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The undecapeptide substance P is thought to mediate both vasodilatation and augmented vascular permeability when released from sensory nerve endings in the skin. Substance P also induces mast cell degranulation in vitro or in vivo. However, the extent to which substance P-induced changes in vascular permeability are mast cell-dependent is unclear. We investigated this issue by injecting substance P and certain related peptides (substance P1-4, substance P4-11) into the skin of genetically mast cell-deficient WBB6F1-W/W or WCB6F1- SI/SId mice the congenic normal (+/+) mice, and W/W mice which had undergone selective local repair of their mast cell deficiency by intradermal injection of IL-3-dependent mast cells generated in vitro from the bone marrow cells of the congenic +/+ mice. Substance P induced significant augmentation of vascular permeability and significant cutaneous swelling when injected into normal mice at doses as low as 2 pmol i.d. Substance P also induced granulocyte infiltration, although the infiltrate were modest and were seen at doses of peptide from 5 to more than 20-fold higher than those required for induction of tissue swelling. The effects of substance P on tissue swelling, vascular permeability, and granulocyte infiltration were virtually entirely mast cell dependent. By contrast, substance P1-4 was inactive in our assays at 25 nmol/site, and substance P4-11 induced modest augmentation of vascular permeability, which was at least in part mast cell independent.

We studied the status of estrogen (ER) and progesterone (PR) receptors in meningiomas removed from 52 patients, comparing dextran-coated charcoal (DCC), nuclear binding (NB), and immunoperoxidase (IP) assays. Each of the assays was performed independently by investigators well-experienced with these assays. The NB assay is a new assay that measures functional steroid receptors--that is, the activation of the receptor and its binding to the nucleus. The assay is very sensitive and requires a relatively small amount of tissue as compared with the DCC assay. In agreement with data from other studies. PR were detected in most meningiomas by all 3 methods: in 69% of the cases by NB, in 76% by DCC, and in 89% by IP. ER were detected in only a few cases: in 33% by NB, in 2% by DCC, and in none by the IP assay. The agreement for PR sites was 62% for all 3 assays; it was 66% between the NB and DCC assays, 67% between the NB and IP assays, and 86% between the DCC and IP assays. Of 26 cases that were positive by the DCC assay, 6 (23%) were negative by NB. The overall agreement for all three ER assays was 65%. The data suggest that the majority of meningiomas contain high-affinity receptors for progesterone, that estrogen receptors are present in only a few meningiomas, and that some of these estrogen and progesterone receptors appear to be functional.

The estrogen receptor content in 100 female breast carcinomas was determined using the biochemical dextran-coated charcoal (DCC) method and the monoclonal estrogen receptor immunocytochemical assay (ER-ICA). Great care was taken to make procedures optimal in order to minimize preanalytical errors. A statistically highly significant correlation was found between the results of the two methods, which were qualitatively in accordance in 91 cases, while nine gave a negative ER-ICA but a weak positive DCC result. The quantitative correlations were also statistically significant but varied considerably in individual cases. Under optimal conditions, ER-ICA provides results similar to the DCC method, but it is still uncertain if it can stand alone in routine diagnosis.

Fundamental aspects of iron metabolism relate to the dynamic processes of metal plasma transport as well as cell storage and efflux. Transferrin not only carries iron in the plasma but also delivers it to the various cells by binding to a diffuse specific cell receptor; it also acts by chelating cell iron. Ferritin co-operates by storing iron in the cell. By a still unknown regulatory mechanism, iron, from the ferritin pool, is redistributed in the cell to a cytosolic, easily chelatable, "transit" pool or to a degradative lysosomal hemosiderin pool from which it is slowly released outside the cell. Iron overload, such as that typical of hyperhemolysis or hemochromatosis, profoundly impairs its metabolism by saturating and/or altering transferrin and ferritin, by freeing iron from any regulated transport, thus allowing parenchymal deposition and damage. An important aspect still awaiting clarification relates to the different storage of excess iron in the parenchymal cells, as in hemochromatosis, or in the reticuloendothelial system such as in hemosiderosis. Studies using cellular models attempt to evaluate such differences in terms of altered properties of the iron proteins or their cell receptors, and of the different cell responsivity to non-transferrin iron. In the expectation of better knowledge, attention should be concentrated, from a clinical standpoint, on precise assessment of iron deposits in the tissues with the aim of preventing its excessive accumulation and parenchymal damage. In hemochromatosis, the risk of iron overload is evaluated by HLA typing.

