Randomized Trial of Sternal Closure for Low Risk Patients: Rigid Fixation versus Wire Closure.

BACKGROUND: A previous retrospective analysis of our cardiac surgery patients showed shortened ventilation time and hospital stay among patients receiving rigid sternal fixation compared to sternal wire fixation. We performed a prospective randomized study to further investigate these outcomes and determine if rigid closure can provide reduced pain after cardiac surgery. METHODS: Patients undergoing cardiac surgery between July 2011 and May 2014 were prospectively randomized into wire closure (Group C) or rigid fixation using sternal plates (Group R) groups. Age above 80, emergency surgery, redo sternotomy, and immunosuppression were among major exclusion criteria precluding randomization.  Intubation time was recorded for all patients. Pain scores were determined daily from postoperative day 1 until day 5 at 6 a.m. using a numeric rating scale. Narcotic pain medication requirements from day 1 to 5 were collected and converted into intravenous morphine equivalents. RESULTS: Of 80 patients, 39 patients were in Group R (average age 65 ± 8; 31 male and 8 female) and 41 patients were in Group C (average age 66 ± 9; 34 male and 7 female).  Group R patients had a higher body mass index than patients in Group C (Group R: 31 ± 5; Group C: 29 ± 5; P = .04). No significant differences in the end points of intubation time and postoperative pain were observed. Conclusion: This randomized study of cardiac surgery patients showed no significant benefits of rigid fixation over conventional sternal wire closure with regard to intubation time, postoperative pain, or length of hospital stay.

A continuous wave 10 W cryogenic fiber amplifier at 1015 nm and frequency quadrupling to 254 nm.

A stable, continuous wave, single frequency fiber amplifier system at 1015 nm with 10 W output power is presented. It is based on a large mode double clad fiber cooled to liquid nitrogen temperature. The amplified light is frequency quadrupled to 254 nm and used for spectroscopy of the 6¹S → 6³P transition in mercury.

Utilization of magnetic and electrostatic separation in the recycling of printed circuit boards scrap.

The progress of the technology is directly related to the growth of production and consumption of electrical/electronics equipment, especially of personal computers. This type of equipment has a relatively short average lifetime, 2-3 years. The amount of defective or obsolete equipment has been increasing substantially; consequently its disposition and/or recycling should be studied. In this work, printed circuit boards, which are used in personal computers, were studied in order to recover the metals in the circuit boards through mechanical processing, such as crushing, screening, as well as magnetic and electrostatic separation. The results obtained demonstrate the feasibility of using these processes to separate metal fractions from polymers and ceramics, and that it is possible to obtain a fraction concentrated in metals containing more than 50% on average of copper, 24% of tin and 8% of lead.

Difluoro ketone peptidomimetics suggest a large S1 pocket for Alzheimer's gamma-secretase: implications for inhibitor design.

The final step in the generation of the amyloid-beta protein (Abeta), implicated in the etiology of Alzheimer's disease, is proteolysis within the transmembrane region of the amyloid precursor protein (APP) by gamma-secretase. Although considered an important target for therapeutic design, gamma-secretase has been neither well-characterized nor definitively identified. Previous studies in our laboratory using substrate-based difluoro ketone and difluoro alcohol transition-state analogue inhibitors suggest that gamma-secretase is an aspartyl protease with loose sequence specificity. To further characterize the active site of gamma-secretase, we prepared a series of difluoro ketone peptide analogues with varying steric bulkiness in the P1 position and tested the ability of these compounds to inhibit Abeta production in APP-transfected cells. Incorporation of bulky, aliphatic P1 side chains, such as sec-butyl or cyclohexylmethyl, led to increased gamma-secretase inhibitory potency, suggesting a large S1 pocket to accommodate these substituents and providing further evidence for loose sequence specificity. The cyclohexylmethyl P1 substituent allowed N-terminal truncation to a low-molecular-weight compound (<600 Da) that effectively blocked Abeta production (IC(50) approximately 5 microM). This finding suggests that optimal S1 binding may allow the development of potent inhibitors with ideal pharmaceutical properties. Moreover, a difluoro alcohol analogue with a cyclohexylmethyl P1 substituent was equipotent with its difluoro ketone counterpart, providing strong evidence that gamma-secretase is an aspartyl protease. All new analogues inhibited total Abeta and Abeta(42) production with the same rank order of potency and increased Abeta(42) production at low concentrations, providing further evidence for distinct gamma-secretases that are nevertheless closely similar with respect to active site topology and mechanism.

