Karyotype variation in the South American aphid genus Neuquenaphis (Hemiptera,Aphididae, Neuquenaphidinae)

The endemic South American aphid genus Neuquenaphis (Hemiptera, Aphididae, Neuquenaphidinae) forms an important component of the phytophagous insect fauna associated with southern beeches, Nothofagus (Nothofagaceae), but has not previously been studied cytologically. As part of ongoing studies of the taxonomy, evolution and host relationships of this genus, the karyotypes of 12 species are described and illustrated. Species are mostly distinguishable by differences in number and/or relative lengths of chromosomes, with 2n (female) numbers ranging from 6 to 16. The taxonomic and evolutionary significance of the karyotype variation in this group are discussed.     

Antibody-mediated control of persistent gamma-herpesvirus infection

The human gamma-herpesviruses, EBV and Kaposi's sarcoma-associated herpesvirus, establish life-long latency and can reactivate in immunocompromised individuals. T cells play an important role in controlling persistent EBV infection, whereas a role for humoral immunity is less clear. The murine gamma-herpesvirus-68 has biological and structural similarities to the human gamma-herpesviruses, and provides an important in vivo experimental model for dissecting mechanisms of immune control. In the current studies, CD28(-/-) mice were used to address the role of Abs in control of persistent murine gamma-herpesvirus-68 infection. Lytic infection was controlled in the lungs of CD28(-/-) mice, and latency was maintained in B cells at normal frequencies. Although class-switched virus-specific Abs were initially generated in the absence of germinal centers, titers and viral neutralizing activity rapidly waned. T cell depletion in CD28(-/-) mice with compromised Ab responses, but not in control mice with intact Ab responses, resulted in significant recrudescence from latency, both in the spleen and the lung. Recrudescence could be prevented by passive transfer of immune serum. These data directly demonstrate an important contribution of humoral immunity to control of gamma-herpesvirus latency, and have significant implications for clinical intervention.     

Bacterial superantigen exposure after resolution of influenza virus infection perturbs the virus-specific memory CD8(+)-T-cell repertoire

Heterologous viral infections have been shown to impact the preexisting memory CD8(+)-T-cell repertoire. Bacterial superantigens are products of common human pathogenic bacteria, including staphylococci and streptococci, that are potent T-cell-stimulatory molecules. In this report, we show that exposure to staphylococcal enterotoxin B, a bacterial superantigen, causes a selective functional deletion of cross-reactive influenza virus-specific CD8(+) memory T cells. This perturbation of the memory repertoire can have a significant impact on viral clearance after secondary challenge.     

A new in vitro model to evaluate differential responses of endothelial cells to simulated arterial shear stress waveforms

In the circulation, flow-responsive endothelial cells (ECs) lining the lumen of blood vessels are continuously exposed to complex hemodynamic forces. To increase our understanding of EC response to these dynamic shearing forces, a novel in vitro flow model was developed to simulate pulsatile shear stress waveforms encountered by the endothelium in the arterial circulation. A modified waveform modeled after flow patterns in the human abdominal aorta was used to evaluate the biological responsiveness of human umbilical vein ECs to this new type of stimulus. Arterial pulsatile flow for 24 hours was compared to an equivalent time-average steady laminar shear stress, using no flow (static) culture conditions as a baseline. While both flow stimuli induced comparable changes in cell shape and alignment, distinct patterns of responses were observed in the distribution of actin stress fibers and vinculin-associated adhesion complexes, intrinsic migratory characteristics, and the expression of eNOS mRNA and protein. These results thus reveal a unique responsiveness of ECs to an arterial waveform and begin to elucidate the complex sensing capabilities of the endothelium to the dynamic characteristics of flows throughout the human vascular tree.     

Structural and biochemical characterization of a fluorogenic rhodamine-labeled malarial protease substrate

Activation of the proenzyme form of the malarial protease PfSUB-1 involves the autocatalytic cleavage of an Asp-Asn bond within the internal sequence motif (215)LVSADNIDIS(224). A synthetic decapeptide based on this sequence but with the N- and C-terminal residues replaced by cysteines (Ac-CVSADNIDIC-OH) was labeled with 5- or 6-isomers of iodoacetamidotetramethylrhodamine (IATR). The doubly labeled peptides have low fluorescence because of ground-state, noncovalent dimerization of the rhodamines. Cleavage of either peptide by recombinant PfSUB-1 results in dissociation of the rhodamine dimers, which abolishes the self-quenching and consequently leads to an approximately 30-fold increase in the fluorescence. This spectroscopic signal provides a continuous assay of proteolysis, enabling quantitative kinetic measurements to be made, and has also enabled the development of a fluorescence-based assay suitable for use in high-throughput screens for inhibitors of PfSUB-1. The structure of the rhodamine dimer in the 6-IATR-labeled peptide was shown by NMR to be a face-to-face stacking of the xanthene rings. Time-resolved fluorescence measurements suggest that the doubly labeled peptides exist in an equilibrium consisting of rhodamines involved in dimers (closed forms) and rhodamines not involved in dimers (open forms). These data also indicate that the rhodamine dimers fluoresce and that the associated lifetimes are subnanosecond.     

