CD4+-T-Cell and CD20+-B-Cell Changes Predict Rapid Disease Progression after Simian-Human Immunodeficiency Virus Infection in Macaques

Simian-human immunodeficiency virus 89.6PD (SHIV_89.6PD) was pathogenic after intrarectal inoculation of rhesus macaques. Infection was achieved with a minimum of 2,500 tissue culture infectious doses of cell-free virus stock, and there was no evidence for transient viremia in animals receiving subinfectious doses by the intrarectal route. Some animals experienced rapid progression of disease characterized by loss of greater than 90% of circulating CD4⁺ T cells, sustained decreases in CD20⁺ B cells, failure to elicit virus-binding antibodies in plasma, and high levels of antigenemia. Slower-progressing animals had moderate but varying losses of CD4⁺ T cells; showed increases in circulating CD20⁺ B cells; mounted vigorous responses to antibodies in plasma, including neutralizing antibodies; and had low or undetectable levels of antigenemia. Rapid progression led to death within 30 weeks after intrarectal inoculation. Plasma antigenemia at 2 weeks after inoculation (P ≤ 0.002), B- and T-cell losses (P ≤ 0.013), and failure to seroconvert (P ≤ 0.005) were correlated statistically with rapid progression. Correlations were evident by 2 to 4 weeks after intrarectal SHIV inoculation, indicating that early events in the host-pathogen interaction determined the clinical outcome.     

Stress symposium in Johannesburg: plants versus humans

On 18 February 1998, a ‘Stress symposium’ was held at the Rand Afrikaans University (RAU) in Johannesburg, South Africa. The meeting brought together people from both the plant and the human oxidative stress field, which was exemplified by a talk entitled ‘Heat shock proteins in host-pathogen interactions: plants versus humans’. There were moments when it appeared as if the main difference between plants and humans was, as sung by Julos Beaucarne, that ‘the human plant is the only one to be able to water itself…’     

Neurite Fasciculation Mediated by Complexes of Axonin-1 and Ng Cell Adhesion Molecule

Neural cell adhesion molecules composed of immunoglobulin and fibronectin type III-like domains have been implicated in cell adhesion, neurite outgrowth, and fasciculation. Axonin-1 and Ng cell adhesion molecule (NgCAM), two molecules with predominantly axonal expression exhibit homophilic interactions across the extracellular space (axonin- 1/axonin-1 and NgCAM/NgCAM) and a heterophilic interaction (axonin-1–NgCAM) that occurs exclusively in the plane of the same membrane (cis-interaction). Using domain deletion mutants we localized the NgCAM homophilic binding in the Ig domains 1-4 whereas heterophilic binding to axonin-1 was localized in the Ig domains 2-4 and the third FnIII domain. The NgCAM–NgCAM interaction could be established simultaneously with the axonin-1–NgCAM interaction. In contrast, the axonin-1–NgCAM interaction excluded axonin-1/axonin-1 binding. These results and the examination of the coclustering of axonin-1 and NgCAM at cell contacts, suggest that intercellular contact is mediated by a symmetric axonin-1₂/NgCAM₂ tetramer, in which homophilic NgCAM binding across the extracellular space occurs simultaneously with a cis-heterophilic interaction of axonin-1 and NgCAM. The enhanced neurite fasciculation after overexpression of NgCAM by adenoviral vectors indicates that NgCAM is the limiting component for the formation of the axonin-1₂/NgCAM₂ complexes and, thus, neurite fasciculation in DRG neurons.     

Lysine 5 and phenylalanine 9 of the factor IX omega-loop interact with phosphatidylserine in a membrane-mimetic environment

The binding of factor IX to cell membranes requires a structured N-terminal omega-loop conformation that exposes hydrophobic residues for a highly regulated interaction with a phospholipid. We hypothesized that a peptide comprised of amino acids Gly4-Gln11 of factor IX (fIX(G4)(-)(Q11)) and constrained by an engineered disulfide bond would assume the native factor IX omega-loop conformation in the absence of Ca(2+). The small size and freedom from aggregation-inducing calcium interactions would make fIX(G4)(-)(Q11) suitable for structural studies for eliciting details about phospholipid interactions. fIX(G4)(-)(Q11) competes with factor IXa for binding sites on phosphatidylserine-containing membranes with a K(i) of 11 microM and inhibits the activation of factor X by the factor VIIIa-IXa complex with a K(i) of 285 microM. The NMR structure of fIX(G4)(-)(Q11) reveals an omega-loop backbone fold and side chain orientation similar to those found in the calcium-bound factor IX Gla domain, FIX(1-47)-Ca(2+). Dicaproylphosphatidylserine (C(6)PS) induces HN, Halpha backbone, and Hbeta chemical shift perturbations at residues Lys5, Leu6, Phe9, and Val10 of fIX(G4)(-)(Q11), while selectively protecting the NHzeta side chain resonance of Lys5 from solvent exchange. NOEs between the aromatic ring protons of Phe9 and specific acyl chain protons of C(6)PS indicate that these phosphatidylserine protons reside 3-6 A from Phe9. Stabilization of the phosphoserine headgroup and glycerol backbone of C(6)PS identifies that phosphatidylserine is in a protected environment that is spatially juxtaposed with fIX(G4)(-)(Q11). Together, these data demonstrate that Lys5, Leu6, Phe9, and Val10 preferentially interact with C(6)PS and allow us to correlate known hemophilia B mutations of factor IX at Lys5 or Phe9 with impaired phosphatidylserine interaction.     

