Vomeronasal phenotype and behavioral alterations in G alpha i2 mutant mice

Several social and reproductive behaviors are under the influence of the vomeronasal (VN) organ; VN neurons detect odorous molecules emitted by individuals of the same species. There are two types of VN neurons, and these differ in their expression of chemosensory receptors and G protein subunits. The significance of this dichotomy is largely unknown. VN neurons express high levels of either G alpha i2 or G alpha o. A mouse line carrying a targeted disruption of the G alpha i2 gene offered the opportunity for studying the effects of a lack of receptor signaling through the heterotrimeric Gi2 protein in one VN cell type. As a consequence of this deficiency, the number of VN neurons that normally express G alpha i2 is decreased by half. These residual neurons are defective in eliciting a response in their target neurons in the accessory olfactory bulb. Moreover, G alpha i2 mutant mice show alterations in behaviors for which an intact VN organ is known to be important. Display of maternal aggressive behavior is severely blunted, and male mice show significantly less aggression toward an intruder. However, male mice show unaltered sexual-partner preference. This suggests that the two types of VN neurons may have separate functions in mediating behavioral changes in response to chemosensory information.     

Nighttime immunoreactive somatostatin content of the median eminence in hypo- and hyperthyroid rats

The median eminence content of immunoreactive somatostatin (IRS) was measured by radioimmunoassay in 107 male albino rats, who were either hypothyroid after surgical thyroidectomy (N = 38), hyperthyroid following a subcutaneous implant of 5 mg of L-thyroxine (N = 36), or otherwise untreated (N = 33). Thyroid function was assessed by determining plasma levels of T4 and TSH from trunk blood obtained at the time of decapitation. Subgroups of animals from the 3 groups were killed either before (1800 hr), during (2200, 0200, 0400 hr), or after the dark portion of their 14:10 LD photoperiod. Although no changes in median eminence IRS content were found throughout the period of study within any of the 3 groups, hypothyroid animals (297.23 +/- 13.47 ng per ME; 620.41 +/- 58 ng IRS/mg protein) had a significantly lower median eminence IRS concentration than untreated rats (355.86 +/- 16.55 ng of IRS per ME, P less than 0.01; 906.86 +/- 96.38 ng IRS/mg protein, P less than 0.05) and hyperthyroid animals (384.12 +/- 14.67 ng per ME, P less than 0.001; 874.1 +/- 104.5 ng IRS@mg protein, P less than 0.05). It is concluded, that the feedback of thyroid hormones on the hypothalamic-pituitary axis during thyroid hormone excess in vivo, contrary to what occurs in hypothyroidism, is probably independent of hypothalamic somatostatin.     

Effect of hypo- and hyperthyroid states on phospholipid composition in developing rat heart

The aim of this study was to examine the effect of hypo- and hyperthyroidism on the phospholipid composition in developing rat heart. The hypothyroid state (PTU) was induced by 0.05% 6-n-propyl-2-thiouracil in drinking water given to nursing mothers from the postnatal day 2–21. The hyperthyroidism (T₃) was made by daily injection of 3,3,5-triiodo-L-thyronine (10 g/100 g body wt) to newborns in the same time period. Age matched intact littermates were taken as euthyroid controls. PTU decreased the concentration of total phospholipids (PL), choline phosphoglycerides (PC), ethanolamine phosphoglycerides (PE) and diphosphatidylglycerol (DPG) and increased the proportion of plasmalogen component of PE (PLPE). T₃ increased the concentration of PL, PC, PE, DPG and decreased PLPE in comparison with euthyroid controls. The ratio of saturated/unsaturated fatty acids (FA) in PE was decreased in PTU and increased in T₃ group. The ratio of n-6/n-3 polyunsaturated FA in PC, PE and phosphatidylinositol (PI) was increased in PTU due to increase of 18:2n-6 and decrease of 22:6n-3 proportion. T₃ decreased this ratio because of decline in 20:4n-6 and rise in 22:6n-3 proportion. Both hypo- and hyperthyroidism decreased the ratio of 20:4n-6/18:2n-6 in the majority of phospholipids. PTU decreased the unsaturation index in PC, PI and phosphatidylserine. It is concluded that thyroid state plays an essential role in the development of membrane phospholipid components in cardiac membranes during the early postnatal period.     

