Dissecting the regions of virion-associated Kaposi's sarcoma-associated herpesvirus complement control protein required for complement regulation and cell binding

Complement, which bridges innate and adaptive immune responses as well as humoral and cell-mediated immunity, is antiviral. Kaposi's sarcoma-associated herpesvirus (KSHV) encodes a lytic cycle protein called KSHV complement control protein (KCP) that inhibits activation of the complement cascade. It does so by regulating C3 convertases, accelerating their decay, and acting as a cofactor for factor I degradation of C4b and C3b, two components of the C3 and C5 convertases. These complement regulatory activities require the short consensus repeat (SCR) motifs, of which KCP has four (SCRs 1 to 4). We found that in addition to KCP being expressed on the surfaces of experimentally infected endothelial cells, it is associated with the envelope of purified KSHV virions, potentially protecting them from complement-mediated immunity. Furthermore, recombinant KCP binds heparin, an analogue of the known KSHV cell attachment receptor heparan sulfate, facilitating infection. Treating virus with an anti-KCP monoclonal antibody (MAb), BSF8, inhibited KSHV infection of cells by 35%. Epitope mapping of MAb BSF8 revealed that it binds within SCR domains 1 and 2, also the region of the protein involved in heparin binding. This MAb strongly inhibited classical C3 convertase decay acceleration by KCP and cofactor activity for C4b cleavage but not C3b cleavage. Our data suggest similar topological requirements for cell binding by KSHV, heparin binding, and regulation of C4b-containing C3 convertases but not for factor I-mediated cleavage of C3b. Importantly, they suggest KCP confers at least two functions on the virion: cell binding with concomitant infection and immune evasion.     

Sex-specific influence of aging on exercising leg blood flow.

Our previous work suggests that healthy human aging is associated with sex-specific differences in leg vascular responses during large muscle mass exercise (2-legged cycling) (Proctor DN, Parker BA. Microcirculation 13: 315-327, 2006). The present study determined whether age x sex interactions in exercising leg hemodynamics persist during small muscle mass exercise that is not limited by cardiac output. Thirty-one young (20-30 yr; 15 men/16 women) and 31 older (60-79 yr; 13 men/18 women) healthy, normally active adults performed graded single-leg knee extensions to maximal exertion. Femoral artery blood velocity and diameter (Doppler ultrasound), heart rate (ECG), and beat-to-beat arterial blood pressure (mean arterial pressure, radial artery tonometry) were measured during each 3-min work rate (4.8 and 8 W/stage for women and men, respectively). The results (means +/- SE) were as follows. Despite reduced resting leg blood flow and vascular conductance, older men exhibited relatively preserved exercising leg hemodynamic responses. Older women, by contrast, exhibited attenuated hyperemic (young: 52 +/- 3 ml.min(-1).W(-1); vs. older: 40 +/- 4 ml.min(-1).W(-1); P = 0.02) and vasodilatory responses (young: 0.56 +/- 0.06 ml.min(-1).mmHg(-1).W(-1) vs. older: 0.37 +/- 0.04 ml.min(-1).mmHg(-1) W(-1); P < 0.01) to exercise compared with young women. Relative (percentage of maximal) work rate comparisons of all groups combined also revealed attenuated vasodilator responses in older women (P < 0.01 for age x sex x work rate interaction). These sex-specific age differences were not abolished by consideration of hemoglobin, quadriceps muscle, muscle recruitment, and mechanical influences on muscle perfusion. Collectively, these findings suggest that local factors contribute to the sex-specific effects of aging on exercising leg hemodynamics in healthy adults.     

Characterization of cross-bridge elasticity and kinetics of cross-bridge cycling during force development in single smooth muscle cells

Force development in smooth muscle, as in skeletal muscle, is believed to reflect recruitment of force-generating myosin cross-bridges. However, little is known about the events underlying cross-bridge recruitment as the muscle cell approaches peak isometric force and then enters a period of tension maintenance. In the present studies on single smooth muscle cells isolated from the toad (Bufo marinus) stomach muscularis, active muscle stiffness, calculated from the force response to small sinusoidal length changes (0.5% cell length, 250 Hz), was utilized to estimate the relative number of attached cross-bridges. By comparing stiffness during initial force development to stiffness during force redevelopment immediately after a quick release imposed at peak force, we propose that the instantaneous active stiffness of the cell reflects both a linearly elastic cross-bridge element having 1.5 times the compliance of the cross-bridge in frog skeletal muscle and a series elastic component having an exponential length-force relationship. At the onset of force development, the ratio of stiffness to force was 2.5 times greater than at peak isometric force. These data suggest that, upon activation, cross-bridges attach in at least two states (i.e., low-force-producing and high-force-producing) and redistribute to a steady state distribution at peak isometric force. The possibility that the cross-bridge cycling rate was modulated with time was also investigated by analyzing the time course of tension recovery to small, rapid step length changes (0.5% cell length in 2.5 ms) imposed during initial force development, at peak force, and after 15 s of tension maintenance. The rate of tension recovery slowed continuously throughout force development following activation and slowed further as force was maintained. Our results suggest that the kinetics of force production in smooth muscle may involve a redistribution of cross-bridge populations between two attached states and that the average cycling rate of these cross-bridges becomes slower with time during contraction.     

