THE PREARGININE IN EDESTIN AND ITS RESISTANCE TO HYDROLYSIS

The titration data of edestin show that all the arginine found on hydrolysis exists in this protein as "prearginine." The extra ionizable groups of histidine, lysine and tyrosine are free in the quantities found on hydrolysis. Part of the extra carboxyl groups of aspartic and glutamic acids are bound as amides, and 50 per cent are bound in some other manner (perhaps anhydride) leaving only about 6 per cent of these groups free to ionize in edestin. The prearginine in edestin is not converted into arginine on hydrolysis with pepsin up to 18 per cent (of the total hydrolysis). In more highly hydrolyzed solutions it is not possible to detect such a conversion, due to high buffering. Complete hydrolysis however converts prearginine into arginine which can be isolated. Hydrolyzed edestin promotes the growth of sarcomatous fibroblasts about equally well whether 5, 14 or 18 per cent hydrolyzed.     

STUDIES ON THE REGULATION OF OSMOTIC PRESSURE : II. THE EFFECT OF INCREASING CONCENTRATIONS OF ALBUMIN ON THE CONDUCTIVITY OF A SODIUM CHLORIDE SOLUTION.

Egg albumin, like gelatin, influences the conductivity of a 0.6 per cent NaCl solution in two ways: (a) At an hydrogen ion concentration of about pH 3.0, increasing concentrations increase the conductivity. (b) Near the isoelectric point of albumin and at the pH of the blood, increasing concentrations of albumin decrease the conductivity of the NaCl solution.     

THE LOCALIZATION OF ALBUMIN AND FIBRINOGEN IN HUMAN LIVER CELLS

Human liver sections were stained with anti-human serum albumin and/or anti-human fibrin monomer fluorescent conjugates. Approximately 10 per cent of the hepatic cells stained specifically for human serum albumin,1 per cent for fibrinogen, and 0.1 per cent for both. Approximately 18 per cent of the Kupffer cells stained specifically for human serum albumin and 33 per cent for fibrinogen. Staining of both cell types was mainly cytoplasmic, although albumin was found in the nuclei of some parenchymal cells, depending on the method of fixation. Cytoplasmic granules staining specifically for fibrinogen could be seen just inside the cell membrane facing the bile caniculi in many more parenchymal cells than the 1 per cent showing diffuse cytoplasmic staining. The technical difficulties involved in preparing fluorescent conjugates against these antigens and in the fixation of these antigens for immunofluorescent staining are discussed.     

MOLECULAR ASSOCIATION OF HEMOCYANIN PRODUCED BY X-RAYS AS OBSERVED IN THE ULTRACENTRIFUGE

1. When normal, monodisperse hemocyanin (60.5S) from Limulus Rolyphemus was irradiated in neutral buffer with x-rays, several new, more rapidly sedimenting ultracentrifugal components (86S, 107S, 122S) were produced, with a corresponding loss in the amount of the unaffected protein. The amount of the effect was roughly proportional to the amount of irradiation. 2. The new resolvable components apparently represented an association of the primary particles into aggregates of 2, 3, and 4 primary particles respectively. 3. The proportional amount of hemocyanin affected decreased almost to the vanishing point as the concentration of the protein was raised to high levels. 4. The absolute effect, i.e. the total number of particles affected in a given volume, increased with the concentration of hemocyanin, at least for concentrations below 15 per cent. 5. The presence of 33 per cent horse serum during irradiation inhibited the effect on the hemocyanin almost completely, with hemocyanin concentrations of both 0.8 and 14 per cent. 6. The presence of 2.8 per cent egg albumin during irradiation lowered the effect by about 70 per cent in the case of dilute preparations (0.8 per cent hemocyanin), but by only about 25 per cent in the case of 14 per cent solutions. 7. A lowering of the solution''s oxygen tension during irradiation enhanced the effect, almost doubling it in some cases. 8. The probable theoretical significance of these and other observations are discussed in the text.     

