The role of aromatic side chain residues in micelle binding by pancreatic colipase. Fluorescence studies of the porcine and equine proteins.

Fluorescence techniques have been employed to study the interaction of porcine and equine colipase with pure taurodeoxycholate and mixed micelles. Nitrotyrosine-55 of porcine colipase is obtained by modification with tetranitromethane (low excess, in the presence of taurodeoxycholate) of the protein followed by gel filtration and ion-exchange chromatography. Verification of the residue modified was obtained by h.p.l.c. peptide purification and sequence analysis. Reduction and quantitative reaction with dansyl chloride yields a fluorescent derivative that is twice as active in conjunction with lipase as is native colipase and that exhibits a strong emission band at 550 nm. Addition of micellar concentrations of taurodeoxycholate causes a 4.3-fold increase in the emission maximum as well as a 70 nm blue shift to 480 nm. Inclusion of oleic acid to form a mixed micelle reduces these spectral effects. Scatchard analysis of the data yield a Kd of 6.8 X 10(-4) M and a single colipase-binding site for taurodeoxycholate micelles. The data, by analogy to a phospholipase system, are consistent with a direct insertion of dansyl-NH-tyrosine-55 into the micelle. The presence of a single tryptophan residue (Trp-52) in equine colipase provides an intrinsic fluorescent probe for studying protein-micelle interaction. The emission maximum of horse colipase at 345 nm indicates a solvent-accessible tryptophan residue which becomes less so on binding of micelles. A blue shift of 8 nm and a 2-fold increase in amplitude is indicative of a more hydrophobic environment for tryptophan induced by taurodeoxycholate micelles. There is also a decrease in KSV for acrylamide quenching in the presence of micelles, which further supports a loss of solvent accessibility. The most dramatic pH effects are observed with KI quenching, and may indicate the presence of negative charges near Trp-52.     

Abnormal binding of soluble IgG immune complexes to hepatic nonparenchymal cells of autoimmune mice

Because the liver is the major organ responsible for removal of soluble immune complexes (IC), the surface binding characteristics of preformed model IC to unstimulated mouse liver nonparenchymal cells (NPC) in suspension were studied. NPC of non-autoimmune C3H/FeJ, C3H/HeJ, A/J, DBA/2 and the autoimmune NZB/W F1 and MRL/lpr female mice of various ages were isolated by perfusion of the portal vein with collagenase followed by separation of NPC from hepatocytes with a metrizamide gradient. Thirty-five percent of NPC of all mouse strains were nonspecific esterase-positive and phagocytosed latex beads. Radiolabeled mouse IgG anti-DNP covalently cross-linked stable IC were separated by gel filtration and bound to NPC under various conditions. Marked differences were noted in maximal number of IC bound per cell between the autoimmune and non-autoimmune mouse strains: 3.3 to 4.0 X 10(5) in the non-autoimmune strains vs 0.3 to 1.4 X 10(5) molecules of IC bound per cell in the autoimmune strains at 1 to 6 mo. Insignificant differences were noted in Ka by Scatchard plot analysis (3.5 to 5.0 X 10(8) M-1) and rate of reversibility of binding as determined by dissociation of surface-bound IC with an excess of heat-aggregated gamma-globulin (T 1/2:1.5 to 2 min). These data demonstrate a decreased number of available binding sites for IC in unstimulated NPC from NZB/W F1 and MRL/lpr female mice throughout their life spans. Although the findings are consistent with saturation of binding sites of the NPC with native IC, the abnormality found in the 1-mo-old autoimmune mice (who do not have detectable autoantibodies) suggests a primary defect in FC receptor expression or an altered state of activation of NPC that may contribute to the disease process.     

Midwife managed delivery unit: a randomised controlled comparison with consultant led care.

