Population studies on the inter-α-trypsin inhibitor (ITI) polymorphism

Serum samples from four European populations — Germans from Jena, Germans from Hannover, Italians from Udine, and Slovakians from Bratislava — were tested for the inter-α-trypsin inhibitor (ITI) polymorphism. The results are compared with those reported for other European, Asiatic and African populations. Reviewing all the hitherto published ITI allele frequencies one can see that the variability among European Populations seems to be rather small, whereas Asiatic and African populations are striking by quite different distribution patterns.     

The effect of intertrial interval food presentions on pigeons' delayed matching to sample accuracy

Parallel effects of temporal variables on autoshaping and on delayed matching to sample performance suggest that delayed matching, like autoshaping, might depend upon the within-trial expectancy of reinforcement relative to the overall expectancy of reinforcement in the session. This possibility was assessed by presenting free food at different times during a 30-sec intertrial interval (ITI) in a delayed matching to sample procedure with pigeons. In three conditions a single free food presentation occurred, either early, mid-way, or late in each ITI; in another condition, three food presentations occurred during each ITI, one at each time location. Relative to a baseline condition, in which free food never occurred during the ITI, only food presentations late in the ITI produced a significant disruption in accuracy, and this effect occurred only at the longest of three delays tested. Three free food presentations in each ITI disrupted accuracy only to the same degree as a single, late, ITI food presentation. Thus, accuracy was affected by the temporal location rather than the frequency of ITI food presentations. These effects appear to differ from those of ITI food presentations on autoshaping and do not seem to be understandable in terms of changes in the background expectancy of reinforcement. It was suggested instead that food presented late in the ITI might disrupt subsequent memory processes.     

中国汉族人群间-α-胰蛋白酶抑制因子(ITI)的遗传多态性

我们建立了测定间-α-胰蛋白酶抑制因子遗传表型的聚丙烯酰胺凝胶等电聚焦免疫固定技术,应用这种方法对居住在成都地区的汉族群体进行了群体研究和家系调查,结果表明中国汉族人群间-α-胰蛋白酶抑制因子具有遗传多态性,它有希望成为法医血液遗传学新的遗传标记。     

A study of the genetic polymorphism of human inter-alpha-trypsin-inhibitor (ITI) in the Han population,Chengdu, China

Phenotypes of inter-alpha-trypsin-inhibitor (ITI) have been determined by isoelectric focusing on polyacrylamide gels followed by immunofixation. The phenotype frequencies of ITI in the Han population in Chengdu, P. R. China have been investigated using this method. In addition, family studies have been conducted in 21 families. The results show that ITI is polymorphic in the Han population in Chengdu, China. The allele frequencies are as follows: ITI*1 = 0.5763. ITI*2 = 0.4107, ITI*3 = 0.0130. ITI is thus a new and promising genetic marker that can be used in the field of forensic haematogenetics.     

Crossed immunoelectrophoresis does not allow accurate determination of inter-alpha-trypsin inhibitor and its derivatives in plasma.

By crossed-immunoelectrophoresis (CIE) of plasma, using anti-inter-alpha-trypsin inhibitor (ITI) immunoglobulins, beside native ITI, related components are visualized as an heterogeneous peak migrating farther than ITI. The area corresponding to this peak is largely increased in case of inflammatory disease. So, quantitative CIE has been previously proposed for discrete evaluation of ITI and its derivatives. We herein present evidence that CIE does not allow a clear separation of ITI from its derivatives: in this system, they are to some extent coprecipitated. Therefore the resulting overestimation of ITI may explain the previously reported absence of inverse relation between relative contents of ITI and derivatives in case of inflammatory disorders. Our data confirm the view that ITI acts as a precursor of smaller immunologically related inhibitors.     

Treatment of midfacial defects using prostheses supported by ITI dental implants

The purpose of this study was to evaluate retrospectively the use of ITI dental implants used for anchoring facial prostheses in the restorative treatment of midface defects. The authors analyzed the clinical data of 26 patients with orbital defects (n = 11), orbitonasal defects (n = 4), orbitonasomaxillary defects (n = 3), and nasal defects (n = 8). Data included age, sex, primary disease, implant position, implant length, implant failure, prosthetic attachment, radiation therapy, and peri-implant skin reactions. Follow-up was at 1, 3, 6, and 12 months and then on a yearly basis. The authors noted the status of healing and complications, if any. In total, 62 implants were placed as follows: 27 (43.5 percent) for orbital prostheses, 12 (19.4 percent) for orbitonasal prostheses, 14 (22.6 percent) for orbitonasomaxillary prostheses, and nine (14.5 percent) for nasal prostheses. Thirty-eight implants (61.3 percent) were placed in previously irradiated areas in 18 patients (69.2 percent). Mild skin reactions together with mild accumulation of sebaceous crusting around implants were recorded in 14.2 percent of the skin observations. No patient experienced severe inflammation requiring administration of systemic antibiotics or surgical revision. Implant success was 100 percent in both irradiated and nonirradiated patients. In conclusion, ITI dental implants result in a high rate of success in retaining midface prostheses and offer good stability and aesthetic satisfaction.     