THE RHEOLOGY OF THE BLOOD. V

A study has been made of those proteins which might offer exceptions to the law that the fluidity of a protein solution is a linear function of the volume concentration; viz., egg albumin, serum albumin, pseudoglobulin, euglobulin, gelatin, and sodium caseinogenate. Solutions of egg albumin below 20 per cent by weight obey the above law but somewhat below 30 per cent the fluidities begin to be too high, presumably due to the contribution to the fluidity made by the deformation of the particles as they come into contact, as the fluidity approaches zero. The fluidity of serum albumin solutions shows a similar behavior, being exceptional above 15 per cent in weight. Pseudoglobulin and euglobulin give fluidity-concentration curves (Fig. 4) which are linear up to about 2.5 per cent each in a total range of 20 and 14 per cent respectively. From this singular point both compounds show a second range which is linear. Pseudoglobulin is the only substance whose solutions seem to show a third linear range. We have also used the data of Chick and Martin for sodium caseinogenate and found evidence for two linear régimes. It is desirable at this time to call attention to the measurements of the flow of glycogen solutions by Botazzi and d''Errico (14) which in Fluidity and See PDF for Structure plasticity, page 207, are expressed in rhes. The data show two linear fluidity curves of different slopes. In this case it was definitely known that the data for each curve were measured with different viscometers which suggested the possibility of an error in viscometry entering in to confuse the issue. We have no suspicions as to the reliability of the data studied in this paper; we only wish to caution the readers that our hypotheses based on these data must be regarded with due reserve until confirmed. We have found a formula (11) based on the supposed linear relation between logarithmic fluidities and concentration which is convenient to use within the range, but close examination reveals that it does not reproduce the data for the higher concentrations at 25° nor does it permit extrapolation to pure water It is not realistic enough because it does not contemplate any change of régime in going from viscous to non-Newtonian or plastic flow. The formula does not apply to any other of the proteins studied in this paper nor to the great majority of proteins already reported as following the linear law. These are serious objections. We have therefore offered as an alternative a simple formula (24) according to which the fluidities are additive in the viscous régime. When the emulsoid particles approach close packing, they are deformed and this deformation contributes to the flow and the fluidity volume concentration curve is again linear. In fact, there may be one or more additional changes of régime.     

AN ATTEMPT AT PEPTIC SYNTHESIS OF INSULIN

1. Synthesis of plastein from the products of peptic hydrolysis of small quantities of egg albumin can be demonstrated with amorphous or crystalline pepsin. 2. Synthesis of plastein from the products of peptic hydrolysis of amorphous or crystalline insulin can be demonstrated with amorphous or crystalline pepsin. 3. The plastein synthesised by pepsin from the products of peptic hydrolysis of insulin is physiologically inactive. 4. The plastein formed in the insulin experiments could not be crystallised by the methods used for the crystallisation of insulin. 5. The physiological activity of insulin is not destroyed by repeated freezing (at about –50°C.) and melting of an aqueous or an alcoholic solution of this hormone. 6. No marked decrease in the physiological activity of insulin after incubation at 37°C. with pepsin at pH 4.0, in dilute or concentrated solutions, was detected.     

COMPLICATIONS OF PEPTIC ULCER IN THE AGED

Peptic ulcer is not an uncommon disease among the aged population. Thirty-five per cent of the patients admitted to the Los Angeles County General Hospital in a period of 12 months for treatment of peptic ulcer were more than 60 years of age.The severity of this disease in the elderly group was evidenced by the high incidence of lethal complications requiring operation—one case in every four.The mortality rate from operation for peptic ulcer complications is related to the factors of concurrent disease, the urgency of operation and the selection of a suitable operation.     

