Regulation of water absorption in the frog skins by two vasotocin-dependent water-channel aquaporins,AQP-h2 and AQP-h3

A new frog aquaporin (AQP) cDNA was cloned from a cDNA library constructed from the ventral skin of the tree frog Hyla japonica. This AQP (Hyla AQP-h2) consisted of 268 amino acid residues with a high homology to mammalian AQP2. The predicted amino acid sequence contained the two conserved Asn-Pro-Ala motifs found in all the major intrinsic protein family members and the putative six transmembrane domains. The sequence also contained a mercurial compound: cysteine, one potential N-glycosylation site at Asn-124, and a putative phosphorylation site recognized by protein kinase A at Ser-262. In a swelling assay using Xenopus oocytes, AQP-h2 facilitated water permeability, especially in response to cAMP. Expression of AQP-h2 mRNA was restricted to several tissues including the ventral skin, kidney, and urinary bladder; but with immunofluorescence staining using an antipeptide antibody (ST-140) against the AQP-h2 protein, immunopositive cells were found only in the ventral skin and urinary bladder. In the ventral pelvic skin, the label for AQP-h2 was localized in the entire plasma membrane of the granular cells beneath the outmost layer of the skin and in the basolateral membrane of the granular cells in this layer. In response to vasotocin, however, the label for AQP-h2 became more intense in the apical membrane in the granular cells of the outermost layer, similar to the case for the earlier studied AQP-h3, which was specifically expressed in the ventral skin. Taken together, these findings suggest that not only AQP-h3, but also AQP-h2 acts as a regulator of the water balance in this frog.     

The effect of heparin on tissue factor and tissue factor pathway inhibitor in patients with acute myocardial infarction

We examined plasma TF and free TFPI levels in 26 consecutive patients with AMI, 26 patients with stable exertional angina, and 25 patients with chest pain syndrome. In patients with AMI, blood samples were obtained immediately after admission and at 4, 8, 16, 24, and 48 h, and the third, fifth, seventh, and fourteenth day after initiation of reperfusion therapy. Plasma TF levels in patients with AMI on admission were significantly higher than in the chest pain syndrome and stable exertional angina groups (248.0+/-117. 4 vs. 179.5+/-29.2 vs. 189.5+/-29.6 pg/ml, P<0.01). In patients with AMI, the level subsequently decreased after heparin administration and was maintained at significantly lower levels compared to those on admission. Plasma free TFPI levels in patients with AMI on admission were significantly higher than in the chest pain syndrome and stable exertional angina groups [33.5+/-12.4 vs. 26.0+/-7.6 ng/ml (P<0.01) vs. 27.5+/-6.3 ng/ml, P<0.05]. In patients with AMI, it reached the maximum level at 4 h after the administration of heparin, and gradually decreased over the time course. These data indicated that continuous administration of a low dose of heparin was effective in decreasing TF levels without affecting TFPI levels. Our results elucidate one of the mechanisms by which the administration of heparin is beneficial in AMI patients undergoing percutaneous revascularization.     

Myeloablative allogeneic hematopoietic stem cell transplantation for non-Hodgkin lymphoma: a nationwide survey in Japan

We retrospectively surveyed the data of 233 patients who underwent myeloablative allogeneic hematopoietic stem cell transplantation (allo-HSCT) for non-Hodgkin lymphoma (NHL). Donors were HLA-matched relatives in 154 patients (66%) or unrelated volunteers in 60 (26%). Ninety patients (39%) were in complete remission. One hundred ninety-three (83%) received a total body irradiation (TBI)-based regimen, and 40 (17%) received a non-TBI-based regimen. Acute graft-versus-host disease (GVHD) occurred in 155 (67%) of the 233 evaluable patients; grade II to IV in 90 (39%), and grade III to IV in 37 (16%). Treatment-related mortality (TRM) was observed in 98 patients (42%), and 68% of them were related to GVHD. In a multivariate analysis, chemoresistance, prior autograft, and chronic GVHD were identified as adverse prognostic factors for TRM. Relapse or progression of lymphoma was observed in 21%. The 2-year overall survival rates of the patients with indolent (n = 38), aggressive (n = 111), and lymphoblastic lymphoma (n = 84) were 57%, 42%, and 41%, respectively. In a multivariate analysis, chemoresistance, prior autograft, and prior radiotherapy were identified as adverse prognostic factors for overall survival. Although myeloablative allo-HSCT represents an effective therapeutic option for patients with NHL, more work is still needed to decrease TRM and relapse.     

