Microtubule self-organization is gravity-dependent

Although weightlessness is known to affect living cells, the manner by which this occurs is unknown. Some reaction-diffusion processes have been theoretically predicted as being gravity-dependent. Microtubules, a major constituent of the cellular cytoskeleton, self-organize in vitro by way of reaction-diffusion processes. To investigate how self-organization depends on gravity, microtubules were assembled under low gravity conditions produced during space flight. Contrary to the samples formed on an in-flight 1 × g centrifuge, the samples prepared in microgravity showed almost no self-organization and were locally disordered.

The paradox of alloreactivity and self MHC restriction: Quantitative analysis and statistics

Although 1–24% of T cells are alloreactive, i.e., respond to MHC molecules encoded by a foreign haplotype, it is generally believed that T cells cannot recognize foreign peptides binding foreign MHC molecules. We show using a quantitative model that, if T cell selection and activation are affinity-driven, then an alloreactivity of 1–24% is incompatible with the textbook notion that self MHC restriction is absolute. If an average of 1% of clones are alloreactive, then according to our model, at most 20-fold more clones should, on average, be activated by antigens presented on self MHC than by antigens presented on foreign MHC. This ratio is at best 5 if alloreactivity is 5%. These results describe average properties of the murine immune system, but not the outcome of individual experiments. Using supercomputer technology, we simulated 100,000 MHC restriction experiments. Although the average restriction ratio was 7.1, restriction was absolute in 10% of the simulated experiments, greater than 100, although not absolute, in 29%, and below 6 in 24%. This extreme variability agrees with experimental estimates. Our analysis suggests that alloreactivity and average self MHC restriction both cannot be high, but that a low average restriction level is compatible with high levels in a significant number of experiments.

The human sex-reversing ATRX gene has a homologue on the marsupial Y chromosome, ATRY: Implications for the evolution of mammalian sex determination

Mutations in the ATRX gene on the human X chromosome cause X-linked α-thalassemia and mental retardation. XY patients with deletions or mutations in this gene display varying degrees of sex reversal, implicating ATRX in the development of the human testis. To explore further the role of ATRX in mammalian sex differentiation, the homologous gene was cloned and characterized in a marsupial. Surprisingly, active homologues of ATRX were detected on the marsupial Y as well as the X chromosome. The Y-borne copy (ATRY) displays testis-specific expression. This, as well as the sex reversal of ATRX patients, suggests that ATRY is involved in testis development in marsupials and may represent an ancestral testis-determining mechanism that predated the evolution of SRY as the primary mammalian male sex-determining gene. There is no evidence for a Y-borne ATRX homologue in mouse or human, implying that this gene has been lost in eutherians and its role supplanted by the evolution of SRY from SOX3 as the dominant determiner of male differentiation.

Self-similar intermediate asymptotics for a degenerate parabolic filtration-absorption equation

The equation ∂tu = u∂xx2u − (c − 1)(∂xu)2 is known in literature as a qualitative mathematical model of some biological phenomena. Here this equation is derived as a model of the groundwater flow in a water-absorbing fissurized porous rock; therefore, we refer to this equation as a filtration-absorption equation. A family of self-similar solutions to this equation is constructed. Numerical investigation of the evolution of non-self-similar solutions to the Cauchy problems having compactly supported initial conditions is performed. Numerical experiments indicate that the self-similar solutions obtained represent intermediate asymptotics of a wider class of solutions when the influence of details of the initial conditions disappears but the solution is still far from the ultimate state: identical zero. An open problem caused by the nonuniqueness of the solution of the Cauchy problem is discussed.

Redox state is a central modulator of the balance between self-renewal and differentiation in a dividing glial precursor cell

We have discovered that intracellular redox state appears to be a necessary and sufficient modulator of the balance between self-renewal and differentiation in dividing oligodendrocyte-type-2 astrocyte progenitor cells. The intracellular redox state of freshly isolated progenitors allows prospective isolation of cells with different self-renewal characteristics. Redox state is itself modulated by cell-extrinsic signaling molecules that alter the balance between self-renewal and differentiation: growth factors that promote self-renewal cause progenitors to become more reduced, while signaling molecules that promote differentiation cause progenitors to become more oxidized. Moreover, pharmacological antagonists of the redox effects of these cell-extrinsic signaling molecules antagonize their effects on self-renewal and differentiation, indicating that cell-extrinsic signaling molecules that modulate this balance converge on redox modulation as a critical component of their effector mechanism.

