Amino acid sequence at the ATP-binding site of cGMP-dependent protein kinase

The amino acid sequence at the ATP-binding site on the cGMP-dependent protein kinase has been determined. For this determination the enzyme was labeled covalently by 5'-p-fluorosulfonyl[14C]benzoyladenosine and fragmented using cyanogen bromide or digested by trypsin after succinylation. The 14C-labeled peptides were purified by gel filtration and high performance liquid chromatography. The amino acid sequence around the site was found to be: -Val-Glu-Leu-Val-Gln-Leu-Lys-Ser-Glu-Glu-Ser-Lys-Thr-Phe-Ala-Met-*Lys-Ile-Leu-Lys--Lys-Arg-His-Ile-Val-Asp-Thr-Arg-Gln-Gln-Glu-His-Ile-Arg-Ser-Glu-Lys-, in which *Lys is the lysine residue that was modified by the affinity reagent. When this sequence was compared with that of the ATP-binding site of the catalytic subunit of cAMP-dependent protein kinase, a high degree of structural homology was observed for this site in the two proteins.     

Dehydrodieugenol and its monomethyl ether from Virola carinata

The constituents of Virola carinata were established as dehydrodieugenol, its monomethyl ether and sitosterol.     

Contribution from the chirality of the growing chain to the enantiomer selectivity in activated-NCA polymerization of α-amino acid N-carboxyanhydride

As a model compound for the growing chain in the activated-NCA type of polymerization of α-amino acid N-carboxyanhydride (NCA), 3-[ω-acetylglycyl-poly(α-amino acid) acyl]-α-amino acid NCA (called the prepolymer) having various degrees of polymerization (DPs) was synthesized by the polymerization of Phe, Val, Glu(OEt), and Asp(OBzl) NCA in the presence of AcGly NCA by the tertiary amine. Activated (S)-Phe, Val, Glu(OEt), and Asp(OBzl) NCA were added to the terminal cyclic group of the corresponding (S)- or (R)- prepolymer, and the enantiomer selectivity in the reaction was investigated. With prepolymers having DPs ranging from 1 to 15, the addition reaction always took place preferentially between species having the same configuration, and the degree of the enantiomer selection increased with increasing DP of the prepolymer. With prepolymers having DP = 1 and 2, we found contributions from the chiral terminal unit and the chiral penultimate unit to the enantiomer selection, respectively. Prepolymer having DP = 5 was shown to take a β-type conformation, which led to higher enantiomer selection; and prepolymers having DP = 10 and 15 were shown to take an α-helix conformation, which led to much higher enantiomer selection than did the β-type conformation. In the present investigation the mechanisms of terminal-unit control, penultimate-unit control and conformational control of the enantiomer selection in the activated-NCA type of polymerization were clearly observed.     

Induction of a novel long-chain acyl-CoA hydrolase in rat liver by administration of peroxisome proliferators

The activity of long-chain acyl-CoA hydrolase in rat liver was increased by the administration of peroxisome proliferators, such as ethyl p-chlorophenoxyisobutyrate, di(2-ethylhexyl)phthalate or acetylsalicylic acid. The induced activity was mainly confined in the soluble fluid after the subcellular fractionation. The enzyme was purified nearly to homogeneity from livers of rats treated with di(2-ethylhexyl)phthalate. The specific activity of the final preparation was 247 mumol palmitoyl-CoA hydrolyzed min-1 mg protein-1. The molecular weight of the native enzyme was estimated to be 150 000 by gel filtration and that of the subunits was 41 000 by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. The activity of the enzyme was not increased but inhibited by bovine serum albumin or Triton X-100. The molecular and catalytic properties of the enzyme suggest that the induced enzyme was different from mitochondrial and microsomal long-chain acyl-CoA hydrolyses in liver.     

Angiostrongylus cantonensis: Rejection of pulmonary arterial transfers of lung stage worms

Experimental transfer of the lung stage worms of Angiostrongylus cantonensis was performed between permissive hosts (rats) and between permissive (rat) and nonpermissive hosts (guinea pigs and rabbits). These worms from rats were rejected when implanted into nonpermissive hosts. Unexpectedly, similar worms did not survive well even in permissive hosts; the majority of recipient rats did not have first-stage larvae (L₁) in their stools and, even when positive for L₁, the number of the larvae shed was few. These findings contrast with the successful pulmonary arterial transfer of younger, intracranial-stage worms. It was shown that differences in rat strain between donor and recipient had no significant effect on the subsequent worm survival in recipient hosts. The alteration of maintaining conditions of the intrapulmonary worms, prior to transfer, in terms of temperature, media, and maintaining period, also showed no profound effect on the subsequent worm survival. The kinetics of precipitating and reaginic antibody levels in rats implanted with the intrapulmonary worms were analogous to those in rats with intracranial-stage worms. The findings indicate that some qualitative differences may exist between the worms obtained from two different sites.     

