Identification of epsilon-N-mono-, epsilon-N-di-, and epsilon-N-trimethyllysine by high-performance liquid chromatography.

Dansyl derivatives of epsilon-N-mono-, epsilon-N-di-, and epsilon-N-trimethyllysine were resolved from other amino acids in proteins by the use of high-performance liquid chromatography. The system was tested with amino acid standard combinations as well as with acid-hydrolyzed proteins known to contain methylated residues. In all cases the methylated lysines were well resolved.     

Comparative biological activities of ANF (Arg 101-Tyr 126) and the synthetic form of circulating ANF (Ser 99-Tyr 126)

The biological activities of ANF (Arg 101-Tyr 126) and of the circulating form, ANF (Ser 99-Tyr 126), were compared in the following assays: precontracted rabbit aortic strip and chick rectum, rat natriuresis, inhibition of aldosterone secretion and receptor affinity in bovine and rat adrenal zona glomerulosa cells, and receptor affinity in rabbit aorta and rat mesenteric artery cells. The results demonstrate that both peptides share the same biological activities. It is concluded that the addition of two amino acids to the N-terminal of ANF (Arg 101-Tyr 126) does not modify its biological characteristics, validating thus previous research employing this peptide.     

A protein kinase copurified with chick oviduct progesterone receptor

A magnesium-dependent protein kinase activity was copurified with both the molybdate-stabilized 8S form of the chick oviduct progesterone receptor (PR) and its B subunit. In each case, purification was performed by hormonal affinity chromatography followed by ion-exchange chromatography. The Km(app) values of the phosphorylation reaction for [gamma-32P]ATP and calf thymus histones were approximately 1.3 X 10(-5) M and approximately 1.6 X 10(-5) M, respectively, and only phosphorylated serine residues were found in protein substrates, including PR B subunit. Physicochemical parameters of the enzyme [pI approximately 5.3, Stokes radius approximately 7.2 nm, sedimentation coefficient (S20,w) approximately 5.6 S, and Mr approximately 200,000] were compared to those of purified forms of PR (B subunit, pI approximately 5.3, Stokes radius approximately 6.1 nm, and Mr approximately 110,000; 8S form, Stokes radius approximately 7.7 nm and Mr approximately 240,000). The results suggest that most of the protein kinase activity copurified with both oligomeric and monomeric forms of PR belongs to an enzyme distinct from currently known receptor components. Its physiological significance remains unknown.     

Interleukin 1 increases the binding of human B and T lymphocytes to endothelial cell monolayers

Lymphocyte binding to specialized high-endothelial venules (HEV) in lymph nodes and Peyer's patches is the first step in normal lymphocyte emigration and recirculation. The development and maintenance of HEV in these lymphoid organs are thought to be immunologically controlled. Because postcapillary venules in chronic inflammatory tissue often resemble the HEV of lymphoid tissue and may also be a site of lymphocyte emigration, examination of the effects of immunologic and inflammatory mediators on endothelial cells (EC) may provide important information about the physiology of both normal lymphocyte recirculation and chronic inflammation. It is reported here that treatment of human umbilical vein EC monolayers in vitro with affinity-purified human interleukin 1 (IL 1) markedly enhances the binding of both B and T lymphocytes. Increased binding was observed within 1 h of treatment of EC with as little as 0.04 U/ml IL 1. This effect of IL 1 was EC-specific, because pretreatment of T cells or human skin fibroblasts with IL 1 did not increase the binding of lymphocytes. Stimulation of binding required active EC metabolism because incubation of EC with IL 1 at 4 degrees C, or prior fixation of EC, prevented enhanced binding. The action of IL 1 was not associated with EC damage. The secretion of IL 1 by macrophages and perhaps other cells in inflammatory lesions may exert a positive feedback signal on EC to enhance further emigration of lymphocytes into the inflammatory focus.     

Involvement of Calcium in the Regulation of Serotonin N-Acetyltransferase in Retina

The possible involvement of calcium in the regulation of retinal serotonin N-acetyltransferase (NAT) activity was investigated using eye cups of Xenopus laevis cultured in defined medium. Omitting CaCl2 from the culture medium completely inhibited the dark-dependent increase of NAT activity at night. Approximately 10(-4)-10(-3) M free Ca2+ was found to be required for the maximal increase of NAT activity in the dark. Other divalent cations--Ba2+, Sr2+, and Mn2+--did not substitute for Ca2+. Antagonists of voltage-sensitive calcium channels, including nifedipine, methoxyverapamil (D600), Co2+, and Mg2+, were found to be effective inhibitors of the dark-dependent increase of retinal NAT activity. Trifluoperazine also decreased retinal NAT activity. These studies indicate that the increase of retinal NAT activity in the dark is mediated by a specific Ca2+-dependent process and that Ca2+ influx through voltage-sensitive calcium channels is involved.     

