Penetration of analogues of H2O and CO2 in proteins studied by room temperature phosphorescence of tryptophan.

The influence of the protein matrix on the reactivity of external molecules with a species buried within the protein interior is considered in two general ways: (1) there may be structural fluctuations that allow for the diffusive penetration of the small molecules and/or (2) the external molecule may react over a distance. As a means to study the protein matrix, a reactive species within the protein can be formed by exciting tryptophan to the triplet state, and then the reaction of the triplet-state molecule with an external molecule can be monitored by a decrease in phosphorescence. In this work, the quenching ability (i.e., reactivity) was examined for H2S, CS2, and NO2- acting on tryptophan phosphorescence in parvalbumin, azurin, horse liver alcohol dehydrogenase, and alkaline phosphatase. A comparison of charged versus uncharged quenchers (H2S vs SH- and CS2 vs NO2-) reveals that the uncharged molecules are much more effective than charged species in quenching the phosphorescence of fully buried tryptophan, whereas the quenching for exposed tryptophan is relatively independent of the charge of the quencher. This is consistent with the view that uncharged triatomic molecules can penetrate the protein matrix to some extent. The energies of activation of the quenching reaction are low for the charged quenchers and higher for the uncharged CS2. A model is presented in which the quenchability of a buried tryptophan is inversely related to the distance from the surface when diffusion through the protein is the rate-limiting step.(ABSTRACT TRUNCATED AT 250 WORDS)     

Monoclonal antibody as a probe for structure and function of an Escherichia coli outer membrane protein

Eight independently derived monoclonal antibodies directed against the LamB protein were produced and characterized. By using these antibodies as probes, we identified four distinct topological and functional regions in the LamB molecule. Four monoclonal antibodies recognize antigenic determinants of the protein exposed on the outer side of the membrane. Two of these have their binding sites located in a region involved in maltose transport. One monoclonal antibody presumably binds to a determinant which is normally hidden in the membrane and three monoclonal antibodies recognize determinants facing the periplasmic space.     

Volatiles from the epicuticular wax of watercress (Rorippa nasturtium-aquaticum)

Volatiles from the epicuticular wax of watercress were collected by ether washing and examined using gas chromatographic and mass spectrometric analysi     

Sexually dimorphic expression of myosin heavy chains in the adult mouse masseter.

Little is known regarding the role of androgenic hormones in the maintenance of myosin heavy chain (MHC) composition of rodent masticatory muscles. Because the masseter is the principal jaw closer in rodents, we felt it was important to characterize the influence of androgenic hormones on the MHC composition of the masseter. To determine the extent of sexual dimorphism in the phenotype of masseter muscle fibers of adult (10-mo-old) C57 mice, we stained tissue sections with antibodies specific to type IIa and IIb MHC isoforms. Females contain twice as many fibers containing the IIa MHC as males, and males contain twice as many fibers containing the IIb MHC as females. There is a modest amount of regionalization of MHC phenotypes in the mouse masseter. The rostral portions of the masseter are composed mostly of type IIa fibers, whereas the midsuperficial and caudal regions contain mostly type IIb fibers. Using immunoblots, we showed that castration results in an increase in the expression of type IIa MHC fibers in males. Ovariectomy has no effect on the fiber type composition in females. We conclude that testosterone plays a role in the maintenance of MHC expression in the adult male mouse masseter.     

Quantifying Biomass Changes of Single CD8+ T Cells during Antigen Specific Cytotoxicity

Existing approaches that quantify cytotoxic T cell responses rely on bulk or surrogate measurements which impede the direct identification of single activated T cells of interest. Single cell microscopy or flow cytometry methodologies typically rely on fluorescent labeling, which limits applicability to primary cells such as human derived T lymphocytes. Here, we introduce a quantitative method to track single T lymphocyte mediated cytotoxic events within a mixed population of cells using live cell interferometry (LCI), a label-free microscopy technique that maintains cell viability. LCI quantifies the mass distribution within individual cells by measuring the phase shift caused by the interaction of light with intracellular biomass. Using LCI, we imaged cytotoxic T cells killing cognate target cells. In addition to a characteristic target cell mass decrease of 20–60% over 1–4 h following attack by a T cell, there was a significant 4-fold increase in T cell mass accumulation rate at the start of the cytotoxic event and a 2–3 fold increase in T cell mass relative to the mass of unresponsive T cells. Direct, label-free measurement of CD8+ T and target cell mass changes provides a kinetic, quantitative assessment of T cell activation and a relatively rapid approach to identify specific, activated patient-derived T cells for applications in cancer immunotherapy.     

The specificity of the syngeneic mixed leukocyte response, a primary anti-I region T cell proliferative response, is determined intrathymically

In previous studies, the syngeneic MLR of peripheral T cells was shown to be predominantly an I region-restricted function. In this report we show that adult thymocytes are also capable of responding to syngeneic irradiated stimulator cells in a syngeneic MLR, provided that TCGF is added to the culture system. Using this assay, it was possible for the first time to examine the pattern of I region restriction within the thymus itself. Analysis of the thymocyte syngeneic MLR in thymuses from radiation-induced bone marrow chimeras demonstrated that the MHC preference seen in the peripheral T cell population also existed in cells resident within the thymus. Experiments utilizing congenitally athymic mice transplanted with allogeneic thymic grafts demonstrated that both peripheral T cells and thymocytes from such animals displayed a strong preferential proliferation toward stimulator cells bearing thymic-type MHC determinants. The results in the nude model thus demonstrate that the thymus by itself is sufficient to impart such restriction specificity on a developing T cell repertoire. These results are consistent with the notion that the thymus exerts selective pressure on maturing T cell populations that results in a skewing of the T cell repertoire toward the recognition of thymic-type I region products, and that this MHC preference exists before expansion of T cells in the periphery.