S100A16, a novel calcium-binding protein of the EF-hand superfamily

S100A16 protein is a new and unique member of the EF-hand Ca(2+)-binding proteins. S100 proteins are cell- and tissue-specific and are involved in many intra- and extracellular processes through interacting with specific target proteins. In the central nervous system S100 proteins are implicated in cell proliferation, differentiation, migration, and apoptosis as well as in cognition. S100 proteins became of major interest because of their close association with brain pathologies, for example depression or Alzheimer's disease. Here we report for the first time the purification and biochemical characterization of human and mouse recombinant S100A16 proteins. Flow dialysis revealed that both homodimeric S100A16 proteins bind two Ca(2+) ions with the C-terminal EF-hand of each subunit, the human protein exhibiting a 2-fold higher affinity. Trp fluorescence variations indicate conformational changes in the orthologous proteins upon Ca(2+) binding, whereas formation of a hydrophobic patch, implicated in target protein recognition, only occurs in the human S100A16 protein. In situ hybridization analysis and immunohistochemistry revealed a widespread distribution in the mouse brain. Furthermore, S100A16 expression was found to be astrocyte-specific. Finally, we investigated S100A16 intracellular localization in human glioblastoma cells. The protein was found to accumulate within nucleoli and to translocate to the cytoplasm in response to Ca(2+) stimulation.     

Three-dimensional in vivo motion of adult hind foot bones

Knowledge of hind foot bone motion is important for understanding gait as well as various foot pathologies, but the three-dimensional (3D) motion of these bones remains incompletely understood. The purpose of this study was to quantify the motion of the talus, calcaneus, navicular, and cuboid in normal adult feet during open chain quasi-static uniplanar plantar flexion motion. Magnetic resonance images of the right feet of six normal young adult males were taken from which 3D virtual models were made of each hind foot bone. The 3D motion of these models was analyzed. Each hind foot bone rotated in the same plane about half as much as the foot (mean 0.54 degrees of bone rotation per degree of foot motion, range 0.40-0.73 degrees per degree of foot motion as measured relative to the fixed tibia). Talar motion was primarily uniaxial, but the calcaneus, navicular, and cuboid bones exhibited biplanar (sometimes triplanar) translation in addition to biaxial rotation. Net translational motions of these bones averaged 0.39 mm of bone translation per degree of foot motion (range 0.06-0.62 mm per degree of foot motion). These data reflect the functional anatomy of the foot, extend the findings of prior studies, provide a standard for comparison to patients with congenital or acquired foot deformities, and establish an objective reference for quantitatively assessing the efficacy of various hind foot therapies.     

Morphometric cranial identity of prehistoric Malawians in the light of sub-Saharan African diversity

Little has been described of the Holocene populations of South‐Central Africa, despite the region demonstrating major subsistence shifts relating to dispersals of agriculturalists at least 2,000 years ago. Seven sites with associated human skeletal remains were selected. Hora, Chencherere, Fingura, and Mtuzi represent the Middle Holocene (2,000–5,000 years ago), and Phwadze, Mtemankhokwe, and Nkudzi Bay represent the Late Holocene and the arrival of agriculturalists between 500–2,000 years ago. Focusing on the identity of Hora and Chencherere specimens, two questions were addressed: are the various Holocene Malawians similar to each other, or do they suggest morphological change over time? What modern populations are closest to the prehistoric specimens? The archaeological sample was compared to modern sub‐Saharan Africans from four regions, plus a historic Khoi‐San foraging group. Factor analyses were performed in order to identify complex patterns of variation in metric traits of the skull. According to the results, prehistoric Malawians showed only slight differences between the Late and Middle Holocene, suggesting a population change without any major discontinuity. Later Stone Age skulls did not exclusively show similarities with the Khoi‐San, as they frequently fit well within the variation of modern Bantu‐speaking groups, especially West‐Central Africa. Therefore, we reject the hypothesis that Middle Holocene South‐Central Africans have an exclusively Khoi‐San ancestry, and support an alternative hypothesis that both Middle and Late Holocene groups share a common biological heritage originating in West‐Central Africa in earlier times. Am J Phys Anthropol, 2006. © 2005 Wiley‐Liss, Inc.     

