Expression of NHE1 and NHE4 in rat pancreatic zymogen granule membranes

We previously characterized a Na+/H+ exchange activity in rat pancreatic zymogen granules [Anderie, I., and Thévenod, F. (1996) J. Membrane Biol, 152, 195-205]. Here we have identified the Na+/H+ exchanger (NHE) isoforms present in zymogen granules by functional studies with NHE inhibitors. The NHE1 specific blocker HOE 694 [3-(methylsulfonyl-4-piperidino-benzoyl)-guanidine methanesulfonate] inhibited zymogen granule Na+/H+ exchange in a concentration dependent manner, maximally to 53 +/- 5% of controls at 100nM. The remaining Na+/H+ exchange activity was inhibitable by EIPA [5-(N-ethyl-N-isopropyl)amiloride] (EC50 approximately 25 microM) or benzamil (EC50 approximately 100 microM). Amiloride inhibited weakly suggesting that "amiloride-resistant" and "amiloride-sensitive" NHE are expressed in zymogen granules. cDNA sequences encoding NHE1- and NHE4-specific transmembrane domains were detected by RT-PCR in rat pancreatic tissue and in the rat pancreatic acinar cell line AR4-2J. The presence of NHE1 and NHE4 in zymogen granule membranes was confirmed by immunoblots of zymogen granule membranes and by pre-embedding immunogold labeling of purified rat pancreatic zymogen granules with polyclonal NHE1 and NHE4 antibodies. Therefore, we propose that NHE1 and NHE4 are expressed in zymogen granule membranes of rat exocrine pancreas.     

Ex vivo staining of metastatic lymph nodes by class I major histocompatibility complex tetramers reveals high numbers of antigen-experienced tumor-specific cytolytic T lymphocytes

Characterization of cytolytic T lymphocyte (CTL) responses to tumor antigens has been impeded by a lack of direct assays of CTL activity. We have synthesized reagents ("tetramers") that specifically stain CTLs recognizing melanoma antigens. Tetramer staining of tumor-infiltrated lymph nodes ex vivo revealed high frequencies of tumor-specific CTLs which were antigen-experienced by surface phenotype. In vitro culture of lymph node cells with cytokines resulted in very large expansions of tumor-specific CTLs that were dependent on the presence of tumor cells in the lymph nodes. Tetramer-guided sorting by flow cytometer allowed isolation of melanoma-specific CTLs and confirmation of their specificity and their ability to lyse autologous tumor cells. Our results demonstrate the value of these novel reagents for monitoring tumor-specific CTL responses and for generating CTLs for adoptive immunotherapy. These data also indicate that strong CTL responses to melanoma often occur in vivo, and that the reactive CTLs have substantial proliferative and tumoricidal potential.     

Perceptions of caring. Comparison of antepartum and postpartum patients

An exploratory, comparative survey design was used to describe and compare similarities and differences in perception of caring behaviors between hospitalized antepartum and short-stay postpartum patients. Forty-two patients completed the 63-item Caring Behavior Assessment instrument (CBA). The scale, based on Watson's theory of caring, contains seven categories. There were no statistically significant differences in age, education, gravida, or para between the two groups. Significantly more women in the postpartum group were married. There were no statistically significant differences or correlations in the total CBA scores and the demographic variables. No statistically significant differences in total CBA scores were found between antepartum participants and postpartum participants. These findings corroborate the results of "caring" studies in other patient populations, that satisfying basic needs--that is, evidence of technical knowledge and skills--is more important to patients than meeting higher order needs.     

The dental status of asthmatic British school children

Pancreatic cancer is a dismal disease. The 5-year overall survival ranges from 1% to 5%. Surgery is the only curative treatment available for this cancer, but it is indicated only in selected patients with a less than 4 cm tumor. In these patients, survival rate is about 30%. We have considered several aspects: the very difficult early diagnosis, the correct diagnostic flow chart, actual surgical procedures and new trends in biologic and genetic research. It is likely that better results can be achieved by defining an "early pancreatic cancer" and establishing how to detect it. This could be the wrigth one way is to significantly improve the survival of these patients.     

Autoimmunity, immunodeficiency and mucosal infections: chronic intestinal inflammation as a sensitive indicator of immunoregulatory defects in response to normal luminal microflora

Despite the fact that target antigens and the genetic basis of several autoimmune diseases are now better understood, the initial events leading to a loss of tolerance towards self-components remain unknown. One of the most attractive explanations for autoimmune phenomena involves various infections as possible natural events capable of initiating the process in genetically predisposed individuals. The most accepted explanation of how infection causes autoimmunity is based on the concept of "molecular mimicry" (similarity between the epitopes of an autoantigen and the epitopes in the environmental antigen). Infectious stimuli may also participate in the development of autoimmunity by inducing an increased expression of stress proteins (hsp), chaperones and transplantation antigens, which leads to abnormal processing and presentation of self antigens. Superantigens are considered to be one of the most effective bacterial components to induce inflammatory reactions and to take part in the development and course of autoimmune mechanisms. It has long been known that defects in the host defense mechanism render the individual susceptible to infections caused by certain microorganisms. Impaired exclusion of microbial antigens can lead to chronic immunological activation which can affect the tolerance to self components. Defects in certain components of the immune system are associated with a higher risk of a development of autoimmune disease. The use of animal models for the studies of human diseases with immunological pathogenesis has provided new insights into the influence of immunoregulatory factors and the lymphocyte subsets involved in the development of disease. One of the most striking conclusion arising from work with genetically engineered immunodeficient mouse models is the existence of a high level of redundancy of the components of the immune system. However, when genes encoding molecules involved in T cell immunoregulatory functions are deleted, spontaneous chronic inflammation of the gut mucosa (similar to human inflammatory bowel disease) develops. Surprisingly, when such immunocompromised animals were placed into germfree environment, intestinal inflammation did not develop. Impairment of the mucosal immune response to the normal bacterial flora has been proposed to play a crucial role in the pathogenesis of chronic intestinal inflammation. The use of immunodeficient models colonized with defined microflora for the analysis of immune reactivity will shed light on the mode of action of different immunologically important molecules responsible for the delicate balance between luminal commensals, nonspecific and specific components of the mucosal immune system.     

