We examined the association between the expression profiles of genes of the activin signalling pathway and ovarian follicular dominance in cattle. In monovular species such as cattle, one ovarian follicle of a cohort is selected to become dominant, whereas all others (i.e. the subordinate follicles) eventually succumb to apoptosis. We showed that Inhibin-betaA, coding for the betaA chain found in the A isoforms of activin, Inh-alpha coding for the inhibin-specific alpha chain, and the activin antagonist follistatin were expressed at higher levels in dominant follicle granulosa cells from Day 3.5 (ovulation=Day 0). Before selection, Inh-betaA but not Inh-alpha was significantly correlated with potential dominant follicles, as defined by high aromatase expression and follicular fluid estrogen concentrations. Follistatin expression marked the largest follicles at Day 1.5, but displayed large variation in levels among cows. The third inhibin gene, Inh-betaB, could only be detected at very low levels from Day 7 and thus was unlikely to play a prominent role in activin/inhibin signalling in cattle during these stages. There was a decrease in activin tone (P=0.07) specifically in the aromatase-high/dominant follicles, as measured by the ratio of Inh-betaA to Inh-alpha plus follistatin transcripts between Days 1.5 and 7. Messenger RNA for both activin type II receptors and the nuclear effector Smad2 were detected in granulosa cells, consistent with an autocrine role for activin signalling. Additionally, expression of the putative activin target genes Smad2 and FSHreceptor were, respectively, either strongly (P<0.001) or weakly (P=0.09) associated with dominant follicles.

Hereditary angioedema (HAE), a rare autosomal dominant genetic disorder, is caused by a deficiency in functional C1 esterase inhibitor (C1-INH). This potentially life-threatening condition manifests as recurrent attacks of subcutaneous and submucosal swelling of the skin, gastrointestinal tract and larynx. The management of HAE includes treatment of acute episodes, short-term prophylaxis in preparation for exposure to known triggers and long-term prophylaxis to decrease the incidence and severity of HAE attacks. Four products are approved in the USA for the treatment of acute attacks of HAE, including one human plasma-derived C1-INH therapy, a recombinant human C1-INH product (rhC1-INH), a plasma kallikrein inhibitor and a bradykinin B2 receptor antagonist. In addition, one human plasma-derived C1-INH therapy and danazol are approved for prophylaxis of HAE attacks. rhC1-INH, extracted from the milk of transgenic rabbits, is a glycoprotein of 478 amino acids with an identical amino acid sequence to the endogenous human C1-INH protein. Population pharmacokinetic analysis of rhC1-INH supports an intravenous dosing strategy of 50 U/kg (maximum 4200 U). The safety and efficacy of rhC1-INH in the treatment of acute attacks in patients with HAE were demonstrated in three randomized, double-blind, placebo-controlled studies and two open-label extension studies. In a pilot prophylaxis study, weekly administration of rhC1-INH 50 U/kg for 8 weeks reduced the incidence of HAE attacks and was well tolerated. Administration of rhC1-INH has not been associated with the development of anti-drug antibodies or antibodies to anti-host-related impurities.

Up to 93% of patients with hereditary angioedema (HAE) experience recurrent abdominal pain. Many of these patients, who often present to emergency departments, primary care physicians, general surgeons, or gastroenterologists, are misdiagnosed for years and undergo unnecessary testing and surgical procedures. Making the diagnosis of HAE can be challenging because symptoms and attack locations are often inconsistent from one episode to the next. Abdominal attacks are common and can occur without other attack locations. An early, accurate diagnosis is central to managing HAE. Unexplained abdominal pain, particularly when accompanied by swelling of the face and extremities, suggests the diagnosis of HAE. A family history and radiologic imaging demonstrating edematous bowel also support an HAE diagnosis. Once HAE is suspected, C4 and C1 esterase inhibitor (C1-INH) laboratory studies are usually diagnostic. Patients with HAE may benefit from recently approved specific treatments, including plasma-derived C1-INH or recombinant C1-INH, a bradykinin B2-receptor antagonist, or a kallikrein inhibitor as first-line therapy and solvent/detergent-treated or fresh frozen plasma as second-line therapy for acute episodes. Short-term or long-term prophylaxis with nanofiltered C1-INH or attenuated androgens will prevent or reduce the frequency and severity of episodes. Gastroenterologists can play a critical role in identifying and treating patients with HAE, and should have a high index of suspicion when encountering patients with recurrent, unexplained bouts of abdominal pain. Given the high rate of abdominal attacks in HAE, it is important for gastroenterologists to appropriately diagnose and promptly recognize and treat HAE, or refer patients with HAE to an allergist.