Transition-state analogue inhibitors of gamma-secretase bind directly to presenilin-1.

The beta-amyloid precursor protein (beta-APP), which is involved in the pathogenesis of Alzheimer's disease, and the Notch receptor, which is responsible for critical signalling events during development, both undergo unusual proteolysis within their transmembrane domains by unknown gamma-secretases. Here we show that an affinity reagent designed to interact with the active site of gamma-secretase binds directly and specifically to heterodimeric forms of presenilins, polytopic proteins that are mutated in hereditary Alzheimer's and are known mediators of gamma-secretase cleavage of both beta-APP and Notch. These results provide evidence that heterodimeric presenilins contain the active site of gamma-secretase, and validate presenilins as principal targets for the design of drugs to treat and prevent Alzheimer's disease.

Toward the characterization and identification of gamma-secretases using transition-state analogue inhibitors.

The amyloid-beta protein (A beta), strongly implicated in the etiology of Alzheimer's disease (AD), is formed from the amyloid-beta precursor protein (APP) through sequential proteolysis by beta- and gamma-secretases. Cleavage by gamma-secretase takes place within the middle of the single transmembrane region of APP and results primarily in 40- and 42-amino acid A beta C-terminal variants, A beta 40 and A beta 42. The latter form of A beta is highly fibrillogenic, is invariably elevated in autosomal-dominant forms of AD, and is the major A beta component found presymptomatically in cerebral deposits. Thus, blocking production of A beta in general and A beta 42 in particular is considered an important therapeutic goal. We have developed transition-state analogue inhibitors of gamma-secretase as molecular probes for characterizing the active site of this enzyme, as pharmacological tools for understanding its role in biology, and as affinity labels toward its definitive identification. Specifically, we found that: (1) difluoro ketone and difluoro alcohol peptidomimetics are effective inhibitors of gamma-secretase activity in APP-transfected cells, strongly suggesting an aspartyl protease mechanism; (2) gamma-secretases that form A beta 40 and A beta 42 are pharmacologically distinct but are nevertheless closely similar; (3) large hydrophobic P1 substituents increase the inhibitory potency of these peptidomimetics, suggesting a large complementary S1 pocket for gamma-secretases; (4) A beta 42 production is increased several fold over control by these gamma-secretase inhibitors after replacement with inhibitor-free media; (5) a bromoacetamide derivative of one of these analogues continues to inhibit total A beta and A beta 42 production hours after replacement with compound-free media and should help identify the target(s) of these protease transition-state mimics.

Protofibrillar intermediates of amyloid beta-protein induce acute electrophysiological changes and progressive neurotoxicity in cortical neurons.

Alzheimer's disease (AD) is a progressive neurodegenerative disorder that is thought to be caused in part by the age-related accumulation of amyloid beta-protein (Abeta). The presence of neuritic plaques containing abundant Abeta-derived amyloid fibrils in AD brain tissue supports the concept that fibril accumulation per se underlies neuronal dysfunction in AD. Recent observations have begun to challenge this assumption by suggesting that earlier Abeta assemblies formed during the process of fibrillogenesis may also play a role in AD pathogenesis. Here, we present the novel finding that protofibrils (PF), metastable intermediates in amyloid fibril formation, can alter the electrical activity of neurons and cause neuronal loss. Both low molecular weight Abeta (LMW Abeta) and PF reproducibly induced toxicity in mixed brain cultures in a time- and concentration-dependent manner. No increase in fibril formation during the course of the experiments was observed by either Congo red binding or electron microscopy, suggesting that the neurotoxicity of LMW Abeta and PF cannot be explained by conversion to fibrils. Importantly, protofibrils, but not LMW Abeta, produced a rapid increase in EPSPs, action potentials, and membrane depolarizations. These data suggest that PF have inherent biological activity similar to that of mature fibrils. Our results raise the possibility that the preclinical and early clinical progression of AD is driven in part by the accumulation of specific Abeta assembly intermediates formed during the process of fibrillogenesis.

