Biomedical subjects

James W Lewis

Publications and source records attributed to James W Lewis.

14 recordsLinked to original sources

PubMed2006

Proton movement and photointermediate kinetics in rhodopsin mutants.

The role of ionizable amino acid side chains in the bovine rhodopsin activation mechanism was studied in mutants E134Q, E134R/R135E, H211F, and E122Q. All mutants exhibited bathorhodopsin stability on the 30 ns to 1 micros time scale similar to that of the wild type. Lumirhodopsin decay was also similar to that of the wild type except for the H211F mutant where early decay (20 micros) to a second form of lumirhodopsin was seen, followed by formation of an extremely long-lived Meta I(480) product (34 ms), an intermediate which forms to a much reduced extent, if at all, in dodecyl maltoside suspensions of wild-type rhodopsin. A smaller amount of a similar long-lived Meta I(480) product was seen after photolysis of E122Q, but E134Q and E134R/R135Q displayed kinetics much more similar to those of the wild type under these conditions (i.e., no Meta I(480) product). These results support the idea that specific interaction of His211 and Glu122 plays a significant role in deprotonation of the retinylidene Schiff base and receptor activation. Proton uptake measurements using bromcresol purple showed that E122Q was qualitatively similar to wild-type rhodopsin, with at least one proton being released during lumirhodopsin decay per Meta I(380) intermediate formed, followed by uptake of at least two protons per rhodopsin bleached on a time scale of tens of milliseconds. Different results were obtained for H211F, E134Q, and E134R/R135E, which all released approximately two protons per rhodopsin bleached. These results show that several ionizable groups besides the Schiff base imine are affected by the structural changes involved in rhodopsin activation. At least two proton uptake groups and probably at least one proton release group in addition to the Schiff base are present in rhodopsin.

Animals↗

PubMed2006

Rhodopsin photointermediates in two-dimensional crystals at physiological temperatures.

Bovine rhodopsin photointermediates formed in two-dimensional (2D) rhodopsin crystal suspensions were studied by measuring the time-dependent absorbance changes produced after excitation with 7 ns laser pulses at 15, 25, and 35 degrees C. The crystalline environment favored the Meta I(480) photointermediate, with its formation from Lumi beginning faster than it does in rhodopsin membrane suspensions at 35 degrees C and its decay to a 380 nm absorbing species being less complete than it is in the native membrane at all temperatures. Measurements performed at pH 5.5 in 2D crystals showed that the 380 nm absorbing product of Meta I(480) decay did not display the anomalous pH dependence characteristic of classical Meta II in the native disk membrane. Crystal suspensions bleached at 35 degrees C and quenched to 19 degrees C showed that a rapid equilibrium existed on the approximately 1 s time scale, which suggests that the unprotonated predecessor of Meta II in the native membrane environment (sometimes called MII(a)) forms in 2D rhodopsin crystals but that the non-Schiff base proton uptake completing classical Meta II formation is blocked there. Thus, the 380 nm absorbance arises from an on-pathway intermediate in GPCR activation and does not result from early Schiff base hydrolysis. Kinetic modeling of the time-resolved absorbance data of the 2D crystals was generally consistent with such a mechanism, but details of kinetic spectral changes and the fact that the residuals of exponential fits were not as good as are obtained for rhodopsin in the native membrane suggested the photoexcited samples were heterogeneous. Variable fractional bleach due to the random orientation of linearly dichroic crystals relative to the linearly polarized laser was explored as a cause of heterogeneity but was found unlikely to fully account for it. The fact that the 380 nm product of photoexcitation of rhodopsin 2D crystals is on the physiological pathway of receptor activation suggests that determination of its structure would be of interest.

Animals↗

PubMed2006

Lefties get it "right" when hearing tool sounds.

Our ability to manipulate and understand the use of a wide range of tools is a feature that sets humans apart from other animals. In right-handers, we previously reported that hearing hand-manipulated tool sounds preferentially activates a left hemisphere network of motor-related brain regions hypothesized to be related to handedness. Using functional magnetic resonance imaging, we compared cortical activation in strongly right-handed versus left-handed listeners categorizing tool sounds relative to animal vocalizations. Here we show that tool sounds preferentially evoke activity predominantly in the hemisphere "opposite" the dominant hand, in specific high-level motor-related and multisensory cortical regions, as determined by a separate task involving pantomiming tool-use gestures. This organization presumably reflects the idea that we typically learn the "meaning" of tool sounds in the context of using them with our dominant hand, such that the networks underlying motor imagery or action schemas may be recruited to facilitate recognition.

Acoustic Stimulation↗

PubMed2006

Cortical networks related to human use of tools.

