Somatostatin analogs which inhibit glucagon and growth hormone more than insulin release

Three analogs of somatostatin, [$\text{D}$-Cys¹⁴] -, [Ala², $\text{D}$-Cys¹⁴] - and [$\text{D}$-Trp⁸, $\text{D}$-Cys¹⁴] - somatostatin, were synthesized by the solid phase method, characterized by several means, and tested for their effects on the release of insulin, glucagon, and growth hormone. The peptides sharply suppressed the release of growth hormone $\text{in vitro}$ and glucagon $\text{in vivo}$, but had less effect on insulin secretion $\text{in vivo}$. These analogs, particularly [$\text{D}$-Trp⁸, $\text{D}$-Cys¹⁴] - somatostatin, could possibly be useful for the treatment of diabetes mellitus.     

The interaction of spectrin · actin and synthetic phospholipids

Using differential scanning calorimetry and freeze fracture electron microscopy interactions were studied between lipids and a spectrin · action complex isolated from human erythrocyte membranes. With dispersions of $\text{1,2-}\text{dimyristoyl}\text{-sn-}\text{glycero}\text{-3-}\text{phosphocholine}$, $\text{1,2-}\text{dimyristoyl}\text{-sn-}\text{glycero}\text{-3-}\text{phosphoglycerol}$ and mixtures of these two compounds, which for experimental reasons were chosen as the lipid counterpart, such an interaction could clearly be deduced from changes in the temperature and the enthalpy of the phase transition. Furthermore it was demonstrated that the interaction with this membrane protein protects the bilayer against the action of Ca²⁺ and Mg²⁺ and prevents fusion of lipid vesicles which easily occurs in some of the systems when divalent ions were added to the pure lipid vesicles.     

Behavioral comparisons of the stereoisomers of tetrahydrocannabinols

The potencies of (?)-$\text{trans}$-Δ⁹-THC, (+)-$\text{trans}$-Δ⁹-THC, (+)-$\text{cis}$-Δ⁹-THC, (?)-$\text{trans}$-Δ⁸-THC and (+)-$\text{trans}$-Δ⁸-THC were compared in several different species. (?)-$\text{trans}$-Δ⁹-THC was 100 times more potent than (+)-$\text{trans}$-Δ⁹-THC in depressing schedule-controlled responding in monkeys. The (+)-$\text{trans}$ isomers were less effective than their corresponding (?)-$\text{trans}$ isomers in the dog static-ataxia test, but potency ratios could not be determined due to a lack of dose-responsiveness of the (+)-$\text{trans}$ isomers. However, it appeared that their potency differed by at least ten fold. The potency of (+)-$\text{cis}$-Δ⁹-THC in the dog static-ataxia test was comparable to that of (+)-$\text{trans}$-Δ⁹-THC. The hypothermia in mice produced by the (?) isomers of $\text{trans}$-Δ⁹-THC and $\text{trans}$-Δ⁸-THC were 9.1 and 30.4 times greater than that produced by their respective (+)-isomers. Also, the potency ratio of the (+)- and (?)-$\text{trans}$-Δ⁹-THC was 5.6 as measured by depression of spontaneous activity in mice. The magnitude of the potency ratios of the THC stereo-isomers is dependent upon the species and the pharmacological test used.     

Membrane fusion of secretory vesicles and liposomes Two different types of fusion

