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Physicochemical and serological studies on immunoglobulins, their Fab and Fc fragments, L and H chains, showed that the strongest differences occurred in case of IgG2, and that the differences were localized in the Fc region.
The scavenger receptor, class B, type 1 receptor (SR-BI) mediates the selective transport of lipids from high density lipoprotein to cells. We describe the structure and subchromosomal location of human SR-BI and provide evidence that it is regulated by the transcription factor, steroidogenic factor 1 (SF-1). SR-BI resides on chromosome 12q24.2-qter, spans approximately 75 kilobase pairs, and contains 13 exons. RNA blot analysis of human tissues reveals an expression pattern similar to that described previously for rodents with the highest levels of mRNA in the adrenal gland, ovary, and liver. Unlike rodents, human SR-BI was expressed at high levels in the placenta. The transcription start site for SR-BI was mapped, and DNA sequence analysis revealed a binding site for SF-1 in the proximal 5'-flanking sequence. SF-1, an orphan member of the nuclear hormone receptor gene family, plays a key role in the regulation of steroidogenesis and is expressed at high levels in steroidogenic tissues. SF-1 binds to the SR-BI promoter in a sequence-specific manner, and efficient transcription from this promoter in adrenocortical Y1 cells is dependent on an intact SF-1 site. These data extend our understanding of SF-1 function within steroidogenic tissues and suggest that SR-BI, which serves to supply selected tissues with lipoprotein-derived lipids, is part of the repertoire of SF-1-responsive genes involved in steroidogenesis.
Lipid-water mixtures were studied by X-ray cryo-diffraction in order to assess the structural changes during freezing. We show that the water of aqueous lipid phases, in the concentration range of 10-30% (water weight/total weight), is vitrified by high-pressure freezing. Vitrified lipid phases can be cryo-sectioned and imaged by electron cryo-microscopy. Both the ideal or average and the real or local structures of the lipid mixtures can be studied at a resolution better than 2 nm. While the average structure of the lipid phases is in good agreement with that determined by X-ray diffraction, the local structure reveals features that might play an important role in the function of biological membranes such as in endo- and exocytosis.
Two simplifying assumptions are frequently used in the biophysical chemistry of aqueous solutions: (i) a dielectric mediates the interactions of polar and ionic molecules in aqueous phases and (ii) the dielectric constant of this medium is high and uniform up to molecular surfaces. Because of their great utility in rationalizing simple electrostatic and dielectric effects in such polar systems, it is important to examine whether these assumptions also lead to deductions that are locally consistent with the solvent restructuring observed in hydrophobic phenomena. In this paper, using a model polar fluid system, these macroscopic assumptions are applied to the rigorous, microscopic nonlinear integral equation for Wki, the potential of mean force between two adjacent polar molecules. In systems of high dielectric constant, linearization of Boltzmann exponentials and approximation of three-molecule potentials of mean force by superposition of two-molecule potentials permit reduction to a linear integral equation for Wki. It is shown that the strictly local electrostatic contributions to Wki exert an effect that is qualitatively similar to the global screening effect of a dielectric medium. Through the relation between Wki and configurational probabilities, it is further found that reducing the polarity of a molecule in a polar fluid shifts local pair probability density from energetically unfavorable to energetically favorable two-molecule configurations. This general effect, which clearly promotes local structure, would augment more specific hydrophobic mechanisms in aqueous systems. Thus, the assumptions upon which the highly successful Debye-Hückel and Onsager models are supported lead also to deductions about local structure that are consistent with hydrophobic structure enhancement.
