1-122) act exclusively as growth-inhibitory binding proteins in the growth cartilage. IGFBP-5, however, can either stimulate (if it remains intact) or inhibit (if amino-terminal forms predominate) IGF-I-stimulated chondrocyte proliferation.

Animals↗