Effects of cGMP and cAMP on light responses of the photosensory pineal neurons in the lamprey, Lampetra japonica.

We examined in this study how external cyclic nucleotides affect the light response mechanism of the pineal photoreceptors and explored the existence of parietal eye type of photoreceptor of which the internal cGMP concentration increased during the light response. Pineal organs of river lampreys, Lampetra japonica, were treated with 8-bromo guanosine 3',5'-cyclic monophosphate (8Br-cGMP) or 8-bromo adenosine 3',5'-cyclic monophosphate (8Br-cAMP) before light stimuli, and the light responses were recorded from the second order neurons, chromatic or achromatic-type neurons. Excitatory and inhibitory light responses of the chromatic-type neuron became obscure by 9 and 3 mM 8Br-cGMP without changing the spontaneous spike discharge in the dark. 8Br-cAMP (3 mM) increased the frequency of spontaneous spike discharge, though it did not inhibit the light responses themselves. The inhibitory light response of the achromatic-type neuron decreased after adding 3 mM 8Br-cGMP, and it was unchanged by 3 mM 8Br-cAMP. The spontaneous spike discharge of the neurons in the dark was not affected by the cyclic nucleotides. The mechanism of these results can be explained if cGMP is an intracellular second messenger of light responses in the pineal photoreceptors and the blocking effect on photoresponses by externally applied 8Br-cGMP is caused by compensating for the reduction in intracellular cGMP by light. However, it does not indicate that the parietal eye type of photoreceptor found in lizard participates in the chromatic and achromatic-type responses in the lamprey pineal organ.

Production and characterization of recombinant Phanerochaete chrysosporium cellobiose dehydrogenase in the methylotrophic yeast Pichia pastoris.

The hemoflavoenzyme cellobiose dehydrogenase (CDH) from the white-rot fungus Phanerochaete chrysosporium has been heterologously expressed in the methylotrophic yeast Pichia pastoris. After 4 days of cultivation in the induction medium, the expression level reached 1800 U/L (79 mg/L) of CDH activity, which is considerably higher than that obtained previously for wild-type CDH (wtCDH) and recombinant CDH (rCDH) produced by P. chrysosporium. Analysis with SDS-PAGE and Coomassie Brilliant Blue (CBB) staining revealed a major protein band with an approximate molecular mass of 100 kDa, which was identified as rCDH by Western blotting. The absorption spectrum of rCDH shows that the protein contains one flavin and one heme cofactor per protein molecule, as does wtCDH. The kinetic parameters for rCDH using cellobiose, ubiquinone, and cytochrome c, as well as the cellulose-binding properties of rCDH were nearly identical to those of wtCDH. From these results, we conclude that the rCDH produced by Pichia pastoris retains the catalytic and cellulose-binding properties of the wild-type enzyme, and that the Pichia expression system is well suited for high-level production of rCDH.

Evidence that endo-1,4-beta-glucanases act on cellulose in suspension-cultured poplar cells.

Suspension-cultured poplar (Populus alba) cells produce two distinct endo-1,4-beta-glucanases, one of which is released in the extracellular culture medium and the other localized in their walls. Two cDNA clones, PopCel1 and PopCel2, isolated from a poplar cDNA library, encode the extracellular and the wall-bound endo-1, 4-beta-glucanases, respectively, based upon deduced amino acid sequences. The products of these two genes contained domains conserved in endo-1,4-beta-glucanase (family 9) and showed 91.5% amino acid identity. The levels of both PopCel1 and PopCel2 mRNAs increased during the lag phase of growth and decreased rapidly during the linear phase. After the levels had decreased, they were again increased by addition of sucrose to the culture medium and further enhanced by the addition of 2,4-dichlorophenoxyacetic acid (2,4-D) in the presence of sucrose. The accumulation of the mRNAs was correlated with the solubilization of cello-oligosaccharides. Cello-oligosaccharides and xyloglucan were also solubilized from the wall preparations of poplar cells incubated with enzyme preparations from the extracellular culture medium and walls. An antibody against both PopCel proteins reduced the production of cello-oligosaccharides by the extracellular enzyme by 90% and that by the wall-bound enzyme by 55%, and also prevented xyloglucan solubilization. The results show that the accumulation of poplar endo-1,4-beta-glucanases is regulated indirectly by auxin in the presence of sucrose and can act on cellulose in suspension-cultured poplar cells.