A brefeldin A-like phenotype is induced by the overexpression of a human ERD-2-like protein, ELP-1.

Brefeldin A (BFA) is a unique drug affecting the molecular mechanisms that regulate membrane traffic and organelle structure. BFA's ability to alter retrograde traffic from the Golgi to the endoplasmic reticulum (ER) led us to ask whether the ERD-2 retrieval receptor, proposed to return escaped ER resident proteins from the Golgi, might either interfere with or mimic the effects of the drug. When either human ERD-2 or a novel human homolog (referred to as ELP-1) is overexpressed in a variety of cell types, the effects are phenotypically indistinguishable from the addition of BFA. These include the redistribution of the Golgi coat protein, beta-COP, to the cytosol, the loss of the Golgi apparatus as a distinct organelle, the mixing of this organelle with the ER, the addition of complex oligosaccharides to resident ER glycoproteins, and the block of anterograde traffic. Thus, these receptors may provide signals that regulate retrograde traffic between the Golgi and the ER.     

Cardiac-specific overexpression of the alpha(1) subunit of the L-type voltage-dependent Ca(2+) channel in transgenic mice. Loss of isoproterenol-induced contraction.

The L-type voltage-dependent calcium channel (L-VDCC) regulates calcium influx in cardiac myocytes. Activation of the beta-adrenergic receptor (betaAR) pathway causes phosphorylation of the L-VDCC and that in turn increases Ca(2+) influx. Targeted expression of the L-VDCC alpha(1) subunit in transgenic (Tg) mouse ventricles resulted in marked blunting of the betaAR pathway. Inotropic and lusitropic responses to isoproterenol and forskolin in Tg hearts were significantly reduced. Likewise, Ca(2+) current augmentation induced by iso- proterenol and forskolin was markedly depressed in Tg cardiomyocytes. Despite no change in betaAR number, isoproterenol-stimulated adenylyl cyclase activity was absent in Tg membranes and NaF and forskolin responses were reduced. We postulate an important pathway for regulation of the betaAR by Ca(2+) channels.     

Paraoxonase 1 overexpression in mice and its effect on high-density lipoproteins.

Paraoxonase (PON1) is a high-density lipoprotein (HDL)-associated enzyme believed to protect against the early events of atherogenesis by its ability to hydrolyze oxidized phospholipids. A transgenic mouse overexpressing PON1 (mPON1) was developed to address the question of whether overexpression of PON1 is important in protecting HDL function during oxidative stress. Transgenic mice were obtained that have up to a 5-fold increase in mPON1 activity measured as arylesterase activity [52.7 +/- 17.3 U/ml versus 251.7 +/- 25.1 U/ml for wild-type (WT) and mPON1 high expressers, respectively]; this increase in mPON1 activity was reflected by a 5.3-fold increase in relative mass of the enzyme. Excess mPON1 was associated solely with HDL but did not alter HDL composition, size, or charge. Lecithin:cholesterol acyltransferase (LCAT) on HDL is a sensitive indicator of oxidative stress; exposure of plasmas from both WT and mPON1 overexpresser mice to 0.4 mM copper ions for 2 h showed a 30-40% protection of LCAT activity in mPON1 overexpressers compared to WT. Excess mPON1 also inhibited lipid hydroperoxide formation on HDL. These data strongly suggest that overexpression of mPON1 protects HDL integrity and function.     

