Thermodynamic stability of wild-type and mutant p53 core domain

Some 50% of human cancers are associated with mutations in the core domain of the tumor suppressor p53. Many mutations are thought just to destabilize the protein. To assess this and the possibility of rescue, we have set up a system to analyze the stability of the core domain and its mutants. The use of differential scanning calorimetry or spectroscopy to measure its melting temperature leads to irreversible denaturation and aggregation and so is useful as only a qualitative guide to stability. There are excellent two-state denaturation curves on the addition of urea that may be analyzed quantitatively. One Zn²⁺ ion remains tightly bound in the holo-form of p53 throughout the denaturation curve. The stability of wild type is 6.0 kcal (1 kcal = 4.18 kJ)/mol at 25°C and 9.8 kcal/mol at 10°C. The oncogenic mutants R175H, C242S, R248Q, R249S, and R273H are destabilized by 3.0, 2.9, 1.9, 1.9, and 0.4 kcal/mol, respectively. Under certain denaturing conditions, the wild-type domain forms an aggregate that is relatively highly fluorescent at 340 nm on excitation at 280 nm. The destabilized mutants give this fluorescence under milder denaturation conditions.     

Acute disseminated encephalomyelitis: the importance of early magnetic resonance imaging

The diagnosis of acute disseminated encephalomyelitis (ADEM) is frequently missed or delayed with consequent delay in instituting therapy in the crucial phase of the illness. The role of MRI in the diagnosis of ADEM is well established, however, the value of its early utilization of treatment on the outcome of patients has not been adequately stressed. Three patients with ADEM are described. Delay in the diagnosis of the first was associated with severe sequelae, while in the other two early diagnosis and institution of corticosteroid therapy which was facilitated by MRI, was associated with a better outcome. MRI should be carried out early once the diagosis of ADEM is entertained.     

Associations between soil lead and childhood blood lead in urban New Orleans and rural Lafourche Parish of Louisiana.

This study evaluates associations between soil lead concentrations (SPb), age of housing, and blood lead levels (BPb) of children in metropolitan New Orleans and Lafourche Parish, Louisiana. The database includes over 2,600 SPb and 6,000 BPb samples paired by their median values and pre-1940 housing percentages for 172 census tracts. Associations were evaluated with Fisher's exact test and Spearman's rho test and modeled with the least sum of absolute deviations regression. Census tracts with low SPb are associated with new housing, but census tracts with high SPb are evenly split between old and new housing [Fisher's exact test, p = 8.60 X 10(-13) for the percentage of housing built before 1940 (percent pre-1940 housing) versus SPb]. The p-value for SPb versus BPb is 12 orders of magnitude stronger than the p-value for percent pre-1940 housing versus BPb. Census tracts with low BPb are associated with new housing, but census tracts with high BPb are split evenly between old and new housing (Fisher's exact test, p = 1. 67 X 10(-12) for percent pre-1940 housing versus BPb). Census tracts with high SPb are associated with high BPb and census tracts with low SPb are associated with low BPb (Fisher's exact test, p = 3.18 X 10(-24) for BPb versus SPb). The Spearman's rho test of the association of SPb and BPb in Orleans and Lafourche Parishes yielded a p-value of 6.12 X 10(-24). The least sum of absolute deviations regression model of the data is BPb = 1. 845 + 0.7215 (SPb)0.4. A comparison of the modeled BPb versus observed BPb has an r(2) of 0.552 and a p-value of 2.83 X 10(-23) that this relation was due to chance. If blood lead in children is more closely associated to soil lead than to the age of housing, then primary lead prevention should also include soil lead.     

Lumbar facet joint tropism does not accelerate degeneration of the facet joints.

PURPOSETo study the relationship of lumbar facet joint tropism to degeneration of the cartilage and subcortical bone in the facet joints and the effect of tropism in intervertebral disk degeneration.METHODSThe orientation of 104 cadaveric lumbar facet joints with respect to sagittal plane was measured on CT scans, and the joints were classified as having no tropism, mild tropism, or severe tropism. On MR images, the severity of cartilage degeneration and bony sclerosis was measured. The correlation between tropism and degeneration was calculated, as was the relationship among age, spinal level, and degeneration.RESULTSWe identified four spinal levels with severe tropism, six with moderate tropism, and 94 without tropism. Cartilage degeneration was not significantly more severe in the joints with tropism than in the joints without. Sclerosis was slightly greater in the joints with tropism than in the joints without it. Sclerosis and cartilage degeneration were significantly related to age and spinal level.CONCLUSIONAge, spinal level, and overall facet joint angle are more important factors in facet joint degeneration than is tropism.     

