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In an attempt to define myosin heavy chain (MHC) gene organization and expression in adult human skeletal muscle, we have isolated and characterized genomic sequences corresponding to different human sarcomeric MHC genes (1). In this report, we present the complete DNA sequence of two different adult human skeletal muscle MHC cDNA clones, one of which encodes the entire light meromyosin (LMM) segment of MHC and represents the longest described MHC cDNA sequence. Additionally, both clones provide new sequence data from a 228 amino acid segment of the MHC tail for which no protein or DNA sequence has been previously available. One clone encodes a "fast" form of skeletal muscle MHC while the other clone most closely resembles a MHC form described in rat cardiac ventricles. We show that the 3' untranslated region of skeletal MHC cDNAs are homologous from widely separated species as are cardiac MHC cDNAs. However, there is no homology between the 3' untranslated region of cardiac and skeletal muscle MHCs. Isotype-specific preservation of MHC 3' untranslated sequences during evolution suggests a functional role for these regions.

The heptapeptide methyl ester Phe-Asn-Glu-Asn-Met-Ala-Tyr-OMe covering the amino acid sequence of the region 213-219 of Escherichia Coli K88 ad protein fimbriae is synthesized using N alpha-t-butyloxycarbonyl-protection and benzyl groups for side-chain-protection. All condensation reactions are performed in 84-97% yield by preactivation of the protected amino acids by dicyclohexylcarbodiimide (DCC) and 1-hydroxybenzotriazole (HOBt), and reaction of the resulting active ester with amine in the presence of 4-methylmorpholine (NMM). A mechanism is proposed for the nitrile formation in the side-chain of activated asparagine, and the suppression of this side-reaction is investigated. The repetitive deprotection is performed in a mixture of trifluoroacetic acid (TFA), phenol and p-cresol to give the TFA salts in virtually quantitatively yields. The final deprotection of the heptapeptide is carried out in a mixture of 25% hydrogen fluoride (HF) and dimethyl sulfide (DMS) in an overall yield of 48%. The serological and conformational properties of the synthetic peptide are under investigation.

The heptapeptide Tyr-Arg-Glu-Asp-Met-Glu-Tyr-OMe, spanning region 213-219 of Escherichia coli K88 ab protein fimbriae, was synthesized with an overall yield of 37% using dicyclohexylcarbodiimide (DCC) and 1-hydroxybenzotriazole (HOBt) preactivation in all condensation reactions. The C-terminal was protected as the methyl ester. The protection scheme of N alpha-tert-butyloxycarbonyl-(Boc) and benzyl-(Bzl) or benzyloxycarbonyl (Z) groups for side chain protection was found to be orthogonal when a mixture of trifluoroacetic acid (TFA), phenol (PhOH) and p-cresol (CrOH) was used for repetitive deprotection. The final deprotection of Boc-Tyr(Bzl)-Arg(Z2)-Glu(Bzl)-Asp(Bzl)-Met-Glu(Bzl+ ++)-Tyr(Bzl)-OMe (17) was accomplished in 80% yield by prolonged treatment with hydrogen fluoride, dimethyl sulfide, p-cresol and p-thiocresol. The BSA-linked synthetic peptide was used in immunisation experiments on rabbits.

Antigenic variants of pseudorabies virus (PRV) containing mutations in a viral glycoprotein with a molecular weight of 82,000 (gIII) were isolated by selecting for resistance to a complement-dependent neutralizing monoclonal antibody (MCA82-2) directed against gIII. These mutants were completely resistant to neutralization with MCA82-2 in the presence of complement. Two mutants selected for further studies either did not express gIII or expressed an improperly processed form of the glycoprotein. The mutations were also associated with an altered plaque morphology (syncytium formation). The gIII gene was mapped by marker rescue of a gIII- mutant with cloned restriction enzyme fragments to the long unique region of the PRV genome between 0.376 and 0.383 map units. This corresponds to the map location of a glycoprotein described by Robbins et al. (J. Mol. Appl. Gen. 2:485-496, 1984). Since gIII is nonessential for viral replication in cell culture and has several other characteristics in common with the herpes simplex virus glycoprotein gC, gIII may represent the PRV equivalent to herpes simplex virus gC.

Mature polymorphonuclear leukocytes (PMN) are capable of mediating phorbol myristate acetate (PMA)- and antibody (A)-dependent cellular cytotoxicity (DCC) against ox red blood cells (ORBC) by using oxidative means. The purpose of the present study was to investigate the acquirement of these cytotoxic functions during PMN ontogeny, using the promyelocytic HL-60 cell line as a model for PMN differentiation. HL-60 cells were induced to differentiate along the PMN pathway by exposure to dimethyl sulfoxide (DMSO). Uninduced HL-60 cells were found to be completely devoid of PMA-DCC and ADCC activity. DMSO-induced cells progressively acquired the capacity to kill ORBC and to undergo the activation of oxidative metabolic burst when triggered by PMA. Despite approximately 40% of them also were capable of binding IgG-sensitized ORBC, no ADCC activity and respiratory burst activation was observed: this finding indicates that maturing HL-60 cells require a more complete maturation than that induced by DMSO to actually exert ADCC. Together the results suggest that: a. the acquirement of both PMA-DCC and ADCC potential is a post-promyelocytic event; b. the cytotoxicity activating stimuli, PMA and IgG-coated targets, follow different post-receptor transductional pathways to trigger the effector cell lytic systems: only the PMA receptor-linked pathway develops during DMSO-driven differentiation of HL-60 cells.

