Analysis of endogenous retinoic acid receptor expression and function in murine melanoma cells

There have been numerous reports over the past several years on the ability of vitamin A analogs (retinoids) to modulate cell proliferation, malignant transformation, morphogenesis, and differentiation in a wide variety of cell types and organisms. Two families of nuclear retinoid-inducible, trans-acting, transcription-enhancing receptors that bear strong DNA sequence homology to thyroid and steroid hormone receptors have recently been discovered. The retinoic acid receptors (RARs) and retinoid X receptors (RXRs) each have at least three types designated $\alpha,$ $\beta,$ and $\gamma,$ which are encoded by separate genes and expressed in a tissue and cell type-specific manner. We have been interested in the mechanism by which retinoids inhibit tumor cell proliferation and induce differentiation. As a model system we have employed several murine melanoma cell lines (S91-C2, K1735P, and B16-F1), which are sensitive to the growth-inhibitory and differentiation-inducing effects of RA, as well as a RA-resistant subclone of one of the cell lines (S91-C154), in order to study the role of the nuclear RARs in these effects. The initial phase of this project consisted of the characterization of the expression pattern of the three known RAR and RXR types in the murine melanoma cell lines in order to determine whether any differences exist which may elucidate a role for any of the receptors in RA-induced growth inhibition and differentiation. The novel finding was made that the RAR-$\beta$ gene is rapidly induced from undetectable levels by RA treatment at the mRNA and protein level, and that the induction of RAR-$\beta$ by other biologically active retinoids correlated with their ability to inhibit the growth of the highly RA-sensitive S91-C2 cell line. This suggests a role for RAR-$\beta$ in the growth inhibiting effect of retinoids. The second phase of this project involves the stable expression of RAR-$\beta$ in the S91-C2 cells and the RAR-$\beta$ receptor-null cell line, K1735P. These studies have indicated an inverse correlation between RAR-$\beta$ expression and proliferation rate. ^

Analysis of galectin expression and identification of endogenous ligands in a human colon carcinoma cell line

The 14.5 kDa (galectin-1) and 31 kDa (galectin-3) lectins are the most well characterized members of a family of vertebrate carbohydrate-binding proteins known as the galectins. Evidence has been obtained implicating these galectins in events as diverse as cell-cell and cell-extracellular matrix interactions, growth regulation, transformation, differentiation, and programmed cell death. In the present study, sodium butyrate was found to be a potent inducer of galectin-1 in the KM12 human colon carcinoma cell line. Prior to treatment with butyrate this cell line expresses only galectin-3. These cells were utilized as an in vitro model system to study galectin expression as well as that of their endogenous ligands. The initial phase of this project involved the examination of the induction of galectin-1 by butyrate at the protein level. These studies indicated that galectin-1 induction by butyrate was relatively rapid reaching nearly maximal levels after only 24 hours. Additionally, the induction was found to be reversible upon the removal of butyrate and to precede the increase in expression of the well characterized differentiation marker, carcinoembryonic antigen (CEA). The second phase of this project involved the characterization of potential glycoprotein ligands for galectin-1 and galectin-3. This work demonstrated that the polylactosaminoglycan-containing glycoproteins laminin, CEA, and the lysosome-associated glycoproteins-1 and -2 (LAMPs-1 and -2) are capable of serving as ligands for both galectin-1 and -3. The third phase of this project involved the analysis of the induction of the galectin-1 promoter by butyrate. Through the analysis of deletion constructs transiently transfected into KM12 cells, the region of the galectin-1 promoter mediating a high level of induction by butyrate was localized primarily within a proximal portion of the promoter containing a CCAAT element and an Sp1 binding site. The CCAAT-binding activity in the KM12 nuclear extracts was subsequently dentified as NF-Y by gel shift analysis. These studies suggest that: (1) the galectins may be involved in modulating adhesive interactions in human colon carcinoma cells through the binding of several polylactosaminoglycans shown to play a role in adhesion and (2) high level induction of the galectin-1 promoter by butyrate can proceed through a discreet, proximal element containing an NF-Y-binding CCAAT box and an Sp1 site. ^