EFFECT OF ELECTRICAL STIMULATION ON THE YIELD AND COMPOSITION OF SYNAPTIC VESICLES FROM THE CHOLINERGIC SYNAPSES OF THE ELECTRIC ORGAN OF TORPEDO: A COMBINED BIOCHEMICAL, ELECTROPHYSIOLOGICAL AND MORPHOLOGICAL STUDY

Abstract— The effect of stimulating the electric organ of Torpedo marmorata , anaesthetized with 0.01% Tricaine methane sulphonate, by means of electrical stimulation (5/s) administered via an electrode placed on the electric lobe has been studied electrophysiologically, biochemically and morphologically. The response of the organ declined to about 50 per cent of its initial value after about 500 stimuli, by a further 10 per cent after another 500 stimuli and then to about 12 per cent of the initial value after a further 1000 stimuli. Thereafter the response fell off progressively. However, even when the response was less than 1 per cent of its initial value, the organ had considerable powers of recuperation during a 30-s rest period, to 30–50 per cent of its initial value.
The fall in response was accompanied by a reduction in vesicle size and number, an increase in the area of the presynaptic membrane and a fall in the protein, total nucleotide, ATP and acetylcholine content of the vesicle fraction isolated from the stimulated tissue. However, whereas vesicle numbers and the protein and total nucleotide content of the vesicle fraction fell by only about 50 per cent, vesicular ATP and acetylcholine levels were reduced to about 10 per cent. An analysis of the covariance of vesicular ATP and acetylcholine showed an initial loss of an acetylcholine-rich (relative to ATP) population of vesicles. The early loss of vesicular protein and nucleotide and vesicle numbers as well as the morphological changes seen would be consistent with a loss of vesicles due to fusion with the external membrane. The preferential loss of acetylcholine and ATP from the vesicle fraction indicates that the vesicles surviving the stimulation procedure have been utilized in a number of cycles causing the progressive fall in vesicle volume, and acetylcholine and ATP content.     

MEASUREMENT OF THE RATE OF ACETYLCHOLINE DIFFUSION THROUGH A BRAIN SLICE AND ITS SIGNIFICANCE IN STUDIES OF THE CELLULAR DISTRIBUTION OF ACETYLCHOLINESTERASE

The rate of ACh diffusion through a 0·8 mm thick slice from the surface of the rat cerebral cortex, under aerobic conditions at 37°C, was determined by bathing the intact surface of the slice (compartment A) with ACh containing buffer and determining the concentration of ACh in buffer bathing the cut surface of the slice (compartment B). With 1 or 5 mM-ACh in compartment A no ACh was detectable in compartment B within 3 h unless at least 95 per cent of the AChE, as assessed on homogenates, was inhibited. With a given level of AChE inhibition, the rate of ACh diffusion was dependent on its concentration in compartment A. With 1 mM-ACh in compartment A the difference between the rates of hydrolysis of ACh during diffusion through slices with an AChE inhibition of 98·3 and 99·4 per cent, as assessed by AChE assays of homogenates made from the slices, was only 6 per cent of the difference between the rates of hydrolysis of 1 m-ACh by the homogenates of that part of the slices through which diffusion took place. For 5 mM-ACh and levels of 95 and 99·2 per cent inhibition the corresponding value was 10-3 per cent. Since the concentration of ACh must fall across the slice it is not possible to calculate from these figures the number of enzyme sites involved in the hydrolysis during diffusion, i.e. the concentration of extracellular AChE. The implications of these observations are discussed, particularly in relation to studies of the release of ACh from the cerebral cortex in vivo     

薄层比色法对953口服液中齐墩果酸的含量测定

本实验采用酸水解氯仿萃取同步法提取齐墩果酸,然后进行薄层一比色测定。展开剂：石油醚－氯仿－冰酸酸（１０：１０：１）显色剂：碘蒸气。定量研究了９５３口服液中主要有效成分齐墩果酸的含量为１６．２％,平均回收率９９．１９％。此法简易,可以作为９５３口服液制剂质量监控指标。     

