Expression of matrix metalloproteinases and their inhibitors in aneurysms and normal aorta

BACKGROUND: Abdominal aortic aneurysms (AAAs) are characterized by degradation of collagen and elastin resulting from increases in matrix metalloproteinase (MMP) activity. Previous authors have identified isolated increases in expression of specific MMPs in AAAs, but none have compared relative levels of expression of particular MMPs to one another or to those of their inhibitors, the tissue inhibitors of metalloproteinases (TIMPs). This study proposes to quantify relative mRNA levels for interstitial collagenase (MMP-1), 72 kd type IV collagenase (MMP-2), 92 kd type IV collagenase (MMP-9), TIMP-1, and TIMP-2 in normal aorta (NA) and AAA to provide insight as to the relative importance of each in aneurysm formation. METHODS: Competitive polymerase chain reactions (PCRs) with gene-specific external standards and cDNA derived from AAAs (n = 8; mean age, 67.4 years) and NA (n = 5; mean age, 40.6 years) were used to quantify mRNA levels. Results were normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA levels, determined by means of competitive PCR, and compared by means of Mann-Whitney statistics. RESULTS: Significant increases in MMP mRNA expression in AAA over NA were observed for MMP-1 (3.64 versus 0.3, p = 0.007), MMP-9 (78.03 versus 3.35, p = 0.003), TIMP-1 (835.32 versus 477.2, p = 0.027), and TIMP-2 (18.09 versus 4.14, p = 0.003). The ratio of MMP to TIMP mRNA levels was higher in AAA than NA (0.135 versus 0.045, p = 0.018). CONCLUSIONS: Increases in expression of MMP-1, MMP-9, and MMP/TIMP ratios may result in increased proteolysis and matrix degradation, which characterize AAAs. MMP-9 appears to be the predominant metalloproteinase expressed in AAA, because its mRNA levels were more than 20 times and 2 times higher than those of MMP-1 and MMP-2, respectively. TIMP-1 mRNA levels were in molar excess to those of any of the metalloproteinases studied.     

Laboratory simulation of hypervelocity debris

A series of hypervelocity damage experiments were performed on spacecraft materials in order to simulate micro-size space debris traveling at 3 to 8 km/s. Two types of impact simulations were investigated: high-power pulsed laser and laser-launched micro-flyer plate. In the first case a laser was used to generate a high-pressure shock wave which propagated into the target by means of rapid ablation of the target surface. The second case used the same laser to accelerate micro-flyer plates at a target. The laser-ablation technique and the apparatus used to propel the micro-flyer plates were compatible with a space environmental chamber equipped with instrumentation capable of analyzing the vapor ejected from the sample. Data obtained from two separate damage effects were of interest in this study: the vapor blow-off produced by the impact and the mechanical damage to the target. The value of the data obtained from both simulation methods was evaluated in terms of likeness to actual space debris damage.

Data for this work were obtained from polysulfone resin and a graphite polysulfone composite. Polysulfone was selected because it was flown on the Long Duration Exposure Facility (LDEF) satellite which spent several years in low earth orbit and experienced many space debris impacts.

The chemistry of the vapor produced by the two simulation techniques was analyzed with a time of flight mass spectrometer (TOFMS) which measured changes in the vapor chemistry as a function of time after impact, obtained a velocity measurement of the vapor, and estimated surface temperature immediately after impact using dynamic gas equations. Samples of the vapor plume were also captured and examined by transmission electron microscopy (TEM).

The mechanical damage effects caused by the simulation methods on a graphit polysulfone composite and a polysulfone resin were studied. Impact craters were examined under optical and scanning electron microscopes (SEM). Based on the two damage effect criteria the micro-flyer method proved to be a useful way to simulate hypervelocity impact of space debris. The laser-ablation method however, had shortcomings and required drastic compromises in the set criteria.     

Preparation and in vitro characterization of gentamycin-impregnated biodegradable beads suitable for treatment of osteomyelitis

A new method for preparing poly(L-lactide) (PLA) biodegradable beads impregnated with an ionic aminoglycoside, gentamycin, is described. The process employs hydrophobic ion pairing to solubilize gentamycin in a solvent compatible with PLA, followed by precipitation with a compressed antisolvent (supercritical carbon dioxide). The resulting precipitate is a homogeneous dispersion of the ion-paired drug in PLA microspheres. The microspheres are approximately 1 microm in diameter and can be compressed into beads (3-6 mm in diameter) strung on surgical sutures for implantation. The bead strings exhibit no significant change in release kinetics upon sterilization with a hydrogen peroxide plasma (Ster-Rad). The kinetics of gentamycin release from the PLA beads are consistent with a matrix-controlled diffusion mechanism. While nonbiodegradable poly(methyl methacrylate) (PMMA) beads initially release gentamycin in a similar manner, the drug release from PMMA ceases after 8 or 9 weeks, while the PLA beads continue to release drug for over 4 months. Moreover, only 10% of the gentamycin is released from the PMMA beads, while PLA beads release more than 60% of their load, if serum is present in the release medium. The PLA system displays improved release kinetics relative to PMMA, is biodegradable, is unaltered by gas sterilization, can be used for a range of antibiotics, and can be manipulated without disintegration. These are all desirable properties for an implantable drug delivery system for the prevention or treatment of osteomyelitis.     