Development of micro-electrospray mass spectrometry for ultra high sensitivity and application in the study of drugs of abuse on endogenous \lbrack Met\rbrack $\sp5$-enkephalin release

A micro-electrospray interface was developed specifically for the neurobiological applications described in this dissertation. Incorporation of a unique nano-flow liquid chromatography micro-electrospray "needle" into the micro-electrospray interface (micro-ES/MS) increased the sensitivity of the mass spectrometric assay by $\sim$1000 fold and thus permitted the first analysis of specific neuroactive compounds in brain extracellular fluid collected by in vivo microdialysis (Md).^ Initial in vivo data presented deals with the pharmacodynamics of a novel GABA$\sb{\rm B}$ antagonist and the availability of the compound in its parent (unmetabolized) form to the brain of the anesthetized rat. Next, the first structurally specific endogenous release of (Met) $\sp5$-enkephalin was demonstrated in unanesthetized freely-moving animals (release of $\sim$6.5 fmole of (Met) $\sp5$-enkephalin into the dialysate by direct neuronal depolarization). The Md/micro-ES/MS system was used to test the acute effects of drugs of abuse on the endogenous release of (Met) $\sp5$-enkephalin from the globus pallidus/ventral pallidum brain region in rats. Four drugs known to be abused by man (morphine, cocaine, methamphetamine and diazepam) were tested. Morphine and cocaine both elicited a two-fold or more increase in the release of (Met) $\sp5$-enkephalin over vehicle controls. Diazepam elicited a small decrease in (Met) $\sp5$-enkephalin levels and methamphetamine showed no significant effect on (Met) $\sp5$-enkephalin. These results imply that (Met) $\sp5$-enkephalin may be involved in the reward pathway of certain drugs of abuse. ^

Endogenous nitric oxide production in canine osteoarthritis: Detection in urine, serum, and synovial fluid specimens

OBJECTIVE To measure nitric oxide (NO) concentrations in serum, urine, and synovial fluid (SF) of dogs with naturally occurring cranial cruciate ligament (CCL) rupture and normal dogs, and to compare these with clinical and histologic changes of osteoarthritis (OA). STUDY DESIGN Prospective clinical study including 2 groups of animals selected from the hospital population. ANIMALS Forty-three dogs (CCL group) with OA secondary to CCL rupture; 30 healthy dogs (control group) without CCL rupture. METHODS Serum, urine, and SF were collected before and during surgery in the CCL group or immediately after euthanasia in the control group. Articular cartilage and synovial membrane tissue specimens were prepared for routine histologic examination. The stable end products of NO, total nitrite and nitrate (NOt) activity, were measured in body fluids and compared with macroscopic and histologic degrees of OA. Urinary NOt concentration was compared with urinary creatinine concentration and stated as urinary NOt:creatinine ratio (UNCR). RESULTS-SF NOt concentrations were not significantly different between the 2 groups. Serum NOt concentrations (45.6 vs 28.9 micromol/L; P =.042) and the UNCR (0.007 vs 0.004; P =.035) were significantly higher in dogs of the CCL group compared with the control population. An association between UNCR and histologic and macroscopical OA grades could be demonstrated. CONCLUSION UNCR might be a useful indicator of nitrite and nitrate production and, therefore, osteoarthritic changes in joints. CLINICAL RELEVANCE UNCR could be used as a tool to evaluate the NOt production by joint tissues over time and might therefore provide a method of evaluating the effects of drugs in the control of osteoarthritis.

Endogenous and synthetic MMP inhibitors in CNS physiopathology.

Matrix metalloproteinases (MMPs, including the membrane-type MMPs (MT-MMPs)), a disintegrin and metalloproteinase (ADAM), and ADAM with thrombospondin motifs belong to the metzincins, a subclass of metalloproteinases that contain a Met residue and a Zn(2+) ion at the catalytic site necessary for enzymatic reaction. MMP proteolytic activity is mainly controlled by their natural tissue inhibitors of metalloproteinase (TIMP). A number of synthetic inhibitors have been developed to control deleterious MMP activity. The roles of MMPs and some of their ECM substrates in CNS physiology and pathology are covered by other chapters of the present volume and will thus not be addressed in depth. This chapter will focus (i) on the endogenous MMP inhibitors in the CNS, (ii) on MMP and TIMP regulations in three large classes of neuropathologic processes (inflammatory, neurodegenerative, and infectious), and (iii) on synthetic inhibitors of MMPs and the perspective of their use in different brain diseases.