Secretory vesicles isolated from adrenal medulla were found to fuse in vitro in response to incubation with Ca²⁺. Intervesicular fusion was detected by electron microscopy and was indicated by the appearance of twinned vesicles in freeze-fractured suspensions of vesicles and in thin-sectioned pellet. Two types of fusion could be distinguished: Type I, occurring between 10^?7 M and 10^?4 M Ca²⁺, was specific for Ca²⁺, was inhibited by other divalent cations and was abolished by pretreatment of vesicles with glutaraldehyde, neuraminidase or trypsin. Fusion type I was linear with temperature. A second type of intervesicular fusion was elicited by Ca²⁺ in concentrations higher than 2.5 mM and was morphologically characterized by multiple fusions of secretory vesicles. This type of fusion was found to be similar to fusion of liposomes prepared from the membrane lipids of adrenal medullary secretory vesicles: Ca²⁺ could be replaced by other divalent cations, the effect of different divalent cations was additive and pretreatments attacking membrane proteins were ineffective. Fusion type II of intact secretory vesicles as well as liposome fusion was discontinuous with temperature. Liposome fusion could be detected within 35 ms and persisted for 180 min. Using liposomes containing defined Ca²⁺ concentrations we have not found a major influence of Ca²⁺ asymmetry on fusion. Incorporation of the ganglioside GM₃, which is present in the membranes of intact adrenal medullary secretory vesicles did not change the properties of liposomes fusion. Using a Ca²⁺-selective electrode we have identified in secretory vesicle membranes both high affinity binding sites for Ca²⁺ ($\text{K}{}_{\mathrm{<mtext>d</mtext>}}\text{= 1.6 · 10}{}^{\mathrm{?6}}\text{M}$) and low affinity sites ($\text{K}{}_{\mathrm{<mtext>d</mtext>}}\text{= 1.2 · 10}{}^{\mathrm{?4}}\text{M}$).     

Biosynthesis of a P1-2-acetamido-2-deoxy-D-glucosyl P2-polyisoprenyl pyrophosphate by calf pancreas microsomes

Incubation of UDP-[¹⁴C]-$\text{N}$-acetylglucosamine with calf pancreas microsomes in the presence of Mn⁺⁺ and potassium thiocyanate gave a labeled glycolipid, tentatively identified as $\text{P}$¹-2-acetamido-2-deoxy-$\text{D}$-glucosyl $\text{P}$²-dolichyl pyrophosphate on the basis of cochromatography with synthetic $\text{P}$¹-2-acetamido-2-deoxy-α-$\text{D}$-glucopyranosyl $\text{P}$²-dolichyl pyrophosphate, similar chemical and enzymic hydrolyses of the biosynthetic and synthetic compounds, and stimulation of the biosynthesis by addition to the incubation mixture o dolichyl phosphate or a crude lipid fraction extracted from microsomes.     

Cytochrome P-450 and 18-oxygenase system from beef adrenocortical mitochondria - inhibitory effect of puromycin.

$\text{Trans}$-3-dehydro-D, L-ornithine and $\text{trans}$-1, 4-diamino-2-butene have been synthesized and shown to be potent competitive inhibitors of ornithine decarboxylase. The K_I′S for $\text{trans}$-3-dehydro-L-ornithine and $\text{trans}$-1, 4-diamino-2-butene are 2.2 and 2.0 μM respectively. Both analogs bind much more tightly to the enzyme than either ornithine or putrescine. Studies of chick embryo muscle cells in culture show results consistent with reversible inhibition of division and/or fusion by addition of $\text{trans}$-3-dehydro-D, L-ornithine to the culture medium.     

Identification of ortho-octopamine and meta-octopamine in mammalian adrenal and salivary gland.

$\text{Ortho}$- and $\text{meta}$- octopamine have been identified in beef and rat adrenal gland and in rat salivary gland by means of gas chromatography-mass spectrometry. The tritrifluoroacetyl derivatives of $\text{ortho}$-, $\text{meta}$- and $\text{para}$- octopamine were resolved by gas chromatography and shown to produce two characteristic ions at $\text{m/e}$ 315 and $\text{m/e}$ 328. The di-O-trimethylsilyl-N-trifluoroacetyl derivatives of these three isomers were also resolved by gas chromatography and shown to produce a characteristic ion at $\text{m/e}$ 267. Biological samples were homogenized in formic acid:acetone, subjected to ion-exchange chromatography and then derivatized. When the derivatized biological extracts were examined for each characteristic ion, peaks were observed at the exact retention times of the standards. The three isomers are present in adrenal gland in concentrations of ～1 μg g^?1 and in rat salivary gland in concentrations of ～0.1 μg g^?1. This evidence confirms a previous report of the presence of $\text{m}$-octopamine in rat salivary gland measured by a radiochemical enzyme assay and is the first report of the presence of $\text{o}$-octopamine in biological tissue.     