Substrate utilization kinetic model for biological treatment process

The applicability of Contois' kinetic equation to aerobic and anaerobic treatments of organic wastes is investigated. A refractory coefficient to account for the nonbiodegradable portion of the organic substrates in the digester is incorporated into the kinetic equation. The kinetic equation is applied to the data for aerobic digestions of organic substrates and for anaerobic treatment of dairy wastes. They all show a very good fit of the kinetic equation to the data. Furthermore, the kinetic parameters and the refractory coefficients are shown to be independent of influent organic substrate concentration. This study confirms previous reports that the effluent quality of biological treatment systems for organic wastes depends on influent organic waste concentration. The effect of temperature on the kinetic parameters and the refractory coefficient for anaerobic treatment of sewage sludge are studied. It shows that the kinetic parameters vary with temperature, while the refractory coefficient remains fairly constant. Equations to predict biodegradable treatment efficiency and volumetric substrate utilization rate are also briefly discussed.     

Effects of oral administration of positively charged insulin liposomes on alloxan diabetic rats: preliminary study.

Insulin encapsulated in liposomes of various lipid compositions were prepared. The amount of insulin trapped in these liposomes increased in the order, negatively charged liposomes less than neutral liposomes less than positively charged liposomes. In positively charged liposomes, the amount of insulin trapped increased with increase in the amount of amphiphile stearylamine. Under the conditions tested, the highest insulin content (about 50%) was obtained with liposomes composed of phosphatidyl choline/cholesterol/stearylamine in a molar ratio of 7/2/2.25. These liposomes were stable on incubation for 3 hr at 37 degrees C in solutions of pepsin, trypsin, and pancreatin, and after these incubations, a considerable amount of insulin was still associated with the liposomes. However, the liposomes released almost all the insulin into the medium on treatment with bile. When the liposomes were administered orally to rats in the 3rd phase of acute alloxan diabetes, reduction of the blood glucose level was observed in 7 of 11 animals, the reduction persisted for several hours and was ranging from 30 to 75%. In alloxan diabetic rats showing hyperglycemia for 3 to 6 months, the liposomes also increased the glucose tolerance in half the animals tested.     

Inhibitory effect of amines on polar filament extrusion byPlistophora anguillarum spores

In in vitro tests, amines were screened for the inhibition of polar filament extrusion by spores ofPlistophora anguillarum, a microsporidian parasite of the eel. Primary amines having C₈ ⁻ C₁₈ alkyl chains were effective, irrespective of the anions bound. Secondary amines having a branched C₈ chain and tertiary amines having C₁₄−C₁₈ chains were also effective. Asymmetry in side chains seemed to be required for inhibitory action. Quaternary ammonium salts having C₁₂−C₁₆ alkyl chains and some germicides or disinfectants such as cetyl pyridinium chloride and benzethonium chloride were also effective. Diamines and amides were ineffective regardless of the length of their alkyl chins. In in vivo tests, eel larvae-fed for 4 days with a commercial feed supplemented with 5×10⁸ cells of spores and 4g of laurylamine aspartate/100 g feed, and thereafter with the usual commercial feed for 26 days—were not infected.     

Stimulation of acyl-CoA:cholesterol acyltransferase activity in rat liver microsomes by phosphatidylcholine

Acyl-CoA:cholesterol acyltransferase (ACCAT) activity of rat liver microsomes was stimulated by phosphatidylcholine. The stimulatory effect varied with the composition of the phosphatide: dimyristyl-, dipalmityl-, distearyl- and dioleylphosphatidylcholine were stimulatory, whereas dicaproyl- and dilinoleylphosphatidylcholine were not. The results suggest that increased fluidity of the membrane induced by phosphatide is probably not involved in the stimulation of cholesterol esterification. Phosphatide exerted its effect directly on the microsomes and did not extract cholesterol or ACCAT from the microsomes to an appreciable extent.Hydrolysis of microsomal phosphatide suppressed ACCAT activity. Enztme activity was restored with the addition of phosphatidylcholine. The results suggest that phosphatide may be required for cholesterol esterification.