Molecular Basis of Calpain Cleavage and Inactivation of the Sodium-Calcium Exchanger 1 in Heart Failure

Cardiac sodium (Na⁺)-calcium (Ca²⁺) exchanger 1 (NCX1) is central to the maintenance of normal Ca²⁺ homeostasis and contraction. Studies indicate that the Ca²⁺-activated protease calpain cleaves NCX1. We hypothesized that calpain is an important regulator of NCX1 in response to pressure overload and aimed to identify molecular mechanisms and functional consequences of calpain binding and cleavage of NCX1 in the heart. NCX1 full-length protein and a 75-kDa NCX1 fragment along with calpain were up-regulated in aortic stenosis patients and rats with heart failure. Patients with coronary artery disease and sham-operated rats were used as controls. Calpain co-localized, co-fractionated, and co-immunoprecipitated with NCX1 in rat cardiomyocytes and left ventricle lysate. Immunoprecipitations, pull-down experiments, and extensive use of peptide arrays indicated that calpain domain III anchored to the first Ca²⁺ binding domain in NCX1, whereas the calpain catalytic region bound to the catenin-like domain in NCX1. The use of bioinformatics, mutational analyses, a substrate competitor peptide, and a specific NCX1-Met³⁶⁹ antibody identified a novel calpain cleavage site at Met³⁶⁹. Engineering NCX1-Met³⁶⁹ into a tobacco etch virus protease cleavage site revealed that specific cleavage at Met³⁶⁹ inhibited NCX1 activity (both forward and reverse mode). Finally, a short peptide fragment containing the NCX1-Met³⁶⁹ cleavage site was modeled into the narrow active cleft of human calpain. Inhibition of NCX1 activity, such as we have observed here following calpain-induced NCX1 cleavage, might be beneficial in pathophysiological conditions where increased NCX1 activity contributes to cardiac dysfunction.     

Functional and morphological differentiation of nonpigmented ciliary body epithelial cells grown on collagen rafts

Summary We have examined the effect of alteration in cell shape on promoting differentiated morphology and physiology in cultured nonpigmented epithelial cells from the ciliary body. We have grown pure populations of nonpigmented cells on collagen gels released from the culture dish to create collagen rafts. Shortly after the gels were detached, the cells shrank in diameter and increased in height while they contracted the gel. Concurrently, the actin cytoskeleton reorganized to the cell cortex as found in vivo. After this differentiated morphology developed, large changes in intracellular Ca²⁺ could be elicited by simultaneous activation of acetylcholine and epinephrine or acetylcholine and somatostatin receptors as seen in intact tissue. Explant cultures of isolated nonpigmented cell layers maintained their actin distribution and also showed synergistic Ca²⁺ increases. Spread cells, grown on rigid substrates, had a disorganized cytoskeleton and rarely showed synergism. These data suggest that the mechanism underlying synergistic Ca²⁺ responses in the ciliary body is functional in nonpigmented cells grown on collagen rafts. In addition, this pathway appears to be sensitive to the disposition of the cell’s cytoarchitecture.     

Immunocytochemical demonstration of neuropeptides and serotonin in the tapeworm Diphyllobothrium dendriticum

Summary The present immunocytochemical study concerns the distribution of four neuropeptides, FMRF-amide, vasotocin, leu-enkephalin and neurotensin, and of the bioamine serotonin in the plerocercoid larva of Diphyllobothrium dendriticum. Anti-FMRF-amide and vasotocin-reactivity occurs in perikarya and nerve fibres in the CNS and PNS of this worm. The peptide-containing fibres surround and seem to innervate the musculature and to terminate beneath the basal lamina of the tegument at the inner surface of the bothridia, suggesting a neurotransmitter function. Antileu-enkephalin reaction occurs in perikarya and fibres in the main nerve cords and in the PNS. Anti-neurotensin reactive fibres were observed in the neuropile of the nerve cords. Serotonin immunoreactivity was found in neurons in the ganglionic commissure of the brain and along the main nerve cords. This study is the first immunocytochemical identification of neuropeptides and serotonin in a parasitic flatworm and the information gained may be of importance for the development of new antihelminthics.