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Biomedical subjects

E M Spencer

Publications and source records attributed to E M Spencer.

At least 19 recordsLinked to original sources
PubMed2001

Insulin-like growth factor-I promotes proliferation and migration of cavernous smooth muscle cells.

To better understand the physiology of cavernous smooth muscle cells (CSMC), particularly their regulation by IGF-I, we isolated CSMC from rats of various ages and grew them as cell cultures. CSMC from very young (1 week of age) and very old (28 months of age) rats secreted the least amounts of IGF-I, and those from 16-week-old rats the most. IGF-I stimulated growth of CSMC at an optimal concentration of 12.5 ng/ml. At this concentration, CSMC from 11-week-old rats showed the highest growth rate and CSMC from 28-month-old rats showed the lowest. The optimal IGF-I concentration for migration of CSMC was 10 ng/ml. At this concentration, CSMC from 4-week-old rats showed the highest migratory rate and CSMC from 28-month-old rats showed the lowest. IGF-I also stimulated VEGF secretion from CSMC at an optimal concentration of 10 ng/ml. At this concentration, CSMC from 16-week-old rats secreted VEGF the most and CSMC from 28-month-old rats secreted the least. The expression levels of IGF-IR paralleled the IGF-I-regulated growth rates of these cells. Expression of IGF-IR was identified in the cavernous smooth muscle and the urethra epithelium of the penis.

Aging↗
PubMed2001

A novel approach to reducing postoperative intraperitoneal adhesions through the inhibition of insulinlike growth factor I activity.

HYPOTHESIS: Interference with insulinlike growth factor I (IGF-I) activity, both systemically and intraperitoneally, reduces postoperative intraperitoneal adhesion severity. SETTING: Experimental animal model. DESIGN, INTERVENTIONS, AND MAIN OUTCOME MEASURES: Adult female rats were subjected to hypophysectomy, sham hypophysectomy (control), IGF binding protein 4 (IGFBP-4) treatment, or albumin treatment (control). All rats underwent laparotomy and uterine horn abrasion with adjacent parietal peritoneal trauma for the purpose of creating postoperative intraperitoneal adhesions. Glucocorticoids and thyroid hormone were replaced in the hypophysectomy group. On postoperative day 10, rats were weighed, subjected to phlebotomy, and killed. Postmortem laparotomies were performed and blinded observers scored uterine-peritoneal adhesions on a 0 to 3 scoring system. Plasma IGFBP-4 levels and organ weights were measured in the IGFBP-4 and albumin treatment groups. Blood samples in all rats were analyzed for IGF-I levels. RESULTS: Rats with low IGF-I levels (hypophysectomy) and inhibited IGF-I activity (IGFBP-4 treatment) formed significantly less severe adhesions than their control counterparts. As expected, rats in the hypophysectomy group displayed greater weight loss and lower plasma IGF-I levels than sham-treated rats. Rats treated with IGFBP-4 and those treated with albumin demonstrated no differences in body weight, organ weights, IGF-I levels, and IGFBP-4 levels. CONCLUSIONS: Both the reduction of systemic IGF-I levels via hypophysectomy and the inhibition of local intraperitoneal IGF-I activity via IGFBP-4 treatment resulted in diminished postoperative adhesion severity. Treatment with IGFBP-4 may play a role in postoperative adhesion prophylaxis in the future.

Animals↗
PubMed2001

A new radioimmunoassay to measure rat insulin-like growth factor binding protein-4 (IGFBP-4) in serum, wound fluid and conditioned media.

