The human lysozyme promoter directs reporter gene expression to activated myelomonocytic cells in transgenic mice.

The 5' region of the human lysozyme gene from -3500 to +25 was fused to a chloramphenicol acetyltransferase (CAT) reporter gene and three transgenic founder mice were obtained. All three transgenic lines showed the same pattern of CAT enzyme expression in adult mouse tissues that was consistent with the targeting of elicited, activated macrophages in tissues and developing and elicited granulocytes. In normal mice high CAT enzyme activity was found in the spleen, lung, and thymus, tissues rich in phagocytically active cells, but not in many other tissues, such as the gut and muscle, which contain resident macrophages. Cultured resident peritoneal macrophages and cells elicited 18 hr (granulocytes) and 4 days (macrophages) after injection of sterile thioglycollate broth expressed CAT activity. Bacillus Calmette-Guérin infection of transgenic mice resulted in CAT enzyme expression in the liver, which contained macrophage-rich granulomas, whereas the liver of uninfected mice did not have any detectable CAT enzyme activity. Although the Paneth cells of the small intestine in both human and mouse produce lysozyme, the CAT gene, under the control of the human lysozyme promoter, was not expressed in the mouse small intestine. These results indicate that the human lysozyme promoter region may be used to direct expression of genes to activated mouse myeloid cells.

The human thyrotropin receptor: a heptahelical receptor capable of stimulating members of all four G protein families.

Thyrotropin is the primary hormone that, via one heptahelical receptor, regulates thyroid cell functions such as secretion, specific gene expression, and growth. In human thyroid, thyrotropin receptor activation leads to stimulation of the adenylyl cyclase and phospholipase C cascades. However, the G proteins involved in thyrotropin receptor action have been only partially defined. In membranes of human thyroid gland, we immunologically identified alpha subunits of the G proteins Gs short, Gs long, Gi1, Gi2, Gi3, G(o) (Go2 and another form of Go, presumably Go1), Gq, G11, G12, and G13. Activation of the thyrotropin (TSH) receptor by bovine TSH led to increased incorporation of the photoreactive GTP analogue [alpha-32P]GTP azidoanilide into immunoprecipitated alpha subunits of all G proteins detected in thyroid membranes. This effect was receptor-dependent and not due to direct G protein stimulation because it was mimicked by TSH receptor-stimulating antibodies of patients suffering from Grave disease and was abolished by a receptor-blocking antiserum from a patient with autoimmune hypothyroidism. The TSH-induced activation of individual G proteins occurred with EC50 values of 5-50 milliunits/ml, indicating that the activated TSH receptor coupled with similar potency to different G proteins. When human thyroid slices were pretreated with pertussis toxin, the TSH receptor-mediated accumulation of cAMP increased by approximately 35% with TSH at 1 milliunits/ml, indicating that the TSH receptor coupled to Gs and G(i). Taken together, these findings show that, at least in human thyroid membranes, in which the protein is expressed at its physiological levels, the TSH receptor resembles a naturally occurring example of a general G protein-activating receptor.

Fixing human factor IX (fIX): correction of a cryptic RNA splice enables the production of biologically active fIX in the mammary gland of transgenic mice.

Transgenic mice and sheep secrete only low levels of human factor IX in their milk because of an aberrant splicing of the transgene RNA in the mammary gland. Removal of the cryptic 3' splice site prevents this splicing and leads to the production of relatively high levels of factor IX. The purified protein is fully active showing that the mammary gland is capable of the efficient post-translational modification of this protein and that transgenic animals are a suitable means of its production.

The human and simian immunodeficiency virus envelope glycoprotein transmembrane subunits are palmitoylated.

The envelope proteins of human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) were found to be modified by fatty acylation of the transmembrane protein subunit gp41. The precursor gp160 was also palmitoylated prior to its cleavage into the gp120 and gp41 subunits. The palmitic acid label was sensitive to treatment with hydroxylamine or 2-mercaptoethanol, indicating that the linkage is through a thioester bond. Treatment with cycloheximide did not prevent the incorporation of [3H]palmitic acid into the HIV envelope protein, indicating that palmitoylation is a posttranslation modification. In contrast to other glycoproteins, which are palmitoylated at cysteine residues within or close to the membrane-spanning hydrophobic domain, the palmitoylation of the HIV-1 envelope proteins occurs on two cysteine residues, Cys-764 and Cys-837, which are 59 and 132 amino acids, respectively, from the proposed membrane-spanning domain of gp41. Sequence comparison revealed that one of these residues (Cys-764) is conserved in the cytoplasmic domains of almost all HIV-1 isolates and is located very close to an amphipathic region which has been postulated to bind to the plasma membrane.