Incorporation of 3H-adenine into free cytokinins by cytokinin-autonomous tobacco callus tissue

Cytokinin-autonomous tobacco callus was incubated in defined mineral medium containing ³H-adenine for 60 minutes. Radioactivity was incorporated into the four predominant free cytokinins, ribosyl-$\text{trans}$-zeatin, $\text{trans}$-zeatin, N⁶-(Δ²-isopentenyl) adenosine and N⁶-(Δ²-isopentenyl) adenine. The bases were more abundant than their respective ribosides, N⁶-(Δ²-isopentenyl) adenine being the most abundant cytokinin. No discrete peaks of radioactivity could be detected on the HPLC column eluate corresponding to the elution volumes of $\text{cis}$-zeatin and ribosyl-$\text{cis}$-zeatin.     

Enzymatic formation of 14,15-leukotriene A and C(14)-sulfur-linked peptides

Human leukocytes converted [³H]-(S)-15-HPETE into [³H]-14,15-LTA. Rat basophilic leukemia cells transformed 14,15-LTA into two bioactive C(14)-S-linked peptides, which have been characterized as 15(S)-hydroxy-14(R)-S-glutathionyl-5,8$\text{Z}$,10,12$\text{E}$-icosatetraenoic acid and 15-(S)-hydroxy-14(R)-S-cysteinylglycyl-5,8$\text{Z}$,10,12$\text{E}$-icosatetraenoic acid by comparison with synthetic specimens.     

Cysteine as a system-specific substrate for transport system ASC in rat hepatocytes.

The rapid transport of $\text{L}$-cysteine into isolated rat hepatocytes escapes detectable inhibition by 2-(methylamino)-isobutyric acid at levels up to 50 mM. The system transporting cysteine instead is convincingly similar to the $\text{ASC}$ system described for the Ehrlich cell in structural and steric specificity and in pH sensitivity. The Na⁺-dependent uptake of 2-aminoisobutyric acid is almost evenly divided between Systems $\text{A}$ and $\text{ASC}$, showing better accommodation of its two α-methyl groups by $\text{ASC}$ than in the Ehrlich cell. The hepatocyte $\text{ASC}$ system tolerates Li⁺-for-Na⁺ substitution better than does System $\text{A}$, although the tolerance depends on amino acid structure. Adaptive regulation and insulin and glucagon stimulation were not seen under conditions producing these effects for System $\text{A}$.     

An antiovulatory decapeptide of higher potency which has an L-amino acid (Ac-Pro) in position 1.

Ac-[Pro¹, $\text{D}\text{-}\text{Phe}{}^2$, $\text{D}$-Trp³, $\text{D}$-Trp⁶]-LH-RH completely inhibited ovulation in cycling rats at 200μg/rat and is comparable in activity to the corresponding D-1-analogue. This Ac-Pro¹-analogue is the most potent antiovulatory peptide yet known having an $\text{L}$-amino acid residue in position 1. This result shows that for the design of potent inhibitors of ovulation, a $\text{D}$-amino acid residue is not essential in position 1. The corresponding Ac-$\text{D}$-Pro¹- and Kic¹-analogues completely inhibited ovulation at 750μg/rat, but not at 200μg/rat, and the Cpc¹-analogue was inactive at these dosages.     

