ELECTROKINETIC PHENOMENA : VII. RELATIONSHIP BETWEEN ELECTRIC MOBILITY,CHARGE, TITRATION CURVE,AND OPTICAL ROTATION OF PROTEIN

The specific rotation of egg albumin, gliadin, and gelatin (40°C.) is discussed in connection with available data on (a) mobility, (b) titration curve, and (c) osmotic pressure. It seems likely that the change in specific rotation with pH of protein solutions is proportional to the change in net charge.     

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 INACTIVATION OF TRYPSIN : II. THE EQUILIBRIUM BETWEEN TRYPSIN AND THE INHIBITING SUBSTANCE FORMED BY ITS ACTION ON PROTEINS.

1. A study has been made of the equilibrium existing between trypsin and the substances formed in the digestion of proteins which inhibit its action. 2. This substance could not be obtained by the hydrolysis of the proteins by acid or alkali. It is dialyzable. 3. The equilibrium between this substance (inhibitor) and trypsin is found to agree with the equation, trypsin + inhibitor ⇌ trypsin-inhibitor The equilibrium is reached instantaneously and is independent of the substrate concentration. If it be further assumed that the rate of hydrolysis is proportional to the concentration of the free trypsin and that the equilibrium conforms to the law of mass action, it is possible to calculate the experimental results by the application of the law of mass action. 4. The equilibrium has been studied by varying (a) the concentration of the inhibiting substance, (b) the concentration of trypsin, (c) the concentration of gelatin, and (d) the concentration of trypsin and inhibitor (the relative concentration of the two remaining the same). In all cases the results agree quantitatively with those predicted by the law of mass action. 5. It was found that the percentage retarding effect of the inhibiting substance on the rate of hydrolysis is independent of the hydrogen ion concentration between pH 6.3 and 10.0. 6. The fact that the experimental results agree with the mechanism outlined under 3, is contrary to the assumption that any appreciable amount of trypsin is combined with the gelatin at any one time; i.e., the velocity of the hydrolysis must depend on the time required for such a compound to form rather than for it to decompose. 7. The experiments may be considered as experimental proof of the validity of Arrhenius'' explanation of Schütz''s rule as applied to trypsin digestion. 8. Inactivated trypsin does not enter into the equilibrium.     

Electroretinogram Characteristics and the Spectral Mechanisms of the Median Ocellus and the Lateral Eye in Limulus polyphemus

Electrical responses (ERG) to light flashes of various wavelengths and energies were obtained from the dorsal median ocellus and lateral compound eye of Limulus under dark and chromatic light adaptation. Spectral mechanisms were studied by analyzing (a) response waveforms, e.g. response area, rise, and fall times as functions of amplitude, (b) slopes of amplitude-energy functions, and (c) spectral sensitivity functions obtained by the criterion amplitude method. The data for a single spectral mechanism in the lateral eye are (a) response waveforms independent of wavelength, (b) same slope for response-energy functions at all wavelengths, (c) a spectral sensitivity function with a single maximum near 520 mµ, and (d) spectral sensitivity invariance in chromatic adaptation experiments. The data for two spectral mechanisms in the median ocellus are (a) two waveform characteristics depending on wavelength, (b) slopes of response-energy functions steeper for short than for long wavelengths, (c) two spectral sensitivity peaks (360 and 530–535 mµ) when dark-adapted, and (d) selective depression of either spectral sensitivity peak by appropriate chromatic adaptation. The ocellus is 200–320 times more sensitive to UV than to visible light. Both UV and green spectral sensitivity curves agree with Dartnall's nomogram. The hypothesis is favored that the ocellus contains two visual pigments each in a different type of receptor, rather than (a) various absorption bands of a single visual pigment, (b) single visual pigment and a chromatic mask, or (c) fluorescence. With long duration light stimuli a steady-state level followed the transient peak in the ERG from both types of eyes.     

