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In the course of studies on the cleavage reaction of S-(isopropylcarboxymethyl) glutathione (GSIV) into isovalthine in kidney homogenate or glutathionase preparation, it has sometimes been observed that the amount of isovalthine formed is far less than that of GSIV decomposed¹. Furthermore, when such reaction mixture is analyzed on an automatic amino acid analyzer, prominent peak corresponding to the reasonable amount of S-(isopropy1carboxymethyl)cysteinylglycine which is an expected intermediate of the GSIV cleavage reaction cannot be found up to 400 effluent ml. Though several reasons may be considered for the explanation of the above curious phenomenon, the effect of cystathionase on isovalthine is at first examined here. But the result was negative. L- and L-Alloisovalthineused as substrate were prepared by the method of OHMORI². Homoserine and purified cystathionase in ammonium sulfate solution prepared according to the method of GREENBERGB³ were kindly furnished by Prof. M. Suda of Osaka University. Incubation mixture contains 0.1 ml of enzyme solution, 1.0 ml of 0.2 M borate buffer (pH 8.0) containing 2×10-³M cysteine, 0.lml of 0.1 M substrate, and 0.8ml of deionized water containing 5×10-4M EDTA. The mixture was shaken at 37°C for 30 minutes in the air. The reaction was terminated by adding 2ml of 10% trichloroacetic acid and the α-keto acids formed were determined by the method of FRIEDEMANN and HAUGEN4 with a following modification: toluene extract was washed once with 8 ml of 10% sodium sulfate. The results obtained are summarized in Table l. When the reaction mixtures are analyzed before or after incubation on an automatic amino acid analyzer, the amount of L- or L-Alloisovalthine is found to be unchanged. Furthermore, as indicated in Table 1, L-isovalthine showed no inhibitory effect on the homoserine cleavage by cystathionase. Since amino acid oxidases have already been reported to have no effect on isovalthine³, the curious phenomenon above cited may have to be explained by other reaction mechanism such as transpeptipation reaction.

The fibrinolytic activity of plasmin present in menstrual blood has been studied by means of the Fibrin-Plate Method, and utilizing the findings of such a study, the identification of menstrual blood stain in legal medicine has been conducted. As the result, it has become clear that the identification of a very small amount of menstrual blood is possible, for an example, only one thread from the menstrual blood stained cloth 1.0 cm2. in size, and with the blood stain left in a room temperature for as long as two years, or with the blood stain left in water for one month, it is possible to identify menstrual blood. Aside from menstrual blood, no other toco-gynecological blood responds to this Fibrin-Plate Method.

We present here a reliable and sensitive method for the determination of acidic opines such as meso-alanopine, beta-alanopine, tauropine and strombine in biological samples. Interfering primary amino acids were eliminated by reaction with o-phthalaldehyde, and the derivatized compounds were passed through Sep-Pak Plus PS-1 cartridges. The acidic opines were recovered by flushing the cartridges with water, then determined by high performance liquid chromatography after a second derivatization with phenylisothiocyanate. All 4 acidic opines were detected within 30 min. This method ensured good separation and guaranteed almost full recovery of all acidic opines. This method was applied to analyze opines in marine animals and to test whether opines are metabolized in the livers of the rat and fish.