Possible contributions of reactive oxygen species and mitogen-activated protein kinase to renal injury in aldosterone/salt-induced hypertensive rats

Studies were performed to test the hypothesis that reactive oxygen species (ROS) and mitogen-activated protein kinase (MAPK) contribute to the pathogenesis of aldosterone/salt-induced renal injury. Rats were given 1% NaCl to drink and were treated with one of the following combinations for 6 weeks: vehicle (0.5% ethanol, SC, n=6); aldosterone (0.75 microg/H, SC, n=8); aldosterone plus a selective mineralocorticoid receptor antagonist; eplerenone (0.125% in chow, n=8); aldosterone plus an antioxidant; and tempol (3 mmol/L in drinking solution, n=8). The activities of MAPKs, including extracellular signal-regulated kinases (ERK)1/2, c-Jun-NH2-terminal kinases (JNK), p38MAPK, and big-MAPK-1 (BMK1) in renal cortical tissues were measured by Western blot analysis. Aldosterone-infused rats showed higher systolic blood pressure (165+/-5 mm Hg) and urinary excretion of protein (106+/-24 mg/d) than vehicle-infused rats (118+/-3 mm Hg and 10+/-3 mg/d). Renal cortical mRNA expression of p22phox, Nox-4, and gp91phox, measured by real-time polymerase chain reaction, was increased in aldosterone-infused rats by 2.3, 4.3, and 3.0-fold, respectively. Thiobarbituric acid-reactive substances (TBARS) content in renal cortex was also higher in aldosterone (0.23+/-0.02) than vehicle-infused rats (0.09+/-0.01 nmol/mg protein). ERK1/2, JNK, and BMK1 activities were significantly elevated in aldosterone-infused rats by 3.3, 2.3, and 3.0-fold, respectively, whereas p38MAPK activity was not changed. Concurrent administration of eplerenone or tempol to aldosterone-infused rats prevented the development of hypertension (127+/-2 and 125+/-5 mm Hg), and the elevations of urinary excretion of protein (10+/-2 and 9+/-2 mg/day) or TBARS contents (0.08+/-0.01 and 0.11+/-0.01 nmol/mg protein). Furthermore, eplerenone and tempol treatments normalized the activities of ERK1/2, JNK, and BMK1. These data suggest that ROS and MAPK play a role in the progression of renal injury induced by chronic elevations in aldosterone.     

Clinicopathological characterization of so-called “cholangiocarcinoma with intraductal papillary growth” with respect to “intraductal papillary neoplasm of bile duct (IPNB)”

Cholangiocarcinoma (CC) of the biliary tract occasionally presents a predominant intraductal papillary growth in the bile ducts, called as biliary tract carcinoma (BTC) of papillary growth (PG) and intrahepatic CC (ICC) of intraductal growth (IG) type. Recently, intraductal papillary neoplasm of bile duct (IPNB) has been proposed as a pre-invasive biliary neoplasm. This study was performed to characterize pathologically BTC of PG type and ICC of IG type with respect to IPNB. It was found that 126 of such 154 CCs (81.8%) fulfilled the criteria of IPNB, while the remaining 28 cases showed different histologies, such as tubular adenocarcinoma and carcinosarcoma. These IPNBs occurred in old aged patients with a male predominance, and the left lobe was rather frequently affected in the liver. A majority of these cases were high grade IPNB (43 cases) and invasive IPNB (77 cases), while low grade IPNB was rare (6 cases). Pancreatobiliary type was predominant (48 cases) followed by gastric (30 cases), intestinal (29 cases) and oncocytic (19 cases) types. Mucus hypersecretion was found in 45 cases, and this was frequent in IPNB at the intrahepatic large bile duct and hilar bile ducts but rare at the extrahepatic bile ducts. Interestingly, 36 cases of high grade and invasive IPNBs contained foci of moderately differentiated adenocacinoma within the intraductal papillary tumor. In conclusion, a majority of ICC of IG type and BTC of PG type could be regarded as a IPNB lineage, and clinically detectable IPNBs were already a malignant papillary lesion.     

Prognostic usefulness of serum uric acid after acute myocardial infarction (the Japanese Acute Coronary Syndrome Study)

Serum uric acid (UA) levels reflect circulating xanthine oxidase activity and oxidative stress production. Hyperuricemia has been identified in patients who have congestive heart failure and is a marker of poor prognosis in such patients. We investigated the relation between serum UA levels and Killip's classification suggestive of the severity of heart failure and whether hyperuricemia influences mortality of patients who have acute myocardial infarction (AMI). Using the Japanese Acute Coronary Syndrome Study database, we evaluated 1,124 consecutive patients who were hospitalized within 48 hours of onset of symptoms of AMI from January to December 2002. There was a close relation between serum UA concentration and Killip's classification. Patients who developed short-term adverse events had high UA concentrations. Serum UA levels, Killip's class, age, and peak creatine phosphokinase level were significant predictors of long-term mortality. The hazard ratio for patients in the highest quartile of UA was 3.7 compared with those in the lowest quartile for death after AMI after adjustment for independent factors that were related to mortality. The combination of the best UA cutoff (447 micromol/L) for predicting survival based on receiver-operating characteristics analysis and Killip's class significantly predicted the prognosis of acute and long-term AMI-related complications. In conclusion, our results suggest that hyperuricemia after AMI is associated with the development of heart failure. Serum UA level is a suitable marker for predicting AMI-related future adverse events, and the combination of Killip's class and serum UA level after AMI is a good predictor of mortality in patients who have AMI.     

