Rapid Oxidation Following Photoreduction in the Avian Cryptochrome4 Photocycle.

Avian magnetoreception is assumed to occur in the retina. Although its molecular mechanism is unclear, magnetic field-dependent formation and the stability of radical-containing photointermediate(s) are suggested to play key roles in a hypothesis called the radical pair mechanism. Chicken cryptochrome4 (cCRY4) has been identified as a candidate magnetoreceptive molecule due to its expression in the retina and its ability to form stable flavin neutral radicals (FADH●) upon blue light absorption. Herein, we used millisecond flash photolysis to investigate the cCRY4 photocycle, in both the presence and absence of dithiothreitol (DTT); detecting the anion radical form of FAD (FAD●-) under both conditions. Using spectral data obtained during flash photolysis and UV-visible photospectroscopy, we estimated the absolute absorbance spectra of the photointermediates, thus allowing us to decompose each spectrum into its individual components. Notably, in the absence of DTT, approximately 37% and 63% of FAD●- was oxidized to FADOX and protonated to form FADH●, respectively. Singular value decomposition analysis suggested the presence of two FAD●- molecular species, each of which was destined to be oxidized to FADOX or protonated to FADH●. A tyrosine neutral radical was also detected; however, it likely decayed concomitantly with the oxidation of FAD●-. On the basis of these results, we considered the occurrence of bifurcation prior to FAD●- generation, or during FAD●- oxidization, and discussed the potential role played by the tyrosine radical in the radical pair mechanism.

THETA system allows one-step isolation of tagged proteins through temperature-dependent protein-peptide interaction.

Tools to control protein-protein interactions by external stimuli have been extensively developed. For this purpose, thermal stimulation can be utilized in addition to light. In this study, we identify a monoclonal antibody termed C13 mAb, which shows an approximately 480-fold decrease in the affinity constant at 37 °C compared to that at 4 °C. Next, we apply this temperature-dependent protein-peptide interaction for one-step protein purifications. We term this THermal-Elution-based TAg system as the THETA system, in which gel-immobilized C13 mAb-derived single-chain variable fragment (scFv) (termed THETAL) is able to bind with proteins tagged by C13 mAb-epitope(s) (THETAS) at 4 °C and thermally release at 37-42 °C. Moreover, to reveal the temperature-dependent interaction mechanism, molecular dynamics simulations are performed along with epitope mapping experiments. Overall, the high specificity and reversibility of the temperature-dependent features of the THETA system will support a wide variety of future applications such as thermogenetics.

Rhodopsin in the Dark Hot Sea: Molecular Analysis of Rhodopsin in a Snailfish, Careproctus rhodomelas, Living near the Deep-Sea Hydrothermal Vent.

Visual systems in deep-sea fishes have been previously studied from a photobiological aspect; however, those of deep-sea fish inhabiting the hydrothermal vents are far less understood due to sampling difficulties. In this study, we analyzed the visual pigment of a deep-sea snailfish, Careproctus rhodomelas, discovered and collected only near the hydrothermal vents of oceans around Japan. Proteins were solubilized from the C. rhodomelas eyeball and subjected to spectroscopic analysis, which revealed the presence of a pigment characterized by an absorption maximum (λmax) at 480 nm. Immunoblot analysis of the ocular protein showed a rhodopsin-like immunoreactivity. We also isolated a retinal cDNA encoding the entire coding sequence of putative C. rhodomelas rhodopsin (CrRh). HEK293EBNA cells were transfected with the CrRh cDNA and the proteins extracted from the cells were subjected to spectroscopic analysis. The recombinant CrRh showed the absorption maximum at 480 nm in the presence of 11-cis retinal. Comparison of the results from the eyeball extract and the recombinant CrRh strongly suggests that CrRh has an A1-based 11-cis-retinal chromophore and works as a photoreceptor in the C. rhodomelas retina, and hence that C. rhodomelas responds to dim blue light much the same as other deep-sea fishes. Because hydrothermal vent is a huge supply of viable food, C. rhodomelas likely do not need to participate diel vertical migration and may recognize the bioluminescence produced by aquatic animals living near the hydrothermal vents.