Insulin-like growth factor binding protein-4 (IGFBP-4) is, like the other five IGFBPs, a critical regulator of the activity of insulin-like growth factor-I (IGF-I) and IGF-II. Because of the possible role of IGFBP-4 in multiple systems including reproduction, pregnancy, bone formation and wound healing, we developed a radioimmunoassay (RIA) to measure the concentration of IGFBP-4 in the serum, extracellular fluid and conditioned media of cells of rodents so that these animal models could be better exploited. Rat IGFBP-4 was expressed in Escherichia coli and purified to homogeneity. The recombinant material was used to raise a rabbit polyclonal antibody against rat IGFBP-4 and for iodination and standards. The RIA developed was sensitive to less than 1 ng/mL of rat IGFBP-4 and IGF-I did not interfere. There was no cross-reactivity with other rat IGFBPs on immuno-Western analysis of serum and wound fluid. IGFBP-4 from mouse serum did cross-react in our assay; however, serum from horse, pig or human showed no cross-reaction. Human IGFBP-1 and IGFBP-3 showed a very weak cross-reactivity. Serum IGFBP-4 levels showed no gender difference but did reveal a significant 66% increase in older rats. During the course of wound healing, which is IGF-I dependent, IGFBP-4 showed no changes. In conclusion an RIA for rat IGFBP-4 has been developed that specifically measures the concentration of IGFBP-4 in the sera, extracellular fluid and conditioned media of rodents. With this assay, the role of IGFBP-4 and IGFs in growth, development and the function of multiple systems can be further investigated using rodent models.

Age Factors↗
PubMed2001

Expression, purification and characterization of the structure and disulfide linkages of insulin-like growth factor binding protein-4.

Insulin-like growth factor binding protein-4 (IGFBP-4), like the other five IGFBPs, is a critical regulator of the activity of insulin-like growth factor (IGF)-I and IGF-II. However IGFBP-4 seems to be the only IGFBP with no potential to enhance the mitogenic actions of the IGFs. IGFBP-1 to -3 and -5 each contain 18 conserved cysteine residues, IGFBP-6 lacks two of the twelve N-terminal cysteines, while IGFBP-4 has two additional cysteines in the central region. A plasmid was constructed to express rat IGFBP-4 as a thioredoxin fusion protein that included a hexahistidine sequence to permit affinity purification. The fusion protein was expressed in E.coli, purified using nickel-chelate affinity chromatography and cleaved by tobacco etch virus (TEV) protease to produce mature rat IGFBP-4 with an additional glycine residue at the N-terminus. Final purification was achieved by further nickel affinity chromatography and reverse phase HPLC. The isoelectric points of the recombinant IGFBP-4 were the same as those of the non-glycosylated isoforms of IGFBP-4 in rat serum. The binding affinities of the recombinant protein and IGFBP-4 secreted by rat cells to IGF-I were compared using a newly developed binding assay. No significant difference could be detected, consistent with proper folding of the recombinant protein. This indicates that glycosylation of IGFBP-4 does not affect its binding to IGF-I. Using mass spectrometry and tandem mass spectrometry no differences between authentic and recombinant IGFBP-4 could be detected. Eight of the ten disulfide linkages have been determined, including linkages of conserved cysteine residues not previously identified in other IGFBPs. Numbering the cysteine residues sequentially from the N-terminus only the disulfide connectivity of C1, C2, C5 and C6 could not be determined. However, C1 is not linked to C1 and C5 is not linked to C6. The established linkages were C3 to C8, C4 to C7, C9 to C 11, and C10 to C12. The two cysteines in the non-conserved mid-region unique to IGFBP-4 (C13 and C14) are linked together. Linkage of the C-terminal cysteine residues is identical to that of IGFBP-2, -5 and -6 (C15 to C16, C17 to C18 and C19 to C20). The central flexible core of IGFBP-4, containing two additional cysteines may contribute to its unique biological action.

Animals↗
PubMed2001

Characterisation of insulin-like growth factor-binding protein-3 binding to a novel receptor on human platelet membranes.