OBJECTIVE--To examine whether intrapartum care and delivery of low risk women in a midwife managed delivery unit differs from that in a consultant led labour ward. DESIGN--Pragmatic randomised controlled trial. Subjects were randomised in a 2:1 ratio between the midwives unit and the labour ward. SETTING--Aberdeen Maternity Hospital, Grampian. SUBJECTS--2844 low risk women, as defined by existing booking criteria for general practitioner units in Grampian. 1900 women were randomised to the midwives unit and 944 to the labour ward. MAIN OUTCOME MEASURES--Maternal and perinatal morbidity. RESULTS--Of the women randomised to the midwives unit, 647 (34%) were transferred to the labour ward antepartum, 303 (16%) were transferred intrapartum, and 80 (4%) were lost to follow up. 870 women (46%) were delivered in the midwives unit. Primigravid women (255/596, 43%) were significantly more likely to be transferred intrapartum than multi-gravid women (48/577, 8%). Significant differences between the midwives unit and labour ward were found in monitoring, fetal distress, analgesia, mobility, and use of episiotomy. There were no significant differences in mode of delivery or fetal outcome. CONCLUSIONS--Midwife managed intrapartum care for low risk women results in more mobility and less intervention with no increase in neonatal morbidity. However, the high rate of transfer shows that antenatal criteria are unable to determine who will remain at low risk throughout pregnancy and labour.     

Role of ADP in the control of actomyosin superprecipitation and ATPase activity.

ATP, in the presence of 0.05–0.15 m KCl and greater than 50 μm Mg²⁺, induces dissociation (clearing) followed by superprecipitation of skeletal muscle actomyosin. Superprecipitation has been studied as a model of muscle contraction, and ATP depletion has been associated with the onset of superprecipitation. Recent studies [Puszkin and Rubin (1975) Science188, 1319–1320] indicate that ADP stimulates superprecipitation without increasing the rate of ATP hydrolysis. We confirm that ADP stimulates superprecipitation; however, contrary to the experience of these investigators, ADP does stimulate ATP hydrolysis in the system studied here. We present evidence that superprecipitation is associated with generation of a critical ADP:ATP ratio but it appears that this ratio is an indirect measure of an associated but uncharacterized phenomenon which signals the onset of superprecipitation. Added ADP decreased the extent and duration of clearing, increased the rate of ATP hydrolysis, and increased the extent of superprecipitation of rat skeletal muscle actomyosin in the presence of excess Mg²⁺. The ADP effect was not mimicked by EDTA or AMP. The duration of clearing was related not to the time required to attain a specific level of any nucleotide phosphate, but to the time required to generate an ADP:ATP ratio of approximately 3.6. Apparently only that ADP generated in the system by ATP hydrolysis was involved in the critical ADP:ATP ratio. Added ADP stimulated myosin ATPase activity in 1.6 or 3.2 mm Mg²⁺. This effect was not mimicked by EDTA or AMP. The results are used to relate studies by others of myosin sulfhydryl modification to a recent model [Burke et al. (1973) Proc. Nat. Acad. Sci. USA70, 3793–3796] in which myosin MgATPase activity is inhibited by formation of a stable cyclic complex of MgATP and the S₁ and S₂ sites of heavy meromyosin.     

Adiposity Is Associated with Gender-Specific Reductions in Left Ventricular Myocardial Perfusion during Dobutamine Stress

Background

Obesity and visceral adiposity are increasingly recognized risk factors for cardiovascular disease. Visceral fat may reduce myocardial perfusion by impairing vascular endothelial function. Women experience more anginal symptoms compared to men despite less severe coronary artery stenosis, as assessed by angiography. Women and men have different fat storage patterns which may account for the observed differences in cardiovascular disease. Therefore, our objective was to evaluate the relationship between visceral adipose tissue distributions and myocardial perfusion in men and women.

Methods

Visceral and subcutaneous fat distributions and myocardial perfusion were measured in 69 men and women without coronary artery disease using magnetic resonance imaging techniques. Myocardial perfusion index was quantified after first-pass perfusion with gadolinium contrast at peak dose dobutamine stress.

Results

We observed inverse relationships between female gender (r = -0.35, p = 0.003), pericardial fat (r = -0.36, p = 0.03), intraperitoneal fat (r = -0.37, p = 0.001), and retroperitoneal fat (r = -0.36, p = 0.002) and myocardial perfusion index. Visceral fat depots were not associated with reduced myocardial perfusion at peak dose dobutamine in men. However, in women, BMI (r = -0.33, p = 0.04), pericardial fat (r = -0.53, p = 0.02), subcutaneous fat (r = -0.39, p = 0.01) and intraperitoneal fat (r = -0.30, p = 0.05) were associated with reduced myocardial perfusion during dobutamine stress.

