The alpha and beta subunits of the metalloprotease meprin are expressed in separate layers of human epidermis, revealing different functions in keratinocyte proliferation and differentiation

The zinc endopeptidase meprin (EC 3.4.24.18) is expressed in brush border membranes of intestine and kidney tubules, intestinal leukocytes, and certain cancer cells, suggesting a role in epithelial differentiation and cell migration. Here we show by RT-PCR and immunoblotting that meprin is also expressed in human skin. As visualized by immunohistochemistry, the two meprin subunits are localized in separate cell layers of the human epidermis. Meprin alpha is expressed in the stratum basale, whereas meprin beta is found in cells of the stratum granulosum just beneath the stratum corneum. In hyperproliferative epidermis such as in psoriasis vulgaris, meprin alpha showed a marked shift of expression from the basal to the uppermost layers of the epidermis. The expression patterns suggest distinct functions for the two subunits in skin. This assumption is supported by diverse effects of recombinant meprin alpha and beta on human adult low-calcium high-temperature keratinocytes. Here, beta induced a dramatic change in cell morphology and reduced the cell number, indicating a function in terminal differentiation, whereas meprin alpha did not affect cell viability, and may play a role in basal keratinocyte proliferation.

Messenger RNA levels and binding sites of muscarinic acetylcholine receptors in gastrointestinal muscle layers from healthy dairy cows

Acetylcholine interacts with muscarinic receptors (M) to mediate gastrointestinal (GI) smooth muscle contractions. We have compared mRNA levels and binding sites of M(1)to M(5) in muscle tissues from fundus abomasi, pylorus, ileum, cecum, proximal loop of the ascending colon (PLAC), and external loop of the spiral colon (ELSC) of healthy dairy cows. The mRNA levels were measured by quantitative RT-PCR. The inhibition of [(3)H]-QNB (1-quinuclidinyl-[phenyl-4-(3)H]-benzilate) binding by M antagonists [atropine (M(1 - 5)), pirenzepine (M(1)), methoctramine (M(2)), 4-DAMP (M(3)), and tropicamide (M(4))] was used to identify receptors at the functional level. Maximal binding (B(max)) was determined through saturation binding with atropine as a competitor. The mRNA levels of M(1), M(2), M(3), and M(5) represented 0.2, 48, 50, and 1.8%, respectively, of the total M population, whereas mRNA of M(4) was undetectable. The mRNA levels of M(2) and of M(3) in the ileum were lower (P < 0.05) than in other GI locations, which were similar among each other. Atropine, pirenzepine, methoctramine, and 4-DAMP inhibited [(3)H]-QNB binding according to an either low- or high-affinity receptor pattern, whereas tropicamide had no effect on [(3)H]-QNB binding. The [(3)H]-QNB binding was dose-dependent and saturable. B(max) in fundus, pylorus, and PLAC was lower (P < 0.05) than in the ELSC, and in the pylorus lower (P < 0.05) than in the ileum. B(max) and mRNA levels were negatively correlated (r = -0.3; P < 0.05). In conclusion, densities of M are different among GI locations, suggesting variable importance of M for digestive functions along the GI tract.

Implementation of a phenomenological evaporation model into a porous network simulation for water management in low temperature fuel cells

A phenomenological transition film evaporation model was introduced to a pore network model with the consideration of pore radius, contact angle, non-isothermal interface temperature, microscale fluid flows and heat and mass transfers. This was achieved by modeling the transition film region of the menisci in each pore throughout the porous transport layer of a half-cell polymer electrolyte membrane (PEM) fuel cell. The model presented in this research is compared with the standard diffusive fuel cell modeling approach to evaporation and shown to surpass the conventional modeling approach in terms of predicting the evaporation rates in porous media. The current diffusive evaporation models used in many fuel cell transport models assumes a constant evaporation rate across the entire liquid-air interface. The transition film model was implemented into the pore network model to address this issue and create a pore size dependency on the evaporation rates. This is accomplished by evaluating the transition film evaporation rates determined by the kinetic model for every pore containing liquid water in the porous transport layer (PTL). The comparison of a transition film and diffusive evaporation model shows an increase in predicted evaporation rates for smaller pore sizes with the transition film model. This is an important parameter when considering the micro-scaled pore sizes seen in the PTL and becomes even more substantial when considering transport in fuel cells containing an MPL, or a large variance in pore size. Experimentation was performed to validate the transition film model by monitoring evaporation rates from a non-zero contact angle water droplet on a heated substrate. The substrate was a glass plate with a hydrophobic coating to reduce wettability. The tests were performed at a constant substrate temperature and relative humidity. The transition film model was able to accurately predict the drop volume as time elapsed. By implementing the transition film model to a pore network model the evaporation rates present in the PTL can be more accurately modeled. This improves the ability of a pore network model to predict the distribution of liquid water and ultimately the level of flooding exhibited in a PTL for various operating conditions.

