Determination of membrane protein topology by red-edge excitation shift analysis: application to the membrane-bound colicin E1 channel peptide

A new approach for the determination of the bilayer location of Trp residues in proteins has been applied to the study of the membrane topology of the channel-forming bacteriocin, colicin E1. This method, red-edge excitation shift (REES) analysis, was initially applied to the study of 12 single Trp-containing channel peptides of colicin E1 in the soluble state in aqueous medium. Notably, REES was observed for most of the channel peptides in aqueous solution upon low pH activation. The extent of REES was subsequently characterized using a model membrane system composed of the tripeptide, Lys-Trp-Lys, bound to dimyristoyl-sn-glycerol-3-phosphatidylserine liposomes. Subsequently, data accrued from the model peptide-lipid system was used to interpret information obtained on the channel peptides when bound to dioleoyl-sn-glycerol-3-phosphatidylcholine/dioleoyl-sn-glycerol-3-phosphatidylglycerol membrane vesicles. The single Trp mutant peptides were divided into three categories based on the change in the REES values observed for the Trp residues when the peptides were bound to liposomes as compared to the REES values measured for the soluble peptides. F-404 W, F-413 W, F-443 W, F-484 W, and W-495 peptides exhibited small and/or insignificant REES changes (Delta REES) whereas W-424, F-431 W, and Y-507 W channel peptides possessed modest REES changes (3 nm< or = Delta REES< or = 7 nm). In contrast, wild-type, Y-367 W, W-460, Y-478 W, and I-499 W channel peptides showed large Delta REES values upon membrane binding (7 nm< Delta REES< or =12 nm). The REES data for the membrane-bound structure of the colicin E1 channel peptide proved consistent with previous data for the topology of the closed channel state, which lends further credence to the currently proposed channel model. In conclusion, the REES method provides another source of topological data for assignment of the bilayer location for Trp residues within membrane-associated proteins; however, it also requires careful interpretation of spectral data in combination with structural information on the proteins being investigated.     

Chromatin motion is constrained by association with nuclear compartments in human cells

BACKGROUND: In comparison with many nuclear proteins, the movement of chromatin in nuclei appears to be generally constrained. These restrictions on motion are proposed to reflect the attachment of chromatin to immobile nuclear substructures. RESULTS: To gain insight into the regulation of chromosome dynamics by nuclear architecture, we have followed the movements of different sites in the human genome in living cells. Here, we show that loci at nucleoli or the nuclear periphery are significantly less mobile than other, more nucleoplasmic loci. Disruption of nucleoli increases the mobility of nucleolar-associated loci. CONCLUSIONS: This is the first report of distinct nuclear substructures constraining the movements of chromatin. These constraints reflect the physical attachment of chromatin to nuclear compartments or steric impairment caused by local ultrastructure. Our data suggest a role for the nucleolus and nuclear periphery in maintaining the three-dimensional organization of chromatin in the human nucleus.     

An arthritogenic monoclonal antibody to type II collagen, CII-C1, impairs cartilage formation by cultured chondrocytes

Antibodies to type II collagen (CII) cause articular damage in collagen-induced arthritis (CIA) in mice as judged by passive transfer to naive animals of mAb to CII. We tested the hypothesis that mAb degrade cartilage structure by reacting with functionally important regions of the collagen molecule by examining the effects of an arthritogenic mAb to CII, CII-C1, on cultured bovine chondrocytes at high density, at days 7 and 14. The effects were compared of CII-C1, an isotype-matched control mAb, or medium alone, on chondrocyte proliferation and viability, cell morphology, matrix structure by light and electron microscopy, and matrix synthesis by metabolic labelling with 3H-proline for collagen or 35SO4 for proteoglycans. Chondrocytes in culture remained viable, proliferated, and produced an extracellular matrix in which CII was the major collagen. The addition of CII-C1, but not a control mAb, increased the synthesis of CII and proteoglycan, and caused disorganization of the extracellular matrix and thin collagen fibrils ultrastructurally. Moreover, using a cell-free assay, CII-C1 inhibited the normal self-assembly of collagen fibrils from CII in solution. The finding that the mAb to CII, CII-C1 has striking degradative effects in vitro on cartilage synthesis suggests that antibodies to collagen perpetuate the chronic phase of CIA and that, in mice at least, such antibodies are an important component of pathogenesis.     

