Genetic control of differential baseline breathing pattern

The purpose of the present study was to determine the genetic control of baseline breathing pattern by examining the mode of inheritance between two inbred murine strains with differential breathing characteristics. Specifically, the rapid, shallow phenotype of the C57BL/6J (B6) strain is consistently distinct from the slow, deep phenotype of the C3H/HeJ (C3) strain. The response distributions of segregant and nonsegregant progeny were compared with the two progenitor strains to determine the mode of inheritance for each ventilatory characteristic. The BXH recombinant inbred (RI) strains derived from the B6 and C3 progenitors were examined to establish strain distribution patterns for each ventilatory trait. To establish the mode of inheritance, baseline breathing frequency (f), tidal volume, and inspiratory time (TI) were measured five times in each of 178 mature male animals from the two progenitor strains and their progeny by using whole body plethysmography. With respect to f and TI, the two progenitor strains were consistently distinct, and segregation analyses of the inheritance pattern suggest that the most parsimonious genetic model for response distributions of f and TI is a two-loci model. In similar experiments conducted on 82 mature male animals from 12 BXH RI strains, each parental phenotype was represented by one or more of the RI strains. Intermediate phenotypes emerged to confirm the likelihood that parental strain differences in f and TI were determined by more than one locus. Taken together, these studies suggest that the phenotypic difference in baseline respiratory timing between male B6 and C3 mice is best explained by a genetic model that considers at least two loci as major determinants.     

HLA-B27-restricted antigen presentation in the absence of tapasin reveals polymorphism in mechanisms of HLA class I peptide loading

Tapasin is a resident ER protein believed to be critical for antigen presentation by HLA class I molecules. We demonstrate that allelic variation in MHC class I molecules influences their dependence on tapasin for peptide loading and antigen presentation. HLA-B*2705 molecules achieve high levels of surface expression and present specific viral peptides in the absence of tapasin. In contrast, HLA-B*4402 molecules are highly dependent upon human tapasin for these functions, while HLA-B8 molecules are intermediate in this regard. Significantly, HLA-B*2705 like HLA-B*4402, requires tapasin to associate efficiently with TAP (transporters associated with antigen processing). The unusual ability of HLA-B*2705 to form peptide complexes without associating with TAP or tapasin confers flexibility in the repertoire of peptides presented by this molecule. We speculate that these properties might contribute to the role of HLA-B27 in conferring susceptibility to inflammatory spondyloarthropathies.     

The A326S mutant of Gialpha1 as an approximation of the receptor-bound state

Agonist-bound heptahelical receptors activate heterotrimeric G proteins by catalyzing exchange of GDP for GTP on their alpha subunits. In search of an approximation of the receptor-alpha subunit complex, we have considered the properties of A326S Gialpha1, a mutation discovered originally in Gsalpha (Iiri, T., Herzmark, P., Nakamoto, J. M., Van Dop, C., and Bourne, H. R. (1994) Nature 371, 164-168) that mimics the effect of receptor on nucleotide exchange. The mutation accelerates dissociation of GDP from the alphai1beta1gamma2 heterotrimer by 250-fold. Nevertheless, affinity of mutant Gialpha1 for GTPgammaS is high in the presence of Mg2+, and the mutation has no effect on the intrinsic GTPase activity of the alpha subunit. The mutation also uncouples two activities of betagamma: stabilization of the GDP-bound alpha subunit (which is retained) and retardation of GDP dissociation from the heterotrimer (which is lost). For wild-type and mutant Gialpha1, beta gamma prevents irreversible inactivation of the alpha subunit at 30 degreesC. However, the mutation accelerates irreversible inactivation of alpha at 37 degreesC despite the presence of beta gamma. Structurally, the mutation weakens affinity for GTPgammaS by steric crowding: a 2-fold increase in the number of close contacts between the protein and the purine ring of the nucleotide. By contrast, we observe no differences in structure at the GDP binding site between wild-type heterotrimers and those containing A326S Gialpha1. However, the GDP binding site is only partially occupied in crystals of G protein heterotrimers containing A326S Gialpha1. In contrast to original speculations about the structural correlates of receptor-catalyzed nucleotide exchange, rapid dissociation of GDP can be observed in the absence of substantial structural alteration of a Galpha subunit in the GDP-bound state.     

