Dramatic aggregation of Alzheimer abeta by Cu(II) is induced by conditions representing physiological acidosis

The cortical deposition of Abeta is an event that occurs in Alzheimer's disease, Down's syndrome, head injury, and normal aging. Previously, in appraising the effects of different neurochemical factors that impact upon the solubility of Abeta, we observed that Zn2+ was the predominant bioessential metal to induce the aggregation of soluble Abeta at pH 7.4 in vitro and that this reaction is totally reversible with chelation. We now report that unlike other biometals tested at maximal biological concentrations, marked Cu2+-induced aggregation of Abeta1-40 emerged as the solution pH was lowered from 7.4 to 6.8 and that the reaction was completely reversible with either chelation or alkalinization. This interaction was comparable to the pH-dependent effect of Cu2+ on insulin aggregation but was not seen for aprotinin or albumin. Abeta1-40 bound three to four Cu2+ ions when precipitated at pH 7.0. Rapid, pH-sensitive aggregation occurred at low nanomolar concentrations of both Abeta1-40 and Abeta1-42 with submicromolar concentrations of Cu2+. Unlike Abeta1-40, Abeta1-42 was precipitated by submicromolar Cu2+ concentrations at pH 7.4. Rat Abeta1-40 and histidine-modified human Abeta1-40 were not aggregated by Zn2+, Cu2+, or Fe3+, indicating that histidine residues are essential for metal-mediated Abeta assembly. These results indicate that H+-induced conformational changes unmask a metal-binding site on Abeta that mediates reversible assembly of the peptide. Since a mildly acidic environment together with increased Zn2+ and Cu2+ are common features of inflammation, we propose that Abeta aggregation by these factors may be a response to local injury. Cu2+, Zn2+, and Fe3+ association with Abeta explains the recently reported enrichment of these metal ions in amyloid plaques in Alzheimer's disease.     

Use of liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) for routine identification of enzymatically digested proteins separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis

A distinct form of aplasia cutis congenita presenting as linear facial skin defects has been described under a variety of names as Xp deletion syndrome. MIDAS (microphthalmia, dermal aplasia and sclerocornea) syndrome, MLS (microphthalmia and linear skin defects) and Gazali-Temple syndrome. The syndrome is lethal in males, and its severity in females varies from a relatively mild residual facial scarring with short stature to lethal developmental organ malformations. A new case with peculiar ultrastructural findings is presented. A review of the literature suggests that these associations represent a series of contiguous-gene syndromes.     

Endothelin-1 levels in induced sputum samples from asthmatic and normal subjects

BACKGROUND: Endothelin-1 (ET-1) is a potent bronchoconstrictor which may have a role in the pathogenesis of asthma. The levels of ET-1 in saliva, induced sputum, and plasma from asthmatic and non-asthmatic subjects were compared. METHODS: Sputum induction was performed on 28 asthmatic subjects and nine normal volunteers. ET-1 levels were measured in plasma, saliva, and sputum samples and reversed phase high performance liquid chromatography (RP-HPLC) was performed on saliva and sputum samples. RESULTS: ET-1 was present in the following order of concentration in both normal and asthmatic subjects: saliva > sputum > plasma (saliva, median 30.1 and 23.9 pg/ ml, respectively; sputum, median 15.5 and 11.2 pg/ml; plasma, median 3.1 and 3.6 pg/ ml). There were no differences between asthmatic and normal subjects in the levels of ET-1 in each fluid. The levels of ET-1 in asthmatic subjects were not influenced by whether or not they were taking inhaled steroids. RP-HPLC of sputum and saliva confirmed the presence of ET-1 in these fluids. CONCLUSIONS: Levels of ET-1 can be measured in saliva and sputum obtained by sputum induction in asthmatic and healthy subjects and, although no difference was found in basal levels of ET-1 in sputum, saliva and plasma between normal subjects and asthmatics without bronchoconstriction, it is apparent that ET-1 is produced or released locally within the respiratory tract in concentrations higher than those in plasma.     

Mask physiotherapy for prevention of pulmonary complications after heart surgery. A controlled study

The objective of this prospective, consecutive, randomized, controlled study was to investigate the effects of mask physiotherapy on postoperative complications after heart surgery. Sixty-six low-risk male patients undergoing coronary artery by-pass graft surgery were evaluated. The patients were treated with routine chest physiotherapy alone or supplied with either positive expiratory pressure (PEP), or inspiratory resistance-positive expiratory pressure (IR-PEP). Postoperative pulmonary complications were assessed by forced vital capacity (FVC), arterial oxygen tension (PaO2), and chest X-ray examination. There was an almost equal decrease and subsequent rise in spirometric and blood gas values in all three groups, but patients treated with the PEP mask had a borderline significantly higher increase in PaO2 from day three to day six compared with patients treated with no mask. There was an almost equal frequency of atelectasis in the three treatment groups. It is concluded that no significant differences in outcome were found between the three groups.     

