The Activation Pathway of Human Rhodopsin in Comparison to Bovine Rhodopsin

Rhodopsin, the photoreceptor of rod cells, absorbs light to mediate the first step of vision by activating the G protein transducin (G_t). Several human diseases, such as retinitis pigmentosa or congenital night blindness, are linked to rhodopsin malfunctions. Most of the corresponding in vivo studies and structure-function analyses (e.g. based on protein x-ray crystallography or spectroscopy) have been carried out on murine or bovine rhodopsin. Because these rhodopsins differ at several amino acid positions from human rhodopsin, we conducted a comprehensive spectroscopic characterization of human rhodopsin in combination with molecular dynamics simulations. We show by FTIR and UV-visible difference spectroscopy that the light-induced transformations of the early photointermediates are very similar. Significant differences between the pigments appear with formation of the still inactive Meta I state and the transition to active Meta II. However, the conformation of Meta II and its activity toward the G protein are essentially the same, presumably reflecting the evolutionary pressure under which the active state has developed. Altogether, our results show that although the basic activation pathways of human and bovine rhodopsin are similar, structural deviations exist in the inactive conformation and during receptor activation, even between closely related rhodopsins. These differences between the well studied bovine or murine rhodopsins and human rhodopsin have to be taken into account when the influence of point mutations on the activation pathway of human rhodopsin are investigated using the bovine or murine rhodopsin template sequences.     

Lack of CCR7 induces pulmonary hypertension involving perivascular leukocyte infiltration and inflammation

The chemokine receptor CCR7 regulates lymphocyte trafficking, and CCR7 deficiency induces infiltration of T and B cells adjacent to vessels in mouse lungs. Perivascular infiltration of T and B cells has also been found in human pulmonary arterial hypertension, and downregulation of the CCR7 receptor in circulating leukocytes of such patients has been observed. To investigate whether changes in the CCR7 system contribute to the pathogenesis of pulmonary hypertension, we utilized mice deficient of the CCR7 receptor. The cardiopulmonary and inflammatory responses of CCR7 depletion were evaluated in CCR7-deficient and wild-type mice. Measurements of cytokines upregulated in the animal model were also performed in patients with pulmonary hypertension and controls and in vascular smooth muscle cells. We found that mice lacking CCR7 had increased right ventricular systolic pressure, reduced pulmonary artery acceleration time, increased right ventricular/tibial length ratio, Rho kinase-mediated pulmonary vasoconstriction, and increased muscularization of distal arteries, indicating pulmonary hypertension. These mice also showed increased perivascular infiltration of leukocytes, consisting mainly of T and B cells, and increased mRNA levels of the inflammatory cytokines interleukin-12 and CX3CL1 within pulmonary tissue. Increased serum levels of interleukin-12 and CX3CL1 were also observed in patients with pulmonary hypertension, particularly in those with pulmonary hypertension associated with connective tissue disorder. In smooth muscle cells, interleukin-12 induced secretion of the angiogenic cytokine interleukin-8. We conclude that these results suggest a role for CCR7 in the development of pulmonary arterial hypertension, at least in some subgroups, possibly via pulmonary infiltration of lymphocytes and secretion of interleukin-12 and CX3CL1.     

Three new species of Lentinus from northern Thailand

There have been few studies on the taxonomy and biodiversity of the genus Lentinus in Thailand, which is a genus of edible mushrooms. Recently, collections from 17 sites in northern Thailand yielded 47 specimens of Lentinus sensu lato. Three were shown to be new species of Lentinus sensu stricto and Lentinus roseus, L. concentricus and L. megacystidiatus are introduced in this paper. The new species are described and illustrated with line drawings and are justified and compared with similar taxa. Furthermore, ITS sequence data do not match closely with any species presently lodged in GenBank.     

Split T cell tolerance against a self/tumor antigen: spontaneous CD4+ but not CD8+ T cell responses against p53 in cancer patients and healthy donors

Analyses of NY-ESO-1-specific spontaneous immune responses in cancer patients revealed that antibody and both CD4(+) and CD8(+) T cell responses were induced together in cancer patients. To explore whether such integrated immune responses are also spontaneously induced for other tumor antigens, we have evaluated antibody and T cell responses against self/tumor antigen p53 in ovarian cancer patients and healthy individuals. We found that 21% (64/298) of ovarian cancer patients but no healthy donors showed specific IgG responses against wild-type p53 protein. While none of 12 patients with high titer p53 antibody showed spontaneous p53-specific CD8(+) T cell responses following a single in vitro sensitization, significant p53-specific IFN-γ producing CD4(+) T cells were detected in 6 patients. Surprisingly, similar levels of p53-specific CD4(+) T cells but not CD8(+) T cells were also detected in 5/10 seronegative cancer patients and 9/12 healthy donors. Importantly, p53-specific CD4(+) T cells in healthy donors originated from a CD45RA(-) antigen-experienced T cell population and recognized naturally processed wild-type p53 protein. These results raise the possibility that p53-specific CD4(+) T cells reflect abnormalities in p53 occurring in normal individuals and that they may play a role in processes of immunosurveillance or immunoregulation of p53-related neoplastic events.     

Sucrose monoester micelles size determined by Fluorescence Correlation Spectroscopy (FCS)

One of the several uses of sucrose detergents, as well as other micelle forming detergents, is the solubilization of different membrane proteins. Accurate knowledge of the micelle properties, including size and shape, are needed to optimize the surfactant conditions for protein purification and membrane characterization. We synthesized sucrose esters having different numbers of methylene subunits on the substituent to correlate the number of methylene groups with the size of the corresponding micelles. We used Fluorescence Correlation Spectroscopy (FCS) and two photon excitation to determine the translational D of the micelles and calculate their corresponding hydrodynamic radius, R(h). As a fluorescent probe we used LAURDAN (6-dodecanoyl-2-dimethylaminonaphthalene), a dye highly fluorescent when integrated in the micelle and non-fluorescent in aqueous media. We found a linear correlation between the size of the tail and the hydrodynamic radius of the micelle for the series of detergents measured.