Transgenic Animal Studies Research Articles

Ethics and Refinement in Animal Research

Based on a case study we conclude that scientists and journals could and should do more to secure the ethical standards of animal use in biomedical research: Scientists often justify animal use referring to the scientific value and biomedical importance (2). However, except from a purely anthropocentric perspective, ethical concern about compromised animal welfare cannot be justified by human benefits alone (1). It is equally important that the benefits are achieved with a minimum of negative effects to the animals involved. Thus scientists must apply ’refinement’, the third R of the socalled 3Rs (3). But here scientists seem sometimes to do less than they should and journals fail in their duty to enforce high ethical standards. We studied the use of animals as models for the neurodegenerative Huntington’s Disease. Here refinement should focus on limiting the negative impact of declining sensorimotor function as the disease progresses: housing adaptations facilitating food and water intake and humane endpoints (using earlier, less severe clinical signs as endpoint parameters rather than awaiting spontaneous death) are two possible approaches (4). We asked to what extent animal welfare problems and refinements addressing them were reported in publications of the most common rodent models by analyzing 90 research reports published in international peer-reviewed journals during 2003-2004. These include quinolinic acid (5,6) and 3-nitropropionic acid (6,7) administration to rats and mice and R6/1, R6/2 and N171-82Q transgenic mice (8,9,10). Our results show that the majority of experiments (36+17) were in the two most severe categories, in which animals have sensorimotor deficits interfering with their capacity to eat and drink normally from the cage top (11, 12, 13). Very few papers reporting such severe experiments included information on refinement measures: Out of 14 survival studies of transgenic animals with a progressive neurodegenerative phenotype, only 6 referred to humane endpoints and only 2 to housing adaptations. Among the majority of studies referring to compliance with official regulations and / or efforts to reduce animal suffering, we also found experiments using far from best practice, such as administration of rising neurotoxin doses until high animal mortalities.

Rapid turnover of apolipoprotein C-III-containing triglyceride-rich lipoproteins contributing to the formation of LDL subfractions

The atherogenicity theory for triglyceride-rich lipoproteins (TRLs; VLDL + intermediate density lipoprotein) generally cites the action of apolipoprotein C-III (apoC-III), a component of some TRLs, to retard their metabolism in plasma. We studied the kinetics of multiple TRL and LDL subfractions according to the content of apoC-III and apoE in 11 hypertriglyceridemic and normolipidemic persons. The liver secretes mainly two types of apoB lipoproteins: TRL with apoC-III and LDL without apoC-III. Approximately 45% of TRLs with apoC-III are secreted together with apoE. Contrary to expectation, TRLs with apoC-III but not apoE have fast catabolism, losing some or all of their apoC-III and becoming LDL. In contrast, apoE directs TRL flux toward rapid clearance, limiting LDL formation. Direct clearance of TRL with apoC-III is suppressed among particles also containing apoE. TRLs without apoC-III or apoE are a minor, slow-metabolizing precursor of LDL with little direct removal. Increased VLDL apoC-III levels are correlated with increased VLDL production rather than with slow particle turnover. Finally, hypertriglyceridemic subjects have significantly greater production of apoC-III-containing VLDL and global prolongation in residence time of all particle types. ApoE may be the key determinant of the metabolic fate of atherogenic apoC-III-containing TRLs in plasma, channeling them toward removal from the circulation and reducing the formation of LDLs, both those with apoC-III and the main type without apoC-III.

On the Relation of Oxidative Stress to Neuroinflammation: Lessons Learned from the G93A-SOD1 Mouse Model of Amyotrophic Lateral Sclerosis