Adenovirus-mediated overexpression of sphingomyelin synthases 1 and 2 increases the atherogenic potential in mice

Sphingomyelin synthase 1 (SMS1) and SMS2 are two isoforms of SMS, the last enzyme for sphingomyelin (SM) biosynthesis. To evaluate the role of SMS in vivo in terms of plasma lipoprotein metabolism, we generated recombinant adenovirus vectors containing human SMS1 cDNA (AdV-SMS1), SMS2 cDNA (AdV-SMS2), or the reporter LacZ cDNA (AdV-LacZ) as a control. On day 7 after intravenous infusion of 2 x 10(11) particles of both AdV-SMS1 and AdV-SMS2 into mice, liver SMS1 and SMS2 mRNA levels as well as SMS activity were significantly increased (2.5-, 2.7-, 2.1-, and 2.3-fold, respectively; P < 0.001). Lipoprotein analysis indicated that AdV-SMS1 and AdV-SMS2 treatment caused no changes of total SM and cholesterol levels but significantly decreased HDL-SM and HDL-cholesterol (42% and 38%, and 27% and 25%, respectively; P < 0.05). It also significantly increased non-HDL-SM and non-HDL-cholesterol levels (50% and 35%, and 64% and 61%, respectively; P < 0.05) compared with AdV-LacZ controls. SDS-PAGE showed a significant increase in apolipoprotein B (apoB; P < 0.01) but no changes in apoA-I levels. Moreover, we found that non-HDL from both AdV-SMS1- and AdV-SMS2-treated mice was significantly aggregated after treatment with a mammalian sphingomyelinase, whereas lipoproteins from control animals did not aggregate. To investigate the mechanism of HDL changes, we measured liver scavenger receptor class B type I (SR-BI) levels by Western blot. We found that AdV-SMS1 and AdV-SMS2 mouse liver homogenates contained 50% and 55% higher SR-BI levels than in controls, whereas no change was observed in hepatic ABCA1 levels. An HDL turnover study revealed an increase of plasma clearance rates for [3H]cholesteryl oleyl ether-HDL but not for [125I]HDL in both AdV-SMS1 and AdV-SMS2 mice compared with controls. In conclusion, adenovirus-mediated SMS1 and SMS2 overexpression increased lipoprotein atherogenic potential. Such an effect may contribute to the increased plasma SM levels observed in animal models of atherosclerosis and in human patients with coronary artery disease.     

Platelet alpha2-macroglobulin and alpha1-antitrypsin.

Subcellular membrane and granule fractions derived from human platelets contain immunologically identifiable alpha2-macroglobulin and alpha1-antitrypsin. These platelet-derived inhibitors show a reaction of immunologic identity when compared to alpha2-macroglobulin and alpha1-antitrypsin purified from human plasma. Further, the platelet protease inhibitors possessed a similar subunit polypeptide chain structure to their plasma counterparts as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis. Studies of the binding of radiolabeled trypsin to the various solubilized platelet subcellular fractions suggest that the granule-associated alpha2-macroglobulin and alpha1-antitrypsin, as well as membrane-associated alpha2-macroglobulin were functionally active. Quantitatively, circulating platelets contain relatively small concentrations of these inhibitors as compared to platelet-associated fibrinogen and factor VIIIAGN. Platelet protease inhibitors may modulate the protease-mediated events involved in the formation of hemostatic plugs and thrombi.     

Left ventricular hypertrophy in mice with a cardiac-specific overexpression of interleukin-1

Recent studies have identified the importance of proinflammatory cytokines in the development of left ventricular (LV) hypertrophy. However, the precise role of interleukin-1 (IL-1), one of the major proinflammatory cytokines, in the myocardium is not fully understood. In this study, we investigated the pathophysiological consequences of cardiac expression of IL-1 in vivo. We generated mice with a cardiac-specific overexpression of human IL-1alpha. We then analyzed their heart morphology and functions. Histological and echocardiographic analyses revealed concentric LV hypertrophy with preserved LV systolic function in the mice. Our results suggest that myocardial expression of IL-1 is sufficient to cause LV hypertrophy.     

Studies on 5'-nucleotidase activity in blood serum, tissues and liver mitochondrial fraction of normal, hypo- and hyperthyroid rats.