Ivabradine as an Adjuvant Agent for Severe Heart Failure Occurring in the Early Phase after Allogeneic Hematopoietic Cell Transplantation

Cardiotoxicity is a critical complication of allogeneic hematopoietic cell transplantation (allo-HCT). In particular, management of severe cardiotoxicity occurring in the early phases of allo-HCT is challenging. We encountered a case of severe cardiotoxicity resulting from AHF six days after allo-HCT, which resisted catecholamines and diuretics. The patient was treated with anthracycline-containing regimens and underwent myeloablative conditioning, including high-dose cyclophosphamide. As invasive circulatory assisting devices were contraindicated because of his immunocompromised status and bleeding tendency, we successfully treated the patient with ivabradine-containing medications. Ivabradine may therefore be considered an alternative drug for the treatment of severe cardiotoxicity induced by cytotoxic agents.     

From antenna to reaction center: Pathways of ultrafast energy and charge transfer in photosystem II

The photosystem II core complex (PSII-CC) is the smallest subunit of the oxygenic photosynthetic apparatus that contains core antennas and a reaction center, which together allow for rapid energy transfer and charge separation, ultimately leading to efficient solar energy conversion. However, there is a lack of consensus on the interplay between the energy transfer and charge separation dynamics of the core complex. Here, we report the application of two-dimensional electronic-vibrational (2DEV) spectroscopy to the spinach PSII-CC at 77 K. The simultaneous temporal and spectral resolution afforded by 2DEV spectroscopy facilitates the separation and direct assignment of coexisting dynamical processes. Our results show that the dominant dynamics of the PSII-CC are distinct in different excitation energy regions. By separating the excitation regions, we are able to distinguish the intraprotein dynamics and interprotein energy transfer. Additionally, with the improved resolution, we are able to identify the key pigments involved in the pathways, allowing for a direct connection between dynamical and structural information. Specifically, we show that C505 in CP43 and the peripheral chlorophyll Chlz_D1 in the reaction center are most likely responsible for energy transfer from CP43 to the reaction center.