Conclusions

Higher visceral fat volumes are associated with reduced left ventricular myocardial perfusion at peak dose dobutamine stress in women but not in men. These findings suggest that visceral fat may contribute to abnormal microcirculatory coronary artery perfusion syndromes, explaining why some women exhibit more anginal symptoms despite typically lower grade epicardial coronary artery stenoses than men.     

A Farnesyltransferase Inhibitor Induces Tumor Regression in Transgenic Mice Harboring Multiple Oncogenic Mutations by Mediating Alterations in Both Cell Cycle Control and Apoptosis

The farnesyltransferase inhibitor L-744,832 selectively blocks the transformed phenotype of cultured cells expressing a mutated H-ras gene and induces dramatic regression of mammary and salivary carcinomas in mouse mammary tumor virus (MMTV)–v-Ha-ras transgenic mice. To better understand how the farnesyltransferase inhibitors might be used in the treatment of human tumors, we have further explored the mechanisms by which L-744,832 induces tumor regression in a variety of transgenic mouse tumor models. We assessed whether L-744,832 induces apoptosis or alterations in cell cycle distribution and found that the tumor regression in MMTV–v-Ha-ras mice could be attributed entirely to elevation of apoptosis levels. In contrast, treatment with doxorubicin, which induces apoptosis in many tumor types, had a minimal effect on apoptosis in these tumors and resulted in a less dramatic tumor response. To determine whether functional p53 is required for L-744,832-induced apoptosis and the resultant tumor regression, MMTV–v-Ha-ras mice were interbred with p53^−/− mice. Tumors in ras/p53^−/− mice treated with L-744,832 regressed as efficiently as MMTV–v-Ha-ras tumors, although this response was found to be mediated by both the induction of apoptosis and an increase in G₁ with a corresponding decrease in the S-phase fraction. MMTV–v-Ha-ras mice were also interbred with MMTV–c-myc mice to determine whether ras/myc tumors, which possess high levels of spontaneous apoptosis, have the potential to regress through a further increase in apoptosis levels. The ras/myc tumors were found to respond nearly as efficiently to L-744,832 treatment as the MMTV–v-Ha-ras tumors, although no induction of apoptosis was observed. Rather, the tumor regression in the ras/myc mice was found to be mediated by a large reduction in the S-phase fraction. In contrast, treatment of transgenic mice harboring an activated MMTV–c-neu gene did not result in tumor regression. These results demonstrate that a farnesyltransferase inhibitor can induce regression of v-Ha-ras-bearing tumors by multiple mechanisms, including the activation of a suppressed apoptotic pathway, which is largely p53 independent, or by cell cycle alterations, depending upon the presence of various other oncogenic genetic alterations.     

Pseudomonas putida and Pseudomonas fluorescens Species Group Recovery from Human Homes Varies Seasonally and by Environment

By shedding light on variation in time as well as in space, long-term biogeographic studies can help us define organisms’ distribution patterns and understand their underlying drivers. Here we examine distributions of Pseudomonas in and around 15 human homes, focusing on the P. putida and P. fluorescens species groups. We describe recovery from 10,941 samples collected during up to 8 visits per home, occurring on average 2.6 times per year. We collected a mean of 141 samples per visit, from sites in most rooms of the house, from the surrounding yards, and from human and pet occupants. We recovered Pseudomonas in 9.7% of samples, with the majority of isolates being from the P. putida and P. fluorescens species groups (approximately 62% and 23% of Pseudomonas samples recovered respectively). Although representatives of both groups were recovered from every season, every house, and every type of environment sampled, recovery was highly variable across houses and samplings. Whereas recovery of P. putida group was higher in summer and fall than in winter and spring, P. fluorescens group isolates were most often recovered in spring. P. putida group recovery from soils was substantially higher than its recovery from all other environment types, while higher P. fluorescens group recovery from soils than from other sites was much less pronounced. Both species groups were recovered from skin and upper respiratory tract samples from healthy humans and pets, although this occurred infrequently. This study indicates that even species that are able to survive under a broad range of conditions can be rare and variable in their distributions in space and in time. For such groups, determining patterns and causes of stochastic and seasonal variability may be more important for understanding the processes driving their biogeography than the identity of the types of environments in which they can be found.     