A proteoglycan related to the urinary trypsin inhibitor (UTI) links the two heavy chains of inter-alpha-trypsin inhibitor

cDNA studies have suggested that inter-alpha-trypsin inhibitor (ITI) is a complex of several different peptide chains; the sequence of the inhibitory part of ITI is in excellent agreement with that of the urinary trypsin inhibitor (UTI). The present report demonstrates that a compound immunologically related to UTI is released by digestion with porcine pancreatic elastase or human leucocyte elastase. Since UTI has been shown to be a proteoglycan, ITI has been treated by chondroitinase. In these conditions, ITI is dissociated and gives rise to two heavy chains (78 and 85 kDa) and one light chain (26 kDa) immunologically related to UTI and which in PAGE moves close to UTIc (produced by chondroitinase treatment of UTI). We suggest that ITI is a non-covalent complex comprising two heavy chains and one light chain immunologically related to UTI and which is also a proteoglycan.     

Cloning of the gene encoding a novel thermostable alpha-galactosidase from Thermus brockianus ITI360.

An alpha-galactosidase gene from Thermus brockianus ITI360 was cloned, sequenced, and expressed in Escherichia coli, and the recombinant protein was purified. The gene, designated agaT, codes for a 476-residue polypeptide with a calculated molecular mass of 53, 810 Da. The native structure of the recombinant enzyme (AgaT) was estimated to be a tetramer. AgaT displays amino acid sequence similarity to the alpha-galactosidases of Thermotoga neapolitana and Thermotoga maritima and a low-level sequence similarity to alpha-galactosidases of family 36 in the classification of glycosyl hydrolases. The enzyme is thermostable, with a temperature optimum of activity at 93 degrees C with para-nitrophenyl-alpha-galactopyranoside as a substrate. Half-lives of inactivation at 92 and 80 degrees C are 100 min and 17 h, respectively. The pH optimum is between 5.5 and 6.5. The enzyme displayed high affinity for oligomeric substrates. The K(m)s for melibiose and raffinose at 80 degrees C were determined as 4.1 and 11.0 mM, respectively. The alpha-galactosidase gene in T. brockianus ITI360 was inactivated by integrational mutagenesis. Consequently, no alpha-galactosidase activity was detectable in crude extracts of the mutant strain, and it was unable to use melibiose or raffinose as a single carbohydrate source.     

Genetic polymorphism of inter-alpha-trypsin-inhibitor (ITI): Formal genetic and linkage analyses

Summary The polymorphism of inter-alpha-trypsin-inhibitor, ITI, was demonstrated by isoelectric focusing in agarose gels (pH 5–8) followed by protein blotting and immunoassay. Segregation in 239 families with 677 children is consistent with the formal hypothesis that there are two common codominant alleles, ITI^*1, ITI^*2, and one rare codominant allele, ITI^*3, at an autosomal locus ITI. Allele frequencies were calculated as ITI^*1=0.600, ITI^*2=0.393, ITI^*3=0.007. Linkage analysis with 36 markers is presented. Slightly positive lod scores were obtained for PGM3 (z=1,35, =0.10) and AK1 (z=1.34, =0.10).     

Plasma proteins immunologically related to inter-alpha-trypsin inhibitor

SDS-polyacrylamide gel electrophoresis and immunoblot were applied to analysis of plasma proteins immunologically related to inter-alpha-trypsin inhibitor (ITI). In this system, anti-ITI sera were able to identify ITI and other components with an Mr near 120 kDa which would be degradation products of ITI by limited proteolysis. An anti-UTI (urinary trypsin-inhibitor) serum could detect, beside these derivatives, two minor components (Mr values near 90 and 60 kDa). Analysis of perchloric acid supernatants of plasma samples, using the same technic, induced visualization of a new component, similar to urinary trypsin inhibitor which could not be detected by direct analysis. This one was also characterized in a higher content in pathological samples (renal failure and infectious diseases).     