Molecular Weight Distribution of Proteins Synthesized in Single, Identified Neurons of Aplysia

Parietovisceral ganglia from Aplysia californica were incubated in medium containing leucine-³H. Single, identified nerve cell somas were isolated from the ganglia, and their proteins extracted and separated by electrophoresis on 5% SDS-polyacrylamide gels. The distribution of total or newly synthesized proteins from the single neurons was determined by staining or slicing and liquid scintillation counting of the gels. Experiments showed that: (a) a number of proteins were being synthesized in abundance in the nerve cells; (b) different, identified neurons showed reproducibly different labeling patterns in the gels; (c) cells R2 and R15, which showed different distributions of radioactivity in the gels, had similar staining patterns; and (d) there was significant incorporation into material of high (>75,000) molecular weight in most of the cells.     

Analytical study of microsomes and isolated subcellular membranes from rat liver. 3. Subfractionation of the microsomal fraction by isopycnic and differential centrifugation in density gradients

Rat liver microsomal fractions have been equilibrated in various types of linear density gradients. 15 fractions were collected and assayed for 27 constituents. As a result of this analysis microsomal constituents have been classified, in the order of increasing median density, into four groups labeled a, b, c, and d. Group a includes: monoamine oxidase, galactosyltransferase, 5''-nucleotidase, alkaline phosphodiesterase I, alkaline phosphatase, and cholesterol; group b: NADH cytochrome c reductase, NADPH cytochrome c reductase, aminopyrine demethylase, cytochrome b ₅, and cytochrome P 450; group c: glucose 6-phosphatase, nucleoside diphosphatase, esterase, β-glucuronidase, and glucuronyltransferase; group d: RNA, membrane-bound ribosomes, and some enzymes probably adsorbed on ribosomes: fumarase, aldolase, and glutamine synthetase. Analysis of the microsomal fraction by differential centrifugation in density gradient has further dissociated group a into constituents which sediment more slowly (monoamine oxidase and galactosyltransferase) than those of groups b and c, and 5''-nucleotidase, alkaline phosphodiesterase I, alkaline phosphatase, and the bulk of cholesterol which sediment more rapidly (group a2). The microsomal monoamine oxidase is attributed, at least partially, to detached fragments of external mitochondrial membrane. Galactosyltransferase belongs to the Golgi complex. Group a2 constituents are related to plasma membranes. Constituents of groups b and c and RNA belong to microsomal vesicles derived from the endoplasmic reticulum. These latter exhibit a noticeable biochemical heterogeneity and represent at the most 80% of microsomal protein, the rest being accounted for by particles bearing the constituents of groups a and some contaminating mitochondria, lysosomes, and peroxisomes. Attention is called to the operational meaning of microsomal subfractions and to their cytological complexity.     

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

1. In pure gelatin solutions the conductivity of the solution increases with increasing concentrations, regardless of the hydrogen ion concentration. The actual value of the specific conductivity is greater at that reaction where the degree of ionization is greater. 2. The addition of gelatin in increasing concentrations to a 0.6 per cent sodium chloride solution affects the conductivity of that solution in two ways: (a) At pH 3.3, (where gelatin is highly ionized) the conductivity increases with each added increment of gelatin. (b) At pH 5.1 and 7.4 (where gelatin is less highly ionized) the conductivity decreases with each added increment of gelatin. A similar study is being made of crystalline egg albumin.     

Conceptual developments in metabolic control, 1924-1974

A brief sketch is given of the development of the understanding of the respiratory mechanism in plants over the past 50 years. Against this background the following aspects of control are discussed: (a) nonreversibility of catabolic sequences; (b) compartmentation of reactions and reactants; (c) control by amount of enzyme; (d) control by NAD and NADP; (e) control by ADP supply; (f) pacemaker reactions in glycolysis; and (g) control at branch points: further examples of allostery.     

Synthesis,characterization, and in vitro antimicrobial activities of 5-alkenyl/hydroxyalkenyl-2-phenylamine-1,3,4-oxadiazoles and thiadiazoles

The long-chain alkenoic acid hydrazides (1a–d) on reaction with phenylisocyanate and phenylthiocyanate gave their corresponding semicarbazides (2a–d) and thiosemicarbazides (4a–d), which on further refluxing with POCl₃ and Ac₂O yielded corresponding 1,3,4-oxadiazoles (3a–d) and thiadiazoles (5a–d), respectively.The structure elucidation of synthesized compounds is based on the elemental analysis and spectral data (IR, ¹H NMR, ¹³C NMR and MS). The synthesized oxadiazoles and thiadiazoles have been screened for antibacterial and antifungal activities. The investigation of antimicrobial screening revealed that compounds 3c, 3d, 5c, 5d and compounds 3b, 5b, showed good antibacterial and antifungal activities, respectively.     