Structure-guided design of peptide-based tryptase inhibitors

Improved peptide-based inhibitors of human beta tryptase were discovered using information gleaned from tripeptide library screening and structure-guided design methods, including fragment screening. Our efforts sought to improve this class of inhibitors by replacing the traditional Lys or Arg P1 element. The optimized compounds display low nanomolar potency against the mast cell target and several hundred-fold selectivity with respect to serine protease off targets. Thus, replacement of Lys/Arg at P1 in a peptide-like scaffold does not need to be accompanied by a loss in target affinity.     

The orientation effect of gramicidin A on bicelles and Eu3+ -doped bicelles as studied by solid-state NMR and FT-IR spectroscopy

We have explored the effect of gramicidin A (gA) on bicelle (Bic) orientation in the absence and presence of Eu(3+) by (31)P and (2)H NMR at different DMPC/gA ratios. FT-IR spectroscopy was used to assess the lipid chain ordering and verify the transmembrane peptide conformation. Our results show a time-dependent flipping of the bilayer normal alignment at high temperatures and high proportion of gA. The results are explained by both the diamagnetic susceptibility anisotropy of the beta(6.3) helical peptides and viscosity of the lipid mixture. The concentration effect of gramicidin on Bic/Eu(3+) is compared to that on Eu(3+)-doped DMPC liposomes. The Bic/Eu(3+) system is no longer oriented in the presence of gA and adopts a vesicular morphology while the peptide incorporation induces the formation of ellipsoidal DMPC/Eu(3+) assemblies aligned with their normal parallel to the magnetic field. The difference is explained in terms of lipid chain disorder and size of the bilayers.     

The effect of differential survivorship on the stability of reproductive queueing

Queues, in which individuals inherit resources in a predictable, temporally stable order, are widespread in animal social groups. We develop an analytic model to explore the effect of differential survivorship on the stability of a reproductive queue. We show that unless fighting for dominance is potentially fatal, future direct benefits are not alone sufficient to stabilize a queue of non-relatives under constant (age-independent) mortality rates, regardless of whether a dominant becomes an isolate or remains a dominant on the death of the first subordinate. In the absence of fatal fighting, stabilization of such a queue by future direct benefits alone requires either the dominant or the subordinate to have age-dependent mortality rates. Even when the queue is stabilized by present direct reproduction, however, the shape of the lifespan distribution can make a significant difference to the size of the required incentive. In contrast to non-relatives, queues of relatives can be stable without age-dependent mortality, so long as relatedness exceeds a critical value; however, age-dependent mortality can lower this critical value.     

Partial delipidation improves the T-cell antigenicity of hepatitis B virus surface antigen

Hepatitis B virus surface antigen (HBsAg) is a complex macromolecular particle composed of glycoproteins and lipids. The latter, representing 25% of the particle mass, are of host origin and determine the solubility, stability, and, indirectly, B-cell immunogenicity of HBsAg. HBsAg is a T-cell-dependent immunogen that does not elicit a detectable humoral immune response in 5% of HBsAg vaccine recipients and in most subjects suffering from chronic hepatitis B. We investigated the influence of the lipid content on the antigenicity of the particle. Lipids were partially removed from HBsAg by treatment with beta-D-octyl glucoside and density centrifugation. Sham treatment consisted of density centrifugation of HBsAg only. We compared the in vitro proliferative responses of established T-cell lines and nonfractionated peripheral blood mononuclear cells (PBMC) from HBsAg vaccinees and chronic HBV patients when stimulated with partially delipidated HBsAg, untreated HBsAg, or sham-treated HBsAg. In all experiments, delipidated HBsAg turned out to be 10 to 100 times more antigenic than its untreated or sham-treated counterpart. Remarkably, PBMC from vaccine nonresponders or chronic HBV patients displayed a proliferative response towards delipidated HBsAg, whereas native HBsAg never induced a response. A series of control experiments demonstrated that this enhancement of T-cell antigenicity was HBsAg specific and directly linked to lipid extraction. Nonspecific adjuvant effects of any kind could be ruled out. In vivo evaluation in mice demonstrated that delipidated particles lose most of their B-cell antigenicity. However, when native and delipidated particles were mixed, these mixtures induced equal or slightly superior anti-HBs responses to those induced by the same quantity of native HBsAg alone. In conclusion, our data show that partial delipidation of HBsAg strikingly increases the T-cell antigenicity of this unique viral antigen.     