Light- and temperature-dependence of the melatonin secretion rhythm in the pineal organ of the lamprey, Lampetra japonica.

To identify the characteristics of the oscillator located in the pineal organ, we examined the effects of temperature and light on melatonin secretion rhythm using pineal organs in cultures. At 20 degrees C, the melatonin rhythm was obvious: low secretion during the daytime and high during the nighttime. When the temperature was lowered from 20 to 10 degrees C, the melatonin rhythm disappeared. When the temperature was returned from 10 to 20 degrees C, the rhythm quickly reappeared. The plasma melatonin level was measured in living lampreys kept at 7 degrees C to establish the melatonin profile at low temperature in vivo: secretion was not significantly different between daytime and nighttime. Under continuous light conditions, the melatonin elevation normally seen during the subjective night became obscure after 72 h. When the LD cycle was shifted by 6 h (phase-advanced or phase-delayed), the melatonin rhythm shifted to remain in the same phase relation to the LD cycle. This re-synchronization took several LD cycles. The results indicate that, in cultures, the melatonin secretion rhythm in the pineal organ of the lamprey is both light- and temperature-sensitive, and that in vivo, the melatonin rhythm is not the critical factor maintaining the locomotor activity rhythm of the lamprey. The role of the pineal organ and melatonin in the circadian organization of the lamprey is discussed.

Ruptured abdominal aortic aneurysms: analysis of factors influencing surgical results in 184 patients.

BACKGROUND: Rupture is often the first manifestation in patients with abdominal aortic aneurysms. Although elective surgery for non-ruptured abdominal aortic aneurysms has provided satisfactory surgical results, operative mortality of ruptured abdominal aortic aneurysms (rAAA) has not improved. The purpose of this study was to identify predictors for early hospital death in patients with rAAA. DESIGN: A retrospective study. SETTING: A university hospital and 20 affiliated hospitals. PATIENTS: PATIENTS undergoing surgical treatment for rAAA (n=183) between 1968 and 1997. INTERVENTIONS: All patients were surgically treated and divided into operative survivors (n=119) and non-survivors (n=64). MEASURES: The patient-related, procedure-related, and postoperative factors were compared between the two groups. A multivariate analysis was also conducted to determine predictors for hospital deaths. RESULTS: In univariate analysis, age at operation (p=0.004), preoperative hemodynamic conditions (p<0.0001), extent of hematoma (p<0.0001), preexistent renal dysfunction (p=0.001), and volumes of blood loss at operation (p=0.001) were significantly different between the two groups. The morbidity of postoperative renal failure (p<0.0001), gut ischemia (p=0.003), heart failure or ischemic heart disease (p<0.0001), and multiple organ dysfunction syndrome (p<0.0001) was higher in the non-survivors' group. Multivariate analysis also identified preoperative hemodynamic conditions, blood loss volume at operation, pre-existent renal dysfunction, postoperative renal failure, heart failure, and multiple organ dysfunction syndrome as incremental risk factors for hospital deaths. CONCLUSIONS: Every effort to maintain preoperative hemodynamic conditions, to reduce volumes of blood loss at operation, and to minimize deterioration of organ functions postoperatively is all essential to improve patient survival.

Occurrence of cello-oligosaccharides in the apoplast of auxin-treated pea stems

Treatment of pea (Pisum sativum L.) hypocotyl segments with indole-3-butyric acid, which promotes segment elongation, increased the solubilization of both xyloglucan and cello-oligosaccharides in the apoplast of auxin-treated pea stems. The cello-oligosaccharides were isolated from the apoplastic solution with a charcoal/Celite column and were identified as cellobiose, cellotriose, and cellotetraose after subsequent thin-layer chromatography and paper electrophoresis. Cello-oligosaccharides in the apoplastic fraction were monitored using cellobiose dehydrogenase. Both xyloglucan and cello-oligosaccharides appeared to be formed concurrently within 30 min after treatment with the auxin, and the cello-oligosaccharides increased with stem elongation even after 2 h. The total activity of cellulase did not increase for up to 4 h.

