C. elegans pro-1 activity is required for soma/germline interactions that influence proliferation and differentiation in the germ line

Strict spatial and temporal regulation of proliferation and differentiation is essential for proper germline development and often involves soma/germline interactions. In C. elegans, a particularly striking outcome of defective regulation of the proliferation/differentiation pattern is the Pro phenotype in which an ectopic mass of proliferating germ cells occupies the proximal adult germ line, a region normally occupied by gametes. We describe a reduction-of-function mutation in the gene pro-1 that causes a highly penetrant Pro phenotype. The pro-1 mutant Pro phenotype stems from defects in the time and position of the first meiotic entry during early germline development. pro-1(RNAi) produces a loss of somatic gonad structures and concomitant reduction in germline proliferation and gametogenesis. pro-1 encodes a member of a highly conserved subfamily of WD-repeat proteins. pro-1(+) is required in the sheath/spermatheca lineage of the somatic gonad in its role in the proper establishment of the proliferation/differentiation pattern in the germline. Our results provide a handle for further analysis of this soma-to-germline interaction.     

Genetic and stochastic influences on the interaction of human immunodeficiency virus type 1 and cytotoxic T lymphocytes in identical twins

Human immunodeficiency virus type 1 (HIV-1) evolves in vivo under selective pressure from CD8+ T-lymphocyte (CTL) responses, which are in turn determined by host and viral genetic factors, such as restricting major histocompatibility complex molecules and the available viral epitope sequences. However, CTL are derived stochastically through the random gene rearrangements to produce T-cell receptors (TCR), and the relative impact of genetic versus stochastic processes on CTL targeting of HIV and immune-driven viral evolution is unclear. Here we evaluate identical twins infected with HIV-1 as neonates from a common blood transfusion, with subsequently similar environmental exposures, thereby allowing controlled comparisons of CTL targeting and viral evolution. Seventeen years after infection, their CTL targeting of HIV-1 was remarkably similar. In contrast, their overall TCR profiles were highly dissimilar, and a dominant epitope was recognized by distinctly different TCR in each twin. Furthermore, their viral epitopes had diverged, and there was ongoing viral phylogenetic divergence between the twins between 12 and 17 years after infection. These results indicate that while CTL targeting is predominately genetically determined, stochastic influences render the interaction of HIV-1 and host immunity, and therefore viral escape and CTL efficacy, unpredictable.     

Molecular organization in striated domains induced by transmembrane alpha-helical peptides in dipalmitoyl phosphatidylcholine bilayers

Transmembrane (TM) alpha-helical peptides with neutral flanking residues such as tryptophan form highly ordered striated domains when incorporated in gel-state 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) bilayers and inspected by atomic force microscopy (AFM) (1). In this study, we analyze the molecular organization of these striated domains using AFM, photo-cross-linking, fluorescence spectroscopy, nuclear magnetic resonance (NMR), and X-ray diffraction techniques on different functionalized TM peptides. The results demonstrate that the striated domains consist of linear arrays of single TM peptides with a dominantly antiparallel organization in which the peptides interact with each other and with lipids. The peptide arrays are regularly spaced by +/-8.5 nm and are separated by somewhat perturbed gel-state lipids with hexagonally organized acyl chains, which have lost their tilt. This system provides an example of how domains of peptides and lipids can be formed in membranes as a result of a combination of specific peptide-peptide and peptide-lipid interactions.     

Tilt angles of transmembrane model peptides in oriented and non-oriented lipid bilayers as determined by 2H solid-state NMR

Solid-state NMR methods employing (2)H NMR and geometric analysis of labeled alanines (GALA) were used to study the structure and orientation of the transmembrane alpha-helical peptide acetyl-GWW(LA)(8)LWWA-amide (WALP23) in phosphatidylcholine (PC) bilayers of varying thickness. In all lipids the peptide was found to adopt a transmembrane alpha-helical conformation. A small tilt angle of 4.5 degrees was observed in di-18:1-PC, which has a hydrophobic bilayer thickness that approximately matches the hydrophobic length of the peptide. This tilt angle increased slightly but systematically with increasing positive mismatch to 8.2 degrees in di-C12:0-PC, the shortest lipid used. This small increase in tilt angle is insufficient to significantly change the effective hydrophobic length of the peptide and thereby to compensate for the increasing hydrophobic mismatch, suggesting that tilt of these peptides in a lipid bilayer is energetically unfavorable. The tilt and also the orientation around the peptide axis were found to be very similar to the values previously reported for a shorter WALP19 peptide (GWW(LA)(6)LWWA). As also observed in this previous study, the peptide rotates rapidly around the bilayer normal, but not around its helix axis. Here we show that these properties allow application of the GALA method not only to macroscopically aligned samples but also to randomly oriented samples, which has important practical advantages. A minimum of four labeled alanine residues in the hydrophobic transmembrane sequence was found to be required to obtain accurate tilt values using the GALA method.     

