Purification and characterization of an antibacterial protein in the skin secretion of rockfish Sebastes schlegeli.

An antibacterial protein in the skin secretion of rockfish (Sebastes schlegeli) was purified by lectin affinity chromatography on Con A-Sepharose and gel filtration on TSKgel G3000SW. The antibacterial protein featured the high molecular mass and selective action against Gram-negative bacteria. The molecular mass of the protein was estimated to be approximately 150 kDa in gel filtration and approximately 75 kDa by SDS-PAGE, suggesting that it is dimeric. The antibacterial principle was an acidic glycoprotein with pI 4.5, 3.4% reducing sugar and 2.8% amino sugar. Its sugar chains had N-type (high mannose-type) oligosaccharide and sialic acid components. It inhibited strongly the growth of Aeromonas salmonicida, Photobacterium damselae and Shewanella putrefaciens with a minimum inhibitory concentration (MIC) of approximately 3 microg/ml, and moderately the growth of Vibrio parahaemolyticus and A. hydrophila with a MIC of 12.5 microg/ml and 25 microg/ml, respectively. The values of the minimum bactericidal concentration were almost equivalent to those of MIC. The potent sensitivity against virulent pathogens such as A. hydrophila, A. salmonicida and P. damselae may contribute considerably to the innate host defense mechanism to combat microbes on the mucosal surfaces of the rockfish.

Occurrence of tetrodotoxin-related substances in the nontoxic puffer Takifugu xanthopterus.

Instead of tetrodotoxin, significant amounts of tetrodotoxin-related substances with no mouse lethality were detected in the nontoxic liver specimen of puffer fish, Takifugu xanthopterus. The tetrodotoxin-related substances, which were demonstrated to be tetrodotoxin derivatives by gas chromatography-mass spectrometry analysis, were similar to tetrodonic acid in HPLC but distinguishable from it in electrophoresis. Our results suggest that nontoxic puffer fish contains nontoxic tetrodotoxin derivatives as precursors or metabolites of tetrodotoxin.

Memory rehabilitation of an amnesic patient following limbic encephalitis and a role of family members: a case report.

We describe problems in daily living that arose in a 46-year-old man with severe amnesia following limbic encephalitis. Amnesic symptoms changed from stage I (difficulty in memory retention) to stage II (loss of continuity of memory) and finally to stage III (paramnesia and confused sequence of events), significantly affecting his ability to function. Questionnaire response assessment, directly observed behavior, neuropsychological testing, and especially interviewing permitted qualitative assessment of clinical changes, promoted patient insight into the memory disturbance, and enhanced motivation to use a memory notebook. Additionally, the family gained a better understanding of the disorder, made appropriate environmental modifications, and provided other necessary assistance. Episodic memory improved, and the memory notebook served as an effective compensatory tool. However, disturbance in prospective memory did not improve, and was not compensated adequately by use of the notebook. Anxiety and significant impairment of everyday functioning resulted. Long-term supportive intervention at home was necessary. The patient's wife in particular needed to make environmental adjustments and aid him in use of the notebook.

Subcellular distribution of tetrodotoxin in puffer fish liver.

The liver homogenate of puffer fish was fractionated into blood cell, nuclear, mitochondrial, microsomal and cytosol fractions by the differential centrifugation method. All the five fractions were toxic to mice, although the toxin amount was significantly high in the cytosol fraction. Analyses by HPLC and LC-FABMS demonstrated that tetrodotoxin is the major toxic principle in each fraction. These results reveal that tetrodotoxin is widely distributed in organelles in liver cells, though predominantly in the cytosol fraction.

Purification and IgE-binding epitopes of a major allergen in the gastropod Turbo cornutus.

