Point mutations in either of the two nucleotide-binding domains (NBD) of Hsp104 (NBD1 and NBD2) eliminate its thermotolerance function in vivo. In vitro, NBD1 mutations virtually eliminate ATP hydrolysis with little effect on hexamerization; analogous NBD2 mutations reduce ATPase activity and severely impair hexamerization. We report that high protein concentrations overcome the assembly defects of NBD2 mutants and increase ATP hydrolysis severalfold, changing Vmax with little effect on Km. In a complementary fashion, the detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate inhibits hexamerization of wild-type (WT) Hsp104, lowering Vmax with little effect on Km. ATP hydrolysis exhibits a Hill coefficient between 1.5 and 2, indicating that it is influenced by cooperative subunit interactions. To further analyze the effects of subunit interactions on Hsp104, we assessed the effects of mutant Hsp104 proteins on WT Hsp104 activities. An NBD1 mutant that hexamerizes but does not hydrolyze ATP reduces the ATPase activity of WT Hsp104 in vitro. In vivo, this mutant is not toxic but specifically inhibits the thermotolerance function of WT Hsp104. Thus, interactions between subunits influence the ATPase activity of Hsp104...

Acute myeloid leukemia (AML) is a heterogeneous group of diseases. Normal cytogenetics (CN) constitutes the single largest group, while trisomy 8 (+8) as a sole abnormality is the most frequent trisomy. How trisomy contributes to tumorigenesis is unknown. We used oligonucleotide-based DNA microarrays to study global gene expression in AML+8 patients with +8 as the sole chromosomal abnormality and AML-CN patients. CD34+ cells purified from normal bone marrow (BM) were also analyzed as a representative heterogeneous population of stem and progenitor cells. Expression patterns of AML patients were clearly distinct from those of CD34+ cells of normal individuals. We show that AML+8 blasts overexpress genes on chromosome 8, estimated at 32% on average, suggesting gene-dosage effects underlying AML+8. Systematic analysis by cellular function indicated up-regulation of genes involved in cell adhesion in both groups of AML compared with CD34+ blasts from normal individuals. Perhaps most interestingly, apoptosis-regulating genes were significantly down-regulated in AML+8 compared with AML-CN. We conclude that the clinical and cytogenetic heterogeneity of AML is due to fundamental biological differences.

Overexpression of both cellular Src (c-Src) and the epidermal growth factor receptor (EGFR) occurs in many of the same human tumors, suggesting that they may functionally interact and contribute to the progression of cancer. Indeed, in murine fibroblasts, overexpression of c-Src has been shown to potentiate the mitogenic and tumorigenic capacity of the overexpressed EGFR. Potentiation correlated with the ability of c-Src to physically associate with the activated EGFR and the appearance of two unique in vivo phosphorylations on the receptor (Tyr-845 and Tyr-1101). Using stable cell lines of C3H10T½ murine fibroblasts that contain kinase-deficient (K−) c-Src and overexpressed wild-type EGFR, we show that the kinase activity of c-Src is required for both the biological synergy with the receptor and the phosphorylations on the receptor, but not for the association of c-Src with the receptor. In transient transfection assays, not only epidermal growth factor but also serum- and lysophosphatidic acid-induced DNA synthesis was ablated in a dominant-negative fashion by a Y845F mutant of the EGFR, indicating that c-Src-induced phosphorylation of Y845 is critical for the mitogenic response to both the EGFR and a G protein-coupled receptor (lysophosphatidic acid receptor). Unexpectedly...

Toxicity prevents the systemic administration of many therapeutic proteins, and attempts at protein targeting via the circulatory system (i.e., “magic bullets”) have failed in all but a few special cases. Direct administration at the target site is a logical alternative, particularly in the central nervous system, but the limits of direct administration have not been defined clearly. Nerve growth factor (NGF) enhances survival of cholinergic neurons and, therefore, has generated considerable interest for the treatment of Alzheimer’s disease. We tested the effectiveness of local delivery by implanting small polymer pellets that slowly released NGF into the central nervous system of adult rats at controlled distances from a target site containing transplanted fetal cholinergic cells. NGF-releasing implants placed within 1–2 mm of the treatment site enhanced the biological function of cellular targets, whereas identical implants placed ≈3 mm from the target site of treatment produced no beneficial effect. Effective NGF therapy required millimeter-scale positioning of the NGF source, and efficacy correlated with the spatial distribution of NGF concentration in the tissue; this result suggests that NGF must be delivered within several millimeters of the target to be effective in treating Alzheimer’s disease. Because the human brain is divided into functional regions that are typically several centimeters in diameter and often irregular in shape...