The central nervous system (CNS) presents both challenges and opportunities to researchers of redox biochemistry. The CNS is sensitive to oxidative damage during aging or disease; excellent transgenic models of specific neurodegenerative diseases have been created that reproduce oxidative stress components of the corresponding human disorder. Mouse models of familial amyotrophic lateral sclerosis (ALS) based on overexpressed mutant human Cu, Zn-superoxide dismutase (SOD1) are cases in point. These animals experience predictably staged, age-dependent motor neuron degeneration with profound cellular and biochemical damage to nerve fibers and spinal cord tissue. Severe protein and lipid oxidation occurs in these animals, apparently as an indirect consequence of protein aggregation or cytopathic protein-protein interactions, as opposed to aberrant redox catalysis by the mutant enzyme. Recent studies of G93A-SOD1 mice and rats suggest that oxidative damage is part of an unmitigated neuroinflammatory reaction, possibly arising in combination from mitochondrial dysfunction plus pathophysiologic activation of both astrocytes and microglia. Lesions to redox signal-transduction pathways in mutant SOD1+ glial cells may stimulate broad-spectrum upregulation of proinflammatory genes, including arachidonic acid-metabolizing enzymes [e.g., cyclooxygenase-II (COX-II) and 5-lipoxygenase (5LOX)]; nitric oxide synthase (NOS) isoforms; cytokines (particularly tumor necrosis factor alpha, TNF-alpha); chemokines; and immunoglobulin Fc receptors (FcgammaRs). The integration of these processes creates a paracrine milieu inconsistent with healthy neural function. This review summarizes what has been learned to date from studies of mutant SOD1 transgenic animals and demonstrates that the G93A-SOD1 mouse in particular is a robust laboratory for the study of neuroinflammation and redox biochemistry.

Prorenin

Prorenin, the precursor of renin, exists in circulating blood at concentrations that are ≈5 to 10× higher than those of renin. For many years, prorenin was considered to be an inactive form of renin with no physiological role. Then, in the mid-80s of the last century, Luetscher et al reported that the levels of circulating prorenin (but not renin) are increased in diabetic subjects.1 Subsequent studies revealed that these high levels correlated with the presence of microvascular complications, and it was proposed that prorenin might be used to predict the occurrence of microalbuminuria.2 Simultaneously, high prorenin levels were observed in pregnant women, and evidence was obtained for the local synthesis of prorenin at extrarenal tissue sites such as the ovary and eye. Until today, however, the exact function of prorenin remains unknown. An attractive concept is that in tissues not synthesizing renin locally, circulating prorenin, after its local conversion to renin, contributes to angiotensin (Ang) generation. This would not only provide a role for prorenin in vivo, but also explain why tissues, in contrast with plasma, contain predominantly renin.3 Studies in transgenic animals support this idea. Veniant et al4 described that transgenic rats expressing prorenin exclusively in the liver (resulting in a 400-fold rise in plasma prorenin) display cardiac hypertrophy and vascular damage in the absence of hypertension. Prescott et al5 detected increased cardiac Ang I levels in mice overexpressing human prorenin in the liver and human angiotensinogen in the heart. The question then arises how tissues sequester and activate circulating prorenin. Sequestration may involve diffusion into the interstitial fluid and/or binding to a receptor.3 …

Chinese red yeast rice (CRYR) inhibits proliferation and stimulates apoptosis in both COX‐2 expressing and non‐expressing human colon cancer cells via inhibition of cholesterogenesis

De novo cholesterogenesis and inflammation are both critical steps in colon carcinogenesis. In population-based studies, statin drug consumption is associated with a reduced risk of colon cancer. Statins are also known to have anti-inflammatory actions which could contribute to their activity in both heart disease and colon cancer prevention. CRYR contains nine Monacolins one of which is lovastatin (MonacolinK). We have studied CRYR in humans demonstrating cholesterol-lowering and have been interested in their potential use for chemoprevention of colon cancer. To determine whether, the primary mechanisms of CRYR in colon cancer was via inhibition of inflammation and/or cholesterogenesis, two human colon cancer cell lines either with (HT-29) or without (HCT-116) expression of cyclooxygenase 2 (COX-2) were treated with CRYR extract (0 to 300 ug/ml) for 24, 48 or 72 h. Cell proliferation and apoptosis were determined using the MTT cell viability assay and TUNEL assay. CRYR treatment (50 ug/ml) for 72 h resulted in 54 % and 44 % of suppression in proliferation in HCT-116 (p<0.001) and HT-29 (p<0.001), respectively. The IC50 on proliferation was 51.6 in HCT-116 and 58.9 in HT-29 cells in the same time point. Apoptosis was enhanced with 48 h incubation of CRYR (100 ug/ml) in both cell lines (p<0.05). The present results demonstrate that CRYR inhibited proliferation and stimulated apoptosis regardless of the expression of COX-2. Studies in transgenic animals and humans will be needed to confirm that CRYR inhibits colon cancer risk primarily via inhibition of de novo cholesterogenesis. Fund: UCLA CNRU CA 42710.