Endogenous α-calcitonin-gene-related peptide promotes exercise-induced, physiological heart hypertrophy in mice.

AIM It is unknown how the heart distinguishes various overloads, such as exercise or hypertension, causing either physiological or pathological hypertrophy. We hypothesize that alpha-calcitonin-gene-related peptide (αCGRP), known to be released from contracting skeletal muscles, is key at this remodelling. METHODS The hypertrophic effect of αCGRP was measured in vitro (cultured cardiac myocytes) and in vivo (magnetic resonance imaging) in mice. Exercise performance was assessed by determination of maximum oxygen consumption and time to exhaustion. Cardiac phenotype was defined by transcriptional analysis, cardiac histology and morphometry. Finally, we measured spontaneous activity, body fat content, blood volume, haemoglobin mass and skeletal muscle capillarization and fibre composition. RESULTS While αCGRP exposure yielded larger cultured cardiac myocytes, exercise-induced heart hypertrophy was completely abrogated by treatment with the peptide antagonist CGRP(8-37). Exercise performance was attenuated in αCGRP(-/-) mice or CGRP(8-37) treated wild-type mice but improved in animals with higher density of cardiac CGRP receptors (CLR-tg). Spontaneous activity, body fat content, blood volume, haemoglobin mass, muscle capillarization and fibre composition were unaffected, whereas heart index and ventricular myocyte volume were reduced in αCGRP(-/-) mice and elevated in CLR-tg. Transcriptional changes seen in αCGRP(-/-) (but not CLR-tg) hearts resembled maladaptive cardiac phenotype. CONCLUSIONS Alpha-calcitonin-gene-related peptide released by skeletal muscles during exercise is a hitherto unrecognized effector directing the strained heart into physiological instead of pathological adaptation. Thus, αCGRP agonists might be beneficial in heart failure patients.

Explaining Endogenous Control Change in Outsourced Software Projects

Given the centrality of control for achieving success in outsourced software projects, past research has identified key exogenous factors that determine the choice of controls. This view of exogenously driven control choice is based on a number of assumptions; particularly, clients and vendors are seen as separate cognitive entities that combat opportunistic threats under environmental uncertainty by one-off choices or infrequent revisions of controls. In this paper we complement this perspective by acknowledging that an outsourced software project may be characterized as a collective, evolving process faced with the challenge of coping with cognitive limitations of both client and vendor through a continuous process of learning. We argue that if viewed in this way, controls are less subject of a deliberate choice but rather are subject of endogenously driven change, i.e. controls evolve in close interaction with the evolving software project. Accordingly, we suggest a complementary model of endogenous control, where controls mediate individual and collective learning processes. Our research contributes to a better understanding of the dynamics in outsourced software projects. It also spells out methodological implications that may help improve cross-section control research.

Endogenous Development as a Social Learning Process

The author perceives endogenous development as a social learning process, which is constructed by all actors involved. To enhance social learning, a methodology called Autodidactic Learning for sustainability is used, in which the perception of both local actors and external actors are highlighted. Reflecting on differences, conflicts and common interests leads to highly motivated debate and shared reflection, which is almost identical with social learning, and flattens the usual hierarchy between local and external actors. The article shows that the energies generated through collective learning can trigger important technical, social and political changes, which take into account the multiple dimensions of local reality.