OBJECTIVE

The plasma levels of prekallikrein, alpha-2-macroglobulin and C1-esterase inhibitor (C1 INH) in patients with previous urticarial reaction to contrast media (CM) were compared to those of a group of nonreacting age- and sex-matched controls. This study evaluated the value of these laboratory variables in predicting acute and delayed urticarial-type reactions.

METHODS

The study comprised 44 patients (reactors) with acute (n = 29) or delayed (n = 15) urticaria after administration of CM, and a group of age- and sex-matched controls.

RESULTS

In the reactors, the levels of prekallikrein and alpha-2-macroglobulin were higher (p < 0.0001) and the level of C1 INH lower (p < 0.0001) than those of the controls. The level of prekallikrein decreased with increasing age (p = 0.02) and women had higher values than men (p = 0.0054). The level of alpha-2-macroglobulin was age-dependent (p = 0.006).

CONCLUSIONS

Although high plasma prekallikrein activity, high plasma alpha-2-macroglobulin activity, and low plasma C1 INH activity are associated with urticaria-type reaction to CM, their value in predicting urticarial reaction is limited because prekallikrein is age- and sex-dependent, and alpha-2-macroglobulin is age-dependent.

Hereditary angioedema (HAE) is an autosomal dominant disease resulting from the deficiency of C1 inhibitor (C1-INH), a glycosylated serine protease inhibitor that plays a regulatory role in the complement system (CS), the contact system and the intrinsic coagulation cascade. HAE disease severity is highly variable and may be influenced by genetic polymorphisms as well as by other factors, such as gender hormone-mediated effects. In HAE, the potential inadequate clearance of immune-complexes (IC) in the presence of reduced levels of CS components and in turn an excess of IC in the tissues results in inflammatory damage and release of autoantigens that may trigger an autoimmune response. Occasional reports link HAE with autoimmune conditions and only few studies have been conducted on large patient populations with controversial results. Although several immunoregulatory disorders have been documented, the prevalence of defined autoimmune diseases in patients with HAE remains debated. The occurrence of autoimmune conditions in HAE patients may worsen the disease severity enhancing the complexity of the comprehensive care.

Angioneurotic edema results from acquired or genetic deficiency of C1 inhibitor (C1 INH), a member of the serpin family of protease inhibitors. C1 INH is the only plasma protease inhibitor of activated C1r and C1s, the serine protease subcomponents of the first complement component. It is also the major inhibitor of plasma kallikrein and of coagulation factor XIIa. C1 INH consists of a single polypeptide chain of 478 amino acid residues. It is the most heavily glycosylated plasma protein; a large portion of the carbohydrate is O-linked to serine and threonine residues. Hereditary angioneurotic edema (HANE) occurs in individuals heterozygous for deficiency of C1 INH. Most patients have absolute deficiency of C1 INH (type 1 HANE), while others (15% of kindred) synthesize a dysfunctional C1 INH protein. The molecular genetic defects in the C1 INH gene in both type 1 and type 2 HANE currently are being defined. Acquired angioneurotic edema (AANE) also is of two types. One of these occurs in individuals with B-cell lymphoproliferative disorders (type 1) and the other is characterized by the presence of autoantibodies directed toward the C1 INH molecule.

We describe a rapid assay of C1-esterase inhibitor (C1-inh) activity in plasma. After adding purified C1s serine protease (EC 3.4.21.42) in excess to plasma, we determine the residual C1s activity towards a new chromogenic tripeptide, CH3CO-Lys(CbO)-Gly-Arg-pNA. Optimal conditions include the addition of methylamine (final concentration 0.12 mol/L) to reduce the potential inhibitory capacity of alpha 2-macroglobulin towards C1s and the addition of heparin (final concentration 3000 int. units/L) to enhance the reaction of C1s with C1-inh. The correlation with C1-inh antigen concentrations in plasma was excellent. The estimated interassay CV was 4.3%, whereas the intra-assay CV was 2.0% for activity concentrations within the range of normal individuals (means +/- 2 SD: 70-124%), 1.3% at lower concentrations. The method is more convenient, rapid, and precise than previous methods, and C1-inh activity in plasma can be assessed within 30 min. We found that concentrations of C1-inh in plasma were low during open-heart bypass surgery.

