The immunoglobulin lambda variable light-chain region in primates has been shaped by multiple, independent, small-scale and large-scale insertion/deletion events

We analyzed genomes of nonhuman primates to determine the ancestral state of a 9.1-kb insertion/deletion polymorphism, located on human chromosome 22. The 9.1-kb+ allele was found in 16 chimpanzees, 3 bonobos, and 2 Bornean orangutans; however, 9 chimpanzees and 6 Sumatran orangutans showed neither the 9.1-kb+ nor the 9.1-kb- allele, but a novel allele, termed 9.1-kbnull. A clone from a chimpanzee BAC library carrying the 9.1-kbnull allele was sequenced: the BAC DNA aligns with the human chromosome 22 reference sequence except for a 75-kb region, suggesting that the 9.1-kbnull allele originated from a deletion. Furthermore, the 9.1-kb+ chromosomes of chimpanzees and bonobos contain a 1030-nucleotide sequence, absent in humans, that may result from a retro-transposition insertion in their common ancestor. Our results provide additional evidence that human chromosome 22 has undergone multiple small-scale and large-scale insertions and deletions since sharing a common ancestor with other primates.     

Frames of reference for eye-head gaze commands in primate supplementary eye fields

The supplementary eye field (SEF) is a region within medial frontal cortex that integrates complex visuospatial information and controls eye-head gaze shifts. Here, we test if the SEF encodes desired gaze directions in a simple retinal (eye-centered) frame, such as the superior colliculus, or in some other, more complex frame. We electrically stimulated 55 SEF sites in two head-unrestrained monkeys to evoke 3D eye-head gaze shifts and then mathematically rotated these trajectories into various reference frames. Each stimulation site specified a specific spatial goal when plotted in its intrinsic frame. These intrinsic frames varied site by site, in a continuum from eye-, to head-, to space/body-centered coding schemes. This variety of coding schemes provides the SEF with a unique potential for implementing arbitrary reference frame transformations.     

Acute and specific collagen type I degradation increases diastolic and developed tension in perfused rat papillary muscle

Collagen degradation is suggested to be responsible for long-term contractile dysfunction in different cardiomyopathies, but the effects of acute and specific collagen type I removal (main type in the heart muscle) on tension have not been studied. We determined the diastolic and developed tension length relations in isometric contracting perfused rat papillary muscles (perfusion pressure 60 cmH(2)O) before and after acute and specific removal of small collagen struts with the use of purified collagenase type I. At 95% of the maximal length (95%L(max)), diastolic tension increased 20.4 +/- 8.1% (P < 0.05, n = 6) and developed tension increased 15.0 +/- 6.7% after collagenase treatment compared with time controls. Treatment increased the diastolic muscle diameter by 7.1 +/- 3.4% at 95%L(max), whereas the change in diameter due to contraction was not changed. Diastolic coronary flow and normalized coronary arterial flow impediment did not change after collagenase treatment. Electron microscopy revealed that the number of small collagen struts, interconnecting myocytes, and capillaries was reduced to approximately 32% after treatment. We conclude that removal of the small collagen struts by acute and specific collagen type I degradation increases diastolic and developed tension in perfused papillary muscle. We suggest that diastolic tension is increased due to edema, whereas developed tension is increased because the removal of the struts poses a lower lateral load on the cardiac myocytes, allowing more myocyte thickening.     

Vasoconstrictor effects of insulin in skeletal muscle arterioles are mediated by ERK1/2 activation in endothelium