Unilateral fusion of the frontosphenoidal suture: a rare cause of synostotic frontal plagiocephaly

Unilateral coronal synostosis is the common appellation for premature, one-sided fusion of the frontoparietal suture-the most common cause of synostotic frontal plagiocephaly. However, frontal asymmetry can also result from isolated fusion across the anterior cranial base without involvement of the frontoparietal suture. This article describes three patients with localized synostosis of the frontosphenoidal suture, the medial extension of the coronal ring. Two patients were initially misdiagnosed as having unilateral coronal synostosis and the other as having deformational frontal plagiocephaly. The patients had variable frontal flattening, with depression and recession of the ipsilateral orbital rim. The nasal root was midline or slightly deviated to the contralateral side. The sagittal position of the ipsilateral malar eminence was slightly retruded in one patient and symmetric in the other two. The auricular position was symmetric in the sagittal plane for all patients. In all three patients, computed tomography examination demonstrated a patent frontoparietal suture and fusion of the frontosphenoidal suture (basilar hemicoronal ring). Two patients had involvement of contiguous sutures: one had fusion extending to the sphenoethmoidal suture and the other's involved part of the sphenozygomatic suture. The sagittal suture was midline in all patients. In summary, synostotic frontal plagiocephaly denotes a relatively broad phenotypic spectrum that includes unilateral coronal synostosis and more isolated fusions in the basilar coronal ring. The physical findings resulting from frontosphenoidal synostosis are unique, yet careful evaluation will minimize confusion with other causes of asymmetric frontal flattening. Proper diagnosis necessitates awareness of this uncommon entity and requires focused computed tomographic assessment.     

Rice Hull Ash and Silicic Acid as Adsorbents for Concentration of Bacteriocins

A model procedure has been developed for the rapid extraction of five bacteriocins (nisin, pediocin RS2, leucocin BC2, lactocin GI3, and enterocin CS1) from concentrated freeze-dried crude culture supernatants by adsorption onto acid or alkaline rice hull ash (RHA) or silicic acid (SA). Bacteriocins were adsorbed onto RHA or SA by a pH-dependent method and desorbed by decreasing the pH to 2.5 or 3.0 and heating at 90°C for 5 min. The maximum adsorption and optimal pH range for different bacteriocins were as follows: nisin, 97% at pH 7.0; lactocin GI3, 94% at pH 6.0; pediocin RS2, 97% at pH 8.0 to 9.0; leucocin BC2, 88% at pH 9.0; and enterocin CS1, 94% at pH 5.0. The desorption level of lactocin GI3 or enterocin CS1 from the surfaces of both RHA and SA was 94%, while the desorption level of pediocin RS2 and leucocin BC2 was 50% or less. Nisin was desorbed readily from SA (91%) but not from RHA (50% or less). The adsorption of bacteriocins onto RHA and SA increased with the increasing concentration of bacteriocins. Analysis of the desorbed bacteriocins after dialysis and sodium dodecyl sulfate–16% polyacrylamide gel electrophoresis showed a single band that gave a single inhibition zone when overlaid with Lactobacillus plantarum for detection of lactocin GI3, enterocin CS1, and nisin. RHA appears useful for extraction, concentration, and partial purification of the five bacteriocins.     

NMR solution structure of a dsRNA binding domain from Drosophila staufen protein reveals homology to the N-terminal domain of ribosomal protein S5.

The double-stranded RNA binding domain (dsRBD) is an approximately 65 amino acid motif that is found in a variety of proteins that interact with double-stranded (ds) RNA, such as Escherichia coli RNase III and the dsRNA-dependent kinase, PKR. Drosophila staufen protein contains five copies of this motif, and the third of these binds dsRNA in vitro. Using multinuclear/multidimensional NMR methods, we have determined that staufen dsRBD3 forms a compact protein domain with an alpha-beta-beta-beta-alpha structure in which the two alpha-helices lie on one face of a three-stranded anti-parallel beta-sheet. This structure is very similar to that of the N-terminal domain of a prokaryotic ribosomal protein S5. Furthermore, the consensus derived from all known S5p family sequences shares several conserved residues with the dsRBD consensus sequence, indicating that the two domains share a common evolutionary origin. Using in vitro mutagenesis, we have identified several surface residues which are important for the RNA binding of the dsRBD, and these all lie on the same side of the domain. Two residues that are essential for RNA binding, F32 and K50, are also conserved in the S5 protein family, suggesting that the two domains interact with RNA in a similar way.     

