H2 metabolism in photosynthetic organisms. II. Light-dependent H2 evolution by preparations from Chlamydomonas, Scenedesmus and spinach.

Light-dependent H₂ evolution from dithiothreitol as electron donor was observed with cell-free preparations of anaerobically adapted $\text{Chlamydomonas reinhardii, Scenedesmus obliquus}$ and from spinach chloroplasts mixed with $\text{Chlamydomonas}$ hydrogenase. NADH substituted for dithiothreitol as electron donor only in the $\text{Chlarmydomonas}$ preparation. Dibromothymoquinone, an antagonist of plastoquinone, selectively inhibited H₂ photoevolution from NADH. These results are interpreted as indicating that 3-(3,4-dichlorophenyl)-1,1-dimethyl urea insensitive H₂ photoevolution by algae containing hydrogenase is due to the capability of NADH to reduce plastoquinone in the electron transport chain, and to evolve H₂ by a low redox potential carrier of photosystem I.     

Solubilization,stabilization and isoelectric focusing of human liver neuraminidase activity

Homogenate preparations of human liver have been prepared and over 75% of the particulate neuraminidase activity (which comprises approx. 90% of the total activity) has been solubilized using 0.85% (w/v) Triton X-100 in 25 mM phosphate buffer (pH 6.8). The solubilized neuraminidase activity is extremely labile, but can be stabilized for at least 4 weeks at 2–4°C, using 10 mM N-acetylneuraminic acid. Kinetic characterization of homogenate and solubilized supernatant fluid neuraminidase activities indicated comparable pH optimum curves (maximum activity at pH 4.5–4.7) and apparent K_m values (0.2–0.4 mM) for the synthetic fluorometric substrate $\text{4-}\text{methylbelliferyl}\text{-α-}\text{D}\text{-N-}\text{acetylneuraminic}$ acid. Isoelectric focusing has been performed on human liver homogenates and Triton X-100-solubilized neuraminidase activities, and the presence of several forms (4–6) with isoelectric points (pI values) between 4.4 and 5.2 has been demonstrated in both preparations. The similar kinetic and isoelectric focusing properties of the two preparations suggest that the solubilized enzyme activity is representative of the homogenate activity and that the solubilized enzyme is suitable for purification purposes.     

Characterization of rauscher leukemia virus (RLV) P40–42, an intermediate cleavage product of the group specific antigen (gag) precursor polypeptide,P65–70

$\text{In vitro}$ cleavage of the murine leukemia virus protein P65–70 (MW app. = 65–70,000; the $\text{g}\text{roup}$ specific $\text{a}\text{nti}\text{g}\text{en}$ or $\text{gag}$ precursor polypeptide) occurs after exposure of virus to 2% nonidet-P40 (NP-40; v/v) at 22°C in 10mM dithiothreitol. The cleavage occurs through an intermediate stage, where a protein P40–42, of MW app. = 40–42,000 is initially formed and then declines. Viral P65–70 contains all four antigenic determinants, while P40–42 contains the determinants for p30 and p10. The results indicate that p30 and p10 are adjacent polypeptides on P65–70 and further suggest that the $\text{in vitro}$ proteolytic cleavage of P65–70 is specific.     

Serotonin action on short-circuit current and ion transport across isolated rabbit ileal mucosa.

Serotonin has previously been implicated as the cause of the diarrhea associated with carcinoid syndrome and the amine has been shown by others to be an intestinal secretagogue in preparations of intestinal loops $\text{in}$$\text{vivo}$. In the present paper the action of serotonin on isolated segments of rabbit ileal mucosa stripped of muscle layers was studied $\text{in}$$\text{vitro}$. Serotonin (10^?4M) caused an abrupt significant rise in short-circuit current (Isc) across the mucosal epithelial cell layer but this effect was transient. No change was observed in tissue conductance. In this preparation, serotonin did not alter ²²Na, ³⁶Cl or residual ion fluxes across the mucosa. High blood serotonin levels for a period of several days also did not alter ion fluxes or Isc in isolated rabbit ileum. Therefore, it is concluded that serotonin must cause its secretory activity observed $\text{in}$$\text{vivo}$ by some mechanism other than a direct action on epithelial cell transport mechanisms.     