Mast cells contain large amounts of the powerful serine proteinases, tryptase and chymase, of which only chymase can be inactivated by serum protease inhibitors. In this study, 20 patients with psoriasis and a control group of 13 with atopic dermatitis were biopsied for lesional and non-lesional skin specimens. The presence of chymase inhibitor alpha 1-proteinase inhibitor (alpha 1-PI), alpha 1-antichymotrypsin (alpha 1-AC), alpha 2-macroglobulin (alpha 2-MG) and C1-esterase inhibitor (C1-Inh) immunoreactivity in mast cells was verified using the sequential double-staining method. Tryptase- and chymase-positive mast cells were stained enzyme-histochemically. Tryptase-positive mast cells were increased in number in the upper dermis of the psoriatic lesion compared with lesion-free psoriatic skin (308 +/- 109 vs. 100 +/- 29 cells/mm2, respectively, mean +/- SD, p < 0.0005, t-test) while the percentage of mast cells showing chymase activity was decreased (76.8 +/- 22.1% vs. 28.6 +/- 14.4%, p < 0.0005). These findings are consistent with our previous ones. In contrast to the decreased percentage of chymase-positive mast cells, a novel finding was that the percentages of alpha 1-AC+ (86.9 +/- 7.2% vs. 59.5 +/- 12.6%, p < 0.0005), alpha 1-PI+ (72.2 +/- 14.9% vs. 33.4 +/- 18.6%, p < 0.0005) and alpha 2-MG+ (16.8 +/- 7.0% vs. 6.2 +/- 3.5%, p < 0.002) mast cells were significantly higher in the psoriatic lesion with the exception of the percentage of C1-Inh+ mast cells (13.7 +/- 10.0% vs. 11.0 +/- 6.1%, p < 0.7). The localization of these inhibitors in mast cells is not a characteristic feature of psoriasis, since mast cells in atopic dermatitis skin also showed immunoreactivity though in slightly lower percentages. Previously, we have shown that MCTC (tryptase+, chymase+) mast cells increase in number in the psoriatic lesion but chymase becomes inactive. The results of this study show again the decreased chymase activity, which could be due to increased levels of its inhibitors (alpha 1-AC, alpha 1-PI and alpha 2-MG) in the same mast cells. Thus, active tryptase could promote inflammation but chymase seems not to be an important mediator in the pathomechanism of psoriasis.

Inhibin alpha is one of the candidate genes that control the ovulation in poultry. To study the genetic effects of inhibin alpha on apoptosis and proliferation of goose granulosa cells cultured in vitro, two RNA interference (RNAi) expression vectors, psiRNA-INH alpha 1 and psiRNA-INH alpha 2, were constructed to knock down inhibin alpha gene expression. After 48 h of transfection, the efficiency of these two RNAi expression vectors was examined by fluorescence microscopy. Meanwhile, inhibin protein expression levels, apoptosis indexes (AI) and proliferation indexes (PI) of granulosa cells were analyzed by flow cytometry. In addition, the supernatants were collected to assay the concentrations of estrogen (E2) and progesterone (P) by radioimmunoassay. The results showed that the expression level of inhibin alpha in the RNAi group were decreased 30%-40% than those in the control groups (P < 0.05) and the apoptosis indexes and proliferation indexes in the RNAi groups were significantly higher than those in the control groups (P < 0.05). However, the E2 concentrations in the RNAi groups were lower than those in the control groups (P < 0.05). These results indicate that inhibin alpha has antagonistic effect on granulosa cell apoptosis.

The biological function of inhibin-alpha subunit (INH alpha) in prostate cancer (PCa) is currently unclear. A recent study associated elevated levels of INH alpha in PCa patients with a higher risk of recurrence. This prompted us to use clinical specimens and functional studies to investigate the pro-tumourigenic and pro-metastatic function of INH alpha. We conducted a cross-sectional study to determine a link between INH alpha expression and a number of clinicopathological parameters including Gleason score, surgical margin, extracapsular spread, lymph node status and vascular endothelial growth factor receptor-3 expression, which are well-established prognostic factors of PCa. In addition, using two human PCa cell lines (LNCaP and PC3) representing androgen-dependent and -independent PCa respectively, we investigated the biological function of elevated levels of INH alpha in advanced cancer. Elevated expression of INH alpha in primary PCa tissues showed a higher risk of PCa patients being positive for clinicopathological parameters outlined above. Over-expressing INH alpha in LNCaP and PC3 cells demonstrated two different and cell-type-specific responses. INH alpha-positive LNCaP demonstrated reduced tumour growth whereas INH alpha-positive PC3 cells demonstrated increased tumour growth and metastasis through the process of lymphangiogenesis. This study is the first to demonstrate a pro-tumourigenic and pro-metastatic function for INH alpha associated with androgen-independent stage of metastatic prostate disease. Our results also suggest that INH alpha expression in the primary prostate tumour can be used as a predictive factor for prognosis of PCa.