The almost universal appreciation for the importance of zinc in metabolism has been offset by the considerable uncertainty regarding the proteins that store and distribute cellular zinc. We propose that some zinc proteins with so-called zinc cluster motifs have a central role in zinc distribution, since they exhibit the rather exquisite properties of binding zinc tightly while remaining remarkably reactive as zinc donors. We have used zinc isotope exchange both to probe the coordination dynamics of zinc clusters in metallothionein, the small protein that has the highest known zinc content, and to investigate the potential function of zinc clusters in cellular zinc distribution. When mixed and incubated, metallothionein isoproteins-1 and -2 rapidly exchange zinc, as demonstrated by fast chromatographic separation and radiometric analysis. Exchange kinetics exhibit two distinct phases (kfast ≃ 5000 min−1·M−1; kslow ≃ 200 min−1·M−1, pH 8.6, 25°C) that are thought to reflect exchange between the three-zinc clusters and between the four-zinc clusters, respectively. Moreover, we have observed and examined zinc exchange between metallothionein-2 and the Gal4 protein (k ≃ 800 min−1·M−1, pH 8.0, 25°C), which is a prototype of transcription factors with a two-zinc cluster. This reaction constitutes the first experimental example of intermolecular zinc exchange between heterologous proteins. Such kinetic reactivity distinguishes zinc in biological clusters from zinc in the coordination environment of zinc enzymes...

The biological activities of the retinoids are mediated by two nuclear hormone receptors: the retinoic acid receptor (RAR) and the retinoid-X receptor (RXR). RXR (and its insect homologue ultraspiracle) is a common heterodimeric partner for many other nuclear receptors, including the insect ecdysone receptor. As part of a continuing analysis of nuclear receptor function, we noticed that, whereas RXR can be readily expressed in Escherichia coli to produce soluble protein, many of its heterodimeric partners cannot. For example, overexpression of RAR results mostly in inclusion bodies with the residual soluble component unable to interact with RXR or ligand efficiently. Similar results are seen with other RXR/ultraspiracle partners. To overcome these problems, we designed a novel double cistronic vector to coexpress RXR and its partner ligand-binding domains in the same bacterial cell. This resulted in a dramatic increase in production of soluble and apparently stable heterodimer. Hormone-binding studies using the purified RXR–RAR heterodimer reveal increased ligand-binding capacity of both components of 5- to 10-fold, resulting in virtually complete functionality. Based on these studies we find that bacterially expressed receptors can exist in one of three distinct states: insoluble...

Computer simulations of the effect of protein dynamics on the long distance tunneling mediated by the protein matrix have been carried out for a Ru-modified (His 126) azurin molecule. We find that the tunneling matrix element is a sensitive function of the atomic configuration of the part of the protein matrix in which tunneling currents (pathways) are localized. Molecular dynamics simulations show that fluctuations of the matrix element can occur on a time scale as short as 10 fs. These short time fluctuations are an indication of a strong dynamic coupling of a tunneling electron to vibrational motions of the protein nuclear coordinates. The latter results in a modification of the conventional Marcus picture of electron transfer in proteins. The new element in the modified theory is that the tunneling electron is capable of emitting or absorbing vibrational energy (phonons) from the medium. As a result, some biological reactions may occur in an activationless fashion. An analytical theoretical model is proposed to account for thermal fluctuations of the medium in long distance electron transfer reactions. The model shows that, at long distances, the phonon-modified inelastic tunneling always dominates over the conventional elastic tunneling.

The fluorescence of a polyanionic conjugated polymer can be quenched by extremely low concentrations of cationic electron acceptors in aqueous solutions. We report a greater than million-fold amplification of the sensitivity to fluorescence quenching compared with corresponding “molecular excited states.” Using a combination of steady-state and ultrafast spectroscopy, we have established that the dramatic quenching results from weak complex formation [polymer(−)/quencher(+)], followed by ultrafast electron transfer from excitations on the entire polymer chain to the quencher, with a time constant of 650 fs. Because of the weak complex formation, the quenching can be selectively reversed by using a quencher-recognition diad. We have constructed such a diad and demonstrate that the fluorescence is fully recovered on binding between the recognition site and a specific analyte protein. In both solutions and thin films, this reversible fluorescence quenching provides the basis for a new class of highly sensitive biological and chemical sensors.

The techniques of optical trapping and manipulation of neutral particles by lasers provide unique means to control the dynamics of small particles. These new experimental methods have played a revolutionary role in areas of the physical and biological sciences. This paper reviews the early developments in the field leading to the demonstration of cooling and trapping of neutral atoms in atomic physics and to the first use of optical tweezers traps in biology. Some further major achievements of these rapidly developing methods also are considered.