In this study, sex determination using polymerase chain reaction (PCR) on tooth material was evaluated from the viewpoint of forensic medicine. The sensitivity of PCR for detection of the Y chromosome-specific alphoid repeat sequence and the X chromosome-specific alphoid repeat sequence was 0.5 pg of genomic DNA. Sex could be determined by PCR of DNA extracted from the pulp of 16 freshly extracted permanent teeth and dentine including the surface of the pulp cavity of 6 freshly extracted milk teeth. Sex could be determined using the pulp in all 20 teeth (10 male and 10 female) preserved at room temperature for 22 years. For the pulp of teeth stored in sea water, the sex could be determined in all 8 teeth immersed for 1 week and in 5 of 6 teeth immersed for 4 weeks. In the remaining 1 tooth, in which sex determination based on the pulp failed, the sex could be determined correctly when DNA extracted from the tooth hard tissue was examined. For teeth stored in soil, the sex could be determined accurately in all 8 teeth buried for 1 week, 7 of 8 teeth buried for 4 weeks, and in all 6 teeth buried for 8 weeks. When teeth were heated for 30 min, sex determination from the pulp was possible in all teeth heated to 100, 150, and 200 degrees C, and even in some teeth heated to 250 degrees C. When this method was applied to actual forensic cases, the sex of a mummified body estimated to have been discovered half a year to 1 year after death could be determined readily by examination of the dental pulp. In the skeletons of 2 bodies placed under water for approximately 1 year and approximately 11 years and 7 months, pulp tissues had been dissolved and lost, but sex determination was possible using DNA extracted from hard dental tissues. These results indicate that this method is useful in forensic practices for sex determination based on teeth samples.

The formation of the swim-bladder gases of some sea and fresh water fishes were investigated and the results may be summarized as follows : 1. As a rule, oxygen content in the swim-bladder is higher in a fish living at greater depth than at shallow, and sea water fishes, than fresh water ones. 2. Oxygen content in the swim-bladder of the fish living at great depth decreases after 1-2 days stay in the aquarium. 3. Carbon dioxide content in the swim-bladder of all fishes examined is very small. 4. Through the poisoning of carbon monoxide, the swim-bladder gas decreases in its oxygen content and increases slightly in its carbon dioxide. 5. Corresponding to the artificial increase or decrease of the external pressures influencing the body surface of the fish, oxygen content of the swim-bladder gases increases or decreases respectively. 6. After the evacuation of the swim-bladder gases, newly formed gases always contain high oxygen percentage. 7. When oxygen or carbon dioxide of high concentration are injected in the swim-bladder, these gases diffuse out easily through the wall of the swim-bladder during 1-2 days. 8. Oxygen dissociation curve of carp blood is remarkably steep compared with the human blood, and influenced very much with the presence of carbon dioxide so as to decrease the affinity of the blood to oxygen. 9. Histological examination of the swim-bladder of Sebastiscus marmoratus and Carassius auratus indicate characteristic structure of the blood capillaries which distributed in the internal layer of membranes and sinus-like dilated. 10. From the above experimental results, some considerations on the gas formation in the swim-bladder were offered.

<p>Iron plays a critical role in the production of activated oxygen species and the activity of chelated iron in the biological system depends on the chemical forms of the chelators. In the present study, we used ferric nitrolotriacetate (Fe-NTA, molar ratio of iron to chelators = 1:3), ferric ethylenediaminetetraacetate (Fe-EDTA, 1:3 complex) and ferric Desferal (Fe-Des, 1:1.1 complex) to see their "free" iron content in aqueous solutions in vitro and in the serum obtained after a single intraperitoneal injection of the chelates to rats (7.5 mg of iron/kg). "Free" iron was measured by the bleomycin-assay system. When Fe-NTA was dissolved in water, "free" iron increased linearly with total iron concentration up to 10 microM, whereas Fe-EDTA and Fe-Des showed no "free" iron with corresponding iron concentrations. When these three ferric chelates were dissolved in normal rat serum, "free" iron in Fe-NTA increased abruptly between 40 microM and 60 microM iron concentrations, then increased slowly up to 100 microM. Fe-Des did not show any "free" iron at comparable iron concentrations. Fe-EDTA had an intermediate "free" iron level in the serum. Among the ferric chelate complexes, Fe-NTA showed a much faster increase of and a higher content of "free" iron in the serum than the other two complexes after a single injection of the chelates into rats.(ABSTRACT TRUNCATED AT 250 WORDS)</p>