Variant BDNF (Val66Met) impact on brain structure and function

Neurotrophins, such as brain-derived neurotrophic factor (BDNF), are a unique family of polypeptide growth factors that influence differentiation and survival of neurons in the developing nervous system. In adults, BDNF is important in regulating synaptic plasticity and connectivity in the brain. Recently, a common single-nucleotide polymorphism in the human BDNF gene, resulting in avaline to methionine substitution in the prodomain (Val66Met), has been shown to lead to memory impairment and susceptibility to neuropsychiatric disorders. An understanding of how this naturally occurring polymorphism affects behavior, anatomy, and cognition in adults is an important first step in linking genetic alterations in the neurotrophin system to definable biological outcomes in humans. We review the recent literature linking this BDNF polymorphism to cognitive impairment in the context of in vitro and transgenic animal studies that have established BDNF's central role in neuronal functioning in the adult brain.

Drug interactions and pharmacogenetic reactions are the basis for chloroquine and mefloquine-induced psychosis

Chloroquine and mefloquine used for prophylaxis and treatment of malaria sometimes causes severe mental status changes, through mechanisms that are poorly understood. Psychosis is caused by interactions with other drugs or by pharmacogenetic vulnerabilities that cause heightened responses to chloroquine or mefloquine alone, mediated through dopamine, acetylcholine, serotonin, P-glycoprotein, inhibited cortical activity, deranged calcium homeostasis, and impaired synaptogenesis. Retrospective studies can identify all other drugs taken coincident with chloroquine or mefloquine psychosis. Various genes from patients could be cloned and compared to those from individuals who tolerated chloroquine and mefloquine, culminating with transgenic animal studies. Identification of candidate genes may be aided by pharmacogenomic analysis of single nucleotide polymorphism maps. Finally, prospective studies with cerebrospinal fluid analysis and PET scanning could help verify the hypothesis. If this hypothesis is correct, the incidence of chloroquine and mefloquine psychosis can be greatly reduced by avoiding interacting medications and by conducting genetic screening prior to initiating chloroquine and mefloquine. Validation of the hypothesis would also provide a paradigm to follow for avoiding neuropsychiatric side effects if antidepressants and neuroleptics are used to overcome chloroquine resistance, if new antimalarial drugs chemically related to chloroquine and mefloquine are developed and if chloroquine and mefloquine are used for non-malarial applications such as HIV and cancer.

244. New Gene Therapy for Parkinson's Disease by Down Regulation of α-Synuclein

Parkinson's disease (PD) is characterized by selective degeneration of substantia nigra dopamine neurons and abnormal cytoplasmic aggregates of |[alpha]|-synuclein. There is substantial evidence from molecular genetics, transgenic animal studies, and aggregation/toxicity studies with recombinant |[alpha]|-synuclein to suggest that conversion of this protein from soluble monomers to aggregated insoluble forms in the brain is a key event in the pathogenesis of PD. In the present study, adeno-associated virus (AAV) vector delivery of |[alpha]|-synuclein ribozyme was tested for its silencing effect on degenerating nigrostriatal neurons in MPP+ model of PD. We designed |[alpha]|-synuclein ribozyme against human |[alpha]|-synuclein gene expression and constructed |[alpha]|-synuclein ribozyme carried rAAV vector (designated rAAV-SynRz). Co-transfection of rAAV-SynRz and rAAV-|[alpha]|- synuclein into HEK293 cells resulted down regulating |[alpha]|-synuclein protein expression in vitro. Then, rAAV-SynRz was injected into SN of the MPP+-treated rats. Cell counts of TH-positive neurons in the substantia nigra revealed that rAAV-SynRz protected a significant number of TH positive cells when compared with cell counts of rAAV-EGFP or no rAAV injected rats. In close agreement, 56.2% of TH-positive cells remained in the nigral rAAV-SynRz group vs only 30.9% of in the EGFP group or 31.8% of no-rAAV treatment. These data indicated that using of rAAV-SynRz could survive more TH-positive neuron in SN in MPP+ model. Down regulation of |[alpha]|-synuclein expression might be a possible therapeutic tool for gene therapy of PD.