Greater manual dexterity and greater conceptual knowledge of tool use represent two main features that distinguish humans from other primates. Studies of human brain lesions suggest that the left hemisphere (at least in right-handed people) includes a system for processing manual skills that is specialized for tool use that interacts with another system involved more with conceptualizing, planning, and accessing knowledge associated with tool use. Growing evidence from recent neuroimaging studies supports this organization, and studies have begun to highlight specific brain regions and pathways that may be necessary for tool use. This review compares and summarizes results from 64 paradigms published over the past decade that have examined cortical regions associated with tool use skills and tool knowledge. A meta-analysis revealed cortical networks in both hemispheres, though with a clear left hemisphere bias, which may be organized to optimally represent action knowledge. Portions of this network appear to represent part of a system that is tightly linked with language systems, which is discussed together with the effects that handedness may have on the cortical organization for tool use.

Cerebral Cortex↗

PubMed2005

Distinct cortical pathways for processing tool versus animal sounds.

Human listeners can effortlessly categorize a wide range of environmental sounds. Whereas categorizing visual object classes (e.g., faces, tools, houses, etc.) preferentially activates different regions of visually sensitive cortex, it is not known whether the auditory system exhibits a similar organization for different types or categories of complex sounds outside of human speech. Using functional magnetic resonance imaging, we show that hearing and correctly or incorrectly categorizing animal vocalizations (as opposed to hand-manipulated tool sounds) preferentially activated middle portions of the left and right superior temporal gyri (mSTG). On average, the vocalization sounds had much greater harmonic and phase-coupling content (acoustically similar to human speech sounds), which may represent some of the signal attributes that preferentially activate the mSTG regions. In contrast, correctly categorized tool sounds (and even animal sounds that were miscategorized as being tool-related sounds) preferentially activated a widespread, predominantly left hemisphere cortical "mirror network." This network directly overlapped substantial portions of motor-related cortices that were independently activated when participants pantomimed tool manipulations with their right (dominant) hand. These data suggest that the recognition processing for some sounds involves a causal reasoning mechanism (a high-level auditory "how" pathway), automatically evoked when attending to hand-manipulated tool sounds, that effectively associates the dynamic motor actions likely to have produced the sound(s).

Acoustic Stimulation↗

PubMed2004

Time-resolved photointermediate changes in rhodopsin glutamic acid 181 mutants.

The role of glutamic acid 181 in the bovine rhodopsin retinylidene chromophore pocket was studied by expressing E181 mutants in COS cells and measuring, as a function of time, the absorbance changes produced after excitation of lauryl maltoside pigment suspensions with 7 ns laser pulses. All mutants studied except E181D showed accelerated decay of bathorhodopsin compared to wild type. Even for E181D, an anomalously large blue shift was observed in the absorption spectrum of the bathorhodopsin decay product, BSI. These observations support the idea that E181 plays a significant role in the earliest stages of receptor activation. E181 mutations have a pronounced effect on the decay of the lumirhodopsin photointermediate, primarily affecting the size of the red shift that occurs in the lumirhodopsin I to lumirhodopsin II transition that takes place on the 10 micros time scale after wild-type photoexcitation. While the spectral change that occurs in the lumirhodopsin I to lumirhodopsin II transition in wild-type rhodopsin is very small ( approximately 2 nm), making it difficult to detect, it is larger in E181D ( approximately 6 nm), making it evident even in the lower signal-to-noise ratio measurements possible with rhodopsin mutants. The change seen is even larger for the E181F mutant where significant amounts of a deprotonated Schiff base intermediate are produced with the 10 micros time constant of lumirhodopsin II formation. The E181Q mutant shows lumirhodopsin decay more similar to wild-type behavior, and no lumirhodopsin I to lumirhodopsin II transition can be resolved. The addition of chloride ion to E181Q increases the lumirhodopsin I-lumirhodopsin II spectral shift and slows the deprotonation of the Schiff base. The latter result is consistent with the idea that a negative charge at position 181 contributes to protonated Schiff base stability in the later intermediates.

Amino Acid Substitution↗

PubMed2004

Human brain regions involved in recognizing environmental sounds.

To identify the brain regions preferentially involved in environmental sound recognition (comprising portions of a putative auditory 'what' pathway), we collected functional imaging data while listeners attended to a wide range of sounds, including those produced by tools, animals, liquids and dropped objects. These recognizable sounds, in contrast to unrecognizable, temporally reversed control sounds, evoked activity in a distributed network of brain regions previously associated with semantic processing, located predominantly in the left hemisphere, but also included strong bilateral activity in posterior portions of the middle temporal gyri (pMTG). Comparisons with earlier studies suggest that these bilateral pMTG foci partially overlap cortex implicated in high-level visual processing of complex biological motion and recognition of tools and other artifacts. We propose that the pMTG foci process multimodal (or supramodal) information about objects and object-associated motion, and that this may represent 'action' knowledge that can be recruited for purposes of recognition of familiar environmental sound-sources. These data also provide a functional and anatomical explanation for the symptoms of pure auditory agnosia for environmental sounds reported in human lesion studies.

