Lysine metabolism in the human and the monkey: Demonstration of pipecolic acid formation in the brain and other organs

Metabolism ofl-[U-¹⁴C]lysine was studied in the human autopsy tissues and the intact monkeys through intracerebroventricular and intravenous injections. The human tissues were more active in the metabolism ofl-[¹⁴C]lysine to [¹⁴C]pipecolate than the rat tissues previously reported. This metabolism was equally active in the phosphate (pH 7) and the glycyl-glycine (pH 8.6) buffers with the brain and the kidney having higher activity than the liver. Besides [¹⁴C]pipecolate, traces of [¹⁴C]saccharopine and -[¹⁴C]aminoadipate were also detected in the liver incubation. Twenty-four hr after intraventricular injection ofl-[¹⁴C]lysine to the monkey, substantial labeling of pipecolate and -aminoadipate was observed in the brain and spinal cord, with the kidney, liver and the plasma having much reduced levels. Radioactivity levels of these two compounds were found low in the organs and plasma of the intravenously injected monkey. The urine of both monkeys contained only traces of [¹⁴C]pipecolate, even though it contained high levels ofl-[¹⁴C]lysine and -[¹⁴C]aminoadipate. It was concluded thatl-lysine is actively metabolized to pipecolate and -aminoadipate in the human and the monkey, that this reaction is most active in the brain whenl-lysine is intraventricularly administered, and that in contrast to the rat, the monkey may have an effective renal reabsorption for pipecolate which is similar to the human.     

The environment of the sulfhydryl group in human plasma albumin as determined by spin labeling

A series of spin labels, varying in chain length between the maleimide attaching group and the nitroxide free radical, has been used to investigate the environment of the sulfhydryl group in human plasma albumin. From the electron spin resonance spectra, the degree of freedom of the nitroxide was determined and the location of the sulfhydryl was assessed. The effect of bound fatty acids on the sulfhydryl environment was also determined. The environment was found to be analogous to that in the bovine protein, that is, a crevice approximately 9.5 Å deep and not affected in the native state by fatty acids.     

Effect of interferon on phospholipid methylation by peripheral blood mononuclear cells

The effect of human interferon (IFN) preparations on the metabolic pathway leading to the synthesis of phosphatidylcholine (PC) by a stepwise addition of methyl groups to phosphatidylethanolamine (PE) was investigated in human peripheral blood mononuclear (PBMN) cells. An inhibition of the synthesis of PC via this pathway was regularly observed with both alpha- (recombinant or natural) and beta-IFN. This inhibition was apparent within the first 5 min of treatment, reached its maximum between 15 min and 1 hr, and persisted at the same level until 6 hr, the last time point examined. Each of the transmethylated products of PE underwent a similar inhibition, as measured by the turnover rate of individual products. The intracellular pool of the methyl donors, methionine and S-adenosyl-methionine (SAM), was shown to be unaffected. The methyltransferase activity of IFN-pretreated cell extracts was unchanged. These findings support the hypothesis that IFN induces a functional change in phospholipid methylation at the level of organized membrane-bound phospholipid methyltransferase enzymes in intact cells.     

The influence of phytoplankton composition on the relative effectiveness of grinding and sonification for chlorophyll extraction

The chlorophyll recovery efficiency was compared between control, ground, and sonified samples. The results showed significant improvement between control and ground samples but not between control and sonified samples. Neither prolonging time of sonification nor using an ice bath during filter grinding improved efficiency. Higher chlorophyll a recovery was obtained from ground samples than from sonified ones, when the water samples contained centric diatoms and filamentous blue-green algae. When total phytoplankton numbers were high, there was a distinct advantage in using grinding rather than sonification for chlorophyll c recovery.Contribution no. 313 of the Great Lakes Research Division, University of Michigan.Contribution no. 313 of the Great Lakes Research Division, University of Michigan.     

Platelet-derived growth factor is a chemoattractant for vascular smooth muscle cells

In previous experiments (Grotendorst et al, 1981), we showed that platelet-derived growth factor promotes the migration of smooth muscle cells in vitro. Using a "checkerboard" analysis, we now establish that platelet-derived growth factor (PDGF) acts as a true chemoattractant for cultured aortic smooth muscle cells. Other growth factors such as epidermal growth factor, fibroblast growth factor, and insulin are not chemoattractants. The chemotactic response occurs before the initiation of DNA synthesis and is not affected by inhibition of DNA synthesis. Chemotaxis occurs at levels of PDGF lower than required for mitogenesis. RNA and protein synthesis are required for the chemotactic response. As found previously in bacteria and leucocytes, we find that methylation reactions are required for the chemotactic response. The possibility is discussed that PDGF acts in vivo at sites of vascular injury to attract smooth muscle cells from the medial layer to the luminal surface, and is involved in the early stages of the formation of atherosclerotic plaques.     

