Technicolour transgenics: imaging tools for functional genomics in the mouse

Over the past decade, a battery of powerful tools that encompass forward and reverse genetic approaches have been developed to dissect the molecular and cellular processes that regulate development and disease. The advent of genetically-encoded fluorescent proteins that are expressed in wild type and mutant mice, together with advances in imaging technology, make it possible to study these biological processes in many dimensions. Importantly, these technologies allow direct visual access to complex events as they happen in their native environment, which provides greater insights into mammalian biology than ever before.     

Role of endothelium-derived relaxing factors in the renal response to vasoactive agents in hypothyroid rats

This study analyzed the role of nitric oxide (NO) and endothelium-derived hyperpolarizing factor (EDHF) in the abnormal renal vascular reactivity of hypothyroid rats. Renal responses to vasoconstrictors [VC: phenylephrine (PHE) and ANG II] and vasodilators [VD: ACh, sodium nitroprusside (SNP), and papaverine (PV)] were studied in kidneys from control and hypothyroid rats under normal conditions and after NO or EDHF blockade. NO was blocked by the administration of Nomega-nitro-l-arginine methyl ester (l-NAME) and EDHF by the administration of tetraethylammonium (TEA) or by an increased extracellular K+. The response to VC was also evaluated after endothelium removal. Hypothyroid kidneys showed reduced responsiveness to PHE and a normal response to ANG II. l-NAME and TEA administration produced an increased sensitivity to PHE and to ANG II in control preparations. l-NAME also increased the response to PHE in hypothyroid kidneys, but the differences between control and hypothyroid kidneys were maintained. TEA administration did not change the response to either VC in hypothyroid preparations. In endothelium-removed preparations, TEA was unable to increase pressor responsiveness to VC. Hypothyroid kidneys showed reduced responsiveness to ACh and SNP and normal response to PV. The differences between hypothyroid and control preparations in the responses to ACh and SNP were maintained after l-NAME or increased K+. In conclusion, this study shows that 1) the attenuated response to PHE in hypothyroidism is not related to an increased production of endothelium-derived relaxing factors NO and EDHF; 2) the response to VC in hypothyroid preparations is insensitive to EDHF blockade; and 3) hypothyroid preparations have a reduced reactivity to the NO donor, and NO-independent vasodilatation remains unaffected.     

Effect of Chronic Manganese Treatment on Adenosine Tissue Levels and Adenosine A2a Receptor Binding in Diverse Regions of Mouse Brain

In the present study the effects of chronic manganese (Mn) treatment on adenosine A_2a receptor binding in mouse brain have been assessed. Male albino mice were divided in two groups: In the Mn-treated group, the animals were injected intraperitoneally (i.p.) with MnCl₂ (5 mg/kg/day) five days per week during 9 weeks; in the control group, they were injected likewise with a saline solution. A significant decrease of the Kd without alteration of Bmax in the cerebellum and, an increase of the Kd and Bmax in hippocampus of mice treated with Mn were found. Also, an increase of Kd in frontal cortex was observed. The binding parameters in caudate nucleus, olfactory bulb and hypothalamus were not altered by Mn. A significant decrease in the adenosine concentration in caudate nucleus, olfactory bulb and hypothalamus, without significant changes in hippocampus, frontal cortex and cerebellum was also detected. These findings suggest that chronic administration of Mn could affect adenosine receptor function and turnover, depending on the brain region analyzed.     

Function and Expression of CD44 during Spreading, Migration, and Invasion of Murine Carcinoma Cells

The cell surface glycoprotein CD44 is proposed as a main participant in cell adhesion and migration. We studied the function, expression, and distribution of CD44 in the invasive and metastatic F3II murine carcinoma cell line during adhesion, spreading, migration, and invasion. A mAb anti-CD44 (KM 201) dramatically blocked F3II cell adhesion on both plastic and hyaluronic acid coatings, as well as spreading on uncoated plastic surfaces (P< 0.01). KM201 mAb significantly inhibited F3II cell migration and invasion in Transwell chambers. Immunocytochemistry of spreading cells revealed that CD44 distributed in bands on the cell surface, particularly in the tip of leading edges and in the perinuclear zones of the cell membrane. CD44 antigen was never detected in filopodia or lamellipodia nor in focal adhesion-like structures, but was also detectable as strong interlamellar bands. Fully spread cells showed a decreased CD44 signal compared to cells in early stages of spreading. This decrease correlated with a reduced expression of CD44 as detected by Western blot. We also investigated the signals that may regulate CD44 expression in F3II cells. Treatment of F3II cells, with phorbol myristate acetate (PMA) or phosphatidic acid (PA, the product of PLD-dependent hydrolysis of phosphatidylcholine), significantly enhanced CD44 expression. Conversely, the treatment of F3II cells with H7, a specific PKC inhibitor, or propranolol, which blocks PA conversion to DAG, significantly decreased CD44 expression levels. These results suggest the involvement of PKC and PLD pathways in CD44 expression. These results demonstrate that CD44 plays an important role during F3II cells adhesion, spreading, migration, and invasion. In addition we provide information linking the PLD- and PKC-dependent pathways with the regulation of CD44 expression.     

