Peroxynitrite increases the degradation of aconitase and other cellular proteins by proteasome

We report that exposure of aconitase to moderate concentrations of peroxynitrite, 3-morpholinosydnonimine (SIN-1; a superoxide- and nitric oxide-liberating substance), or hydrogen peroxide, inhibits the enzyme and enhances susceptibility to proteolytic digestion by the isolated 20 S proteasome. Exposure to more severe levels of oxidative stress, from these same agents, causes further inhibition of the enzymatic activity of aconitase but actually decreases its proteolytic breakdown by proteasome. It should be noted that the superoxide and nitric oxide liberated by SIN-1 decomposition react to form a steady flux of peroxynitrite. S-Nitroso-N-acetylpenicillamine, a compound that liberates nitric oxide alone, causes only a small loss of aconitase activity (25% or less) and has no effect on the proteolytic susceptibility of the enzyme. Proteasome also seems to be the main protease in cell lysates that can degrade aconitase after it has been oxidatively modified by exposure to peroxynitrite, SIN-1, or hydrogen peroxide. Using cell lysates isolated from K562 cells treated for several days with an antisense oligodeoxynucleotide to the initiation codon region of the C2 subunit of proteasome (a treatment which diminishes proteasome activity by 50-60%), the enhanced degradation of moderately damaged aconitase was essentially abolished. Other model proteins as well as complex mixtures of proteins, such as cell lysates, also exhibit enhanced proteolytic susceptibility after moderate SIN-1 treatment. Therefore we conclude that peroxynitrite reacts readily with proteins and that mild modification by peroxynitrite results in selective recognition and degradation by proteasome.     

A newly identified polymorphism in the apolipoprotein E enhancer gene region is associated with Alzheimer's disease and strongly with the epsilon 4 allele

Apolipoprotein E allele 4 (apoE epsilon 4) is a major risk factor for late-onset AD. Inheritance of this allele is associated with an earlier age of onset of dementia in individuals with AD. It is unknown whether other polymorphisms in the apoE gene may influence the effect of apoE epsilon 4 on AD. We screened portions of the promoter enhancer element and of the apoE receptor binding domain for other polymorphisms that could affect risk of AD. In particular, a C/G polymorphism at position +113 of the apoE mRNA in the apoE intron 1 enhancer element (IE1) has been recently identified. We found no other polymorphisms. We studied the relationship of the two alleles of the IE1 polymorphism with AD and found an apparent association between IE1 G and AD (n = 94; p = 0.0515). However, the IE1 G allele is also closely associated with apoE epsilon 4 (p < 0.0001). When the presence of apoE epsilon 4 is covaried, the association between the IE1 G allele and AD is no longer statistically significant (odds ratio = 1.29, 95% confidence interval: 0.44, 3.78). In contrast, epsilon 4 is still highly associated with AD when IE1 G is controlled for (odds ratio = 5.91, 95% confidence interval: 3.29, 10.63). Furthermore, there is no significant association between the age of onset of dementia and the inheritance of the G allele. We believe that the apparent association between IE1 G and AD is a consequence of the association between the epsilon 4 and IE1 G alleles.     

The prognostic significance of tissue-specific antibodies in the extraction of cataracts of different etiologies

The authors demonstrate a high informative value of preoperative lacrimal fluid examinations to predict postoperative uveitis in patients with complicated cataracts. Detection of antituberculin antibodies in titers 1:128 or higher is a factor of risk of development of a postoperative inflammatory reaction. The risk of postoperative uveitis is increased to 100% if antibodies to tuberculin, tissue-specific retinal S antigen, and lenticular proteins are simultaneously detected in the lacrimal fluid.     

A nested PCR for detection of North American isolates of bluetongue virus based on NS1 genome sequence analysis of BTV-17

A nested polymerase chain reaction (PCR)-based assay, for detection of bluetongue virus (BTV) ribonucleic acid in cell culture and tissue samples, was developed. Two pairs of oligonucleotide primers (BTV1 and BTV4 and BTV2 and BTV3), selected from non-structural protein 1 (NS1) gene of BTV-17, were used for the nested PCR in two amplification steps. First a 826-bp product was amplified using an outer primer pair BTV1 and BTV4. The second amplification, using nested or internal primer pair BTV2 and BTV3, produced a 517-bp PCR product. RNA from North American prototype serotypes 2, 10, 11, 13 and 17, propagated in cell cultures, were detected by this nested PCR-based assay. The nested primers BTV2 and BTV3 increased the sensitivity of the BTV PCR assay, and as little as 0.1 fg of BTV RNA (equivalent to 5 viral particles) could be detected. Amplification products were not detected when the PCR-based assay was applied to RNA from a closely related orbivirus, epizootic hemorrhagic disease virus (EHDV) prototype serotypes 1 and 2; total nucleic acid extracts from uninfected BHK-21 cells; or whole blood from calves and deer that were BTV-seronegative and virus isolation negative. Application of this nested BTV PCR-based assay to clinical samples resulted in detection of BTV RNA from a variety of tissues collected from calves and deer with natural and experimental BTV infections. The described BTV PCR-based assay provides a valuable tool to study the epidemiology of BTV infection in susceptible wild ruminants and domestic livestock.     