The nucleotide sequence of the mercuric resistance operons of plasmid R100 and transposon Tn501: further evidence for mer genes which enhance the activity of the mercuric ion detoxification system

Summary The DNA sequences of the mercuric resistance determinants of plasmid R100 and transposon Tn501 distal to the gene (merA) coding for mercuric reductase have been determined. These 1.4 kilobase (kb) regions show 79% identity in their nucleotide sequence and in both sequences two common potential coding sequences have been identified. In R100, the end of the homologous sequence is disrupted by an 11.2 kb segment of DNA which encodes the sulfonamide and streptomycin resistance determinants of Tn21. This insert contains terminal inverted repeat sequences and is flanked by a 5 base pair (bp) direct repeat. The first of the common potential coding sequences is likely to be that of the merD gene. Induction experiments and mercury volatilization studies demonstrate an enhancing but non-essential role for these merA-distal coding sequences in mercury resistance and volatilization. The potential coding sequences have predicted codon usages similar to those found in other Tn501 and R100 mer genes.     

Superoxide Radical-Mediated Alteration of Synaptosome Membrane Structure and High-Affinity γ-[14C]Aminobutyric Acid Uptake

Mouse cortical synaptosomal structure and function are altered when exposed to hypoxanthine/xanthine oxidase (HPX/XOD)-generated active oxygen/free radical species. The structure of both the synaptic vesicle and plasma membrane systems are altered by HPX/XOD treatment. The alteration of synaptic vesicle structure is exhibited by a significant increase in the cumulative length of nonsynaptic vesicle membrane per nerve terminal. With respect to the nerve terminal plasma membrane, the length of the perimeter of the synaptosome is increased as the membrane pulls away from portions of the terminal in blebs. The functional lesion generated by HPX/XOD treatment results in a reduction in selective high-affinity gamma-[14C]aminobutyric acid (GABA) uptake. Kinetic analysis of the reduction in high-affinity uptake reveals that the Vmax is significantly altered whereas the Km is not. Preincubation with specific active oxygen/free radical scavengers indicates that the super-oxide radical is directly involved. This radical, most probably in the protonated perhydroxyl form, initiates lipid peroxidative damage of the synaptosomal membrane systems. Low-affinity [14C]GABA transport is unaltered by the HPX/XOD treatment. The apparent ineffectiveness of free radical exposure on low-affinity [14C]GABA transport coupled with its effectiveness in reducing high-affinity transport supports the idea that two separate and different amino acid uptake systems exist in CNS tissue, with the high-affinity being more sensitive (lipid-dependent) and/or more energy-dependent (Na+,K+-ATPase) than the low-affinity system.     

Characteristics of the β-Adrenoreceptor from Neuronal and Glial Cells in Primary Cultures of Rat Brain

The cellular characteristics of the beta-adrenoreceptor in glial and neuronal cells from the newborn rat brain were determined by (-)-[125I]iodocyanopindolol binding. In membranes from both cell types, the binding was saturable and from competition assays the potency series of (-)-isoproterenol greater than (-)-epinephrine = (-)-norepinephrine greater than (+)-isoproterenol was observed. 5'-Guanylyl-imidodiphosphate reduced the affinity of (-)-isoproterenol for the beta-adrenoreceptor from glial cells but had no effect on agonist affinity in neuronal cells. Chronic treatment of both cell types with (-)-isoproterenol reduced the receptor content and the capacity of the agonist to increase the cellular cyclic AMP content. However, the receptor recovery after chronic agonist treatment was faster in glial cells (72 h) than neuronal cells (120 h) and was blocked by cycloheximide. Treatment of both types with the irreversible beta-blocker bromoacetylalprenololmentane (2 microM) reduced the receptor content by 78% but no receptor recovery was observed for 120 h after the initial receptor loss. The data indicated that the majority of beta-adrenoreceptors in both cell types are the beta-1 subtype, but show some differences in receptor-agonist interactions. Furthermore, these CNS cells may be useful models for regulatory studies on the beta-adrenoreceptor.     

Possible Involvement of Phage-Like Structures in Antagonism of Cowpea Rhizobia by Rhizobium trifolii

A reduction in the viability of cowpea rhizobia was observed when Rhizobium trifolii IARI and cowpea Rhizobium strain 3824 were inoculated together in soil. The reduction in number of cowpea rhizobia in soil was found to be associated with the reduction in number of nodules per plant and retardation in plant growth. An antimicrobial substance was isolated from R. trifolii which, on electron microscopic investigation, demonstrated the presence of several phage-like structures.