Mass spectrometric identification of various molecular forms of dynorphin in bovine adrenal medulla

Deliberate miscompartmentalization of liver outer mitochondrial membrane (OMM) proteins and liver mitochondrial proteins has been achieved by polyethylene-glycol mediated OMM vesicle-hepatocyte or mitochondrial-hepatocyte fusion. Reductively methylated OMM and mitochondrial proteins (³H) are destroyed at rates remarkably similar to those for OMM ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$, 60–70 h) or mitochondrial proteins ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$, 84–104 h) in liver $\text{in vivo}$ when studied over 4–5 days in hepatocyte monolayers cultured in conditions giving stabilized endogenous protein catabolic rates mimicking endogenous $\text{in vivo}$ rates. Destruction of transplanted OMM proteins is partially sensitive to chloroquine, supporting some lysosomally mediated autophagic destruction of long-lived transplanted OMM proteins in hepatocyte monolayers.     

Receptor-like stereospecific binding of a pyrethroid insecticide to mouse brain membranes

A heterogeneous particulate fraction of mouse brain homogenates binds NRDC 157 (3-phenoxybenzyl [1$\text{R}$,$\text{cis}$]-3-(2,2-dibromovinyl)-2,2- dimethylcyclopropanecarboxylate), a potent pyrethroid insecticide, stereospecifically and with high affinity. Stereospecific binding is a minor component of total binding (2.8%); the remainder of observed binding is predominantly nonspecific and unsaturable. Stereospecific binding is half-saturated at 4×10^?8$\text{M}$ and fully saturated at concentrations in excess of 1×10^?7$\text{M}$. The stereospecific binding capacity of this preparation was 200–250 pmoles of NRDC 157 per gram equivalent of brain tissue (2.3–2.8 pmol/mg protein). This binding site may represent the neural receptor involved in the stereospecific toxic action of pyrethroids.     

DNA synthesis and the cell cycle in cultures of normal and mutant (mdg/mdg) embryonic mouse muscle cells.

The relationship between cell fusion, DNA synthesis and the cell cycle in cultured embryonic normal and dysgenic ($\text{mdg}\text{mdg}$) mouse muscle cells has been determined by autoradiography. The experimental evidence shows that the homozygous mutant myotubes form by a process of cell fusion and that nuclei within the myotubes do not synthesize DNA or undergo mitotic or amitotic division. The duration of the total cell cycle and its component phases was statistically the same in 2-day normal and mutant ($\text{mdg}\text{mdg}$) myogenic cultures with the approximate values: T, 21.5 hr; G₁, 10.5 hr; S, 7.5 hr; and G₂, 2.5 hr. In both kinds of cultures, labeled nuclei appeared in myotubes 15–16 hr after mononucleated cells were exposed to [³H]thymidine, and the rate of incorporation of labeled nuclei into multinucleated muscle cells was comparable in control and dysgenic cultures. Thus, homozygous $\text{mdg}\text{mdg}$ muscle cells in culture are similar to control cells with respect to their mechanism of myotube formation and the coordinate regulation of DNA synthesis and the cell cycle during myogenesis.     

Glutathione S-transferase activities in rat and mouse sperm and human semen

Glutathione $\text{S}$-transferase activity was found in sperm of the rat and $\text{DBA}\text{2J}$ and C57 $\text{BL}\text{6J}$ mice. In rat sperm activities with benzo(a)pyrene 4,5-oxide, styrene 7,8-oxide, and 1-chloro-2,4-dinitrobenzene were 0.88, 1.07, and 26.1 nmoles/min/mg protein, respectively. Δ⁵-3-Ketosteroid isomerase activity of rat sperm was 4.9 nmoles/min/mg protein. These specific glutathione $\text{S}$-transferase and Δ⁵-3-ketosteroid isomerase activities in sperm represent 0.4–4.1% of rat liver cytosol values. Human semen also contained significant glutathione $\text{S}$-transferase activity. It is postulated that these enzymes could function in the metabolism and detoxification of certain electrophilic xenobiotics, if present in sperm.     

Lithosperm inhibition of anterior pituitary hormones.