The objective was to investigate the potential role of the oocyte in modulating proliferation and basal, FSH-induced and insulin-like growth factor (IGF)-induced secretion of inhibin A (inh A), activin A (act A), follistatin (FS), estradiol (E(2)), and progesterone (P(4)) by mural bovine granulosa cells. Cells from 4- to 6-mm follicles were cultured in serum-free medium containing insulin and androstenedione, and the effects of ovine FSH and IGF analogue (LR3-IGF-1) were tested alone and in the presence of denuded bovine oocytes (2, 8, or 20 per well). Medium was changed every 48 h, cultures were terminated after 144 h, and viable cell number was determined. Results are based on combined data from four independent cultures and are presented for the last time period only when responses were maximal. Both FSH and IGF increased (P < 0.001) secretion of inh A, act A, FS, E(2), and P(4) and raised cell number. In the absence of FSH or IGF, coculture with oocytes had no effect on any of the measured hormones, although cell number was increased up to 1.8-fold (P < 0.0001). Addition of oocytes to FSH-stimulated cells dose-dependently suppressed (P < 0.0001) inh A (6-fold maximum suppression), act A (5.5-fold), FS (3.6-fold), E(2) (4.6-fold), and P(4) (2.4-fold), with suppression increasing with FSH dose. Likewise, oocytes suppressed (P < 0.001) IGF-induced secretion of inh A, act A, FS, and E(2) (P < 0.05) but enhanced IGF-induced P(4) secretion (1.7-fold; P < 0.05). Given the similarity of these oocyte-mediated actions to those we observed previously following epidermal growth factor (EGF) treatment, we used immunocytochemistry to determine whether bovine oocytes express EGF or transforming growth factor (TGF) alpha. Intense staining with TGFalpha antibody (but not with EGF antibody) was detected in oocytes both before and after coculture. Experiments involving addition of TGFalpha to granulosa cells confirmed that the peptide mimicked the effects of oocytes on cell proliferation and on FSH- and IGF-induced hormone secretion. These experiments indicate that bovine oocytes secrete a factor(s) capable of modulating granulosa cell proliferation and responsiveness to FSH and IGF in terms of steroidogenesis and production of inhibin-related peptides, bovine oocytes express TGFalpha but not EGF, and TGFalpha is a prime candidate for mediating the actions of oocytes on bovine granulosa cells.

This work reports an estimate of the inhibition rate constant (k(inh)) for alpha-tocopherol (alpha-TOH) in low-density lipoproteins (LDL) based on cholesteryl linoleate hydroperoxide products formed during autoxidation of intact lipoproteins. The ratio of cis,trans/trans,trans product hydroperoxides was determined during the consumption of the antioxidant. For a reasonable determination of k(inh) in LDL, the pro-oxidant behavior of alpha-TOH was minimized by oxidizing LDL with an unsymmetrical amphiphilic azo initiator which significantly reduces phase-transfer mediated pro-oxidant effects of alpha-TOH. This initiator delivers a more constant flux of initiator radicals into LDL lipid regions and permits determination of alpha-TOH k(inh) in LDL. Development of a tocopherol-mediated peroxidation (TMP) model and analysis of cholesteryl linoleate hydroperoxide cis,trans/trans,trans product ratios provided an estimated value for the inhibition rate constant of alpha-TOH in a lipoprotein of k(inh) = 5.9 +/- 0.5 x 10(5) M(-)(1) s(-)(1)

Phenolic antioxidants of the hydroxychroman class, alpha-tocopherol (alpha-TOC) and 2,2,5,6,7-pentamethyl-6-hydroxychroman (PMHC), and the hindered phenols 2,3-dihydro-5-hydroxy-2,2,4-trimethylnaphtho[1,2-b]furan (NFUR), 2,6-di-tert-butyl-4-methoxyphenol (DBHA), and 2,6-di-tert-butyl-4-methyl phenol (BHT), were delivered into oxidizable (ACCEPTOR) liposomes of dilinoleoylphosphatidylcholine (DLPC) or 1-palmitoyl-2-linoleoyl-phosphatidylcholine (PLPC) from saturated DONOR liposomes of dimyristoylphosphatidylcholine (DMPC) by liposomal transfer. The antioxidant activities, k(inh), by the inhibited oxygen uptake method were compared with the k(inh)s determined when the antioxidants were introduced into the liposomes by coevaporation from organic solvents. The peroxidations were initiated using either thermal initiators, water-soluble azo-bis-amidinopropane hydrochloride (ABAP), lipid-soluble azo-bis-2,4-dimethylvaleronitrile (ADVN) and di-tert-butylhyponitrite (DBHN), or the photoinitiator benzophenone. The antioxidants PMHC, NFUR, DBHA, and BHT transferred rapidly between liposomes, but several hours of incubation were needed to transfer alpha-TOC. The average k(inh)s in liposomes, in the relative order NFUR approximately DBHA>> PMHC>> BHT approximately alpha-TOC, were markedly lower than known values in organic solvent. k(inh) values in liposomes appear to be controlled by effects of hydrogen bonding with water and by restricted diffusion of antioxidants, especially in the case of alpha-TOC. Product studies of the hydroperoxides formed during inhibited oxygen consumption were carried out. The cis,trans/trans,trans (c,t/t,t) product ratios of the 9- and 13-hydroperoxides formed from PLPC during inhibited peroxidation by PMHC were similar for both the coevaporated and liposomal transfer procedures. The c,t/t,t ratio for the same concentration of alpha-TOC, 1.52, compares to a value of 1.69 for PMHC at the start of the inhibition period. The higher c,t/t,t ratio observed for NFUR in DLPC, which varied between values of 7.0 at the start of the inhibition to about 1.8 after the break in the induction period, is a reflection of the increased hydrogen atom donating ability of the antioxidant plus the increased concentration of oxidizable lipid provided by DLPC.