Renin, Prorenin and the Putative (Pro)renin Receptor

Renin is an aspartic protease that consists of 2 homologous lobes. The cleft in between contains the active site with 2 catalytic aspartic residues.1 Unlike other aspartic proteases such as pepsin or cathepsin D, renin is monospecific and only cleaves angiotensinogen, to generate angiotensin (Ang) I. Ang I is the precursor of the active end-product of the renin-angiotensin system (RAS), Ang II. Renin has also been called active renin to underline that an enzymatically inactive form of renin exists. In 1971, Lumbers found that amniotic fluid, left at low pH in the cold, acquired renin activity.2 Later, Skinner described a similar phenomenon in plasma.3 Acidification was not strictly necessary for this increase in Ang I-generating activity, because incubation at low temperature also increased renin activity, albeit to only 15% of activity after acidification. Soon it was postulated that this inactive, but activatable “big” renin (its molecular weight was 5 kDa higher than that of renin) was the biosynthetic precursor of renin. Hence, it was named prorenin. Only with the cloning of the renin gene in 1984 was prorenin definitively proved to be the precursor of renin.4 For reasons that are unknown, prorenin circulates in human plasma in excess to renin, sometimes at concentrations that are 100-times higher.5 Prorenin has also been demonstrated in plasma of cat, dog, cattle, pig, horse, sheep, rabbit, rat, and mouse. A 43-amino acid N-terminal propeptide explains the absence of enzymatic activity of prorenin. This propeptide covers the enzymatic cleft and obstructs access of angiotensinogen to the active site of renin. (Pro)renin is synthesized as a preprohormone. It contains a signal peptide that directs the protein to the endoplasmic reticulum and ultimately to the exterior of the cell. Both renin and prorenin can be fractionated into multiple species by isoelectric focusing. …

The great MYC escape in tumorigenesis

Increased wild-type MYC expression occurs frequently in human cancers, except in Burkitt's lymphoma, where the translocated MYC allele is frequently mutated at several hotspots, including a major one at threonine-58. Acute MYC expression increases p53 or ARF levels and induces apoptosis, and previous transgenic animal studies revealed frequent inactivating mutations of p53 or p19ARF in transgenic Myc-induced lymphomas. Lowe and coworkers (Hemann et al., 2005) demonstrate that wild-type MYC can also trigger apoptosis by inducing Bim, which neutralizes Bcl-2. In contrast, the MYC point mutants failed to induce Bim, promoting murine lymphomas that escaped both wild-type p53 and p19ARF, and in doing so, evaded apoptosis.

Contraction signaling to glucose transport in skeletal muscle

Contracting skeletal muscles acutely increases glucose transport in both healthy individuals and in people with Type 2 diabetes, and regular physical exercise is a cornerstone in the treatment of the disease. Glucose transport in skeletal muscle is dependent on the translocation of GLUT4 glucose transporters to the cell surface. It has long been believed that there are two major signaling mechanisms leading to GLUT4 translocation. One mechanism is insulin-activated signaling through insulin receptor substrate-1 and phosphatidylinositol 3-kinase. The other is an insulin-independent signaling mechanism that is activated by contractions, but the mediators of this signal are still unknown. Accumulating evidence suggests that the energy-sensing enzyme AMP-activated protein kinase plays an important role in contraction-stimulated glucose transport. However, more recent studies in transgenic and knockout animals show that AMP-activated protein kinase is not the sole mediator of the signal to GLUT4 translocation and suggest that there may be redundant signaling pathways leading to contraction-stimulated glucose transport. The search for other possible signal intermediates is ongoing, and calcium, nitric oxide, bradykinin, and the Akt substrate AS160 have been suggested as possible candidates. Further research is needed because full elucidation of an insulin-independent signal leading to glucose transport would be a promising pharmacological target for the treatment of Type 2 diabetes.