The enzyme system for the synthesis of the pteridine pigment, sepiapterin, from $\text{2-amino-4-hydroxy-6-(D-}\text{erythro}\text{-1',2',3'-trihydroxyprophyl)}$ triphosphate (dihydroneopterin triphosphate) has been found in extracts of $\text{Drosophila melanogaster}$. NADP⁺ or NADPH and Mg²⁺ are required for this enzymatic transformation. No sepiapterin is produced when dihydroneopterin is supplied as substrate in place of dihydroneopterin triphosphate.     

Chemical structure and absolute configuration of a juvenile hormone from grasshopper corpora allata in vitro

One juvenile hormone was isolated from culture medium containing isolated corpora allata of the grasshopper $\text{Schistocerca vaga}$ (Orthoptera: Acrididae) and was shown by microchemical methods to be methyl (2$\text{E}$, 6$\text{E}$) - (10$\text{R}$) - 10, 11-epoxy-3, 7, 11-trimethyldodeca-2, 6-dienoate. This compound (JH III), which occurs in a sphingid moth $\text{Manduca sexta}$, is the first juvenile hormone identified in an insect order other than the Lepidoptera. Grasshopper organs incorporate both [2?¹⁴C] acetate and [methyl-¹⁴C] methionine into JH III showing $\text{de novo}$ biosynthesis, but no indication of the synthesis of JH I or JH II was seen.     

5' Cap methylation of homologous poly A(+) RNA by a RNA (guanine-7) methyltransferase from Neurospora crassa.

RNA (guanine-7) methyltransferase, partially purified from $\text{N.}\text{crassa}$ mycelia, catalyzed the transfer of the methyl group from S-adenosylmethionine to the 5′ terminus of both $\text{N.}\text{crassa}$ poly A(+) RNA and reovirus unmethylated mRNA. RNase T₂ digestion of the $\text{in}\text{vitro}$ methylated poly A(+) RNA from $\text{N.}\text{crassa}$ yielded the “cap” structures m ⁷G(5′)pppAp and m ⁷G(5′)pppGp in a ratio of 2:1 respectively. RNase T₂ digestion of the $\text{in}\text{vitro}$ methylated reovirus mRNA yielded m ⁷G(5′)pppGp exclusively. The absence of mRNA 2′-0-methyltransferase activity in the enzyme preparation is consistent with the absence of 2′-0-methylation in $\text{N.}\text{crassa}$ mRNA [Seidel, B. L. and Somberg, E. W. (1978) Arch. Biochem. Biophys. $\text{187}$, 108–112]. This is the first isolation of an eucaryotic, cellular RNA (guanine-7) methyltransferase that has been shown to methylate homologous substrate.     

Expression and subcellular distribution of mouse cytochrome P1-450 mRNA as determined by molecular hybridization with cloned P1-450 DNA

Cytochrome P₁-450 (P₁-450) is defined as that cytochrome most closely associated with 3-methylcholanthrene (MC)-induced aryl hydrocarbon hydroxylase (AHH) activity. Recently a cloned DNA sequence (clone 46) was shown to represent a portion of the P₁-450 structural gene [Negishi $\text{et}\text{al.}\text{,}\text{Proc. Nat. Acad. Sci. U.S.A.}\text{78}$: 800–804 (1981)]. Poly(A⁺)-enriched RNA was isolated from total liver homogenate, membrane-bound polysomes and from free polysomes at various times after MC treatment of $\text{Ah}$-responsive $\text{C57BL}\text{6N}$ (B6) and $\text{Ah}$-nonresponsive $\text{DBA}\text{2N}$ (D2) inbred mice. The poly(A⁺)-enriched RNA was separated by methylmercury-agarose gel electrophoresis and hybridized to nick-translated [³²P]DNA from clone 46. By means of this RNA-DNA hybridization, only 6% of total polysomal P₁-450 mRNA exists in free polysomes after 24 h of MC treatment. The data indicate that the endoplasmic reticulum is the principal site of synthesis for this integral microsomal protein.Studies of induction kinetics following MC treatment provided the evidence of the rapid increase of total liver and membrane bound P₁-450 mRNA preceding the synthesis of apo-P₁-450 and the increase of AHH activity.