To investigate the physiologic function of heparin cofactor II (HCII), endothelial cells from human umbilical vein were incubated in vitro for 20 min with 0.5 NIH U/ml thrombin in the presence of HCII or antithrombin III (ATIII), and prostacyclin production determined by radioimmunoassay for 6-keto-prostaglandin F1 alpha, the stable metabolite of prostacyclin. Although ATIII at 20 mInh.U/ml slightly but significantly inhibited thrombin-induced prostacyclin production, neither unfractionated heparin (UFH) nor low molecular weight heparin (LMWH) at 1 U/ml accelerated the inhibitory effect of ATIII. HCII at 10 and 20 mInh.U/ml did not decrease thrombin stimulation of prostacyclin production in the presence or absence of UFH or LMWH. However, HCII caused a marked decrease in the thrombin-stimulated prostacyclin prostacyclin production in the presence of 2 mg/ml dermatan sulfate (DS). The significant inhibition by HCII occurred when the DS concentrations were 0.2 microgram/ml and higher. From these results we suggest that HCII may prevent a prostacyclin-induced inhibition of platelet aggregation for hemostasis when plasma is exposed to vascular smooth muscle cells or fibroblasts which synthesize a significant amount of DS.

The postsynaptic actions of acetylcholine, adenosine, gamma-aminobutyric acid, histamine, norepinephrine, and serotonin were analyzed in human cortical pyramidal cells maintained in vitro. The actions of these six putative neurotransmitters converged onto three distinct potassium currents. Application of acetylcholine, histamine, norepinephrine, or serotonin all increased spiking by reducing spike-frequency adaptation, in part by reducing the current that underlies the slow after hyperpolarization. In addition, application of muscarinic receptor agonists to all neurons or of serotonin to middle-layer cells substantially reduced or blocked the M-current (a K+ current that is voltage and time dependent). Inhibition of neuronal firing was elicited by adenosine, baclofen (a gamma-aminobutyric acid type B receptor agonist), or serotonin and appeared to be due to an increase in the same potassium current by all three agents. These data reveal that individual neuronal currents in the human cerebral cortex are under the control of several putative neurotransmitters and that each neurotransmitter may exhibit more than one postsynaptic action. The specific anatomical connections of these various neurotransmitter systems, as well as their heterogeneous distribution of postsynaptic receptors and responses, allows each to make a specific contribution to the modulation of cortical activity.

To investigate side reactions during the activation of side chain unprotected asparagine in Fmoc-solid phase peptide synthesis the peptide Met-Lys-Asn-Val-Pro-Glu-Pro-Ser was synthesized using different coupling conditions for introduction of the asparagine residue. Asparagine was activated by DCC/HOBt, BOP (Castro's reagent) or introduced as the pentafluorophenyl ester. The resulting peptide products were analyzed by HPLC, mass spectrometry and Edman degradation. In the crude products varying amounts of beta-cyano alanine were found, which had been formed by dehydration of the side chain amide during carboxyl activation of Fmoc-asparagine. A homogeneous peptide was obtained by using either side chain protected asparagine derivatives with BOP mediated activation or by coupling of Fmoc-Asn-OPfp. Fmoc-Asn(Mbh)-OH and Fmoc-Asn(Tmob)-OH were coupled rapidly and without side reactions with BOP. For the side chain protected derivatives the coupling was as fast as that of other Fmoc-amino acid derivatives, whereas couplings of Fmoc-Asn-OH proceed more slowly. However, during acidolytic cleavage both protection groups, Mbh and Tmob, generate carbonium ions which readily alkylate tryptophan residues in a peptide. Tryptophan modification was examined using the model peptide Asn-Trp-Asn-Val-Pro-Glu-Pro-Ser. Alkylation could be reduced by addition of scavengers to the TFA during cleavage and side chain deprotection. A homogeneous peptide containing both, asparagine and tryptophan, was obtained only by coupling of Fmoc-Asn-OPfp.

Pseudomonas putida F1 contains a multicomponent enzyme system, toluene dioxygenase, that converts toluene and a variety of substituted benzenes to cis-dihydrodiols by the addition of one molecule of molecular oxygen. Toluene-grown cells of P. putida F1 also catalyze the monohydroxylation of phenols to the corresponding catechols by an unknown mechanism. Respirometric studies with washed cells revealed similar enzyme induction patterns in cells grown on toluene or phenol. Induction of toluene dioxygenase and subsequent enzymes for catechol oxidation allowed growth on phenol. Tests with specific mutants of P. putida F1 indicated that the ability to hydroxylate phenols was only expressed in cells that contained an active toluene dioxygenase enzyme system. 18O2 experiments indicated that the overall reaction involved the incorporation of only one atom of oxygen in the catechol, which suggests either a monooxygenase mechanism or a dioxygenase reaction with subsequent specific elimination of water.

