The conformational and property space of acetylcholine bound to muscarinic receptors: an entropy component accounts for the subtype selectivity of acetylcholine

The conformational behavior of receptor-bound acetylcholine (ACh) was investigated by molecular dynamics simulations. Based on the great similarity among muscarinic receptors, the study was focused on the human M(1), M(2), and M(5) receptors as previously modeled by us. The results showed that receptor-bound ACh was not frozen in a single preferred conformation but preserved an unexpected fraction of its conformational space. However, there were marked differences between the three receptors since the ligand was mostly trans in the M(1) receptor, equally distributed among trans and gauche conformers in M(2), and exclusively gauche in the M(5); the greater flexibility of M(2)-bound ACh was paralleled by the greater flexibility of the occupied M(2) binding site. By contrast, the property space of receptor-bound ACh, and particularly its virtual (computed, conformation-dependent) lipophilicity, was restricted to relatively narrow ranges optimal for successful interaction. Experimental binding investigations to the individual human M(1), M(2), and M(5) muscarinic receptors showed ACh to have a 10-fold higher affinity for the M(2) compared to the M(1) and M(5) receptors. This selectivity was not confirmed by the calculated binding scores, a fact postulated to be caused by the absence of an entropy component in such binding scores. Indeed, the Shannon entropy of all geometric and physicochemical properties monitored were markedly higher in M(2)-bound ACh compared to M(1)-bound and M(5)-bound ACh. This finding suggests that the selectivity profile of acetylcholine for the M(2) receptor is largely entropy-driven, a fact that might explain the intrinsic difficulty to design subtype-selective muscarinic agonists.     

Endothelin-1 response to mental stress in early ischemic lesions of the extremities due to systemic sclerosis

We studied circulating levels of endothelin-1, catecholamines and nitric oxide after a mental arithmetic test in 14 patients with early ischemic lesions of the extremities due to systemic sclerosis and slightly impaired peripheral vascular flow. The test induced an increase (P < 0.01) in blood pressure, heart rate, endothelin-1 and catecholamine levels, whereas it did not change the low basal levels of nitric oxide. In healthy subjects (n = 20) the test significantly (P < 0.01) decreased endothelin-1 without affecting nitric oxide. The low basal levels of nitric oxide and the high plasma concentration of endothelin-1 after psychological stress cannot be explained by an impaired release from the limited ischemic lesions alone. This suggests a diffuse microvascular derangement that aggravates the course of peripheral microvascular ischemic lesions.     

Gleason grading of prostate carcinoma in needle biopsies vs. radical prostatectomy specimens

The Gleason grading system for prostatic carcinoma is the dominant method used around the world in research and in daily practice. It is based on glandular architecture. The grading system should be applied to all prostatic tissue samples, including needle core biopsies and radical prostatectomy specimens. Its prognostic value was tested in a large population with long-term follow-up that included use of survival as an end point. The Gleason grading system shows a reasonable degree of correlation between biopsy and radical prostatectomy specimens. Several sources of discrepancy between these 2 types of specimen have been identified. Further educational endeavors are needed to arrive at a greater consensus and accuracy in the use of the Gleason system.     

Human dorsal and ventral auditory streams subserve rehearsal-based and echoic processes during verbal working memory

To hear a sequence of words and repeat them requires sensory-motor processing and something more-temporary storage. We investigated neural mechanisms of verbal memory by using fMRI and a task designed to tease apart perceptually based ("echoic") memory from phonological-articulatory memory. Sets of two- or three-word pairs were presented bimodally, followed by a cue indicating from which modality (auditory or visual) items were to be retrieved and rehearsed over a delay. Although delay-period activation in the planum temporale (PT) was insensible to the source modality and showed sustained delay-period activity, the superior temporal gyrus (STG) activated more vigorously when the retrieved items had arrived to the auditory modality and showed transient delay-period activity. Functional connectivity analysis revealed two topographically distinct fronto-temporal circuits, with STG co-activating more strongly with ventrolateral prefrontal cortex and PT co-activating more strongly with dorsolateral prefrontal cortex. These argue for separate contributions of ventral and dorsal auditory streams in verbal working memory.     

Two Plasmodium rhomboid proteases preferentially cleave different adhesins implicated in all invasive stages of malaria

Invasion of host cells by the malaria pathogen Plasmodium relies on parasite transmembrane adhesins that engage host-cell receptors. Adhesins must be released by cleavage before the parasite can enter the cell, but the processing enzymes have remained elusive. Recent work indicates that the Toxoplasma rhomboid intramembrane protease TgROM5 catalyzes this essential cleavage. However, Plasmodium does not encode a direct TgROM5 homolog. We examined processing of the 14 Plasmodium falciparum adhesins currently thought to be involved in invasion by both model and Plasmodium rhomboid proteases in a heterologous assay. While most adhesins contain aromatic transmembrane residues and could not be cleaved by nonparasite rhomboid proteins, including Drosophila Rhomboid-1, Plasmodium falciparum rhomboid protein (PfROM)4 (PFE0340c) was able to process these adhesins efficiently and displayed novel substrate specificity. Conversely, PfROM1 (PF11_0150) shared specificity with rhomboid proteases from other organisms and was the only PfROM able to cleave apical membrane antigen 1 (AMA1). PfROM 1 and/or 4 was thus able to cleave diverse adhesins including TRAP, CTRP, MTRAP, PFF0800c, EBA-175, BAEBL, JESEBL, MAEBL, AMA1, Rh1, Rh2a, Rh2b, and Rh4, but not PTRAMP, and cleavage relied on the adhesin transmembrane domains. Swapping transmembrane regions between BAEBL and AMA1 switched the relative preferences of PfROMs 1 and 4 for these two substrates. Our analysis indicates that PfROMs 1 and 4 function with different substrate specificities that together constitute the specificity of TgROM5 to cleave diverse adhesins. This is the first enzymatic analysis of Plasmodium rhomboid proteases and suggests an involvement of PfROMs in all invasive stages of the malaria lifecycle, in both the vertebrate host and the mosquito vector.     

Structural analysis of a rhomboid family intramembrane protease reveals a gating mechanism for substrate entry

Intramembrane proteolysis regulates diverse biological processes. Cleavage of substrate peptide bonds within the membrane bilayer is catalyzed by integral membrane proteases. Here we report the crystal structure of the transmembrane core domain of GlpG, a rhomboid-family intramembrane serine protease from Escherichia coli. The protein contains six transmembrane helices, with the catalytic Ser201 located at the N terminus of helix alpha4 approximately 10 A below the membrane surface. Access to water molecules is provided by a central cavity that opens to the extracellular region and converges on Ser201. One of the two GlpG molecules in the asymmetric unit has an open conformation at the active site, with the transmembrane helix alpha5 bent away from the rest of the molecule. Structural analysis suggests that substrate entry to the active site is probably gated by the movement of helix alpha5.     

Occurrence of A-kinase anchor protein and associated cAMP-dependent protein kinase in the inner compartment of mammalian mitochondria

Evidence showing the existence in the inner compartment of rat-heart mitochondria of AKAP121 and associated PKA is presented. Immunoblotting analysis and trypsin digestion pattern show that 90% or more of mitochondrial C-PKA, R-PKA and AKAP121 is localized in the inner mitochondrial compartment, when prepared both from isolated mitochondria or cardiomyocyte cultures. This localization is verified by measurement of the specific catalytic activity of PKA, radiolabelling of R-PKA by (32)P-phosphorylated C-PKA and of AKAP by (32)P-phosphorylated R-PKA and electron microscopy of mitochondria exposed to gold-conjugated AKAP121 antibody.