The human papilloma virus 16E6 gene sensitizes human mammary epithelial cells to apoptosis induced by DNA damage.

Programmed cell death (apoptosis) is a normal physiological process, which could in principle be manipulated to play an important role in cancer therapy. The key importance of p53 expression in the apoptotic response to DNA-damaging agents has been stressed because mutant or deleted p53 is so common in most kinds of cancer. An important strategy, therefore, is to find ways to induce apoptosis in the absence of wild-type p53. In this paper, we compare apoptosis in normal human mammary epithelial cells, in cells immortalized with human papilloma virus (HPV), and in mammary carcinoma cell lines expressing wild-type p53, mutant p53, or no p53 protein. Apoptosis was induced with mitomycin C (MMC), a DNA cross-linking and damaging agent, or with staurosporine (SSP), a protein kinase inhibitor. The normal and HPV-transfected cells responded more strongly to SSP than did the tumor cells. After exposure to MMC, cells expressing wild-type p53 underwent extensive apoptosis, whereas cells carrying mutated p53 responded weakly. Primary breast cancer cell lines null for p53 protein were resistant to MMC. In contrast, two HPV immortalized cell lines in which p53 protein was destroyed by E6-modulated ubiquitinylation were highly sensitive to apoptosis induced by MMC. Neither p53 mRNA nor protein was induced in the HPV immortalized cells after MMC treatment, although p53 protein was elevated by MMC in cells with wild-type p53. Importantly, MMC induced p21 mRNA but not p21 protein expression in the HPV immortalized cells. Thus, HPV 16E6 can sensitize mammary epithelial cells to MMC-induced apoptosis via a p53- and p21-independent pathway. We propose that the HPV 16E6 protein modulates ubiquitin-mediated degradation not only of p53 but also of p21 and perhaps other proteins involved in apoptosis.

A human RNase E-like activity that cleaves RNA sequences involved in mRNA stability control.

We have detected an endoribonucleolytic activity in human cell extracts that processes the Escherichia coli 9S RNA and outer membrane protein A (ompA) mRNA with the same specificity as RNase E from E. coli. The human enzyme was partially purified by ion-exchange chromatography, and the active fractions contained a protein that was detected with antibodies shown to recognize E. coli RNase E. RNA containing four repeats of the destabilizing motif AUUUA and RNA from the 3' untranslated region of human c-myc mRNA were also found to be cleaved by E. coli RNase E and its human counterpart in a fashion that may suggest a role of this activity in mammalian mRNA decay. It was also found that RNA containing more than one AUUUA motif was cleaved more efficiently than RNA with only one or a mutated motif. This finding of a eukaryotic endoribonucleolytic activity corresponding to RNase E indicates an evolutionary conservation of the components of mRNA degradation systems.

The human plakoglobin gene localizes on chromosome 17q21 and is subjected to loss of heterozygosity in breast and ovarian cancers.

The gene encoding human plakoglobin was mapped to chromosome 17q12-q22. An intragenic restriction fragment length polymorphism was used to localize the plakoglobin gene distal to locus KRT10 and proximal to the marker D17S858. The plakoglobin gene colocalizes with the polymorphic 17q21 marker UM8 on the same cosmid insert. This subregion of chromosome 17 is known to be particularly subjected to genetic alterations in sporadic breast and ovarian tumors. We show loss of heterozygosity of the plakoglobin gene in breast and ovarian tumors. We have identified a low-frequency polymorphism in the plakoglobin coding sequence which results in an arginine to histidine substitution at amino acid position 142 of the protein, as well as a silent mutation at nucleotide position 332 of the coding sequence. This polymorphism allowed us to demonstrate an allelic association of plakoglobin with predisposition to familial breast and ovarian cancers. Our results, together with the present knowledge about the biological function of plakoglobin, suggest that plakoglobin might represent a putative tumor suppressor gene for breast and ovarian cancers.

Decreased human immunodeficiency virus type 1 plasma viremia during antiretroviral therapy reflects downregulation of viral replication in lymphoid tissue.