Cellobiose dehydrogenase from the fungi Phanerochaete chrysosporium and Humicola insolens. A flavohemoprotein from Humicola insolens contains 6-hydroxy-FAD as the dominant active cofactor.

Cellobiose dehydrogenases (CDH) were purified from cellulose-grown cultures of the fungi Phanerochaete chrysosporium and Humicola insolens. The pH optimum of the cellobiose-cytochrome c oxidoreductase activity of P. chrysosporium CDH was acidic, whereas that of H. insolens CDH was neutral. The absorption spectra of the two CDHs showed them to be typical hemoproteins, but there was a small difference in the visible region. Limited proteolysis between the heme and flavin domains was performed to investigate the cofactors. There was no difference in absorption spectrum between the heme domains of P. chrysosporium and H. insolens CDHs. The midpoint potentials of heme at pH 7.0 were almost identical, and no difference in pH dependence was observed over the range of pH 3-9. The pH dependence of cellobiose oxidation by the flavin domains was similar to that of the native CDHs, indicating that the difference in the pH dependence of the catalytic activity between the two CDHs is because of the flavin domains. The absorption spectrum of the flavin domain from H. insolens CDH has absorbance maxima at 343 and 426 and a broad absorption peak at 660 nm, whereas that of P. chrysosporium CDH showed a normal flavoprotein spectrum. Flavin cofactors were extracted from the flavin domains and analyzed by high-performance liquid chromatography. The flavin cofactor from H. insolens was found to be a mixture of 60% 6-hydroxy-FAD and 40% FAD, whereas that from P. chrysosporium CDH was normal FAD. After reconstitution of the deflavo-proteins it was found that flavin domains containing 6-hydroxy-FAD were clearly active but their cellobiose oxidation rates were lower than those of flavin domains containing normal FAD. Reconstitution of flavin cofactor had no effect on the optimum pH. From these results, it is concluded that the pH dependence is not because of the flavin cofactor but is because of the protein molecule.

Unidirectional processive action of cellobiohydrolase Cel7A on Valonia cellulose microcrystals.

On the basis of the 'parallel-up' structure of the cellulose crystal, a crystallographic approach to study the mode of action of cellobiohydrolase Cel7A on Valonia cellulose microcrystal has been carried out. After incubation with Cel7A, most of the initially smooth and well defined Valonia microcrystals displayed fibrillation. However, as the hydrolysis reaction was rather heterogeneous, some microcrystals remained superficially intact. Close investigation on such crystals revealed polar morphology: one end was narrowed extremely or pointed. Electron microdiffraction analysis of these crystals evidenced that the narrowing of the microcrystals occurs at their reducing end side. This was also confirmed by the visualization of selective reducing end labeling at the pointed ends of microcrystals. These lines of investigation are the first demonstration that the processivity of Cel7A action against insoluble highly crystalline celluloses is unambiguously toward non-reducing ends from reducing ends.

Cellobiose dehydrogenase enhances Phanerochaete chrysosporium cellobiohydrolase I activity by relieving product inhibition.

The interaction of cellobiose dehydrogenase (CDH) with cellobiohydrolase I (CBH I) in cellulose-grown cultures of Phanerochaete chrysosporium was investigated to clarify the role of CDH in cellulose degradation. Decomposition of bacterial microcrystalline cellulose by CBH I was enhanced significantly in the presence of the CDH/ferricyanide redox-system compared with CBH I alone. To explain this phenomenon, a model system, using p-nitrophenyl-beta-D-cellobioside as a substrate, was elaborated for measurement of CBH I activity with and without the CDH redox-system. The activity of CBH I for hydrolysis of p-nitrophenyl-beta-D-cellobioside was also enhanced in the presence of the redox system. It was found that Km for hydrolysis of p-nitrophenyl-beta-D-cellobioside by CBH I was lower in the presence than in the absence of the CDH/ferricyanide redox-system, 142 microM and 384 microM, respectively, while no significant difference was observed between the k(cat) values. These results indicate that cellulase activity is enhanced by an increased affinity for p-nitrophenyl-beta-D-cellobioside, rather than by an increased hydrolysis rate. This shows that cellobiose, the hydrolysis product, acts as a competitive inhibitor of the interaction between CBH I and p-nitrophenyl-beta-D-cellobioside. This was confirmed by addition of cellobiose, which was found to competitively inhibit hydrolysis of p-nitrophenyl-beta-D-cellobioside by CBH I in the absence of the CDH redox system, and the Ki value for cellobiose inhibition was estimated to be 65 microM. However, this inhibition did not occur if cellobiose was incubated with CDH before addition of CBH I. It was concluded from these results that the reason for the enhancement of CBH I activity in the presence of the CDH redox system was that it relieves competitive inhibition of cellobiose by its oxidation to cellobionolactone.