Rotavirus anti-VP6 secretory immunoglobulin A contributes to protection via intracellular neutralization but not via immune exclusion

Immunoglobulin A (IgA) monoclonal antibodies (MAbs) directed at the conserved inner core protein VP6 of rotavirus, such as the IgA7D9 MAb, provide protective immunity in adult and suckling mice when delivered systemically. While these antibodies do not have traditional in vitro neutralizing activity, they could mediate their antiviral activity either by interfering with the viral replication cycle along the IgA secretory pathway or by acting at mucosal surfaces as secretory IgA and excluding virus from target enterocytes. We sought to determine the critical step at which antirotaviral activity was initiated by the IgA7D9 MAb. The IgA7D9 MAb appeared to directly interact with purified triple-layer viral particles, as shown by immunoprecipitation and immunoblotting. However, protection was not conferred by passively feeding mice with the secretory IgA7D9 MAb. This indicates that the secretory IgA7D9 MAb does not confer protection by supplying immune exclusion activity in vivo. We next evaluated the capacity of polymeric IgA7D9 MAb to neutralize rotavirus intracellularly during transcytosis. We found that when polymeric IgA7D9 MAb was applied to the basolateral pole of polarized Caco-2 intestinal cells, it significantly reduced viral replication and prevented the loss of barrier function induced by apical exposure of the cell monolayer to rotavirus, supporting the conclusion that the antibody carries out its antiviral activity intracellularly. These findings identify a mechanism whereby the well-conserved immunodominant VP6 protein can function as a target for heterotypic antibodies and protective immunity.     

Rectal immunization with rotavirus virus-like particles induces systemic and mucosal humoral immune responses and protects mice against rotavirus infection

To evaluate whether the rectal route of immunization may be used to provide appropriate protection against enteric pathogens such as rotaviruses (RV), we studied the antibody response and the protection induced by rectal immunization of mice with RV virus-like particles (VLP). For this purpose, 6-week-old BALBc mice were rectally immunized twice with RV 8-2/6/7-VLP derived from the bovine RV RF81 strain either alone or combined with various adjuvants including four toxins [cholera toxin (CT) and three attenuated Escherichia coli-derived heat-labile toxins (LTs), LT(R192G), LT(R72), and LT(K63)] and two Toll-like receptor-targeting adjuvants (CpG and resiquimod). Six weeks after the second immunization, mice were challenged with murine RV strain ECw. RV VLP administered alone were not immunogenic and did not protect mice against RV challenge. By contrast, RV VLP combined with any of the toxin adjuvants were immunogenic (mice developed significant titers of anti-RV immunoglobulin A [IgA] in both serum and feces and of anti-RV IgG in serum) and either efficiently induced complete protection of the mice (no detectable fecal virus shedding) or, for LT(K63), reduced the amount of fecal virus shedding after RV challenge. When combined with RV VLP, CpG and resiquimod failed to achieve protection, although CpG efficiently induced an antibody response to RV. These results support the consideration of the rectal route for the development of new immunization strategies against RV infection. Rectal delivery of a VLP-based vaccine might allow the use of adjuvants less toxic than, but as efficient as, CT.