Acoustic Stimulation↗

PubMed2003

Two intermediates appear on the lumirhodopsin time scale after rhodopsin photoexcitation.

Absorbance difference spectra were recorded at 20 degrees C with a dense sequence of delay times from 1 to 128 micros after photolysis of lauryl maltoside suspensions of rhodopsin prepared from hypotonically washed bovine rod outer segments. Data were best fit by two-exponential components with a small, fast component (tau = 12 micros) occurring during the period that lumirhodopsin has been presumed to be stable. The shape of the spectral change corresponds to an approximately 2 nm red shift of the lumirhodopsin spectrum. Measurements with linearly polarized light verified that no absorbance changes associated with rotational diffusion were present in these preparations on this time scale, and experiments designed to enhance isorhodopsin production during photolysis showed no effect on the relative amplitude of the fast process. A similar process was previously observed in membrane suspensions of rhodopsin, but there the similarity of the change to rotational diffusion artifacts made conclusive identification of a second lumirhodopsin difficult. However, reexamination of polarized light measurements on rhodopsin in membrane supports the fact that the fast process seen here in detergent also takes place there. The new absorbance process occurs when time-resolved resonance Raman experiments have shown that the protonated Schiff base is moving from one hydrogen bond acceptor to another. The results are discussed in the context of possibly related processes on the same time scale that have been observed recently in artificial visual pigments with synthetic retinylidene chromophores and in a related rhodopsin mutant. The details of lumirhodopsin behavior are important because it is the last protonated Schiff base intermediate that occurs under physiological conditions.

Animals↗

PubMed2003

Photophysical behaviour of an opp-dibenzoporphyrin (2,12-diethyl-3,13-dimethyldibenzo[g,q]porphyrin) in micelles and organic solvents.

Photophysical parameters relevant to photodynamic therapy have been studied for a novel lipophilic opp-dibenzoporphyrin (DBP), 2,12-diethyl-3,13-dimethyldibenzo[g,q]porphyrin, in N,N-dimethylformamide and TX-100 micelles. The structure of DBP is intermediate between a porphyrin and a phthalocyanine and is associated with higher molar extinction coefficients in the red Q-bands than those found in the haematoporphyrin derivative currently used for photodynamic therapy. The ultrafast measurements on DBP in DMF revealed a fast (ps) lifetime for the second excited singlet state. Observed lifetimes of the first excited singlet state were found to be similar in DMF and TX-100 (12.2 and 14.7 ns), but excited triplet lifetimes were different in the two solvents (0.46 and 2.86 micros). The fluorescent quantum yields of DBP in DMF and TX-100 were twice that of free-base tetraphenylporphyrin and the singlet oxygen quantum yield in DMF and TX-100 was high (0.56-0.65). The combination of stable chemical structure, stronger red-absorption, high singlet oxygen quantum yields, and high fluorescent quantum yields suggests that DBP is an potential chromophore for applications in photodynamic therapy.

Journal Article↗

PubMed2002

Intramolecular proton transfers and structural changes during the photocycle of the LOV2 domain of phototropin 1.

The phototropins are a family of membrane-associated flavoproteins that function as the primary blue light receptors regulating phototropism, chloroplast movements, stomatal opening, and leaf expansion in plants. Phot1, a member of this family, contains two FMN-binding domains, LOV1 and LOV2, within the N-terminal region and a C-terminal serine-threonine protein kinase domain. Light irradiation of oat phot1 LOV2 produces a cysteinyl adduct (Cys-39) at the flavin C(4a) position, which decays thermally back to the dark state. We measured pH and isotope effects on the photocycle. Between pH 3.7 and 9.5, adduct formation showed minimal pH dependence, and adduct decay showed only slight pH dependence, indicating that the pK values of mechanistically relevant groups are outside this range. LOV2 showed a nearly 5-fold slowing of adduct formation in D(2)O relative to H(2)O, indicating that the rate-limiting step involves proton transfer(s). Light-induced changes in the far UV CD spectrum of LOV2 revealed putative protein structural perturbations. The light minus dark CD difference spectrum resembles an inverted alpha-helix spectrum, suggesting that alpha-helicity is reversibly lost upon light irradiation. Decay kinetics for CD spectral changes in the far UV region occur at the same rate as those in the visible region, indicating synchronous relaxation of protein and chromophore structures.

Amino Acid Sequence↗

PubMed2002

Bleaching kinetics of artificial visual pigments with modifications near the ring-polyene chain connection.

