Characterization of cross-bridge elasticity and kinetics of cross-bridge cycling during force development in single smooth muscle cells

Force development in smooth muscle, as in skeletal muscle, is believed to reflect recruitment of force-generating myosin cross-bridges. However, little is known about the events underlying cross-bridge recruitment as the muscle cell approaches peak isometric force and then enters a period of tension maintenance. In the present studies on single smooth muscle cells isolated from the toad (Bufo marinus) stomach muscularis, active muscle stiffness, calculated from the force response to small sinusoidal length changes (0.5% cell length, 250 Hz), was utilized to estimate the relative number of attached cross-bridges. By comparing stiffness during initial force development to stiffness during force redevelopment immediately after a quick release imposed at peak force, we propose that the instantaneous active stiffness of the cell reflects both a linearly elastic cross-bridge element having 1.5 times the compliance of the cross-bridge in frog skeletal muscle and a series elastic component having an exponential length-force relationship. At the onset of force development, the ratio of stiffness to force was 2.5 times greater than at peak isometric force. These data suggest that, upon activation, cross-bridges attach in at least two states (i.e., low-force-producing and high-force-producing) and redistribute to a steady state distribution at peak isometric force. The possibility that the cross-bridge cycling rate was modulated with time was also investigated by analyzing the time course of tension recovery to small, rapid step length changes (0.5% cell length in 2.5 ms) imposed during initial force development, at peak force, and after 15 s of tension maintenance. The rate of tension recovery slowed continuously throughout force development following activation and slowed further as force was maintained. Our results suggest that the kinetics of force production in smooth muscle may involve a redistribution of cross-bridge populations between two attached states and that the average cycling rate of these cross-bridges becomes slower with time during contraction.     

Enhanced biodegradation of phenanthrene in oil tar-contaminated soils supplemented with Phanerochaete chrysosporium.

In recent years, the white rot fungus Phanerochaete chrysosporium has shown promise as an organism suitable for the breakdown of a broad spectrum of environmental pollutants, including polynuclear aromatic hydrocarbons (PAHs). The focus of this study was to determine whether P. chrysosporium could effectively operate in an actual field sample of oil tar-contaminated soil. The soil was loaded with [14C]phenanthrene to serve as a model compound representative of the PAHs. Soil with the native flora present under static, aerobic conditions with buffering (pH 5.0 to 5.5) displayed full mineralization on the order of 20% in 21 days. The addition of P. chrysosporium was synergistic, with full mineralization on the order of 38% in 21 days. In addition to full mineralization, there was an increase in the proportion of radiolabelled polar extractives, both soluble and bound, in the presence of P. chrysosporium. From this study, it is apparent that the native soil microflora can be prompted into full mineralization of PAHs in some contaminated soils and that this mineralization can be enhanced when supplemented with the white rot fungus P. chrysosporium. With further refinement, this system may prove an effective bioremediation technology for soils contaminated with PAHs.     

Elemental microanalysis of fibroblasts by a scanning proton microprobe and application to Menkes’ disease

A scanning proton microprobe has been used for the elemental microanalysis of individual fibroblast cells. Both normal fibroblasts and fibroblasts cultured from patients with Menkes' disease, an X-linked genetic disorder known to be associated with defective copper metabolism, were examined by the probe. The cells were cultured on a thin ultra-clean nylon foil and retained on that surface for analysis. The focused high-energy proton beam was used to irradiate selected individual cells and elemental information was derived from X-ray and backscattered proton data. The sensitivity of the scanning proton microprobe to trace concentrations of heavy elements has allowed this elemental information to be used to identify individual cells as being either normal or a Menkes' mutant. The cell identification was based on the application of discriminate analysis to a data set formed from the ratios of copper to each of the macroelements present in the cell. This method of cell identification offers the promise of rapid diagnosis of Menkes' disease.     