Implementation of porous silicon technology for a fluidic flow-through optical sensor for pH measurements

This work presents an innovative integration of sensing and nano-scaled fluidic actuation in the combination of pH sensitive optical dye immobilization with the electro-osmotic phenomena in polar solvents like water for flow-through pH measurements. These flow-through measurements are performed in a flow-through sensing device (FTSD) configuration that is designed and fabricated at MTU. A relatively novel and interesting material, through-wafer mesoporous silica substrates with pore diameters of 20 -200 nm and pore depths of 500 µm are fabricated and implemented for electro-osmotic pumping and flow-through fluorescence sensing for the first time. Performance characteristics of macroporous silicon (> 500 µm) implemented for electro-osmotic pumping include, a very large flow effciency of 19.8 µLmin-1V-1 cm-2 and maximum pressure effciency of 86.6 Pa/V in comparison to mesoporous silica membranes with 2.8 µLmin-1V-1cm-2 flow effciency and a 92 Pa/V pressure effciency. The electrical current (I) of the EOP system for 60 V applied voltage utilizing macroporous silicon membranes is 1.02 x 10-6A with a power consumption of 61.74 x 10-6 watts. Optical measurements on mesoporous silica are performed spectroscopically from 300 nm to 1000 nm using ellipsometry, which includes, angularly resolved transmission and angularly resolved reflection measurements that extend into the infrared regime. Refractive index (n) values for oxidized and un-oxidized mesoporous silicon sample at 1000 nm are found to be 1.36 and 1.66. Fluorescence results and characterization confirm the successful pH measurement from ratiometric techniques. The sensitivity measured for fluorescein in buffer solution is 0.51 a.u./pH compared to sensitivity of ~ 0.2 a.u./pH in the case of fluorescein in porous silica template. Porous silica membranes are efficient templates for immobilization of optical dyes and represent a promising method to increase sensitivity for small variations in chemical properties. The FTSD represents a device topology suitable for application to long term monitoring of lakes and reservoirs. Unique and important contributions from this work include fabrication of a through-wafer mesoporous silica membrane that has been thoroughly characterized optically using ellipsometry. Mesoporous silica membranes are tested as a porous media in an electro-osmotic pump for generating high pressure capacities due to the nanometer pore sizes of the porous media. Further, dye immobilized mesoporous silica membranes along with macroporous silicon substrates are implemented for continuous pH measurements using fluorescence changes in a flow-through sensing device configuration. This novel integration and demonstration is completely based on silicon and implemented for the first time and can lead to miniaturized flow-through sensing systems based on MEMS technologies.

Development and characterization of fluorescent pH sensors based on porous silica and hydrogel support matrices

The hydrogen ion activity (pH) is a very important parameter in environment monitoring, biomedical research and other applications. Optical pH sensors have several advantages over traditional potentiometric pH measurement, such as high sensitivity, no need of constant calibration, easy for miniaturization and possibility for remote sensing. Several pH indicators has been successfully immobilized in three different solid porous materials to use as pH sensing probes. The fluorescent pH indicator fluorescein-5-isothiocyanate (FITC) was covalently bound onto the internal surface of porous silica (pore size ~10 nm) and retained its pH sensitivity. The excited state pK* a of FITC in porous silica (5.58) was slightly smaller than in solution (5.68) due to the free silanol groups (Si-OH) on the silica surface. The pH sensitive range for this probe is pH 4.5 - 7.0 with an error less than 0.1 pH units. The probe response was reproducible and stable for at least four month, stored in DI water, but exhibit a long equilibrium of up to 100 minutes. Sol-gel based pH sensors were developed with immobilization of two fluorescent pH indicators fluorescein-5-(and-6)-sulfonic acid, trisodium salt (FS) and 8-hydroxypyrene- 1,3,6-trisulfonic acid (HPTS) through physical entrapment. Prior to immobilization, the indicators were ion-paired with a common surfactant hexadecyltrimethylammonium bromide (CTAB) in order to prevent leaching. The sol-gel films were synthesized through the hydrolysis of two different precursors, ethyltriethoxysilane (ETEOS) and 3- glycidoxypropyltrimethoxysilane (GPTMS) and deposited on a quartz slide through spin coating. The pK a of the indicators immobilized in sol-gel films was much smaller than in solutions due to silanol groups on the inner surface of the sol-gel films and ammonium groups from the surrounding surfactants. Unlike in solution, the apparent pK a of the indicators in sol-gel films increased with increasing ionic strength. The equilibrium time for these sensors was within 5 minutes (with film thickness of ~470 nm). Polyethylene glycol (PEG) hydrogel was of interest for optical pH sensor development because it is highly proton permeable, transparent and easy to synthesize. pH indicators can be immobilized in hydrogel through physical entrapment and copolymerization. FS and HPTS ion-pairs were physically entrapped in hydrogel matrix synthesized via free radical initiation. For covalent immobilization, three indicators, 6,8-dihydroxypyrene-1,3- disulfonic acid (DHPDS), 2,7-dihydroxynaphthalene-3,6-disulfonic acid (DHNDS) and cresol red were first reacted with methacrylic anhydride (MA) to form methacryloylanalogs for copolymerization. These hydrogels were synthesized in aqueous solution with a redox initiation system. The thickness of the hydrogel film is controlled as ~ 0.5 cm and the porosity can be adjusted with the percentage of polyethylene glycol in the precursor solutions. The pK a of the indicators immobilized in the hydrogel both physically and covalently were higher than in solution due to the medium effect. The sensors are stable and reproducible with a short equilibrium time (less than 4 minutes). In addition, the color change of cresol red immobilized hydrogel is vivid from yellow (acidic condition) to purple (basic condition). Due to covalently binding, cresol red was not leaching out from the hydrogel, making it a good candidate of reusable "pH paper".