Prolactin is an autocrine or paracrine growth factor for human myometrial and leiomyoma cells.

OBJECTIVE: To test the hypothesis that prolactin (PRL) acts as a mitogenic growth factor for human leiomyoma and myometrial cells. METHODS: To test this hypothesis, we performed three different types of experiments. First, we assessed whether exogenous PRL acted as a mitogen for cultured uterine smooth muscle cells. Second, we examined the role of endogenous PRL by assessing the cell number after exposure of the cultures to a neutralizing antibody to PRL. Finally, we examined both fresh tissues and cultured cells for expression of the PRL receptor messenger ribonucleic acid using the techniques of reverse-transcriptase polymerase chain reaction and Southern blotting. RESULTS: A significant suppression in cell number was seen after 5 days of culture for leiomyoma cells but not for myometrial cells after treatment with exogenous PRL. Both cell types showed a significant decrease in cell number after treatment with anti-PRL antibody. A 893-bp segment consistent with the cytoplasmic domain of the long form of the PRL receptor was amplified from both fresh and cultured tissues and confirmed by Southern blotting and sequencing. CONCLUSIONS: PRL appears to be an autocrine or paracrine growth factor for both leiomyoma and myometrial cells. However, there are some differences between tissues in their sensitivity to this growth factor.

Two transmembrane aspartates in presenilin-1 required for presenilin endoproteolysis and gamma-secretase activity.

Accumulation of the amyloid-beta protein (Abeta) in the cerebral cortex is an early and invariant event in the pathogenesis of Alzheimer's disease. The final step in the generation of Abeta from the beta-amyloid precursor protein is an apparently intramembranous proteolysis by the elusive gamma-secretase(s). The most common cause of familial Alzheimer's disease is mutation of the genes encoding presenilins 1 and 2, which alters gamma-secretase activity to increase the production of the highly amyloidogenic Abeta42 isoform. Moreover, deletion of presenilin-1 in mice greatly reduces gamma-secretase activity, indicating that presenilin-1 mediates most of this proteolytic event. Here we report that mutation of either of two conserved transmembrane (TM) aspartate residues in presenilin-1, Asp 257 (in TM6) and Asp 385 (in TM7), substantially reduces Abeta production and increases the amounts of the carboxy-terminal fragments of beta-amyloid precursor protein that are the substrates of gamma-secretase. We observed these effects in three different cell lines as well as in cell-free microsomes. Either of the Asp --> Ala mutations also prevented the normal endoproteolysis of presenilin-1 in the TM6 --> TM7 cytoplasmic loop. In a functional presenilin-1 variant (carrying a deletion in exon 9) that is associated with familial Alzheimer's disease and which does not require this cleavage, the Asp 385 --> Ala mutation still inhibited gamma-secretase activity. Our results indicate that the two transmembrane aspartate residues are critical for both presenilin-1 endoproteolysis and gamma-secretase activity, and suggest that presenilin 1 is either a unique diaspartyl cofactor for gamma-secretase or is itself gamma-secretase, an autoactivated intramembranous aspartyl protease.

Additive effects of PS1 and APP mutations on secretion of the 42-residue amyloid beta-protein.