Hydrogen peroxide is the major oxidant product of xanthine oxidase

Xanthine oxidase (XO) is a critical source of reactive oxygen species (ROS) in inflammatory disease. Focus, however, has centered almost exclusively on XO-derived superoxide (O₂^??), whereas direct H₂O₂ production from XO has been less well investigated. Therefore, we examined the relative quantities of O₂^?? and H₂O₂ produced by XO under a range (1–21%) of O₂ tensions. At O₂ concentrations between 10 and 21%, H₂O₂ accounted for ～75% of ROS production. As O₂ concentrations were lowered, there was a concentration-dependent increase in H₂O₂ formation, accounting for 90% of ROS production at 1% O₂. Alterations in pH between 5.5 and 7.4 did not affect the relative proportions of H₂O₂ and O₂^?? formation. Immobilization of XO, by binding to heparin–Sepharose, further enhanced relative H₂O₂ production by ～30%, under both normoxic and hypoxic conditions. Furthermore, XO bound to glycosaminoglycans on the apical surface of bovine aortic endothelial cells demonstrated a similar ROS production profile. These data establish H₂O₂ as the dominant (70–95%) reactive product produced by XO under clinically relevant conditions and emphasize the importance of H₂O₂ as a critical factor when examining the contributory roles of XO-catalyzed ROS in inflammatory processes as well as cellular signaling.     

Pericardial fat and myocardial perfusion in asymptomatic adults from the Multi-Ethnic Study of Atherosclerosis

Background

Pericardial fat has adverse effects on the surrounding vasculature. Previous studies suggest that pericardial fat may contribute to myocardial ischemia in symptomatic individuals. However, it is unknown if pericardial fat has similar effects in asymptomatic individuals.

Methods

We determined the association between pericardial fat and myocardial blood flow (MBF) in 214 adults with no prior history of cardiovascular disease from the Minnesota field center of the Multi-Ethnic Study of Atherosclerosis (43% female, 56% Caucasian, 44% Hispanic). Pericardial fat volume was measured by computed tomography. MBF was measured by MRI at rest and during adenosine-induced hyperemia. Myocardial perfusion reserve (PR) was calculated as the ratio of hyperemic to resting MBF.

Results

Gender-stratified analyses revealed significant differences between men and women including less pericardial fat (71.9±31.3 vs. 105.2±57.5 cm³, p<0.0001) and higher resting MBF (1.12±0.23 vs. 0.93±0.19 ml/min/g, p<0.0001), hyperemic MBF (3.49±0.76 vs. 2.65±0.72 ml/min/g, p<0.0001), and PR (3.19±0.78 vs. 2.93±0.89, p = 0.03) in women. Correlations between pericardial fat and clinical and hemodynamic variables were stronger in women. In women only (p = 0.01 for gender interaction) higher pericardial fat was associated with higher resting MBF (p = 0.008). However, this association was attenuated after accounting for body mass index or rate-pressure product. There were no significant associations between pericardial fat and hyperemic MBF or PR after multivariate adjustment in either gender. In logistic regression analyses there was also no association between impaired coronary vasoreactivity, defined as having a PR <2.5, and pericardial fat in men (OR, 1.18; 95% CI, 0.82–1.70) or women (OR, 1.11; 95% CI, 0.68–1.82).

Conclusions

Our data fail to support an independent association between pericardial fat and myocardial perfusion in adults without symptomatic cardiovascular disease. Nevertheless, these findings highlight potentially important differences between asymptomatic and symptomatic individuals with respect to the underlying subclinical disease burden.     

A nuclear RNA is cut out for translation

In this issue of Cell, Prasanth et al. (2005) provide evidence that an inosine-containing RNA that is normally retained in the nucleus is cleaved within its 3' untranslated region following cellular stress. It is then transported to the cytoplasm and translated into protein. These findings suggest that the nucleus may store RNAs destined for translation that then can be released, as needed, in response to specific cellular signals.