Identification of conserved amino acids in the human granulocyte-macrophage colony-stimulating factor receptor alpha subunit critical for function. Evidence for formation of a heterodimeric receptor complex prior to ligand binding

A superfamily of growth factor and cytokine receptors has recently been identified, which is characterized by four spatially conserved cysteine residues, a tryptophan-serine motif (WSXWS) in the extracellular domain, and a proline-rich cytoplasmic domain. The high affinity human granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor (hGM-CSFR) consists of two subunits, alpha (hGM-CSFR alpha) and beta (hGM-CSFR beta), both of which are members of the receptor superfamily. In this study, we prepared mutations in conserved amino acids of the receptor subunit necessary for GM-CSF binding (hGM-CSFR alpha) and analyzed mutant receptors for low affinity binding, internalization, and high affinity binding when complexed with the beta subunit. Mutations in the cytoplasmic domain did not affect GM-CSF binding or receptor internalization. Mutation of a single conserved serine residue within the WSXWS motif diminishes cell surface receptor expression but not ligand binding. Mutation of either the second or third conserved cysteine residue of hGM-CSFR alpha resulted in complete loss of low affinity binding; however, co-expression of the cysteine 2 mutant with hGM-CSFR beta yielded a high affinity receptor complex. Since neither the cysteine 2 mutant nor the beta subunit can bind ligand alone, this result suggests that hGM-CSFR alpha and hGM-CSFR beta exist in a preformed heterodimeric protein complex on the plasma membrane.     

The role of lymphocyte subsets in accelerated diabetes in nonobese diabetic-rat insulin promoter-B7-1 (NOD-RIP-B7-1) mice

B7-1 transgene expression on the pancreatic islets in nonobese diabetic (NOD) mice leads to accelerated diabetes, with >50% of animals developing diabetes before 12 wk of age. The expression of B7-1 directly on the pancreatic beta cells, which do not normally express costimulator molecules, converts the cells into effective antigen-presenting cells leading to an intensified autoimmune attack. The pancreatic islet infiltrate in diabetic mice consists of CD8 T cells, CD4 T cells, and B cells, similar to diabetic nontransgenic NOD mice. To elucidate the relative importance of each of the subsets of cells, the NOD-rat insulin promoter (RIP)-B7-1 animals were crossed with NOD.beta2microglobulin -/- mice which lack major histocompatibility complex class I molecules and are deficient in peripheral CD8 T cells, NOD.CD4 -/- mice which lack T cells expressing CD4, and NOD.muMT -/- mice which lack B220-positive B cells. These experiments showed that both CD4 and CD8 T cells were necessary for the accelerated onset of diabetes, but that B cells, which are needed for diabetes to occur in normal NOD mice, are not required. It is possible that B lymphocytes play an important role in the provision of costimulation in NOD mice which is unnecessary in the NOD-RIP-B7-1 transgenic mice.     

Western blotting analysis of heat shock proteins of Rickettsiales and other eubacteria

Promyelocytic leukemia HL-60 cells promoted by PMA to differentiate along the monocyte pathway adhere to tissue culture plates. To explore the regulation of adhesion molecules in cells promoted to differentiate, the expression and secretion of osteopontin (OPN) and expression of associated cell surface receptors, CD44 and integrin subunits alpha(v), beta3, beta1, were examined. Results were as follows: 1) PMA induced OPN mRNA and OPN secretion into media; 2) untreated cells expressed beta1 and CD44 mRNA, and PMA induced alpha(v), and beta3 mRNA and increased beta1 and CD44 mRNA expression; 3) PMA increased levels of alpha(v), beta3, beta1 and CD44 protein on the cell surface; and 4) retinoic acid, which promotes granulocytic differentiation of HL-60 cells, did not affect OPN, alpha(v), beta3, beta1, or CD44 mRNA or protein expression. These data suggest that induction of OPN and associated receptors may play a role during monocytic differentiation of HL-60 cells.     