Humans harboring missense mutations in the presenilin 1 (PS1) gene undergo progressive cerebral deposition of the 42-residue amyloid beta-protein (A beta 42) at an early age and develop severe Alzheimer's disease. A beta 42 is selectively elevated in the conditioned media of cells expressing mutant but not wild-type PS1, indicating that presenilin mutations alter APP processing. Here we analyze the effects of various PS1 mutant constructs on the cellular production of A beta 42. A construct expressing only the PS1 N-terminal endoproteolytic fragment with the mutation Y115H causes no significant increase in A beta 42, whereas a full-length PS1 construct with the same mutation does. This result suggests that the pathogenic effect of mutant presenilins is produced by the full-length molecule even though only a minor proportion of total PS1 occurs as holoprotein in tissues and cell lines. We demonstrate that the effects of two different PS1 mutations are additive when engineered into the same PS1 molecule. Therefore, two mutations alter gamma-secretase processing of APP more than one and the PS1 mutations described to date do not cause the maximum possible PS1-mediated rise in A beta 42. When a PS1 mutation was expressed in cells carrying the APPV717I mutation, A beta 42 rose dramatically to become the predominant secreted A beta species, an observation of interest for transgenic modeling of AD. Our results are consistent with the hypothesis that presenilin is a major regulator of the proteolytic processing of APP by gamma-secretases.

Increased expression of stromelysin 3 mRNA in leiomyomas (uterine fibroids) compared with myometrium.

OBJECTIVE: To determine the level of expression of selected matrix metalloproteinases in uterine leiomyoma compared with unaffected myometrium in an effort to explain the abnormal accumulation of extracellular matrix in the leiomyoma. METHODS: The levels of matrix metalloproteinase (MMP) mRNA in leiomyoma and myometrium were measured in samples from 22 patients during either proliferative (n = 6) or secretory phases (n = 16) of the menstrual cycle. Relative amounts of collagenase (MMP-1) and stromelysin (MMP-3) mRNAs were measured by Northern blot analysis, and amounts of stromelysin 3 (MMP-11) and matrilysin (MMP-7) mRNA from each sample were determined by semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR) and normalized to beta-actin mRNA. RESULTS: The levels of MMP-1 and MMP-3 mRNAs were similar in both leiomyoma and unaffected myometrium. The levels of MMP-11 mRNA were twofold greater in leiomyoma compared with myometrium throughout the menstrual cycle, and the differences in the levels of MMP-11 were significantly different during the secretory phase. The level of MMP-7 mRNA expression was similar in leiomyoma, myometrium, and endometrium. CONCLUSIONS: Among the metalloproteinases examined in this study, only the levels of MMP-11 mRNA were elevated in leiomyoma compared with myometrium. The increased expression of MMP-11 in uterine leiomyoma compared with myometrium is analagous to previously reported elevations of MMP-11 mRNA in dermatofibromas compared with unaffected skin. The increased expression of MMP-11 mRNA in fibroid tumors suggests that this MMP may be involved in the formation of a more fibrous extracellular matrix in leiomyoma relative to unaffected myometrium.

Presenilin proteins undergo heterogeneous endoproteolysis between Thr291 and Ala299 and occur as stable N- and C-terminal fragments in normal and Alzheimer brain tissue.

Humans inheriting missense mutations in the presenilin (PS)1 and -2 genes undergo progressive cerebral deposition of the amyloid beta-protein at an early age and develop a clinically and pathologically severe form of familial Alzheimer's disease (FAD). Because PS1 mutations cause the most aggressive known form of AD, it is important to elucidate the structure and function of this multitransmembrane protein in the brain. Using a panel of region-specific PS antibodies, we characterized the presenilin polypeptides in mammalian tissues, including brains of normal, AD, and PS1-linked FAD subjects, and in transfected and nontransfected cell lines. Very little full-length PS1 or -2 was detected in brain and untransfected cells; instead the protein occurred as a heterogeneous array of stable N- and C-terminal proteolytic fragments that differed subtly among cell types and mammalian tissues. Sequencing of the major C-terminal fragment from PS1-transfected human 293 cells showed that the principal endoproteolytic cleavage occurs at and near Met298 in the proximal portion of the large hydrophilic loop. Full-length PS1 in these cells is quickly turned over (T1/2 approximately 60 min), in part to the two major fragments. The sizes and amounts of the PS fragments were not significantly altered in four FAD brains with the Cys410Tyr PS1 missense mutation. Our results indicate that presenilins are rapidly processed to N- and C-terminal fragments in both neural and nonneural cells and that interference with this processing is not an obligatory feature of FAD-causing mutations.