Archaeal-type rhodopsins can accommodate either all-trans- or 13-cis,15-syn-retinal in their chromophore binding site in the dark, but only the former isomer is functionally important. In contrast, Anabaena sensory rhodopsin (ASR), an archaeal-type rhodopsin found in eubacteria, exhibits a photochromic interconversion of both forms, suggesting that ASR functions as a photosensor which interacts with its 14 kDa soluble transducer differently in the all-trans and 13-cis,15-syn forms. In this study, we applied low-temperature Fourier transform infrared (FTIR) spectroscopy to the 13-cis,15-syn form of ASR (13C-ASR) at 77 K and compared the local structure around the chromophore and its structural changes upon retinal photoisomerization with those of the all-trans form (AT-ASR) [Furutani, Y., Kawanabe, A., Jung, K. H., and Kandori, H. (2005) Biochemistry 44, 12287-12296]. By use of [zeta-15N]lysine-labeled ASR, we identified the N-D stretching vibrations of the Schiff base (in D2O) at 2165 cm(-1) for 13C-ASR and at 2163 and 2125 cm(-1) for AT-ASR. The frequencies indicate strong hydrogen bonds of the Schiff base with a water molecule for both 13C-ASR and AT-ASR. In contrast, the N-D stretching vibration appears at 2351 cm(-1) and at 2483 cm(-1) for the K states of 13C-ASR (13C-ASR(K)) and AT-ASR (AT-ASR(K)), respectively, indicating that the Schiff base still forms a hydrogen bond in 13C-ASR(K). Rotational motion of the Schiff base upon retinal isomerization is probably smaller for 13C-ASR than for AT-ASR, the latter altering hydrogen bonding of the Schiff base similar to bacteriorhodopsin (BR), a light-driven proton pump. Appearance of several hydrogen-out-of-plane vibrations and amide I vibrations in 13C-ASR(K), but not in AT-ASR(K), suggests that structural changes are distributed widely along the polyene chain for 13C-ASR. On the other hand, retinal photoisomerization in AT-ASR breaks the hydrogen bond of the Schiff base, and localized structural changes in the Schiff base region are induced.
Plague, the disease caused by the bacterium Yersinia pestis, has greatly impacted human civilization. Y. pestis is a successful global pathogen, with active foci on all continents except Australia and Antarctica. Because the Y. pestis genome is highly monomorphic, previous attempts to characterize the population genetic structure within a single focus have been largely unsuccessful. Here we report that highly mutable marker loci allow determination of Y. pestis population genetic structure and tracking of transmission patterns at two spatial scales within a single focus. In addition, we found that in vitro mutation rates for these loci are similar to those observed in vivo, which allowed us to develop a mutation-rate-based model to examine transmission mechanisms. Our model suggests there are two primary components of plague ecology: a rapid expansion phase for population growth and dispersal followed by a slower persistence phase. This pattern seems consistent across local, regional, and even global scales.
Homozygotes of the mutant mouse spastic exhibit reduced binding of 3H-strychnine to homogenates from various regions of the CNS compared with unaffected littermates (White and Heller, 1982). Here we report evidence that the spastic mutation coincides with a reduced concentration and an unaltered structure of the glycine receptor in spinal cord. Scatchard analysis of 3H-strychnine binding revealed a single binding site with a Bmax of 267 +/- 62 fmol/mg protein for spastic and of 864 +/- 220 fmol/mg protein for control mice; no difference was found for the corresponding KD values. Also Ki values of glycine for 3H-strychnine binding and displacement of 3H-strychnine by beta-alanine and taurine were indistinguishable for both preparations. Photoaffinity labeling of synaptic membranes with 3H-strychnine identified an Mr = 48,000 polypeptide in both control and spastic mouse membranes. Tryptic digestion of these membranes produced radiolabeled peptide fragments of identical molecular weights, suggesting that the proteolytic cleavage sites around the antagonist binding site are conserved in the mutant glycine receptor protein. Glycine receptors from both control and mutant mice were purified by affinity chromatography on aminostrychnine agarose. SDS/PAGE revealed three polypeptides of Mr = 48,000, 58,000, and 93,000 in both receptor preparations. Monoclonal antibodies directed against different subunits of the glycine receptor were applied to an enzyme-linked immunosorbent assay. The same pattern of immunoreactivity was obtained for glycine receptor from spinal cord of spastic homozygotes, control mice, and rats, suggesting conservation of the antigenic epitopes in the mutant receptor.(ABSTRACT TRUNCATED AT 250 WORDS)
We have performed molecular-dynamics simulations of CO(2) system along the gas-liquid coexistence curve and on the isochore 94.22 cm(3) mol(-1) (which corresponds to the critical isochore). The calculation has been carried out in order to analyze the diffusion of CO(2) and particularly to figure out how the diffusion coefficient may be decomposed along the molecular axes. This makes it possible to analyze the anisotropy of the diffusion along these axes and to shed light on the microscopic changes which accompany such behavior. This anisotropy is traced back to the effect of the translation-rotation coupling (TRC) along the molecular axes. Along the liquid-gas coexistence curve, the pseudolongitudinal diffusion is found to be more rapid than the transverse one. The opposite trend is found along the isochore 94.22 cm(3) mol(-1). The role of the local structure was explored by calculating intermediate scattering function and the autocorrelation functions for the forces acting along the molecular axes. It is shown that the strength of the TRC effect is correlated to the difference between the relaxation times of the local structure, that of the reorientation along the molecular axes, and that of the translational motion. The analysis of the correlation time and the average mean square force along the longitudinal and transverse directions confirms the anisotropy of the local environment that determines the translational dynamics of a molecule.