SRF-dependent gene expression in isolated cardiomyocytes: Regulation of genes involved in cardiac hypertrophy

Serum response factor (SRF) is a transcription factor required for the regulation of genes important for cardiac structure and function. Notably, the "fetal gene expression profile" that is characteristic of cardiac hypertrophy consists of genes known to be regulated by SRF. Transgenic animal studies suggest that cardiac-specific overexpression of SRF induces this pattern of hypertrophic genes and subsequently causes the progression of pathologic adaptations. Furthermore, studies examining cardiac tissues from patients with severe heart failure indicate significant alterations in SRF expression that correspond with alterations in expression of SRF-dependent genes. Based on these observations, it has been postulated that SRF may be critical for stimulating pathologic gene expression at the onset of hypertrophic adaptation. To address the role of SRF in cardiac hypertrophy we investigated whether SRF is necessary and sufficient for the expression of genes associated with the hypertrophic response. We used isolated cardiomyocytes from both neonatal rats, and transgenic mice containing floxed SRF alleles, to examine cardiac gene expression in response to overexpression and absence of SRF. Using this approach, we demonstrate that SRF is required for the induction of atrial naturetic factor (ANF), c-fos, NCX1, BNP, alpha-actins, alpha-myosin heavy chain, and beta-myosin heavy chain genes. However, overexpression of exogenous SRF in isolated cardiomyocytes is only sufficient to induce NCX1 and alpha-myosin heavy chain. These results indicate that SRF is critical for the regulation and induction of genes associated with the progression of pathologic cardiac hypertrophy, however, the pattern of genes induced by overexpression of SRF in isolated cardiomyocytes is different from those genes expressed in hypertrophic transgenic hearts. This suggests that SRF-dependent gene expression is modulated in a complex manner by in vivo physiologic systems prior to and during heart failure as the organism adapts to cardiac stress.

Apolipoprotein gene and its interaction with the environmentally driven risk factors: molecular, genetic and epidemiological studies of Alzheimer’s disease

Herein we review the role of apolipoprotein E (ApoE) in Alzheimer's disease (AD) and how ApoE interacts with various risk factors. ApoE is localized with the major pathological hallmarks of AD, extracellular amyloid deposits and intracellular neurofibrillary tangles. The ApoE4 allele is associated with the development of late-onset familial and sporadic AD. ApoE4 has a gene dose effect on the risk and age of onset of AD. ApoE mRNA and protein are found predominantly in astrocytes within the CNS. There is also a high expression of ApoE mRNA in the brains of people with sporadic AD. ApoE acts as a cholesterol transporter in the brain. Cholesterol controls amyloid production and deposition by regulating beta-secretase. In transgenic animal studies, ApoE4 expression causes neuropathology and behavioral deficits. We also discuss data from three different cohorts for AD in the general population, in different racial and ethnic groups and the role of the 4 allele in the clinical onset of the disease. Although the 4 allele is an important genetic risk factor for AD, it accounts for a fairly small fraction of disease in the population. The effect of the 4 allele on annual decline in episodic memory is significantly stronger than its effect on decline in other cognitive systems. Notably, the 2 allele has an equal and opposite effect. Thus, ApoE allele status influences risk of AD by a relatively selective effect on episodic memory. Mechanistically, the role of APoE in AD needs to be established in terms of its gene expression, which ultimately controls levels of various ApoE isoforms. Transcriptional regulation suggests complex regulation of this gene and the resultant ApoE protein in injured neurons. We discuss the characteristics of ApoE regulatory elements, including their interactions with different transcription factors, to understand ApoE gene expression. Thus, ApoE4 contributes to the pathogenesis of AD, but additional environmental risk factors will also be identified independent of ApoE and other genetic polymorphisms.

The central melanocortin system and the integration of short- and long-term regulators of energy homeostasis.

The importance of the central melanocortin system in the regulation of energy balance is highlighted by studies in transgenic animals and humans with defects in this system. Mice that are engineered to be deficient for the melanocortin-4 receptor (MC4R) or pro-opiomelanocortin (POMC) and those that overexpress agouti or agouti-related protein (AgRP) all have a characteristic obese phenotype typified by hyperphagia, increased linear growth, and metabolic defects. Similar attributes are seen in humans with haploinsufficiency of the MC4R. The central melanocortin system modulates energy homeostasis through the actions of the agonist, alpha-melanocyte-stimulating hormone (alpha-MSH), a POMC cleavage product, and the endogenous antagonist AgRP on the MC3R and MC4R. POMC is expressed at only two locations in the brain: the arcuate nucleus of the hypothalamus (ARC) and the nucleus of the tractus solitarius (NTS) of the brainstem. This chapter will discuss these two populations of POMC neurons and their contribution to energy homeostasis. We will examine the involvement of the central melanocortin system in the incorporation of information from the adipostatic hormone leptin and acute hunger and satiety factors such as peptide YY (PYY(3-36)) and ghrelin via a neuronal network involving POMC/cocaine and amphetamine-related transcript (CART) and neuropeptide Y (NPY)/AgRP neurons. We will discuss evidence for the existence of a similar network of neurons in the NTS and propose a model by which this information from the ARC and NTS centers may be integrated directly or via adipostatic centers such as the paraventricular nucleus of the hypothalamus (PVH).

