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1.
In this work, it was investigated the effect of different moisture contents on PVA-gelatin films by means of dielectric properties, infrared spectroscopy, microwave response and gravimetric method. The films were elaborated from a blend of gelatin and PVA, with 0 and 25?% glycerol. The sorption isotherms were determined by gravimetric methods, at 25?°C. A capacimeter was used for dielectric measurements, and a device called SOLFAN setup was used for microwave measurements. The sorption isotherms were markedly affected by the glycerol content and relative humidity, due to the hygroscopic nature of the films. The dielectric constant and the microwave response signal were also strongly affected by the moisture and glycerol content in the films. Finally, Infrared spectra showed some changes in the amide peak positions, attributed to the modifications in the interactions between the macromolecules. The behaviors obtained in this work were explained on the basis the way the water enters in the film matrix. 相似文献
2.
Amsden JJ Kralj JM Bergo VB Spudich EN Spudich JL Rothschild KJ 《Biochemistry》2008,47(44):11490-11498
We examine the structural changes during the primary photoreaction in blue-absorbing proteorhodopsin (BPR), a light-driven retinylidene proton pump, using low-temperature FTIR difference spectroscopy. Comparison of the light-induced BPR difference spectrum recorded at 80 K to that of green-absorbing proteorhodopsin (GPR) reveals that there are several differences in the BPR and GPR primary photoreactions despite the similar structure of the retinal chromophore and all-trans --> 13-cis isomerization. Strong bands near 1700 cm(-1) assigned previously to a change in hydrogen bonding of Asn230 in GPR are still present in BPR. However, additional bands in the same region are assigned on the basis of site-directed mutagenesis to changes occurring in Gln105. In the amide II region, bands are assigned on the basis of total (15)N labeling to structural changes of the protein backbone, although no such bands were previously observed for GPR. A band at 3642 cm(-1) in BPR, assigned to the OH stretching mode of a water molecule on the basis of H2(18)O substitution, appears at a different frequency than a band at 3626 cm(-1) previously assigned to a water molecule in GPR. However, the substitution of Gln105 for Leu105 in BPR leads to the appearance of both bands at 3642 and 3626 cm(-1), indicating the waters assigned in BPR and GPR exist in separate distinct locations and can coexist in the GPR-like Q105L mutant of BPR. These results indicate that there exist significant differences in the conformational changes occurring in these two types proteorhodopsin during the initial photoreaction despite their similar chromophore structures, which might reflect a different arrangement of water in the active site as well as substitution of a hydrophilic for hydrophobic residue at residue 105. 相似文献
3.
S. Bergoñón C. Codina J. Bastida F. Viladomat E. Melé 《Plant Cell, Tissue and Organ Culture》1996,45(3):191-199
Galanthamine (GAL) is increasingly used in the treatment of Alzheimer's disease. We have attempted to develop a method of producing this alkaloid using in vitro cultures of Narcissus confusus plants. The “shoot-clump” culture in liquid medium was shown to be an appropriate method for the micropropagation of this bulbous plant. The complete process included three steps:
- culture of “twin-scales” starting from the bulbs;
- culture of the newly formed shoots in a medium for bud proliferation (Murashige Skoog+1 mg l-1 of 2,4-dichlorophenoxyacetic acid+5 mg l-1 of benzyladenine), and
- culture of “shoot-clumps” in a liquid-shake medium. Here we describe the effect of the addition of trans-cinnamic acid, a precursor in the biosynthesis of the Amaryllidaceae alkaloids, on the production of galanthamine and related alkaloids, and also on the growth of the “shoot-clump” culture. The production of galanthamine was found to be inhibited by the addition of the precursor, which promoted the production of the other alkaloid in the same biosynthetic pathway, N-formyl-norgalanthamine. The total production of galanthamine in the control cultures in day-long photoperiod was 2.50 mg per culture, of which 1.97 mg per culture were released into the medium.
4.