The major allergen (Tur c 1) in the muscle of the gastropod, Turbo cornutus, was isolated by Sephacryl S-300, Mono Q HR 5/5 and TSKgel Phenyl-5PW RP column chromatography. ELISA showed Tur c 1 to react strongly with sera from three individuals sensitive to both mollusks and crustaceans. SDS-PAGE showed Tur c 1 to produce a major band corresponding to a molecular mass of 35 kDa under the reduced condition. Its amino acid composition was characterized by the abundance of Glx, followed by Leu, Ala and Lys in decreasing abundance, and the absence of Trp. In addition to these properties, the determined partial amino acid sequence identified Tur c 1 to be a tropomyosin, as in the case of the known mollusk and crustacean allergens. However, the results of competitive ELISA inhibition experiments suggest that Tur c 1 has an IgE-binding epitope in the C-terminal region which is dissimilar to those proposed for Cra g 1 (the oyster Crassostrea gigas allergen) and Pen i 1 (the shrimp Penaeus indicus allergen).

Tigloylcholine: a new choline ester toxin from the hypobranchial gland of two species of muricid gastropods (Thais clavigera and Thais bronni).

Crude extracts from hypobranchial glands of two species of muricid gastropods, Thais clavigera and Thais bronni, were highly lethal to mice. Regardless of species, a new choline ester was isolated as the major toxic principle and elucidated to be tigloylcholine, a structural isomer of senecioylcholine widely found in gastropod hypobranchial glands, by 1H- and 13C-NMR as well as FAB-MS. The i.v. LD50 (mouse) of tigloylcholine was estimated to be 0.92 mg/kg.

Purification and properties of phospholipases A2 from the crown-of-thorns starfish (Acanthaster planci) venom.

Two phospholipases A < inf2 (named AP-PLA2-I and II) were purified from the crown-of-thorns starfish (Acanthaster planci) venom. Both enzymes were confirmed to be PLA2s, based on the results that they showed hemolytic activity only in the presence of phosphatidylcholine (PC) and also released fluorescent fatty acids from PC with labeled fatty acids at the sn-2 position. The enzyme activity of both PLA2s was enhanced by Ca2+ but reduced by Cu2+ and Zn2+. The molecular mass of AP-PLA2-I was estimated to be 28 kDa by gel filtration and 15 kDa by SDS-PAGE, indicating that AP-PLA2-I is a dimer composed of the same subunit. In contrast, AP-PLA2-II was judged to be a monomer with a molecular mass of 12 kDa (gel filtration) or 15 kDa (SDS-PAGE). The amino acid compositions of the two enzymes were comparable to each other; Asx, Glx and Gly were rich in both molecules, while Met, His and Trp were poor. Analyses by a sequencer determined the first 62 amino acid residues for both PLA2s. In the AP-PLA2-I preparation, minor amino acids were additionally found at 17 positions, suggesting the coexistence of another PLA2-component. As compared to the N-terminal sequences of the known PLA2s, both AP-PLA2-I and II were identified as class I enzymes not only because they have Cys-11 and lack Cys-51 but also because they contain the elapid loop in the region 53-61.

Pulmonary edema induced by angiotensin I in rats.

This study was performed to demonstrate an experimental procedure of pulmonary edema induced by angiotensin I (AT I) in rats and to elucidate the mechanism of hemodynamic pulmonary edema. In the previous pilot study, 20 microg/kg of AT I was found to be an adequate dose for inducing pulmonary edema. To elucidate the mechanism of AT I pulmonary edema and protective measures against it, we observed the effects of captopril (CAP, 5 and 10 mg/kg), an angiotensin converting enzyme inhibitor; losartan (LOS, 10 mg/kg), an angiotensin II (AT II)-receptor antagonist; and phentolamine (PHE, 10 mg/kg), an alpha-adrenergic receptor blocker, on AT I-induced pulmonary edema in rats. Similarly, we also observed the effects of CAP (10 and 20 mg/kg) on pulmonary edema induced by 25 microg/kg of adrenaline (ADR) in rats. The development of AT I-induced pulmonary edema was significantly suppressed by CAP and LOS, but was unaffected by PHE. In contrast, the development of ADR-induced pulmonary edema was not suppressed by CAP. These results suggest that AT I-induced pulmonary edema is developed via the AT II and a specific AT II-receptor, without the indirect action of adrenaline.

