Case Report of Extensive Isolated Spontaneous Celiac Trunk Dissection After Liver Transplantation.

Arterial dissection is a rare complication after liver transplantation (LT). We report a case of extensive isolated spontaneous celiac trunk dissection (ISCTD) up to the proper hepatic artery, left gastric artery, and splenic artery after living donor liver transplantation. A 48-year-old woman with cryptogenic liver cirrhosis underwent living donor liver transplantation. Intraoperative and postoperative Doppler ultrasound revealed sufficient flow in the hepatic artery, portal vein, and hepatic vein. On postoperative day (POD) 10, Doppler ultrasound showed reduction of hepatic arterial flow. On POD 16, a contrast-enhanced computed tomography scan showed that the ISCTD extended to the proper hepatic artery, left gastric artery, and splenic artery with an entry tear on the proximal side of the celiac trunk. Although the computed tomography scan showed ischemia of a small part of the liver, blood flow to the liver was kept to some extent. Because all false lumens were occluded by thrombi and the liver enzyme levels normalized, we chose conservative therapy with antiplatelet agents. The patient was discharged on POD 53. She remains well without any liver dysfunction after 18 months with reduction in all false lumens and a patent hepatic artery. Several cases of ISCTD have been reported apart from LT, most of which were treated with conservative therapy. We conclude that conservative therapy could be the first choice in ISCTD even after LT.

Accurate and rapid novel genetic diagnosis for detection of sentinel lymph node metastasis in breast cancer patients.

BACKGROUND: The transcription-reverse transcription concerted reaction (TRC) test is a novel molecular-based procedure, which can assess nodal metastasis accurately and quickly. We examined the usefulness of the TRC test with a double marker, cytokeratin 19 (CK19) and carcinoembryonic antigen (CEA) mRNA, to detect sentinel lymph nodes (SLN) metastasis in breast cancer patients. METHODS: A total of 264 SLNs from 131 breast cancer patients were assigned to a training set (109 SLNs from 50 patients) and validation set (155 SLNs from 81 patients). Cytokeratin 19 and CEA mRNA were detected by TRC tests, and the sensitivity and specificity of the SLN metastasis between the TRC and histology cohorts were compared. RESULTS: Mean copy numbers of CK19 and CEA by TRC tests were increased according to the metastatic size. In the training set, TRC test showed 100% sensitivity, specificity and concordance rates against the permanent histopathology test. In the validation set, sensitivity was 97.1%, specificity was 99.2% and the concordance rate was 99.4%. CONCLUSION: Our results showed that the detection of CK19 and CEA mRNA using the TRC test is, an accurate and rapid method for detection of SLN metastasis and can be applied as an intraoperative molecular diagnosis in breast cancer patients.

Sotrastaurin, a protein kinase C inhibitor, ameliorates ischemia and reperfusion injury in rat orthotopic liver transplantation.

Sotraustaurin (STN), a small molecule, targeted protein kinase C (PKC) inhibitor that prevents T-lymphocyte activation via a calcineurin-independent pathway, is currently being tested in Phase II renal and liver transplantation clinical trials. We have documented the key role of activated T cells in the inflammation cascade leading to liver ischemia/reperfusion injury (IRI). This study explores putative cytoprotective functions of STN in a clinically relevant rat model of hepatic cold ischemia followed by orthotopic liver transplantation (OLT). Livers from Sprague-Dawley rats were stored for 30 h at 4°C in UW solution, and then transplanted to syngeneic recipients. STN treatment of liver donors/recipients or recipients only prolonged OLT survival to >90% (vs. 40% in controls), decreased hepatocellular damage and improved histological features of IRI. STN treatment decreased activation of T cells, and diminished macrophage/neutrophil accumulation in OLTs. These beneficial effects were accompanied by diminished apoptosis, NF-κB/ERK signaling, depressed proapoptotic cleaved caspase-3, yet upregulated antiapoptotic Bcl-2/Bcl-xl and hepatic cell proliferation. In vitro, STN decreased PKCθ/IκBα activation and IL-2/IFN-γ production in ConA-stimulated spleen T cells, and diminished TNF-α/IL-1ß in macrophage-T cell cocultures. This study documents positive effects of STN on liver IRI in OLT rat model that may translate as an additional benefit of STN in clinical liver transplantation.