Overexpression in yeast, photocycle, and in vitro structural change of an avian putative magnetoreceptor cryptochrome4.

Cryptochromes (CRYs) have been found in a wide variety of living organisms and can function as blue light photoreceptors, circadian clock molecules, or magnetoreceptors. Non-mammalian vertebrates have CRY4 in addition to the CRY1 and CRY2 circadian clock components. Though the function of CRY4 is not well understood, chicken CRY4 (cCRY4) may be a magnetoreceptor because of its high level of expression in the retina and light-dependent structural changes in retinal homogenates. To further characterize the photosensitive nature of cCRY4, we developed an expression system using budding yeast and purified cCRY4 at yields of submilligrams of protein per liter with binding of the flavin adenine dinucleotide (FAD) chromophore. Recombinant cCRY4 dissociated from anti-cCRY4 C1 mAb, which recognizes the C-terminal region of cCRY4, in a light-dependent manner and showed a light-dependent change in its trypsin digestion pattern, suggesting that cCRY4 changes its conformation with light irradiation in the absence of other retinal factors. Combinatorial analyses with UV-visible spectroscopy and immunoprecipitation revealed that there is chromophore reduction in the cCRY4 photocycle and formation of a flavosemiquinone radical intermediate that is likely accompanied by a conformational change in the carboxyl-terminal region. Thus, cCRY4 seems to be an intrinsically photosensitive and photoswitchable molecule and may exemplify a vertebrate model of cryptochrome with possible function as a photosensor and/or magnetoreceptor.

Total hip arthroplasty using a cylindrical cementless stem in patients with a small physique.

We performed total hip arthroplasty using an anatomic medullary locking cementless stem for small-physique patients from 1988 to 1995. We conducted a retrospective study of 50 joints in 44 cases, including 40 developmentally dysplastic hips followed for 12 to 20 years (average, 15.1 years). Average height and body weight were 152 cm and 56 kg (5.0 ft and 124 lb), respectively, with an average body mass index of 24.2. Twelve joints (24%) were revised for acetabular-sided failures. Forty-eight stems (96%) showed bone ingrowth fixation, and there were no unstable stems. The simple cylindrical shape of the distal portion of the AML stem was less affected by deformity of the proximal femur of developmental dysplasia of the hip in patients with a small physique, and both clinically and radiologically good results were confirmed at long-term follow-up.

Femoral nerve palsy associated with iliacus hematoma following pseudoaneurysm after revision hip arthroplasty.

Iliacus hematoma associated with femoral nerve palsy is a very rare but considerable complication of hip arthroplasty. A 76-year-old woman had femoral nerve palsy 6 months after revision hip arthroplasty using a reinforcement plate. Computed tomography and selective angiography revealed the pseudoaneurysm on an extension of one of the screws fixing the hardware. Transcatheter arterial embolization proximal to the aneurysm was performed to resolve the aneurysm and to prevent further bleeding. Surgical removal of the hematoma was successfully achieved, as was full functional recovery. This case report describes our diagnosis and treatment of this rare condition and also the therapeutic usefulness of the radiologic intervention to control further bleeding before and after surgical removal of the hematoma.

Combined rotational osteotomy and vascularized iliac bone graft for advanced osteonecrosis of the femoral head.

This paper describes the rationale and technique for a combined procedure of transtrochanteric rotational osteotomy and vascularized iliac bone grafting in cases of osteonecrosis of the femoral head with an extensive and widely collapsed necrotic lesion in young patients. The authors performed this combined operation for 12 joints in 12 patients over 14 years from 1984 until 1997. At a mean of 81 months after the operation, in eight patients who had no pain, the average Harris hip score was 90.0 points. Postoperative radiographic evaluation revealed that collapse (more than 2 mm) of the femoral head was prevented in ten patients. Conversion to an endoprosthesis was avoided in all patients except one in this series. The authors recommend this procedure for advanced and extensive osteonecrosis of the femoral head, especially in young patients, to preserve the joint, even though it is a relatively complex procedure.