Mutant presenilins of Alzheimer's disease increase production of 42-residue amyloid beta-protein in both transfected cells and transgenic mice.

The mechanism by which mutations in the presenilin (PS) genes cause the most aggressive form of early-onset Alzheimer's disease (AD) is unknown, but fibroblasts from mutation carriers secrete increased levels of the amyloidogenic A beta 42 peptide, the main component of AD plaques. We established transfected cell and transgenic mouse models that coexpress human PS and amyloid beta-protein precursor (APP) genes and analyzed quantitatively the effects of PS expression on APP processing. In both models, expression of wild-type PS genes did not alter APP levels, alpha- and beta-secretase activity and A beta production. In the transfected cells, PS1 and PS2 mutations caused a highly significant increase in A beta 42 secretion in all mutant clones. Likewise, mutant but not wildtype PS1 transgenic mice showed significant overproduction of A beta 42 in the brain, and this effect was detectable as early as 2-4 months of age. Different PS mutations had differential effects on A beta generation. The extent of A beta 42 increase did not correlate with presenilin expression levels. Our data demonstrate that the presenilin mutations cause a dominant gain of function and may induce AD by enhancing A beta 42 production, thus promoting cerebral beta-amyloidosis.

Evidence that the 42- and 40-amino acid forms of amyloid beta protein are generated from the beta-amyloid precursor protein by different protease activities.

Cerebral deposition of the amyloid beta protein (A beta) is an early and invariant feature of Alzheimer disease (AD). Whereas the 40-amino acid form of A beta (A beta 40) accounts for approximately 90% of all A beta normally released from cells, it appears to contribute only to later phases of the pathology. In contrast, the longer more amyloidogenic 42-residue form (A beta 42), accounting for only approximately 10% of secreted A beta, is deposited in the earliest phase of AD and remains the major constituent of most amyloid plaques throughout the disease. Moreover, its levels have been shown to be increased in all known forms of early-onset familial AD. Thus, inhibition of A beta 42 production is a prime therapeutic goal. The same protease, gamma-secretase, is assumed to generate the C termini of both A beta 40 and A beta 42. Herein, we analyze the effect of the compound MDL 28170, previously suggested to inhibit gamma-secretase, on beta-amyloid precursor protein processing. By immunoprecipitating conditioned medium of different cell lines with various A beta 40- and A beta 42-specific antibodies, we demonstrate a much stronger inhibition of the gamma-secretase cleavage at residue 40 than of that at residue 42. These data suggest that different proteases generate the A beta 40 and A beta 42 C termini. Further, they raise the possibility of identifying compounds that do not interfere with general beta-amyloid precursor protein metabolism, including A beta 40 production, but specifically block the generation of the pathogenic A beta 42 peptide.

[Experience with the use of a blood culture system for demonstration of clinically relevant bacteria in veterinary medicine diagnosis]. / Erfahrungen mit dem Einsatz eines Blutkultur-systems zum Nachweis klinisch relevanter Bakterien in der veterinärmedizinischen Diagnostik.

268 diagnostic samples from dogs, cats, horses and cattle were examined in a commercially available blood culture system. Samples of blood, liquor, ascites, thorax punctate, synovia and urine were examined with a blood culture system (Oxoid) over a period of two years in cooperation with the veterinary clinical institutes of internal medicine and surgery, Ludwig-Maximilians-University, Munich and different veterinarians. It was shown that this blood culture system, which has been initially developed for the requirements of human bacteriology, can be used for isolation of clinical important microorganisms in veterinary medicine. In 29% of examined samples isolation of bacteria was possible. Even bacteria, which are not often cultivated and bacteria, which could not be identified biochemically, could be isolated. Because of experience in human bacteriology and in conclusion of our results, the use of blood culture systems can be recommended for veterinary diagnosis, in particular when sepsis is suspected.

Inhibition of amyloid beta-protein production in neural cells by the serine protease inhibitor AEBSF.