Evaluation of the SCA instrument for measuring patient satisfaction with cancer care administered via paper or via the Internet.

BACKGROUND: Patients' perspectives provide valuable information on quality of care. This study evaluates the feasibility and validity of Internet administration of Service Satisfaction Scale for Cancer Care (SCA) to assess patient satisfaction with outcome, practitioner manner/skill, information, and waiting/access. PATIENTS AND METHODS: Primary data collected from November 2007 to April 2008. Patients receiving cancer care within 1 year were recruited from oncology, surgery, and radiation clinics at a tertiary care hospital. An Internet-based version of the 16-item SCA was developed. Participants were randomised to Internet SCA followed by paper SCA 2 weeks later or vice versa. Seven-point Likert scale responses were converted to a 0-100 scale (minimum-maximum satisfaction). Response distribution, Cronbach's alpha, and test-retest correlations were calculated. RESULTS: Among 122 consenting participants, 78 responded to initial SCA. Mean satisfaction scores for paper/Internet were 91/90 (outcome), 95/94 (practitioner manner/skill), 89/90 (information), and 86/86 (waiting/access). Response rate and item missingness were similar for Internet and paper. Except for practitioner manner/skill, test-retest correlations were robust r = 0.77 (outcome), 0.74 (information), and 0.75 (waiting/access) (all P < 0.001). CONCLUSIONS: Internet SCA administration is a feasible and a valid measurement of cancer care satisfaction for a wide range of cancer diagnoses, treatment modalities, and clinic settings.

A simple and sensitive method for detection of Bacillus anthracis by loop-mediated isothermal amplification.

AIMS: To develop a rapid and simple system for detection of Bacillus anthracis using a loop-mediated isothermal amplification (LAMP) method and determine the suitability of LAMP for rapid identification of B. anthracis infection. METHODS AND RESULTS: A specific LAMP assay targeting unique gene sequences in the bacterial chromosome and two virulence plasmids, pXO1 and pXO2, was designed. With this assay, it was possible to detect more than 10 fg of bacterial DNA per reaction and obtain results within 30-40 min under isothermal conditions at 63 degrees C. No cross-reactivity was observed among Bacillus cereus group and other Bacillus species. Furthermore, in tests using blood specimens from mice inoculated intranasally with B. anthracis spores, the sensitivity of the LAMP assay following DNA extraction methods using a Qiagen DNeasy kit or boiling protocol was examined. Samples prepared by both methods showed almost equivalent sensitivities in LAMP assay. The detection limit was 3.6 CFU per test. CONCLUSIONS: The LAMP assay is a simple, rapid and sensitive method for detecting B. anthracis. SIGNIFICANCE AND IMPACT OF THE STUDY: The LAMP assay combined with boiling extraction could be used as a simple diagnostic method for identification of B. anthracis infection.

Internal water molecules of pharaonis phoborhodopsin studied by low-temperature infrared spectroscopy.

In the Schiff base region of bacteriorhodopsin (BR), a light-driven proton-pump protein, three internal water molecules are involved in a pentagonal cluster structure. These water molecules constitute a hydrogen-bonding network consisting of two positively charged groups, the Schiff base and Arg82, and two negatively charged groups, Asp85 and Asp212. Previous infrared spectroscopy of BR revealed stretching vibrations of such water molecules under strong hydrogen-bonding conditions using spectral differences in D2O and D2(18O) [Kandori and Shichida (2000) J. Am. Chem. Soc. 122, 11745-11746]. The present study extends the infrared analysis to another archaeal rhodopsin, pharaonis phoborhodopsin (ppR; also called pharaonis sensory rhodopsin-II, psR-II), involved in the negative phototaxis of Natronobacterium pharaonis. Despite functional differences between ppR and BR, similar spectral features of water bands were observed before and after photoisomerization of the retinal chromophore at 77 K. This implies that the structure and the structural changes of internal water molecules are similar between ppR and BR. Higher stretching frequencies of the bridged water in ppR suggest that the water-containing pentagonal cluster structure is considerably distorted in ppR. These observations are consistent with the crystallographic structures of ppR and BR. The water structure and structural changes upon photoisomerization of ppR are discussed here on the basis of their infrared spectra.