The development of two major subdivisions of the vertebrate nervous system, the midbrain and the cerebellum, is controlled by signals emanating from a constriction in the neural primordium called the midbrain/hindbrain organizer (Joyner, A. L. (1996) Trends Genet. 12, 15–201). The closely related transcription factors Pax-2 and Pax-5 exhibit an overlapping expression pattern very early in the developing midbrain/hindbrain junction. Experiments carried out in fish (Krauss, S., Maden, M., Holder, N. & Wilson, S. W. (1992) Nature (London) 360, 87–89) with neutralizing antibodies against Pax-b, the orthologue of Pax-2 in mouse, placed this gene family in an regulatory cascade necessary for the development of the midbrain and the cerebellum. The targeted mutation of Pax-5 has been reported to have only slight effects in the development of structures derived from the isthmic constriction, whereas the Pax-2 null mutant mice show a background-dependent phenotype with varying penetrance. To test a possible redundant function between Pax-2 and Pax-5 we analyzed the brain phenotypes of mice expressing different dosages of both genes. Our results demonstrate a conserved biological function of both proteins in midbrain/hindbrain regionalization. Additionally...

The Central American isthmus was a major dispersal route for plant taxa originally brought under cultivation in the domestication centers of southern Mexico and northern South America. Recently developed methodologies in the archaeological and biological sciences are providing increasing amounts of data regarding the timing and nature of these dispersals and the associated transition to food production in various regions. One of these methodologies, starch grain analysis, recovers identifiable microfossils of economic plants directly off the stone tools used to process them. We report on new starch grain evidence from Panama demonstrating the early spread of three important New World cultigens: maize (Zea mays), manioc (Manihot esculenta), and arrowroot (Maranta arundinacea). Maize starch recovered from stone tools at a site located in the Pacific lowlands of central Panama confirms previous archaeobotanical evidence for the use of maize there by 7800–7000 cal BP. Starch evidence from preceramic sites in the less seasonal, humid premontane forests of Chiriquí province, western Panama, shows that maize and root crops were present by 7400–5600 cal BP, several millennia earlier than previously documented. Several local starchy resources...

It is generally known that bacterial genes working in the same biological pathways tend to group into operons, possibly to facilitate cotranscription and to provide stoichiometry. However, very little is understood about what may determine the global arrangement of bacterial genes in a genome beyond the operon level. Here we present evidence that the global arrangement of operons in a bacterial genome is largely influenced by the tendency that a bacterium keeps its operons encoding the same biological pathway in nearby genomic locations, and by the tendency to keep operons involved in multiple pathways in locations close to the other members of their participating pathways. We also observed that the activation frequencies of pathways also influence the genomic locations of their encoding operons, tending to have operons of the more frequently activated pathways more tightly clustered together. We have quantitatively assessed the influences on the global genomic arrangement of operons by different factors. We found that the current arrangements of operons in most of the bacterial genomes we studied tend to minimize the overall distance between consecutive operons of a same pathway across all pathways encoded in the genome.

The DNA mismatch repair protein PMS2 was recently found to encode a novel endonuclease activity. To determine the biological functions of this activity in mammals, we generated endonuclease-deficient Pms2E702K knock-in mice. Pms2EK/EK mice displayed increased genomic mutation rates and a strong cancer predisposition. In addition, class switch recombination, but not somatic hypermutation, was impaired in Pms2EK/EK B cells, indicating a specific role in Ig diversity. In contrast to Pms2−/− mice, Pms2EK/EK male mice were fertile, indicating that this activity is dispensable in spermatogenesis. Therefore, the PMS2 endonuclease activity has distinct biological functions and is essential for genome maintenance and tumor suppression.

Flies are one of four superradiations of insects (along with beetles, wasps, and moths) that account for the majority of animal life on Earth. Diptera includes species known for their ubiquity (Musca domestica house fly), their role as pests (Anopheles gambiae malaria mosquito), and their value as model organisms across the biological sciences (Drosophila melanogaster). A resolved phylogeny for flies provides a framework for genomic, developmental, and evolutionary studies by facilitating comparisons across model organisms, yet recent research has suggested that fly relationships have been obscured by multiple episodes of rapid diversification. We provide a phylogenomic estimate of fly relationships based on molecules and morphology from 149 of 157 families, including 30 kb from 14 nuclear loci and complete mitochondrial genomes combined with 371 morphological characters. Multiple analyses show support for traditional groups (Brachycera, Cyclorrhapha, and Schizophora) and corroborate contentious findings, such as the anomalous Deuterophlebiidae as the sister group to all remaining Diptera. Our findings reveal that the closest relatives of the Drosophilidae are highly modified parasites (including the wingless Braulidae) of bees and other insects. Furthermore...