The Benzodiazepine/GABA Receptor Complex: Molecular Size in Brain Synaptic Membranes and in Solution

Abstract: The molecular size of the benzodiazepine (BZ) receptor in the synaptic membrane of brain cortex (bovine or rat) was determined by an improved version of the radiation inactivation method to be 220,000. An identical size was found simultaneously for the associated γ-aminobutyric acid (GABA) receptor and for the component binding β-carboline esters. It is proposed that all three activities reside in a single protein or protein complex in the membrane. The size in solution, after extraction into Triton X-100 medium from exhaustively washed membranes, was estimated by sedimentation constant (9.4S) and by gel filtration (∼230,000 apparent MW), again with the BZ and GABA binding activities behaving identically. This size applies to the component that undergoes photoaffinity labelling by [³H]flunitrazepam in the membrane, and contains a 51,000 M_r polypeptide as the BZ-binding subunit. It is concluded that a protein complex or oligomer of 200,000–220,000 MW carries a class of BZ-binding sites and an associated class of GABA_A sites.     

Selective killing of Leishmania infected mouse macrophages by 6-methylpurine 2′-deoxyriboside

6-methylpurine 2′-deoxyriboside killed mouse macrophages infected with amastigotes of $\text{Leishmania donovani}$ and $\text{Leishmania mexicana}$, but did not affect the growth of non-parasitized cells. $\text{Leishmania}$ extracts cleaved the non-toxic 6-methylpurine 2′-deoxyriboside to 6-methylpurine, a potent adenine antimetabolite for mammalian cells. By eliminating macrophages latently infected with $\text{Leishmania donovani}$ amastigotes, 6-methylpurine 2′-deoxyriboside could augment the effects of leishmanicidal agents $\text{in vivo}$.     

Pre- and postsynaptic adrenergic activation by norepinephrine reuptake inhibitors in the field-stimulated rat vas deferens

Desipramine (DMI), protriptyline, chlorpromazine, amitriptyline and cocaine, alone or in the presence of prazosin, produced a dose-related inhibition of contractions induced by field stimulation of the rat vas deferens. The inhibition of contractions was readily reversed by yohimbine. In contrast, when yohimbine was first added to the bath, all agents, except chlorpromazine, produced a dose-related enhancement of contractions which were readily reversed by prazosin. The potencies of these agents for induction of contractile inhibition, after prazosin, and contractile enhancement, after yohimbine, were similar. Both of the latter contractile responses of DMI were markedly attenuated or absent in tissues taken from rats pretreated with reserpine and alpha-methyl-para-tyrosine. The data indicate that, in the rat vas deferens, inhibition of norepinephrine reuptake results primarily in presynaptic (α₂) receptor activation. Postsynaptic (α₁) adrenergic activation by inhibition of norepinephrine reuptake can be demonstrated in this tissue only after presynaptic (α₂) receptor blockade. The possible implications of the present studies to the delayed clinical onset of action of tricyclic antidepressants is discussed.     

Solid-phase peptide synthesis of somatostatin using mild base cleavage of N alpha-9-fluorenylmethyloxycarbonylamino acids

Experimental details for the "Fmoc solid phase peptide synthesis" of somatostatin are described. The 9-fluorenylmethyloxycarbonyl group was rapidly and quantitatively cleaved by 55% piperidine in dimethylformamide and monitored (u.v.) manually. For a kinetic study, a centrifugal reactor with a photometric control system and reference cell was used at each stage. The symmetrical anhydride coupling reaction was rapid and either acetic anhydride or fluorescamine termination was incorporated to minimize formation of deletion peptides. Anchor-bond cleavage was effected with trifluoroacetic acid which simultaneously removed all the acid labile tert.-butyl side chain protecting groups. N alpha-9-fluorenylmethyloxycarbonyl peptides may be obtained by omitting the piperidine deprotection step after the last cycle of synthesis. From several syntheses, analytically pure di-S-protected somatostatin 14-peptide was obtained in 55-60% overall yield. The S-protecting groups were removed and the product was purified by gel filtration to give homogeneous dihydrosomatostatin (91%) yield. Oxidation of dihydrosomatostatin with potassium ferricyanide and purification by countercurrent distribution provided analytically pure homogeneous somatostatin.