Although several immunologic and virologic markers measured in peripheral blood are useful for predicting accelerated progression of human immunodeficiency virus (HIV) disease, their validity for evaluating the response to antiretroviral therapy and their ability to accurately reflect changes in lymphoid organs remain unclear. In the present study, changes in certain virologic markers have been analyzed in peripheral blood and lymphoid tissue during antiretroviral therapy. Sixteen HIV-infected individuals who were receiving antiretroviral therapy with zidovudine for > or = 6 months were randomly assigned either to continue on zidovudine alone or to add didanosine for 8 weeks. Lymph node biopsies were performed at baseline and after 8 weeks. Viral burden (i.e., HIV DNA copies per 10(6) mononuclear cells) and virus replication in mononuclear cells isolated from peripheral blood and lymph node and plasma viremia were determined by semiquantitative polymerase chain reaction assays. Virologic and immunologic markers remained unchanged in peripheral blood and lymph node of patients who continued on zidovudine alone. In contrast, a decrease in virus replication in lymph nodes was observed in four of six patients who added didanosine to their regimen, and this was associated with a decrease in plasma viremia. These results indicate that decreases in plasma viremia detected during antiretroviral therapy reflect downregulation of virus replication in lymphoid tissue.

Mitochondrial impairments contribute to Spinocerebellar ataxia type 1 progression and can be ameliorated by the mitochondria-targeted antioxidant MitoQ

Spinocerebellar ataxia type 1 (SCA1), due to an unstable polyglutamine expansion within the ubiquitously expressed Ataxin-1 protein, leads to the premature degeneration of Purkinje cells (PCs), decreasing motor coordination and causing death within 10-15 years of diagnosis. Currently, there are no therapies available to slow down disease progression. As secondary cellular impairments contributing to SCA1 progression are poorly understood, here, we focused on identifying those processes by performing a PC specific proteome profiling of Sca1154Q/2Q mice at a symptomatic stage. Mass spectrometry analysis revealed prominent alterations in mitochondrial proteins. Immunohistochemical and serial block-face scanning electron microscopy analyses confirmed that PCs underwent age-dependent alterations in mitochondrial morphology. Moreover, colorimetric assays demonstrated impairment of the electron transport chain complexes (ETC) and decrease in ATPase activity. Subsequently, we examined whether the mitochondria-targeted antioxidant MitoQ could restore mitochondrial dysfunction and prevent SCA1-associated pathology in Sca1154Q/2Q mice. MitoQ treatment both presymptomatically and when symptoms were evident ameliorated mitochondrial morphology and restored the activities of the ETC complexes. Notably, MitoQ slowed down the appearance of SCA1-linked neuropathology such as lack of motor coordination as well as preventing oxidative stress-induced DNA / RNA damage and PC loss. Our work identifies a central role for mitochondria in PC degeneration in SCA1 and provides evidence for the supportive use of mitochondria-targeted therapeutics in slowing down disease progression.

Spatio-temporal evaluation of neck muscle activation during postural perturbations in healthy subjects

The purpose of this study was to examine the spatio-temporal activation of the sternocleidomastoid (SCM) and cervical extensor (CE) muscles with respect to the deltoid muscle onset during rapid voluntary upper limb movement in healthy volunteers. The repeatability and reliability of the spatio-temporal aspects of the myoelectric signals were also examined. Ten subjects performed bilateral and unilateral rapid upper limb flexion, abduction and extension in response to a visual stimulus. EMG onsets and normalised root mean square (nRMS) values were calculated for the SCM and CE muscles. Subjects attended three testing sessions over non-consecutive days allowing the repeatability and reliability of these measures to be assessed. The SCM and CE muscles demonstrated feed-forward activation (activation within 50 ms of deltoid onset) during rapid arm movements in all directions. The sequence and magnitude of neck muscle activation displayed directional specificity, however, the neck flexor and extensor muscles displayed co-activation during all perturbations. EMG onsets demonstrated high repeatability in terms of repeated measure precision (nSEM in the range 1.9-5.7%). This was less evident for the repeatability of nRMS values. The results of this study provide a greater understanding of cervical neuromotor control strategies. During bilateral and unilateral upper limb perturbations, the SCM and CE muscles demonstrate feed-forward co-activation. It seems apparent that feed-forward activation of neck muscles is a mechanism necessary to achieve stability for the visual and vestibular systems, whilst ensuring stabilisation and protection of the cervical spine. (C) 2004 Elsevier Ltd. All rights reserved.

Feedforward activity of the cervical flexor muscles during voluntary arm movements is delayed in chronic neck pain

The objective of this study was to compare onset of deep and superficial cervical flexor muscle activity during rapid, unilateral arm movements between ten patients with chronic neck pain and 12 control subjects. Deep cervical flexor (DCF) electromyographic activity (EMG) was recorded with custom electrodes inserted via the nose and fixed by suction to the posterior mucosa of the oropharynx. Surface electrodes were placed over the sternocleidomastoid (SCM) and anterior scalene (AS) muscles. While standing, subjects flexed and extended the right arm in response to a visual stimulus. For the control group, activation of DCF, SCM and AS muscles occurred less than 50 ms after the onset of deltoid activity, which is consistent with feedforward control of the neck during arm flexion and extension. When subjects with a history of neck pain flexed the arm, the onsets of DCF and contralateral SCM and AS muscles were significantly delayed (p<0.05). It is concluded that the delay in neck muscle activity associated with movement of the arm in patients with neck pain indicates a significant deficit in the automatic feedforward control of the cervical spine. As the deep cervical muscles are fundamentally important for support of the cervical lordosis and the cervical joints, change in the feedforward response may leave the cervical spine vulnerable to reactive forces from arm movement.