Clinical and experimental studies have previously shown the effect of thyroid state on the activity of non-specific phosphohydrolases. This research was undertaken to determine the influence of thyroid hormones on the activity of specific phosphohydrolase-5'-nucleotidase. The activity of 5'-nucleotidase was estimated in blood serum, tissues (liver, kidney, lung, brain) and mitochondrial fraction of liver of normal, methylothiouracil-induced hypothyroid and thyroxin-induced hyperthyroid rats of Wistar strain. The experiments have shown that the hypothyroid state induced decrease of the activity of 5'-nucleotidase in all studied materials, and on the contrary, the hyperthyroidism induced the increase of the enzyme activity. The hypothetical mechanism of the effect of thyroid hormones on the processes of feed back regulation of oxydative phosphorylation and delivery of substrates and oxygen is presented. The key role of 5'-nucleotidase in this mechanism is discussed.     

Surface phenotype of responder cells in the syngeneic mixed lymphocyte reaction in mice

The organ distribution and surface phenotype of SMLR responder cells has been investigated. Nylon-wool-passed spleen cells, which proliferate in response to mitomycin-C-treated syngeneic spleen cells, are Thy 1.2⁺ Ly 1⁺2^?3^?. SMLR responder cells are not confined to the spleen since nylon-wool-nonadherent lymph node cells as well as unfractionated thoracic duct lymphocytes show activity. Responder cells have characteristics of mature T cells since cortisone-resistant thymocytes, but not thymocytes from untreated mice, are capable of SMLR response. In addition, naturally occurring thymocytotoxic antibody (NTA), which in our experiments exhibits cytotoxicity only for thymocytes, does not appear to affect the subpopulation of the T cells which respond in the SMLR.     

Molecular cloning, overexpression and characterization of human interleukin 1alpha

Interleukin-1 alpha (IL-1alpha) regulates a wide range of important cellular processes. In this study for the first time, we report the cloning, expression, biophysical, and biological characterization of the human interleukin-1alpha. Human IL-1alpha has been expressed in Escherichia coli in high yields ( approximately 4mg per liter of the bacterial culture). The protein was purified to homogeneity ( approximately 98% purity) using affinity chromatography and size exclusion chromatography. Results of the steady-state fluorescence and 2D NMR experiments show that the recombinant IL-1alpha is in a folded conformation. Far-UV circular dichroism (CD) data suggest that IL-1alpha is an all beta-sheet protein with a beta-barrel architecture. Isothermal titration calorimetry (ITC) experiments show that the recombinant IL-1alpha binds strongly (K(d) approximately 5.6 x 10(-7) M) to S100A13, a calcium binding protein that chaperones the in vivo release of IL-1alpha into the extracellular compartment. Recombinant IL-1alpha was observed to exhibit strong cytostatic effect on human umbilical vascular endothelial cells. The findings of the present study not only pave way for an in-depth structural investigation of the molecular mechanism(s) underlying the non-classical release of IL-1alpha but also provide avenues for the rational design of potent inhibitors against IL-1alpha mediated pathogenesis.     

Thymosin alpha 1 potentiates interleukin 2-induced cytotoxic activity in mice

We have investigated the effects of interleukin 2 (IL-2) on cytotoxic activity of spleen lymphocytes, from normal and cyclophosphamide (200 mg/kg) or B-16 melanoma suppressed mice, after in vitro or in vivo pretreatment with thymosin alpha 1 (TA1). The results of this study indicate that pretreatment in vitro (100 ng/ml for 1 hr) or in vivo (200 micrograms/kg/day for 4 days) with thymosin alpha 1 (TA1), significantly increased the IL-2 (from 100 to 500 U/ml) in vitro induced cytotoxic activity of spleen lymphocytes, collected from both normal and cyclophosphamide and tumor-suppressed animals, against both YAC-1 (NK sensitive) and MBL-2 (NK resistant) cell lines. The potential use in combination of these two different biological response modifiers, useful in enhancing the immunological responses to IL-2 of lymphocytes, may provide a novel model of immunotherapeutic intervention in cancer.     

Subtypes of alpha 1- and alpha 2-adrenergic receptors.