Absorbance difference spectra were recorded at 20 degrees C from 30 ns to milliseconds after photolysis of lauryl maltoside suspensions of artificial visual pigments derived from 9-cis isomers of 5-ethylretinal, 8,16-methanoretinal (a 6-s-trans-bicyclic analogue), or 5-demethyl-8-methylretinal. In all three pigments, the earliest intermediate that was detected had the characteristics of a mixture of bathorhodopsin and a blue-shifted intermediate, BSI, which is the first decay product of bathorhodopsin in bovine rhodopsin. The first decays resolved on the nanosecond time scale were the formation of the lumirhodopsin analogues. Subsequent decays were able to be fit with a mechanistic scheme which has been shown to apply to both membrane and detergent suspensions of rhodopsin. Large increases were seen in the amount of metarhodopsin I which appeared after photolysis of 5-ethylisorhodopsin and the bicyclic isorhodopsin analogue, while 5-demethyl-8-methylisorhodopsin more closely followed native rhodopsin in decaying through meta I380, a 380 nm absorbing precursor to metarhodopsin II. In addition to forming more metarhodopsin I, the bicyclic analogue stabilized the metarhodopsin I-metarhodopsin II equilibrium similarly to what has been previously reported for 9-demethylrhodopsin in detergent, introducing the possibility that the bicyclic analogue could similarly be defective in transducin activation. These observations support the idea that long after initial photolysis, structural details of the retinylidene chromophore continue to play a decisive role in processes leading to the activated form, metarhodopsin II.

Animals↗

PubMedDate not supplied

Visual object agnosia and pure word alexia: correlation of functional magnetic resonance imaging and lesion localization.

We present a case of a 64-year-old, right-handed female with a metastatic breast cancer lesion involving the left posterior inferior temporal lobe causing complete loss of the ability to recognize visually common objects and words. After her symptoms resolved on corticosteroid therapy, functional magnetic resonance imaging (fMRI) mapping demonstrated strong left-hemispheric dominance for word recognition and right-hemispheric dominance for object recognition. The case illustrates the relationships among ventral occipito-temporal cortical activation, lesion localization, and lesion-induced deficits of higher visual function. The relationship between hemispheric dominance determined by fMRI and risk of postoperative deficit depends on the specific visual function of interest.

Agnosia↗

PubMedDate not supplied

Effect of digitonin on the rhodopsin meta I-meta II equilibrium.

Absorbance difference spectra were recorded from 10 micros to 540 ms after photoexcitation of sonicated suspensions of hypotonically washed bovine rod outer segments with varying amounts of the detergent digitonin added (0 to 2%) at 20 degrees C. Metarhodopsin I480 and metarhodopsin II displayed the expected anomalous pH dependence at pH 6 and 8 (i.e. opposite to that expected from direct protonation of the chromophore Schiff base). However, increasing levels of digitonin eliminated the pH dependence of the equilibrium, and at 2% digitonin the pH 6 and pH 8 data were both similar to the data collected at pH 8 without digitonin. Addition of 0.5% azolectin restored approximately 50% of the anomalous pH dependence at pH 6 in the 2% digitonin sample. The possibility that digitonin induced large-scale aggregation of rhodopsin in the disk membrane that could be reversed by azolectin was tested using time-resolved linear dichroism. Those results showed that even 0.3% digitonin disrupted the membrane, and no large aggregates were detectable under any conditions. Thus, digitonin reduces the activity of a component of the disk membrane required for metarhodopsin II formation, and that deficiency can be compensated for by azolectin.

Animals↗

PubMedDate not supplied

Lumi I --> Lumi II: the last detergent independent process in rhodopsin photoexcitationt.

Time-resolved absorbance difference spectra were collected at delays from 1 to 128 micros after photolysis of membrane and detergent suspensions of rhodopsin at 20 degrees C. Fitting both sets of data with two exponential decays plus a constant showed a similar fast process (lifetime 11 micros in membrane, 12 micros in 5% dodecyl maltoside) with a small but similar spectral change. This demonstrates that the Lumi I - Lumi II process, previously characterized in detergent suspensions, has similar properties in membrane without significant effect of detergent. The slower exponential process detected in the data is quite different in membrane compared to detergent solubilized samples, showing that the pronounced effect of detergent on the later rhodopsin photointermediates begins fairly abruptly near 20 micros. Besides affecting the late processes, the data collected here shows that detergent induces a small blue shift in the 1 micros difference spectrum (the Lumi I minus rhodopsin difference spectrum). The blue shift is similar to one induced by chloride ion in the E181Q rhodopsin mutant and may indicate that the ionization state of Glu181 in rhodopsin is affected by detergent.

Animals↗