Protein secretion from rat submandibular acini and granular ducts: effects of exogenous VIP and substance P during parasympathetic nerve stimulation

The influences of exogenous vasoactive intestinal peptide (VIP) and substance P on the release of peroxidase from acini and true tissue kallikrein (rK1) from granular ducts of the rat submandibular gland were studied during continuous parasympathetic stimulation. Parasympathetic nerve impulses caused a moderate flow of saliva (mean +/- SD, 108+/-26 microl/g tissue/min) that had a low protein concentration (174+/-88 microg/ml). The outputs of peroxidase and rK1 were minimal (14.3+/-11.8 pmol DCF/g tissue/min and 6.5+/-3.4 nmol AFC/g tissue/min, respectively). When administered intravenously, VIP had no apparent effect on the overall flow rate, but caused a significant increase in the output of peroxidase; 450% at 1 microg/kg and a further 10-fold increase at 10 microg/kg. In contrast, substance P (1 microg/kg) evoked a marked increase in flow rate (68%), and peroxidase secretion increased only 3-fold. The output of rK1 was unaffected by either VIP or substance P. Our results support the hypothesis that acinar, but not granular duct, protein secretion is evoked by non-adrenergic, non-cholinergic peptides released from parasympathetic nerve terminals.     

Factors That Influence the Extensional Rheological Property of Saliva

The spinnbarkeit of saliva reflects the ability of saliva to adhere to surfaces within the mouth, thereby serving as a protective role and aiding in lubrication. Therefore, alterations in the extensional rheology of saliva may result in the loss in adhesiveness or the ability to bind onto surfaces. Mucin glycoproteins and their structures are known to be important factors for the extensional rheological properties of saliva. The conformation of mucin depends on factors such as pH and ionic strength. Chewing is one of the main stimuli for salivary secretion but creates significant sheer stress on the salivary film which could influence mouthfeel perceptions. The current study investigates the possible factors which affect the extensional rheological properties of saliva by comparing submandibular/sublingual saliva with different oral stimuli within the same group of subjects. Unstimulated and stimulated saliva (chew, smell and taste) salivas were collected primarily from submandibular/sublingual glands. The saliva samples were measured for Spinnbarkeit followed by the measuring mucin, total protein, total calcium and bicarbonate concentrations. The results indicated correlations between rheological properties and mucin/ion concentrations. However, chewing stimulated submandibular/sublingual saliva is shown to have significantly lower Spinnbarkeit, but factors such as mucin, protein and calcium concentrations did not account for this variation. Analysis of the concentration of bicarbonate and pH appears to suggest that it has a prominent effect on extensional rheology of saliva.     

FUM9 Is Required for C-5 Hydroxylation of Fumonisins and Complements the Meitotically Defined Fum3 Locus in Gibberella moniliformis

Deletion of the Gibberella moniliformis FUM9 gene resulted in mutants that produce only fumonisins that lack a C-5 hydroxyl group. This phenotype is identical to that of previously described mutants with defective alleles at the meiotically defined Fum3 locus. Transformation with a wild-type FUM9 gene into a Fum3-defective mutant restored wild-type fumonisin production. These results indicate that the FUM9 protein catalyzes the C-5 hydroxylation of fumonisins and that FUM9 and the Fum3 locus are the same gene.     

Comparative trial of treatment with Prioderm lotion and Kwellada shampoo in children with head lice.

The safety and efficacy of treatment with a new pediculicide lotion, Prioderm (0.5% malathion in isopropanol), were compared with those of treatment with Kwellada (1% lindane) shampoo in a randomized trial of children with head lice. The children''s scalps were examined for live lice immediately, 7 days and 4 to 6 weeks following treatment. To determine the in-vitro ovicidal effect of treatment, samples of hair with nits were removed before and immediately following treatment; the subsequent rates of hatching were compared. No live lice were present immediately following either treatment. At 7 days after treatment 2 of the 29 children treated with Prioderm lotion and 4 of the 33 children treated with Kwellada shampoo were infested with live lice, whereas at 4 to 6 weeks after treatment 5 and 3 children respectively were infested. The initial effectiveness of treatment was 93% in the children treated with Prioderm lotion and 88% in those treated with Kwellada shampoo; however, a large difference in efficacy could have been missed owing to the small number of children in the study. Both preparations demonstrated in-vitro ovicidal activity, but neither totally abolished post-treatment hatching. No side effects were reported from either preparation. Because ovicidal activity was incomplete two treatments with the same pediculicides should be given about 7 days apart.