Relationship between the Physical Properties and Surface Area of Cellulose Derived from Adsorbates of Various Molecular Sizes.

An aqueous suspension of bacterial cellulose (BC) has such physical properties as higher viscosity, emulsion-stabilizing effect and filler retention than cellulose of other origins. The specific surface areas of BC, microfibrillated cellulose and wood pulp were evaluated by determining the maximum amounts of adsorption of Congo red, cellobiose dehydrogenase (CDH) and xyloglucan. There was a positive linear correlation between the above-mentioned physical properties of each cellulose sample and the specific surface area derived from the maximum amount of CDH adsorbed. The highest physical property values for BC result from the largest external surface area of the fibrils of BC to which CDH was adsorbed.

Enhanced production of cellobiose dehydrogenase in cultures of Phanerochaete chrysosporium supplemented with bovine calf serum.

The enzyme cellobiose dehydrogenase (CDH), produced by many wood-degrading fungi has, in recent years, attracted considerable interest for its possible role in both cellulose and lignin degradation. To characterize the enzyme better and to identify its role in the degradation of wood and wood components, it is desirable to produce it in higher amounts. We report here that the addition of bovine calf serum to cellulose-grown cultures of Phanerochaete chrysosporium enhances the production of certain enzymes, CDH in particular. The highest CDH production was obtained with 45 ml of serum/litre of medium added on day 3 or 4. The resultant CDH yield was approx. 700-800 units/litre, which was 3.5-4 times higher than that in cultures without serum. Serum addition also enhanced the production of beta-glucosidase. However, the impact on CDH production was the most dramatic. The enhanced enzyme production cannot be explained by increased rates of spore germination, simple nutrient effects or cofactor effects. Fractionation of serum by Cohn's fractionation technique showed that the albumin (BSA) fraction had almost the same effect as whole serum. However, purified BSA had less effect than crude BSA (fraction V of Cohn's fractions), suggesting that an additional factor, probably a protease inhibitor in serum, also contributed to the effect of serum.

Three-dimensional reconstruction of serotonin-immunoreactive photoreceptors in the pineal organ of the river lamprey, Lampetra japonica.

Serotonin-immunoreactive (5-HT IR) photoreceptors are present in the pineal complex (pineal and parapineal organ) of the river lamprey, Lampetra japonica. They are so-called modified pineal photoreceptors and have been regarded as photoneuroendocrine cells which secrete melatonin. We reconstructed 5-HT IR cells with a computer to demonstrate their three-dimensional structures from optical sections taken by a confocal laser scanning microscope. The 5-HT IR cell possesses a basal process, and it appears that the process does not branch out. These processes contact each other at the basal region of the end vesicle, and a process extends to the soma of the neighboring 5-HT IR cell. These findings were obtained by three-dimensional analysis with a computer, which is a useful technique to demonstrate the interaction between cells. We suggest that the 5-HT IR photoreceptors interact with one another.

Melatonin excretion rhythms in the cultured pineal organ of the lamprey, Lampetra japonica.

Pineal organ of the lamprey, Lampetra japonica, is essential to keep the circadian locomotor activity rhythm as previously reported. In this paper, we tried to show that an endogenous oscillator is located and is working in the pineal organ. When the pineal organs were excised and cultured in a plastic tube with M199 medium at 20 degrees C, melatonin secretion rhythms were clearly observed under both light-dark and continuous dark conditions. The circadian secretion of melatonin continued for more than five cycles under the continuous dark condition. This indicates that the pineal organ has an endogenous oscillator and that the melatonin secretion rhythm is controlled by this oscillator. These findings suggest the possibility that the locomotor activity rhythm of the lamprey is under the control of the oscillator in the pineal organ.