Neuropeptides in idiopathic chronic constipation (slow transit constipation)

Tissue specimens from the large bowel of 18 patients with long-standing slow transit constipation were investigated to determine the distribution and density of several neuropeptides and amines in the enteric nerve system, and also of endocrine cells in comparison to normal individuals. CGRP (calcitonin gene-related peptide), galanin, glucagon, GRP (gastrin-releasing peptide), metenkephalin, motilin, neuropeptide Y (NPY), PACAP, peptide YY (PYY), serotonin, somatostatin, substance P and VIP were studied by immunohistochemistry. Tissue concentrations of VIP, substance P and galanin were also measured by radioimmunoassay. Significantly increased VIP, SP and galanin contents were found in specimens from the ascending colon. Levels of VIP and galanin were also increased in the transverse colon. Immunohistochemistry revealed only marginal changes with an increased density of PACAP nerve fibres in the smooth muscle and of VIP and PACAP nerves in the myenteric plexus of the transverse colon. In the descending colon substance P and NPY immunoreactivity were also increased in the myenteric plexus while the density of VIP nerve fibres was reduced in the mucosa/submucosa. The frequency of PYY-containing cells and the 5-HT-containing cells in the ascending colon was significantly increased in the constipated patients.     

The effect of outdoor fungal spore concentrations on daily asthma severity.

The relationship between day-to-day changes in asthma severity and combined exposures to community air pollutants and aeroallergens remains to be clearly defined. We examined the effects of outdoor air pollutants, fungi, and pollen on asthma. Twenty-two asthmatics ages 9-46 years were followed for 8 weeks (9 May-3 July 1994) in a semirural Southern California community around the air inversion base elevation (1,200 ft). Daily diary responses included asthma symptom severity (6 levels), morning and evening peak expiratory flow rates (PEFR), and as-needed beta-agonist inhaler use. Exposures included 24-hr outdoor concentrations of fungi, pollen, and particulate matter with a diameter < 10 microns (PM10; maximum = 51 micrograms/m3) and 12-hour day-time personal ozone (O3) measurements (90th percentile = 38 ppb). Random effects longitudinal regression models controlled for autocorrelation and weather. Higher temperatures were strongly protective, probably due to air conditioning use and diminished indoor allergens during hot, dry periods. Controlling for weather, total fungal spore concentrations were associated with all outcomes: per minimum to 90th percentile increase of nearly 4,000 spores/m3, asthma symptom scores increased 0.36 (95% CI, 0.16-0.56), inhaler use increased 0.33 puffs (95% CI, -0.02-0.69), and evening PEFR decreased 12.1 l/min (95% CI, -1.8-22.3). These associations were greatly enhanced by examining certain fungal types (e.g., Alternaria, basidiospores, and hyphal fragments) and stratifying on 16 asthmatics allergic to tested deuteromycete fungi. There were no significant associations to low levels of pollen or O3, but inhaler use was associated with PM10 (0.15 inhaler puffs/10 micrograms/m3; p < 0.02). These findings suggest that exposure to fungal spores can adversely effect the daily respiratory status of some asthmatics.     

Covalent binding of 7,12-dimethylbenz[a]anthracene to lymphoid and nonlymphoid tissues following oral administration to B6C3F1 mice.

Previous studies have shown that 7,12-dimethylbenz[a]anthracene (DMBA) is cytotoxic to various murine lymphoid tissues, including the spleen, thymus, mesenteric lymph nodes (MLNs), and Peyer's patches (PPs). In the present studies, we measured the amount of covalent binding of [3H]DMBA to lymphoid and nonlymphoid tissues and correlated these findings with the overall levels of [3H]DMBA (and derived substances) present in various tissues following a single oral administration to mice. Results show that [3H]DMBA was taken up relatively rapidly from the GI tract and that it was nearly completely eliminated within 24 hr via the feces. Peak plasma levels were obtained approximately 6 hr after gavage, and most organs (including brain, heart, liver, lung, kidney, spleen, and thymus) achieved their peak level of DMBA at this time. Maximal concentrations of DMBA were detected in gut-associated lymphoid tissues (i.e., PPs and MLNs) at 4 hr, during which time covalent binding of [3H]DMBA was also maximal. The time course for covalent binding was different in the liver, lung, thymus, and spleen, peaking at 6-12 hr. The amount of covalent binding of [3H]DMBA and derived metabolites in the spleen was more than twice that seen in the other tissues examined. Since the spleen has previously been found to be less sensitive to DNA fragmentation induced by DMBA than the PPs, these results suggest that covalent binding may not be the primary determinant of lymphotoxicity in these organs.     

The Membrane Lipid Cholesterol Modulates Anesthetic Actions on a Human Brain Ion Channel

Background: Molecular theories of general anesthesia often are divided into two categories: (l) Anesthetics may bind specifically to proteins, such as ionic channels, and alter their function directly, and (2) anesthetics may alter the functions of integral membrane proteins indirectly through modification of the physical properties of the membrane. Recent studies have provided evidence that anesthetics can bind to proteins and modify their function directly, bringing into question the role of the membrane in anesthetic interactions. To reexamine the role of membrane lipids in anesthetic interactions, an experimental approach was used in which the membrane lipid composition could be systematically altered and the impact on anesthetic interactions with potential targets examined.

Methods: Sodium channels from human brain cortex were incorporated into planar lipid bilayers with increasing cholesterol content. The anesthetic suppression of these channels by pentobarbital was quantitatively examined by single channel measurements under voltage-clamp conditions.

Results: Changes in cholesterol content had no effect on measured channel properties in the absence of anesthetic. In the presence of pentobarbital, however, cholesterol inhibited anesthetic suppression of channel ionic currents, with 1.9% (weight/weight, corresponding to 3.5 mol%) cholesterol decreasing anesthetic suppression of sodium channels by half.