Asp85 is the only internal aspartic acid that gets protonated in the M intermediate and the purple-to-blue transition of bacteriorhodopsin. A solid-state 13C CP-MAS NMR investigation.

High-resolution solid-state 13C NMR spectra of the ground state and M intermediate of the bacteriorhodopsin mutant D96N with the isotope label at [4-13C]Asp and [11-13C]Trp were recorded. The NMR spectra show that Asp85 is protonated in the M intermediate. The environment of Asp85 is quite hydrophobic. On the other hand, Asp212 remains deprotonated and a slight shift to lower field indicates a more hydrophilic environment. Asp85 also protonates in the purple-to-blue transition of bacteriorhodopsin in the deionized membrane, where it experiences a similar environment to M. The shift of Trp resonances in M reflect a conformational change of the protein in forming the M intermediate.     

Role of endogenous peptides in murine allogenic cytotoxic T cell responses assessed using transfectants of the antigen-processing mutant 174xCEM.T2.

One model to explain the high frequency of alloreactive T cells proposes that allogeneic MHC molecules are recognized together with host cell-derived peptides. A model system was developed to investigate the relevance of this mechanism by expression of H-2Dd or H-2Ld in 174xCEM.T2 (T2) cells. This human cell line contains a mutation in its Ag-processing pathway that should restrict the association of endogenous peptides with cell surface class I molecules. CTL generated by stimulating C57BL/6 (H-2b) responder cells with H-2Dd or H-2Ld transfectants of the human B cell line C1R or the murine T cell lymphoma EL4 were assayed for their ability to recognize alloantigenic determinants on these transfectants. The major fraction of the H-2Dd-specific allogeneic CTL response, generated in a MLC or under clonal limiting dilution conditions, was composed of T cells that recognized H-2Dd expressed on C1R or EL4 cells, but failed to recognize this molecule on T2 cells. Clonal analysis indicated that approximately one-third of these CTL recognized determinants that were unique to H-2Dd expressed on C1R stimulator cells whereas the remainder recognized determinants that were also found on EL4 transfectants. Less than 10% of H-2Dd-reactive CTL recognized the T2 transfectant, and these clones also killed C1R-Dd and EL4-Dd. This result suggests that the great majority of H-2Dd-specific alloreactive CTL recognize determinants that are formed by a complex of H-2Dd with endogenous peptides that are absent or significantly reduced in T2 cells. Based on recognition of human or murine transfectants, these CTL exhibit some level of specificity for the structure or composition of the bound peptides. Examination of allogeneic CTL specific for H-2Ld revealed populations similar to those described for H-2Dd. In addition, a major new population was present that recognized determinants shared between C1R-Ld and T2-Ld but not present on EL4-Ld. These results are consistent with the idea that the alloreactive response to H-2Ld is also largely dependent on the presence of bound peptide. However, they also may indicate that the H-2Ld molecule expressed on T2 cells is occupied by one or more peptides that are shared with other human, but not murine, cells. The significance of these results to current models of alloreactivity is discussed.     

Production of cyclic AMP from extracellular ATP by intact LM cells.

Intact LM cells, a line of cultured mouse fibroblasts, exhibited an adenylate cyclase (ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1) activity in the presence of exogenous [alpha-32P]ATP which was 20--30% of that observed with comparable preparations of lysed cells. The extent of NaF and prostaglandin E1 stimulation was comparable in intact cells and lysed cells. 96% of the added ATP and 92% of the cyclic AMP produced by intact cells could be isolated extracellularly in the incubation medium. Cellular integrity under assay conditions was monitored by trypan blue exclusion. These data suggest that LM cells contain an adenylate cyclase activity which is accessible to extracellular ATP.     

Biodeterioration of Mayan buildings at uxmal and tulum,Mexico

Uxmal and Tulum are two important Mayan sites in the Yucatan peninsula. The buildings are mainly composed of limestone and grey/black discoloration is seen on exposed walls and copious greenish biofilms on inner walls. The principal microorganisms detected on interior walls at both Uxmal and Tulum were cyanobacteria; heterotrophic bacteria and filamentous fungi were also present. A dark‐pigmented mitosporic fungus and Bacillus cereus, both isolated from Uxmal, were shown to be acidogenic in laboratory cultures. Cyanobacteria belonging to rock‐degrading genera Synechocystis and Gloeocapsa were identified at both sites. Surface analysis previously showed that calcium ions were present in the biofilms on buildings at Uxmal and Tulum, suggesting the deposition of biosolubilized stone. Apart from their potential to degrade the substrate, the coccoid cyanobacteria supply organic nutrients for bacteria and fungi, which can produce organic acids, further increasing stone degradation.     