Characterization of the gene for pyruvate,orthophosphate dikinase from rice, a C3 plant, and a comparison of structure and expression between C3 and C4 genes for this protein.

To investigate the molecular changes that might have occurred in genes for pyruvate,orthophosphate dikinase (PPDK) during the evolution of C4 plants from C3 plants, we isolated a full-length cDNA and the corresponding gene for a C4-like PPDK from rice, a C3 gramineous plant and compared their structures and promoter activities to those of the corresponding gene from maize, a C4 gramineous plant. As in maize, there are at least two ppdk genes in rice and one of them was very similar to the maize C4-type ppdk. The deduced amino acid sequence of the rice PPDK was 88% homologous to the maize C4-type PPDK in the mature peptide region and 56% homologous in the transit peptide region. The C4-like ppdk in rice contained 21 exons, which were interrupted by twenty introns, and the positions of the introns were essentially the same as those in the gene from maize, with the except in that the gene from rice had two extra introns. Such extra introns were also found in the C4-type ppdk from a dicot, Flaveria, at the same positions. These results strongly suggest that the two introns were present in an ancestral gene before the divergence of monocot and dicot plants. The C4-like ppdk in rice contained two functionally independent promoters had generated a larger transcript with the transit peptide region and a smaller transcript without this region. The unusual dual-promoter system for transcription has been conserved in the C4-type ppdk gene from maize, indicating that the dual-promoter system is a common feature of ppdk genes in both C3 and C4 plants. The patterns of expression of the two transcripts in rice were different: the larger transcript was expressed exclusively in green leaves at a low level whereas the smaller transcript was expressed in some reproductive organs at a high level. Essentially the same patterns of expression were observed in maize, but the level of expression of the larger transcript in maize green leaves was much higher than that in green leaves of rice. The promoter activities of the rice and maize genes for PPDK were examined directly in a transient expression assay in maize mesophyll protoplasts after electroporation with promoter::beta-glucuronidase chimeric genes. The rice promoter for the smaller transcript was very active in the protoplasts but the rice promoter for the larger transcript had relatively low activity. By contrast, both promoters of the maize gene had high activity. Taken together, these results demonstrate that the rice C4-like ppdk is very similar to the maize C4-type ppdk, not only in terms of primary structure but also in terms of the regulation of expression, with the exception that the strength of the maize promoter for the larger transcript is higher. The results strongly suggest that the genetic alterations required to give rise to the C4-type ppdk gene were relatively limited.

Localization of cellobiose dehydrogenase in cellulose-grown cultures of Phanerochaete chrysosporium.

To elucidate the function of cellobiose dehydrogenase (CDH) in cellulose degradation by Phanerochaete chrysosporium, production and localization of CDH were investigated and compared with those in shaking and aerated static cultures grown on cellulose. Substantial CDH activity was detected in the medium of the shake cultures after 8 days of incubation, while no CDH activity was detected in the medium of static cultures at any point during the incubation period. Light microscopy clearly showed that many cellulose particles were adsorbed on the surface of the hypha in static cultures, whereas no cellulose particles were absorbed to the hypha is shake cultures. The addition of laminarinase to static cultures was very effective in detaching cellulose particles from the hypha surfaces. Using a potentiometric assay performed with an oxidation-reduction potential electrode, some CDH activity could be detected on the hypha/cellulose complexes in static cultures. Thus, CDH is produced also in static cultures, albeit in lower amounts that in shake cultures, but the enzyme is not released into the medium. It seem likely that the beta-1,3-glucan layer plays an important role in CDH localization and cellulose degradation. Immunocytochemical confocal laser scanning microscopy for the static cultures demonstrated that most CDH was adsorbed on the surface of the cellulose, especially around the cracks, which were formed by the action of cellulases during the course of incubation. From these observations, we conclude a direct participation of CDH in the degradation of cellulose in cooperation with cellulases.

Effect of iron as a new type of phosphate binder in hemodialysis patients.