Heparin preparations with different anticoagulant and antilipemic (fat-clearing) activities were oxidized with periodate under conditions of cleavage of all the C(2)-C(3) bonds of non-sulfated uronic acid residues, while preserving the original molecular weight of the polysaccharide. Periodate-oxidised heparins (oxyheparins, O-HEP) and the corresponding borohydride-reduced products (reduced oxyheparins, RO-HEP) were compared with the original heparins for their content in trisulfated disaccharide sequences (as determined by 13C-nuclear magnetic resonance) and in active sites for antithrombin-III (as determined indirectly by affinity chromatography), and for their anticoagulant and antilipemic (lipoprotein lipase-releasing) activities. The drop of anticoagulant activity induced by periodate oxidation was paralleled by a substantial decrease of affinity for antithrombin, and is thought to arise from glycol splitting at the level of the D-glucuronic acid residue that is part of the active site for antithrombin. The trisulfated disaccharide sequences and the associated antilipemic activities were substantially unaffected by periodate oxidation. The residual anticoagulant activity of periodate-oxidized heparins obtained from preparations - such as those from beef lung - rich in trisulfated disaccharide sequences is discussed in terms of the influence of charge density on heparin-protease interactions not mediated by antithrombin.

Comparative studies in early treatment for DIC caused by circulatory arrest were carried out on three groups of dogs during 300 min from the recovery of circulatory arrest: a heparin group, a FOY (gabexate mesilate) group, and a FUT-175 (nafamostat mesilate) group. The parameters employed were platelet count, prothrombin time (PT), activated partial prothrombin time (APTT), fibrinogen, antithrombin-III (AT-III), and fibrin or fibrinogen degradation products (FDP). In the heparin group, there was less of a drop in the platelet count and the level of AT-III than in the control group, but the FDP levels were the same as in the control group. The PT and APTT remained within normal limits in the FOY group and no decrease was observed in either platelet count or AT-III levels. In addition, FDP levels were kept within normal limits. In the FUT-175 group, prolongation of APTT, no decline in the platelet count and AT-III levels, and normal levels of FDP were observed. The results of these experiments indicate the importance of early treatment for DIC. Judging from the parameters, better results were obtained in the FOY and FUT-175 group than in the heparin group.

In 1969 McCord and Fridovich discovered superoxide dismutase, which converts the oxygen free radical O(2) (-) to hydrogen peroxide H(2)O(2). In the presence of excess O(2) (-), H(2)O(2) may then undergo further reduction to the highly toxic hydroxyl radical, OH(*). Since the description of this enzymatic process, there has been explosive growth in related biochemical research, which has now percolated through to clinical investigation. The hypoxanthine-xanthine oxidase system originally used as a radical production model has a close counterpart in the ischemia-reperfusion phenomenon purported to cause diseases of heart, brain and gastrointestinal tract, and free radicals are now known to have a critical role in postphagocytic bacterial killing. Prototypic deficiency diseases such as chronic granulomatous disease are now recognized. Some evidence indicates that excess states such as perhaps Batten's disease also occur, and environmental influences such as selenium and vitamin E deficiency may augment free radical levels. Many disorders including microvasculopathies, noncardiogenic pulmonary edema, glomerulopathies and radiation damage may owe part of their proximate pathogenesis to free radicals. Control of tissue free radical levels is now pharmacologically feasible and perhaps justified for specific diseases.

In cervical cancers, the estrogen receptor (ER) and progestin receptor (PR) were characterized in the cytosol protein. Receptor levels were determined in cytosol, nuclear KCl extract, and nuclear KCl unextractable fraction in various cervical cancer tissues. The androgen receptor (AR) was also similarly characterized. In the cytosol, the estrogen (E2)-ER complex and the promegesterone (R5020)-PR complex were sedimented at approximately the 5S and 8S or the 4S and 8S regions, respectively, by 5-20% linear sucrose gradient centrifugation. A steroid specificity study showed the presence of an estrogen-specific and a progestin-specific binder. The dissociation constant of the specific binder for estrogen (ER), progestin (PR), or androgen (AR) was approximately 10(-10) to 10(-9)M in cytosol, nuclear KCl, and nuclear KCl unextractable fraction in detected cases. Levels were determined by Scatchard analyses, using the dextran-coated charcoal (DCC) adsorption method for the former two and the washing method for the latter one. ER, PR, or AR was detected in some cases of determined cervical cancers (27 cases of squamous cell carcinoma, and 7 cases of adenocarcinoma); regardless of the histological types, ER was detected in almost all cases. PR was not detected in the given cases of cervical adenocarcinoma. It is suggested that estrogen stimulation of PR synthesis in cervical adenocarcinoma may be damaged.

A rapid and simple one-pot method for O-sulfation of nonsulfated cholecystokinin octapeptide (CCK-8) was developed using sulfuric acid and dicyclohexylcarbodiimide (DCC) without protection of the amino acid side chains. The extent of sulfation was increased with increasing the amount of reactants, sulfuric acid, and DCC, and reached maximum (40%) with fourfold molar excess of sulfuric acid and 40-fold molar excess of DCC. The excess of nonsulfated peptide inhibited the sulfation. The sulfation product was purified by HPLC or TLC to give a pure sulfated substance which showed exactly the same behavior as that of an authentic O-sulfated CCK-8 on HPLC or TLC. The purified sulfated peptide was active in stimulating amylase secretion from rat pancreatic fragments, and amino acid analysis showed that the tyrosine residue in the peptide existed in O-sulfated form. Sulfation with [35S]sulfuric acid-DCC produced a radioactive substance, from which O-[35S]sulfated CCK-8 could be easily purified by two-dimensional TLC.