We examined the effect of food deprivation for three days on hypothalamic arginine vasopressin (AVP) mRNA in rats. Simultaneously the effect of water deprivation for the same period was examined as a model of dehydration. Levels of AVP mRNA in the supraoptic nucleus (SON) and the paraventricular nucleus (PVN) were determined by semiquantitative in situ hybridization histochemistry. Water deprivation increased AVP mRNA in both nuclei as previously reported. In contrast, food deprivation decreased AVP mRNA in these nuclei. The changes in AVP mRNA levels in the PVN were observed in the magnocellular subdivision of the nucleus. Plasma levels of ACTH and corticosterone were greatly increased in both treated groups of rats. Plasma AVP and osmolality levels were significantly elevated in water-deprived rats but not in food-deprived rats. These observations indicated that both food deprivation and water deprivation stimulated the pituitary-adrenal axis and that a reduction in AVP mRNA levels in food-deprived rats was caused by food deprivation but not by glucocorticoid feedback suppression nor by altered plasma osmolality.

The role of hyperammonemia in the pathogenesis of cerebral edema was investigated using mongrel dogs to develop a treatment for cerebral edema in acute hepatic failure. Intravenous infusion of ammonium acetate alone into dogs did not induce brain edema, although blood ammonia reached unphysiologically high levels. However, ammonium acetate infusion during mannitol-induced reversible (osmotic) opening of the blood-brain barrier (BBB) effectively induced cytotoxic brain edema. Pretreatment with a branched-chain amino acid (BCAA; valine, leucine and isoleucine) solution prevented an increase in intracranial pressure (ICP) and brain water content, and caused a decrease in brain ammonia content and an increase in brain BCAA and glutamic acid. The results suggest that ammonia plays an important role in the pathogenesis of cerebral edema during acute hepatic failure and that BCAAs accelerate ammonia detoxification in the brain.

In such animals not having any organic changes in their brains during the initial stage showed a descendence of convulsive threshold. abnormal findings in their electroencephalogram and ascending activity of ChE. But what is the cause of these functional changes? First, from the fact that though there was no organic changes, they were sensitized and reiniected by a known antigen, which is obviously an antigen-antibody reaction. Second, from the fact that we got a histological.change, which was acknowledged as C.L.A. changes by increasing the concentration of these solution and the number of injections, it could be thought that these functional changes were caused by what I called latent C.L.A.. That is, it seems it could be thought that it would give functionally a permanent hypersensitivity, which is called convulsive arrangement. Furthermore, a similar histological findings as seen in old epileptics were made experimentally after prolonged and repeated injections of very diluted antigens. I believe it can be said, also from this histological point that they are experimental epileptics. But I am not trying to say that idiopathic epilepsy is the same allergic disease as asthma. If it was so, it should offer clinically a problem of eosinophilia in the blood of epileptics. But actually there is no eosinophilia in epileptics. Also, in adult epileptics, convulsive attacks is not often seen soon after introduction of antigens. Consequently, my theory that epilepsy is allergic, does not mean that allergy is the direct cause of epileptic attacks. What I mean is, the causal genesis of idiopathic epilepsy is hypersensitivity of nerve cells in the brain. This hypersensitivity was attained as a tissue reaction by some allergic mechanism without any organic changes. This functional change gives the nerve cell a hypersensitive state, which becomes the base of the beginnihg of convulsion. Its inducement of attack could be water stagnation in the body, anemic state of the brain, alkalosis, or introduction of allergens. In short, the cause of attack does not always come from allergic reactions.