Environmental and Endogenous Factors Influencing Emigration in Juvenile Anadromous Alewives

We analyzed juvenile anadromous alewife migration at Bride Lake, a coastal lake in Connecticut, during summer 2006 and found that migration on 24-hour and seasonal timescales was influenced by conditions of the environment and characteristics of the individual. To identify environmental cues of juvenile migration, we continuously video recorded fish at the lake outflow and employed information-theoretic model selection to identify the best predictors of daily migration rate. More than 80% of the approximately 320,000 juveniles that migrated from mid-June to mid-August departed in three pulses lasting one or two days. Pulses of migration were associated with precipitation events, transient decreases in water temperature and transient increases in stream discharge. Diel timing of migration shifted over the summer. Early in the season most migration occurred around dawn; late in the season migration occurred at night. To identify individual characteristics associated with migratory behavior, we compared migrating juveniles that we collected as they were exiting Bride Lake to non-migrating juveniles that we collected from the center of the lake. Migrants were a non-random subset of the population; they were on average 1 – 12 mm larger, 2 – 14 d older, had grown more rapidly (11% greater length-at-age), and were in better condition (14% greater mass-at-length) than non-migrant fish. We infer that the amount of accumulated energy has a positive effect on the net benefit of migration at any time in the migratory season.

The role of endogenous IFN-beta in cutaneous melanoma: Consequences of epidermal hyperplasia

The purpose of this study was to characterize epidermal hyperplasia overlying malignant melanoma, to determine the mitogenic factor responsible for the induction of this hyperplasia and to investigate its biological consequence. Whether increased keratinocyte proliferation overlying melanoma is due to production of growth factors by the tumor cells or to other mechanisms is unknown. Epidermal hyperplasia overlying human melanoma was found overlying thick (>4.0mm), but not thin (<1.0mm) tumors. Immunostaining of the sections for growth factors related to angiogenesis revealed that epidermal hyperplasia was associated with loss of IFN-β production by the keratinocytes directly overlying the tumors. Since previous studies from our laboratory have demonstrated that exogenous administration of IFN-β negatively regulates angiogenesis, we hypothesize that tumors are able to produce growth factors which stimulate the proliferation of cells in the surrounding tissues. This hyperplasia leads to a decrease in the endogenous negative regulator of angiogenesis, IFN-β. ^ The human melanoma cell line, DM-4 and several of its clones were studied to identify the mitogenic factor for keratinocytes. The expression of TGF-α directly correlated with epidermal hyperplasia in the DM-4 clones. A375SM, a human melanoma cell line that produces high levels of TGF-α, was transfected with a plasmid encoding full-length antisense TGF-α. The parental and transfected cells were implanted intradermally into nude mice. The extent of epidermal hyperplasia directly correlated with expression of TGF-α and decreased production of IFN-β, hence, increased angiogenesis. ^ In the next set of experiments, we determined the role of IFN-β on angiogenesis, tumor growth and metastasis of skin tumors. Transgenic mice containing a functional mutation in the receptor for IFN α/β were obtained. A375SM melanoma cells were implanted both s.c. and i.v. into IFN α/βR −/− mice. Tumors in the IFN α/β R −/− mice exhibited increased angiogenesis and metastasis. IFN α/βR −/− mice were exposed to chronic UV irradiation. Autochthonous tumors developed earlier in the transgenic mice than the wild-type mice. ^ Collectively, the data show that TGF-α produced by tumor cells induces proliferation of keratinocytes, leading to epidermal hyperplasia overlying malignant melanoma associated with loss of IFN-β and enhanced angiogenesis, tumorigenicity and metastasis. ^

A Tort for Risk and Endogenous Bankruptcy

Conventional tort law bars victims of exposure to a toxic substance from filing suit until they actually develop symptoms of illness. Practically speaking, this rule often bars recovery due to bankruptcy and causal uncertainty. One solution is to allow victims to file at exposure for expected damages (a tort for risk). The trade-off is that such a rule may trigger a race to file among exposure victims, thereby itself inducing bankruptcy. This paper characterizes the conditions under which such a race will occur in equilibrium and examines the implications for social welfare.

Endogenous Financing and the Long Run Impact of Money Growth on Output and Prices

Most monetary models make use of the quantity theory of money along with a Phillips curve. This implies a strong correlation between money growth and output in the short run (with little or no correlation between money and prices) and a strong long run correlation between money growth and inflation and inflation (with little or no correlation between money growth and output). The empirical evidence between money and inflation is very robust, but the long run money/output relationship is ambiguous at best. This paper attempts to explain this by looking at the impact of money growth on firm financing.