The objective in this work is to determine the antioxidant capacity and effectiveness of icariin (2-(4'-methoxylphenyl)-3-rhamnosido-5-hydroxyl-7-glucosido-8-(3'-methyl-2-butylenyl)-4-chromanone), the major component in herba epimedii being used widely in traditional Chinese medicine for the treatment of artherosclerosis and neuropathy, in which 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH)-induced peroxidation of linoleic acid (LH) in sodium dodecyl sulfate (SDS) acts as the experimental system. By containing an intramolecular hydrogen bond, icariin protects LH against AAPH-induced peroxidation of LH only in SDS, an anionic micelle. The number of trapping peroxyl radicals (LOO(*)), n, by icariin is just 0.0167 whereas alpha-tocopherol (TOH) and L-ascorbyl-6-laurate (VC-12) are 2.14 and 1.25, respectively, with reference to the n of 6-hydroxyl-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox), 2.00. This is also related to how the intramolecular hydrogen bond enhances the bond dissociation enthalpy (BDE) of O-H in icariin. However, calculation of the inhibition rate constant, k(inh), a kinetic parameter to describe the reaction between the antioxidant and LOO(*), results in a k(inh) of icariin at about one magnitude larger than those of Trolox, TOH, and VC-12. This fact reveals that, by the view of kinetics, icariin is an antioxidant with much higher effectiveness. In addition, the antioxidant capacities of icariin used together with other antioxidants have been determined and the results indicate that the n of icariin decreases markedly while the n values of Trolox and TOH increase, even if the n of icariin is a negative value in the presence of VC-12. Furthermore, an analysis of k(inh) in this case reveals that the k(inh)(icariin) increases nearly one magnitude with the decrease of k(inh)(Trolox) and no remarkable change occurs for k(inh)(TOH). The negative value of k(inh)(icariin) in the presence of VC-12 can be regarded as the icariin functions as a prooxidant that can be rectified by VC-12 effectively. These findings implicate that the evaluation of antioxidant activity should not only focus on an n value, a thermodynamic possibility, but k(inh) and the charge property of the micelle should be also taken into account. To some extent, the latter factors are more important than the thermodynamic possibility.

OBJECTIVE

We investigated the vascular effects of agmatine (decarboxylated arginine=AGM), an endogenous ligand for alpha(2)-adrenoceptors and imidazoline receptors, present in endothelium and smooth muscle, using the diabetic rat aortae.

METHODS

Studies were performed in control group (0.2 ml i.p. saline, n=10), streptozotocin (STZ)-diabetic control group (60 mg kg(-1) STZ i.p., n=10), agmatine (AGM)-control group (5 mg kg(-1)day(-1) i.p. AGM for 1 month, n=10), citrate-control group (0.2 ml 0.01 M, n=10), insulin-treated diabetic group ((3 U kg(-1) NPH+1 U kg(-1) regular insulin) twice per day, for 1 month, n=10) and AGM-treated diabetic group (5 mg kg(-1)day(-1) i.p. for 1 month, n=10). All values are expressed as means+/-S.E.M. Statistical analysis of the data was performed using ANOVA followed by Tukey multiple comparisons test.

RESULTS

One-month AGM-treatment significantly decreased the blood glucose levels of diabetic rats (502+/-44 mg dl(-1) to 343+/-31 mg dl(-1), P<0.001). Fast, slow and total components of responses to noradrenaline in all the experimental groups were not significantly affected by AGM-treatment. AGM reversed the decreased responses of acetylcholine (pD(2) and Inh.%, P<0.001 and P<0.05) in diabetic rats although it did not affect the responses of sodium nitroprusside in all groups. The contraction values of KCl in all groups were not affected by AGM-treatment.

CONCLUSIONS

AGM-treatment could improve the increased blood glucose level, reverse the endothelial dysfunction and normalize the endothelium-dependent relaxation responses in STZ-diabetic rats.