Novel polypeptide toxins with crab lethality from the sea anemone Anemonia erythraea.

The sea anemone Anemonia erythraea was found to contain polypeptide toxins with crab lethality as well as hemolysins. Three polypeptide toxins (AETX I, II and III) were isolated by gel filtration on Sephadex G-50 and reverse-phase HPLC on TSKgel ODS-120T. A geographic variation in toxin composition was suggested. The LD50 against crabs of AETX I, II and III were estimated to be 2.2, 0.53 and 0.28 microg/kg, respectively, but none of the toxins showed lethality in mice. The amino acid sequences of the three toxins were deduced from sequencings of the whole molecules and their enzymatic fragments. Amino acid analyses and molecular mass determinations supported the accuracy of the deduced sequences. AETX I, comprising 47 amino acid residues including 6 half-Cys residues, is an analog of sea anemone type I toxins. On the other hand, AETX II and III, which are highly homologous with each other, are quite distinct from the known sea anemone polypeptide toxins in that they are composed of 59 residues including 10 half-Cys residues. Interestingly, both toxins have sequence similarities with neurotoxins isolated from the Brazilian 'armed' spider Phoneutria nigriventer.

Halcurin, a polypeptide toxin from the sea anemone Halcurias sp., with a structural resemblance to type 1 and 2 toxins.

The aqueous extract of the sea anemone Halcurias sp. belonging to the suborder Endocoelantheae was found to be potently lethal to crabs, although it showed neither lethal activity in mice nor hemolytic activity. A polypeptide toxin (named halcurin) with a LD50 of 5.8 micrograms/kg against crabs was isolated by gel filtration on Sephadex G-50 and reverse-phase high-performance liquid chromatography on TSKgel ODS-120T. The complete amino acid sequence of halcurin comprising 47 residues was elucidated by sequence analysis of the native molecule and its enzymatic fragment. Comparison with the known sea anemone polypeptide toxins (types 1-3), which are all from members of the suborder Nynantheae, revealed a high sequence homology (49-74%) of halcurin with type 2 toxins. Also, halcurin has several residues conserved for only type 1 toxins. These results, together with the fact the Halcurias sp. is a more primitive species than members of Nynantheae, suggest that type 1 and 2 toxins have evolved from a common ancestor with a sequence more similar to halcurin.

Angiotensin II-induced pulmonary edema in a rabbit model.

We conducted the present study to propose a rabbit model of pulmonary edema (PE) induced by angiotensin II (AII) and to test the preventive effect of losartan on this form of PE. AII was administered to rabbits intravenously at 50, 100, 150 or 300 microg/kg, either by continuous infusion (10 min) or by bolus injection (30 sec). Continuously administered AII (150 microg/kg) induced PE in most cases, while a bolus injection of the same dosage did not. Additionally, the incidence of PE increased with higher dosages of AII when it was infused continuously. A newly established parameter, the area under the systolic blood pressure-time curve corrected by baseline (cAUC), was prone to rise as the incidence of PE increased. Moreover, cAUC signficantly correlated with the wet-dry lung weight ratio (r=0.66, P<0.05). Subsequently, 0.5 or 3.0 mg/kg of losartan was given before continuous infusion of 150 microg/kg of AII. The higher dosage of losartan prevented PE completely, while the lower one did so moderately. We concluded that intravenous administration of AII induces PE, probably as a result of increasing afterload. Furthermore, an adequate dosage of losartan can prevent PE because it reduces the pressor effect of AII.

Isolation and amino acid sequences of two Kunitz-type protease inhibitors from the sea anemone Anthopleura aff. xanthogrammica.