The adrenergic receptors are members of the superfamily of G protein-coupled receptors. There are three major types of adrenergic receptors: alpha 1, alpha 2, and beta. Each of these three major types can be divided into three subtypes. Within the alpha 1-adrenergic receptors, alpha 1A and alpha 1B subtypes have been defined pharmacologically on the basis of reversible antagonists, such as WB4101 and phentolamine, and the irreversible antagonist chloroethylclonidine. In at least some tissues the mechanism of action of the alpha 1A subtype is related to activation of a calcium channel, whereas the alpha 1B receptor exerts its effect through the second messenger inositol trisphosphate. Both of these receptor subtypes as well as a third, the alpha 1C, have been identified by molecular cloning. Three pharmacological subtypes of the alpha 2-adrenergic receptor have also been identified. Prototypic tissues and cell lines in continuous culture have been developed for each of these subtypes, which facilitated their study. The definition of the alpha 2 subtypes has been based on radioligand binding data and more limited functional data. All three subtypes have been shown to inhibit the activation of adenylate cyclase and thus reduce the levels of cAMP. Three alpha 2-adrenergic receptor subtypes have been identified by molecular cloning in both the human and rat species. There is reasonable agreement between the pharmacological identified subtypes and those identified by molecular cloning.     

Net-targeted mutant mice develop a vascular phenotype and up-regulate egr-1

The ternary complex factors (TCFs) Net, Elk-1 and Sap-1 regulate immediate early genes through serum response elements (SREs) in vitro, but, surprisingly, their in vivo roles are unknown. Net is a repressor that is expressed in sites of vasculogenesis during mouse development. We have made gene-targeted mice that express a hypomorphic mutant of Net, Net delta, which lacks the Ets DNA-binding domain. Strikingly, homozygous mutant mice develop a vascular defect and up-regulate an immediate early gene implicated in vascular disease, egr-1. They die after birth due to respiratory failure, resulting from the accumulation of chyle in the thoracic cage (chylothorax). The mice have dilated lymphatic vessels (lymphangiectasis) as early as E16.5. Interestingly, they express more egr-1 in heart and pulmonary arteries at E18.5. Net negatively regulates the egr-1 promoter and binds specifically to SRE-5. Egr-1 has been associated with pathologies involving vascular stenosis (e.g. atherosclerosis), and here egr-1 dysfunction could possibly be associated with obstructions that ultimately affect the lymphatics. These results show that Net is involved in vascular biology and egr-1 regulation in vivo.     

Ablation of renin-expressing juxtaglomerular cells results in a distinct kidney phenotype

Renin-expressing cells are peculiar in that they act as differentiated cells, producing the hormone renin, while they also seem to act as progenitors for other renal cell types. As such, they may have functions independent of their ability to generate renin/angiotensin. To test this hypothesis, we ablated renin-expressing cells during development by placing diphtheria toxin A chain (DTA) under control of the Ren1d mouse renin promoter by homologous recombination in a two-renin gene strain (Ren2 and Ren1d). Renin-expressing cells are essentially absent from kidneys in homozygotes (DTA/DTA) which, unlike wild-type mice, are unable to recruit renin-expressing cells when homeostasis is threatened. In contrast, renin staining in the submandibular gland (SMG), which expresses mainly Ren2, is normal. Homozygous mice survive normally, but the kidneys are small and have morphological abnormalities: 25% of the glomeruli are hyperplastic or atrophic, tubules are dilated and atrophic, and areas of undifferentiated cells exist near the atrophic glomeruli and tubules. However, in contrast to the very abnormal renal vessels found when renin-angiotensin system genes are deleted, the kidney vessels in homozygotes have normal wall thickness and no decrease in lumen size. Homozygotes have severely reduced kidney and plasma renin concentrations and females have reduced blood pressure. Homozygotes have elevated blood urea nitrogen and potassium levels, which are suggestive of altered renal function. We conclude that renin cells per se are necessary for the morphological integrity of the kidney and may have a role in maintenance of normal kidney function.