Cytoskeletal Polarization of T Cells Is Regulated by an Immunoreceptor Tyrosine-based Activation Motif–dependent Mechanism

Abstract. Binding of a T cell to an appropriate antigen-presenting cell (APC) induces the rapid reorientation of the T cell cytoskeleton and secretory apparatus towards the cell–cell contact site in a T cell antigen receptor (TCR) and peptide/major histocompatibility complex–dependent process. Such T cell polarization directs the delivery of cytokines and cytotoxic mediators towards the APC and contributes to the highly selective and specific action of effector T cells. To study the signaling pathways that regulate cytoskeletal rearrangements in T lymphocytes, we set up a conjugate formation assay using Jurkat T cells as effectors and cell-sized latex beads coated with various antibodies as artificial APCs. Here, we report that beads coated with antibodies specific for the TCR-CD3 complex were sufficient to induce T cell polarization towards the bead attachment site, as judged by reorientation of the microtubule-organizing center (MTOC) and localized actin polymerization. Thus, these cytoskeletal changes did not depend on activation of additional coreceptors. Moreover, single subunits of the TCR complex, namely TCR-ζ and CD3ε, were equally effective in inducing cytoskeletal polarization. However, mutagenesis of the immunoreceptor tyrosine-based activation motifs (ITAMs), present three times in TCR-ζ and once in CD3ε, revealed that the induction of cytoskeletal rearrangements required the presence of at least one intact ITAM. In agreement with this result, lack of functional Lck, the protein tyrosine kinase responsible for ITAM phosphorylation, abolished both MTOC reorientation and polarized actin polymerization. Both inhibitor and transient overexpression studies demonstrated that MTOC reorientation could occur in the absence of Ras activation. Our results suggest that APC-induced T cell polarization is a TCR-mediated event that is coupled to the TCR by the same signaling motif as TCR-induced gene activation, but diverges in its distal signaling requirements.     

Two different primate species express an identical functional MHC class I allele

 The products of the highly polymorphic and variable major histocompatibility complex (MHC) class I loci play a crucial role in host defenses against infectious disease. While similar alleles have been found in closely related species, sharing of a functional MHC class I allele between two species has never been reported. Here we show that an identical functional MHC class I molecule is present in two different primate species with an approximate divergence time of 0.7 million years. Lymphocytes from the red-crested tamarin (Saguinus geoffroyi) expressed an MHC class I allele (Sage-G ^* 01) that was identical in coding sequence to an MHC class I allele (Saoe-G ^* 08) found in the cotton-top tamarin (Saguinus oedipus). Furthermore, influenza virus-specific cytotoxic T lymphocytes (CTLs) generated in the cotton-top tamarin killed lymphocytes expressing the influenza virus nucleoprotein (NP) from the red-crested tamarin. Since the influenza virus NP epitope is bound by Saoe-G^*08 in the cotton-top tamarin, it is likely that this molecule is functional in both species. These data provide the first evidence that functional MHC class I molecules can be maintained entirely intact in two separate species. Received: 6 June 1997 / Revised: 21 July 1997     

Functional Activity of Blood Polymorphonuclear Leukocytes as an Oxidative Stress Biomarker in Human Subjects

In the present work, we studied the role of polymorphonuclear leukocytes (PMN) in aged individuals and coronary heart disease (CHD)-bearing patients, two physiopathological processes associated with overproduction of reactive oxygen species (ROS). The effects of antioxidant supplementation on the functional activity of PMN from CHD patients were also determined. The function of PMNs was evaluated by measuring of phagocytosis, killing activity, and ROS production. Luminol amplified chemiluminescence (CL) was used to estimate ROS production by stimulated PMNs. Total cholesterol and the LDL-cholesterol fraction from CHD patients were found to be higher than those recommended, returning to normal levels after antioxidant therapy. PMN CL of CHD patients was found to be higher than the associated control groups. Antioxidant therapy administrated to CHD patients lead to an increase in the killing activity accompanied by a decrease in PMN CL of these subjects. The study also showed that killing activity of PMN from human subjects over 60 years was significantly lower than the activity measured in younger subjects. PMN CL produced after stimulation was found to be positively correlated with the increasing age of human subjects (r = .946, p < .01).