We report results from molecular dynamics simulations of the freezing transition of TIP5P water molecules confined between two parallel plates under the influence of a homogeneous external electric field, with magnitude of 5 V/nm, along the lateral direction. For water confined to a thickness of a trilayer we find two different phases of ice at a temperature of T=280 K. The transformation between the two, proton-ordered, ice phases is found to be a strong first-order transition. The low-density ice phase is built from hexagonal rings parallel to the confining walls and corresponds to the structure of cubic ice. The high-density ice phase has an in-plane rhombic symmetry of the oxygen atoms and larger distortion of hydrogen bond angles. The short-range order of the two ice phases is the same as the local structure of the two bilayer phases of liquid water found recently in the absence of an electric field [J. Chem. Phys. 119, 1694 (2003)]. These high- and low-density phases of water differ in local ordering at the level of the second shell of nearest neighbors. The results reported in this paper, show a close similarity between the local structure of the liquid phase and the short-range order of the corresponding solid phase. This similarity might be enhanced in water due to the deep attractive well characterizing hydrogen bond interactions. We also investigate the low-density ice phase confined to a thickness of 4, 5, and 8 molecular layers under the influence of an electric field at T=300 K. In general, we find that the degree of ordering decreases as the distance between the two confining walls increases.
PURPOSE: Robot assisted prostatectomy (RAP) is more costly than traditional radical retropubic prostatectomy (RRP) under the cost structures at certain hospitals. However, this finding may not be the case in all care settings. We investigated the sensitivity of RAP and RRP inpatient costs to variations in length of stay (LOS), local hospitalization costs and robotic case volume in the specialist and generalist settings. MATERIALS AND METHODS: We developed a model of RAP vs RRP costs in the specialist and generalist settings using published data on operative time and LOS, and cost data from our academic medical center. All inpatient cost centers were included, namely surgery costs, professional fees, postoperative care, robotic equipment and service. Extensive 1 and 2-way sensitivity analyses were performed. RESULTS: Our base case model demonstrated a cost premium for RAP vs RRP of USD $783 and $195 in the specialist and generalist settings, respectively. Sensitivity analysis of our model assumptions demonstrated that RAP could achieve cost equivalence with RRP at a surgical volume of 10 cases weekly. If case volume increased to 14 cases weekly, RAP would be less expensive than RRP in some practice settings in which RAP LOS was less than 1.5 days. CONCLUSIONS: The inpatient costs of robotic assisted prostatectomy are volume dependent and cost equivalence with generalist radical retropubic prostatectomy is possible at higher volume RAP specialty centers. While RAP may be cost competitive with RRP at high cost hospitals or high volume RAP specialist centers, this procedure would exist at a cost premium to RRP in other practice settings.
