Structure-activity relationships of novel hematoregulatory peptides.

Hematopoiesis is a lifelong cell renewal process regulated by a family of lineage specific hematopoietic growth factors. Several hematopoietic growth factors such as G-CSF, GM-CSF, and M-CSF have been clinically evaluated for enhancement of host defense in normal and immunocompromised patients and for the treatment of infectious diseases. This paper reports the structure-activity relationships of low molecular weight hematoregulatory peptides based on a nonapeptide (1, SK&F 107647). Like the macromolecular growth factors, these peptides modulate host defense. A molecular target for this class of compounds has not yet been identified. However, the structure-activity relationships established by this study implicate a very specific molecular recognition event that is pivotal for the biological activities of 1 and its analogues.

Inhibition of acetylcholinesterase by distigmine bromide (Ubretid).

A randomized phase-I study was performed in a clinical setting in 24 healthy young male subjects, aged 20 to 35 years, to investigate the influence of Ubretid on AChE inhibition following oral and i.m. administration in one of three medication schemes: -single oral (10 mg) and i.m. (0.5 mg) medication (randomized crossover), -multiple oral dosing (5 mg on trial days 1, 2, 3, 5, 7, 9, 11 and 13), -multiple oral dosing (5 mg on trial days 5 to 14) with initial i.m. loading doses (0.5 mg i.m. on trial days 1, 2 and 4). The multiple dosing schemes were chosen as they are both frequently used in clinical practice. The results of the AChE inhibition after Ubretid can be summarized as follows: repeated Ubretid administration as used in this trial did not lead to a cumulation of AChE inhibition. Statistical testing (page test) of maximum AChE inhibition on the last medication days gave no indication of an increased AChE inhibition towards the end of treatment. Compared with the i.m. administration, the Ubretid tablet had a bioavailability of 2.2 +/- 1.1% (mean +/- STD).

Indirect stimulation of hemopoiesis by hemoregulatory peptide (HP5b) dimer in murine long-term bone marrow cultures.

The hemoregulatory dipeptide (pEEDCK)2 was shown to stimulate production of a synergistic activity (SA) in 6- to 8-week-old primary stromal cell cultures. The SA increased colony formation by murine bone marrow cells (approximately 50% above control levels) in cultures stimulated by optimal concentrations of L-cell-derived macrophage colony-stimulating factor (M-CSF). An increased number of granulocyte-macrophage colony-forming cells (GM-CFC) was also observed in long-term bone marrow cell cultures following daily administration of dipeptide for 5 days. The increase in GM-CFC was approximately 90% above control as assessed by colony formation in soft agar and coincided with SA production. It appears that the dipeptide augments the production of myeloid progenitor cells through an indirect mechanism mediated by accessory cells.

Biosynthesis of S-methyl-N-oleoylmercaptoethylamide from oleoyl coenzyme A and S-adenosylmethionine.

Addition of oleoyl-CoA to incubations containing rat lung membranes and S-adenosyl [methyl-3H]methionine resulted in the formation of a previously unidentified nonpolar methylated lipid. The product was formed enzymatically, with an apparent Km for S-adenosylmethionine (AdoMet) of about 0.3 microM and half-maximal activity using about 0.1 mM oleoyl-CoA. Activity was highest in microsomes but present in other membranous fractions, including plasma membranes from mature human erythrocytes. Intact red blood cells formed the nonpolar methylated lipid intracellularly upon incubation with [methyl-3H]methionine and oleoyl-CoA. Product formation differed among membranes from various tissues. The nonpolar methylated lipid was analyzed by TLC, high performance liquid chromatography, and gas chromatography with radiodetection. It was identified as S-methyl-N-oleoylmercaptoethylamide by gas chromatography-mass spectrometry. Products obtained from oleoyl-CoA or palmitoyl-CoA, incubated with nonradioactive or [methyl-14C]AdoMet, were compared using electron impact and/or chemical ionization mass spectrometry. Inferred structures were confirmed using authentic standards. The methylated product was apparently formed by tissue as follows: (a) cleavage of oleoyl-CoA by an amidase to form S-oleoylmercaptoethylamine; (b) spontaneous rearrangement to form N-oleoylmercaptoethylamide; and (c) enzymatic methylation of the free thiol by AdoMet. Participation of the amidase was suggested by the biosynthesis of the amide (free thiol) using [1-14C]oleoyl-CoA.

Stimulation of fatty acid methylation in human red cell membranes by phospholipase A2 activation.

Nonpolar methylated products comprise approximately 50% of the radioactive material extractable into chloroform/methanol after incubation of human red cell membranes with S-[methyl-3H]adenosylmethionine. One of these nonpolar products is fatty acid methyl ester. The enzyme which synthesizes fatty acid methyl ester had an apparent Km for S-adenosylmethionine of about 0.6 micro M and a Vmax of about 0.6 pmol/mg protein per 30 min. Half-maximal activity was achieved upon addition of about 20 micro M sodium oleate. Of the fatty acids tested, sodium oleate increased activity most effectively (6-fold) and arachidonic acid was ineffective. Evidence indicated that fatty acid methylation takes place on the cytoplasmic side of the plasma membrane. The reaction was demonstrable in intact cells incubated with [methyl-3H]methionine, and increased upon addition of sodium oleate. Incubation of intact cells with melittin, a potent membrane phospholipase A2 activator from bee venom, increased fatty acid methylation several-fold. Fatty acid methylation appears to be one of the consequences of phospholipase A2 action in plasma membranes.