Activated T lymphocytes within human solid tumors

Summary The majority of lymphocytes separated from tumor cell suspensions were T cells. Conjugates of T lymphocytes and tumor cells were often seen. Variable numbers of T cells exhibited signs of activation such as the ability to form stable E rosettes and attachment to normal and malignant cells (a phenomenon designated natural attachment: NA). A proportion of T cells activated in vitro by allogeneic stimulation regularly exhibit these properties. The T cell-tumor conjugates in the suspensions may represent the NA phenomenon, but they could also be the product of T cells that adhere on the basis of specific recognition of cell surface antigens.Abbreviations BBS balanced salt solution - E rosettes rosettes formed with sheep erythrocytes - EA rosettes rosettes formed with ox erythrocytes coated with anti-ox IgG - FCS fetal calf serum - MLC mixed lymphocyte cultures - NA natural attachment - PBL peripheral blood lymphocytes - SRBC sheep erythrocytes - T lymphocytes thymusderived lymphocytes     

The stimulatory effect of human chorionic gonadotropin on amino acid uptake by amphibian follicles.

Human chorionic gonadotropin (hCG) stimulates the uptake of eight different amino acids and four nucleosides by Xenopus laevis ovarian follicles. This hormone also stimulates amino acid uptake in the follicles of another amphibian, Callyptocephallela caudiverbera. The stimulation of uptake is due to a reduction in the amino acid concentration required for half-maximal uptake velocity and not to an increment in V_max. The effect of hCG does not require protein synthesis but requires physiological conditions of temperature and pH. Incorporation of radioactive exogenous amino acid into proteins is also stimulated by the hormone, but high-resolution electrophoresis shows that there are no drastic qualitative changes in the pattern of proteins synthesized at early times after hCG treatment. The effect of hCG on the uptake of exogenous amino acids does not appear to be required for oocyte maturation because other hormones such as progesterone and testosterone which induce maturation do not increase amino acid uptake. Also the concentration of hCG required for oocyte maturation is significantly lower than that required for an effect on amino acid transport. Inhibitors of oocyte maturation such as theophylline and cycloheximide do not inhibit the action of hCG on amino acid uptake by the amphibian follicles.     

The reciprocal exclusion by L-dopa (L-3,4-dihydroxyphenylalanine) and L-tyrosine of their incorporation as single units into a soluble rat brain protein.

Several compounds, structurally and metabolically related to phenylalanine and tyrosine, were tested for their effects on the incorporations of phenylalanine and tyrosine as single units into a protein of the soluble subcellular fraction of rat brain. Of the compounds tested, only L-dopa (L-3,4-dihydroxyphenylalanine) inhibited these incorporations. Further, L-dopa was incorporated into a protein of the same fraction in such a way that it excluded the incorporation of tyrosine as a single unit. Conversely, tyrosine inhibited and excluded the incorporation of L-dopa. The incorporation of L-dopa required ATP (apparent Km = 0.23mM), KCl (apparent Km = 20mM) and MgCl2 (optimal concentration range, 5-16mM). These requirements were similar to those previously determined for the incorporation of tyrosine and phenylalanine. The inactivation rate of the enzymic systems for L-tyrosine and L-dopa incorporations, when kept at 37 degrees C, was the same for both amino acids (half-life = 80 min). It is suggested that the acceptor for the incorporation of dopa is the same as that for the incorporation of tyrosine.     

Cbp1, a fungal virulence factor under positive selection,forms an effector complex that drives macrophage lysis