OBSERVATIONS ON THE BIOSYNTHESIS OF RAT BRAIN CERAMIDE AND CEREBROSIDE IN VIVO

Abstract— The incorporation in vivo of l -[¹⁴C]serine into ceramide and cerebroside of young rat brain has been studied. Acid hydrolysis of labelled ceramide and galactosyl-ceramide followed by selective partitioning of the resulting components indicated that 88 per cent of the radioactivity was present in the long-chain base portion. At early time points (10 min, 20 min) the precursor was incorporated into ceramide and to a lesser degree into glucosyl-ceramide. During time intervals of 5 and 10 h, the specific activity values (d.p.m./μmol) for ceramide and glucosyl-ceramide decreased, while values for galactosyl-ceramide, containing either unsubstituted fatty acids (NFA) or α-hydroxy fatty acids (HFA), increased 50 and 30 per cent, respectively. Analysis of labelled ceramide at all time points studied (10 min-10 h) indicated that l -[¹⁴C]serine was incorporated onto the NFA type. This observation suggests that HFA-ceramide may not be the physiological precursor of HFA-galactosyl-ceramide. In this context, the postulated precursor roles of both ceramide and psychosine in the biosynthesis of brain cerebrosides are discussed.     

THE EFFECT OF BILIRUBIN ON THE ENERGY METABOLISM OF BRAIN MITOCHONDRIA

Abstract— Unconjugated bilirubin caused uncoupling of oxidative phosphorylation in brain mitochondria prepared from mature or weanling rats. Human serum albumin in an amount sufficient to bind 100 per cent of the added bilirubin was able to protect the mitochondria from the inhibitory effects of bilirubin. Bilirubin inhibited both the endogenous and DNP-activated ATPase activities but had no effect upon the Mg²⁺-stimulated ATPase; albumin prevented the inhibition.     

Mineralization of nitrogen in two soil-organic-matter fractions extracted with formic acid

Summary Extraction of a calcareous meadow soil with boiling formic acid containing 10 per cent acetylacetone yielded a fraction of soil organic matter representing about 40 per cent of the total. This fraction contained only 2.2 per cent N. Less than 18 per cent of this was mineralized during incubation for 8 weeks at 28°C. Further extraction of the soil with a formic acid and hydrofluoric acid mixture yielded a second fraction. This represented about 37 per cent of the total organic matter and contained 6.0 per cent N of which up to 41 per cent was mineralized during incubation. Preincubation and leaching had a stabilizing effect on subsequent mineralization of native N of a standard sandy loam soil.     

CHANGES IN THE APPARENT CYTOPLASMIC HYDROGEN ION CONCENTRATION OF AMEBA DUBIA ON INJECTION OF EGG ALBUMIN

1. Egg albumin when injected into an ameba or discharged into the solution about it raises the apparent pH of the cytoplasm of the ameba. 2. With time the cytoplasm returns to the original pH 6.9 if the nucleus is present. Amebae that have received repeated injections of albumin in some cases extrude their nuclei. In these cells the cytoplasm remains at the more alkaline pH induced by the albumin for at least 12 hours. 3. When a 2 per cent solution of albumin is introduced into a suspension of amebae there is a temporary marked rise in the rate at which CO₂ is given off with no corresponding rise in O₂ uptake. 4. The results observed can be explained if the albumin discharged onto the surface of the ameba rapidly enters the cell and there becomes distributed in a phase of the cytoplasm other than the one which contains the phenol red.     

RESULTS OF IRRADIATION IN CANCER OF THE LIP,TONGUE AND EAR

Cancer of the lip: The primary lesion can be controlled by irradiation in approximately 80 per cent of cases. For lesions with metastases there is only about a 25 per cent chance of five-year arrest (irradiation of the primary lesion followed by excision of involved nodes).Cancer of the tongue: Lesions in the anterior two-thirds are controllable by irradiation in about 50 per cent of cases if the nodes are not involved; the salvage is only about 15 per cent if the nodes are involved (nodes treated surgically). Lesions in the posterior third of the tongue are seldom controlled in the author''s experience.Cancer of the ear (auricle): Five-year arrest of basal-cell lesions should be attained by irradiation in about 80 per cent of cases; of squamous-cell lesions in about 60 per cent. If the lesion is extensive, radiation does not offer a superior cosmetic result to operation and entails danger of late chondronecrosis. Therefore extensive lesions are probably best treated surgically. In either event, it appears probable that results of irradiation can be improved by the use of more adequate fields and greater fractionation.     