Two protease inhibitors (AXPI-I and -II) were isolated from the sea anemone Anthopleura aff. xanthogrammica by a combination of acetone precipitation, gel filtration on Sephadex G-75, cation-exchange fast protein liquid chromatography (FPLC) on Mono S and reverse-phase HPLC on TSKgel ODS-120T. Both inhibitors are basic polypeptides, and their amino acid compositions are characterized by the presence of six half-Cys residues and the absence of Met and Trp. They are potently active against trypsin; inhibition of other serine proteases (alpha-chymotrypsin and elastase) is also displayed by only AXPI-I. However, the inhibitors show no affinity for metallo-proteases and cysteine proteases. Analyses of the N-terminal portion and enzymatic fragments established their complete amino acid sequences comprising 58 residues. The overall sequence homology and the conserved location of all half-Cys residues confirmed that the A. aff. xanthogrammica inhibitors belong to the Kunitz-type family.

Serum albumin for estimating creatinine clearance in the elderly with muscle atrophy.

The present study was conducted to develop a special formula to predict creatinine clearance (CCr, ml/min) in the elderly with chronic muscle atrophy using serum albumin (Alb, g/dl). We obtained 90 data sets including actual body weight (BW, kg), urinary creatinine excretion (UCr, mg/24 h), serum creatinine (SCr, mg/dl), and Alb from 90 inpatients aged 60-92 years. Regression equations were determined between the dependent variable of UCr/BW and the explanatory variable of Alb as follows: For males Ucr/BW = 2.695 Alb + 4.665 For females Ucr/BW = 1.827 Alb + 4.146. Then, the new predictive formula was derived from the equations: For males CCr = (19Alb + 32)BW/(100 x SCr) For females CCr = (13Alb + 29)BW/(100 x SCr). Evaluations for the predictive error (predicted CCr- measured CCr) showed that the new formula could provide more accurate and less biased estimates of CCr than the Cockcroft and Gault formula could, even in patients with renal insufficiency and in those with Alb < or = 2.8 g/dl.

Pulmonary edema induced by angiotensin II in rats.

We performed this study to demonstrate the experimental procedure for inducing pulmonary edema by angiotensin II (AT II) in rats and to determine the mechanism of hemodynamic pulmonary edema. In the pilot study, 10 micrograms/ml of AT II was found to be adequate as the edematogenic dose for inducing pulmonary edema. The edematogenic dose of AT II was intravenously given to rats pretreated with 20 mg/kg of an AT II-receptor antagonist, E 4177 (3-[(2'-carboxybiphenyl-4-yl)methyl]-2-cyclopropyl-7-methyl-3H- imidazo[4,5-b]pyridine), and to rats given 10 mg/kg of an alpha-adrenergic blocker, phentolamine. Similarly, pulmonary edema was induced by 25 micrograms/ml of adrenaline in rats pretreated with E 4177 (20 mg/kg) and rats with no pretreatment. E 4177 completely suppressed the development of AT II-induced pulmonary edema, whereas phentolamine could not. On the contrary, E 4177 could not suppress the development of adrenaline-induced pulmonary edema. We concluded that AT II-induced pulmonary edema will develop via the specific AT II receptor without the indirect action of adrenaline.

Rapid and accurate estimation of creatinine clearance in the muscle-wasted elderly by computed tomography.

To develop a new method for accurately predicting creatinine clearance (Ccr) in the debilitated elderly without urine collections, the total muscle volume of both thighs (MV thigh), as a new predictive parameter, was calculated using computed tomography in 68 debilitated patients aged 61-92 years. A regression line between 24-hour urinary creatinine excretion and MV thigh was divided by serum creatinine (Scr), and then the following formula was derived: Ccr (ml/min) = 9 x [MV thigh (cm3) - 100]/[1,000 x Scr (mg/dl)] Predicted Ccr by the new method more closely correlated with measured Ccr (r = 0.862) than did predicted Ccr by Cockcroft and Gault's method (the C-G method) (r = 0.727). Statistical analysis of prediction error (= predicted Ccr-measured Ccr) suggested that the new method could provide more accurate and less biased estimations of Ccr than did the C-G method, even in patients with renal insufficiency. By using computed tomography, we have proposed a more accurate method for predicting Ccr of the debilitated elderly than the C-G method, although the new method has inherent disadvantages of radiation exposure and high cost.