A simple breathing circuit minimizing changes in alveolar ventilation during hyperpnoea

Many clinical and research situations require maintenance of isocapnia, which occurs when alveolar ventilation (V'A) is matched to CO2 production. A simple, passive circuit that minimizes changes in V'A during hyperpnoea was devised. It is comprised of a manifold, with two gas inlets, attached to the intake port of a nonrebreathing circuit or ventilator. The first inlet receives a flow of fresh gas (CO2=0%) equal to the subject's minute ventilation (V'E). During hyperpnoea, the balance of V'E is drawn (inlet 2) from a reservoir containing gas, the carbon dioxide tension (PCO2) approximates that of mixed venous blood and therefore contributes minimally to V'A. Nine normal subjects breathed through the circuit for 4 min at 15-31 times resting levels. End-tidal PCO2 (Pet,CO2) at rest, 0, 1.5 and 3.0 min were (mean+/-SE) 5.1+/-0.1 kPa (38.1+/-1.1 mmHg), 4.9+/-0.1 kPa (36.4+/-1.1 mmHg), 5.0+/-0.2 kPa (37.8+/-1.6 mmHg) and 5.0+/-0.2 kPa (37.6+/-1.4 mmHg) (p=0.53, analysis of variance (ANOVA)), respectively; without the circuit, Pet,CO2 would be expected to have decreased by at least 2.7 kPa (20 mmHg). Six anaesthetized, intubated dogs were first ventilated at control levels and then hyperventilated by stepwise increases in either respiratory frequency (fR) from 10 to 24 min(-1) or tidal volume (VT) from 400 to 1,200 mL. Increases in fR did not significantly affect arterial CO2 tension (Pa,CO2) (p=0.28, ANOVA). Only the highest VT decreased Pa,CO2 from control (-0.5 +/- 0.3 kPa (-3.4 +/- 2.3 mmHg), p<0.05). In conclusion, this circuit effectively minimizes changes in alveolar ventilation and therefore arterial carbon dioxide tension during hyperpnoea.     

Inhibition of rat lung mixed-function oxidase activity following repeated low-level toluene inhalation: possible role of toluene metabolites

Toluene is a commonly used solvent that has been shown to alter mixed-function oxidase (MFO) activity, in an organ- and isozyme-specific pattern, following intraperitoneal administration. The purpose of this study was to determine whether similar changes occurred following repeated, low-level inhalation exposure, and to investigate the role of toluene metabolites in these alterations. Exposure to 375 ppm toluene, 6 h/d for up to 5 d, resulted in significant inhibition of the activity of pulmonary arylhydrocarbon hydroxylase (AHH), cytochrome P-4502B1 (CYP2B1), and CYP4B1, but not CYP1A1. After exposure to lower toluene levels (125 ppm, 6 h/d, 3 d), the activities of lung AHH, CYP2B1, and CYP4B1 were also significantly decreased, but in a dose-related manner. MFO activity was not consistently altered in liver. Control pulmonary or liver microsomes were incubated with various concentrations (0.01-10 mM) of toluene or its metabolites and CYP2B1, CYP1A1, and/or CYP4B1 activities were subsequently determined. Benzaldehyde produced a significant dose-related inhibition in the activity of all three lung P-450s examined (IC50 10(-3) M). Toluene was found to be a more potent inhibitor of lung CYP2B1 and CYP1A1 (IC50, 10(-4) M) than benzaldehyde, but neither toluene nor benzyl alcohol was an effective inhibitor of lung CYP4B1. Toluene and its metabolites were weaker inhibitors of CYP1A1 than of CYP2B1. For CYP2B1 and CYP1A1, the order of inhibitory potency was toluene > benzaldehyde > benzyl alcohol and suggests that both the parent molecule and its metabolites may act in concert to inhibit catalytic activity of these cytochromes. The MFO inhibition seen after repeated low-level toluene inhalation exposure could result in altered metabolic profiles of other xenobiotics in an organ-specific fashion.     

Placental pathology and antiphospholipid antibodies: a descriptive study

We described placental pathology in antiphospholipid antibody (APL) syndrome, APL and no history of recurrent pregnancy loss, and in treated and untreated pregnancies of APL syndrome. Thirty-nine pregnancies of 28 patients were studied: 23 placentas delivered from 23 women with APL (13 with APL syndrome and 10 with serological APL); 8 untreated miscarriages before APL diagnosis from 6 of the 13 patients with APL syndrome and 1 of 10 with serological APL; and 8 miscarriages by 5 additional women before APL syndrome diagnosis. Histopathology was reviewed by a pathologist blinded except to gestational age. Contingency tables and analysis of variance (ANOVA) considered p < 0.05 significant. Comparing the placentas delivered at > 18 weeks' gestation, excessive perivillous coagulation, avascular terminal villi, and chronic villitis/uteroplacental vasculitis tended to be more common in treated APL syndrome than serological APL cases (p = 0.07). Of the 16 miscarriages before diagnosis of APL, 11 were lost at < 18 weeks' gestation. None had pathology typical of APL, but 4 of 11 (36%) had chronic intervillositis. Five of 16 miscarriages before the diagnosis of APL were miscarried between 18-22 weeks. Three of 5 (60%) miscarried after 18 weeks had multifocal uteroplacental thromboses, compared to 6 of 13 (46%) treated pregnancies with APL syndrome and 0 of 10 cases with serological APL.     