Focal adhesion kinase (FAK) expression and phosphorylation in sea urchin embryos

We have cloned three cDNA isoforms of focal adhesion kinase (FAK) from the sea urchin, Lytechinus variegatus. The sea urchin FAK is more closely related to FAK from other deuterostomes than from invertebrate protostomes or to cell adhesion kinase beta (CAKbeta/Pyk2/FAK2). FAK is expressed in all cells of sea urchin embryos by the 120-cell stage and strongly in blastulae. Phospho-FAK concentrates on basal surfaces of epithelial cells in early blastulae and occurs in syncytial cables of primary mesenchyme cells (PMC). Inhibition of FAK by constructs of FAK-related non-kinase delays blastocoel expansion and early PMC ingression. These results suggest that FAK has roles in cell adhesion and in the shape and integrity of the epithelial cells in sea urchin embryos.     

Chronic hypoxia potentiates capacitative Ca2+ entry in type-I cortical astrocytes

Prolonged hypoxia exerts profound effects on cell function, and has been associated with increased production of amyloid beta peptides (A beta Ps) of Alzheimer's disease. Here, we have investigated the effects of chronic hypoxia (2.5% O2, 24 h) on capacitative Ca2+ entry (CCE) in primary cultures of rat type-I cortical astrocytes, and compared results with those obtained in astrocytes exposed to A beta Ps. Chronic hypoxia caused a marked enhancement of CCE that was observed after intracellular Ca2+ stores were depleted by bradykinin application or by exposure to thapsigargin (1 microM). Exposure of cells for 24 h to 1 microM A beta P(1-40) did not alter CCE. Enhancement of CCE was not attributable to cell hyperpolarization, as chronically hypoxic cells were significantly depolarized as compared with controls. Mitochondrial inhibition [by FCCP (10 microM) and oligomycin (2.5 microg/mL)] suppressed CCE in all three cell groups, but more importantly there were no significant differences in the magnitude of CCE in the three astrocyte groups under these conditions. Similarly, the antioxidants melatonin and Trolox abolished the enhancement of CCE in hypoxic cells. Our results indicate that chronic hypoxia augments CCE in cortical type-I astrocytes, a finding which is not mimicked by A beta P(1-40) and appears to be dependent on altered mitochondrial function.     

Different data from different labs: lessons from studies of gene-environment interaction

It is sometimes supposed that standardizing tests of mouse behavior will ensure similar results in different laboratories. We evaluated this supposition by conducting behavioral tests with identical apparatus and test protocols in independent laboratories. Eight genetic groups of mice, including equal numbers of males and females, were either bred locally or shipped from the supplier and then tested on six behaviors simultaneously in three laboratories (Albany, NY; Edmonton, AB; Portland, OR). The behaviors included locomotor activity in a small box, the elevated plus maze, accelerating rotarod, visible platform water escape, cocaine activation of locomotor activity, and ethanol preference in a two-bottle test. A preliminary report of this study presented a conventional analysis of conventional measures that revealed strong effects of both genotype and laboratory as well as noteworthy interactions between genotype and laboratory. We now report a more detailed analysis of additional measures and view the data for each test in different ways. Whether mice were shipped from a supplier or bred locally had negligible effects for almost every measure in the six tests, and sex differences were also absent or very small for most behaviors, whereas genetic effects were almost always large. For locomotor activity, cocaine activation, and elevated plus maze, the analysis demonstrated the strong dependence of genetic differences in behavior on the laboratory giving the tests. For ethanol preference and water escape learning, on the other hand, the three labs obtained essentially the same results for key indicators of behavior. Thus, it is clear that the strong dependence of results on the specific laboratory is itself dependent on the task in question. Our results suggest that there may be advantages of test standardization, but laboratory environments probably can never be made sufficiently similar to guarantee identical results on a wide range of tests in a wide range of labs. Interpretations of our results by colleagues in neuroscience as well as the mass media are reviewed. Pessimistic views, prevalent in the media but relatively uncommon among neuroscientists, of mouse behavioral tests as being highly unreliable are contradicted by our data. Despite the presence of noteworthy interactions between genotype and lab environment, most of the larger differences between inbred strains were replicated across the three labs. Strain differences of moderate effects size, on the other hand, often differed markedly among labs, especially those involving three 129-derived strains. Implications for behavioral screening of targeted and induced mutations in mice are discussed.     