The ITI system in South Koreans and iranians analysed by an improved classification procedure

Summary Phenotype and gene frequency distributions of the inter--trypsin inhibitor (ITI) system were analysed in populations from southern Korea and from Iran. The gene frequencies of the common alleles ITI^*I and ITI^*2 were 0.532 and 0.422, respectively, in southern Korea, and 0.612 and 0.354, respectively, in Iran. The postulated third allele, ITI^*3, was found in the homozygous form. Gene frequencies of this rare allele were calculated to be 0.042 and 0.029 in Korea and Iran, respectively. Two additional rare alleles, ITI^*4 and ITI^*5, determine further phenotypes found in the population from Taejon (Korea) and Iran, respectively, in combination with the common ITI^*2 allele. Gene frequencies of ITI^*4 and ITI^*5 were calculated to be 0.006 and 0.005, respectively. For phenotype classification, untreated sera were separated by isoelectric focusing (IEF) on polyacrylamide gels followed by immunoblotting.     

cDNA cloning of human inter-alpha-trypsin inhibitor discloses three different proteins

Inter-alpha-trypsin inhibitor (ITI) is a serum protein of unknown function. Part of the molecule (formerly called HI30) is closely related to a tumor-derived protein acting as a growth factor for endothelial cells. We screened a human liver cDNA expression library with antibodies raised against human ITI and isolated several clones which could be divided into three groups according to their DNA sequences. The cDNA of the first group codes for a protein composed of alpha 1-microglobulin (alpha 1M) and urinary trypsin inhibitor (UTI) and is identical to that encoded by a clone originally found by screening a human liver cDNA library with oligonucleotides derived from amino-acid sequences of the two Kunitz-type domains of UTI. The proteins derived from the cDNA of the second and the third group of clones are distantly related to each other, but unrelated to the protein derived from group 1 clones. Partial amino-acid sequencing of ITI isolated from serum allowed the verification of large parts of the cDNA-derived amino-acid sequences. The results favour the view that ITI is not a single chain protein, but rather a very tight complex of several components or a mixture of such complexes.     

Immunohistochemical distribution of inter-alpha-trypsin inhibitor chains in normal and malignant human lung tissue.

The inter-alpha-trypsin inhibitor (ITI) family is a group of plasma proteins built up from heavy (HC1, HC2, HC3) and light (bikunin) chains synthesized in the liver. In this study we determined the distribution of ITI constitutive chains in normal and cancerous lung tissues using polyclonal antibodies. In normal lung tissue, H2, H3, and bikunin chains were found in polymorphonuclear cells, whereas H1 and bikunin proteins were found in mast cells. Bikunin was further observed in bronchoepithelial mucous cells. In lung carcinoma, similar findings were obtained on infiltrating polymorphonuclear and mast cells surrounding the tumor islets. Highly differentiated cancerous cells displayed strong intracytoplasmic staining with H1 and bikunin antiserum in both adenocarcinoma and squamous cell carcinoma. Moreover, weak but frequent H2 expression was observed in adenocarcinoma cells, whereas no H3-related protein could be detected in cancer cells. Local lung ITI expression was confirmed by RT-PCR. Although the respective role of inflammatory and tumor cells in ITI chain synthesis cannot be presently clarified, these results show that heavy chains as well as bikunin are involved in malignant transformation of lung tissue.(J Histochem Cytochem 47:1625-1632, 1999)     

Defect in SHAP-hyaluronan complex causes severe female infertility. A study by inactivation of the bikunin gene in mice

Hyaluronan (HA) associates with proteins and proteoglycans to form the extracellular HA-rich matrices that significantly affect cellular behaviors. So far, only the heavy chains of the plasma inter-alpha-trypsin inhibitor (ITI) family, designated as SHAPs (serum-derived hyaluronan-associated proteins), have been shown to bind covalently to HA. The physiological significance of such a unique covalent complex has been unknown but is of great interest, because HA and the ITI family are abundant in tissues and in plasma, respectively, and the SHAP-HA complex is formed wherever HA meets plasma. We abolished the formation of the SHAP-HA complex in mice by targeting the gene of bikunin, the light chain of the ITI family members, which is essential for their biosynthesis. As a consequence, the cumulus oophorus, an investing structure unique to the oocyte of higher mammals, had a defect in forming the extracellular HA-rich matrix during expansion. The ovulated oocytes were completely devoid of matrix and were unfertilized, leading to severe female infertility. Intraperitoneal administration of ITI, accompanied by the formation of the SHAP-HA complex, fully rescued the defects. We conclude that the SHAP-HA complex is a major component of the HA-rich matrix of the cumulus oophorus and is essential for fertilization in vivo.     