Preferential localization of self-stimulation sites in striosomes/patches in the rat striatum

Histological sections of the mammalian striatum reveal a “matrix” that is histochemically distinguishable from patches, or “striosomes”. The latter are cross sections of a compartment that consists primarily of tube-shaped structures radiating through the matrix. As a test of the hypothesis that the function of the striosome/patch compartment includes the mediation of behaviors related to reward, the present study examined electrical self-stimulation of the caudoputamen in rats with electrodes in either of the two compartments. Rats acquired and maintained bar-pressing responses that were contingent on stimulation through electrodes making contact with striosomes/patches more reliably than animals with electrodes terminating exclusively in the matrix. The results provide in vivo evidence that the striosome/patch compartment is functionally differentiated from the matrix compartment: Stimulation centered in or around the striosome/patch compartment but not in the matrix led to rapid acquisition of a new behavior.

Determination of gene organization in individual haplotypes by analyzing single DNA fragments from single spermatozoa

To determine human Ig heavy chain variable region (VH) gene segment organization on individual homologous chromosomes, an efficient approach has been developed. Single spermatozoa were used as subjects for the study. Upon sperm lysis, VH regions in each sperm were randomly sheared into fragments by the random Brownian force. The fragments were separated from each other by aliquoting the lysate into a certain number of tubes. The gene segments in the VH1 and VH4 families in each tube were identified by denaturing gradient gel electrophoresis after PCR amplification. The polymorphic VH sequences were used to determine the parental origins of the analyzed sperm. VH segment organization in the parental haplotypes was determined by aligning the overlapping fragments from the spermatozoa with the corresponding haplotypes. Based on this comparison between the resulting haplotype maps and the composite map reported previously, the VH region on chromosome 14 could be subdivided into four portions. The numbers and compositions of the VH gene segments differ considerably among the maps in two portions, but are highly conserved in the other two. The data also indicate that the VH region on chromosome 15 may contain a large duplicated block with copy number varying among haplotypes. The approach used in the present study may be used to construct high-resolution haplotype maps without molecular cloning.

Involvement of a cytosine side chain in proton transfer in the rate-determining step of ribozyme self-cleavage

Ribozymes of hepatitis delta virus have been proposed to use an active-site cytosine as an acid-base catalyst in the self-cleavage reaction. In this study, we have examined the role of cytosine in more detail with the antigenomic ribozyme. Evidence that proton transfer in the rate-determining step involved cytosine 76 (C76) was obtained from examining cleavage activity of the wild-type and imidazole buffer-rescued C76-deleted (C76Δ) ribozymes in D2O and H2O. In both reactions, a similar kinetic isotope effect and shift in the apparent pKa indicate that the buffer is functionally substituting for the side chain in proton transfer. Proton inventory of the wild-type reaction supported a mechanism of a single proton transfer at the transition state. This proton transfer step was further characterized by exogenous base rescue of a C76Δ mutant with cytosine and imidazole analogues. For the imidazole analogues that rescued activity, the apparent pKa of the rescue reaction, measured under kcat/KM conditions, correlated with the pKa of the base. From these data a Brønsted coefficient (β) of 0.51 was determined for the base-rescued reaction of C76Δ. This value is consistent with that expected for proton transfer in the transition state. Together, these data provide strong support for a mechanism where an RNA side chain participates directly in general acid or general base catalysis of the wild-type ribozyme to facilitate RNA cleavage.

Distinct role of gp130 activation in promoting self-renewal divisions by mitogenically stimulated murine hematopoietic stem cells

Previous studies have demonstrated hematopoietic stem cell amplification in vitro after the activation of three cell-surface receptors: flt3/flk2, c-kit, and gp130. We now show flt3-ligand and Steel factor alone will stimulate >85% of c-kit+Sca-1+lin− adult mouse bone marrow cells to proliferate in single-cell serum-free cultures, but concomitant retention of their stem cell activity requires additional exposure to a ligand that will activate gp130. Moreover, this response is restricted to a narrow range of gp130-activating ligand concentrations, above and below which hematopoietic stem cell activity is lost. These findings indicate a unique contribution of gp130 signaling to the maintenance of hematopoietic stem cell function when these cells are stimulated to divide with additional differential effects dictated by the intensity of gp130 activation.