Morphologic comparison of atherosclerotic lesions in native coronary arteries and saphenous vein graphs with intracoronary angioscopy in patients with unstable angina

The purpose of this study was to investigate platelet effects on postischemic heart function in conjunction with adenosine effects on intracoronary platelet adhesion. Homologous platelets were infused into the coronaries of isolated guinea pig hearts, either during low-flow ischemia or during reperfusion, and external heart work (EHW) and intracoronary platelet adhesion were determined. In most experiments, thrombin was added to the perfusate. The influence of endogenous adenosine was studied by use of the uptake blocker dipyridamole and the unspecific adenosine-receptor blocker theophylline, the A1-receptor blocker 8-cyclopentyl-1,3-dipropylxanthine (DPCPX), and the A2-receptor blocker 3,7-dimethyl-1-propargylxanthine (DMPX). The importance of nitric oxide and prostaglandin I2 (PGI2) was tested by using nitro-L-arginine (NOLAG) and indomethacin, respectively. When platelets were applied with thrombin during low-flow ischemia, EHW recovered to only 63 +/- 4% of the preischemic value, as compared with 89 +/- 3% without platelets (p < 0.05). Despite thrombin, platelets incurred no significant functional loss when applied in the first minute of reperfusion (but again in the fifth minute); however, when theophylline was also present, recovery of EHW amounted to only 42 +/- 12%. Intracoronary adhesion of platelets was negligible without thrombin, and highest during low-flow ischemia with thrombin (35 +/- 3% of the applied number). No adhesion occurred during the first minute of reperfusion, whereas in the fifth minute, adhesion was again 20.8 +/- 4%. Dipyridamole increased adenosine release and attenuated adhesion at this time. Theophylline increased adhesion in the first minute of reperfusion (33 +/- 6.4%), whereas NOLAG and indomethacin proved to be ineffective. DPCPX and DMPX each increased platelet retention during the first minute of reperfusion, their effects being additive. Intracoronary adhesion of platelets induced by thrombin in isolated hearts can reduce postischemic recovery of heart function. During reperfusion, but not during low-flow, endogenous adenosine can prevent platelet adhesion and loss of myocardial function, an action mediated both by A1- and A2-receptor-dependent mechanisms.     

Monitoring of lineage-specific chimaerism allows early prediction of response following donor lymphocyte infusions for relapsed chronic myeloid leukaemia

Thrombogenesis in the left atrial appendage (LAA) has been related to the special morphology of this cavity and to its size and degree of dysfunction. However, no study has focused on LAA function in conjunction with left atrial (LA) function in both sinus rhythm (SR) and nonrheumatic idiopathic atrial fibrillation (AF) in relation to clinical status (cardioembolic stroke). Forty-three patients in SR (14 patients with stroke, 29 control subjects) and 45 patients in AF (27 patients with stroke, 18 control subjects) were examined by transthoracic and transesophageal echocardiography. Baseline clinical characteristics and standard transthoracic and transesophageal measurements of the LA and LAA (size, fractional area change, flow measurements, spontaneous echo contrast, and thrombus) were recorded and compared in relation to cardiac rhythm. Patients in the stroke-SR group showed a significant decrease of fractional area change in the LA (32%+/-15%) and LAA (34%+/-15%) in relation to control subjects (43%+/-10%, p = 0.035, 49%+/-13%, p = 0.006, respectively). Patients in the stroke-AF group showed significant reduction of appendage flow measurements (outward velocity = 22+/-13 vs 33+/-19 cm/sec, p = 0.036), whereas no differences were detected in the center of the LA. In multiple regression analysis, the presence of cardioembolic stroke was positively associated with the presence of spontaneous echo contrast (p = 0.0253) and spontaneous echo contrast negatively associated with appendage inward flow velocity (p<0.001). Cardioembolic stroke in patients in SR is associated with a global decrease of shortening in both cavities and in patients with AF, with a reduction of LAA flow parameters. Patients with spontaneous echo contrast, thrombus, or both showed further reduction of shortening and flow velocities in both cavities, indicating a more advanced stage of dysfunction.     

Relaxin and decidualization in mice: a reappraisal

The nature of the physiological stimulus inducing decidualization in the endometrium is unknown. In this study we attempted to verify a recent report that relaxin can induce decidualization in intact mice primed with a high dose of estradiol valerate (5 micrograms) and a low dose (10 micrograms) of medroxyprogesterone acetate. In our study, neither s.c. nor intrauterine relaxin, nor intraluminal arachis oil, (an established deciduogenic stimulus) were able to induce decidualization. In addition, while oil was able to induce decidualization (increased uterine weight, and positive Pontamine Sky Blue and stromal alkaline phosphatase reactions) in ovariectomized mice treated with a regimen of estradiol and medroxyprogesterone acetate designed to produce optimum uterine sensitivity, no decidualization occurred in response to either s.c. or intraluminal relaxin. This study fails to provide any support for a role for relaxin as a deciduogenic stimulus.     

Novel diffusion cell for in vitro transdermal permeation, compatible with automated dynamic sampling

The development of a new diffusion cell for in vitro transdermal permeation is described. The so-called Kelder cells were used in combination with the ASPEC system (Automatic Sample Preparation with Extraction Columns), which is designed for the automation of solid-extractions (SPE). Instead of SPE columns, 20 Kelder cells were placed in the racks. This allowed automatic sampling of up to 20 cells for 24 h in a dynamic mode. The cells consist of an inlet compartment, a donor compartment and a receptor compartment. The size and the depth of the inlet compartment were important to avoid entrapment of air bubbles in the receptor compartment. The Kelder cells mimic blood flow beneath the skin by replacement of the permeating drug every 2 min. Hence sink condition are more easily maintained than with the static Franz diffusion cell. The performance of the cells was tested with permeation experiments using atropine as a model drug permeating through an artificial membrane (Silastic). The use of this skin model minimized the variability in permeation of atropine as compared with human skin.     