Parameters of nonspecific defence--complement protein function, circulating immune complex level, neutrophil phagocytic activity, completeness of the phagocytic reaction, C3c level, were evaluated in 162 carriers of hemochromatosis gene (114 homozygotes and 48 heterozygotes with respect to hereditary hemochromatosis) aged from 7 to 64 years. The analysis of the results obtained has permitted a conclusion that the suppression of the complement system function, according to the classic or alternative type, is associated with the disease pathogenesis--hyperferremia, and is caused by the damage of the immunocompetent organs and cells synthesizing individual proteins of the complement, as well as by infectious complications leading to hypocomplementemia with respect to separate components.

We studied the effects of FR-860 on coagulative and fibrinolytic activities in human plasma compared to conventional unfractionated heparin (UF-heparin). Both FR-860 and UF-heparin dose-dependently prolonged the recalcification time, activated partial thromboplastin time, prothrombin time, factor Xa (F.Xa) clotting time and thrombin time. These effects of FR-860 were weaker than that of UF-heparin. FR-860 showed equipotent efficacy on the anti-F.Xa activity, and weak antithrombin activity compared to UF-heparin. FR-860 had no effects on the activity of ATIII and fibrinolytic activity. UF-heparin shortened the urokinase-activated euglobulin lysis time and showed antiplasmin activity, but did not influence the activities of ATIII, plasminogen and alpha 2-plasmin inhibitor. UF-heparin decreased the fibrinogen level at higher doses. These efficacies of FR-860 were weaker than that of UF-heparin. These results suggest that FR-860 is more efficient and lower in bleeding risk than UF-heparin in clinical use.

We tested the hypothesis that the hepatic siderosis that characterizes sporadic porphyria cutanea tarda is due to the presence of HLA-linked hemochromatosis alleles. We studied 21 probands with sporadic porphyria cutanea tarda and 135 of their relatives by determining HLA haplotypes and measuring transferrin saturation and serum ferritin concentration. Liver biopsies were performed in all probands and in relatives when appropriate. Seventeen pedigrees were available and were studied by both likelihood analysis and by a gene counting method. We estimated that 10 of the 17 probands with available living relatives possessed at least one hemochromatosis allele. Thirteen of the 21 probands (62%) possessed at least one HLA-A3 alloantigen. Eighteen of 69 relatives who shared an HLA haplotype with a proband (26%) had an elevation of transferrin saturation or serum ferritin concentration. Only one first-degree relative not sharing an HLA haplotype with a proband had an elevated transferrin saturation or serum ferritin concentration. These findings indicate that HLA-linked hemochromatosis alleles are far more common in patients with sporadic porphyria cutanea tarda than in individuals in the general population and may be responsible for the hepatic siderosis associated with most cases of sporadic porphyria cutanea tarda.

The genes of seven structural mutants of antithrombin III (ATIII), presenting either defective serine protease reactivity or abnormal heparin binding, were analyzed. The polymerase chain reaction (PCR) was used to amplify the corresponding gene exon and the mutation was identified by either dot blot analysis using a battery of allele-specific oligonucleotide probes or sequencing. Variants Paris and Paris 2 were identified as Arg 47 Cys mutations, and Clichy, Clichy 2, and Franconville were found to be Pro 41 Leu mutations. All five are heparin binding-site variants. ATIII Avranches is an Arg 393 His mutation and ATIII Charleville is an Ala 384 Pro mutation. These two mutations impair the reactive site of the molecule. ATIII Charleville is a new mutation of the reactive center, as predicted by previous biochemical data. The position of this new mutation, together with the other previously described mutations of the reactive center, sheds light on the molecular function of this site in inhibiting thrombin. Finally, genomic amplification by PCR is a powerful technique for the fast identification of antithrombin III mutations and their homozygous/heterozygous status, and should be useful for predicting thrombotic risk.

Plasma antithrombin-III activity was quantitated in plasma samples obtained from 165 clinically normal horses 3 years old or younger. In the horses as a group, antithrombin-III activity ranged from 63 to 131% of a species-specific reference plasma. Thoroughbred horses had significantly higher antithrombin-III activity (103.3 +/- 18.3; mean +/- SD) than did Standardbred horses (92.3 +/- 14.2). The plasma antithrombin-III activities were significantly lower in horses younger than 16 months old when compared with those in more mature horses (3 years old). There was no statistically significant gender-related effect on plasma antithrombin-III activity. Breed and age factors should therefore be taken into consideration when interpreting plasma antithrombin-III activity in horses.