Insulin exerts both NO-dependent vasodilator and endothelin-dependent vasoconstrictor effects on skeletal muscle arterioles. The intracellular enzymes 1-phosphatidylinositol 3-kinase (PI3-kinase) and Akt have been shown to mediate the vasodilator effects of insulin, but the signaling molecules involved in the vasoconstrictor effects of insulin in these arterioles are unknown. Our objective was to identify intracellular mediators of acute vasoconstrictor effects of insulin on skeletal muscle arterioles. Rat cremaster first-order arterioles (n=40) were isolated, and vasoreactivity to insulin was studied using a pressure myograph. Insulin induced dose-dependent vasoconstriction of skeletal muscle arterioles (up to -22 +/- 3% of basal diameter; P <0.05) during PI3-kinase inhibition with wortmannin (50 nmol/l). Insulin-induced vasoconstriction was abolished by inhibition of extracellular signal-regulated kinase 1/2 (ERK1/2) with PD-98059 (40 micromol/l). In addition, inhibition of ERK1/2 without PI3-kinase inhibition uncovered insulin-mediated vasodilatation in skeletal muscle arterioles (up to 37 +/- 10% of baseline diameter; P <0.05). Effects of insulin on ERK1/2 activation in arterioles were then investigated by Western blot analysis. Insulin induced a transient 2.4-fold increase in ERK1/2 phosphorylation (maximal at approximately 15 min) in skeletal muscle arterioles (P <0.05). Removal of the arteriolar endothelium abolished insulin-induced vasoconstriction, which suggests that activation of ERK1/2 in endothelial cells is involved in acute insulin-mediated vasoconstriction. To investigate this, acute effects of insulin on ERK1/2 phosphorylation were studied in human microvascular endothelial cells. In support of the findings in skeletal muscle arterioles, insulin induced a 1.9-fold increase in ERK1/2 phosphorylation (maximal at approximately 15 min) in microvascular endothelial cells (P <0.05). We conclude that acute vasoconstrictor effects of insulin in skeletal muscle arterioles are mediated by activation of ERK1/2 in endothelium. This ERK1/2-mediated vasoconstrictor effect antagonizes insulin-induced, PI3-kinase-dependent vasodilatation in skeletal muscle arterioles. These findings provide a novel mechanism by which insulin may determine blood flow and glucose disposal in skeletal muscle.     

Characteristics of intermittent mitochondrial transport in guinea pig enteric nerve fibers

Enteric neurons controlling various gut functions are prone to oxidative insults that might damage mitochondria (e.g., intestinal inflammation). To resume local energy supply, mitochondria need to be transported. We used MitoTracker dyes and confocal microscopy to investigate basic characteristics of mitochondrial transport in guinea pig myenteric neurites. During a 10-s observation of 1 mm nerve fiber, on average, three mitochondria were transported at an average speed of 0.41 +/- 0.02 microm/s. Movement patterns were clearly erratic, and velocities were independent of mitochondrial size. The velocity oscillated periodically ( approximately 6 s) but was not consistently affected by structures such as en route boutons, bifurcations, or stationary mitochondria. Also, mitochondria transported in opposite directions did not necessarily affect each others' mobility. Transport was blocked by microtubule disruption (100 microM colchicine), and destabilization (1 microM cytochalasin-D) or stabilization (10 microM phalloidin) of actin filaments, respectively, decreased (0.22 +/- 0.02 microm/s, P < 0.05) or increased (0.53 +/- 0.02 microm/s, P < 0.05) transport speed. Transport was inhibited by TTX (1 microM), and removal of extracellular Ca(2+) (plus 2 mM EGTA) had no effect. However, depletion of intracellular stores (thapsigargin) reduced (to 33%) and slowed the transport significantly (0.18 +/- 0.02 microm/s, P < 0.05), suggesting an important role for stored Ca(2+) in mitochondrial transport. Transport was also reduced (to 21%) by the mitochondrial uncoupler FCCP (1 microM) in a time-dependent fashion and slowed by oligomycin (10 microM). We conclude that mitochondrial transport is remarkably independent of structural nerve fiber properties. We also show that mitochondrial transport is TTX sensitive and speeds up by stabilizing actin and that functional Ca(2+) stores are required for efficient transport.     

Alkylated derivatives of poly(ethylacrylic acid) can be inserted into preformed liposomes and trigger pH-dependent intracellular delivery of liposomal contents

Poly(ethylacrylic acid) (PEAA) is a pH-sensitive polymer that undergoes a transition from a hydrophilic to a hydrophobic form as the pH is lowered from neutral to acidic values. In this work we show that pH sensitive liposomes capable of intracellular delivery can be constructed by inserting a lipid derivative of PEAA into preformed large unilamellar vesicles (LUV) using a simple one step incubation procedure. The lipid derivatives of PEAA were synthesized by reacting a small proportion (3%) of the carboxylic groups of PEAA with C10 alkylamines to produce C10-PEAA. Incubation of C10-PEAA with preformed LUV resulted in the association of up to 8% by weight of derivatized polymer with the LUV without inducing aggregation. The resulting C10-PEAA-LUV exhibited pH-dependent fusion and leakage of LUV contents on reduction of the external pH below pH 6.0 as demonstrated by lipid mixing and release of calcein encapsulated in the LUV. In addition, C10-PEAA-LUV exhibited pH dependent intracellular delivery properties following uptake into COS-7 cells with appreciable delivery to the cell cytoplasm as evidenced by the appearance of diffuse intracellular calcein fluorescence. It is demonstrated that the cytoplasmic delivery of calcein by C10-PEAA-LUV could be inhibited by agents (bafilomycin or chloroquine) that inhibit acidification of endosomal compartments, indicating that this intracellular delivery resulted from the pH-dependent destabilization of LUV and endosomal membranes by the PEAA component of the C10-PEAA-LUV. It is concluded that C10-PEAA-LUV represents a promising intracellular delivery system for in vitro and in vivo applications.     