Role of acyl-coenzyme a: cholesterol acyltransferase activity in the processing of the amyloid precursor protein.

Alzheimer's disease (AD) is a neurodegenerative disorder characterized by progressive memory deficit, cognitive impairment, and personality changes accompanied by specific structural abnormalities in the brain. Deposition of amyloid-beta (Abeta) peptide into senile plaques is a consistent feature of the brains of patients affected by AD. Studies with both animal and cellular models of AD have shown that cholesterol homeostasis and distribution regulate Abeta generation. We have provided genetic, biochemical, and metabolic evidence that implicates intracellular cholesterol distribution, rather than total cholesterol levels, in the regulation of Abeta generation. This minireview focuses on the role of acyl-coenzyme A: cholesterol acyltransferase activity (ACAT) in Abeta generation. In genetically mutant cell lines that overproduce cholesterol but cannot synthesize cholesteryl esters (CEs) because of deficient ACAT activity, Abeta production is almost completely inhibited. Acyl-coenzyme A: cholesterol acyltransferase activity (ACAT) inhibitors, currently being developed for the treatment and prevention of atherosclerosis, reduce CE levels and Abeta generation by up to 50% in cell culture models of AD. Future mechanistic and transgenic animal studies are needed to evaluate the potential use of ACAT inhibitors in the therapeutic treatment or prevention of AD.

CRH, Stress, and Major Depression: A Psychobiological Interplay

Major depressive disorder (MDD) is a complex disease and is one of the leading causes of disability in our society. The provoking factors are multiple; acute and chronic psychological stress, severe early trauma experiences, somatic disease, and genetic factors all play a role. This review focuses on hyperdrive of corticotropin-releasing hormone (CRH) as the fundamental neurobiological correlate of MDD. CRH plays a key role in the adaptation to acute stress, but chronic CRH hyperdrive leads to a number of disadvantageous emotional and somatic effects. The evidence that the HPA axis is hyperactive in MDD, probably as a result of a primary hyperdrive of CRH, comes from multiple sources: biochemical studies, functional HPA axis tests, neuroimaging and postmortem studies, and clinical trials with HPA axis-related compounds. The liability to develop CRH hyperdrive is probably partly genetic. For a number of relevant genes, transgenic animal studies and human association studies indicate a role in HPA axis regulation and the liability to develop CRH hyperdrive. These data are reviewed. Finally, early adverse experience can produce a lasting effect on HPA axis regulation as well, probably leading to a lifelong tendency to develop chronic CRH hyperdrive in response to stress. This has been shown in a number of animal studies, and recently some data in humans with early trauma have become available as well. Taken together, these findings allow formulating an integrative hypothesis, with CRH hyperdrive at the core, bridging the old dichotomy between biology and psychology in our thinking about MDD.

Inducible expression of green fluorescent protein in porcine tracheal epithelial cells by the bovine tracheal antimicrobial peptide promoter.

Tracheal antimicrobial peptides (TAP) are expressed primarily in respiratory epithelial cells of cattle. The TAP expression is inducible upon challenge with bacteria and bacterial lipopolysaccharide (LPS). In pigs, a promoter that can be activated by bacterial infection has yet to be identified. The objective of this study was to use green fluorescent protein (GFP) as a reporter gene to determine the function and inducibility of the bovine TAP promoter in porcine primary tracheal epithelial cells. Thus, evaluating the feasibility of using this promoter to direct transgene expression in porcine cells. The percentage of GFP expressing cells increased in response to LPS challenge in both a dose-dependent and time-dependent manner (p < 0.05). Moreover, when the intensity of the GFP fluorescence was measured, it was observed that the percentage of cells that have a high intensity of GFP fluorescence, also increased gradually as LPS dose increased, the difference between the unchallenged (control) and challenged group become statistically significant at the concentration of 100 ng/mL after 36 h LPS challenge (p < 0.05). The level of induced-expression driven by the TAP promoter was 67.8 +/-12.2% that of the cytomegalovirus (CMV) promoter. The intensity of GFP fluorescence by the TAP promoter was 39.8 +/- 7.6% when compared to the expression driven by the CMV promoter. These data suggest the TAP promoter functions at a lower, but comparable, level to the strong CMV promoter. Our data demonstrated that the bovine TAP promoter was functional in porcine primary tracheal epithelial cells. The ability of the TAP promoter to control gene expression in an inducible manner in the porcine respiratory tract presents an important application potential in transgenic animal studies.