Insulin-like growth factor-binding protein-3 (IGFBP-3) is an important regulator of insulin-like growth factor (IGF) bioavailability and IGF-independent growth responses. IGFBP-3 is stored within the alpha granules of platelets, permitting its rapid and concentrated delivery at sites of platelet lysis. Previous studies have demonstrated a lack of mRNA for IGFBP-3 in platelets and in the megakaryocytes from which platelets are formed, indicating that IGFBP-3 is endocytosed from the extracellular milieu. In this study, the binding of IGFBP-3 to platelet membranes was characterised to determine whether specific cell-surface IGFBP-3 receptors exist that might account for IGFBP-3 uptake into the alpha granules by megakaryocytes. IGFBP-3 binding to platelets was saturable, requiring at least 4.6 nM (125)I-labelled IGFBP-3 to occupy all binding sites present on 100 microg of platelet membranes. Non-linear regression analysis revealed the presence of a single class of high-affinity binding sites for IGFBP-3 on platelets, with a K(d) between 2.6x10(-10) and 8.0x10(-10) M and 1.51-4.89x10(11) binding sites/mg of platelet membrane. Kinetic analysis of (125)I-IGFBP-3 binding to platelet membranes demonstrated a forward rate (k(on)) of 8.1x 10(8) per M per min. The reverse rate constant (k(off)) was calculated to be 1.6x10(-1) per min (t(1/2)=4.2 min) and confirmed experimentally to be 3.3x10(-1) per min (t(1/2)=2.1 min). Binding of (125)I-IGFBP-3 to platelet membranes was inhibited in a dose-dependent manner by recombinant Escherichia coli IGFBP-3. In contrast, rat IGFBP-4 was not able to compete with (125)I-IGFBP-3 for platelet binding sites. Additionally, concentrations of IGF-I ranging from a 15-fold to a 40 000-fold molar excess caused a consistent 20% reduction in (125)I-IGFBP-3 binding. The mechanism of this slight reduction is unknown, but suggests that IGF-I does not compete directly with IGFBP-3 for receptor binding sites. However, it does not preclude the possibility that IGF-I may be endocytosed into the alpha granules as part of an IGF-I-IGFBP-3 complex. These results demonstrate the presence of high-affinity binding sites for IGFBP-3 on human platelet membranes. The nature and kinetics of the binding reaction are characteristic of a receptor-ligand interaction. This receptor may be involved in the endocytosis of circulating IGFBP-3 by megakaryocytes for packaging within the alpha granules of platelets. It is unknown if it is present in other tissues.

Animals↗
PubMed2000

Characterization of the enzymatic specificity of the IGF-dependent insulin-like growth factor binding protein-4 (IGFBP-4) protease.

The insulin-like growth factor (IGF)-dependent IGF binding protein-4 protease secreted by cultured adult human fibroblasts was recently identified as pregnancy-associated plasma protein-A (PAPP-A). In this study we showed that in addition to human IGFBP-4 the IGF-dependent IGFBP-4 protease also digests recombinant rat IGFBP-4 into two fragments by specifically cleaving at the carboxyl-terminal side of methionine at position 131 for rat IGFBP-4. Thus the cleavage site is at the KMKV site, which is not represented in other IGFBPs. While kallikrein may cleave at this site, its action is not specific.

Animals↗
PubMed1998

Physician's conscience and HECs: friends or foes?

No matter the future of healthcare financing and management, physicians of conscience and integrity must still be an important force in the consideration of ethical issues. The traditional role for the conscientious physician--being the only or even the major determinant of the morality of specific clinical decisions--is, for better or worse, no longer in effect. Much of this authority now belongs to patients and HECs are the mechanism within HCOs to help maintain this authority and to observe, comment on, recommend, and occasionally "regulate" the ethics of the healthcare arena. It is natural that these mechanisms for addressing areas of moral uncertainty create a certain tension. This tension should be acknowledged by conscientious physicians and HEC members. Total agreement on all moral issues in the clinical setting is impossible and should not be a goal. However, the respectful recognition of the importance of each perspective by both HEC members and conscientious physicians, and cooperation in developing effective mechanisms to address real differences, are possible and desirable. All who are interested in the ethics of healthcare now and in the future should support these endeavors.

Committee Membership↗
PubMed1998

Megakaryocytes endocytose insulin-like growth factor (IGF) I and IGF-binding protein-3: a novel mechanism directing them into alpha granules of platelets.

