Exercise intensity influences cardiac baroreflex function at the onset of isometric exercise in humans.

We sought to examine the influence of exercise intensity on carotid baroreflex (CBR) control of heart rate (HR) and mean arterial pressure (MAP) at the onset of exercise in humans. To accomplish this, eight subjects performed multiple 1-min bouts of isometric handgrip (HG) exercise at 15, 30, 45 and 60% maximal voluntary contraction (MVC), while breathing to a metronome set at eupneic frequency. Neck suction (NS) of -60 Torr was applied for 5 s at end expiration to stimulate the CBR at rest, at the onset of HG (<1 s), and after approximately 40 s of HG. Beat-to-beat measurements of HR and MAP were recorded throughout. Cardiac responses to NS at onset of 15% (-12 +/- 2 beats/min) and 30% (-10 +/- 2 beats/min) MVC HG were similar to rest (-10 +/- 1 beats/min). However, HR responses to NS were reduced at the onset of 45% and 60% MVC HG (-6 +/- 2 and -4 +/- 1 beats/min, respectively; P < 0.001). In contrast to HR, MAP responses to NS were not different from rest at exercise onset. Furthermore, both HR and MAP responses to NS applied at approximately 40s of HG were similar to rest. In summary, CBR control of HR was transiently blunted at the immediate onset of high-intensity HG, whereas MAP responses were preserved demonstrating differential baroreflex control of HR and blood pressure at exercise onset. Collectively, these results suggest that carotid-cardiac baroreflex control is dynamically modulated throughout isometric exercise in humans, whereas carotid baroreflex regulation of blood pressure is well-maintained.     

Molecular model for the binary complex of uropepsin and pepstatin

The three-dimensional structure of human uropepsin complexed with pepstatin has been modelled using human pepsin as a template. Uropepsin is an aspartic proteinase from the urine, produced in the form of pepsinogen A in the gastric mucosa. The structure is bilobal, consisting of two predominantly beta-sheet lobes which, as observed in other aspartic proteinases, are related by a pseudo twofold axis. A structural comparison between binary complexes of pepsin:pepstatin and uropepsin:pepstatin is discussed.     

Calreticulin affects focal contact-dependent but not close contact-dependent cell-substratum adhesion.

We used two cell lines expressing fast (RPEfast) and slow (RPEslow) attachment kinetics to investigate mechanisms of cell-substratum adhesion. We show that the abundance of a cytoskeletal protein, vinculin, is dramatically decreased in RPEfast cells. This coincides with the diminished expression level of an endoplasmic reticulum chaperone, calreticulin. Both protein and mRNA levels for calreticulin and vinculin were decreased in RPEfast cells. After RPEfast cells were transfected with cDNA encoding calreticulin, both the expression of endoplasmic reticulum-resident calreticulin and cytoplasmic vinculin increased. The abundance of other adhesion-related proteins was not affected. RPEfast cells underexpressing calreticulin displayed a dramatic increase in the abundance of total cellular phosphotyrosine suggesting that the effects of calreticulin on cell adhesiveness may involve modulation of the activities of protein tyrosine kinases or phosphatases which may affect the stability of focal contacts. The calreticulin and vinculin underexpressing RPEfast cells lacked extensive focal contacts and adhered weakly but attached fast to the substratum. In contrast, the RPEslow cells that expressed calreticulin and vinculin abundantly developed numerous and prominent focal contacts slowly, but adhered strongly. Thus, while the calreticulin overexpressing RPEslow cells "grip" the substratum with focal contacts, calreticulin underexpressing RPEfast cells use close contacts to "stick" to it.     

Effect of 50 Hz, 0.2 mT magnetic fields on RBC properties and heart functions of albino rats

In this work the effect of sinusoidal 50 Hz, 0.2 mT magnetic fields on the red blood cells (RBCs) and heart functions of Albino rats were investigated. Twenty-four male Albino rats were equally divided into four groups, A, B, C, and D. Animals from groups B were continuously exposed to the magnetic field for 15 days; and groups C and D, for 30 days. Group A was used as control. Animals from group D were kept after exposure to the magnetic field for a period of 45 days for delayed effect studies. The osmotic fragility and shape of RBCs' membrane and hemoglobin (Hb) structure tests were carried out for all groups. The dielectric relaxation of Hb molecules was measured in the frequency range of 0.1-10 MHz and the dielectric increment (Deltaepsilon), relaxation time (tau), molecular radius (r), and Cole-Cole parameter (alpha) were calculated for all groups. The ECG was measured for all animals before and after exposure to the magnetic field. The results indicated that exposure of the animals to 50 Hz, 0.2 mT magnetic fields resulted in the decrease of RBCs membrane elasticity and permeability and changes in the molecular structure of Hb. The ECG of the exposed animals was considerably altered. The data also indicated that there was no sign of repair in the newly generated RBCs structure and the ECG after removing the animals from the magnetic field, which indicates that the blood generating system was severely injured. The injuries in the heart of the animals were attributed to the loss of some physiological functions of the RBCs as a result of exposures of the rats to the magnetic field.     