Alcohol-induced potentiation of the neurally evoked contractions of the retractor unguis muscle of the locust, Schistocerca gregaria

Each of a series of seven monohydroxyl alcohols caused an increase in the force of the neurally evoked contractions of the innervated retractor unguis muscle isolated from the metathoracic femurs of the locust, Schistocerca gregaria. The negative logarithm of equipotent concentrations of the alcohols was proportional to the logarithm of the partition coefficient (1-octanol/water) of the alcohols: $\text{log}\text{1}\text{C}\text{= 1·282}\text{log}\text{P+1·684}$, where C is concentration, and P is the partition coefficient.Ethylene glycol and glycerol did not potentiate the contractions, possibly due to their very low lipophilic character. Acetone (10^?2 M) and benzene (10^?5 M) also potentiated the neurally evoked contractions of the insect muscle.     

Membrane-associated reactions in ubiquinone biosynthesis: 2-octaprenyl-3-methyl-5-hydroxy-6-methoxy-1,4-benzoquinone methyltransferase

The O-methylation of 2-octaprenyl-3-methyl-5-hydroxy-6-methoxy-1,4-benzoquinone, which has been previously postulated to be the final reaction in the biosynthesis of ubiquinone was demonstrated in vitro using cell extracts of Escherichia coli. $\text{S-}\text{Adenosyl}\text{-}\text{l}\text{-}\text{methionine}$ was active as the methyl donor for the reaction. The enzyme concerned, S-adenosyl-l-methionine: 2-octaprenyl-3-methyl-5-hydroxy-6-methoxy-1,4-benzoquinone-O- methyltransferase, was partially purified and shown to have a molecular weight of about 50 000 and to require a divalent metal and dithiothreitol for optimal acitivity in vitro. The methyltransferase was absent from extracts from ubiG^? mutants suggesting that the ubiG gene is the structural gene coding for the methyltransferase. The enzyme, although not firmly membrane-bound, showed some affinity for the cell membrane in broken cell preparations and could utilize the benzoquinone substrate when the latter was free or bound to the cell membrane, with about equal efficiency. It is concluded that in vivo, the methyltransferase reaction probably occurs at the internal surface of the cytoplasmic membrane.     

Immunological properties of membrane fractions from wild type and dnaA mutants of Escherichia coli

Membrane vesicles from Escherichia coli wild type and an otherwise isogenic $\text{dna}\text{A}$ mutant were used to immunize rabbits. In addition, a membrane protein fraction, containing the material found deficient in $\text{dna}$A mutants, was purified by preparative polyacrylamide gel electrophoresis in sodium dodecylsulfate, and used for immunization. The antisera produced were analyzed by immunoelectrophoresis and immunofluorescence microscopy. The antisera obtained by immunization with membrane vesicles from either wild type or $\text{dna}\text{A}$ mutant membrane preparations were qualitatively similar in the precipitin bands seen after immunoelectrophoresis. The antisera obtained by immunization with the purified protein fraction contained a subset of the antibodies seen when whole vesicles were used for immunization. In a semiquantitative precipitin assay, the antisera prepared against whole membrane vesicles or the isolated protein fraction both caused the precipitation of more protein from sodium dodecylsulfate-solubilized membranes of wild type than of $\text{dna}\text{A}$ mutants. No difference was seen by immunoelectrophoresis between the protein composition of wild type or $\text{dna}\text{A}$ membrane preparations. Thus, the $\text{dna}\text{A}$ mutant appears to differ from the wild type in the quantitative composition of its membrane proteins, whereas no qualitative differences were detected.Fluorescein-conjugated antiserum preparations were employed to assess the reactivity of intact cells, spheroplasts and membrane vesicles with the antisera studied above. Wild type cells of E. coli have a barrier to reaction with the antisera; this barrier is removed when the cells are converted to spheroplasts or to membrane vesicle. Similarly, a highly permeable mutant of E. coli permits reaction of the antisera with unaltered cells. Antisera to both whole membrane vesicles and to the isolated protein fraction react identically with the cellular and subcellular preparations. Thus, antisera prepared from membrane proteins isolated after sodium dodecylsulfate-polyacrylamide gel electrophoresis can still recognize some antigens present in membrane vesicle preparations.     