INACTIVATION OF PEPSIN BY IODINE AND THE ISOLATION OF DIIODO-TYROSINE FROM IODINATED PEPSIN

In the presence of iodine at pH 5.0–6.0 a solution of pepsin absorbs iodine and the specific proteolytic activity of the solution decreases. The activity is less than 1 per cent of the original activity when the number of iodine atoms per mol of pepsin is 35–40. If the pH is 4.5 or less, iodine reacts very slowly and there is a correspondingly slower loss in activity. Glycyl tyrosine reacts with iodine in a manner similar to pepsin. Experiments were performed to determine the extent to which oxidation of pepsin by iodine occurs during iodination, and if such oxidation were responsible for the loss in enzymatic activity. Although the results were not absolutely decisive, there seems to be no appreciable oxidation taking place during iodination and no relationship between the slight oxidation and loss in peptic activity. From a dialyzed preparation of completely iodinated pepsin which was inactive and contained 13.4 per cent bound iodine, 82 per cent of the iodine was obtained in a solution which analyzed as a solution of diiodo-tyrosine. Because of the presence of a material which contained no iodine and prevented quantitative crystallization, only 53 per cent of the iodine containing substance could be crystallized. This 53 per cent was, however, identified as diiodo-tyrosine. The part of the titration curve which in pepsin and most proteins represents the phenolic group of tyrosine was, in the curve for iodinated pepsin, shifted toward the acid region as expected. From these results, it appears that the loss in proteolytic activity of pepsin, when treated with iodine under the specified conditions, is due to the reaction of the iodine with the tyrosine in pepsin.     

THE EFFECT OF WATER CONTENT UPON THE RATE OF HEAT DENATURATION OF CRYSTALLIZABLE EGG ALBUMIN

1. The denaturation rate of partially dried crystallizable egg albumin is greatly decreased by decreasing its water content. 2. The temperature of denaturation, defined as the temperature at which half of the protein becomes insoluble in distilled water after a definite time of heating, is a linear function of the relative humidity with which the protein is in equilibrium. 3. By applying the Arrhenius equation it is shown that the rate of heat denaturation at a given temperature is an exponential function of the relative humidity. 4. The application of the observed relations to the analysis of the mechanism of thermal death of microorganisms is suggested. 5. The water content of native and heat-denatured egg albumin is determined as a function of the relative humidity of water vapor. It is shown that the heat-denatured modification takes up approximately 80 per cent as much water at all relative humidities as does native egg albumin.     

AXOPLASMIC TRANSPORT IN THE OPTIC NERVE AND TRACT OF THE RABBIT

The axonal transport of labelled proteins was studied in the optic system of adult rabbits after an intraocular injection of [³H]Ieucine. It was demonstrated that the precursor was incorporated into protein, which was transported along the axons of the retinal ganglion cells. Intraocularly injected puromycin inhibited protein synthesis in the retina and markedly inhibited the appearance of labelled protein in the optic nerve and tract. It was further demonstrated by intracisternal injection of [³H]leucine that an intraocular injection of puromycin did not affect the local protein synthesis in the optic nerve and tract. Cell fractionation studies of the optic nerve and tract showed that the rapidly migrating component, previously described as moving at an average rate of 110-150 mm/day, was largely associated with the microsomal fraction. About 40 per cent of the total protein-bound radioactivity in this component was found in the microsomal fraction and about 15 per cent was recovered in the soluble protein fraction. Most of the labelled material moving at a rate of 1-5-2 mm/day was soluble protein. The specific radioactivity of this component was about ten times greater than that of the fast one. In the slow component about 50 per cent of the radioactivity was found in the soluble protein fraction and about 10 per cent of the radioactivity was recovered in the microsomal fraction. Radioautography demonstrated incorporated label in the neuropil structures in the lateral geniculate body as early as 4-8 hr after intraocular injection. The labelling of the neuropil increased markedly during the first week, and could be observed after 3 weeks.     

