Effect of albumin on the estimation, in vitro, of phenytoin Vmax and Km values: implications for clinical correlation

The effect of bovine serum albumin (BSA) on human liver metabolism, in vitro, of 14C-phenytoin (PHT) was studied. Michaelis Menten parameters were determined for the conversion of PHT to p-hydroxy phenytoin in seven different microsomal preparations with the addition of 0, 2, and 4% BSA. The unbound Km (Kmu) values were 30.8 +/- 18.6, 1.57 +/- 0.21 and 1.50 +/- 0.17 microM (mean +/- S.D.), respectively; however, there was excellent agreement among the Vmax values (29.1, 31.8 and 31.5 pmol/min/mg). With intact tissue slices, BSA (4%) added to incubations of PHT had a minimal effect on the Vmax values in two of the four livers studied and resulted in a mean Kmu value of 2.20 +/- 0.59 microM, although the Kmu in the absence of BSA was 6.64 +/- 3.17. In scaling-up to the whole body, Vmax values were 3.9 and 1.0 mg/kg/day for microsomes and slices, respectively, compared to 5.9 mg/kg/day, in vivo. The Kmu values determined in the presence of albumin in both microsomes and slices were similar to those based on in vivo human steady state data (Kmu = 2-3 microM), and the intersubject variation, in vitro, was decreased in the presence of BSA. These findings for phenytoin metabolism suggest that the addition of albumin to incubation media for slices or microsome experiments may yield Km estimates that are more representative of in vivo values.     

Cell mediated immunity in rheumatoid arthritis: Imparied lymphocyte responsiveness, humoral immunosuppressants, and correlations with clinical status in patients off drug therapy

Cell mediated immune functions were studied in 17 patients with rheumatoid arthritis (RA) at times off all drug therapy, and were correlated with clinical findings. Cellular immunity was evaluated by (1) skin testing to antigens and (2) measuring peripheral blood lymphocyte (PBL) and effusion lymphocyte tritiated thymidine (3H-TdR) uptake in in vitro cultures containing 20 percent autologous or 20 percent AB plasma and various concentrations of mitogens or antigens. Cutaneous and in vitro reactivity were decreased in RA patients: (1) RA PBL spontaneously incorporated more 3H-TdR than normals; (2) 3H-TdR uptake by RA PBL, cultured in AB plasma, in response to mitogens and antigens was markedly reduced when compared with normals; (3) RA PBL responses were further diminished when cells were cultured in autologous plasmas; (4) effusion lymphocytes similarly often had high unstimulated 3H-TdR uptakes and poor responses to stimulation; and (5) decreased proliferative responses of RA PBL (in AB plasma) correlated with class and stage III-IV, increasing age, number of tender or swollen joints, rheumatoid factor titer, total protein, decreased grip strength, and poor skin test reactivity.     

A Hexagonal Grid-Sampling Scheme for Improving X-Ray Diffraction-Retained Austenite Measurements of Sheet Steels

Metallurgical and Materials Transactions A - Advanced High-Strength Steels make use of retained austenite to improve mechanical properties. The benefits of the austenitic phase are directly tied to...     

Microelectromechanical System Measurement of Platelet Contraction: Direct Interrogation of Myosin Light Chain Phosphorylation

Myosin Light Chain (MLC) regulates platelet contraction through its phosphorylation by Myosin Light Chain Kinase (MLCK) or dephosphorylation by Myosin Light Chain Phosphatase (MLCP). The correlation between platelet contraction force and levels of MLC phosphorylation is unknown. We investigate the relationship between platelet contraction force and MLC phosphorylation using a novel microelectromechanical (MEMS) based clot contraction sensor (CCS). The MLCK and MLCP pair were interrogated by inhibitors and activators of platelet function. The CCS was fabricated from silicon using photolithography techniques and force was validated over a range of deflection for different chip spring constants. The force of platelet contraction measured by the clot contraction sensor (CCS) was compared to the degree of MLC phosphorylation by Western Blotting (WB) and ELISA. Stimulators of MLC phosphorylation produced higher contraction force, higher phosphorylated MLC signal in ELISA and higher intensity bands in WB. Inhibitors of MLC phosphorylation produced the opposite. Contraction force is linearly related to levels of phosphorylated MLC. Direct measurements of clot contractile force are possible using a MEMS sensor platform and correlate linearly with the degree of MLC phosphorylation during coagulation. Measured force represents the mechanical output of the actin/myosin motor in platelets regulated by myosin light chain phosphorylation.     