Intracellular pathogens secrete effectors to manipulate their host cells. Histoplasma capsulatum (Hc) is a fungal intracellular pathogen of humans that grows in a yeast form in the host. Hc yeasts are phagocytosed by macrophages, where fungal intracellular replication precedes macrophage lysis. The most abundant virulence factor secreted by Hc yeast cells is Calcium Binding Protein 1 (Cbp1), which is absolutely required for macrophage lysis. Here we take an evolutionary, structural, and cell biological approach to understand Cbp1 function. We find that Cbp1 is present only in the genomes of closely related dimorphic fungal species of the Ajellomycetaceae family that lead primarily intracellular lifestyles in their mammalian hosts (Histoplasma, Paracoccidioides, and Emergomyces), but not conserved in the extracellular fungal pathogen Blastomyces dermatitidis. We observe a high rate of fixation of non-synonymous substitutions in the Cbp1 coding sequences, indicating that Cbp1 is under positive selection. We determine the de novo structures of Hc H88 Cbp1 and the Paracoccidioides americana (Pb03) Cbp1, revealing a novel “binocular” fold consisting of a helical dimer arrangement wherein two helices from each monomer contribute to a four-helix bundle. In contrast to Pb03 Cbp1, we show that Emergomyces Cbp1 orthologs are unable to stimulate macrophage lysis when expressed in the Hc cbp1 mutant. Consistent with this result, we find that wild-type Emergomyces africanus yeast are able to grow within primary macrophages but are incapable of lysing them. Finally, we use subcellular fractionation of infected macrophages and indirect immunofluorescence to show that Cbp1 localizes to the macrophage cytosol during Hc infection, making this the first instance of a phagosomal human fungal pathogen directing an effector into the cytosol of the host cell. We additionally show that Cbp1 forms a complex with Yps-3, another known Hc virulence factor that accesses the cytosol. Taken together, these data imply that Cbp1 is a fungal virulence factor under positive selection that localizes to the cytosol to trigger host cell lysis.     

Uncoupling protein-3 (UCP3) mRNA expression in reconstituted human muscle after myoblast transplantation in RAG2-/-/gamma c/C5(-) immunodeficient mice

Uncoupling protein-3 (UCP3), which is expressed abundantly in skeletal muscle, is one of the carrier proteins dissipating the transmitochondrial electrochemical gradient as heat and has therefore been implicated in the regulation of energy metabolism. Myoblasts or differentiated muscle cells in vitro expressed little if any UCP3, compared with the levels detected in biopsies of skeletal muscle. In the present report, we sought to investigate UCP3 mRNA expression in human muscle generated by myoblast transplantation in the skeletal muscle of an immunodeficient mouse model. Time course experiments demonstrated that 7-8 weeks following transplantation fully differentiated human muscle fibers were formed. The presence of differentiated human muscle fibers was assessed by quantitative PCR measurement of the human alpha-actin mRNA together with immunohistochemical staining using specific antibodies for spectrin and the slow adult myosin heavy chain. Interestingly, we found that the expression of UCP3 mRNA was dependant on human muscle differentiation and that the UCP3 mRNA level was comparable with that found in human muscle biopsies. Moreover, the human UCP3 (hUCP3) promoter seems to be fully functional, since triiodothyronine treatment of the mice not only stimulated the mouse UCP3 (mUCP3) mRNA expression but also strongly stimulated the hUCP3 mRNA expression in human fibers formed after myoblast transplantation. To our knowledge, this is the first time that primary myoblasts could be induced to express the UCP3 gene at a level comparable of that found in human muscle fibers.     

The sensitivity of ABI2 to hydrogen peroxide links the abscisic acid-response regulator to redox signalling

ABI1 and ABI2 are two protein serine/threonine phosphatases of type 2C (EC 3.1.3.16) that act as key regulators in the responses of Arabidopsis thaliana (L.) Heynh. to abscisic acid (ABA). They are involved in the control of ABA-mediated seed dormancy, stomatal closure and vegetative growth inhibition. Analysis of the enzymatic properties of ABI2 revealed high sensitivities towards protons and unsaturated fatty acids. Furthermore, the protein phosphatase activity of ABI2 is very sensitive to H2O2, which has recently emerged as a secondary messenger of ABA signalling. Upon H2O2 challenge, ABI2 is rapidly inactivated with an IC50 value of 50 microM in the presence of reduced glutathione. Inhibitor studies with phenylarsine oxide and manipulation of the redox status of ABI2 in vitro indicate that oxidation of critical cysteine residue(s) is responsible for inactivation. The levels of the major cellular thiol compounds cysteine and glutathione in leaves and seedlings of A. thaliana are compatible with a physiological role of H2O2 in regulating ABI2 activity. ABI2 is considered to exert negative regulation on ABA action. Thus, transient inactivation of this protein phosphatase by H2O2 would allow or enhance the ABA-dependent signalling process. In conclusion, ABI2 represents a likely target for redox-regulation of a hormonal signalling pathway in higher plants.