Molecular characteristics of fibroblast growth factor–fibroblast growth factor receptor–heparin-like glycosaminoglycan complex

Fibroblast growth factor (FGF) family plays key roles in development, wound healing, and angiogenesis. Understanding of the molecular nature of interactions of FGFs with their receptors (FGFRs) has been seriously limited by the absence of structural information on FGFR or FGF–FGFR complex. In this study, based on an exhaustive analysis of the primary sequences of the FGF family, we determined that the residues that constitute the primary receptor-binding site of FGF-2 are conserved throughout the FGF family, whereas those of the secondary receptor binding site of FGF-2 are not. We propose that the FGF–FGFR interaction mediated by the ‘conserved’ primary site interactions is likely to be similar if not identical for the entire FGF family, whereas the ‘variable’ secondary sites, on both FGF as well as FGFR mediates specificity of a given FGF to a given FGFR isoform. Furthermore, as the pro-inflammatory cytokine interleukin 1 (IL-1) and FGF-2 share the same structural scaffold, we find that the spatial orientation of the primary receptor-binding site of FGF-2 coincides structurally with the IL-1β receptor-binding site when the two molecules are superimposed. The structural similarities between the IL-1 and the FGF system provided a framework to elucidate molecular principles of FGF–FGFR interactions. In the FGF–FGFR model proposed here, the two domains of a single FGFR wrap around a single FGF-2 molecule such that one domain of FGFR binds to the primary receptor-binding site of the FGF molecule, while the second domain of the same FGFR binds to the secondary receptor-binding site of the same FGF molecule. Finally, the proposed model is able to accommodate not only heparin-like glycosaminoglycan (HLGAG) interactions with FGF and FGFR but also FGF dimerization or oligomerization mediated by HLGAG.

Dynamic characteristics and adaptability of mouse vestibulo-ocular and optokinetic response eye movements and the role of the flocculo-olivary system revealed by chemical lesions

The dynamic characteristics of reflex eye movements were measured in two strains of chronically prepared mice by using an infrared television camera system. The horizontal vestibulo-ocular reflex (HVOR) and horizontal optokinetic response (HOKR) were induced by sinusoidal oscillations of a turntable, in darkness, by 10° (peak to peak) at 0.11–0.50 Hz and of a checked-pattern screen, in light, by 5–20°at 0.11–0.17 Hz, respectively. The gains and phases of the HVOR and HOKR of the C57BL/6 mice were nearly equivalent to those of rabbits and rats, whereas the 129/Sv mice exhibited very low gains in the HVOR and moderate phase lags in the HOKR, suggesting an inherent sensory-motor anomaly. Adaptability of the HOKR was examined in C57BL/6 mice by sustained screen oscillation. When the screen was oscillated by 10° at 0.17 Hz, which induced sufficient retinal slips, the gain of the HOKR increased by 0.08 in 1 h on average, whereas the stimuli that induced relatively small or no retinal slips affected the gain very little. Lesions of the flocculi induced by local applications of 0.1% ibotenic acid and lesions of the inferior olivary nuclei induced by i.p. injection of 3-acetylpyridine in C57BL/6 mice little affected the dynamic characteristics of the HVOR and HOKR, but abolished the adaptation of the HOKR. These results indicate that the olivo-floccular system plays an essential role in the adaptive control of the ocular reflex in mice, as suggested in other animal species. The data presented provide the basis for analyzing the reflex eye movements of genetically engineered mice.

Comparison of the ion channel characteristics of proapoptotic BAX and antiapoptotic BCL-2

The BCL-2 family of proteins is composed of both pro- and antiapoptotic regulators, although its most critical biochemical functions remain uncertain. The structural similarity between the BCL-XL monomer and several ion-pore-forming bacterial toxins has prompted electrophysiologic studies. Both BAX and BCL-2 insert into KCl-loaded vesicles in a pH-dependent fashion and demonstrate macroscopic ion efflux. Release is maximum at ≈pH 4.0 for both proteins; however, BAX demonstrates a broader pH range of activity. Both purified proteins also insert into planar lipid bilayers at pH 4.0. Single-channel recordings revealed a minimal channel conductance for BAX of 22 pS that evolved to channel currents with at least three subconductance levels. The final, apparently stable BAX channel had a conductance of 0.731 nS at pH 4.0 that changed to 0.329 nS when shifted to pH 7.0 but remained mildly Cl− selective and predominantly open. When BAX-incorporated lipid vesicles were fused to planar lipid bilayers at pH 7.0, a Cl−-selective (PK/PCl = 0.3) 1.5-nS channel displaying mild inward rectification was noted. In contrast, BCL-2 formed mildly K+-selective (PK/PCl = 3.9) channels with a most prominent initial conductance of 80 pS that increased to 1.90 nS. Fusion of BCL-2-incorporated lipid vesicles into planar bilayers at pH 7.0 also revealed mild K+ selectivity (PK/PCl = 2.4) with a maximum conductance of 1.08 nS. BAX and BCL-2 each form channels in artificial membranes that have distinct characteristics including ion selectivity, conductance, voltage dependence, and rectification. Thus, one role of these molecules may include pore activity at selected membrane sites.