Analysis of the Sephacryl S-200 fractionated type IV collagen domains from bovine and human glomerular basement membranes (GBM) and calf anterior lens capsule (ALC) indicated that Asn-linked oligosaccharides are primarily or exclusively localized in the 7 S region, whereas the hydroxylysine-linked Glc alpha 1----2Gal disaccharides (Glc-Gal-Hyl) are present in all the major segments of the molecule (7 S, NC1, and helical domain); no Ser/Thr-linked saccharide were detected. The Asn-linked carbohydrate units observed in the 7 S domain (Mr approximately 300,000) occurred in a number equal to the 12 polypeptide chains constituting this cross-linked region, and this was consistent with lectin blots of the reduced electrophoretically resolved 7 S components. Fractionation of the N-glycanase and endo-beta-N-acetylglucosaminidase-released oligosaccharides by concanavalin A affinity and high performance liquid chromatography indicated that the Asn-linked carbohydrate occurred predominantly in the form of complex tri- and biantennary units, although submolar amounts of polymannose variants (Man5-7GlcNAc2) were also present in calf ALC and bovine GBM. Structural studies of the complex N-linked oligosaccharides employing hydrazine/nitrous acid fragmentation and glycosidase digestions indicated a pattern in which there was complete fucosylation of the innermost GlcNAc residue of the Man3GlcNAc2 core but only sparse substitution with capping groups of the nonrepeating N-acetyllactosamine branches. Whether tri- or biantennary, the oligosaccharides from bovine GBM contained only one capping residue, in the form of either NeuAc or alpha-D-Gal, whereas those from ALC had only a single alpha-D-Gal and no NeuAc; human GBM oligosaccharides were devoid of both NeuAc and alpha-D-Gal. The absence of terminal alpha-D-Gal in the human 7 S domain was reflected in its lack of reactivity with Bandeiraea simplicifolia I and from its failure to yield Gal alpha 1----3Gal beta 1----4 [3H]anhydromannitol after hydrazine/nitrous acid/NaB3H4 treatment. Application of the latter procedure to the collagen domains yielded, in addition to fragments from the N-linked oligosaccharides, a disaccharide (Glc alpha 1----2[3H]galactitol) derived from the Glc-Gal-Hyl units. The localization of Asn-linked carbohydrate units in the evolutionarily conserved 7S domain of type IV collagens suggests that these oligosaccharides may play a role in the assembly of the collagen network of basement membranes.
CD53 is a pan-leukocyte surface glycoprotein which spans the plasma membrane four times and is a member of the transmembrane 4 superfamily (TM4SF). The protein sequence and gene structure of mouse CD53 (Cd53) were determined by isolation of both genomic and cDNA clones. CD53 is highly conserved in evolution, as mouse Cd53 was 91% identical to rat CD53 and 82% identical to human CD53. The mouse Cd53 gene spanned approximately 9.0 kb of DNA and encoded the 219 amino acid residues of CD53 over seven exons. The Cd53 gene produced a 1.8 kb transcript which was dramatically upregulated after cell activation. The mouse Cd53 gene was mapped to chromosome 3, whereas the locus of another TM4SF member, CD37 (Cd37), was mapped to mouse chromosome 7. Three lines of evidence suggest that the TM4SF arose divergently from an ancestral gene. First, the gene structure of CD53 was strikingly similar to two other members of the TM4SF, CD63 and TAPA-1; second, Cd37 mapped to the same chromosome as Tapa-1; and, third, Cd37 mapped to a segment of chromosome 7 that contains a number of genes that are structurally or functionally related to genes closely linked to Cd53 on chromosome 3.
The human language faculty has been claimed to be grounded in the ability to process hierarchically structured sequences. This human ability goes beyond the capacity to process sequences with simple transitional probabilities of adjacent elements observable in non-human primates. Here we show that the processing of these two sequence types is supported by different areas in the human brain. Processing of local transitions is subserved by the left frontal operculum, a region that is phylogenetically older than Broca's area, which specifically holds responsible the computation of hierarchical dependencies. Tractography data revealing differential structural connectivity signatures for these two brain areas provide additional evidence for a segregation of two areas in the left inferior frontal cortex.
The Fau gene is the cellular homolog of the fox sequence of the Finkel-Biskis-Reilly murine sarcoma virus (FBR-MuSV). FBR-MuSV acquired the Fau gene by transduction in a transcriptional orientation opposite to that of the genomic Fau gene. The genomic structure of the mouse Fau gene (MMFAU) and its upstream elements have been determined and are similar to those of the human FAU gene. The gene consists of five exons and is located on chromosome 19. The first exon is not translated. The promoter region has no well-defined TATA box but contains the polypyrimidine initiator flanked by regions of high GC content (65%) and shows all of the characteristics of a housekeeping gene. The 5' end of the mRNA transcript was determined by 5' RACE analysis and is located, as expected, in the polypyrimidine initiator site. Furthermore, the sequences of two retropseudogenes (Fau-ps1 and Fau-ps2) are reported. Both pseudogenes are approximately 75% identical to the Fau cDNA, but both are shorter due to a deletion at the 5' end and do not encode a functional protein. Fau-prs is interrupted by an AG-rich region of about 350 bp within the S30 region of the Fau cDNA. Fau-ps1 was localized on chromosome 1 and Fau-ps2 on chromosome 7.