AN APPARATUS FOR DETERMINING THE ANNEALING CONSTANTS OF GLASS1

An apparatus is described by means of which annealing data on glass may be rapidly and accurately obtained. Essentially, the apparatus consists of a mechanical means straining the glass, an optical means of measuring the stresses, and a means of heating the sample uniformly while making the measurement. The results are consistent and expressed in definite terms. The specimens for test may be quite small. The apparatus may be used for control purposes in place of measuring softening temperatures.     

Guidance of intracoronary radiation therapy based on dose-volume histograms derived from quantitative intravascular ultrasound

BACKGROUND/AIMS: The risk factors for esophageal variceal rebleeding are little known. Variceal pressure is one of the major determinants of variceal rupture, but the relationship between variceal pressure and variceal rebleeding during maintenance sclerotherapy has not been determined. This study was undertaken to evaluate the relationship between variceal pressure/gradient change and variceal rebleeding during maintenance sclerotherapy. METHODS: Patients with liver cirrhosis and recent esophageal variceal hemorrhage underwent consecutive variceal pressure measurements by direct puncture of the varices before each elective sclerotherapy. RESULTS: In 46 patients, the initial variceal pressure was no different regardless of age, sex, underlying etiology or hepatic reserve. Variceal pressure was higher in large varices, varices with more severe red wale markings, and varices with slower reduction in size during maintenance sclerotherapy. A larger volume of sclerosant was required to eradicate large varices, varices with more severe red wale markings, and varices with slower reduction in size during maintenance sclerotherapy. There was a positive correlation between initial variceal pressure and total amount of sclerosant (r=0.485, p=0.001). Initial variceal pressure was not related to rebleeding. Variceal pressure increased more in patients with rebleeding from varices per se (n=7) than in those without rebleeding (n= 24). There was no difference in pressure change between patients without rebleeding (n=24) and those with rebleeding from variceal ulcers (n=7). CONCLUSIONS: Large varices, severe red color signs and slow reduction in variceal size were associated with higher initial variceal pressure, and more sclerosant was required to eradicate the varices. An increase in variceal pressure during maintenance sclerotherapy indicates a higher risk of variceal rebleeding, but not of variceal ulcer rebleeding.     

Anatomy and histotopography of human pericardial lymphoid tissue

Data concerning microscopic structure of the layers of human serous pericardium, localization of lymphoid structures as related to them and their interrelations with connective tissue elements and vessels are represented. Findings on the thickness of serous pericardium in different layers are also given.     

Transthoracic vertebrectomy for metastatic spinal tumors

Protein kinase C is an important second messenger system, which is translocated from the cytosol to the cell membrane upon cell stimulation. We used confocal microscopy to study the spatial distribution of protein kinase C isoforms after stimulation of cultured vascular smooth muscle cells with different agonists. First, we analysed the effects of angiotensin II and platelet-derived growth factor (PDGF). Confocal microscopy showed a rapid assembly of PKC alpha along cytosolic fibres followed by a translocation towards the nucleus with angiotensin II. PDGF engendered a similar, but much slower response; however, a cytoskeletal distribution was not observed. We then investigated the effects of thrombin and bFGF on nuclear translocation. bFGF induced a rapid translocation of the isoform towards the perinuclear region and into the nucleus. bFGF had a similar effect on PKC epsilon. In contrast, thrombin had a smaller effect on nuclear translocation of PKC alpha and did not influence PKC epsilon, but instead induced a rapid nuclear translocation of PKC zeta. Thus, tyrosine kinase receptor activation via bFGF induces a rapid association of PKC alpha and epsilon within nuclear structures. Our results show that agonists cause, not only a translocation of protein kinase C isoforms into the cell membrane but also into the cell nucleus. Lastly, we analyzed the nuclear immunoreactivity of the PKC isoforms, alpha, delta, epsilon and zeta in vascular smooth muscle cells during the cell cycle. Resting cells were stimulated with foetal calf serum (FCS, 10%), which translocated PKC alpha and epsilon to the perinuclear region and into the nucleus, while PKC delta and zeta showed no increase in nuclear immunoreactivity. After 4 h of FCS, the nuclear immunoreactivity for PKC alpha and epsilon was reduced to or below control values. At 8 h, increased nuclear expression of isoforms alpha, epsilon and zeta was observed, while isoform delta was not affected. Our results demonstrate a complex spatial and temporal regulation of PKC isoforms in response to vasoactive hormones and growth factors. We suggest that protein kinase C may be important for nuclear signaling and demonstrate that nuclear translocation of PKC isoforms is differentially regulated during the cell cycle.