The effect of isonicotinic acid hydrazide (INH), a potent haem inhibitor, on globin chain synthesis was studied in reticulocytes from the following groups of patients: four non-thalassaemic patients (group i); five beta thalassaemia heterozygotes (group ii); three Hb S/beta thalassaemia heterozygotes (group iii); and two additional patients--one with homozygous beta thalassaemia and the other with thalassaemia intermedia (group iv). This was done to determine whether haem inhibitors depress alpha globin chain synthesis. The progressive increase of INH concentration (10-40 mmol l-1) in reticulocytes from a beta thalassaemia heterozygote resulted in a remarkable decrease of the alpha and beta chain synthesis, ranging from 80% to 97% and from 74% to 96% of control values, respectively, and in a gradual drop of alpha:beta ratio from 1.87 to 1.38. Furthermore, in the samples incubated with 40 mmol l-1 INH, a pronounced inhibition of globin chain synthesis 77 (19%) for alpha chain and 67 (27%) for beta or beta S chain) and a substantial drop of the alpha:beta or beta S ratio in samples with INH (median 1.16) compared with that in samples without INH (median 1.70) were observed. The inhibitory effect of INH was significantly or completely corrected by adding exogenous haem. It is suggested that haem inhibition and the resulting preferential diminution of alpha chain synthesis could provide a new approach to the treatment of homozygous beta thalassaemia with an excess of detrimental free alpha chain in erythroid cells.

Proteinase inhibitor members of the SERPIN superfamily are characterized by the presence of a proteolytically sensitive reactive-site loop. Cleavage within this region results in a conformational transition from an unstable "stressed" native protein to a more stable "relaxed" cleaved molecule. In order to identify the principal molecular aspects of this transition, 1H nuclear magnetic resonance (n.m.r.) and FT-IR spectroscopy were applied to the study of four SERPINs. 1H n.m.r. spectra of approximately 20 high-field ring-current-shifted methyl signals exhibited slightly different chemical shifts in the native and cleaved forms of alpha 1-antitrypsin (alpha 1-AT), alpha 1-antichymotrypsin (alpha 1-ACT) and C1 inhibitor (C1-INH), but not ovalbumin, between 20 degrees C and 90 degrees C. Ring current calculations based on crystal co-ordinates for cleaved alpha 1-AT and alpha 1-ACT and native ovalbumin showed that these signals originate from highly localized interactions between different buried residues corresponding to alpha-helix and beta-sheet segments of the SERPIN fold. The small shift changes correspond to small relative conformational side-chain rearrangements of about 0.01 nm to 0.05 nm in the protein hydrophobic core, i.e. the tertiary structure interactions in the two forms of the SERPIN fold are well-preserved, and changes in this appear unimportant for the stabilization found after reactive centre cleavage. Fourier transform infrared (FT-IR) spectroscopic studies of the amide I band showed that the native and cleaved forms of alpha 1-AT, alpha 1-ACT and C1-INH contain 28% to 36% alpha-helix and 38% to 44% beta-sheet. Second derivative FT-IR spectra using H2O and 2H2O buffers revealed very large differences in the amide I band between the native and cleaved forms of alpha 1-AT, alpha 1-ACT and C1-INH, but not for ovalbumin. The alpha-helix band was most sensitive to 1H-2H exchange, while the beta-sheet bands were not, and greater amounts of antiparallel beta-sheet were detected in the cleaved form. 1H n.m.r. showed that polypeptide amide 1H-2H exchange was greater in the native forms of alpha 1-AT, alpha 1-ACT and C1-INH than in their cleaved forms, whereas for ovalbumin it was unchanged. The FT-IR and 1H-2H exchange data show that alterations in the secondary structure are central to the stabilization of the cleaved SERPIN structure.(ABSTRACT TRUNCATED AT 400 WORDS)

Immunohistochemical studies of human fetal Sertoli cells (SCs) have shown transient expression of cytokeratin (CK) and desmin (DES) that is replaced after birth by expression of vimentin (VIM) and inhibin-α (INH-α). Human Sertoli cell tumours (SCTs) are characterized by re-expression of CK and DES. The aim of the present study was to evaluate immunohistochemically the expression of VIM, INH-α, CK and DES in normal and neoplastic canine SCs. Normal testicular tissue from three adult dogs, one 6-month-old puppy and two neonatal pups was examined in addition to samples from 21 canine SCTs. VIM was not expressed by neonatal SCs, but was present in SCs from the puppy, the adult dogs and in all SCTs. Conversely, INH-α was expressed by neonatal SCs and most SCTs, but not by normal SCs of adult dogs and the puppy. DES and CK were expressed only by some SCTs. These results show that, contrary to findings in man, canine SCs do not express VIM at the time of birth. SCs from neonatal dogs do express INH-α, but such expression was lost in the puppy and the adult dogs. Canine SCs therefore differ from human SCs, as expression of INH-α characterizes immature SCs, whereas the expression of VIM characterizes mature SCs. Canine SCTs may express CK and DES, suggesting that the neoplastic cells undergo de-differentiation during transformation.

