Radial dispersion of red blood cells in blood flowing through glass capillaries: the role of hematocrit and geometry

The flow properties of blood in the microcirculation depend strongly on the hematocrit (Hct), microvessel geometry, and cell properties. Previous in vitro studies have measured the radial displacement of red blood cells (RBCs) at concentrated suspensions using conventional microscopes. However, to measure the RBCs motion they used transparent suspensions of ghost red cells, which may have different physical properties than normal RBCs. The present study introduces a new approach (confocal micro-PTV) to measure the motion of labeled RBCs flowing in concentrated suspensions of normal RBCs. The ability of confocal systems to obtain thin in-focus planes allowed us to measure the radial position of individual RBCs accurately and to consequently measure the interaction between multiple labeled RBCs. All the measurements were performed in the center plane of both 50 and 100 microm glass capillaries at Reynolds numbers (Re) from 0.003 to 0.005 using Hcts from 2% to 35%. To quantify the motion and interaction of multiple RBCs, we used the RBC radial dispersion (D(yy)). Our results clearly demonstrate that D(yy) strongly depends on the Hct. The RBCs exhibited higher D(yy) at radial positions between 0.4 and 0.8R and lower D(yy) at locations adjacent to the wall (0.8-1R) and around the middle of the capillary (0-0.2R). The present work also demonstrates that D(yy) tends to decrease with a decrease in the diameter. The information provided by this study not only complements previous investigations on microhemorheology of both dilute and concentrated suspensions of RBCs, but also shows the influence of both Hct and geometry on the radial dispersion of RBCs. This information is important for a better understanding of blood mass transport mechanisms under both physiological and pathological conditions.     

Listerial invasion protein internalin B promotes entry into ileal Peyer's patches in vivo

Listeria monocytogenes (Lm) invades the host intestine using listerial invasion proteins, internalins. The in vivo role of internalin A (InlA) and internalin B (InlB) is reported here. Intragastric (i.g.) administration and ligated loop assays with ΔinlB-Lm demonstrated that a lack of InlB significantly attenuates the invasive ability of Lm into various organs. On the other hand, InlA(m)-Lm expressing a mutant InlA with two substitutions, S192N and Y369S, which has been reported to increase the affinity of InlA to mouse E-cadherin, resulted in little increase in intestinal infection according to both ligated loop and i.g. infection assays. Lm preferentially enters ileal Peyer's patch (PP) via M cells and ΔinlB-Lm showed severely reduced ability to invade though these cells. The present results reveal the importance of InlB, which accelerates listerial invasion into M cells on ileal PPs in vivo.     

Different Rifampicin Inactivation Mechanisms in Nocardia and Related Taxa

Mycolic acid-containing bacteria inactivate rifampicin in a variety of ways such as glucosylation, ribosylation, phosphorylation and decolorization. These inactivations were found to be a species-specific phenomena in Nocardia and related taxa. Gordona, Tsukamurella and fast-growing Mycobacterium modified rifampicin by ribosylation of the 23-OH group of the antibiotic. Such ribosylation was not observed in Rhodococcus and Corynebacterium, but phosphorylation of the 21-OH group of rifampicin was observed in one strain of Rhodococcus. Nocardia modified the antibiotic by glucosylation (23-OH group) and phosphorylation, but ribosylation was not observed.     

Advances on genetic rat models of epilepsy

Considering the suitability of laboratory rats in epilepsy research, we and other groups have been developing genetic models of epilepsy in this species. After epileptic rats or seizure-susceptible rats were sporadically found in outbred stocks, the epileptic traits were usually genetically-fixed by selective breeding. So far, the absence seizure models GAERS and WAG/Rij, audiogenic seizure models GEPR-3 and GEPR-9, generalized tonic-clonic seizure models IER, NER and WER, and Canavan-disease related epileptic models TRM and SER have been established. Dissection of the genetic bases including causative genes in these epileptic rat models would be a significant step toward understanding epileptogenesis. N-ethyl-N-nitrosourea (ENU) mutagenesis provides a systematic approach which allowed us to develop two novel epileptic rat models: heat-induced seizure susceptible (Hiss) rats with an Scn1a missense mutation and autosomal dominant lateral temporal epilepsy (ADLTE) model rats with an Lgi1 missense mutation. In addition, we have established episodic ataxia type 1 (EA1) model rats with a Kcna1 missense mutation derived from the ENU-induced rat mutant stock, and identified a Cacna1a missense mutation in a N-Methyl-N-nitrosourea (MNU)-induced mutant rat strain GRY, resulting in the discovery of episodic ataxia type 2 (EA2) model rats. Thus, epileptic rat models have been established on the two paths: ‘phenotype to gene’ and ‘gene to phenotype’. In the near future, development of novel epileptic rat models will be extensively promoted by the use of sophisticated genome editing technologies.     