Appearance of contractile activity in muscular dysgenesis (mdgmdg) mouse myotubes during coculture with normal spinal cord cells

Muscular dysgenesis (mdg) in the mouse is an autosomal recessive mutation expressed in the homozygous mutant as lack of skeletal muscle contraction. To test the ability of normal neurons to form neuromuscular contacts with, and/or possibly induce contractions in $\text{mdg}\text{mdg}$ muscle, dispersed cell cultures of normal and dysgenic muscle from newborn mice were cocultured with normal embryonic rat, mouse, and chick dissociated spinal cord cells. Contraction was induced in $\text{mdg}\text{mdg}$ muscle 1 to 10 days (depending upon the species of the neuronal source) following establishment of the cocultures. Control experiments indicated that the dispersed spinal cord preparations were free of myoblasts capable of fusing with $\text{mdg}\text{mdg}$ muscle. The establishment of neuromuscular contacts in the rat neuron cocultures was monitored by cytochemical staining of acetylcholinesterase (AChE), autoradiography of ¹²⁵I-α-bungarotoxin-bound acetylcholine receptors (AChR), and electrophysiological study of muscle membrane activity. Patches of high AChE activity were similar in size and distribution to high-density clusters of AChR on both control and $\text{mdg}\text{mdg}$ myotubes cocultured with rat neurons. The resting membrane potentials of normal myotubes and those of $\text{mdg}\text{mdg}$ myotubes in the presence of neurons were similar (? ?52 mV). The mepp frequency and the mepp amplitude distribution were the same for both control and mutant cocultured muscle. Thus, normal rat spinal cord neurons were capable of forming normal, functional neuromuscular junctions with $\text{mdg}\text{mdg}$ myotubes, and contractions were induced under coculture conditions, in otherwise noncontracting mutant muscle.     

Comparison of invivo and invitro uterine contractility in the ewe

Uterine contractions were observed $\text{in}$$\text{vivo}$ by laparotomy and exposure of the uterus. Ten hours after the beginning of estrus, an average of 39 contractions per 10 min originated in the posterior ends of the uterine horns and moved toward the oviducts, while an average of, 13 contractions originated in the anterior ends of the horns and moved toward the cervix. Two days later (58 hr after the beginning of estrus), the contractions had changed in origin and direction; only 6 contractions originated in the posterior ends of the horns and moved anteriorly, while 39 originated in the anterior ends and moved posteriorly.Experiments were done to determine whether the change in origin of contractions $\text{in}$$\text{vivo}$ was reflected in the contractility of strips of myometrium in a tissue bath. The number and amplitude of contractions were recorded from strips of circular and of longitudinal myometrium taken from the posterior and anterior ends of the uterine horns at 10 and 58 hr after the beginning of estrus. The myometrial strips contracted approximately 3 to 4 times per minute regardless of the time after the beginning of estrus, the end of the uterine horn from which the tissue was taken, or whether the contracting muscle was circular or longitudinal. Thus, the physiological mechanisms that controlled the number and origin of uterine contractions $\text{in}$$\text{vivo}$ did not maintain that control over myometrial tissue $\text{in}$$\text{vitro}$.     

