Regulation and role of REST and REST4 variants in modulation of gene expression in in vivo and in vitro in epilepsy models.

Repressor element-1 silencing transcription factor (REST) is a candidate modulator of gene expression during status epilepticus in the rodent. In such models, full-length REST and the truncated REST4 variant are induced and can potentially direct differential gene expression patterns. We have addressed the regulation of these REST variants in rodent hippocampal seizure models and correlated this with expression of the proconvulsant, substance P encoding, PPT-A gene. REST and REST4 were differentially regulated following kainic acid stimulus both in in vitro and in vivo models. REST4 was more tightly regulated than REST in both models and its transient expression correlated with that of the differential regulation of PPT-A. Consistent with this, overexpression of a truncated REST protein (HZ4, lacking the C-terminal repression domain) increased expression of the endogenous PPT-A gene. Similarly the proximal PPT-A promoter reporter gene construct was differentially regulated by the distinct REST isoforms in hippocampal cells with HZ4 being the major inducer of increased reporter expression. Furthermore, REST and REST4 proteins were differentially expressed and compartmentalized within rat hippocampal cells in vitro following noxious stimuli. This differential localization of the REST isoforms was confirmed in the CA1 region following perforant path and kainic acid induction of status epilepticus in vivo. We propose that the interplay between REST and REST4 alter the expression of proconvulsant genes, as exemplified by the PPT-A gene, and may therefore regulate the progression of epileptogenesis.

'Double hit' from streptococcal pneumonia and hypersensitivity pneumonitis associated with anagrelide.

We report a case of unresolved streptococcal pneumonia in a patient taking anagrelide for essential thrombocythaemia. Resolution only occurred after discontinuing anagrelide and the institution of corticosteroid therapy, which resulted in dramatic improvement. There has been only one previous case report of anagrelide causing hypersensitivity pneumonia. We postulate this patient suffered a 'double hit' from streptococcal infection and anagrelide.

Effect of insulin-like growth factor-1 and insulin-like growth factor binding protein-3 complex in cavernous nerve cryoablation.

The purpose of this work was to study the effect of insulin-like growth factor 1 (IGF-1) and its binding protein (IGFBP-3) on the recovery of erectile function in a rat model for neurogenic impotence. In all, 28 male Sprague-Dawley rats were divided into four groups: seven underwent a sham operation; seven underwent bilateral cavernous nerve freezing (control group); seven underwent bilateral cavernous nerve freezing followed by intraperitoneal injection of IGF-1; and seven underwent bilateral cavernous nerve freezing followed by intraperitoneal injection of IGFBP-3. Erectile response was assessed by cavernous nerve electrostimulation at 3 months, and samples of penile tissue were evaluated histochemically for nitric oxide synthase (NOS)-containing fibers. In the sham and IGF-1 group, there were significantly higher maximal intracavernous pressures compared to the IGFBP-3 complex and the control group. Correspondingly in the cavernosum, there were significantly more NOS-containing nerve fibers in the sham and IGF-1 groups. In conclusion, administration of IGF-1 can facilitate the regeneration of NOS-containing nerve fibers in penile tissue and enhance the recovery of erectile function after bilateral cavernous nerve cryoablation. The reverse effect was noted with the IGFBP-3 complex injection.

Seasonal control of penile development of Siberian hamsters (Phodopus sungorus) by daylength and testicular hormones.

Seasonal variation in prepubertal penile growth has not previously been studied. The present study assessed the influence of daylength and androgens on penile development in the Siberian hamster (Phodopus sungorus). Adult penile masses were achieved at 18 and 8 weeks of age in hamsters maintained from birth under short (10 h light:14 h dark) versus long (14 h light:10 h dark) daylengths, respectively. Insulin-like growth factor I concentrations, previously implicated in penile growth, did not differ between hamsters maintained in short versus long daylengths. Gonadectomized juvenile males maintained in short and long daylengths and administered testosterone attained adult penile masses well in advance of untreated gonad-intact males maintained in short daylengths. Hamsters from both photoperiods, castrated as juveniles and first treated with testosterone in adulthood, also achieved adult penile masses. The photoinhibited gonad is insufficient to promote penile growth, and prepubertal gonadal secretions during short daylengths are not necessary for eventual penile development. Among young born near the end of the mating season, onset of neuroendocrine refractoriness to short daylengths at about 100 days of age and subsequent gonadal development induces growth in all reproductive tissues. Timing of puberty and increased androgen secretion controlled by daylength are the primary determinants of postnatal penile growth, which may also be affected by prenatal and early postnatal organizational actions of androgens.

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.

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.

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.

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.

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.

Organization ethics or compliance: which will articulate values for the United States' healthcare system?

There have been two major stimuli leading to the institution of compliance/ethics programs. One has been Justice Department attention and the need for a government approved compliance program. The other has been the JCAHO's promulgation of standards addressing organization ethics issues. Many healthcare organizations have seen the requirements imposed on them by JCAHO (and the necessity to have a mechanism to address federal compliance as separate issues) and have developed two compartmentalized programs. But the intensity with which the government has pursued and will continue to pursue its "fraud and abuse" program means that most healthcare organizations now support and encourage the institution of compliance programs, while allowing organization ethics initiatives to become marginalized within the organization. This approach may ignore consideration of the larger issue of what each of these activities means for a healthcare organization that is attempting to define itself based on its stated values. Although using two separate groups to focus on these particular issues may fulfill the letter of the law for both the Federal Government and the JCAHO, we believe attention to these issues is better accomplished under the umbrella of a single committee. This pathway for attention to accreditation and compliance issues within an HCO is one that may serve the HCO better than two committees working on similar issues but with separate agendas. Nothing here should be construed as a suggestion that the goal of developing a comprehensive values-oriented organization ethics program should be abandoned. Instead, we have offered an assessment of the current environment and current direction of the healthcare organization--an assessment that leads us to the conclusion that instituting organization ethics programs that are not linked to or incorporated somehow within compliance programs will probably fail. This is a frustrating position for those who believe that compliance programs may ultimately undermine the goals of an organization ethics program. Nevertheless, it is important to realize that the legal compliance of an HCO, like its compliance to high standards of ethics, are considerations that determine the ethical climate of an organization. In the absence of a consensus concerning the values that should inform the larger healthcare system, it may be the only consideration that all healthcare organization stakeholders can agree upon, and so may represent the only stable and consistent platform ethically to evaluate the activities of the healthcare organization at the present time. This can be the basis for each HCO to develop its values-oriented ethical program which will define and support the organization's ethical climate for the organization, its staff, its patients, and its community.

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.

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.

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.

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.

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.

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.