Effect of Human Serum on the pH-Induced Rheological Properties of Polymethacrylic Acid

The use of polyelectrolytes have been proposed for the production of pH sensitive microcapsules and liposomes. Polymethacrylic acid (PMA) has been synthesized for this purpose and has been shown to be extremely effective in increasing the release from these delivery systems a pH's below 6.0 in distilled water. The current studies show that serum and ionic strength conditions dramatically effect the rate and extent of any PMA conformational changes. The rate of conformational change was found to be proportional to time in contrast to the cube root of time reported within molecular probe investigations. Conformational changes in this study have been monitored using a cone/plate rotational viscometer. The results of this study suggest that under biologically relevant conditions, PMA (Mv 400,000) may not be suitable for use in pH sensitive delivery systems but may have other important uses.     

Linking non-culturable (qPCR) and culturable enterococci densities with hydrometeorological conditions

Quantitative polymerase chain reaction (qPCR) measurement of enterococci has been proposed as a rapid technique for assessment of beach water quality, but the response of qPCR results to environmental conditions has not been fully explored. Culture-based E. coli and enterococci have been used in empirical predictive models to characterize their responses to environmental conditions and to increase monitoring frequency and efficiency. This approach has been attempted with qPCR results only in few studies. During the summer of 2006, water samples were collected from two southern Lake Michigan beaches and the nearby river outfall (Burns Ditch) and were analyzed for enterococci by culture-based and non-culture-based (i.e., qPCR) methods, as well as culture-based E. coli. Culturable enterococci densities (log CFU/100 ml) for the beaches were significantly correlated with enterococci qPCR cell equivalents (CE) (R = 0.650, P < 0.0001, N = 32). Enterococci CE and CFU densities were highest in Burns Ditch relative to the beach sites; however, only CFUs were significantly higher (P < 0.0001). Culturable enterococci densities at Burns Ditch and the beaches were significantly correlated (R = 0.565, P < 0.0001, N = 32). Culturable E. coli and enterococci densities were significantly correlated (R = 0.682, P < 0.0001, N = 32). Regression analyses suggested that enterococci CFU could be predicted by lake turbidity, Burns Ditch discharge, and wind direction (adjusted R² = 0.608); enterococci CE was best predicted by Burns Ditch discharge and log-transformed lake turbidity × wave height (adjusted R² = 0.40). In summary, our results show that analytically, the qPCR method compares well to the non-culture-based method for measuring enterococci densities in beach water and that both these approaches can be predicted by hydrometeorological conditions. Selected predictors and model results highlight the differences between the environmental responses of the two method endpoints and the potentially high variance in qPCR results.     

Population structure of Cladophora-borne Escherichia coli in nearshore water of Lake Michigan

We previously reported that the macrophytic green alga Cladophora harbors high densities (up to 10(6) colony-forming units/g dry weight) of the fecal indicator bacteria, Escherichia coli and enterococci, in shoreline waters of Lake Michigan. However, the population structure and genetic relatedness of Cladophora-borne indicator bacteria remain poorly understood. In this study, 835 E. coli isolates were collected from Cladophora tufts (mats) growing on rocks from a breakwater located within the Indiana Dunes National Lakeshore in northwest Indiana. The horizontal fluorophore enhanced rep-PCR (HFERP) DNA fingerprinting technique was used to determine the genetic relatedness of the isolates to each other and to those in a library of E. coli DNA fingerprints. While the E. coli isolates from Cladophora showed a high degree of genetic relatedness (92% similarity), in most cases, however, the isolates were genetically distinct. The Shannon diversity index for the population was very high (5.39). Both spatial and temporal influences contributed to the genetic diversity. There was a strong association of isolate genotypes by location (79% and 80% for lake- and ditch-side samplings, respectively), and isolates collected from 2002 were distinctly different from those obtained in 2003. Cladophora-borne E. coli isolates represented a unique group, which was distinct from other E. coli isolates in the DNA fingerprint library tested. Taken together, these results indicate that E. coli strains associated with Cladophora may be a recurring source of indicator bacteria to the nearshore beach.     