Photochemistry and photoinduced proton-transfer by pharaonis phoborhodopsin.

Phoborhodopsin (pR or sensory rhodopsin II, sRII) is a photoreceptor of the negative phototaxis of Halobacterium salinarum, and pharaonis phoborhodopsin (ppR or pharaonis sensory rhodopsin II, psRII) is a corresponding protein of Natronobacterium pharaonis. The photocycle of ppR is essentially as follows: ppR(498) --> ppRK(approximately 540) --> ppRKL(512) --> ppRL(488) --> ppRM(390) --> ppRO(560) --> ppR (numbers in parenthesis denote the maximum absorbance). The photocycle is very similar to that of bacteriorhodopsin, but the rate of initial pigment recovery is about two-orders of magnitude slower. By low-temperature spectroscopy, two K-intermediates were found but the L intermediate was not detected. The lack of L indicates extraordinary stability of K at low temperature. ppRM is photoactive similar to M of bR. The ground state ppR contains only all-trans retinal whereas ppRM and ppRO contain 13-cis and all-trans, respectively. ppR has the ability of light-induced proton transport from the inside to the outside. Proton uptake occurs at the formation of ppRO and the release at its decay. ppR associates with its transducer and this complex transmits a signal to the cytoplasm. The proton transport ability is lost when the complex forms, but the proton uptake and release still occur, suggesting that the proton movement is non-electrogenic (release and uptake occur from the same side). The stoichiometry of the complex between ppR and the transducer is 1 : 1. ppR or pR has absorption maximum at approximately 500 nm, which is blue-shifted from those of other archaeal rhodopsins. The molecular mechanism of this color regulation is not yet solved.

Environment around the chromophore in pharaonis phoborhodopsin: mutation analysis of the retinal binding site.

Phoborhodopsin (pR or sensory rhodopsin II, sRII) and pharaonis phoborhodopsin (ppR or pharaonis sRII, psRII) have a unique absorption maximum (lambda(max)) compared with three other archaeal rhodopsins: lambda(max) of pR and ppR is approx. 500 nm and of others (e.g. bacteriorhodopsin, bR) is 560-590 nm. To determine the residue contributing to the opsin shift from ppR to bR, we constructed various ppR mutants, in which a single residue was substituted for a residue corresponding to that of bR. The residues mutated were those which differ from that of bR and locate within 5 A from the conjugated polyene chain of the chromophore or any methyl group of the polyene chain. The shifts of lambda(max) of all mutants were small, however. We constructed a mutant in which all residues which differ from those of bR in the retinal binding site were simultaneously substituted for those of bR, but the shift was only from 499 to 509 nm. Next, we constructed a mutant in which 10 residues located within 5 A from the polyene as described above were simultaneously substituted. Only 44% of the opsin shift (lambda(max) of 524 nm) from ppR to bR was obtained even when all amino acids around the chromophore were replaced by the same residues as bR. We therefore conclude that the structural factor is more important in accounting for the difference of lambda(max) between ppR and bR rather than amino acid substitutions. The possible structural factors are discussed.

Comparison of the effects of different antiviral treatments on the antioxidant systems of stroma-free hemoglobin.

