Intimate view of a kinetic protein folding intermediate: residue-resolved structure, interactions, stability, folding and unfolding rates, homogeneity

A cytochrome c kinetic folding intermediate was studied by hydrogen exchange (HX) pulse labeling. Advances in the technique and analysis made it possible to define the structured and unstructured regions, equilibrium stability, and kinetic opening and closing rates, all at an amino acid-resolved level. The entire N-terminal and C-terminal helices are formed and docked together at their normal native positions. They fray in both directions from the interaction region, due to a progression in both unfolding and refolding rates, leading to the surprising suggestion that helix propagation may proceed very slowly in the condensed milieu. Several native-like beta turns are formed. Some residues in the segment that will form the native 60s helix are protected but others are not, suggesting energy minimization to some locally non-native conformation in the transient intermediate. All other regions are unprotected, presumably dynamically disordered. The intermediate resembles a partially constructed native state. It is early, on-pathway, and all of the refolding molecules pass through it. These and related results consistently point to distinct, homogeneous, native-like intermediates in a stepwise sequential pathway, guided by the same factors that determine the native structure. Previous pulse labeling efforts have always assumed EX2 exchange during the labeling pulse, often leading to the suggestion of heterogeneous intermediates in alternative parallel pathways. The present work reveals a dominant role for EX1 exchange in the high pH labeling pulse, which will mimic heterogeneous behavior when EX2 exchange is assumed. The general problem of homogeneous versus heterogeneous intermediates and pathways is discussed.     

Connective-tissue macromolecules in Golgi chicken tendon organs and at their interface with muscle fibers and adjoining tendinous structures.

Tendon organs from leg and forearm muscles of white leghorn chickens were examined with a library of monoclonal antibodies to determine the composition of their connective-tissue framework and the types of connective-tissue macromolecules that occur at the sites where muscle fibers attach to the receptors. The capsules of the tendon organs were positive for connective-tissue macromolecules typical of basal lamina (collagen type IV, laminin, and heparin sulfate proteoglycan) and for tenascin, collagen types III and VI, and fibronectin. Connective-tissue bundles in the lumen of a receptor reacted primarily with antibodies against collagen type I and 4-chondroitin sulfate. The narrow partitions that divide each lumen into compartments stained for collagen type III. Toward its tendinous end, a receptor made few contacts with muscle fibers. Instead, the capsule and the collagenous bundles blended gradually with the intermuscular portions of tendons. At the muscular end, the connections were more complex. Muscle fibers that attached in series to tendon organs split to produce basal lamina-covered, finger-like extensions, which were separated from each other by fissures. Tongues of connective tissue containing tenascin, collagen types I and VI, and fibronectin extended into the fissures. Distally the tongues were continuous with the tenascin in the capsule and just internal to the capsule, fibronectin and basal lamina macromolecules in the capsule, and collagen type I in the collagenous bundles. The uninterrupted presence of these macromolecules around terminating muscle fibers and in the capsule and/or the intraluminal collagen bundles suggests that muscle fibers that attach in series at the muscular end exert a force during muscular contraction on the intraluminal collagen bundles and on the receptor capsule.     

Studies of CD40L expression by lymphoid cells from experimentally and naturally SIV-infected nonhuman primate species.

Dysfunction of T lymphocytes is well documented in HIV-1-infected individuals; however, the mechanisms responsible for the noted dysfunction are not well understood. CD40L is an important costimulatory molecule that helps initiate immune responses, and there is controversy regarding whether or not expression of CD40L is compromised in HIV-1-infected individuals. We have utilized the SIV infection of experimentally infected (disease-susceptible) and naturally infected (disease-resistant) nonhuman primates as animal models of human AIDS to address this issue. Little is known concerning the expression of CD40L in nonhuman primates. Studies were conducted to determine the frequency, density, phenotype, and kinetics of CD40L expression by in vitro activated peripheral blood mononuclear cells (PBMCs) from different species of uninfected and SIV-infected monkeys. Data obtained show marked differences in the density and phenotypic lineage that expresses CD40L in lymphoid cells from the three species examined. However, no detectable differences were noted in the frequency and density of CD40L expression by in vitro activated lymphoid cells from uninfected and SIV-infected disease-susceptible rhesus macaques and seropositive as compared to seronegative disease-resistant sooty mangabeys. These data suggest that phenotypic expression of CD40L is not compromised due to SIV infection.     

The subcellular localization of the pteridines in strain R-26 of Rhodopseudomonas spheroides

Pteridines were detected in strain R-26 of Rhodopseudomonas spheroides and the pteridine concentrations in subcellular fractions were measured by a sensitive fluorimetric method. The whole cell fraction contained nearly equal concentrations of pteridine and reaction center bacteriochlorophyll. The soluble subcellular fraction contained the majority of the pteridine and the photochemically active chromatophore preparations contained very little. No pteridine was detected in purified reaction center complex preparations. This subcellular localization of pteridines was not consistent with a pteridine functioning as the primary electron acceptor in this photosynthetic system.     

The effect of embryo extract on the types of collagen synthesized by cultured chick chondrocytes.

Growth of embryonic chick chondrocytes in dialyzed embryo extract results in both a change in morphology of the cells toward that of a fibroblast and a change in the type of collagen synthesized from the cartilage-specific Type II collagen (chain composition [α1(II)]₃) to a mixture of Type I collagen (chain composition [α1(I)]₂α2) and the Type I trimer (chain composition [α1(I)]₃). Analyses after 6 days of growth in embryo extract show that the synthesis of only Type I collagen and the Type I trimer can be detected. However, on subculturing the cells to a low density and allowing a period of growth without embryo extract, colonies of chondrocytes reappear and the synthesis of Type II collagen apparently resumes. It is suggested that the observed changes represent a “modulation” in cell behavior, this being expressed not only by the morphological changes but also by changes in cell-specific protein synthesis as demonstrated by the changes in the type of collagen synthesized.