Preferential incorporation of an exogenous cytokinin, N6-benzyladenine, into 18S and 25S ribosomal RNA of tobacco cells in suspension culture

Cytokinin-requiring tobacco cells were incubated for 10 h in the presence of a labeled cytokinin. N6-benzyl-[2-3H]Ade, and of [8-14C]Ado. After alkaline hydrolysis of total RNA and fractionation of the resulting nucleotides, 80 per cent of the 3H radioactivity of RNA were recovered as the N6-benzyl-Ado nucleotide, covalently inserted into polynucleotidic chains. The N6-benzyl-Ado nucleotide was not significantly labled by 14C: at most one part of this nucleotide per 10 000 may result from a transfer of the benzyl moiety to adenyl residues in preformed RNA. Thus, the covalent insertion of N6-benzyl-Ade into RNA involves the intact N6-substituted base. Total RNA was fractionated either by sucrose density gradient centrifugation or by polyacrylamide gel electrophoresis. All identified RNA species were shown to contain N6-benzyl-Ade. The insertion frequency, measured as the molecular proportion of N6-benzyl-Ade to the total base content, was 3 to 4 times larger in 25S and 18S rRNA than in 5S and 4S RNA. The amount of N6-benzyl-Ade inserted into cytoplasmic ribosomal RNA accounted for about 90 per cent of the amount incorporated into total RNA. Electrophoresis of denatured RNA in the presence of formamide provided additional evidence that N6-benzyl-Ade was indeed incorporated into RNA molecules.     

ISOLATION AND CHARACTERIZATION OF A SOLUBLE GLUCOSE-CONTAINING SIALOGLYCOPROTEIN FROM THE CORTICAL GREY MATTER OF CALF BRAIN

A sialoglycoprotein has been isolated from the cortical grey matter of calf brain after homogenization in 0.32 M-sucrose or in 0.15 M-NaCl. The sialoglycoprotein is present in the supernatant obtained after centrifugation at 100,000 g for 60 min. It is designated GP-350 on account of its elution with 350 mM-NaCl on a DEAE-cellulose column. From DEAE-cellulose chromatography it is evident that compounds comparable to GP-350 occur in the brain of calf and sheep, whereas they seem to be absent in calf liver and kidney. After purification, with polyacrylamide gel electrophoresis only one band can be shown both at pH 8.9 and 7.5. GP-350 consists of about 83 percent of protein and about 17 per cent of carbohydrate. The polypeptide core has an acidic character: amino acid analysis gives 26 per cent for glutamic acid plus aspartic acid and their amides, with a ratio of acidic to basic amino acids of 3.3. The carbohydrate moiety contains 2.4% sialic acid, 5.5 % hexosamine and 9.4% hexose. It is remarkable that this brain sialoglycoprotein comprises 4% glucose. Care was taken to prevent contamination with glucose-containing materials during the purification procedure of GP-350. The complete absence of other glucose-containing compounds which occur in brain, Le. glycogen and gangliosides, was demonstrated. GP-350 accounts for at least 3 per cent of the total saline-extractable protein and about 20 per cent of the total saline-extractable protein-bound sialic acid of the cortical grey matter of calf brain. These percentages correspond to 390 pg of protein and to 14 μg of sialic acid per g wet weight. GP-350 remains soluble when the pH is brought to 3.9 or when ethanol is added to 70 % (v/v).