Lysis of platelets releases the contents of the alpha-granules, which contain growth factors, including insulin-like growth factor I (IGF-I) and IGF-binding protein-3 (IGFBP-3). We investigated the mechanism by which IGF-I and IGFBP-3 appeared in the alpha-granules with a goal of modulating their levels in platelets to affect platelet functions. Reverse transcription-PCR was initially used to test whether megakaryocytes contained IGFBP-3 and IGF-I messenger RNA transcripts. We found that megakaryocytes did not express the IGFBP-3 gene, but did have IGF-I messenger RNA. We subsequently investigated whether they incorporated IGFBP-3 and IGF-I by the process of endocytosis and packaged them into the alpha-granules. This hypothesis was tested in two ways. 1) We examined whether during pregnancy in the rat the alpha-granule content for IGFBP-3 paralleled the changes in plasma IGFBP-3 levels caused by the pregnancy-induced IGFBP-3 protease. The alpha-granule contents of both IGFBP-3 and IGF-I declined in parallel to the plasma changes in pregnant rats and returned to normal postpartum. As the binding protein protease acts extracellularly, endocytosis of the IGF-I:IGFBP-3 complex from the extracellular fluid by megakaryocytes was suggested. 2) We tested whether an IGF-I:IGFBP-3 complex comprised of human IGF-I and IGFBP-3 (recombinant 28.7 kDa) injected i.v. appeared in rat platelet alpha-granules. Hypophysectomized rats were injected i.v. with 5.24 mg of a 1:1 complex of IGF-I:IGFBP-3. After 24 h, platelet lysates were prepared and analyzed for IGFBP-3 by Western ligand blotting, and IGF-I was determined by RIA. Platelet lysates of the treated animals showed a prominent new band at approximately 28 kDa, whereas control rats were negative. In addition, the alpha-granule IGF-I concentration increased from 0.38 to 1.9 ng/1 x 10(9) platelets. These results indicate that the IGF-I:IGFBP-3 complex is taken up by megakaryocytes and packaged into the alpha-granules of platelets and demonstrate how the contents of IGF-I and IGFBP-3 in platelets can be modulated by their plasma concentrations. As reverse transcription-PCR has shown that the IGF-I, but not the IGFBP-3, gene is expressed by megakaryocytes, there may be two mechanisms for directing IGF-I into the alpha-granules of platelets.

Animals↗
PubMed1998

Growth hormone enhances regeneration of nitric oxide synthase-containing penile nerves after cavernous nerve neurotomy in rats.

PURPOSE: As growth hormone has been reported to improve nerve regeneration, we studied the effect of rat growth hormone (GH) on the regeneration of nitric oxide synthase (NOS)-containing penile nerves and the neurons in the pelvic ganglia after unilateral cavernous nerve neurotomy in rats. MATERIALS AND METHODS: Male rats were divided into three groups: sham operation (n = 14); unilateral neurotomy of a 5 mm. segment of the cavernous nerve (n = 14) with subsequent injection of buffer solution only; and unilateral neurotomy with GH injection (n = 14). Electrostimulation of the intact cavernous nerve was performed at 1 and 3 months. Nicotinamide adenine dinucleotide phosphate (NADPH) diaphorase staining was used to identify NOS in penile nerve fibers of the mid-shaft segment and in neurons of the pelvic ganglia. RESULTS: One month after unilateral neurotomy, both the buffer alone and GH-treated groups showed a significant decrease in NOS-containing nerve fibers in the dorsal and intracavernosal nerves on the side of neurotomy. At 3 months, the number of NOS-containing nerve fibers in the buffer alone group did not increase, while the GH-treated group showed a significant increase. In the GH-treated group at 3 months, more NOS-positive neurons in the pelvic ganglia were found on the intact side than on the side of neurotomy (p <0.034), indicating that the regeneration derives from pelvic ganglion neurons on the intact side. Furthermore, electrostimulation in the GH-treated group revealed a greater maximal intracavernosal pressure and a shorter latency period at 3 months than in those given buffer alone. CONCLUSIONS: Our results show that GH injection significantly enhances the regeneration of NOS-containing fibers in the dorsal and intracavernosal nerves after unilateral cavernous nerve injury. We believe that GH administration may present a new and more physiologic approach to the treatment of erectile dysfunction after radical pelvic surgery.

Animals↗
PubMed1997

General aspects of insulin-like growth factor binding proteins.