In this study, we tested brain surface cooling as a new method of inducing selective brain hypothermia, and evaluated its effects on focal cerebral ischemia using a cat model of transient middle cerebral artery (MCA) occlusion. Cats underwent 1 h of MCA occlusion followed by 5 h of reperfusion. Brain surface cooling was induced for 4 h during and after MCA occlusion in the hypothermia group, but not in the normothermia group. Brain surface cooling was performed using saline perfusion into the subdural space. Rectal temperature, brain surface temperature, and deep brain temperature were monitored, and regional cerebral blood flow (rCBF) and somatosensory evoked potential (SEP) were serially measured. After 5 h of reperfusion, water content was also measured. Although the rectal temperature was maintained at about 37 degrees C, the brain surface temperature decreased rapidly to 33 degrees C and was maintained at that temperature. For 3 h following reperfusion, the rCBF was lower in the hypothermia group than in the normothermia group. At 4 and 5 h after reperfusion, the recovery of SEP amplitude was significantly more enhanced in the hypothermia group than in the normothermia group. In the gray matter, the water content was significantly more diminished in the hypothermia group than in the normothermia group. These results demonstrate that our method is useful for protecting the ischemic brain from a transient MCA occlusion. This method may be adapted for neurological surgery.

Separation of the urinary ester-form bilirubin was attempted, and the results obtained may be summarized as follows: 1. A brown pigment was obtained from jaundiced urine by the following procedures; namely, salting out, methanol extraction, chloroform flocculation, and separation on cellulose column. The pigment has been found to be easily soluble in water, displaying the absorption maximum at 420 - 410 mμ at pH 7.0, and it also gave a positive reaction both to GMELIN's and EHRLICH's diazo reagents within a minute without the addition of alcohol. These characteristics agree well with those of the socalled ester-form bilirubin. 2. On the basis of the results of paper chromatography and paper electrophoresis, the pigment has been determined to contain no amino acid, steroid, nor reducing substance. Moreover, no glucuronic acid could be detected whether examined in vitro or by paper chromatography together with paper electrophoresis, either.

Problems with infusion therapy for correcting fluid and sodium imbalance in decompensated liver cirrhosis (DLC) were investigated by establishing the safety zone of Talbot et al. for parenteral fluid therapy in 4 DLC patients infused with over 900 ml of fluid each day for at least 9 days. The safety zone was different in each case. The safe infusion volume decreased and the safe electrolyte concentration shifted to a lower osmolality when there was ascites with renal failure than ascites without renal failure. Infusion therapy was performed without deterioration of the water and sodium balance in those patients whose infusion volume and fluid osmolality were in the safety zone. In contrast, ascites retention increased and peripheral edema appeared in patients whose infusion volume and osmolality were out of the safety zone. Therefore, the safety zone should be determined repeatedly during infusion therapy.

The results obtained in this investigation may be summarized as follows : 1. The CO2-output of the male muscle and other tissues is greater than that of the female. 2. The female muscle contains larger amount of water than the female muscle. 3. The muscle immersed 1/2 Ringer solution (or 1/2.5 R.) gave out smaller amount of CO2 per minute than the muscle in 2-Ringer's solution (or 2.5 R.). In spite of the difference in the water content of tissue between different sex, the salt content of the tissue liquid seems to be the same. In other words, larger the water content means larger content of tissue liquid in the tissue. Artificial introduction of water in the tissue or reduction of water content by immersing the tissue in 1/2 or 2-Ringer's solution is quite different from the natural condition occurring between different sex. However both of these conditions influence the gaseous metabolism in the same manner. On an assumption that the gas diffusion in liquid is proportional to the solubility of that gas, the above mentioned difference of CO2-output should be just reversed. Therefore it is not possible to interpret how the water content influences the gaseous metabolism. It may only be stated that the muscle which has a small amount of water to an extent which does not abolish excitability, gives out much CO2 and vice versa.

The excretion of bilirubin and therefore the relief of jaundice is dependent upon at least three factors. First, the bilirubin must be conjugated and thus converted into a water soluble compound: this means its conversion to an ester glucuronide although other conjugates may also be formed. Secondly, there is the problem of the transport of bilirubin through the hepatic cell. A defect in either the up-take of bilirubin or the secretion of conjugated bilirubin may result in jaundice such as is seen in the various types of familial hyperbilirubinemia. Thirdly, there is the possibility of alternative catabolic pathways for bilirubin: this approach to the problem has, however, not yet received the attention of investigators.