Iterative sequence/secondary structure search for protein homologs: comparison with amino acid sequence alignments and application to fold recognition in genome databases

MOTIVATION: Sequence alignment techniques have been developed into extremely powerful tools for identifying the folding families and function of proteins in newly sequenced genomes. For a sufficiently low sequence identity it is necessary to incorporate additional structural information to positively detect homologous proteins. We have carried out an extensive analysis of the effectiveness of incorporating secondary structure information directly into the alignments for fold recognition and identification of distant protein homologs. A secondary structure similarity matrix based on a database of three-dimensionally aligned proteins was first constructed. An iterative application of dynamic programming was used which incorporates linear combinations of amino acid and secondary structure sequence similarity scores. Initially, only primary sequence information is used. Subsequently contributions from secondary structure are phased in and new homologous proteins are positively identified if their scores are consistent with the predetermined error rate. RESULTS: We used the SCOP40 database, where only PDB sequences that have 40% homology or less are included, to calibrate homology detection by the combined amino acid and secondary structure sequence alignments. Combining predicted secondary structure with sequence information results in a 8-15% increase in homology detection within SCOP40 relative to the pairwise alignments using only amino acid sequence data at an error rate of 0.01 errors per query; a 35% increase is observed when the actual secondary structure sequences are used. Incorporating predicted secondary structure information in the analysis of six small genomes yields an improvement in the homology detection of approximately 20% over SSEARCH pairwise alignments, but no improvement in the total number of homologs detected over PSI-BLAST, at an error rate of 0.01 errors per query. However, because the pairwise alignments based on combinations of amino acid and secondary structure similarity are different from those produced by PSI-BLAST and the error rates can be calibrated, it is possible to combine the results of both searches. An additional 25% relative improvement in the number of genes identified at an error rate of 0.01 is observed when the data is pooled in this way. Similarly for the SCOP40 dataset, PSI-BLAST detected 15% of all possible homologs, whereas the pooled results increased the total number of homologs detected to 19%. These results are compared with recent reports of homology detection using sequence profiling methods. AVAILABILITY: Secondary structure alignment homepage at http://lutece.rutgers.edu/ssas CONTACT: anders@rutchem.rutgers.edu; ronlevy@lutece.rutgers.edu Supplementary Information: Genome sequence/structure alignment results at http://lutece.rutgers.edu/ss_fold_predictions.     

Characterization of SPACR, a sialoprotein associated with cones and rods present in the interphotoreceptor matrix of the human retina: immunological and lectin binding analysis

Rod and cone photoreceptors project from the outer retinal surface into a carbohydrate-rich interphotoreceptor matrix (IPM). Unique IPM glycoconjugates are distributed around rods and cones. Wheat germ agglutinin (WGA) strongly decorates the rod matrix domains and weakly decorates the cone matrix domains. This study characterizes the major WGA-binding glycoprotein in the human IPM, which we refer to as SPACR (sialoprotein associated with cones and rods). SPACR, which has a molecular weight of 147 kDa, was isolated and purified from the IPM by lectin affinity chromatography. A polyclonal antibody to SPACR was prepared that colocalizes in tissue preparations with WGA-binding domains in the IPM. Sequential digestion of SPACR with N- and O- glycosidases results in a systematic increase in electrophorectic mobility, indicating the presence of both N- and O-linked glycoconjugates. Complete deglycosylation results in a reduction in the relative molecular mass of SPACR by about 30%. Analysis of lectin binding allowed us to identify some of the structural characteristics of SPACR glycoconjugates. Treatment with neuraminidase exposes Galbeta1- 3GalNAc disaccharide as indicated by positive peanut agglutinin (PNA) staining, accompanied by the loss of WGA staining. Maackia amurensis agglutinins (MAA-1 and MAA-2), specific for sialic acid in alpha2-3 linkage to Gal, bind SPACR, while Sambucus nigra agglutinin (SNA), specific for alpha2-6 linked sialic acid, does not, indicating that the dominant glycoconjugate determinant on SPACR is the O-linked carbohydrate, NeuAcalpha2-3Galbeta1-3GalNAc. The abundance of sialic acid in SPACR suggests that this glycoprotein may contribute substantially to the polyanionic nature of the IPM. The carbohydrate chains present on SPACR could also provide sites for extensive crosslinking and participate in the formation of the ordered IPM lattice that surrounds the elongate photoreceptors projecting from the outer retinal surface.      

The structure, catalytic mechanism and regulation of adenylyl cyclase

The recent structure determinations of the mammalian effector enzyme adenylyl cyclase reveal the structure of its catalytic core, provide new insights into its catalytic mechanism and suggest how diverse signaling molecules regulate its activity.     

Cytosolic Access of Mycobacterium tuberculosis: Critical Impact of Phagosomal Acidification Control and Demonstration of Occurrence In Vivo

Mycobacterium tuberculosis (Mtb) uses efficient strategies to evade the eradication by professional phagocytes, involving—as recently confirmed—escape from phagosomal confinement. While Mtb determinants, such as the ESX-1 type VII secretion system, that contribute to this phenomenon are known, the host cell factors governing this important biological process are yet unexplored. Using a newly developed flow-cytometric approach for Mtb, we show that macrophages expressing the phagosomal bivalent cation transporter Nramp-1, are much less susceptible to phagosomal rupture. Together with results from the use of the phagosome acidification inhibitor bafilomycin, we demonstrate that restriction of phagosomal acidification is a prerequisite for mycobacterial phagosomal rupture and cytosolic contact. Using different in vivo approaches including an enrichment and screen for tracking rare infected phagocytes carrying the CD45.1 hematopoietic allelic marker, we here provide first and unique evidence of M. tuberculosis-mediated phagosomal rupture in mouse spleen and lungs and in numerous phagocyte types. Our results, linking the ability of restriction of phagosome acidification to cytosolic access, provide an important conceptual advance for our knowledge on host processes targeted by Mtb evasion strategies.