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In a longitudinal study the plasma levels of antithrombin-III, alpha 2-macroglobulin, alpha 2-antiplasmin, histidine-rich glycoprotein, and protein C were followed in two groups of patients with acute myocardial infarction (AMI), one with and one without deep vein thrombosis (DVT). None of the sequentially studied periods revealed significant differences between the two groups of patients. However, small but consistently higher levels of histidine-rich glycoprotein in patients with DVT suggested the existence among patients submitted for myocardial infarction of a subgroup with increased thrombophilic potential. It was concluded that the inhibitors studied are of little value as possible indicators of the presence of DVT at early stages of the disease when clinical signs are absent and when antithrombotic prophylaxis should preferably be initiated.

Antithrombin III (ATIII) plays an integral role in the coagulation system by inhibiting thrombin and several other activated clotting factors. Inherited deficiency of ATIII is quite common and can result in life-threatening thrombotic complications. In order to understand the basis of ATIII deficiency, we have isolated and characterized the normal human ATIII gene from a recombinant Charon 4A bacteriophage genomic library. The ATIII gene contains six exons and five introns distributed over approximately 19 kilobases of DNA. The positions of introns in the ATIII gene were compared with other members of the serine protease inhibitor family which share 17-31% amino acid homology. When aligned to achieve maximal protein homology, only one of the ATIII introns corresponded to the four introns of rat angiotensinogen or human alpha 1-antitrypsin. Similarly, only one ATIII intron was homologous to the seven introns of chicken ovalbumin. We present two testable models to explain the discrepancy in intron positions among members of the serine protease inhibitor superfamily of genes.

Phospholipids are synthesized and concomitantly inserted on the cytoplasmic surface of the endoplasmic reticulum. Assembly of the phospholipid bilayer requires translocation to the lumenal monolayer. The hypothesis that rat liver microsomes contain a protein transporter, or "flippase," for phosphatidylcholine was tested by measuring the transport of sn-1,2,-dibutyroylphosphatidylcholine (diC4PC). This homolog retains the polar head group, the portion of the phospholipid unable to undergo spontaneous transmembrane movement in vesicles, and its water solubility permits application of standard transport methods. DiC4PC entered the lumenal compartment of microsomal vesicles. Transport was saturable and was dependent on time, amount of microsomes, and an intact permeability barrier. DiC4PC transport was inhibited by structural analogs (but not by sn-2,3-diC4PC) and by treatment of microsomes with proteases, N-ethylmaleimide, and trinitrobenzenesulfonic acid. These data suggest that the transmicrosomal movement of diC4PC is protein mediated. DiC4PC was not transported across PC vesicles or red cell membranes, where PC translocation is slow.

As a result of the use of synthetic luteinizing-hormone-releasing hormone (LHRH) (and its analogs), significant advances in modern clinical practice are being realized. We studied the use of LHRH as a test for pituitary reserve for gonadotropin secretion in different reproductive disorders. Synthetic LHRH was used as a diagnostic test for discriminating pituitary from hypothalamic disorders. After appropriate LHRH priming of the pituitary, LHRH was used to document hypothalamic dysfunction in patients with Kallmann's syndrome who had normal gonadotropin responsiveness to LHRH. The gonadotropin responsiveness to 100 micrograms of LHRH was impaired or absent in patients with panhypopituitarism, craniopharyngiomas, hemochromatosis and acromegaly accompanied by abnormal lactation. In women with gonadal dysgenesis, the absence of gonadal steroid feedback exacerbated the pituitary responsiveness to LHRH. Women with hyperprolactinemia are also known to have a blunted gonadotropin response to endogenous and exogenous LHRH. An experimental rat model was developed in our laboratory to study the site of prolactin action on gonadotropin secretion. LHRH challenge tests during perphenazine-induced hyperprolactinemia in rats indicated that prolactin may decrease pituitary sensitivity to LHRH. Additional experiments indicated that the increased progesterone produced in these hyperprolactinemic (pseudopregnant) rats was probably responsible for the decreased pituitary responsiveness to LHRH. Further studies will be necessary to determine whether prolactin, which can alter ovarian steroidogenesis in vitro, interferes with ovulation directly in addition to affecting the hypothalamic-pituitary axis.

Peptides corresponding to sequences 122-133, 136-147, and 154-164 of the heavy chain of hemagglutinin of the A/Aichi/2/68 (H3N2) influenza virus have been synthesized by stepwise elongation of the peptide chain with Boc-amino acid activated esters or by condensation of peptide blocks by DCC/HOBt-method. A coloured C-protecting group, 2-[4-(phenylazo)-benzylsulfonyl]ethyl (PSE), was used, which is convenient in purification of synthetic peptides. After removal of terminal N-and C-protecting groups the side-protecting residues were cleaved off with 1 M trifluoromethanesulfonic acid in trifluoroacetic acid containing 10% thioanisole. Crude products were purified by preparative reversed-phase liquid chromatography. Synthesized peptides were conjugated with BSA.

A novel synthesis of thymosin alpha 1 by classical methods using seven tert.-butyl side chain protected fragments is described. Optimum conditions were found for the final DCC/HOBt coupling of the two key intermediates; decapeptide and octadecapeptide. Thymosin alpha 1 was purified by two stages of preparative HPLC (partial purification with C8 and final purification with C18 reverse phase silica gel) to give a 30% overall yield for the final four stages of synthesis (including catalytic hydrogenation of octadecapeptide, coupling, deprotection and purification). The product was shown to be homogeneous by thin-layer and paper high voltage electrophoresis, isoelectric focusing analysis, thin-layer chromatography and high performance liquid chromatography. Amino acid analysis, optical rotation, 1H-n.m.r. spectroscopy, FAB mass spectroscopy and peptide mapping after tryptic digestion confirmed the structure of thymosin alpha 1. Three minor stereoisomer contaminants were isolated by HPLC and characterized as [D-Lys14]-thymosin alpha 1, [D-Lys17]-thymosin alpha 1 and [D-Ala3]-thymosin alpha 1 resulting from racemization at Lys14, Lys17 and Ala3 during the coupling of the fragments. A final contaminant, isolated by HPLC, was characterized as N alpha-isobutyloxycarbonyl-thymosin alpha 1 (15-28), which results from "wrong way opening" of an activated mixed anhydride.