The effect of virus inactivation by 1,9-dimethylmethylene blue (DMMB) phototreatment, methylene blue (MB) phototreatment or heat on the activities of antioxidant systems of stroma-free hemoglobin (SFH) was studied. DMMB photoinactivated human immunodeficiency virus by > 3.69 log10 under conditions that inactivated 3.33 log10 of vesicular stomatitis virus (VSV). Under conditions which inactivated VSV by 6.10 log10 (1.37 J/cm2 irradiation and 2 microM DMMB), there was little change in the methemoglobin (Met-Hb) formation, concentration of reduced glutathione (GSH), or superoxide dismutase (SOD), catalase (CAT) or glutathione peroxidase (GPX) activities. However, the activity of glutathione reductase (GR) was decreased by 77%. Under conditions that inactivated VSV by 5.69 log10 (1.37 J/cm2 irradiation and 24 microM MB) there was little effect of MB phototreatment on SOD, CAT, GPX and GSH activities. However, GR activity was decreased by 74% and Met-Hb content reached 3.98%. Under conditions that inactivated VSV by more than 6.20 log10 (60 degrees C for 2 min), virucidal heat treatment resulted in 27% Met-Hb formation and decreased GPX activity by 43%. No significant decline in SOD, CAT or GR activities or GSH concentration was observed. These results suggest that, compared with heat treatment and MB phototreatment, virucidal DMMB treatment preserves not only the oxidative state of hemoglobin but also the antioxidant systems against superoxide and hydrogen peroxide, although the reduced GR activity may limit the quenching capacity of antioxidants in DMMB-treated SFH.

Pharaonis phoborhodopsin binds to its cognate truncated transducer even in the presence of a detergent with a 1:1 stoichiometry.

Pharaonis phoborhodopsin (ppR) (also pharaonis sensory rhodopsin II) is a receptor of the negative phototaxis of Natronobacterium pharaonis. ppR forms a complex with its pharaonis halobacterial transducer (pHtrII), and this complex transmits the light signal to the sensory system in the cytoplasm. The expressed C-terminal-His tagged ppR and C-terminal-His tagged truncated pHtrII (t-Htr) in Escherichia coli (His means the 6x histidine tag) form a complex even in the presence of 0.1% of n-dodecyl-beta-D-maltoside, and the M-decay of the complex became about twice slower than that of ppR alone. The photocycling rates under varying concentration ratios of ppR to t-Htr in the presence of detergent were measured. The data were analyzed on the following assumptions: (1) the M-decay of both ppR alone and the complex followed a single exponential decay with different time constants; and (2) the M-decay under varying concentration ratios of ppR to t-Htr, therefore, followed a biexponential decay function which combined the decay of the free ppR and that of the complex as photoreactive species. From these analyses we estimated the dissociation constant (15.2 +/- 1.8 microM) and the number of binding sites (1.2 +/- 0.08).

Selective reaction of hydroxylamine with chromophore during the photocycle of pharaonis phoborhodopsin.

Phoborhodopsin (pR; also called sensory rhodopsin II, sRII) is a receptor of negative phototaxis of Halobacterium salinarum, and pharaonis phoborhodopsin (ppR; also pharaonis sensory rhodopsin II, psRII) is a corresponding protein of Natronobacterium pharaonis. These receptors contain retinal as a chromophore which binds to a lysine residue via Schiff base. This Schiff base can be cleaved with hydroxylamine to loose their color (bleaching). In dark, the bleaching rate of ppR was very slow whereas illumination accelerated considerably the bleaching rate. Addition of azide accelerated the decay of the M-intermediate while its formation (decay of the L-intermediate) is not affected. The bleaching rate of ppR under illumination was decreased by addition of azide. Essentially no reactivity with hydroxylamine under illumination was observed in the case of D75N mutant which lacks the M-intermediate in its photocycle. Moreover, we provided illumination by flashes to ppR in the presence of varying concentrations of azide to measure the bleaching rate per one flash. A good correlation was obtained between the rate and the mean residence time, MRT, which was calculated from flash photolysis data of the M-decay. These findings reveal that water-soluble hydroxylamine reacts selectively with the M-intermediate and its implication was discussed.

Structural changes of pharaonis phoborhodopsin upon photoisomerization of the retinal chromophore: infrared spectral comparison with bacteriorhodopsin.