[Comparison of formulas for calculating average skin temperature and their characteristics].

In order to obtain data of skin temperatures experiments were carried out using three healthy young Japanese males. The subjects were exposed to each of the four environments with dry bulb temperatures of 15 degrees C, 19 degrees C, 25 degrees C and 33 degrees C. At each of these air temperatures, relative humidity and air movement were set at 50% and 0.15m/s respectively. The subjects wore only athletic shorts, seated on the meshed chair. Each subject was measured with thermisters continuously for one hour under these conditions to obtain twenty-nine regional skin temperature. The above experiments were made with one subject at a time in the test chamber. The data of skin temperatures observed were substituted into twenty-eight different weighting formulas for comparison. The present analysis revealed that the calculation from the 12-point and the 7-point skin area formulas by Hardy-DuBois showed approximate mean values of the twenty eight. Moreover, the values calculated from the formula by Nadel et al, which was weighted by skin area and thermal sensitivity, are similar to the values calculated by the formula of Mochida, which was weighted by skin area, heat transfer coefficients and thermal sensitivity. Furthermore, the authors verified that the area-mean weighting factor was derived from the Teichner's definition in which a limiting value of arithmetical mean of skin temperatures gave a value of average skin temperature.

[Values of wettedness observed in clothed subject and the theoretical equal line of average skin temperature].

In order to obtain the characteristics of the change of wettedness under constant average skin temperature 36 degrees C, the experiments were carried out using two young male subjects in a test chamber. From the basic measurements of both environmental parameters and human physiological responses, the authors found the wettedness value changeable even though the average skin temperature was constant. The following results were obtained under the conditions of resting-sitting, summer clothing worn, still air movement and constant average skin temperature 36 degrees C; 1. there is a positive correlation between wettedness and the environmental vapor pressure. 2. There is a negative correlation between wettedness and the air temperature. 3. There is a positive correlation between the evaporative heat loss from skin surface and the air temperature. 4. There is a negative correlation between the evaporative heat loss and the vapor pressure. 5. There is a negative correlation between wettedness and the evaporative heat loss. 6. Both maximum and minimum values of wettedness correspond to the average skin temperature. Considering the afore-mentioned results, the equal line of the average skin temperature does not form a straight line, but a curved line on the psychrometric chart.

Affinity chromatographic purification of tetrodotoxin by use of tetrodotoxin-binding high molecular weight substances in the body fluid of shore crab (Hemigrapsus sanguineus) as ligands.

A purification method for tetrodotoxin (TTX), based on affinity chromatography using the TTX-binding high mol. wt substances in the body fluid of shore crab (Hemigrapsus sanguineus) as ligands, was developed. This method was particularly useful for analysis of TTX in biological samples with low concentrations of TTX. The affinity gel prepared was highly specific for TTX, having no ability to bind 4-epi-TTX and anhydro-TTX as well as saxitoxin.

Factors affecting the patency of stents in malignant biliary obstructive disease: univariate and multivariate analysis.

To investigate the factors affecting the patency of biliary stents, we performed univariate and multivariate analyses in 370 straight stents with diameters of 10- to 12-French gauge which had been inserted singly as palliative treatment in 228 patients with malignant biliary obstructive disease. In the analysis of 226 first stents, advanced age of patients and higher serum bilirubin levels before insertion were found to be significant unfavorable factors limiting the duration of stent function. Overall, older age, male sex, no stent cleaning, and insertion on three or more occasions were significant unfavorable prognostic factors for patency. The risk of occlusion for stents inserted on three or more occasions was 2.32 times that of the first stent insertion. The median duration of patency of first, second, and subsequent stents was 177, 98, and 97 days, respectively. Diagnosis and the area of stenosis were unrelated to patency. No predictive significance of length or size of stent between 10- and 12-French gauge was found in our study.