Substance P antagonists: meet the new drugs, same as the old drugs? Insights from transgenic animal models.

Antidepressants that primarily target the reuptake of monoamines have been highly successful treatments. However, therapies with these drugs still have several drawbacks, namely severe side effects, delays in the onset of action, and a significant percentage of non-responders. Recently, non-peptidic antagonists of the neurokinin 1 receptor, or substance P antagonists, have emerged as a novel class of drugs with antidepressant efficacy that is comparable to current drugs, but a potentially reduced side effect profile. This review summarizes the pre-clinical evidence derived from pharmacological and transgenic animal studies that suggests an important role for the substance P/neurokinin 1 system in anxiety and depression. Also, potential mechanisms by which substance P antagonists may produce their therapeutic effects are discussed.

Podocan, a Novel Small Leucine-rich Repeat Protein Expressed in the Sclerotic Glomerular Lesion of Experimental HIV-associated Nephropathy

Growing evidence suggests that human immunodeficiency virus (HIV)-1 infection of podocytes plays a central role in the glomerular disease of HIV-associated nephropathy (HIVAN). As an approach to identify host genes involved in the pathogenesis of the sclerotic glomerular lesion in HIVAN, representational difference analysis of cDNA was used to identify differentially expressed genes in HIV-1 transgenic and nontransgenic podocytes. We isolated a novel member of the small leucine-rich repeat (SLR) protein family, podocan, that is expressed at high levels in the HIV-1 transgenic podocytes. In normal embryonic kidney, a 3.2-kb podocan transcript was detected at low levels, and expression increased dramatically within 24 h following birth. Expression of a 2.3-kb transcript became evident after birth and gradually increased to 50% of the total podocan RNA in the mature kidney. Phylogenetically, podocan represents a new class in the SLR protein gene family, an expanding protein family sharing homology with the small leucine-rich repeat proteoglycans. The 3.2-kb transcript encodes a predicted 611-amino acid secretory protein with 20 leucine-rich repeats, a unique N-terminal cysteine-rich cluster pattern and a highly acidic C-terminal domain. In situ hybridization of normal kidney revealed podocan mRNA expression in podocytes and likely vascular endothelial cells within the glomerulus. The immunohistochemical staining pattern of podocan protein in normal kidney glomeruli was consistent with that of the glomerular basement membrane, and staining was markedly increased in sclerotic glomerular lesions in the transgenic HIVAN model. Thus, podocan defines a new class within the SLR protein family and is a previously unrecognized component of the sclerotic glomerular lesion that develops in the course of experimental HIVAN.

Superoxide dismutases in the lung and human lung diseases.

The lungs are directly exposed to higher oxygen concentrations than most other tissues. Increased oxidative stress is a significant part of the pathogenesis of obstructive lung diseases such as asthma and chronic obstructive pulmonary disease, parenchymal lung diseases (e.g., idiopathic pulmonary fibrosis and lung granulomatous diseases), and lung malignancies. Lung tissue is protected against these oxidants by a variety of antioxidant mechanisms among which the superoxide dismutases (SODs) are the only ones converting superoxide radicals to hydrogen peroxide. There are three SODs: cytosolic copper-zinc, mitochondrial manganese, and extracellular SODs. These enzymes have specific distributions and functions. Their importance in protecting lung tissue has been confirmed in transgenic and knockout animal studies. Relatively few studies have been conducted on these enzymes in the normal human lung or in human lung diseases. Most human studies suggest that there is induction of manganese SOD and, possibly, extracellular SOD during inflammatory, but not fibrotic, phases of parenchymal lung diseases and that both copper-zinc SOD and manganese SOD may be downregulated in asthmatic airways. Many previous antioxidant therapies have been disappointing, but newly characterized SOD mimetics are being shown to protect against oxidant-related lung disorders in animal models.