Relationships of dehydroepiandrosterone sulfate in the elderly with functional, psychological, and mental status, and short-term mortality: A French community-based study

In human beings of both sexes, dehydroepiandrosterone sulfate (DHEAS) circulating in blood is mostly an adrenally secreted steroid whose serum concentration (in the micromolar range and 30–50% higher in men than in women) decreases with age, toward ≈20–10% of its value in young adults during the 8th and 9th decades. The mechanism of action of DHEA and DHEAS is poorly known and may include partial transformation into sex steroids, increase of bioavailable insulin-like growth factor I, and effects on neurotransmitter receptors. Whether there is a cause-to-effect relationship between the decreasing levels of DHEAS with age and physiological and pathological manifestations of aging is still undecided, but this is of obvious theoretical and practical interest in view of the easy restoration by DHEA administration. Here we report on 622 subjects over 65 years of age, studied for the 4 years since DHEAS baseline values had been obtained, in the frame of the PAQUID program, analyzing the functional, psychological, and mental status of a community-based population in the south-west of France. We confirm the continuing decrease of DHEAS serum concentration with age, more in men than in women, even if men retain higher levels. Significantly lower values of baseline DHEAS were recorded in women in cases of functional limitation (Instrumental Activities of Daily Living), confinement, dyspnea, depressive symptomatology, poor subjective perception of health and life satisfaction, and usage of various medications. In men, there was a trend for the same correlations, even though not statistically significant in most categories. No differences in DHEAS levels were found in cases of incident dementia in the following 4 years. In men (but not in women), lower DHEAS was significantly associated with increased short-term mortality at 2 and 4 years after baseline measurement. These results, statistically established by taking into account corrections for age, sex, and health indicators, suggest the need for further careful trials of the administration of replacement doses of DHEA in aging humans. Indeed, the first noted results of such “treatment” are consistent with correlations observed here between functional and psychological status and endogenous steroid serum concentrations.

Conserved characteristics of heterochromatin-forming DNA at the 15q11-q13 imprinting center

Nuclear matrix binding assays (NMBAs) define certain DNA sequences as matrix attachment regions (MARs), which often have cis-acting epigenetic regulatory functions. We used NMBAs to analyze the functionally important 15q11-q13 imprinting center (IC). We find that the IC is composed of an unusually high density of MARs, located in close proximity to the germ line elements that are proposed to direct imprint switching in this region. Moreover, we find that the organization of MARs is the same at the homologous mouse locus, despite extensive divergence of DNA sequence. MARs of this size are not usually associated with genes but rather with heterochromatin-forming areas of the genome. In contrast, the 15q11-q13 region contains multiple transcribed genes and is unusual for being subject to genomic imprinting, causing the maternal chromosome to be more transcriptionally silent, methylated, and late replicating than the paternal chromosome. We suggest that the extensive MAR sequences at the IC are organized as heterochromatin during oogenesis, an organization disrupted during spermatogenesis. Consistent with this model, multicolor fluorescence in situ hybridization to halo nuclei demonstrates a strong matrix association of the maternal IC, whereas the paternal IC is more decondensed, extending into the nuclear halo. This model also provides a mechanism for spreading of the imprinting signal, because heterochromatin at the IC on the maternal chromosome may exert a suppressive position effect in cis. We propose that the germ line elements at the 15q11-q13 IC mediate their effects through the candidate heterochromatin-forming DNA identified in this study.