In studying chimeras of NR2A and NR2C subunits of the NMDA receptor, we have found that glycine-independent desensitization depends on two regions of the extracellular N-terminal domain. One corresponds to a stretch of approximately 190 amino acids preceding the glutamate-binding domain S1. The other localizes at the interface between the N-terminal segment and the first transmembrane domain of NR2A subunits and involves A555 and S556. Both regions support desensitization in the absence of the other with different time courses. Desensitization did not develop with time in receptors containing the entire N-terminal region of NR2C. The introduction of A555 into the corresponding position of NR2C subunits enabled the receptors to manifest time-dependent increase in desensitization. Thus, this determinant behaves as an allosteric effector for glycine-independent desensitization.
Prediction of the degree of drug-like character in small molecules is of great industrial interest. The major barrier, however, is the lack of a definition for drug-like character. We used the concept of the multilevel chemical compatibility (MLCC) between a compound and a drug library as a measure of the drug-like character of a compound. The rationale is that the local chemical environment of each atom or group of atoms in a compound largely contributes to the stability, toxicity, and metabolism in vivo. A systematic comparison of the local environments within a compound and those within the existing drugs provides a basis for determining whether and how much a compound is drug-like. We applied the MLCC calculations to four test sets: top selling drugs, compounds under biological testing prior to the preclinical test, anticancer drugs, and compounds known to have poor drug-like character. The following conclusions were obtained: (1) A convergent number of unique local structure types were found in the analysis of the library of the existing drugs. It suggests that the current drug library contains about 80% of all the viable types; therefore, discovery of a drug with new local structures is only an event of relatively small probability. (2) The method is highly selective in discerning drug-like compounds: most of the top drugs are predicted to be drug-like, about one-quarter of the biological testing compounds are drug-like, and about one-fifth of the anticancer drugs are drug-like. (3) The method also correctly predicted that none of the known problematic compounds are drug-like. (4) The method is fast enough for computational screening of virtual combinatorial chemistry libraries and databases of available compounds.
The germline DNA sequence of the human CD79 alpha (Ig-alpha/mb-1) gene was determined by polymerase chain reaction sequencing of a cosmid clone derived from an arrayed human chromosome 19 library. The CD79 alpha gene was localized to chromosome 19q13.2; this localization places the gene within the CEA-like gene cluster with the following gene order: -CEA-CGM1-CD79 alpha-RPS11-ATP1A3-BGP-CGM9-. The genomic organization of the human CD79 alpha gene resembles the mouse counterpart with five exons interrupted by four introns. Computer analyses suggest the presence of transcription regulatory elements known to be important in the regulation of mouse CD79 alpha (AP-1, EBF, AP-2, MUF2, and SP-1 sites), as well as elements not found in the mouse gene (an NK-kappa B binding site and a series of E-box motifs). Similar to the mouse gene, the 5' flanking region of human CD79 alpha lacks a TATA box; however, unlike mouse CD79 alpha, a classical octamer motif could not be identified in the human gene. Finally, a new Rsa I restriction fragment length polymorphism was defined in the non-coding regions of the human gene.
Insulin-like growth factors (IGFs) are peptide mitogens, structurally related to insulin, whose biological actions in the CNS are incompletely known. The retina is largely uncharacterized with respect to IGF receptors. We, therefore, studied IGF receptors in bovine and murine retinal tissues by immunohistochemistry, autoradiographic localization, and affinity labeling. Notable IGF-II receptor immunoreactivity was found in retinal pigment epithelium (RPE), with intermediate levels in choroid, low levels in the inner and outer plexiform layers and outer nuclear layer, and very low levels in other regions. Autoradiographic localization using [125I]IGF-II confirmed the IGF-II receptor immunohistochemistry. Autoradiographic localization using [125I]IGF-I labeled the nuclear layers and the photoreceptor region. Affinity labeling disclosed differences in the apparent mol wt of IGF-I and IGF-II receptors from bovine eye tissues and those from liver and brain. IGF-I receptor alpha-subunits (the IGF-binding subunit) migrated at: liver, 139,000; brain, 125,000; RPE, 125,000 and 135,000 (two sizes); and retina, 125,000 and 135,000. IGF-II receptors migrated at: liver, 245,000; brain, 235,000; RPE, 240,000; and retina, 230,000. We conclude that mammalian retina contains both IGF-I and -II receptors, which differ from those found in other tissues and have a characteristic spatial distribution within the retina.