Hyperphosphatemia is one of the major problems requiring management in the majority of hemodialysis patients and they require phosphate-binding agents to control the hyperphosphatemia. Aluminum hydroxide and calcium compounds are used currently as phosphate-binding agents to treat hyperphosphatemia, but these compounds can cause undesirable side effects. Therefore, the development of new phosphate-binding agents is imperative. It is well known that oral and intravenous administration of iron causes hypophosphatemia. We hypothesized that this side effect of iron may be beneficial for the treatment of hyperphosphatemia in hemodialysis patients. Consequently, we conducted a fundamental and clinical investigation of the effects of iron administration. Membrane permeability of phosphorus in a mixture of sodium ferrous citrate and dessicated aluminium hydroxide in the presence of hydrogenated lecithin as a phosporic compound was examined. Fifteen patients undergoing hemodialysis were treated with 150 mg of sodium ferrous citrate given orally for eight weeks. The permeability of the filtering membrane to phosphorus decreased in accordance with the dosage of sodium ferrous citrate and dessicated aluminum hydroxide. The degree of phosphate-binding effect of sodium ferrous citrate was larger than that of dessicated aluminum hydroxide. Serum phosphorus decreased significantly during the experiment. These results suggest that the oral administration of sodium ferrous citrate as a new phosphate binder is a useful therapeutic method for hemodialysis in patients with hyperphosphatemia.

Distribution of cholinergic and dopaminergic receptors in rainbow trout pineal gland.

The involvement of multiple receptors in modulating the function of the pineal gland was investigated by searching for dopaminergic and cholinergic receptors in trout pineal gland. Dopamine D1 and D2 receptors were measured using [3H]SCH23390 and [3H]spiperone, respectively. Muscarinic and nicotinic cholinergic receptors were measured using quinuclidinyl benzilate ([3H]QNB) and [3H]methylcarbamyl choline, respectively. High-affinity choline uptake sites were measured using [3H]hemicholinium-3. The distribution of dopaminergic receptors varied throughout the pineal gland in that the density of D2 receptors, which was higher than that of D1 receptors, was most abundant in the distal region, exhibiting a value of 112 +/- 17 fmol/mg tissue. The distribution of both muscarinic and nicotinic receptors was uniform throughout the pineal gland. However, the highest value for the high-affinity choline transporter (106 +/- 17 fmol/mg tissue) occurred in the proximal portion of the trout pineal gland. The results of these studies indicate that the pineal gland should not be viewed as a homogeneous tissue possessing identical density of various receptors. Furthermore, these results, along with previous data, are interpreted to suggest that different regions of pineal gland may indeed possess unique functions.

Release of the FAD domain from cellobiose oxidase by proteases from cellulolytic cultures of Phanerochaete chrysosporium.

Evidence has previously suggested that cellobiose:quinone oxidoreductase (CBQ) in cellulolytic cultures of Phanerochaete chrysosporium might be produced from cellobiose oxidase (CBO) by proteolytic cleavage. This study demonstrates that the ratio of CBO activity to (CBO + CBQ) activity declines with decreasing culture pH, while protease activity increases. Furthermore, we demonstrate that endogenous P. chrysosporium proteases can only cleave CBO when the enzyme is bound to cellulose. This is the first demonstration that the proteases produced in cellulolytic cultures of P. chrysosporium can release the FAD domain from CBO.

Identification of vasoactive intestinal polypeptide (VIP) binding protein in bovine pineal gland.

Vasoactive intestinal polypeptide (VIP) containing nerves are present in close proximity to epithelial, endocrine, and vascular smooth muscle cells. The pineal gland, known also as a "neuroendocrine transducer organ" contains a high content of VIP which prompted us to characterize the binding sites for VIP in this organ. [Tyr10-125I]VIP was bound selectively and specifically to pineal membrane preparations in a time-dependent fashion. Scatchard analysis demonstrated a single class of high affinity binding sites with a dissociation constant (Kd) value of 5.7 +/- 0.52 nmol/1 and a receptor density (Bmax) value of 440 +/- 35 fmol/mg protein. A Hill Plot with a slope of 1.013 indicated the absence of cooperativity. Covalent crosslinking with [Tyr10-125I]VIP followed by SDS electrophoresis and autoradiography, revealed that the VIP binding protein exhibited a molecular weight of 51.8 +/- 0.5 kDa. The precise function of pineal VIP binding protein remains to be delineated.