Loss of Individual Mitochondrial Ribonuclease P Complex Proteins Differentially Affects Mitochondrial tRNA Processing In Vivo

Over a thousand nucleus-encoded mitochondrial proteins are imported from the cytoplasm; however, mitochondrial (mt) DNA encodes for a small number of critical proteins and the entire suite of mt:tRNAs responsible for translating these proteins. Mitochondrial RNase P (mtRNase P) is a three-protein complex responsible for cleaving and processing the 5′-end of mt:tRNAs. Mutations in any of the three proteins can cause mitochondrial disease, as well as mutations in mitochondrial DNA. Great strides have been made in understanding the enzymology of mtRNase P; however, how the loss of each protein causes mitochondrial dysfunction and abnormal mt:tRNA processing in vivo has not been examined in detail. Here, we used Drosophila genetics to selectively remove each member of the complex in order to assess their specific contributions to mt:tRNA cleavage. Using this powerful model, we find differential effects on cleavage depending on which complex member is lost and which mt:tRNA is being processed. These data revealed in vivo subtleties of mtRNase P function that could improve understanding of human diseases.     

Detection of multiple naturally occurring Salmonella serotypes from commercial broiler carcasses with conventional methods

Many laboratories sampling foods for Salmonella are interested only in presence or absence of Salmonella, so only one colony may be selected. The objectives of this study were to use two selective enrichment broths and two selective agar plating media for Salmonella recovery from naturally contaminated broiler carcass rinsates and evaluate these media combinations on Salmonella serotypes recovered from each carcass. Broiler carcasses (n = 52) from a commercial processing plant prior to chilling were rinsed with buffered peptone water and after incubation subcultured to gram-negative and tetrathionate, and after inoculation to Rappaport Vassiliadis broth which was incubated and then streaked onto plates of Brilliant Green Sulfa and Xylose-Lysine-Tergitol-4 agar. On 11/49 positive carcasses, both plating media yielded the same serotypes; for the other 38 positive samples different serotypes were found on the different plating media. Enrichment and plating media combinations influence the serotypes recovered and demonstrates bias even in a limited study such as this.     

The influence of temperature and humidity on the life-cycle of Ectomyelois ceratoniae (Zeller) (Lepidoptera: Phycitidae)

Adults of Ectomyelois ceratoniae would not mate in small containers kept in constant conditions. Mating occurred and fertile eggs were laid when the adults were placed in a large conical cage, kept in regularly fluctuating conditions of light, temperature and humidity resembling their native habitat. The dusk/dawn light intensity changes were particularly important. Most mating and oviposition occurred from dusk until the following dawn, with adults motionless during daylight.At 70% r.h. eggs hatched on average in 4·2 days at 25°C, 3·0 days at 30°C and 3·6 days at 35°C. At 30°C, this period varied from 3·4 days at 30% r.h. to 3·0 days at 90% r.h. On a diet of soybean meal, sucrose and water at 30°C, the larval period averaged 30 days at 30% r.h., 13 days longer than at 70% r.h., and the low relative humidity also increased larval mortality. At 90% r.h., no larvae emerged from food that became very mouldy. The total developmental period from egg hatch to adult emergence at 70% r.h. averaged 48 days at 20°C, 30 days at 25°C and 23 days at 30°C.     

Analysis and tests of boned insulted rail joints subjected to vertical wheel loads

In traditional railroad tracks, joint bars are being used to connect the ends of adjoining rails. Because the vertical bending stiffness of two bars is generally much lower than of the rails, the passing wheels generate larger deflections in the joint region. This in turn leads to larger wheel forces caused by the dynamics of the passing vehicles and to accelerated track deterioration. In recent decades these expansion joints are being eliminated by the introduction of the continuously welded rails (CWRs). However, the retention of the automatic block signaling system created the need for electrically insulated joints is that the bending stiffness of the insulated joints bars is even smaller than of the non-insulated bars that are being eliminated. There are very few published analyses of joints in track; especilly of insulated joints. The purpose of this paper is to contribute to a better understanding of the mechanics of rail joints. At first an analysis is presented for the joint tests to be conducted. Then a test program is described that utilizes actual bonded joints. The obtained test results are then compared with the corresponding analytical results. The agreement is good. This confirms the validity of the assumed joint model and of the presented analysis. The paper concludes by analyzing the effect of a rail joints in a CWR track caused by a vertical wheel load.