Visualization by cryo-electron microscopy of genomic RNA that binds to the protein capsid inside bacteriophage MS2

The icosahedrally symmetrized structure of bacteriophage MS2 as determined by cryo-electron microscopy (EM) reveals the presence of genomic RNA that attaches to coat-protein dimers. Earlier X-ray diffraction studies revealed similar interactions between the unique operator hairpin of the MS2 genomic RNA and the coat-protein dimer. This observation leads us to conclude that not only the operator, but also many other RNA sequences in the genome of MS2, are able to bind to the coat-protein dimer. A substantial number of potential coat-protein-dimer binding sites are present in the genome of MS2 that can account for the observed RNA densities in the EM map. Moreover, it appears that these stem-loop structures are able to bind in a similar fashion to the coat protein dimer as the wild-type operator hairpin. The EM map also shows additional density between the potential operator-binding sites, linking the RNA stem-loops together to form an icosahedral network around the 3 and 5-fold axes. This RNA network is bound to the inside of the MS2 capsid and probably influences both capsid stability and formation, supporting the idea that capsid formation and RNA packaging are intimately linked to each other.     

Identification of a functionally critical protein kinase C phosphorylation residue of cardiac troponin T

Cardiac Troponin T (cTnT) is one prominent substrate through which protein kinase C (PKC) exerts its effect on cardiomyocyte function. To determine the specific functional effects of the cTnT PKC-dependent phosphorylation sites (Thr197, Ser201, Thr206, and Thr287) we first mutated these residues to glutamate (E) or alanine (A). cTnT was selectively mutated to generate single, double, triple, and quadruple mutants. Bacterially expressed mutants were evaluated in detergent-treated mouse left ventricular papillary muscle fiber bundles where the endogenous troponin was replaced with a recombinant troponin complex containing either cTnT phosphorylated by PKC-alpha or a mutant cTnT. We simultaneously determined isometric tension development and actomyosin Mg-ATPase activity of the exchanged fiber bundles as a function of Ca2+ concentration. Our systematic analysis of the functional role of the multiple PKC phosphorylation sites on cTnT identified a localized region that controls maximum tension, ATPase activity, and Ca2+ sensitivity of the myofilaments. An important and novel finding of our study was that Thr206 is a functionally critical cTnT PKC phosphorylation residue. Its exclusive phosphorylation by PKC-alpha or replacement by Glu (mimicking phosphorylation) significantly decreased maximum tension, actomyosin Mg-ATPase activity, myofilament Ca2+ sensitivity, and cooperativity. On the other hand the charge modification of the other three residues together (T197/S201/T287-E) had no functional effect. Fibers bundles containing phosphorylated cTnT-wt (but not the T197/S201/T206/T287-E) exhibited a significant decrease of tension cost as compared with cTnT-wt.     

Processing of seminal plasma hCAP-18 to ALL-38 by gastricsin: a novel mechanism of generating antimicrobial peptides in vagina

The human cathelicidin, hCAP-18, is expressed both in neutrophils and in epithelial cells. hCAP-18 is processed to the antimicrobial peptide LL-37 by proteinase 3 in neutrophils. hCAP-18 is highly expressed in the epididymis with a subsequent high concentration in seminal plasma where the protein is present in its unprocessed and antimicrobially inactive form. We report here that hCAP-18 in seminal plasma is processed to generate a 38-amino acid antimicrobial peptide ALL-38 by the prostate-derived protease gastricsin when incubated at a pH corresponding to the vaginal pH. In accordance with this, seminal plasma derived hCAP-18 was found in its processed form in the vagina following sexual intercourse. The antimicrobial activity of ALL-38 against a variety of microorganisms tested is equal to that of LL-37. This enzymatic activation of a proantimicrobial substance in seminal plasma following exposure to the vaginal milieu represents a novel mechanism to prevent infection following sexual intercourse.