Differential activity of regions of transversus abdominis during trunk rotation

The role of the abdominal muscles in trunk rotation is not comprehensively understood. This study investigated the electromyographic (EMG) activity of anatomically distinct regions of the abdominal muscles during trunk rotation in six subjects with no history of spinal pain. Fine-wire electrodes were inserted into the right abdominal wall; upper region of transversus abdominis (TrA), middle region of TrA, obliquus internus abdominis (OI) and obliquus externus abdominis (OE), and lower region of TrA and OI. Surface electrodes were placed over right rectus abdominis (RA). Subjects performed trunk rotation to the left and right in sitting by rotating their pelvis relative to a fixed thorax. EMG activity was recorded in relaxed supine and sitting, and during an isometric hold at end range. TrA was consistently active during trunk rotation, with the recruitment patterns of the upper fascicles opposite to that of the middle and lower fascicles. During left rotation, there was greater activity of the lower and middle regions of contralateral TrA and the lower region of contralateral OI. The upper region of ipsilateral TrA and OE were predominately active during right rotation. In contrast, there was no difference in activity of RA and middle OI between directions (although middle OI was different between directions for all but one subject). This study indicates that TrA is active during trunk rotation, but this activity varies between muscle regions. These normative data will assist in understanding the role of TrA in lumbopelvic control and movement, and the effect of spinal pain on abdominal muscle recruitment.

The vacuous concept of dignity

Rights talk dominates contemporary moral discourse. It is also having a growing impact on the development of legal principle and doctrine. One of the best known general arguments in support of rights-based moral theories is the one given by John Rawls, who claims that only rights-based theories take seriously the distinction between human beings; only they can be counted on to protect certain rights and interests that are so paramount that they are beyond the demands of net happiness (Rawls 1971). Charges and assertions of this nature have been extremely influential. After the Second World War, there was an immense increase in rights talk, both in the sheer volume of that talk and in the number of supposed rights being claimed. Rights doctrine has progressed a long way since its original modest aim of providing “a legitimization of … claims against tyrannical or exploiting regimes” (Benn 1978: 61). As Tom Campbell points out: The human rights movement is based on the need for a counter-ideology to combat the abuses and misuses of political authority by those who invoke, as a justification for their activities, the need to subordinate the particular interests of individuals to the general good (Campbell 1996: 13).

Hijos de víctimas del terrorismo de Estado : Justicia, identidad y memoria en el movimiento de derechos humanos en Argentina, 1995-2008

Este trabajo analiza la agrupación HIJOS, organismo de derechos humanos conformado por hijos de víctimas del terrorismo de Estado argentino. El objetivo consiste en realizar un estudio de algunas de las características distintivas de este grupo, teniendo en cuenta especialmente sus rupturas y continuidades con respecto a las prácticas dele derechos humanos. Se analiza el surgimiento del grupo, la conformación de su identidad colectiva, su demanda de justicia y la elaboración de relatos acerca del pasado reciente

HIJOS La Plata. Memorias tensionadas. Análisis de los relatos sobre el pasado reciente que elaboran los militantes de HIJOS

El presente trabajo propone mostrar y analizar cómo los integrantes de la agrupación HIJOS La Plata (organismos de derechos humanos conformado por hijos de víctimas del terrorismo de Estado argentino) han construido sus memorias acerca del pasado reciente. Interesan, más específicamente, las maneras en que elaboran relatos referidos al terror estatal llevado a cabo por la última dictadura (1976-1983) y al período de radicalización política inmediatamente anterior. Mientras la memoria humanitaria, propia del los organismos de derechos humanos que nacieron como respuesta defensista frente al terror estatal, centra sus denuncias en el carácter humano de las personas cuyos derechos fueron violados y omite o silencia sus trayectorias políticas; la memoria militante de los HIJOS rescata en clave reivindicatoria las experiencias políticas de sus padres y pretende no recordarlos sólo como víctimas, sino también como luchadores políticos. El objetivo de este trabajo es indagar las tensiones que resultan de la pertenencia de HIJOS al movimiento de derechos humanos, muchas de cuyas prácticas reproducen, y los conflictos que supone su intención de reivindicar la lucha revolucionaria de sus padres