Syndemics and public health: reconceptualizing disease in bio-social context

The world of public health has undergone dramatic changes since the emergence of AIDS in the early 1980s. The appearance and global spread in recent years of wave after wave of new and renewed infectious diseases and their entwinement with each other and with the social conditions and biopsychological consequences of disparity, discrimination, and structural violence has produced a new significant threat to public health internationally. The term syndemic has been introduced recently by medical anthropologists to label the synergistic interaction of two or more coexistent diseases and resultant excess burden of disease. This article provides the fullest examination of this new concept to date, including a review of relevant new literature and recent research finds concerning coinfection and synergistic interaction of diseases and social conditions at the biological and population levels.     

Microsatellite instability and suppressed DNA repair enzyme expression in rheumatoid arthritis

Reactive oxygen and nitrogen are produced by rheumatoid arthritis (RA) synovial tissue and can potentially induce mutations in key genes. Normally, this process is prevented by a DNA mismatch repair (MMR) system that maintains sequence fidelity during DNA replication. Key members of the MMR system include MutSalpha (hMSH2 and hMSH6) and MutSbeta (hMSH2 and hMSH3). To provide evidence of DNA damage in inflamed synovium, we analyzed synovial tissues for microsatellite instability (MSI). MSI was examined by PCR on genomic DNA of paired synovial tissue and peripheral blood cells of RA patients using specific primer sequences for five key microsatellites. Surprisingly, abundant MSI was observed in RA synovium compared with osteoarthritis tissue. Western blot analysis for the expression of MMR proteins demonstrated decreased hMSH6 and increased hMSH3 in RA synovium. To evaluate potential mechanisms of MMR regulation in arthritis, fibroblast-like synoviocytes (FLS) were isolated from synovial tissues and incubated with the NO donor S-nitroso-N-acetylpenicillamine. Western blot analysis demonstrated constitutive expression of hMSH2, 3, and 6 in RA and osteoarthritis FLS. When FLS were cultured with S-nitroso-N-acetylpenicillamine, the pattern of MMR expression in RA synovium was reproduced (high hMSH3, low hMSH6). Therefore, oxidative stress can relax the DNA MMR system in RA by suppressing hMSH6. Decreased hMSH6 can subsequently interfere with repair of single base mutations, which is the type observed in RA. We propose that oxidative stress not only creates DNA adducts that are potentially mutagenic, but also suppresses the mechanisms that limit the DNA damage.     

Hypoxic remodeling of Ca2+ stores in type I cortical astrocytes

Prolonged periods of hypoxia are deleterious to higher brain functions and increase the likelihood of developing dementias. Here, we have used fluorimetric techniques to investigate the effects of chronic hypoxia (2.5% O(2), 24 h) on Ca(2+) stores in type I cortical astrocytes, because such stores are crucial to various astrocyte functions, including Ca(2+)-dependent modulation of neuronal activity. Rises of [Ca(2+)](i) evoked by exposure of astrocytes to bradykinin were enhanced following chronic hypoxia, as were transient increases in [Ca(2+)](i) recorded in Ca(2+)-free perfusate. The enhanced responses were due partly to impaired plasmalemmal Na(+)/Ca(2+) exchange following chronic hypoxia. More importantly, chronic hypoxia increased the Ca(2+) content of mitochondria (as determined by exposing cells to mitochondrial inhibitors), such that they were unable to act as Ca(2+) buffers following bradykinin-evoked Ca(2+) release from the endoplasmic reticulum. Hypoxic enhancement of mitochondrial Ca(2+) content was also observed in confocal images of cells loaded with the mitochondrial Ca(2+) indicator, Rhod-2. Confocal imaging of cells loaded with tetramethylrhodamine ethyl ester, an indicator of mitochondrial membrane potential, indicated that mitochondria were hyperpolarized in astrocytes following chronic hypoxia. Our findings indicate that hypoxia disturbs Ca(2+) signaling in type I astrocytes, primarily by causing mitochondrial Ca(2+) overload.