Multimodal pressure-flow method to assess dynamics of cerebral autoregulation in stroke and hypertension

Background

This study evaluated the effects of stroke on regulation of cerebral blood flow in response to fluctuations in systemic blood pressure (BP). The autoregulatory dynamics are difficult to assess because of the nonstationarity and nonlinearity of the component signals.

Methods

We studied 15 normotensive, 20 hypertensive and 15 minor stroke subjects (48.0 ± 1.3 years). BP and blood flow velocities (BFV) from middle cerebral arteries (MCA) were measured during the Valsalva maneuver (VM) using transcranial Doppler ultrasound.

Results

A new technique, multimodal pressure-flow analysis (MMPF), was implemented to analyze these short, nonstationary signals. MMPF analysis decomposes complex BP and BFV signals into multiple empirical modes, representing their instantaneous frequency-amplitude modulation. The empirical mode corresponding to the VM BP profile was used to construct the continuous phase diagram and to identify the minimum and maximum values from the residual BP (BP_R) and BFV (BFV_R) signals. The BP-BFV phase shift was calculated as the difference between the phase corresponding to the BP_R and BFV_R minimum (maximum) values. BP-BFV phase shifts were significantly different between groups. In the normotensive group, the BFV_R minimum and maximum preceded the BP_R minimum and maximum, respectively, leading to large positive values of BP-BFV shifts.

Conclusion

In the stroke and hypertensive groups, the resulting BP-BFV phase shift was significantly smaller compared to the normotensive group. A standard autoregulation index did not differentiate the groups. The MMPF method enables evaluation of autoregulatory dynamics based on instantaneous BP-BFV phase analysis. Regulation of BP-BFV dynamics is altered with hypertension and after stroke, rendering blood flow dependent on blood pressure.

     

Ciliary neurotrophic factor upregulates ubiquitin-proteasome components in a rat model of neuronal injury

Neuronal injury triggers the release of ciliary neurotrophic factor (CNTF), promoting local neuronal repair but producing systemic effects of anorexia and lean body weight loss. Due to the rapid rate of systemic protein loss stimulated by CNTF, we hypothesized involvement of the hepatic ubiquitin-proteasome proteolytic (UPP) pathway in CNTF-induced proteolysis. To assess the role of central CNTF in systemic UPP regulation, we measured hepatic UPP mRNA and proteasome activity in a rat model of neuronal injury and determined alterations induced by intracerebroventricular (ICV) administration of CNTF-neutralizing antibody or additional exogenous CNTF. We also assessed proteolytic parameters and nutritional status by measuring caloric intake, body weight, and protein levels. We produced neuronal injury by implanting a lateral ventricle cannula and giving daily ICV saline bolus injections, which increased hepatic 20S proteasome mRNA and enzymatic activity while reducing caloric intake, body weight, and protein levels compared to controls. Administration of ICV anti-CNTF antibodies (but not control antibodies) prevented these effects. Addition of exogenous CNTF augmented the weight loss along with the increases in 20S proteasome mRNA and proteolytic activity induced by neuronal injury. We conclude that CNTF decreases lean body weight through a combination of appetite inhibition and UPP pathway activation.     

Probing the proton channel and the retinal binding site of Natronobacterium pharaonis sensory rhodopsin II

The sensory rhodopsin II from Natronobacterium pharaonis (NpSRII) was mutated to try to create functional properties characteristic of bacteriorhodopsin (BR), the proton pump from Halobacterium salinarum. Key residues from the cytoplasmic and extracellular proton transfer channel of BR as well as from the retinal binding site were chosen. The single site mutants L40T, F86D, P183E, and T204A did not display altered function as determined by the kinetics of their photocycles. However, the photocycle of each of the subsequent multisite mutations L40T/F86D, L40T/F86D/P183E, and L40T/F86D/P183E/T204A was quite different from that of the wild-type protein. The reprotonation of the Schiff base could be accelerated approximately 300- to 400-fold, to approximately two to three times faster than the corresponding reaction in BR. The greatest effect is observed for the quadruple mutant in which Thr-204 is replaced by Ala. This result indicates that mutations affecting conformational changes of the protein might be of decisive importance for the creation of BR-like functional properties.