CD1d-restricted natural killer T (NKT) cells are emerging as critical regulators of the immune response to infectious agents, including Pseudomonas aeruginosa; and therapies to augment NKT-cell activation may represent a novel approach to treat chronic, antibiotic-resistant bacterial infections. We examined the capacity of dendritic cells (DCs) from people with cystic fibrosis (CF) to activate NKT cells. Our study was motivated by three lines of evidence: (i) NKT cells play a critical role in clearing P. aeruginosa infection; (ii) activation of NKT cells requires acidification-dependent processing of glycolipid antigens within the endolysosomal compartment; and (iii) endolysosomal acidification may be reduced in CF. We demonstrated that NKT-cell activation was dependent upon intact organelle acidification as inhibitors of the vacuolar (H(+))-ATPases prevented DCs from activating NKT cells with two glycolipid antigens, alpha-galactosylceramide and galactose-galactosylceramide. In contrast, cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel dysfunction had no significant biological impact on the capacity of DCs to activate NKT cells. Dendritic cells from subjects with CF and DCs treated with the thiazolidinone CFTR(inh)-172 inhibitor showed no reduction in their ability to activate NKT cells. Based on these data, we find no evidence for an inherent defect in glycolipid antigen presentation to NKT cells in CF subjects.

The enoyl-ACP reductase enzyme (InhA) from M. tuberculosis is recognized as the primary target of isoniazid (INH), a first-line antibiotic for tuberculosis treatment. To identify the specific interactions of INH-NAD adduct and its derivative adducts in InhA binding pocket, molecular docking calculations and quantum chemical calculations were performed on a set of INH derivative adducts. Reliable binding modes of INH derivative adducts in the InhA pocket were established using the Autodock 3.05 program, which shows a good ability to reproduce the X-ray bound conformation with rmsd of less than 1.0 A. The interaction energies of the INH-NAD adduct and its derivative adducts with individual amino acids in the InhA binding pocket were computed based on quantum chemical calculations at the MP2/6-31G (d) level. The molecular docking and quantum chemical calculation results reveal that hydrogen bond interactions are the main interactions for adduct binding. To clearly delineate the linear relationship between structure and activity of these adducts, CoMFA and CoMSIA models were set up based on molecular docking alignment. The resulting CoMFA and CoMSIA models are in conformity with the best statistical qualities, in which r2cv is 0.67 and 0.74, respectively. Structural requirements of isoniazid derivatives that can be incorporated into the isoniazid framework to improve the activity have been identified through CoMFA and CoMSIA steric and electrostatic contour maps. The integrated results from structure-based, ligand-based design approaches and quantum chemical calculations provide useful structural information facilitating the design of new and more potentially effective antitubercular agents as follow: the R substituents of isoniazid derivatives should contain a large plane and both sides of the plane should contain an electropositive group. Moreover, the steric and electrostatic fields of the 4-pyridyl ring are optimal for greater potency.

The versatile transgenic (TG) techniques allow the production of in vivo animal models for a variety of diseases, including malignant tumors, through tissue-specific expression of oncogenes. We have created a TG mouse model for gonadal somatic cell tumors by expressing the powerful viral oncogene, Simian virus 40 T-antigen (Tag) under regulation of the murine inhibin alpha-subunit promoter (inh alpha). Ovarian granulosa and theca cell tumors were formed in the female, and those of testicular Leydig cells, in the male TG mice at the age of 5-6 months, with 100% penetrance. The tumors produced high levels of inhibin peptides, especially the alpha-subunit, and were steroidogenically active, mainly producing progesterone. The gonadal tumorigenesis was gonadotropin-dependent, since TG mice rendered gonadotropin-deficient by crossbreeding them into the hypogonadotropic hpg genetic background, or by treating them with a gonadotropin-releasing hormone (GnRH) antagonist, did not develop tumors. In order to study the possibility of using the tumor mouse model for testing gene therapy, we created another TG mouse model expressing under the same inhibin-alpha promoter the Herpes Simplex virus (HSV) thymidine kinase (TK) transgene. The inh alpha/HSV-TK mice were crossbred with the inh alpha/Tag mice and the double mutant mice also developed gonadal tumors. When they were treated with antiherpes drugs (acyclovir or gancyclovir), further growth of the tumors was blocked. These preliminary findings prove the principle that tumor ablation in our TG mouse model can be achieved by transduction of the HSV-TK gene into the tumor cells. Besides studies of formation, regulation and therapy of the tumors in vivo, immortalized cell lines derived from them provide models for studies of gonadal somatic cell functions in vitro.