Archaeal rhodopsins possess a retinal molecule as their chromophores, and their light energy and light signal conversions are triggered by all-trans to 13-cis isomerization of the retinal chromophore. Relaxation through structural changes of the protein then leads to functional processes, proton pump in bacteriorhodopsin and transducer activation in sensory rhodopsins. In the present paper, low-temperature Fourier transform infrared spectroscopy is applied to phoborhodopsin from Natronobacterium pharaonis (ppR), a photoreceptor for the negative phototaxis of the bacteria, and infrared spectral changes before and after photoisomerization are compared with those of bacteriorhodopsin (BR) at 77 K. Spectral comparison of the C--C stretching vibrations of the retinal chromophore shows that chromophore conformation of the polyene chain is similar between ppR and BR. This fact implies that the unique chromophore-protein interaction in ppR, such as the blue-shifted absorption spectrum with vibrational fine structure, originates from both ends, the beta-ionone ring and the Schiff base regions. In fact, less planer ring structure and stronger hydrogen bond of the Schiff base were suggested for ppR. Similar frequency changes upon photoisomerization are observed for the C==N stretch of the retinal Schiff base and the stretch of the neighboring threonine side chain (Thr79 in ppR and Thr89 in BR), suggesting that photoisomerization in ppR is driven by the motion of the Schiff base like BR. Nevertheless, the structure of the K state after photoisomerization is different between ppR and BR. In BR, chromophore distortion is localized in the Schiff base region, as shown in its hydrogen out-of-plane vibrations. In contrast, more extended structural changes take place in ppR in view of chromophore distortion and protein structural changes. Such structure of the K intermediate of ppR is probably correlated with its high thermal stability. In fact, almost identical infrared spectra are obtained between 77 and 170 K in ppR. Unique chromophore-protein interaction and photoisomerization processes in ppR are discussed on the basis of the present infrared spectral comparison with BR.

Significance of substrate hydrophobicity for recognition by an oligopeptide transporter (PEPT1).

Our previous paper [(1999) Bioconjugate Chem. 10, 24-31] pointed out that hydrophobicity of substrates/inhibitors plays an important role in the recognition by an oligopeptide transporter (PEPT1) expressed in the human intestinal epithelial cell line Caco-2. To determine the significance of that hydrophobicity, we have now synthesized dipeptide analogues conjugating the epsilon-amino group of Lys in Val-Lys with aliphatic carboxylic acids: acetic acid (C2), propanoic acid (C3), pentanoic acid (C5), hexanoic acid (C6), and decanoic acid (C10). The affinities of these conjugates were estimated by their inhibition of the accumulation rate of Gly-Sar, a well-established substrate for PEPT1. With the increase in length of the hydrocarbon chain of the conjugates, i.e., in the hydrophobicity of the conjugates, the inhibition strengthened. Dixon-Webb plot analysis of the inhibition by the C10-conjugated dipeptide showed competitive inhibition. The trans-stimulation effect of Val-Lys conjugated to C10 or C5 on the uptake of Ceftibuten was observed using rat brush border membrane vesicles. This findings showed that these conjugates are transportable substrates. These results confirmed that the hydrophobicity of substrates/inhibitor is one of the factors in the recognition by PEPT1.

Ion-selective electrode for transmembrane pH difference measurements.

A triethylammonium-sensitive electrode was constructed using sodium tetrakis[3,5-bis(2-methoxyhexafluoro-2-propyl)phenyl]borate as an ion-exchanger and benzyl 2-nitrophenyl ether as a solvent mediator in a poly(vinylchloride) membrane matrix and was used to determine the pH difference across a cell membrane. The method is based on monitoring of the pH gradient-induced uptake of triethylammonium in situ. The triethylammonium electrode exhibited a near-Nernstian response to triethylammonium in the concentration range of 5 x 10(-6)-1 x 10(-2) M with a slope of 58.5 mV per concentration decade in a buffer solution composed of 150 mM NaCl and 10 mM NaH2PO4/Na2HPO4 (pH 7.5). The limit of detection was 1 microM. In experiments using liposomes, the uptake of triethylammonium into liposomes was quantitatively induced according to the pH difference across the liposomal membrane. The transmembrane pH differences in Escherichia coli cells and the light-induced pH differences across the envelope vesicles of Halobacterium halobium were successfully determined by the present method.

Effect of liposome type and membrane fluidity on drug-membrane partitioning analyzed by immobilized liposome chromatography.