Lymphocyte activation gene-3 (CD223) regulates the size of the expanding T cell population following antigen activation in vivo

Lymphocyte activation gene-3 (LAG-3) is a CD4-related, activation-induced cell surface molecule that binds to MHC class II with high affinity. In this study, we used four experimental systems to reevaluate previous suggestions that LAG-3(-/-) mice had no T cell defect. First, LAG-3(-/-) T cells exhibited a delay in cell cycle arrest following in vivo stimulation with the superantigen staphylococcal enterotoxin B resulting in increased T cell expansion and splenomegaly. Second, increased T cell expansion was also observed in adoptive recipients of LAG-3(-/-) OT-II TCR transgenic T cells following in vivo Ag stimulation. Third, infection of LAG-3(-/-) mice with Sendai virus resulted in increased numbers of memory CD4(+) and CD8(+) T cells. Fourth, CD4(+) T cells exhibited a delayed expansion in LAG-3(-/-) mice infected with murine gammaherpesvirus. In summary, these data suggest that LAG-3 negatively regulates T cell expansion and controls the size of the memory T cell pool.     

L-Carnitine and acetyl-L-carnitine in the treatment of complications associated with HIV infection and antiretroviral therapy

L-Carnitine (LC) and acetyl-L-carnitine (ALC) play major roles in cell energy and lipid metabolism. Supplementation with these nutrients, which are highly popular in USA, has been associated with favorable effects, including anti-oxidant action, neuro- and cardioprotection, immunomodulation, and cognitive enhancement. Patients with HIV infection and undergoing highly active antiretroviral therapy (HAART) often develop complications, such as polyneuropathy, skeletal myopathy, dyslipidemia and lipodystrophy, which have been linked to mitochondrial dysfunction. Moreover, these patients are often LC-deficient. Thus, they may benefit from LC and ALC supplementation. Indeed, oral, i.v., or i.m. administration of large doses of LC and/or ALC to HIV positive subjects untreated/treated with HAART was shown to: (1) increase the number of CD4 cells and reduce lymphocyte apoptosis; (2) improve symptoms of polyneuropathy; (3) prevent cardiovascular damage from wasting and diarrhea syndromes; (4) decrease serum levels of triglycerides and TNFalpha. No significant toxicities were associated with LC and ALC treatment. Although promising, most of these findings derive from small uncontrolled clinical trials. Further research is warranted to prove the efficacy and safety of LC and ALC supplementation in patients with complications of HIV infection and HAART.     

Subtilisin-like proteases of the malaria parasite

Proteases play critical roles in the life cycle of the malaria parasite, Plasmodium spp. Within the asexual erythrocytic cycle, responsible for the clinical manifestations of malaria, substantial interest has focused on the role of parasite serine proteases as a result of indications that they are involved in red blood cell invasion. Over the past 6 years, three Plasmodium genes encoding serine proteases of the subtilisin-like clan, or subtilases, have been identified. All are expressed in the asexual blood stages and, in at least two cases, the gene products localize to secretory organelles of the invasive merozoite. They may have potential as novel drug targets. Here, we review progress in our understanding of the maturation, specificity, structure and function of these Plasmodium subtilases.     

Proteolytic processing and primary structure of Plasmodium falciparum apical membrane antigen-1

Plasmodium falciparum apical membrane antigen-1 (PfAMA-1) is a malaria merozoite integral membrane protein that plays an essential but poorly understood role in invasion of host erythrocytes. The PfAMA-1 ectodomain comprises three disulfide-constrained domains, the first of which (domain I) is preceded by an N-terminal prosequence. PfAMA-1 is initially routed to secretory organelles at the apical end of the merozoite, where the 83-kDa precursor (PfAMA-1(83)) is converted to a 66-kDa form (PfAMA-1(66)). At about the time of erythrocyte invasion, PfAMA-1(66) selectively translocates onto the merozoite surface. Here we use direct microsequencing and mass spectrometric peptide mass fingerprinting to characterize in detail the primary structure and proteolytic processing of PfAMA-1. We have determined the site at which processing takes place to convert PfAMA-1(83) to PfAMA-1(66) and have shown that both species possess a completely intact and unmodified transmembrane and cytoplasmic domain. Following relocation to the merozoite surface, PfAMA-1(66) is further proteolytically cleaved at one of two alternative sites, either between domains II and III, or at a membrane-proximal site following domain III. As a result, the bulk of the ectodomain is shed from the parasite surface in the form of two soluble fragments of 44 and 48 kDa. PfAMA-1 is not detectably modified by the addition of N-linked oligosaccharides.