Increased CD8+ cytotoxic T cell responses to myelin basic protein in multiple sclerosis

Autoreactive T cells of CD4 and CD8 subsets recognizing myelin basic protein (MBP), a candidate myelin autoantigen, are thought to contribute to and play distinct roles in the pathogenesis of multiple sclerosis (MS). In this study we identified four MBP-derived peptides that had high binding affinity to HLA-A2 and HLA-A24 and characterized the CD8(+) T cell responses and their functional properties in patients with MS. There were significantly increased CD8(+) T cell responses to 9-mer MBP peptides, in particular MBP(111-119) and MBP(87-95) peptides that had high binding affinity to HLA-A2, in patients with MS compared with healthy individuals. The resulting CD8(+) T cell lines were of the Th1 phenotype, producing TNF-alpha and IFN-gamma and belonged to a CD45RA(-)/CD45RO(+) memory T cell subset. Further characterization indicated that the CD8(+) T cell lines obtained were stained with MHC class I tetramer (HLA-A2/MBP(111-119)) and exhibited specific cytotoxicity toward autologous target cells pulsed with MBP-derived peptides in the context of MHC class I molecules. These cytotoxic CD8(+) T cell lines derived from MS patients recognized endogenously processed MBP and lysed COS cells transfected with genes encoding MBP and HLA-A2. These findings support the potential role of CD8(+) CTLs recognizing MBP in the injury of oligodendrocytes expressing both MHC class I molecules and MBP.     

Functional reconstitution of thymopoiesis after human immunodeficiency virus infection

We have utilized combination antiretroviral therapy following human immunodeficiency virus type 1-induced human CD4(+) thymocyte depletion in the SCID-hu mouse to examine the immune competence of reconstituting thymocytes which appear following administration of combination therapy. These cells express a normal distribution of T-cell receptor variable gene families and are responsive to costimulatory signals. These results suggest that normal thymic function may be restored following antiretroviral treatment.     

The effect of protein stability on protein-monoglyceride interactions

We have previously shown that proteins such as beta-lactoglobulin and lysozyme insert into monoglyceride monolayers and are able to induce an L(beta) to coagel phase transition in monoglyceride bilayers. These studies gave a first indication that protein stability could be an important factor for these interactions. This study therefore aims at further investigating the potential role of protein stability on protein-monoglyceride interactions. To this end we studied the interaction of stable and destabilized alpha-lactalbumin with monostearoylglycerol. Our results show that protein stability is important for the insertion of proteins into a monostearoylglycerol monolayer, such that the lower the stability of the protein the better the protein inserts. In marked contrast to beta-lactoglobulin and lysozyme we found that destabilized alpha-lactalbumin does not induce the L(beta) to coagel phase transition in monoglyceride bilayers. We propose that this is due to an increased surface coverage by the protein which could result from the unfolding of the protein upon binding to the interface.     

Characterization of the thermotropic behavior and lateral organization of lipid-peptide mixtures by a combined experimental and theoretical approach: effects of hydrophobic mismatch and role of flanking residues

A combined experimental and theoretical study was performed on a series of mixtures of dipalmitoylphosphatidylcholine (DPPC) and synthetic peptides to investigate their thermotropic behavior and lateral organization. The experimental study was based on differential scanning calorimetry (DSC) and phosphorous nuclear magnetic resonance ((31)P-NMR) techniques; the theoretical study was based on calculations on a microscopic molecular interaction model, where the lipid-peptide interaction is built on the hydrophobic matching principle. The chosen peptides, WALP and KALP, consist of a hydrophobic stretch, of variable length, of alternating leucine and alanine residues, flanked on both ends with tryptophan and lysine residues, respectively. By systematically varying the peptide hydrophobic length it was thus possible to explore different matching conditions between the peptide's hydrophobic length and the lipid bilayer hydrophobic thickness, and to investigate the potential role of flanking residues. The results show that both the WALP and the KALP peptides tend to favor the liquid-crystalline (or fluid) phase of the system; i.e., they tend to depress the main-transition temperature, T(m), of pure DPPC. However, the detailed effects of both peptides on the lateral phase behavior of the lipid-peptide system are dependent on the peptide length and the type of flanking residues. The results suggest that below T(m), the shortest among the WALP and KALP peptides induce gel-fluid phase separation in the system within an extensive temperature-composition region. The longer the hydrophobic length of the peptides is, the more narrow this region appears to become.     

Effect of thoracoabdominal breathing patterns on inspiratory effort sensation

The present study examined the effects of elastase-induced emphysema on the structure of the external oblique and transverse abdominis muscles and a non-respiratory muscle, the extensor digitorum longus. Muscle structure was assessed from the cross-sectional area (CSA) and percent of individual fiber types in histochemically stained sections and from the number of sarcomeres arranged in series along the length of individual fibers. Data were obtained in eight hamsters with emphysema and nine saline-injected controls. In the normal (control) animals the external oblique was thicker but contained fewer sarcomeres than the transverse abdominis. Fiber size was similar in the two muscles. In the transverse abdominis the percents of fast-glycolytic and fast-oxidative fibers were greater and smaller, respectively, than in the external oblique. Lung volume of emphysematous hamsters was 168% of control values (P less than 0.001). In emphysematous compared with control animals, the CSA of fast-twitch fibers in the external oblique and transverse abdominis was significantly reduced. Fiber length and sarcomere number were significantly decreased in the transverse abdominis but not in the external oblique in emphysematous hamsters. In contrast, fiber size and composition of the extensor digitorum longus was similar in emphysematous and control animals. These data indicate that cellular responses of the ventilatory muscles to chronic hyperinflation and altered thoracic geometry induced by emphysema are not present in limb skeletal muscle. We speculate that changes in fiber length and CSA of fast fibers in the abdominal expiratory muscles reflect responses to chronic alterations in the mechanics of breathing that may affect muscle load, length, or the pattern of activity.