Attempts were made to identify menstrual blood by means of paper electrophoresis with preparation of extracts of menstrual blood isolated under various conditions and mixed with human fibrin. Also similar analyses were conducted with blood aspirated from the median cubital vein of a woman during menstruation as well as from a man as the control, also with extracts of lochial blood from a woman after normal delivery, and of the blood obtained at arrtificial abortion. Animal fibrins (from rabbit, mouse, steer, and guinea pig) were also used to see the lytic action of the bloods. The following are the results of the present experiments. 1. The identification of menstrual blood by means of paper electrophoresis is a simple method in legal medicine and its electrophorogram is an excellent method to offer an evidence of proof for mentrual blood. 2. By this method it is possible to identify the menstrual bloodstain even after the lapse of time as much as 6 months. 3. It is possible to identify even putrefied menstrual bloodstain. 4. In the case where the material stained with menstrual blood is found in water, it is not possible to identify the menstrual blood by this method. 5. When the menstrual blood is heated at 60°C over 30 minutes, it becomes impossible to identify it by this method. 6. In the case of venous blood during menstruation fibrinolytic product can be detected only on the first day of menstruation, but since it appears only in trace, it is easy to differentiate it from menstrual blood. 7. As for lochial blood the fibrinolytic product can be detected only in the blood obtained on the first and second days of puerperium, but the amount being so slight that it can readily be distinguished from menstrual blood. 8. In the case of the blood obtained at artificial abortion fibrinolytic product appears just as much as in the case of menstrual blood, and thus it is impossible to differentiate it from mentrual blood by this method. 9. As for the use of human fibrin it is best to employ it while it isdresh, but the human fibrin up to 6 days old can be used. However, the older is the human fibrin the lesser the fibrinolytic product detectable. 10. In the case using animal fibrins mixed with the extract of menstrual blood some do produce fibrinolytic product in trace, but since there is a danger of also producing the fibrinolytic product-like substance in venous blood, it is advisable not to use animal fibrins.

Through the use of an automatic photo tube dew-point hygrometer, the author succeeded in measuring dew point of gas flows continuously in anesthetic circuits. Simultaneous thermometries were done on the nasal or oral mucosa, on the respiratory gas flows in the anesthetic mask or the endotracheal tube, and on the gas in the inhaling conduit. Experiments were performed on ten adults patients undergoing various types of surgery under general inhalation anesthesia. Anesthetic technics were varied intentionally during the measurements. Thus, both absolute and relative humidities of exhaled and inhaled gases, and respiratory water and heat losses were calculated under various anesthetic conditions, and physiological and clinical considerations were discussed. The conclusions obtained from this research are as follows: (1) When a non-rebreathing system is applied, moisture content of exhaled gas is minimal, and respiratory losses of both water and heat are maximum. With a semi-closed circle method, according to decreasing fresh gas flows, the humidity of the inspiratory and expiratory gases becomes higher, and both heat and water losses through respiration are lessened. When a closed circle method, with carbon dioxide absorption, is employed, temperature and humidity of gas in the inhaling conduit are highest, and the expired gas offers the maximum temperature and moisture contenL Both water and heat losses from anesthesia become minimal when administered in a closed system. (2) While the water and heat that a patient loses through respiration increase with increasing breathing capacity, they are still small parts of the total water and heat losses of the patient. Water and heat losses via anesthesia systems are not so predominant in maintaining water balance and heat regulation of patients during anesthesia and surgery.