Cerebral deposition of amyloid beta protein (A beta) is an early and critical feature of Alzheimer's disease. A beta production requires the proteolytic release of A beta from the beta-amyloid precursor protein (beta APP). Thus, inhibition of A beta release is a prime therapeutic goal. Here, we show that the broad spectrum, irreversible serine protease inhibitor, AEBSF, inhibits the constitutive production of A beta in five different human cell lines, both neural and nonneural. AEBSF also stabilizes full-length beta APP and enhances alpha-secretion, as shown by an increase in the proteolytic derivative, alpha-APPS. Further, we demonstrate that the inhibitory effect of AEBSF is specific for A beta proteins starting at Aspartate 1, suggesting that AEBSF directly inhibits beta-secretase, the Methionine-Aspartate (Met-Asp)-cleaving enzyme. These results indicate that specific inhibition of this A beta-generating protease is possible in living human neural cells and provide information about the characteristics of this as yet unidentified enzyme.

GTP gamma S loading of endothelial cells stimulates phospholipase C and uncouples ATP receptors.

The effects of GTP gamma S, a stable GTP analogue that can activate guanine nucleotide-binding proteins, on phospholipase C activation/calcium mobilization were studied in intact cultured bovine aortic endothelial cells (BAEC). Phosphoinositide metabolism and cytosolic free Ca2+ concentration [( Ca2+]i; fura-2 fluorescence) were studied after the cells were transiently permeabilized, loaded with different guanine nucleotides, and then allowed to reseal and recover. Intracellular GTP gamma S stimulated a dose-dependent [median effective concentration (EC50) 2.5 microM] decrease in basal [3H]phosphoinositide content. Phosphatidylinositol 4,5-bisphosphate, phosphatidylinositol 4-bisphosphate, and phosphatidylinositol levels decreased to 57 +/- 9, 63 +/- 8, and 74 +/- 8% control levels, respectively, in BAEC loaded with approximately 85 microM GTP gamma S. Basal inositol trisphosphate (IP3) and [Ca2+]i were increased in GTP gamma S-loaded BAEC when compared with sham-loaded BAEC. In control BAEC, the purinergic receptor agonist ATP (100 microM) induced rapid increases in [Ca2+]i and IP3. However, BAEC that had been intracellularly loaded with GTP gamma S [median inhibitory constant (IC50) 1 microM] or 5'-guanylyl-imidodiphosphate exhibited decreased calcium mobilization in response to ATP. Ionomycin (calcium ionophore)-releasable pools of calcium were similar in sham- and GTP gamma S-loaded cells, suggesting that total intracellular calcium had not been depleted by the permeabilization protocol. The diminished calcium mobilization in response to ATP was associated with decreases in ATP-stimulated PIP2 hydrolysis and IP3 formation. In addition, GTP gamma S loading did not increase basal levels of cyclic AMP. Intracellular GDP beta S, GDP, or GTP did not inhibit ATP-stimulated increases in [Ca2+]i or IP3.(ABSTRACT TRUNCATED AT 250 WORDS)

Breast masses in adolescent females.

Conservative management of breast masses in adolescents is generally advocated in consideration of the low incidence of breast cancer. A retrospective chart review of 130 female patients seen in a general adolescent clinic over a two-year period was performed. The mean age was 17.5 years (range 12-21). One hundred and seven (88% of available data) had self-discovered lesions. Fibrocystic disease was clinically diagnosed in 66 (51%) patients, while 19 (15%) had fibroadenomas and 17 (13%) had a normal breast examination. Of the remaining patients, six (5%) had mastalgia, five (4%) had mastitis/abscess, three (2%) has asymmetry, three (2%) had hypertrophy, two (1%) had breast changes of early pregnancy, two (1%) had hematoma, one (1%) had axillary lymphadenopathy, and six (5%) had unknown. Excisional biopsy was performed on eleven patients; it revealed fibroadenoma in eight, and one each had a hematoma, granular cell myoblastoma, and breast abscess. Improvement or complete resolution of breast masses was documented in 31 (47%) of the patients with fibrocystic disease.