The detergent and salt effect on the light-harvesting chlorophyll ab complex from green plants

The light-harvesting accessory pigment-protein complex (LHC) with a chlorophyll (Chl) $\text{a}\text{b}$ ratio of 1.2 was isolated by treating pea chloroplasts with Triton X-100. The LHC was used to investigate the action of ionic (sodium dodecyl sulfate) and non-ionic (Triton X-100) detergents. By optical methods (absorption and fluorescence spectra, measurements of fluorescence yield, ?, and lifetime, τ) two successive stages of the process were demonstrated, namely (1) interaction between detergent monomers and proteins and (2) solubilization of pigments into detergent micelles, which is facilitated by the presence of salts. The concentration ranges, characteristic of these stages, differ by 1.5–2 orders of magnitude for SDS, but slightly overlap for Triton X-100. At the second stage, certain changes occur in LHC absorption and fluorescence spectra. Several stable states of the LHC were established: (1) an aggregated state formed in the presence of 10 mM MgSO₄ with τ ≈ 0.6 ns; (2) the dialyzed LHC with τ ≈ 0.9 ns; (3) the states of the LHC in detergent solution with τ ≈ 2.3, 2.9, 3.4 ns; (4) a 30 kilodalton monomer obtained by SDS-polyacrylamide gel electrophoresis with τ ≈ 4.1 ns. The fluorescence parameters of the LHC states were compared with those of Chl a in detergent micelles (for the micelles τ = 5.6–6.0 ns. The $\text{τ}\text{?}$ ratio (the criterion for emission heterogeneity) for the LHC in the absence of a detergent was shown to be higher at least by a factor of 3.5 than that for Chl a in the presence of a detergent. Successive additions of the detergent to the LHC cause gradual decrease in the $\text{τ}\text{?}$ ratio, and for the LHC monomer it reaches practically the same value as for Chl a in detergent micelles. The results are discussed on the basis of the data obtained previously. It is suggested that in vivo LHCs do not form such aggregates as in water solution without a detergent.     

Phosphatidylinositol as the endogenous activator of the (Na+ + K+)-ATPase in microsomes of rabbit kidney

Incubation of rabbit kidney microsomes with pig pancreatic phospholipase A₂ produced residual membrane preparations with very low $\text{(Na}{}^{\mathrm{+}}\text{+ K}{}^{\mathrm{+}}\text{)-ATPase}$ activity. The activity could be restored by recombination with lipid vesicles of negatively-charged glycerophospholipids. Vesicles of pure phosphatidylcholine and phosphatidylethanolamine were virtually inactive in this respect, but could reactivate in the presence of cholate.Incubation of the microsomes with a combination of phospholipase C (Bacillus cereus) and sphingomyelinase C (Staphylococcus aureus) resulted in 90–95% release of the phospholipids. The residual membrane contained only phosphatidylinositol and still showed 50–100% of the $\text{(Na}{}^{\mathrm{+}}\text{+ K}{}^{\mathrm{+}}\text{)-ATPase}$ activity.     

Repair of deaminated cytosine residues of DNA: biological significance of the absence of uracil from DNA.

Uracil-DNA glycosylase of $\text{Bacillus}\text{subtilis}$ is involved in repair of deaminated cytosine residues of DNA. Survivals of SPO2 phage after treatment with bisulfite and weak alkali are considerably higher in wild type strains than in $\text{urg}$ mutants, which are deficient in the enzyme activity, whereas survivals of bisulfite/alkali-treated PBS1 phage in the two types of cells are essentially the same. The spontaneous mutation frequency of a $\text{urg}$ mutant is three fold higher than is that of a wild type strain.     

Glycodeoxycholate transport in brush border membrane vesicles isolated from rat jejunum and ileum

The transport of the bile salt, glycodeoxycholate, was studied in vesicles derived from rat jejunal and ileal brush border membranes using a rapid filtration technique. The uptake was osmotically sensitive, linearly related to membrane protein and resembled d-glucose transport. In ileal, but not jejunal, vesicles glycodeoxycholate uptake showed a transient vesicle/medium ratio greater than 1 in the presence of an initial sodium gradient. The differences between glycodeoxycholate uptake in the presence and absence of a Na⁺ gradient yielded a saturable transport component. Kinetic analysis revealed a $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ value similar to that described previously in everted whole intestinal segments and epithelial cells isolated from the ileum. These findings support the existence of a transport system in the brush border membrane that: (1) reflects kinetics and characteristics of bile salt transport in intact intestinal preparations, and (2) catalyzes the co-transport of Na⁺ and bile salt across the ileal membrane in a manner analogous to d-glucose transport.     