Laminin-like glycoproteins in extracellular matrix of endodermal cells

Extracellular matrix from a mouse endodermal cell line consisted mainly of two polypeptides with molecular weights of about 200,000 (200K) and 400,000 (400K). Both poly-peptides incorporated radioactivity from [³H]proline and [³H]glucosamine and were solubilized from the matrix by treatment with bacterial collagenase or 0.5 m sodium chloride. These polypeptides appeared similar to those of laminin (R. Timpl, H. Rohde, P. G. Robey, S. I. Rennard, J.-M. Foidart, and G. R. Martin, 1979, J. Biol. Chem., 254, 9933–9937) in polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate but the laminin polypeptides seemed slightly larger than the 200K and 400K polypeptides, respectively. The amino acid compositions of the isolated 200K and 400K polypeptides resembled one another and the previously published amino acid composition of laminin. Antibodies prepared against the solubilized extracellular matrix protein (mixture of 200K and 400K components) as well as those against the isolated 400K component precipitated both the 400K component and the 200K component from culture media. These antisera and antisera to laminin showed identical reactivities in immunodiffusion and in immunofluorescence of tissue sections where they stained basement membranes. The immunofluorescent staining pattern was similar to that obtained with antifibronectin except in the liver where antifibronectin stained the biliary ducts and the liver sinusoids, while laminin-like immunoreactivity was not present in the sinusoidal areas. Such differences in distribution of matrix components could be involved in generation of signals for differentiation and growth of the adjacent cells.     

Assay method for antitumor L-methionine gamma-lyase: comprehensive kinetic analysis of the complex reaction with L-methionine

L-Methionine gamma-lyase (EC 4.4.1.11) is a pyridoxal 5'-phosphate-dependent multifunctional enzyme. Measuring the initial velocity of alpha-ketobutyrate production by alpha,gamma-elimination of L-methionine catalyzed by L-methionine gamma-lyase is not very feasible, because the enzyme simultaneously catalyzes both gamma-replacement and alpha,gamma-elimination. To develop an accurate enzyme assay, the comprehensive enzyme kinetics needed to be elucidated by progress curve analysis on the basis of a reaction model for conversion of L-methionine to alpha-ketobutyrate, methanethiol, and ammonia with pyridoxal 5'-phosphate as a cofactor. Kinetic parameters were determined by linear transformation using an approximation of a Maclaurin series from the whole velocity of alpha-ketobutyrate production including alpha,gamma-elimination and gamma-replacement. The significance of gamma-replacement was revealed both theoretically and practically by the kinetic analysis. The enzyme activity was standardized and represented as the Vmax value taking into consideration gamma-replacement in the presence of L-methionine at 37 degrees C and pH 8.0. The novel method that we proposed is accurate, sensitive, reproducible, and linear over a wide range for the determination of L-methionine gamma-lyase activity.     

Poduction of Biotin by Microbial Transformation of dl-cis-Tetrahydro-2-oxo-4-n-pentyl-thieno-(3,4-d)-imidazoline (dl-TOPTI)

The conditions for biotin production were investigated. Urea was a more effective nitrogen source than ammonium chloride and ammonium sulfate. About 60% conversion from dl-cis-tetrahydro-2-oxo-4-n-pentyl-thieno-(3,4-d)-imidazoline (dl-TOPTI) to biotinol and biotin occurred using Corynebacterium sp. B–321. Strain M–6318 which derived from B–321 as a mutant incapable of assimilating n-alkane produced large amounts of dl-biotin from dl-TOPTI. The inability of the microbe to assimilate n-alkane resulted in repression of biotin degradation. Maximum conversion (80%) was obtained by growing cultures of strain M–6318 in the constant presence of n-paraffin.     

Synthesis and spectroscopic properties of Co(salen) derivatives containing pendant thioether groups and their dioxygen adducts

Two Co(salen) derivatives, Co(sal-ipsen) and Co(sal-bsen), containing pendant (CH₂)₂S(i-C₃H₇) and (CH₂)₂SC₆H₅ groups were synthesized. Electronic and ESR spectra in methylene chloride show that the former is five-coordinate with pendant thioether coordination at 198 K or below whereas the latter is four-coordinate at 198 K and becomes a mixture of the four- and five-coordinate species at liquid nitrogen temperature. Upon oxygenation at low temperatures, both complexes form dioxygen adducts in which the pendant thioether groups are coordinated to the trans position to dioxygen. Resonance Raman spectra show that Co(sal-ipsen) yields an equilibrium mixture of the 1:1 and 1:2(O₂/ Co) adducts at 190 K while Co(sal-bsen) forms only the 1:1 adduct under similar conditions. These differences between Co(sal-ipsen) and Co(sal-bsen) can be attributed to the variance in basicity of their pendant sulfur atoms.     

Cell-permeable carboxyl-terminal p27(Kip1) peptide exhibits anti-tumor activity by inhibiting Pim-1 kinase

The incidence and death rate of prostate cancer is increasing rapidly. In addition, the low sensitivity of prostate cancer to chemotherapy makes it difficult to treat this condition. The serine/threonine kinase Pim-1 plays an important role in cell cycle progression and apoptosis inhibition, resulting in prostate tumorigenesis. Therefore, Pim-1 inhibition has been expected to be an attractive target for developing new anti-cancer drugs. However, no small compounds targeting Pim-1 have progressed to clinical use because of their lack of specificity. Here, we have reported a new cell-permeable Pim-1 inhibitory p27(Kip1) peptide that could interfere with the binding of Pim-1 to its substrates and act as an anti-cancer drug. The peptide could bind to Pim-1 and inhibit phosphorylation of endogenous p27(Kip1) and Bad by Pim-1. Treatment of prostate cancer with the peptide induces G(1) arrest and subsequently apoptosis in vitro. However, the peptide showed almost no growth inhibitory or apoptosis-inducing effects in normal cells. The peptide could inhibit tumor growth in in vivo prostate cancer xenograft models. Moreover, the peptide treatment could overcome resistance to taxol, one of the first line chemotherapeutic agents for prostate cancer, and a combination of the peptide with taxol synergistically inhibited prostate cancer growth in vivo. These results indicate that a Pim-1 inhibitory p27(Kip1) peptide could be developed as an anti-cancer drug against prostate cancer.