Characteristics of virus-specific CD8+ T cells in the liver during the control and resolution phases of influenza pneumonia

Dissection of the primary and secondary response to an influenza A virus established that the liver contains a substantial population of CD8+ T cells specific for the immunodominant epitope formed by H-2Db and the influenza virus nucleoprotein peptide fragment NP366–374 (DbNP366). The numbers of CD8+ DbNP366+ cells in the liver reflected the magnitude of the inflammatory process in the pneumonic lung, though replication of this influenza virus is limited to the respiratory tract. Analysis of surface phenotypes indicated that the liver CD8+ DbNP366+ cells tended to be more “activated” than the set recovered from lymphoid tissue but generally less so than those from the lung. The distinguishing characteristic of the lymphocytes from the liver was that the prevalence of the CD8+ DbNP366+ set was always much higher than the percentage of CD8+ T cells that could be induced to synthesize interferon γ after short-term, in vitro stimulation with the NP366–374 peptide, whereas these values were generally comparable for virus-specific CD8+ T cells recovered from other tissue sites. Also, the numbers of apoptotic CD8+ T cells were higher in the liver. The results overall are consistent with the idea that antigen-specific CD8+ T cells are destroyed in the liver during the control and resolution phases of this viral infection, though this destruction is not necessarily an immediate process.

The Escherichia coli MG1655 in silico metabolic genotype: Its definition, characteristics, and capabilities

The Escherichia coli MG1655 genome has been completely sequenced. The annotated sequence, biochemical information, and other information were used to reconstruct the E. coli metabolic map. The stoichiometric coefficients for each metabolic enzyme in the E. coli metabolic map were assembled to construct a genome-specific stoichiometric matrix. The E. coli stoichiometric matrix was used to define the system's characteristics and the capabilities of E. coli metabolism. The effects of gene deletions in the central metabolic pathways on the ability of the in silico metabolic network to support growth were assessed, and the in silico predictions were compared with experimental observations. It was shown that based on stoichiometric and capacity constraints the in silico analysis was able to qualitatively predict the growth potential of mutant strains in 86% of the cases examined. Herein, it is demonstrated that the synthesis of in silico metabolic genotypes based on genomic, biochemical, and strain-specific information is possible, and that systems analysis methods are available to analyze and interpret the metabolic phenotype.

Multiple independent origins of Shigella clones of Escherichia coli and convergent evolution of many of their characteristics

The evolutionary relationships of 46 Shigella strains representing each of the serotypes belonging to the four traditional Shigella species (subgroups), Dysenteriae, Flexneri, Boydii, and Sonnei, were determined by sequencing of eight housekeeping genes in four regions of the chromosome. Analysis revealed a very similar evolutionary pattern for each region. Three clusters of strains were identified, each including strains from different subgroups. Cluster 1 contains the majority of Boydii and Dysenteriae strains (B1–4, B6, B8, B10, B14, and B18; and D3–7, D9, and D11–13) plus Flexneri 6 and 6A. Cluster 2 contains seven Boydii strains (B5, B7, B9, B11, B15, B16, and B17) and Dysenteriae 2. Cluster 3 contains one Boydii strain (B12) and the Flexneri serotypes 1–5 strains. Sonnei and three Dysenteriae strains (D1, D8, and D10) are outside of the three main clusters but, nonetheless, are clearly within Escherichia coli. Boydii 13 was found to be distantly related to E. coli. Shigella strains, like the other pathogenic forms of E. coli, do not have a single evolutionary origin, indicating convergent evolution of Shigella phenotypic properties. We estimate the three main Shigella clusters to have evolved within the last 35,000 to 270,000 years, suggesting that shigellosis was one of the early infectious diseases of humans.

Antigenicity of fullerenes: Antibodies specific for fullerenes and their characteristics

The recent interest in using Buckminsterfullerene (fullerene) derivatives in biological systems raises the possibility of their assay by immunological procedures. This, in turn, leads to the question of the ability of these unprecedented polygonal structures, made up solely of carbon atoms, to induce the production of specific antibodies. Immunization of mice with a C60 fullerene derivative conjugated to bovine thyroglobulin yielded a population of fullerene-specific antibodies of the IgG isotype, showing that the immune repertoire was diverse enough to recognize and process fullerenes as protein conjugates. The population of antibodies included a subpopulation that crossreacted with a C70 fullerene as determined by immune precipitation and ELISA procedures. These assays were made possible by the synthesis of water-soluble fullerene derivatives, including bovine and rabbit serum albumin conjugates and derivatives of trilysine and pentalysine, all of which were characterized as to the extent of substitution and their UV-Vis spectra. Possible interactions of fullerenes with the combining sites of IgG are discussed based on the physical chemistry of fullerenes and previously described protein-fullerene interactions. They remain to be confirmed by the isolation of mAbs for x-ray crystallographic studies.