Opioid properties of beta-lipotropin fragment 60-65, H-Arg-Try-Gly-Gly-Phe-Met-OH.

The pharmacologic activity of the hexapeptide fragment corresponding to the amino acid fragment 60–65 in β-lipotropin, (β-LPH-(60–65)) was studied $\text{in vitro}$ and $\text{in vivo}$. In binding assays on synaptosomal plasma membrane the peptide was found to be equipotent to met-enkephalin, but behaved differently to cations; in contrast to met-enkephalin both Mn⁺² and Na⁺ enhanced the binding of β-LPH-(60–65) to synaptosomal plasma membrane. On both the quinea pig ileum and mouse vas deferens β-LPH-(60–65) inhibited contractions elicited by electrical stimulation and each effect was reversible by naloxone. On the guinea pig ileum β-LPH-(60–65) was equipotent to met-enkephalin and 0.5 as potent as normorphine but on the vas deferens it was 4.6 times more potent than normorphine. The activities of β-LPH-(60–65) appear to be due to the intact compound rather than to its conversion to met-enkephalin, since the peptide extracted from the ileum assay was found to behave identically as β-LPH-(60–65) with high pressure liquid chromatography. When β-LPH-(60–65) was administered centrally to mice and rats, no overt central actions were observed and an antinociceptive effect could not be demonstrated. Nor did β-LPH-(60–65) antagonize morphine action or precipitate the withdrawal syndrome in morphine dependent animals. It is concluded that the good agreement which generally exists between $\text{in vitro}$ and $\text{in vivo}$ assay procedures for opiate-like activity of morphine and its surrogates does not necessarily hold for the endogenous peptides with similar actions.     

Ultraviolet effects on presumptive primordial germ cells (pPGCs) in Xenopus laevis after the cleavage stage

The presumptive primordial germ cell (pPGC) number with development after the cleavage stage and the fate of pPGCs damaged by uv irradiation were studied in successive Epon sections (0.5 μm thick) with the light microscope in both uv-irradiated and unirradiated Xenopus embryos. taking survival rate and sterility into consideration. The pPGCs of the uv-irradiated embryos occupy nearly the same location in the embryos as those of the unirradiated embryos at stages 12, 17, 23, and 28 [see Ikenishi, K., and Kotani, M. (1975). Develop. Growth Different. 17, 101–110]. At stage $\text{33}\text{34}$ they are found in the central part of the endoderm cell mass in the uv-irradiated embryos, while they are situated in the lateral or dorsal part of the endoderm cell mass in the unirradiated. In the uv-irradiated embryos, a cavity which was never found in the unirradiated embryos was observed in the endoderm cell mass beneath the archenteron cavity and in the almost-median part of the posterior endoderm cell mass at stages 17 and 23, respectively, and some vacuoles in pPGCs as well as in somatic cells around those pPGCs were noticed at stages $\text{17–}\text{33}\text{34}$. The number of pPGCs of the unirradiated enbryos increases about three- or fourfold during stages 12–46, while the pPGCs of the uv-irradiated embryos slowly increase in number from stage 17 to stage 28, indicating that the division occurs in pPGCs, then decrease with development and finally disappear from the tadpole.     

Heterogeneity in the performance of outbred sprague-dawley rats in an elevated-plus maze test: A possible animal model for anxiety disorder