In situ localization and quantification of mRNA for 92-kD type IV collagenase and its inhibitor in aneurysmal, occlusive, and normal aorta

Ninety-two-kilodalton type IV collagenase (MMP-9) is present in aortic aneurysms and may be important to the pathogenesis of this disease. Alteration in expression of MMP-9 or its inhibitor, the tissue inhibitor of metalloproteinase type 1 (TIMP-1), could increase degradation of extracellular matrix and lead to aneurysm formation. The purpose of this study was (1) to measure tissue levels of MMP-9 and TIMP-1 mRNA in aneurysmal (AAA), atherosclerotic occlusive (AOD), and normal (NL) human infrarenal aorta; (2) to test for their expression by cultured AAA and NL vascular smooth muscle cells (VSMCs); and (3) to locate in situ the cells responsible for mRNA production within AAA, AOD, and NL aortic wall. Total RNA extracted from AAA (n = 8), AOD (n = 8), and NL (n = 7) tissue was subjected to Northern analysis. Signals for MMP-9 and TIMP-1 were normalized to alpha-tubulin. Mean values +/- SEM were compared by ANOVA. NL and AAA VSMCs were cultured, passaged, and grown to confluence before RNA extraction and Northern analysis. In situ hybridization with digoxigenin-labeled RNA probes localized cells responsible for MMP-9 and TIMP-1 mRNA expression within sections of AAA (n = 5), AOD (n = 2), and NL (n = 2) aorta. MMP-9 mRNA levels were significantly greater in AAA (0.855 +/- 0.180) than NL (0.046 +/- 0.23) (P < .02), but differences between AOD (0.406 +/- 0.196) and AAA or AOD and NL were not significant.(ABSTRACT TRUNCATED AT 250 WORDS)     

Human carcinoembryonic antigen and biliary glycoprotein can serve as mouse hepatitis virus receptors

Receptors for murine coronavirus mouse hepatitis virus (MHV) are members of the murine carcinoembryonic antigen (CEA) gene family. Since MHV can also infect primates and cause central nervous system lesions (G. F. Cabirac et al., Microb. Pathog. 16:349-357, 1994; R. S. Murray et al., Virology 188:274-284, 1992), we examined whether human CEA-related molecules can be used by MHV as potential receptors. Transfection of plasmids expressing human carcinoembryonic antigen (hCEA) and human biliary glycoprotein into COS-7 cells, which lack a functional MHV receptor, conferred susceptibility to two MHV strains, A59 and MHV-2. Domain exchange experiments between human and murine CEA-related molecules identified the immunoglobulin-like loop I of hCEA as the region conferring the virus-binding specificity. This finding expands the potential MHV receptors to primate species.     

Color discrimination in the cat.

Conducted an experiment with 4 experienced and 2 naive cats in which Ss' ability to discriminate relatively long wavelength distributions of light from relatively short ones was demonstrated. Results suggest that the spectrum for the cat may be divided into 3 discriminable regions: long, middle, and short wavelengths. Ss appeared to learn color discriminations between 2 illuminated panels on the basis of the relative spectral distributions of the illumination and not on the basis of any absolute discrimination of unique colors. Ss which had previously learned a color discrimination showed a persistent influence of this learning in subsequent attempts to train them in a heterochromatic brightness discrimination. (22 ref.) (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

The systemic arterial pattern of the guinea pig: the pelvis and pelvic limb

The changes of heart rate in response to i.v. administration of methylatropine (0.5 mg/kg) and/or propranolol (2 mg/Kg) or practolol (2.5 mg/Kg) were studied in conscious trained dogs. Cholinergic blockade alone or combined blockade of sympathetic and parasympathetic effector systems resulted in cardiac acceleration. Conversely, beta-adrenoceptor antagonism with either propranolol or practolol reduced heart rate. The data were analysed by means of a new method, whereby the heart (HRN) of the dog is considered to be the product of the intrinsic heart rate (HR0) and 3 further factors: HRN-HR0 times S times V times W (Multiplicative model). 2 of these factors represent the tonic sympathetic (S) and parasympathetic (V) influences, whereas the third (W) represents the sympathetic-parasympathetic interaction. This type of analysis reveals that W was approximately 1, i.e., the sympathetic-parasympathetic interaction did not play any significant role in determining the heart rate of conscious resting dogs (HRN = HRO-S-V-W = HRO-S-V). The change of heart rate due to the action of parasympathetic system (-53% of the intrinsic heart rate) was more important than the change caused by the action of the sympathetic system (26% of the intrinsic heart rate).