A series of experiments have been conducted with ten adult rabbits, drowning them to death in a ditch those water contains diatoms in abundance. The bones (selected ones are the femur, humerus, riHand vertebra) of these drowned rabbits have been buried underground, wrapped tightly in cellophane bags and left there for three years, and the detection of diatoms has been conducted with these bones either as they are or after cremating them in the electric'oven at 300°C, 500°C, 800°C or 1,000°C, for 20 minutes. As the results it has been clarified that diatoms can be detected in a considerable number in the bones of four limbs, and of these detectable diatoms some of them can be found even after cremation at 1,000°C for 20 minutes. This clearly proves diatoms are detectable from the bones even after a long period of time·after burial and even after cremation at high temperatures.

To study the effect of partial liquid ventilation (PLV) with perfluorocarbon on acute respiratory failure, 3 groups of 17 rabbits were examined to compare. After acute respiratory failure was induced by lung lavage with sea water in 12 of the 17 rabbits, 7 of the 12 rabbits were treated with conventional mechanical ventilation (AC group) and 5 of the 12 rabbits were treated with PLV using perfluorocarbon (AP group). The remaining 5 normal rabbits without acute respiratory failure were treated with PLV with perfluorocarbon as a control group (PL group). In the PL group, PaO2, PaCO2, blood pH, pulmonary compliance or pathological findings were not so changed after PLV. In the AC and AP groups, PaCO2 significantly increased, and in contrast, PaO2 and pulmonary compliance significantly decreased after lung lavage. However, these findings improved to almost the same levels as those of a control group within 2 h after the PLV treatment in the AP group, but in the AC group, these gradually deteriorated over time. As for the pathological findings, pulmonary vascular congestion, alveolar hemorrhage and inflammatory infiltration were observed in the AC group. However, these findings were not observed in the specimens of the AP group. From these results, PLV with perfluorocarbon was shown to be useful to improve gas exchange and pulmonary functions without major side effects.

In our studies on the hypotensive effect of Diamox by intravenous injection, we have arrived at the following conclusions. 1. Ocular tension falls and the flow of aqueous humor becomes sluggish. 2. Diamox inhibits the activity of carbonic anhydrase, and the concentrations of HCO3-, K+, Cl- and glucose are markedly altered. 3. Protein increases both in blood and aqueous humor, but no change in protein fraction can be observed in blood. 4. Diamox in no way affects the metabolism. 5. It seems that Diamox brings about the change in the specific gravity of blood, making the latter either more diluted or more concentrated. From these, we conclude that the mechanism of the loweing of ocular tension by Diamox seems to lie in the fact that it inhibits the activity of carbonic anhydrase, and that consequent alteration in the concentrations of HCO3- and other ions accompanied by the change in osmotic pressure as well as a slight decrease of water in tissue all bring about the fall in the ocular tension. However, Diamox seems to have nothing to do with aqueous humor in so far as active transport or permeability are concerned.

For the purpose to investigate the physiological functions of microvillus ATPase, general properties of the enzyme were studied on the microvillus membranes isolated from rabbit intestinal epithelial cells. 1) ATPase of the microvillus membranes was activated with Mg2+. Mg.ATP complex was thought to be a subStrate of the enzyme. The Michaelis constant for ATP of the ATPase was a value of 0.8 to I .0 mM. 2) The microvillus ATPase was also activated with Ca2+, but the affinity was lower than a half of that of Mg2+. 3) The optimum pH of the ATPase was about 7.8. 4) Activity of the microvillus ATPase was markedly inhibited by treating with deoxycholate (DOC), and the activity inhibited was partially restored by washing the microvillus membrane with distilled water. The structure of the membranes destroyed by treating with DOC was also partially restored by the same procedure. 5) Ultrasonic treatment also markedly destroyed the microvillus membrane and inhibited ATPase activity. Damaged ultrastructure and ATPase activity both were partially restored by treating with phospholipid, EPL. 6) Simultaneous presence of Na+ and K + stimulated scarcely the ATPase of purified microvillus membranes. 7) The microvillus ATPase was slightly activated in the presence of n-glucose. Phloridin gave little effect on the activity of the microvillus ATPase.