Marital therapy in the treatment of depression: toward a third generation of therapy and research

Two generations of outcome research demonstrate the potential efficacy of marital therapy in the treatment of depression. After reviewing treatment outcome studies on marital therapy for depression, we examine basic research linking aspects of the marital relationship to depressive symptoms. In doing so, we highlight a number of theoretical perspectives and research findings that can inform work with couples in which one spouse is depressed. Finally, we identify potential innovations that may lead to a third generation of marital interventions for depression and several avenues of inquiry for a third generation of outcome research on marital therapy for depression.     

Attachment staining: a novel method for flow cytometric quantitation of protein expression in limited numbers of adherent cells

Malignant tumors are known to exhibit high rates of glycolytic activity leading to high production of lactic acid. Hence, neoplastic cells have elevated activity of enzymes responsible for glycolysis. Echitamine chloride, an indole alkaloid extracted from the bark of Alstonia scholaris, has been reported to have a highly promising anticancer activity against fibrosarcoma in rats. In the present study, the effect of echitamine chloride on energy metabolism of S-180 cells is investigated to have a better understanding on the mode of action of echitamine chloride. The effect of echitamine chloride on the mitochondrial and cellular respiration of S-180 cells was studied. Also, the effects on glucose utilization, pyruvate utilization and lactate formation were studied on whole S-180 cells and S-180 cell-free homogenate. The levels of glycolytic enzymes such as hexokinase and lactate dehydrogenase were estimated in which particular emphasis has been laid on hexokinase which occurs both in cytosolic and particulate forms in neoplastic cells. Hence the differential effect of echitamine chloride on the levels of total, cytosolic and particulate hexokinase has been investigated. In conclusion, echitamine chloride affects both cellular and mitochondrial respiration, leading to reduction of the cellular energy pool and thereby resulting in the loss of viability of S-180 cells.     

Cerebrovascular ischemic events with high positive anticardiolipin antibodies

BACKGROUND AND PURPOSE: The aim of our study was to characterize the patient profile and prognostic value associated with high positive IgG (>100 GPL) anticardiolipin antibodies (aCL). METHODS: We studied the clinical, laboratory, radiological, and prospective historical features of ischemic cerebrovascular disease in patients with >100 GPL titers. From our neurology department, 27 consecutive patients were prospectively identified and followed up (mean follow-up time, 34 months). RESULTS: The mean age of our cohort was 41 years. Lupuslike illness occurred in 3; 23 had primary antiphospholipid syndrome, including 3 who met criteria for Sneddon's syndrome; 1 patient had progressive systemic sclerosis. Cerebral infarcts occurred in 74% and were recurrent in 37%. Systemic ischemic events, most commonly deep vein thrombosis, occurred in 37%. Tobacco use was documented in 85%, hyperlipidemia in 74%, hypertension in 44%, and diabetes mellitus in 7% of patients. A prominent headache history was present in 67%. Lupus anticoagulant (LA) was present in 72%, approximately one half had positive antinuclear antibodies and thrombocytopenia, and one quarter had a false-positive VDRL. We compared mean GPL levels in patients testing positive for specific laboratory features of antiphospholipid syndrome with those testing negative for these parameters. Only the LA(+) group had a significantly higher mean GPL than the LA(-) group (P=0.006). Brain imaging showed nonlacunar infarcts in 73% and lacunes in 12%. Of 19 cerebral angiograms, 5 (26%) showed large-vessel occlusive disease and 6 (32%) branch obstruction. Echocardiograms were abnormal in 75%: thickened left-sided valves in 33% and vegetations in 12%. Recurrent cerebrovascular ischemic events were observed in 96%, with transient events (mean rate, 25%/y) occurring 5 times more frequently than strokes (mean rate, 5%/y). Using a standardized disability scale blinded to aCL titer, neurological impairment was severe in 7%, moderate in 30%, and mild or nonexistent in 63%, and unrelated to mean GPL value (P=0.567). Titers fluctuated greatly for individual patients, and most did not consistently test as highly positive. An analysis of fluctuation in symptom severity with concurrent GPL values did not show a statistically significant correlation. Compared with historical controls having a wide range of positive titers, the presence of high IgG aCL titers did not confer a worse prognosis for disability and recurrent ischemic events. CONCLUSIONS: Our data suggest that cerebrovascular events associated with high positive GPL are frequently multiple and minor (with no disability-titer correlation), present in relatively young patients, and often associated with tobacco abuse, hyperlipidemia, LA, systemic ischemic events, and occult cardiac disease.