Immobilized liposome chromatography (ILC) has been proven to be a useful method for the study or rapid screening of drug-membrane interactions. To obtain an adequate liposomal membrane phase for ILC, unilamellar liposomes were immobilized in gel beads by avidin-biotin binding. The retardation of 15 basic drugs on the liposome column could be converted to membrane partitioning coefficients, K(LM). The effects of small or large unilamellar liposomes and multilamellar liposomes on the drug-membrane partitioning were compared. The K(LM) values for both small and large liposomes were similar, but higher than those for the multilamellar liposomes. The basic drugs showed stronger partitioning into negatively charged liposomes than into either neutral liposomes or positively charged liposomes. The membrane fluidity of the immobilized liposomes was modulated by incorporating cholesterol into the liposomal membranes, by changing the acyl chain length and degree of unsaturation of the phospholipids, and by changing the temperature for ILC runs. Our data show that K(LM) obtained using ILC correlated well with those reported by batch studies using free liposomes. It is concluded that negatively charged or cholesterol-containing large unilamellar liposomes are suitable models for the ILC analysis of drug-membrane interactions.

Inactivation of parvovirus B19 in coagulation factor concentrates by UVC radiation: assessment by an in vitro infectivity assay using CFU-E derived from peripheral blood CD34+ cells.

BACKGROUND: Nonenveloped and thermostable viruses such as parvovirus B19 (B19) can be transmitted to patients who are receiving plasma-derived coagulation factor concentrates treated by the S/D method for inactivating enveloped viruses. Therefore, it is important to develop and validate new methods for the inactivation of nonenveloped viruses. STUDY DESIGN AND METHODS: Suspensions of B19 in coagulation factor concentrates (FVIII) were irradiated with UVC light. B19 infectivity was determined by an indirect immunofluorescence assay using CFU-E, as a host cell, derived from peripheral blood CD34+ cells. The effects of catechins on B19 infectivity and on FVIII activity after UVC illumination were also examined. RESULTS: The indirect immunofluorescence assay estimated the B19 infectivity of samples containing virus copies of 10(5) to 10(11) per 10 microL to be a median tissue culture-infectious dose of 10(0.3) to 10(5.4) per 10 microL. B19 was inactivated by 3 log at 750 J per m(2) of UVC radiation and was undetectable after 1000 or 2000 J per m(2) of irradiation. However, FVIII activity decreased to 55 to 60 percent of pretreatment activity after 2000 J per m(2) of UVC radiation. This was inhibited in the presence of rutin or catechins. Epigallocatechin gallate could maintain FVIII activity at almost 100 percent of pretreatment activity after 2000 J per m(2) of UVC radiation, while B19 infectivity was decreased to undetectable levels, which resulted in >3.9 log inactivation. CONCLUSION: UVC radiation in the presence of catechins, especially epigallocatechin gallate, appears to be an effective method of increasing the viral safety of FVIII concentrates without the loss of coagulation activity.

Occurrence of D-Amino Acids and a pyridoxal 5'-phosphate-dependent aspartate racemase in the acidothermophilic archaeon, Thermoplasma acidophilum.

Free D-amino acid content in some archaea was investigated and D-forms of several amino acids were found in them. In the acidothermophilic archaeon, Thermoplasma acidophilum, the proportion of D-aspartate (D-Asp) to total Asp was as high as 39.7%. Crude extracts of Thermoplasma acidophilum had Asp-specific racemase activity that was pyridoxal 5'-phosphate (PLP)-dependent. The relative insensitivity to a SH-modifying reagent distinguished this activity from those of the PLP-independent Asp racemases found in other hyperthermophilic archaea (Matsumoto, M., et al., J. Bacteriol. 181, 6560-6563 1999). Thus, high levels of d-Asp should be produced by a new type(s) of Asp-specific racemase in Thermoplasma acidophilum, although the function of d-Asp in this archaeon remains unknown.

Photo-induced proton transport of pharaonis phoborhodopsin (sensory rhodopsin II) is ceased by association with the transducer.