Insulin-like growth factor binding proteins (IGFBPs) belong to a family of at least six homologous proteins that bind insulin-like growth factors (IGFs) and modulate many of their biological actions. IGFBPs are produced by a wide variety of tissues and their circulating levels are regulated by both hormonal and metabolic factors. The binding activity of some IGFBPs is affected by phosphorylation, proteolysis, or association with extracellular matric (ECM) and plasma membrane. The IGFBPs may exert either inhibitory or potentiating effects on IGF actions. Recent evidence indicate that some IGFBPs are capable of direct actions on cells independent of the IGFs.

Aging↗
PubMed1997

Insulin-like growth factor I is suppressed in socially subordinate male baboons.

Insulin-like growth factor (IGF) I is a potent growth-promoting and anabolic hormone with major roles in cellular growth and differentiation, protein metabolism and muscle physiology, wound healing, erythropoiesis, and immune stimulation. Few, if any, studies have examined social or psychological factors that could give rise to individual differences in IGF-I levels. As part of a long-term psychoendocrine study of a population of male baboons living freely in a national reserve in East Africa, we examined the relationship between social rank and IGF-I concentrations. We observed that social subordinance was associated with a relative suppression of IGF-I concentrations; no rank-related differences in concentrations of IGF-II or IGF-binding protein were observed. Extensive psychoendocrine literature suggests that the individual differences in IGF-I profiles were a consequence, rather than a cause, of the rank difference. We were able to rule out a number of possible proximal explanations for the rank-IGF-I correlation: 1) the correlation was not a function of age (which involves both an adolescent spurt in IGF-I concentrations as well as a decline in concentrations in aged individuals); 2) the IGF-I suppression in subordinate individuals could not be explained by the basal hypercortisolism typical of such subordinate animals; and 3) neither differences in the quality or quantity of food consumed, in basal testosterone concentrations, nor in genetics could explain the rank difference. Although the mediating mechanisms responsible for this rank difference were not discernible in this study, the magnitude of difference in IGF-I levels among baboons of differing ranks might be of physiological significance.

Animals↗
PubMed1997

A new role for institutional ethics committees: organizational ethics.

New methods for financing and managing healthcare organizations have caused concerns regarding how these changes will affect the ethics of the care of patients. To address these concerns, the JCAHO has, for the first time, promulgated a standard requiring that each HCO develop and operate under a code of organizational ethics. This standard is now a part of the requirements that JCAHO will consider in its accreditation of HCOs. Development of the required code is mandatory for JCAHO subscribers and will, by necessity, need to be addressed by each institution. An IEC is the proper forum for undertaking this work.

Codes of Ethics↗
PubMed1995

A 3-dimensional model for the insulin-like growth factor binding proteins (IGFBPs); supporting evidence using the structural determinants of the IGF binding site on IGFBP-3.

It is generally accepted but not established that the insulin-like growth factor (IGF) binding site on the IGF binding proteins (IGFBPs) is in the N-terminal region. However, other workers have reported C-terminal fragments with IGF binding determinants. Therefore, we tested the hypothesis that both the N- and C-terminal regions of IGFBPs are involved in binding. Using a protein A gene fusion system, a cDNA encoding residues 1-147 (N147) was cloned into the plasmid pRIT2T and expressed in E. coli as a fusion protein. Since an Asn N-terminal to the gly of IGFBP-3 had been engineered into the cDNA construct, protein A was cleaved from N147 by hydroxylamine. Purified N147 was refolded in a DTT/cystamine redox system at pH 8.4 under nitrogen atmosphere. Both ligand binding Westerns and solution binding assays demonstrated that the recombinant derived N147 bound IGFs. The 147 and 176 residue N-terminal fragments, including a C-terminal fragment (residues 151-263) of IGFBP-3 were also expressed in pichia (yeast) as glycosylated proteins. Solution binding assays showed that they all bound labelled IGF-1. In conclusion, IGFBP-3 contains at least two binding determinants, one on the N- and one on the C-terminal domain. There may also be a possible contribution from the intermediate (I) domain. Our molecular genetic approach to mapping the binding region for IGFs on IGFBP-3 can now be tested on the other mutants we have prepared. Subsequently, site directed mutagenesis can be used to pinpoint key functional residues.

Binding Sites↗