BACKGROUND

Acute psychotic access is defined as the occurrence of a single, acute, and intense psychotic episode in a subject without a neurological or psychiatric history. Isoniazid (INH), a major antibacillar, is the drug most often involved in the occurrence of this psychiatric disorder. However, this side effect is rare and only a few cases have been reported in the literature.

METHODS

A 57-year-old female patient with diabetes mellitus presented miliary tuberculosis for which an antibacillar treatment was prescribed. Three days later, she presented an acute psychotic access requiring the withdrawal of the INH and the prescription of neuroleptic drugs, without any pyridoxine supplementation. The lab tests were normal. The good clinical evolution after the INH withdrawal confirmed its imputabilty.

CONCLUSIONS

Acute psychotic access is a severe and exceptional complication following the administration of INH. Emergency treatment is the only guarantee of a good outcome.

BACKGROUND

Acquired deficiency in C1-inhibitor (C1-INH) associated with malignancy is often asymptomatic because clinical manifestations are not dependent on a critical complement threshold (in contrast to hereditary C1-INH deficiency). Increased complement consumption involving different kinds of antibodies is the postulated mechanism for this disease, but other factors must play an important role.

METHODS

A 76-year-old woman with unremarkable medical history experienced three episodes of angioedema over 6 months. Investigations revealed a complement profile characteristic of acquired deficiency in C1-INH, a hemolytic anemia, and a signet ring cell adenocarcinoma (linitis plastica). A gastrectomy and a splenectomy were performed. The postoperative course was characterized by a complete disappearance of the symptoms of angioedema and hemolytic anemia. A local recurrence of the tumor 5 months later could not be resected. The patient died 17 months after the initial surgery was performed.

RESULTS

Quantitative and functional analyses of the complement factors showed persistent excessive complement consumption. Markers of hemolytic anemia disappeared after tumor removal but recurred in the second part of the disease evolution. Immunohistochemical findings in tumor tissue showed loss of normal blood group antigens but expression of Lea antigen, as well as C1q deposition.

CONCLUSIONS

To explain the whole clinical and laboratory picture, we hypothesize a connection between tumor immunohistochemical profile, complement consumption, and hemolytic anemia. Tumor cell surface antigens might lead to a permanent but asymptomatic complement consumption that is worsened and becomes clinically manifest by superimposed hemolytic anemia caused by cross-reactive antibodies to newly expressed blood group antigens on tumor cells. This hypothesis should be confirmed by other observations.

BACKGROUND

Meconium aspiration syndrome has a complex pathophysiology. Meconium activates the complement system and meconium-induced cytokine formation is differentially mediated by complement and CD14. C1-inhibitor (C1-INH) regulates complement and contact-system activation mainly by protease inhibition, but may reduce inflammation by other mechanisms as well.

OBJECTIVE

The aim of the study was to investigate the initial mechanisms of meconium-induced complement activation and to study the effect of C1-INH on the meconium-induced inflammatory reaction.

METHODS

Human serum from five donors was preincubated with an anti-MBL monoclonal antibody and then incubated with meconium for 30 min at 37 degrees C. Human cord whole blood, anticoagulated with lepirudin, from six donors was preincubated with C1-INH and then incubated with meconium for 30 min and 4h at 37 degrees C. Complement activation products specific for the different pathways were measured by ELISAs: classical pathway C1rs/C1-INH complexes, classical and lectin pathway C4d, alternative pathway C3bBbP, and terminal pathway sC5b-9 complex (TCC). A Bio-Plex Array Reader was used to measure 27 inflammatory mediators.

RESULTS

The anti-MBL monoclonal antibody significantly reduced meconium-induced formation of C4d by 63% (p=0.0159) and TCC by 27% (p=0.0079). C1-INH dose-dependently inhibited meconium-induced formation of C1rs/C1-INH complexes, C4d, C3bBbP, and TCC compared to albumin (p<0.002 for all). C1-INH induced a dose-dependent and substantial inhibition of meconium-induced formation of the proinflammatory cytokines TNFalpha, IL-1 beta, IL-6 and IFN-gamma (p<0.01 for all), the chemokines IL-8, MCP-1, MIP-1 alpha, MIP-1 beta, and eotaxin (p<0.02 for all), the growth factors G-CSF, GM-CSF, basic FGF, and PDGFbb (p<0.05 for all), and the anti-inflammatory cytokine IL-1ra (p<0.001).

CONCLUSIONS

Meconium activated the lectin complement pathway as well as the alternative pathway. C1-INH efficiently reduced a broad spectrum of inflammatory mediators even at the lowest concentration. Administration of C1-INH may thus reduce the inflammatory response in MAS.