His-65 in the proton-sucrose symporter is an essential amino acid whose modification with site-directed mutagenesis increases transport activity

The proton-sucrose symporter that mediates phloem loading is a key component of assimilate partitioning in many higher plants. Previous biochemical investigations showed that a diethyl pyrocarbonate-sensitive histidine residue is at or near the substrate-binding site of the symporter. Among the proton-sucrose symporters cloned to date, only the histidine residue at position 65 of AtSUC1 from Arabidopsis thaliana is conserved across species. To test whether His-65 is involved in the transport reaction, we have used site-directed mutagenesis and functional expression in yeast to determine the significance of this residue in the reaction mechanism. Symporters with mutations at His-65 exhibited a range of activities; for example, the H65C mutant resulted in the complete loss of transport capacity, whereas H65Q was almost as active as wild type. Surprisingly, the H65K and H65R symporters transport sucrose at significantly higher rates (increased Vmax) than the wild-type symporter, suggesting His-65 may be associated with a rate-limiting step in the transport reaction. RNA gel blot and protein blot analyses showed that, with the exception of H65C, the variation in transport activity was not because of alterations in steady-state levels of mRNA or symporter protein. Significantly, those symporters with substitutions of His-65 that remained transport competent were no longer sensitive to inactivation by diethyl pyrocarbonate, demonstrating that this is the inhibitor-sensitive histidine residue. Taken together with our previous results, these data show that His-65 is involved in sucrose binding, and increased rates of transport implicate this region of the protein in the transport reaction.     

X-linked retinitis pigmentosa in two families with a missense mutation in the RPGR gene and putative change of glycine to valine at codon 60

OBJECTIVE: This study describes the ophthalmic findings in two unrelated white families with X-linked retinitis pigmentosa (XLRP) caused by a missense mutation in the retinitis pigmentosa GTPase regulator (RPGR) gene. DESIGN: Genetic screening and clinical correlation. PARTICIPANTS: Thirty-six families with XLRP seen by the authors were screened for a possible mutation in the RPGR gene to identify three affected hemizygotes with retinitis pigmentosa and four heterozygote carriers in one family and one hemizygote and one carrier in a second family. INTERVENTION: All nine patients underwent a routine ocular examination, including slit-lamp biomicroscopy and a dilated fundus examination. Goldmann visual field kinetic perimetry, static threshold perimetry, and electroretinography also were obtained. The DNA screening was performed on the three affected male patients and four obligate carriers examined from one family and the two examined patients, plus an additional male and obligate carrier, from the second family to determine the presence of any causative mutation in the RPGR gene. MAIN OUTCOME MEASURES: Findings on fundus examination, static threshold and kinetic perimetry, and electroretinography testing were the main outcome measures. RESULTS: A G-->T nucleotide change at position 238 in exon 3 of the RPGR gene resulting in a putative substitute of Gly-->Val at codon 60 was shown to segregate with RP in affected males and the carrier state in female heterozygotes in these two families. The ophthalmologic findings in hemizygotes as well as the carriers in this family were within the spectrum of findings characteristically noted in XLRP families. A tapetal-like reflex was not observed in any of the five female carriers. Psychophysical and electrophysiologic testing on the carriers indicated that cone and rod functions were impaired equivalently. When present in the carriers, visual field restriction was most apparent in, or limited to, the superotemporal quadrant, which corresponded to the retinal pigmentary changes that tended to occur in the inferonasal retina. CONCLUSIONS: A mutation in exon 3 of the RPGR gene, which would result in a putative glycine to valine substitution at codon 60, is associated with a severe clinical phenotype in male patients and a patchy retinopathy without a tapetal-like reflex in carrier females. In these families, heterozygote carriers showed equivalent impairment of their cone and rod function.     

Comparison of push-pull versus discard method from central venous catheters for blood testing

This article evaluates recent research results comparing two different methods for obtaining complete blood cell counts and chemistry profiles from central venous catheters. Complete blood cell count and chemistry profile samples were obtained from 25 patients at three hospital-based infusion clinics. The results indicate no appreciable difference between these two methods. Minimizing risks of blood loss, blood exposure to healthcare personnel, potential specimen contamination, and erroneous reporting are benefits of the push-pull method.     