Immobilization of tyrosinase for use in nonaqueous media: Enzyme deactivation phenomena

Summary We have investigated features for minimizing the inactivation of tyrosinase (E.C. 1.14.18.1) that is caused by immobilization on glass beads and Celite^®. The degree of inactivation is dependent on the enzyme loading and the carrier's surface area. Addition of a sacrificial protein during the immobilization procedure offers a protective effect with increased residual activity on the basis of comparable enzyme loading.     

Biotransformation of dopamine to norlaudanosoline by Aspergillus niger

Norlaundanosoline is a key intermediate in the synthesis of the benzylisoquinoline alkaloids providing the upper isoquinoline portion of the morphinan skeleton. This study evaluates the feasibility of using Aspergillus niger as an in situ biotransformation system to produce norlaudanosoline from dopamine. A. niger was chosen because monoamine oxidase can be readily induced in this organism. Monoamine oxidase catalyzes the conversion of dopamine to 3,4-dihydroxyphenylacetaldehyde. In the presence of dopamine, this aldehyde will then undergo a spontaneous Picket-Spengler condensation to form norlaudanosoline. Fermentation condition to form norlaudanosoline. Fermentation conditions were optimized for the production monoamine oxidase by using a two-stage process consisting of a growth stage and an induction stage. pH control was found to be important, and at pH 4.5 dopamine accumulation in the cells was high as was the level of monoamine oxidase. With pH control at 4.5, up to 21% of the cellular dopamine was converted to norlaudanosoline. It is proposed that with further protein engineering improvements, this system may prove suitable for the in situ bio-transformation of dopamine to norlaudanosoline.     

Monitoring of an antigen manufacturing process

Fluorescence spectroscopy in combination with multivariate statistical methods was employed as a tool for monitoring the manufacturing process of pertactin (PRN), one of the virulence factors of Bordetella pertussis utilized in whopping cough vaccines. Fluorophores such as amino acids and co-enzymes were detected throughout the process. The fluorescence data collected at different stages of the fermentation and purification process were treated employing principal component analysis (PCA). Through PCA, it was feasible to identify sources of variability in PRN production. Then, partial least square (PLS) was employed to correlate the fluorescence spectra obtained from pure PRN samples and the final protein content measured by a Kjeldahl test from these samples. In view that a statistically significant correlation was found between fluorescence and PRN levels, this approach could be further used as a method to predict the final protein content.     

Introduced cats Felis catus eating a continental fauna: inventory and traits of Australian mammal species killed

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Neonatal immunity develops in a transgenic TCR transfer model and reveals a requirement for elevated cell input to achieve organ-specific responses

In recent years, it has become clear that neonatal exposure to Ag induces rather than ablates T cell immunity. Moreover, rechallenge with the Ag at adult age can trigger secondary responses that are distinct in the lymph node vs the spleen. The question addressed in this report is whether organ-specific secondary responses occur as a result of the diversity of the T cell repertoire or could they arise with homogeneous TCR-transgenic T cells. To test this premise, we used the OVA-specific DO11.10 TCR-transgenic T cells and established a neonatal T cell transfer system suitable for these investigations. In this system, neonatal T cells transferred from 1-day-old DO11.10/SCID mice into newborn (1-day-old) BALB/c mice migrate to the host's spleen and maintain stable frequency. The newborn BALB/c hosts were then given Ig-OVA, an Ig molecule carrying the OVA peptide, and challenged with the OVA peptide in CFA at the age of 7 wk; then their secondary responses were analyzed. The findings show that the lymph node T cells were deviated and produced IL-4 instead of IFN-gamma and the splenic T cells, although unable to proliferate or produce IFN-gamma, secreted a significant level of IL-2. Supply of exogenous IL-12 during Ag stimulation restores both proliferation and IFN-gamma production by the splenic T cells. This restorable form of splenic unresponsiveness referred to as IFN-gamma-dependent anergy required a transfer of a high number of neonatal DO11.10/SCID T cells to develop. Thus, the frequency of neonatal T cell precursors rather than repertoire diversity exerts control on the development of organ-specific neonatal immunity.