Kinetics of Vibrio cholerae sialidase action on gangliosidic substrates at different supramolecular-organizational levels

G_d1a, G_d1b and G_t1b gangliosides were dispersed in the following membrane-mimicking systems: (a) homogeneous micelles; (b) mixed micelles with G_m1 ganglioside (which is resistant to the enzyme action), Triton X-100 or bovine serum albumin; (c) small unilamellar vesicles of egg phosphatidylcholine. The effect of dispersion on sialic acid release by Vibrio cholerae sialidase was studied. As reference substrates freely interacting with the enzyme the lipid-free carbohydrates of G_d1a and 3′-sialosyl-lactose were employed. The apparent V_max. of the enzyme was, with all the gangliosides, dependent on the type of ganglioside dispersion. It was lowest for homogeneous micelles and mixed micelles with ganglioside G_m1, and increased about 6-fold for ganglioside/bovine serum albumin lipoprotein micelles, 15-fold for mixed-ganglioside/Triton X-100 micelles (optimal molar ratio 1:7.5) and 30-fold for phosphatidylcholine vesicles containing 2.5 mol% ganglioside (this proportion was optimal for enzyme activity on the vesicles). For ganglioside G_d1a, the activity on Triton X-100 mixed micelles and on mixed vesicles was even greater (3- and 6-fold respectively) than that displayed on G_d1a lipid-free carbohydrate. With each of the used gangliosides the apparent K_m values were very similar values for homogeneous micelles and vesicular dispersions, but showed marked increases for Triton X-100 mixed micelles, approaching the values exhibited by reference oligosaccharides. Triton X-100 micelles and phosphatidylcholine vesicles did not appreciably alter the kinetics of sialidase action on 3′-sialosyl-lactose and on G_d1a lipid-free carbohydrate, indicating that the above effects are dependent on the intrinsic characteristics of the membrane-like systems containing gangliosides.     

ADENINE NUCLEOTIDE-INDUCED CONTRACTION OF THE INNER MITOCHONDRIAL MEMBRANE : II. Effect of Bongkrekic Acid

In bovine heart mitochondria bongkrekic acid at concentrations as low as about 4 nmol/mg protein (a) completely inhibits phosphorylation of exogenous adenosine diphosphate (ADP) and dephosphorylation of exogenous adenosine triphosphate (ATP), (b) completely reverses atractyloside inhibition of inner membrane contraction induced by exogenous adenine nucleotides, and (c) decreases the amount of adenine nucleotide required to elicit maximal exogenous adenine nucleotide-induced inner membrane contraction to a level which appears to correspond closely with the concentration of contractile, exogenous adenine nucleotide binding sites Bongkrekic acid at concentrations greater than 4 nmol/mg protein induces inner membrane contraction which seems to depend on the presence of endogenous ADP and/or ATP. The findings appear to be consistent with the interpretations (a) that the inner mitochondrial membrane contains two types of contractile, adenine nucleotide binding sites, (b) that the two sites differ markedly with regard to adenine nucleotide affinity, (c) that the high affinity site is identical with the adenine nucleotide exchange carrier, (d) that the low affinity site is accessible exclusively to endogenous adenine nucleotides and is largely unoccupied in the absence of bongkrekic acid, and (e) that bongkrekic acid increases the affinity of both sites in proportion to the amount of the antibiotic bound to the inner membrane.     

Studies on creatine determination by alpha-naphthol-diacetyl reaction

1.

1. For estimation of creatine in the presence of other guanidino compounds the following factors are important: (a) time, (b) temperature, (c) wavelength, and (d) addition of alcohol.     