A wide variation in the performance of inbred rats measured in the elevated plus maze test suggests a possible genetic basis for anxiety response (AR). To gain further insight into the role of genetics in AR, we have characterized AR in male outbred S-D rats. Rats were placed in the black compartment (BC) facing the wall opposite the aperture and time needed for the animal to exit BC was noted. All rats underwent 3 successive trials 1–1.5 hrs apart. Naive rats showed a wide variation in their AR in trial 1(mean = 89 ± 19 sec, range = 5–360 sec). Sixty-eight % of the rats exhibiting low AR exited BC in <30sec, whereas 16% stayed in for the entire 360 sec (high AR). On successive testing, there was a progressive increase in AR which reached to max on second trial (Trial 1: 89±19, Trial 2: 171± 23, Trial 3: 210± 22 sec, p<0.0001). The time spent in BC on successive trials increased for most rats ($\text{33}\text{44}$), decreased for some ($\text{2}\text{44}$), showed min to no change ($\text{5}\text{44}$) or erratic response ($\text{4}\text{44}$) for others. In conclusion wide variation in the AR in outbred rats could be exploited to study genetic and neurochemical mechanisms of anxiety.     

A novel radioiodinated high affinity ligand for the D2-dopamine receptor: Characterization of its binding in bovine anterior pituitary membranes

A novel high affinity dopaminergic ligand, N-(p-aminophenethyl)spiroperidol, has been synthesized and radioiodinated to a specific radioactivity of 2175 $\text{Ci}\text{mmol}$. Binding of this ligand to bovine anterior pituitary membranes is: (i) rapid (40–60 min to equilibrium at 25°C) and reversible t_{$\text{1}\text{2}$} = 1 h at 25°C); (ii) saturable and of high affinity (K_D ~ 20 pM) and (iii) displays a typical D₂-dopaminergic specificity. The ligand, which identifies the same number of receptor sites as other tritiated antagonist ligands, can be used in different tissues and preparations to delineate the characteristics of the D₂ receptor. Thus, this high affinity, high specific radioactivity ligand (N-(p-amino-m-[¹²⁵I]iodophenethyl)spiroperidol) represents a tool which until now had not been available for the characterization of the D₂-dopamine receptor.     

Effects of adrenal steroid activator protein on the conversion of various 20- and 22-hydroxycholesterols to pregnenolone by adrenal mitochondrial enzymes

A heat-stable protein activator from bovine adrenal cortex mitochondria stimulates the conversion of cholesterol to pregnenolone in crude extracts of adrenal mitochondria, and resembles in some of its properties, the sterol carrier protein of liver (Kan $\text{et}\text{al}$. $\text{Biochem}$. $\text{Biophys}$. $\text{Res}$. $\text{Commun}$. $\text{48}$, 423–429, 1972). We have shown that activator preparations also stimulate highly purified adrenal enzyme preparations comprising four components: cytochrome P-450 specific for side chain cleavage, adrenodoxin, adrenodoxin reductase, and an NADPH-generating system. Furthermore, this activator stimulates the conversion not only of cholesterol, but also of (20S)-20-hydroxycholesterol, (22$\text{R}$)-22-hydroxycholesterol, and (20$\text{R}$, 22$\text{R}$)-20,22-dihydroxycholesterol to pregnenolone. Our findings provide additional evidence that the steroid-activator complexes are the substrates for the side chain cleavage enzyme and that the monohydroxy and dihydroxycholesterols are true intermediates in the conversion of cholesterol to pregnenolone by bovine adrenal cortex mitochondria.     

Antenna function of a chlorophyll ab protein complex of Photosystem I

The functional role of a chlorophyll $\text{a}\text{b}$ complex associated with Photosystem I (PS I) has been studied. The rate constant for P-700 photooxidation, K_P-700, which under light-limiting conditions is directly proportional to the size of the functional light-harvesting antenna, has been measured in two PS I preparations, one of which contains the chlorophyll $\text{a}\text{b}$ complex and the other lacking the complex. K_P-700 for the former preparation is half of that of the preparation which has the chlorophyll $\text{a}\text{b}$ complex present. This difference reflects a decrease in the functional light-harvesting antenna in the PS I complex devoid of the chlorophyll $\text{a}\text{b}$ complex. Experiments involving reconstitution of the chlorophyll $\text{a}\text{b}$ complex with the antenna-depleted PS I preparation indicate a substantial recovery of the K_P-700 rate. These results demonstrate that the chlorophyll $\text{a}\text{b}$ complex functions as a light-harvesting antenna in PS I.