Photosynthesis is the process through which solar energy is converted into chemical energy (1–3). Photosystem II (PSII), a pigment–protein complex found in cyanobacteria, algae, and land plants, is the site of water splitting and is therefore crucial for photosynthetic function (4–6). It is connected with a large light-harvesting antenna system that collects solar energy and transfers the energy to the reaction center (RC), where charge separation (CS) occurs. Unlike the antenna system of purple bacteria that has a clear energy funnel, the PSII antenna system has a more complicated composition and a very complex energy landscape (4–7). These features allow for regulation that responds to rapid environmental fluctuations and protect the organisms in, for example, excess light, while maintaining highly efficient electronic energy transfer (EET) under optimal conditions (8). To understand the intricate interactions between the subunits that allow for the robustness of this photosynthetic system, the first step is to understand how the antenna system is connected to the RC. The PSII core complex (PSII-CC) is the smallest unit in which the RC is connected to the antenna proteins. It is a dimeric pigment–protein complex in which each monomer contains an RC and two core antenna proteins, namely, CP43 and CP47 (1, 7). These core antennas not only harvest solar energy but also act as the crucial bridge between the peripheral light-harvesting antenna system and the RC. Fig. 1A shows the pigment arrangement of the PSII-CC. The RC, consisting of the D1 and D2 branches, binds the following pigments: 1) two special pair chlorophyll a (P_D1 and P_D2), 2) two accessory chlorophyll a (Chl_D1 and Chl_D2), 3) two pheophytin a (Pheo_D1 and Pheo_D2), and 4) two peripheral chlorophyll a (Chlz_D1 and Chlz_D2) (9, 10). Despite the similarity between the D1 and D2 branches, CS occurs only along the D1 branch (11, 12). CP43, one of the two core antenna proteins, contains 13 chlorophyll a (Chls) and is located closer to the D1 active branch. CP47 contains 16 Chls and is located closer to the D2 branch (10). Together, these proteins provide highly effective EET and CS, which are key to the high quantum yield of CS in the RC.Open in a separate windowFig. 1.(A) Pigment arrangement of monomeric PSII-CC (whereas it is typically found as a dimer) depicted based on the cryoelectron microscopy structure (3JCU) reported by Wei et al. (10). The pigments of CP43, RC, and CP47 are shown in green, blue, and red, respectively. (B) Corresponding excitonic energy levels of monomeric PSII-CC color coded to match pigments in A (55–57). The gray shaded regions in the background represent the three groups based on similar characteristic dynamics. Note that the boundaries between the groups provide only a rough separation region as the dynamical behaviors change gradually along ω_exc. The asterisk (for the RC state) indicates an optically dark state.Despite the importance of the PSII-CC, its early time dynamics is not fully understood—specifically the competition between EET and CS (5, 7). This is largely due to the highly congested excitonic manifold (Fig. 1B) and ultrafast EET timescales, which challenge ultrafast spectroscopic techniques. Two distinct models have been put forth to try to describe the function of the PSII-CC. These two models are the “exciton/radical pair equilibrium” (ERPE) model (13–17) and the “transfer-to-trap limited” (TTTL) model (18–22). An early fluorescence decay experiment (13, 14) suggested that rapid EET allows the excitonic states to reach an equilibrium between the core antennas and the RC before CS occurs (k_EET ≫ k_CS), which is the basis for the ERPE model. This model was later supported by improved time-resolved fluorescence (15) and transient absorption experiments (16). However, a major discrepancy in this model arose with the measurement of the X-ray crystal structure of the PSII-CC (18). It was suggested that the large distances (center-to-center distance, >20 Å) between antenna and RC pigments resolved in the crystal structure would mean that ultrafast EET between the antenna proteins and the RC is unlikely. A model was then put forth that instead suggested that the EET from the core antenna to the RC is slow compared to CS (k_EET ≪ k_CS), and therefore, the EET to the trap becomes a kinetic bottleneck (18). This TTTL model was later supported by transient infrared (IR) (19) and time-resolved fluorescence experiments (20, 21) as well as structure-based simulations (22). Additionally, Kaucikas et al. (23) performed a polarized transient IR experiment on an oriented single PSII-CC crystal. The decay of the polarization-dependent signature (50–100 ps) observed in their experiment suggests that equilibration between different subunits is slow, consistent with the TTTL model. However, it has been pointed out that satisfactory fitting of the spectral evolution to this model does not necessarily imply that it is correct (24, 25), especially as others have shown that the EET dynamics cannot be adequately described by a single hopping scheme (26, 27). A recent two-dimensional electronic spectroscopy (2DES) experiment (28) with improved time resolution has also revealed the existence of ultrafast EET (<100 fs) that was not predicted by theoretical calculations. In their work, Pan et al. (28) attributed the origin of this unexpectedly fast EET pathway to polaron formation. Vibronic effects on the ultrafast EET and CS dynamics of other photosynthetic proteins have also been discussed (29–38).The lack of detailed understanding of the PSII-CC early time dynamics, in particular the EET between the core antennas and the RC, highlights the need for further experimental input with the ability to make specific assignments of the dynamical pathways. This, however, requires simultaneous high temporal and spectral resolution, which remains a challenge for ultrafast spectroscopic techniques. Here, we describe the application of two-dimensional electronic-vibrational (2DEV) spectroscopy (39–41) to the PSII-CC. The combination of both spectral dimensions provides an improved resolution that allows us to obtain much more detailed dynamical information in complex systems. The excitonic energy landscapes generated by electronic coupling in photosynthetic complexes, combined with site-dependent and charge state–dependent vibrational spectra, allow the resolution along both axes of 2DEV spectra to provide a direct connection between energetic space (via visible excitation) and physical space (via IR detection). This advantage has proven to be useful for the studies of dynamics in photosynthetic pigment–protein complexes (33, 40–45). Specifically, the resolution along the electronic excitation axis allows for the separation of the contributions from different pathways, while the resolution along the vibrational detection axis provides a way to identify the protein subunits or even specific states involved in the dynamics. As we will show, this unique feature of 2DEV spectroscopy provides insight into the complex dynamics of the PSII-CC.In the following text, we will show that the sub-100-ps dynamics of the PSII-CC extracted from spinach are highly dependent on the excitation frequency range. The resolution along the detection axis allows different dominant dynamics to be identified. In addition, we will demonstrate how 2DEV spectroscopy allows us to connect the observed dynamics to specific excitonic states. This connection allows us to obtain a more specific pigment assignment for the EET pathways and therefore provides a more detailed understanding of the finely tuned interactions between the RC and the core antennas (specifically CP43, which is closer to the active D1 branch). We will conclude with a comparison between our results and the existing models in order to provide a path forward in the understanding of this critical photosynthetic component.