Synthesis of 4-substituted pyrido[2,3-d]pyrimidin-4(1H)-one as analgesic and anti-inflammatory agents

4-Substituted-pyrido[2,3-d]pyrimidin-4(1H)-ones 4a–c were synthesized by oxidation of 4-substituted-dihydropyrido[2,3-d]pyrimidin-4(1H)-ones 3a–c which were in turn prepared from arylidenemalononitriles 1a–c and 6-aminothiouracil 2. The reactivity of compounds 4a–c towards some reagents such as formamide, carbon disulfide, urea, thiourea, formic and acetic acids were studied. All the synthesized compounds were characterized by spectroscopic means and elemental analysis. Compound 4c exhibited 64% and 72% analgesic activity. Also, compound 4b showed 50% and 65% anti-inflammatory activity. Interestingly these compounds showed one-third of ulcer index of the reference aspirin and diclofenac.     

Methylation of the ß-positions of the furan ring in F-acids

Major incubation products in feeding experiments with the sodium salt of 7-(5-butyl-furan-2-yl)heptanoic acid (3) on suspension cultures of Saccharum spec. are the unusual F-acids (4a) and (4b). They possess in contrast to natural monomethyl substituted F-acids a methyl substituent in the 4-position of the furan ring. Unexpectedly, the dimethyl substituted F-acids (4c) and (4d) were found only in very small amounts. The detection and structure elucidation of the methylation products (4a)–(4d) was achieved predominantly by GC-MS analysis of the corresponding tetrahydrofuran derivatives (5a)–(5d).     

The effects of air ions of the living mammalian trachea

Studies on the effects of air ions on the functional efficiency of the extirpated tracheal strip have been extended to the trachea of the living rabbit, rat, and mouse. Animals exposed to high mobility (+) air ions administered via a tracheotomy aperture displayed: (a) Decreased ciliary activity. (b) Decline in mucus flow rate, sometimes reversed by prolonged exposure to (+) ions; a frequent drop in the volume of mucous secretion. (c) Contraction of the membranous posterior tracheal wall. (d) Increased vulnerability to trauma of cilia and mucosal blood vessels. Similar treatment with (-) air ions reversed (+) ion effects on ciliary activity, mucus flow, contraction of the tracheal smooth muscle. Continued (-) ion treatment raised the ciliary rate (invariably) and the mucus flow rate (often) above their initial levels. (+) Air ions administered to unoperated resting mice and rats increased the respiratory rate; (-) ions reversed this effect. Long exposure of unoperated ambulatory mice to (+) air ions produced: (a) Decreased ciliary activity. (b) No clear cut effect on mucus flow. (c) Contraction of the posterior tracheal wall. (d) Increased vulnerability of the mucosa to trauma. (-) Air ions increased ciliary activity but had no clear-cut effect on the mucus flow rate.     

Design and synthesis of novel 1,25-dihydroxyvitamin D3 analogues having a spiro-oxetane fused at the C2 position in the A-ring

Four structurally novel stereoisomeric analogues of 1,25-dihydroxyvitamin D₃ (3a–d) bearing a spiro-oxetane fused at the C2 position of the A-ring have been designed and synthesised in a convergent manner. The requisite A-ring enyne precursors (13a,b) for the vitamin D analogues (3a,b) and (3c,d), respectively, were synthesised from pentaerythritol according to an eleven-step procedure. Preliminary biological evaluation of the analogues using the bovine thymus vitamin D receptor (VDR) suggested that the incorporation of the spiro-oxetane moiety instead of a gem-dimethyl group at the C2 position had a beneficial effect on the VDR affinity.     

Role of the side chain stereochemistry in the α-glucosidase inhibitory activity of kotalanol,a potent natural α-glucosidase inhibitor. Part 2

To examine the role of the side chain of kotalanol (2), a potent natural α-glucosidase inhibitor isolated from Salacia reticulata, on inhibitory activity, four diastereomers (11a–11d) with reversed configuration (S) at the C-4′ position in the side chain were synthesized and evaluated. Two of the four (11b and 11d) significantly lost their inhibitory activity against both maltase and sucrase, while the other two (11a and 11c) sustained the inhibitory activity to a considerable extent, showing distinct activity in response to the change of stereochemistry of the hydroxyls at the 5′and 6′ positions. Different activities were rationalized with reference to in silico docking studies on these inhibitors with hNtMGAM. Against isomaltase, all four analogs showed potent inhibitory activity as well as 2, and 11b and 11d exhibited enzyme selectivity.     