Integrin receptor signaling: the propagation of an alpha-helix model

Improper assessment and treatment of asthma attacks have been identified as causes of increased morbidity and mortality: several pneumological societies have therefore created and published guidelines for facilitating decision making and for preventing unnecessary failures of therapy. The objective of this study was to examine emergency department compliance with such guidelines in our hospital, comparing the performance of pneumologists and other specialists. We reviewed the records of 117 patients treated for acute asthma attacks in 1994 (87 women and 30 men, mean age 46 years); 37 patients were treated by pneumologists and 80 by other specialists. The two physician groups differed significantly with respect to initial assessment of severity, particularly in the recording of vital signs (p < 0.05) and in the examination of some signs such as the use of accessory musculature (38% versus 10%, for pneumologists and other specialists, respectively) or the presence of cyanosis (81% versus 55%). Other factors associated with risk of death were noted only occasionally. Peak flow meters were used with only 5 patients, all examined by pneumologists; on the other hand, arterial blood samples for gasometric measurements were taken from 97%, although only 24% met the criteria stipulated in the guidelines. Treatment evaluated against the guidelines was incorrect in 24%, with no significant differences between pneumologists and other specialists. We conclude that: 1) the emergency clinical assessment and treatment of patients presenting with acute asthma attack is inadequate for a large proportion of patients, as the recommendations of consensual guidelines are habitually ignored, and 2) although there are differences in the management of these patients by pneumologists and other emergency room specialists, the former do not generally do a better job of following the guidelines.     

Hispanics and worksite health promotion: review of the past, demands for the future

During the last 15 years, reports, books, and published papers have demonstrated the positive health benefits of comprehensive health promotion and disease prevention interventions at the worksite. Although the progress made in worksite health promotion should not be understated, experts agree that it is time to step forward and address the new demands of the changing labor force. One area of intervention that needs to be more aggressively addressed is that of minority populations. Because work force projections show the high participation rate that Hispanics will have in the future labor force, the Hispanic population should be one of the targets of worksite health promotion. This paper reviews how employee health promotion programs have addressed Hispanic workers in the past and establishes directions for the future.     

Oral mucositis with features of psoriasis: report of a case and review of the literature

There complete 18S ribosomal RNA gene sequences from the rumen ciliates, Entodinium caudatum (1,639 bp), Epidinium caudatum (1,638 bp), and Polyplastron multivesiculatum (1,640 bp) were determined and confirmed in the opposite direction. Trees produced using maximum parsimony and distance-matrix methods (least squares and neighbour-joining), with strong bootstrap support, depict the rumen ciliates as a monophyletic group. Entodinium caudatum is the earliest branching rumen ciliate. However, Entodinium simplex does not pair with En. caudatum, but rather with Polyplastron multivesiculatum. Signature sequences for these rumen ciliates reveal that the published SSrRNA gene sequence from En. simplex is in fact a Polyplastron species. The free-living haptorian ciliates, The Loxophyllum, Homalozoon and Spathidium (Subclass Haptoria), are monophyletic and are the sister group to the rumen ciliates. The litostomes (Class Litostomatea), consisting of the haptorians and the rumen ciliates, are also a monophyletic group.     

Autoimmune polyglandular syndrome type I. A case report

Calcineurin is a Ca2+ calmodulin dependent protein phosphatase which has an important role in the control of intracellular Ca2+ signalling. The protein is a heterodimer of one catalytic (CnA) subunit and one regulatory (CnB) subunit. As suggested by the protein sequence and confirmed by the crystallographic structure, the catalytic subunit of calcineurin (CnA) has high homologies with other protein phosphatases. The regulatory subunit (CnB) belongs to the EF-hand Ca2+ binding protein family. Despite its similarity with calmodulin, it has a different tertiary structure. Calcineurin is the target of two important immunosuppressant drugs: cyclosporin A and FK506. Subsequently, a detailed clarification of the role of calcineurin in the cytokine mediated activation of the T-cells has been possible. The understanding of the role of calcineurin in other cells, in particular neurons, is also progressing rapidly.