The cost of sex revisited: effects of male gamete output of hermaphrodites that are asexual in their female capacity

The genetic cost of sexual reproduction has been attributed to two causes in mathematical formulations: male function or genome dilution. We develop and analyse a genetic model that shows that both costs occur, depending upon the conditions. The model differs from previous formulations in that the level of output and fertilization success of male gametes produced by hermaphrodites that are asexual in their female function (henceforth "parthenogenetic hermaphrodites") are treated as variables, rather than constants fixed at 0 or 1, as has previously been the case. By expressing the cost of sex in terms of per capita egg loss of sexual individuals and parthenogenetic hermaphrodites, we partition the cost into components due to male function and genome dilution. Which component dominates the cost of sex depends upon the relative male gamete output of the parthenogenetic hermaphrodites. The cost of sex is observed to increase, or remain unchanged in some marginal cases, with increases in (i) frequency of parthenogenetic hermaphrodites, (ii) fertilization success of male gametes produced by parthenogenetic hermaphrodites and (iii) potential eggs lost by diverting resources to male gamete production. In certain situations, parthenogenetic hermaphrodites with an intermediate level of male gamete output have the greatest fitness advantage over sexual individuals. If heritable variation for levels of male gamete output exists among parthenogenetic hermaphrodites, this raises the possibility of the evolution of optimal levels of male gamete production by parthenogenetic hermaphrodites through natural selection, in situations of recurring invasion of asexual populations by propagules from sexual populations, a scenario that is increasingly being appreciated as potentially fairly likely to occur in nature.     

The "Heart Area" of Juan Canalejo Hospital Complex. A new approach to clinical management

The present work describes the process by which the pilot project of clinical management of the Hospital Complex Juan Canalejo, designated as "Heart Area", was implemented. In the first section, the needs and reasons that led to the undertaking of this project are explained. The project's objectives and operative strategies are listed. In the Material and Methods section, three basic aspects of the "Heart Area" are described: selection criteria of the "Area", its structure and function, and its foundation and development. In the Results section, we compare the activity undertaken in the "Area" to the situation present prior to its implementation, in relation to quality and costs. Finally, in the Conclusions, we comment on the important implications that our project can have within the Hospital Complex Juan Canalejo as well as in the health care field in general.     

Compensation for gaze perturbation during inactivation of the caudal fastigial nucleus in the head-unrestrained cat

Muscimol injection in the caudal part of the fastigial nucleus (cFN) leads, in the head-unrestrained cat, to a characteristic dysmetria of saccadic gaze shifts toward visual targets. The goal of the current study was to test whether this pharmacological cFN inactivation impaired the ability to compensate for unexpected perturbations in gaze position during the latency period of the saccadic response. Such perturbations consisted of moving gaze away from the target by a transient electrical microstimulation in the deep layers of the superior colliculus simultaneously with extinction of the visual target. After injection of muscimol in the cFN, targets located in the contralesional hemifield elicited gaze shifts that fell short of the target in both "perturbed" and "unperturbed" trials. The amplitude of the compensatory contraversive gaze shifts in perturbed trials coincided with the predicted amplitude of unperturbed responses starting from the same position. Targets located in the opposite hemifield elicited hypermetric gaze shifts in both trial types, and the error of compensatory responses was not statistically different from that of unperturbed gaze shifts. These results indicate that inactivation of the cFN does not interfere with the ability of the head-unrestrained cat to compensate for ipsiversive or contraversive perturbations in gaze position. Thus the gaze-related feedback signals that are used to compute a reference signal of desired gaze displacement are not impaired by cFN inactivation.     

Proline isomerization-independent accumulation of an early intermediate and heterogeneity of the folding pathways of a mixed alpha/beta protein, Escherichia coli thioredoxin

Oxidized Escherichia coli thioredoxin (Trx) is a small protein of 108 residues with one disulfide bond (C32-C35 essentially involved in the activity) and no prosthetic moieties, which folds into a structural motif containing a central twisted beta-sheet flanked by helices that is found in many larger proteins. The kinetics of refolding of Trx in vitro have been investigated using a newly developed active site titration assay and continuous or stopped-flow (SF) methods in conjunction with circular dichroism (CD) and fluorescence (Fl) spectroscopy. These studies revealed the presence of early folding intermediates with "molten globule or pre-molten globule" characteristics. Measurements of the ellipticity at 222 nm indicated that about 68% of the total change associated with refolding occurred during the dead time (4 ms) of the stopped-flow instrument, suggesting the formation of substantial secondary structure. The reconstruction of the far-UV CD spectrum of the burst intermediate using combined continuous and stopped-flow methods showed the formation of a defined secondary structure that contains more beta-structure than the native state. Kinetic measurements using SF far-UV CD and Fl over a wide range (0.087-6 M) of GuHCl concentrations at two temperatures (6 and 20 degreesC) demonstrated that the population formed during the 4 ms dead time contained multiple species that are stabilized mainly by hydrophobic interactions and undergo further folding along alternative pathways. One of these species leads directly and rapidly to the native state as demonstrated by active site titration, while the two others fold into a fourth intermediate that is slowly converted to the native protein. Double-jump experiments suggest that the heterogeneity in folding behavior results from proline isomerizations occurring in the unfolded state. Conversely, the accumulation of the burst intermediate does not depend on proline isomerizations.