Phoborhodopsin (pR; also sensory rhodopsin II, sRII) is a retinoid protein in Halobacterium salinarum and works as a receptor of negative phototaxis. Pharaonis phoborhodopsin (ppR; also pharaonis sensory rhodopsin II, psRII) is a corresponding protein of Natronobacterium pharaonis. In bacterial membrane, ppR forms a complex with its transducer pHtrII, and this complex transmits the light signal to the sensory system in the cytoplasm. We expressed pHtrII-free ppR or ppR-pHtrII complex in H. salinarum Pho81/wr(-) cells. Flash-photolysis experiments showed no essential changes between pHtrII-free ppR and the complex. Using SnO2 electrode, which works as a sensitive pH electrode, and envelope membrane vesicles, we showed the photo-induced outward proton transport. This membranous proton transport was also shown using membrane vesicles from Escherichia coli in which ppR was functionally expressed. On the other hand, the proton transport was ceased when ppR formed a complex with pHtrII. Using membrane sheet, it was shown that the complex undergoes first proton uptake and then release during the photocycle, the same as pHtrII-free ppR, although the net proton transport ceases. Taking into consideration that the complex of sRII (pR) and its transducer undergoes extracellular proton circulation (J. Sasaki and J. L., Biophys. J. 77:2145-2152), we inferred that association with pHtrII closes a cytoplasmic channel of ppR, which lead to the extracellular proton circulation.

Involvement of two groups in reversal of the bathochromic shift of pharaonis phoborhodopsin by chloride at low pH.

Pharaonis phoborhodopsin (ppR; or pharaonis sensory rhodopsin II, psRII) is a photophobic receptor of the halobacterium Natronobacterium pharaonis. Its lambdamax is at 496 nm, but upon acidification in the absence of chloride, lambdamax shifted to 522 nm. This bathochromic shift is thought to be caused by the protonation of Asp75, which corresponds to Asp85 of bacteriorhodopsin (bR). The D75N mutant, in which Asp75 was replaced by Asn, had its lambdamax at approximately 520 nm, supporting this mechanism for the bathochromic shift. A titration of the shift yielded a pKa of 3.5 for Asp75. In the presence of chloride, the spectral shifts were different: with a decrease in pH, a bathochromic shift was first observed, followed by a hypsochromic shift on further acidification. This was interpreted as: the disappearance of a negative charge by the protonation of Asp75 was compensated by the binding of chloride, but it is worthy to note that the binding requires the protonation of another proton-associable group other than Asp75. This is supported by the observation that in the presence of chloride, upon acidification, the lambdamax of D75N even showed a blue shift, showing that the protonation of a proton-associable group (pKa = 1.2) leads to the chloride binding that gives rise to a blue shift.

Effects of three characteristic amino acid residues of pharaonis phoborhodopsin on the absorption maximum.

Phoborhodopsin (pR or sensory rhodopsin II, sRII) or pharaonis phoborhodopsin (ppR or pharaonis sensory rhodopsin II, psRII) has a unique absorption maximum (lambda max) compared with three other archaeal rhodopsins: lambda max of pR or ppR at ca 500 nm and others at 560-590 nm. Alignment of amino acid sequences revealed three sites characteristic of the shorter wavelength-absorbing pigments. The amino acids of these three sites are conserved completely among archaeal rhodopsins having longer lambda max, and are different from those of pR or ppR. We replaced these amino acids of ppR with amino acids corresponding to those of bacteriorhodopsin, Val-108 to Met, Gly-130 to Ser and Thr-204 to Ala. The lambda max of V108M mutant was 502 nm with a slight redshift. G130S and T204A mutants had lambda max of 503 and 508 nm, respectively. Thus, each site contributes only a small effect to the color tuning. We then constructed three double mutants and one triple mutant. The opsin-shifts of these mutants suggest that Val-108 and Thr-204 or Gly-130 are synergistic, and that Gly-130 and Thr-204 work additively. Even in the triple mutant, the lambda max was 515 nm, an opsin-shift only ca 30% of the shift value from 500 to 560 nm. This means that there is another yet unidentified factor responsible for the color tuning.