Opioid inhibition of hippocampal interneurons via modulation of potassium and hyperpolarization-activated cation (Ih) currents

The actions of mu- and delta-opioid agonists (DAMGO and DPDPE, respectively) on GABAergic interneurons in stratum oriens of area CA1 of the hippocampus were examined by using whole-cell voltage-clamp recordings in brain slices. Both agonists consistently generated outward currents of similar magnitude (15-20 pA) in the majority of cells. However, under control conditions, current-voltage (I/V) relationships revealed that only a small number of these cells (3 of 77) demonstrated clear increases in membrane conductance, associated with the activation of the potassium current known as Girk. These interneurons also exhibited a slowly activating, inwardly rectifying current known as Ih on hyperpolarizing step commands. Ih was blocked by the extracellular application of cesium (3-9 mM) or ZD 7288 (10-100 microM) but was insensitive to barium (1-2 mM). In an effort to determine whether the holding current changes were attributable to the modulation of Girk and/or Ih, we used known blockers of these ion channels (barium or cesium and ZD 7288, respectively). Extracellular application of cesium (3-9 mM) or ZD 7288 (25-100 microM) blocked Ih and significantly reduced the opioid-induced outward currents by 58%. Under these conditions the opioid agonists activated a potassium current with characteristics similar to Girk. Similarly, during barium (1-2 mM) application the opioid-induced outward currents were reduced by 46%, and a clear reduction in Ih and the whole-cell conductance was revealed. These data suggest that the opioids can modulate both Ih and Girk in the same population of stratum oriens interneurons and that the modulation of these ion channels can contribute to the inhibition of interneuron activity in the hippocampus.     

Adenosine: potential modulator for vasovagal syncope

OBJECTIVES: This study examined the hypothesis that adenosine could provoke a vasovagal response in susceptible patients. Mechanisms of the vasovagal response were further explored by studying the adenosine-mediated reactions. BACKGROUND: Increased sympathetic activity is frequently observed before vasovagal syncope. Recent studies have demonstrated that adenosine, in addition to its direct bradycardiac and vasodilatory effects, can increase sympathetic discharge by activating cardiovascular afferent nerves. METHODS: The effects of adenosine and head-up tilt-table testing with or without isoproterenol were prospectively evaluated in 85 patients examined for syncope after negative results of electrophysiologic testing (51 men and 34 women, mean [+/- SD] age 61 +/- 17 years). Adenosine bolus injections of 6 mg and 12 mg were sequentially administered to patients in the upright position. The same protocol was implemented in 14 normal control subjects (7 men and 7 women, mean [+/- SD] age 38 +/- 10 years). RESULTS: Transient hypertension or tachycardia was observed in 57 (67%) and 20 (24%) patients after administration of 6 mg and 12 mg of adenosine, respectively, during the immediate phase (first 15 s), suggesting direct sympathetic activation. Hypotension and reflex tachycardia were observed in all patients during the delayed phase (15 to 60 s after adenosine injection), suggesting baroreceptor unloading. A vasovagal response was induced in 22 (26%) and 29 (34%) patients after adenosine administration and during tilt-table testing. Inducibility of a vasovagal response by these two methods was comparable (p = 0.12). Of the control subjects, one (7%) had a vasovagal response after adenosine administration and one (7%) had a positive response during tilt-table testing. CONCLUSIONS: These observations support the idea that adenosine is an endogenous modulator of the cardiac excitatory afferent nerves. Sympathetic activation by adenosine can be direct (i.e., cardiac excitatory afferent nerves) and indirect (i.e., vasodilation and reflex sympathetic activation). Adenosine could be an important modulator in triggering a vasovagal response in susceptible patients during examination for syncope.     

Cardiac muscle cell hypertrophy and apoptosis induced by distinct members of the p38 mitogen-activated protein kinase family

p38 mitogen-activated protein (MAP) kinase activities were significantly increased in mouse hearts after chronic transverse aortic constriction, coincident with the onset of ventricular hypertrophy. Infection of cardiomyocytes with adenoviral vectors expressing upstream activators for the p38 kinases, activated mutants of MAP kinase kinase 3b(E) (MKK3bE) and MAP kinase kinase 6b(E) (MKK6bE), elicited characteristic hypertrophic responses, including an increase in cell size, enhanced sarcomeric organization, and elevated atrial natriuretic factor expression. Overexpression of the activated MKK3bE in cardiomyocytes also led to an increase in apoptosis. The hypertrophic response was enhanced by co-infection of an adenoviral vector expressing wild type p38 beta, and was suppressed by the p38 beta dominant negative mutant. In contrast, the MKK3bE-induced cell death was increased by co-infection of an adenovirus expressing wild type p38 alpha, and was suppressed by the dominant negative p38 alpha mutant. This provides the first evidence in any cell system for divergent physiological functions for different members of the p38 MAP kinase family. The direct involvement of p38 pathways in cardiac hypertrophy and apoptosis suggests a significant role for p38 signaling in the pathophysiology of heart failure.