Additive effect of heparin on the photoinactivation of Escherichia coli using tricationic P-porphyrins

Polycationic porphyrins have received substantial attention in developing singlet oxygen-sensitizers for biological use such as in the photoinactivation of bacteria and photodynamic therapy (PDT) of tumor cells because they have strong binding affinities for DNA and proteins. However, these strong cellular interactions can retard elimination of the drug after PDT. Therefore, the studies on the interactions of porphyrins with other molecules present much interest, in order to modulate the sensitizers’ activity or even remove them from the human body after PDT. Here, we studied the additive effect of heparin on the photoinactivation by polycationic porphyrins using Escherichia coli as a model cell. Tricationic P-porphyrin sensitizers substituted with an N-alkylpyridinium group (alkyl?=?pentyl (1a), hexyl (1b), and heptyl (1c)) or N-hexylammonium (1d) as the axial ligand were used. Additionally, dicationic Sb-porphyrin substituted with an N-hexylpyridinium group (1e) was prepared. We studied the additive effect of heparin on the photoinactivation of E. coli by 1a–1e. The bactericidal activities were evaluated using the half-life (T_1/2 in min) of E. coli and the minimum effective concentrations ([P]) of the porphyrin sensitizers. In the absence of heparin, the [P] values were determined to be 0.4–0.5?μM for 1a?1c and 2.0?μM for 1d?1e. The bactericidal activity of 1a?1c was completely retarded by the addition of heparin (1.0?μM). However, the addition of heparin (1.0?μM) could not completely retard the bactericidal activity of 1d?1e whose [P] values were relatively large. It is suggested that tricationic 1a?1c adsorbed onto the anionic heparin through electrostatic interactions. The adsorption of 1 on heparin disturbs the uptake of 1 into E. coli cells. Thus, the addition of heparin was found to be a useful method for retarding photoinactivation.     

Potassium retention in crab nerve

Spectrophotometric determinations of the potassium content of small volumes of sea water brought into contact with crab nerves over half-hour intervals demonstrate (a) a continuous loss of potassium from the fibers while at rest in oxygen; (b) an appreciable increase in the leakage during anoxia; (c) a reabsorption of potassium during postanoxic recovery; and (d) a reduction in the amplitude of the anoxic and postanoxic changes in the presence of glucose.     

Reaction of 2-amino-2-deoxyheptoses with cyclic β-dicarbonyl compounds

The reaction between 2-amino-2-deoxyaldoses and β-dicarbonyl compounds yields polyhydroxyalkylpyrroles. Thus, 6,6-dimethyl-2-(D-galacto-pentitol-1-yl)-4,5,6,7-tetrahydroindol-4-one (4a), 6,6-dimethyl-2-(D-gluco-pentitol-1-yl)-4,5,6,7-tetrahydroindol-4-one (4b), and 6,6-dimethyl-2-(D-manno-pentitol-1-yl)-4,5,6,7-tetrahydroindol-4-one (4c) have been obtained from 5,5-dimethylcyclohexane-1,3-dione (2) and 2-amino-2-deoxyheptoses having D-glycero-L-gluco (1a), D-glycero-D-ido (1b), and D-glycero-D-talo (1c) configurations, respectively. 2-Amino-2-deoxy-D-glycero-L-manno-heptose (1d), the epimer of 1a, also reacts with 2, to yield 4a. In a similar way, 1a, 1b, and 1c react with cyclohexane-1,3-dione (3), to give 2-(D-galacto-pentitol-1-yl)-4,5,6,7-tetrahydroindol-4-one (5a), 2-D-gluco-pentitol-1-yl)-4,5,6,7-tetrahydroindol-4-one (5b), and 2-(D-manno-pentitol-1-yl)-4,5,6,7-tetrahydroindol-4-one (5c), respectively.