Ana C. Coan Brunno M. Campos Clarissa L. Yasuda Bruno Y. Kubota Felipe PG. Bergo Carlos AM. Guerreiro Fernando Cendes 《PloS one》2014,9(1)
Objective
Patients with temporal lobe epilepsy (TLE) with hippocampal sclerosis (HS) have diffuse subtle gray matter (GM) atrophy detectable by MRI quantification analyses. However, it is not clear whether the etiology and seizure frequency are associated with this atrophy. We aimed to evaluate the occurrence of GM atrophy and the influence of seizure frequency in patients with TLE and either normal MRI (TLE-NL) or MRI signs of HS (TLE-HS).Methods
We evaluated a group of 172 consecutive patients with unilateral TLE-HS or TLE-NL as defined by hippocampal volumetry and signal quantification (122 TLE-HS and 50 TLE-NL) plus a group of 82 healthy individuals. Voxel-based morphometry was performed with VBM8/SPM8 in 3T MRIs. Patients with up to three complex partial seizures and no generalized tonic-clonic seizures in the previous year were considered to have infrequent seizures. Those who did not fulfill these criteria were considered to have frequent seizures.Results
Patients with TLE-HS had more pronounced GM atrophy, including the ipsilateral mesial temporal structures, temporal lobe, bilateral thalami and pre/post-central gyri. Patients with TLE-NL had more subtle GM atrophy, including the ipsilateral orbitofrontal cortex, bilateral thalami and pre/post-central gyri. Both TLE-HS and TLE-NL showed increased GM volume in the contralateral pons. TLE-HS patients with frequent seizures had more pronounced GM atrophy in extra-temporal regions than TLE-HS with infrequent seizures. Patients with TLE-NL and infrequent seizures had no detectable GM atrophy. In both TLE-HS and TLE-NL, the duration of epilepsy correlated with GM atrophy in extra-hippocampal regions.Conclusion
Although a diffuse network GM atrophy occurs in both TLE-HS and TLE-NL, this is strikingly more evident in TLE-HS and in patients with frequent seizures. These findings suggest that neocortical atrophy in TLE is related to the ongoing seizures and epilepsy duration, while thalamic atrophy is more probably related to the original epileptogenic process. 相似文献5.
Vladislav B. Bergo Oleg A. Sineshchekov Joel M. Kralj Ranga Partha Elena N. Spudich Kenneth J. Rothschild John L. Spudich 《The Journal of biological chemistry》2009,284(5):2836-2843
Proteorhodopsins (PRs), photoactive retinylidene membrane proteins
ubiquitous in marine eubacteria, exhibit light-driven proton transport
activity similar to that of the well studied bacteriorhodopsin from halophilic
archaea. However, unlike bacteriorhodopsin, PRs have a single highly conserved
histidine located near the photoactive site of the protein. Time-resolved
Fourier transform IR difference spectroscopy combined with visible absorption
spectroscopy, isotope labeling, and electrical measurements of light-induced
charge movements reveal participation of His-75 in the proton translocation
mechanism of PR. Substitution of His-75 with Ala or Glu perturbed the
structure of the photoactive site and resulted in significantly shifted
visible absorption spectra. In contrast, His-75 substitution with a positively
charged Arg did not shift the visible absorption spectrum of PR. The mutation
to Arg also blocks the light-induced proton transfer from the Schiff base to
its counterion Asp-97 during the photocycle and the acid-induced protonation
of Asp-97 in the dark state of the protein. Isotope labeling of histidine
revealed that His-75 undergoes deprotonation during the photocycle in the
proton-pumping (high pH) form of PR, a reaction further supported by results
from H75E. Finally, all His-75 mutations greatly affect charge movements
within the PR and shift its pH dependence to acidic values. A model of the
proteorhodopsin proton transport process is proposed as follows: (i) in the
dark state His-75 is positively charged (protonated) over a wide pH range and
interacts directly with the Schiff base counterion Asp-97; and (ii)
photoisomerization-induced transfer of the Schiff base proton to the Asp-97
counterion disrupts its interaction with His-75 and triggers a histidine
deprotonation.A variety of unicellular microorganisms contain primary proton pumps that
convert solar energy into a transmembrane electrochemical proton gradient,
which is subsequently used by membrane ATP synthases to generate chemical
energy. Well known examples of such pumps are the haloarchaeal rhodopsins,
photoactive, seven-helix membrane proteins, which include the well studied
proton pump bacteriorhodopsin
(BR)4 from
Halobacterium salinarum and BR homologs in other haloarchaea.
Recently, a much larger new family of light-driven proton pumps, the
proteorhodopsins (PRs), was identified in marine proteobacteria throughout the
oceans
(1–3).