Nanohedra: Using symmetry to design self assembling protein cages, layers, crystals, and filaments

A general strategy is described for designing proteins that self assemble into large symmetrical nanomaterials, including molecular cages, filaments, layers, and porous materials. In this strategy, one molecule of protein A, which naturally forms a self-assembling oligomer, An, is fused rigidly to one molecule of protein B, which forms another self-assembling oligomer, Bm. The result is a fusion protein, A-B, which self assembles with other identical copies of itself into a designed nanohedral particle or material, (A-B)p. The strategy is demonstrated through the design, production, and characterization of two fusion proteins: a 49-kDa protein designed to assemble into a cage approximately 15 nm across, and a 44-kDa protein designed to assemble into long filaments approximately 4 nm wide. The strategy opens a way to create a wide variety of potentially useful protein-based materials, some of which share similar features with natural biological assemblies.

One ring or two? Determination of ring number in carotenoids by lycopene ɛ-cyclases

Carotenoids in the photosynthetic membranes of plants typically contain two β-rings (e.g., β-carotene and zeaxanthin) or one ɛ- and one β-ring (e.g., lutein). Carotenoids with two ɛ-rings are uncommon. We reported earlier that the Arabidopsis thaliana lycopene ɛ-cyclase (LCYe) adds one ɛ-ring to the symmetrical linear substrate lycopene, whereas the structurally related lycopene β-cyclase (LCYb) adds two β-rings. Here we describe a cDNA encoding LCYe in romaine lettuce (Lactuca sativa var. romaine), one of the few plant species known to accumulate substantial quantities of a carotenoid with two ɛ-rings: lactucaxanthin. The product of the lettuce cDNA, similar in sequence to the Arabidopsis LCYe (77% amino acid identity), efficiently converted lycopene into the bicyclic ɛ-carotene in a heterologous Escherichia coli system. Regions of the lettuce and Arabidopsis ɛ-cyclases involved in the determination of ring number were mapped by analysis of chimeric ɛ-cyclases constructed by using an inverse PCR approach. A single amino acid was found to act as a molecular switch: lettuce LCYe mutant H457L added only one ɛ-ring to lycopene, whereas the complementary Arabidopsis LCYe mutant, L448H, added two ɛ-rings. An R residue in this position also yields a bi-ɛ-cyclase for both the lettuce and Arabidopsis enzymes. Construction and analysis of chimera of related enzymes with differing catalytic activities provide an informative approach that may be of particular utility for studying membrane-associated enzymes that cannot easily be crystallized or modeled to existing crystal structures.

Direct observation of the enhancement of noncooperative protein self-assembly by macromolecular crowding: Indefinite linear self-association of bacterial cell division protein FtsZ

Recent measurements of sedimentation equilibrium and sedimentation velocity have shown that the bacterial cell division protein FtsZ self-associates to form indefinitely long rod-like linear aggregates in the presence of GDP and Mg2+. In the present study, the newly developed technique of non-ideal tracer sedimentation equilibrium was used to measure the effect of high concentrations—up to 150 g/liter—of each of two inert “crowder” proteins, cyanmethemoglobin or BSA, on the thermodynamic activity and state of association of dilute FtsZ under conditions inhibiting (−Mg2+) and promoting (+Mg2+) FtsZ self-association. Analysis of equilibrium gradients of both FtsZ and crowder proteins indicates that, under the conditions of the present experiment, FtsZ interacts with each of the two crowder proteins essentially entirely via steric repulsion, which may be accounted for quantitatively by a simple model in which hemoglobin, albumin, and monomeric FtsZ are modeled as effective spherical hard particles, and each oligomeric species of FtsZ is modeled as an effective hard spherocylinder. The functional dependence of the sedimentation of FtsZ on the concentrations of FtsZ and either crowder indicates that, in the presence of high concentrations of crowder, both the weight-average degree of FtsZ self-association and the range of FtsZ oligomer sizes present in significant abundance are increased substantially.