Characteristics of glycine receptors expressed by embryonic rat brain mRNAs

A study was made of glycine (Gly) and γ-aminobutyric acid (GABA) receptors expressed in Xenopus oocytes injected with rat mRNAs isolated from the encephalon, midbrain, and brainstem of 18-day-old rat embryos. In oocytes injected with encephalon, midbrain, or brainstem mRNAs, the Gly-current amplitudes (membrane current elicited by Gly; 1 mM Gly) were respectively 115 ± 35, 346 ± 28, and 389 ± 22 nA, whereas the GABA-currents (1 mM GABA) were all ≤40 nA. Moreover, the Gly-currents desensitized faster in oocytes injected with encephalon or brainstem mRNAs. The EC50 for Gly was 611 ± 77 μM for encephalon, 661 ± 28 μM for midbrain, and 506 ± 18 μM for brainstem mRNA-injected oocytes, and the corresponding Hill coefficients were all ≈2. Strychnine inhibited all of the Gly-currents, with an IC50 of 56 ± 3 nM for encephalon, 97 ± 4 nM for midbrain, and 72 ± 4 nM for brainstem mRNAs. During repetitive Gly applications, the Gly-currents were potentiated by 1.6-fold for encephalon, 2.1-fold for midbrain, and 1.3-fold for brainstem RNA-injected oocytes. Raising the extracellular Ca2+ concentration significantly increased the Gly-currents in oocytes injected with midbrain and brainstem mRNAs. Reverse transcription–PCR studies showed differences in the Gly receptor (GlyR) α-subunits expressed, whereas the β-subunit was present in all three types of mRNA. These results indicate differential expression of GlyR mRNAs in the brain areas examined, and these mRNAs lead to the expression of GlyRs that have different properties. The modulation of GlyRs by Ca2+ could play important functions during brain development.

Characteristics of the deep ocean carbon system during the past 150,000 years: ΣCO2 distributions, deep water flow patterns, and abrupt climate change

Studies of carbon isotopes and cadmium in bottom-dwelling foraminifera from ocean sediment cores have advanced our knowledge of ocean chemical distributions during the late Pleistocene. Last Glacial Maximum data are consistent with a persistent high-ΣCO2 state for eastern Pacific deep water. Both tracers indicate that the mid-depth North and tropical Atlantic Ocean almost always has lower ΣCO2 levels than those in the Pacific. Upper waters of the Last Glacial Maximum Atlantic are more ΣCO2-depleted and deep waters are ΣCO2-enriched compared with the waters of the present. In the northern Indian Ocean, δ13C and Cd data are consistent with upper water ΣCO2 depletion relative to the present. There is no evident proximate source of this ΣCO2-depleted water, so I suggest that ΣCO2-depleted North Atlantic intermediate/deep water turns northward around the southern tip of Africa and moves toward the equator as a western boundary current. At long periods (>15,000 years), Milankovitch cycle variability is evident in paleochemical time series. But rapid millennial-scale variability can be seen in cores from high accumulation rate series. Atlantic deep water chemical properties are seen to change in as little as a few hundred years or less. An extraordinary new 52.7-m-long core from the Bermuda Rise contains a faithful record of climate variability with century-scale resolution. Sediment composition can be linked in detail with the isotope stage 3 interstadials recorded in Greenland ice cores. This new record shows at least 12 major climate fluctuations within marine isotope stage 5 (about 70,000–130,000 years before the present).

Euler characteristics and elliptic curves

Let E be a modular elliptic curve over ℚ, without complex multiplication; let p be a prime number where E has good ordinary reduction; and let F∞ be the field obtained by adjoining to ℚ all p-power division points on E. Write G∞ for the Galois group of F∞ over ℚ. Assume that the complex L-series of E over ℚ does not vanish at s = 1. If p ⩾ 5, we make a precise conjecture about the value of the G∞-Euler characteristic of the Selmer group of E over F∞. If one makes a standard conjecture about the behavior of this Selmer group as a module over the Iwasawa algebra, we are able to prove our conjecture. The crucial local calculations in the proof depend on recent joint work of the first author with R. Greenberg.