Structural basis of fullerene derivatives as novel potent inhibitors of protein acetylcholinesterase without catalytic active site interaction: insight into the inhibitory mechanism through molecular modeling studies

Abstract

Acetylcholinesterase (AChE) is an important kind of esterase that plays a key biological role in the central and peripheral nervous systems. Recent research has demonstrated that some fullerene derivatives serve as a new nanoscale class of potent inhibitors of AChE, but the specific inhibition mechanism remains unclear. In the present article, several molecular modeling methods, such as molecular docking, molecular dynamics simulations and molecular mechanics/generalized Born surface area calculations, were used for the investigation of the binding mode and inhibition mechanism of fullerene inhibitions for AChE. Results revealed that fullerene inhibitors block the entrance of substrates by binding with the peripheral anionic site (PAS) region. Thus, fullerene derivatives might mainly serve as competitive inhibitors. The interactions of a fullerene backbone with AChE are at the same level in different single side chain systems and seem to be related to the length or aromaticity of the side chain. The inhibitor with multihydroxyl side chains shows an obviously large electrostatic interaction as it forms additional hydrogen bonds with AChE. Moreover, fullerene derivatives might exhibit noncompetitive inhibition partly by affecting some secondary structures around them. Thus, the destructions of these secondary structures can lead to conformational changes in some important regions, such as the catalytic triad and acyl pocket. The investigation is of great importance to the discovery of good fullerene inhibitors.

Communicated by Ramaswamy H. Sarma     

Intertrial interval as a contextual stimulus

Four experiments with rats investigated whether the time between appetitive conditioning trials can serve as a discriminative cue for responding during the next conditional stimulus (CS). In Experiment 1, rats that received extinction trials with a 4-min intertrial interval (ITI) showed spontaneous recovery after a retention interval of 16 min, whereas rats that received extinction with a 16-min ITI did not. Experiments 2 and 3 investigated more explicit discriminations between the 4- and 16-min ITIs. When a 16-min ITI signaled that the CS would be reinforced and a 4-min ITI signaled that it would not, the ITIs modulated responding to the CS. But when the 4-min ITI signaled reinforcement and the 16-min ITI did not, there was less evidence of modulation by the ITIs. This asymmetry was due at least partly to a difficulty in performance rather than learning. Experiment 4 investigated similar discriminations with 1- and 4-min ITIs. Here the results took a different form: time in the reinforced ITI elicited responding directly, but did not modulate responding to the CS. ITI can function as a contextual cue, and the results suggest new similarities between the processes behind interval timing and associative learning.     

The crystal structures of human alpha-thrombin complexed with active site-directed diamino benzo[b]thiophene derivatives: a binding mode for a structurally novel class of inhibitors

The crystal structures of four active site-directed thrombin inhibitors, 1-4, in a complex with human alpha-thrombin have been determined and refined at up to 2.0 A resolution using X-ray crystallography. These compounds belong to a structurally novel family of inhibitors based on a 2,3-disubstituted benzo[b]thiophene structure. Compared to traditional active-site directed inhibitors, the X-ray crystal structures of these complexes reveal a novel binding mode. Unexpectedly, the lipophilic benzo[b]thiophene nucleus of the inhibitor appears to bind in the S1 specificity pocket. At the same time, the basic amine of the C-3 side chain of the inhibitor interacts with the mostly hydrophobic proximal, S2, and distal, S3, binding sites. The second, basic amine side chain at C-2 was found to point away from the active site, occupying a location between the S1 and S1' sites. Together, the aromatic rings of the C-2 and C-3 side chains sandwich the indole ring of Trp60D contained in the thrombin S2 insertion loop defined by the sequence "Tyr-Pro-Pro-Trp." [The thrombin residue numbering used in this study is equivalent to that reported for chymotrypsinogen (Hartley BS, Shotton DM, 1971, The enzymes, vol. 3. New York: Academic Press. pp 323-373).] In contrast to the binding mode of more classical thrombin inhibitors (D-Phe-Pro-Arg-H, NAPAP, Argatroban), this novel class of benzo[b]thiophene derivatives does not engage in hydrogen bond formation with Gly216 of the thrombin active site. A detailed analysis of the three-dimensional structures not only provides a clearer understanding of the interaction of these agents with thrombin, but forms a foundation for rational structure-based drug design. The use of the data from this study has led to the design of derivatives that are up to 2,900-fold more potent than the screening hit 1.     

Effects of intertrial reinforcers on rats' timing behavior

Two experiments were conducted to assess the effects of non-contingent intertrial interval (ITI) reinforcers on rats' discrimination of duration. In the first experiment, rats' discrimination of a 2 vs. 8 s of light was significantly disrupted when reinforcers were presented in the ITI. Disruption was not different on short (2 s) and long (8 s) trials. The second experiment showed that this disruptive effect was not specific to trials preceded by ITI reinforcers; responding on empty ITI trials run in the same session as ITI-reinforcer trials was also disrupted. This disruption however was not as great as on the ITI-reinforcer trials. The results of these experiments show that ITI reinforcers affect timing discriminations in much the same way they affect classical conditioning and delayed matching to sample. However, detailed examination of the results suggests that the deleterious effects of ITI reinforcers in these different paradigms might be produced by different rather than the same mechanism. The results also support the conclusion that pre-trial reinforcement “priming” produces disruption rather than facilitation in complex tasks.