Self-assembled monolayers from a designed combinatorial library of de novo β-sheet proteins

A variety of naturally occurring biomaterials owe their unusual structural and mechanical properties to layers of β-sheet proteins laminated between layers of inorganic mineral. To explore the possibility of fabricating novel two-dimensional protein layers, we studied the self-assembly properties of de novo proteins from a designed combinatorial library. Each protein in the library has a distinct 63 amino acid sequence, yet they all share an identical binary pattern of polar and nonpolar residues, which was designed to favor the formation of six-stranded amphiphilic β-sheets. Characterization of proteins isolated from the library demonstrates that (i) they self assemble into monolayers at an air/water interface; (ii) the monolayers are dominated by β-sheet secondary structure, as shown by both circular dichroism and infrared spectroscopies; and (iii) the measured areas (500- 600 Å2) of individual protein molecules in the monolayers match those expected for proteins folded into amphiphilic β-sheets. The finding that similar structures are formed by distinctly different protein sequences suggests that assembly into β-sheet monolayers can be encoded by binary patterning of polar and nonpolar amino acids. Moreover, because the designed binary pattern is compatible with a wide variety of different sequences, it may be possible to fabricate β-sheet monolayers by using combinations of side chains that are explicitly designed to favor particular applications of novel biomaterials.

How amoeboids self-organize into a fruiting body: Multicellular coordination in Dictyostelium discoideum

When individual amoebae of the cellular slime mold Dictyostelium discoideum are starving, they aggregate to form a multicellular migrating slug, which moves toward a region suitable for culmination. The culmination of the morphogenesis involves complex cell movements that transform a mound of cells into a globule of spores on a slender stalk. The movement has been likened to a “reverse fountain,” whereby prestalk cells in the upper part form a stalk that moves downwards and anchors to the substratum, while prespore cells in the lower part move upwards to form the spore head. So far, however, no satisfactory explanation has been produced for this process. Using a computer simulation that we developed, we now demonstrate that the processes that are essential during the earlier stages of the morphogenesis are in fact sufficient to produce the dynamics of the culmination stage. These processes are cAMP signaling, differential adhesion, cell differentiation, and production of extracellular matrix. Our model clarifies the processes that generate the observed cell movements. More specifically, we show that periodic upward movements, caused by chemotactic motion, are essential for successful culmination, because the pressure waves they induce squeeze the stalk downwards through the cell mass. The mechanisms revealed by our model have a number of self-organizing and self-correcting properties and can account for many previously unconnected and unexplained experimental observations.

Medial prefrontal cortex and self-referential mental activity: Relation to a default mode of brain function

Medial prefrontal cortex (MPFC) is among those brain regions having the highest baseline metabolic activity at rest and one that exhibits decreases from this baseline across a wide variety of goal-directed behaviors in functional imaging studies. This high metabolic rate and this behavior suggest the existence of an organized mode of default brain function, elements of which may be either attenuated or enhanced. Extant data suggest that these MPFC regions may contribute to the neural instantiation of aspects of the multifaceted “self.” We explore this important concept by targeting and manipulating elements of MPFC default state activity. In this functional magnetic resonance imaging (fMRI) study, subjects made two judgments, one self-referential, the other not, in response to affectively normed pictures: pleasant vs. unpleasant (an internally cued condition, ICC) and indoors vs. outdoors (an externally cued condition, ECC). The ICC was preferentially associated with activity increases along the dorsal MPFC. These increases were accompanied by decreases in both active task conditions in ventral MPFC. These results support the view that dorsal and ventral MPFC are differentially influenced by attentiondemanding tasks and explicitly self-referential tasks. The presence of self-referential mental activity appears to be associated with increases from the baseline in dorsal MPFC. Reductions in ventral MPFC occurred consistent with the fact that attention-demanding tasks attenuate emotional processing. We posit that both self-referential mental activity and emotional processing represent elements of the default state as represented by activity in MPFC. We suggest that a useful way to explore the neurobiology of the self is to explore the nature of default state activity.