Despite the diverse properties of PRs, including different visible absorption
maxima and photocycle rates
(4–6),
they all share with BR several key conserved residues as well as an
all-trans-retinylidene chromophore in their unphotolyzed state, which
is covalently bound to transmembrane helix G via a protonated Schiff base
linkage.Many of the molecular events that occur in PRs following light activation
are similar to those of BR, including an initial ultrafast
all-trans→13-cis-retinal isomerization, which triggers
a sequence of protein conformational changes, including several intramolecular
proton transfer reactions. The two key carboxylate groups involved in proton
pumping in helix C of BR are conserved in PRs, and in the first found and most
commonly studied PR, the Monterey Bay variant eBAC31A08, also known as
green-absorbing proteorhodopsin (GPR), the helix C residues Asp-97 and Glu-108
undergo protonation changes during the photocycle similar to those of the
homologous carboxylate residues in BR. Initial FTIR studies on GPR identified
the role of Asp-97 as the Schiff base counterion and proton acceptor during
Schiff base deprotonation and concomitant M formation and Glu-108 as the
proton donor that reprotonates the Schiff base during N formation
(7,
8). Studies of other variants
indicate these roles of the two carboxylic acid residues are general in the
proteorhodopsin
family.5One major difference between BR and the PRs is the presence of a highly
conserved histidine residue at position 75, near the middle of transmembrane
helix B in the latter pigments. The His-75 homolog is not present in BR nor
thus far found in other microbial rhodopsins
(9). The proximity of His-75 to
the protein active site and specifically to the Schiff base counterion Asp-97
inferred from the x-ray crystal structure of BR suggests its involvement in
spectral tuning of the visible absorption
(10) and potentially PR
photochemical reactions. Because the pKa of histidine in
solution is close to neutral pH
(11), its imidazole group
often plays a major role in intramolecular proton transfers in enzymes,
including NADPH oxidase (12),
alcohol dehydrogenase (13),
carbonic anhydrase II (14),
and serine proteases (15).In this study we have used a combination of time-resolved FTIR difference
spectroscopy, visible absorption spectroscopy, isotope labeling, kinetic
charge displacement measurements, and site-directed mutagenesis to study the
role of His-75 in GPR. We report evidence that protonated His-75 interacts
directly with Asp-97 in the unphotolyzed protein and during the photocycle
undergoes a deprotonation in response to the protonation of Asp-97. 相似文献
6.
7.
Yang SH Chang SY Tu Y Lawson GW Bergo MO Fong LG Young SG 《Journal of lipid research》2012,53(1):77-86
Protein farnesyltransferase (FTase) and protein geranylgeranyltransferase-I (GGTase-I) add 15- or 20-carbon lipids, respectively, to proteins that terminate with a CaaX motif. These posttranslational modifications of proteins with lipids promote protein interactions with membrane surfaces in cells, but the in vivo importance of the CaaX prenyltransferases and the protein lipidation reactions they catalyze remain incompletely defined. One study concluded that a deficiency of FTase was inconsequential in adult mice and led to little or no tissue pathology. To assess the physiologic importance of the CaaX prenyltransferases, we used conditional knockout alleles and an albumin-Cre transgene to produce mice lacking FTase, GGTase-I, or both enzymes in hepatocytes. The hepatocyte-specific FTase knockout mice survived but exhibited hepatocellular disease and elevated transaminases. Mice lacking GGTase-I not only had elevated transaminases but also had dilated bile cannaliculi, hyperbilirubinemia, hepatosplenomegaly, and reduced survival. Of note, GGTase-I-deficient hepatocytes had a rounded shape and markedly reduced numbers of actin stress fibers. Hepatocyte-specific FTase/GGTase-I double-knockout mice closely resembled mice lacking GGTase-I alone, but the disease was slightly more severe. Our studies refute the notion that FTase is dispensable and demonstrate that GGTase-I is crucial for the vitality of hepatocytes. 相似文献
8.
Hutchinson–Gilford progeria syndrome (HGPS) is caused by an LMNA mutation that results in the production of the abnormal progerin protein. Children with HGPS display phenotypes of premature aging and have an average lifespan of 13 years. We found earlier that the targeting of the transmembrane protein PLA2R1 overcomes senescence and improves phenotypes in a mouse model of progeria. PLA2R1 is regulating the JAK/STAT signaling, but we do not yet know whether targeting this pathway directly would influence cellular and in vivo progeria phenotypes. Here, we show that JAK1/2 inhibition with ruxolitinib rescues progerin‐induced cell cycle arrest, cellular senescence, and misshapen nuclei in human normal fibroblasts expressing progerin. Moreover, ruxolitinib administration reduces several premature aging phenotypes: bone fractures, bone mineral content, grip strength, and a trend to increase survival in a mouse model of progeria. Thus, we propose that ruxolitinib, an FDA‐approved drug, should be further evaluated as a drug candidate in HGPS therapy. 相似文献
9.
10.