The functional role of IL-1 family member 10, recently renamed IL-38, remains unknown. In the present study we aimed to elucidate the biological function of IL-38 and to identify its receptor. Heat-killed Candida albicans was used to stimulate memory T-lymphocyte cytokine production in freshly obtained human peripheral blood mononuclear cells from healthy subjects. The addition of recombinant IL-38 (152 amino acids) inhibited the production of T-cell cytokines IL-22 (37% decrease) and IL-17 (39% decrease). The reduction in IL-22 and IL-17 caused by IL-38 was similar to that caused by the naturally occurring IL-36 receptor antagonist (IL-36Ra) in the same peripheral blood mononuclear cells cultures. IL-8 production induced by IL-36γ was reduced by IL-38 (42% decrease) and also was reduced by IL-36Ra (73% decrease). When human blood monocyte-derived dendritic cells were used, IL-38 as well as IL-36Ra increased LPS-induced IL-6 by twofold. We screened immobilized extracellular domains of each member of the IL-1 receptor family, including the IL-36 receptor (also known as “IL-1 receptor-related protein 2”) and observed that IL-38 bound only to the IL-36 receptor, as did IL-36Ra. The dose–response suppression of IL-38 as well as that of IL-36Ra of Candida-induced IL-22 and IL-17 was not that of the classic IL-1 receptor antagonist (anakinra)...

Using cryoelectron microscopy of vitreous sections, we investigated in situ the ultrastructure of biological membranes, selected from several cell types for their diverse biological functions. Here we describe how to visualize the two membrane leaflets and tightly apposed membranes, lying as close as 1.1 nm apart, by tuning the imaging conditions. We show how defects in membrane stacks may be clues to resolving their structure. Details of membrane proteins are also resolved, as well as protein lattices with correlations between stacked membranes. Imaging the cell in its native hydrated state can now be done in the nanometer resolution range, which should open unique routes for investigating structure–function relationships.

Zero-mode waveguides (ZMWs) provide a powerful technology for studying single-molecule real-time dynamics of biological systems. However, difficulties in instrumental implementation and ZMW fabrication prevented their widespread use. Here, we modify a commercially available ZMW-based DNA sequencer for use as a multipurpose single-molecule fluorescence instrument. The instrumentation presented here allows access to ZMWs for the general biophysics community for high-throughput multiplexed dynamics of single biological molecules.

Cell-based soft robotic devices could have a transformative impact on our ability to design machines and systems that can dynamically sense and respond to a range of complex environmental signals. We demonstrate innovative advancements in biomaterials, tissue engineering, and 3D printing, as well as an integration of these technologies, to forward engineer a controllable centimeter-scale biological machine capable of locomotion on a surface in fluid. Due in part to their elastic nature and the living components that can permit a dynamic response to environmental and applied stimuli, these biological machines can have diverse applications and represent a significant advancement toward high-level functional control over soft biorobotic systems.

Ants are the world's most conspicuous and important eusocial insects and their diversity, abundance, and extreme behavioral specializations make them a model system for several disciplines within the biological sciences. Here, we report the discovery of a new ant that appears to represent the sister lineage to all extant ants (Hymenoptera: Formicidae). The phylogenetic position of this cryptic predator from the soils of the Amazon rainforest was inferred from several nuclear genes, sequenced from a single leg. Martialis heureka (gen. et sp. nov.) also constitutes the sole representative of a new, morphologically distinct subfamily of ants, the Martialinae (subfam. nov.). Our analyses have reduced the likelihood of long-branch attraction artifacts that have troubled previous phylogenetic studies of early-diverging ants and therefore solidify the emerging view that the most basal extant ant lineages are cryptic, hypogaeic foragers. On the basis of morphological and phylogenetic evidence we suggest that these specialized subterranean predators are the sole surviving representatives of a highly divergent lineage that arose near the dawn of ant diversification and have persisted in ecologically stable environments like tropical soils over great spans of time.

VirB8-like proteins are essential components of type IV secretion systems, bacterial virulence factors that mediate the translocation of effector molecules from many bacterial pathogens into eukaryotic cells. Based on cell biological, genetic, and x-ray crystallographic data, VirB8 was proposed to undergo multiple protein–protein interactions to mediate assembly of the translocation machinery. Here we report the results of a structure–function analysis of the periplasmic domain of VirB8 from the mammalian pathogen Brucella suis, which identifies amino acid residues required for three protein–protein interactions. VirB8 variants changed at residues proposed to be involved in dimerization, and protein–protein interactions were purified and characterized in vitro and in vivo. Changes at M102, Y105, and E214 affected the self-association as measured by analytical ultracentrifugation and gel filtration. The interaction with B. suis VirB10 was reduced by changes at T201, and change at R230 inhibited the interaction with VirB4 in vitro. The in vivo functionality of VirB8 variants was determined by complementation of growth in macrophages by a B. suis virB8 mutant and by using a heterologous assay of type IV secretion system assembly in Agrobacterium tumefaciens. Changes at Y105...