Transient response of serpinA3 during cellular stress.

We demonstrated that serpinA3c/k relocates from the cytoplasm to the apical tubular membrane (ATM) in chronic kidney disease (CKD), suggesting its secretion in luminal space in pathophysiological contexts. Here, we studied serpinA3c/k expression and secretion under different stressful conditions in vitro and in vivo. HEK-293 cells were transfected with a FLAG-tagged serpinA3c/k clone and exposed to H2 O2 or starvation. Both stressors induced serpinA3c/k secretion but with a higher molecular weight. Glycanase treatment established that serpinA3c/k is glycosylated. Site-directed mutagenesis for each of the four glycosylation sites was performed. During cellular stress, serpinA3c/k secretion increased with each mutant except in the quadruple mutant. In rats and patients suffering acute kidney injury (AKI), an atypical urinary serpinA3c/k excretion (uSerpinA3c/k) was observed. In rats with AKI, the greater the induced kidney damage, the greater the uSerpinA3 c/k, together with relocation toward ATM. Our findings show that: (1) serpinA3c/k is glycosylated and secreted, (2) serpinA3c/k secretion increases during cellular stress, (3) its appearance in urine reveals a pathophysiological state, and (4) urinary serpinA3 excretion could become a potential biomarker for AKI.

Design of an allosterically modulated doxycycline and doxorubicin drug-binding protein.

The allosteric interplay between distant functional sites present in a single protein provides for one of the most important regulatory mechanisms in biological systems. While the design of ligand-binding sites into proteins remains challenging, this holds even truer for the coupling of a newly engineered binding site to an allosteric mechanism that regulates the ligand affinity. Here it is shown how computational design algorithms enabled the introduction of doxycycline- and doxorubicin-binding sites into the serine proteinase inhibitor (serpin) family member α1-antichymotrypsin. Further engineering allowed exploitation of the proteinase-triggered serpin-typical S-to-R transition to modulate the ligand affinities. These design variants follow strategies observed in naturally occurring plasma globulins that allow for the targeted delivery of hormones in the blood. By analogy, we propose that the variants described in the present study could be further developed to allow for the delivery of the antibiotic doxycycline and the anticancer compound doxorubicin to tissues/locations that express specific proteinases, such as bacterial infection sites or tumor cells secreting matrix metalloproteinases.

NewBG: A surrogate corticosteroid-binding globulin with an unprecedentedly high ligand release efficacy.

The introduction of ligand-binding sites into proteins and the engineering of molecular allosteric coupling pathways are topical issues in protein design. Here, we show that these issues can be addressed concurrently, using the serpin human α1-antichymotrypsin (ACT) as a model. We have introduced up to 15 amino acid substitutions into ACT, converting it into a surrogate corticosteroid-binding globulin (CBG), thereby creating a new binding globulin (NewBG). Human CBG and ACT share 46% sequence identity, and CBG served as the blue-print for our design, which was guided by side-chain-packing calculations, ITC measurements and crystal structure determinations. Upon transfer of ligand-interacting residues from CBG to ACT and mutation of specific second shell residues, a NewBG variant was obtained, which binds cortisol with 1.5 µM affinity. This novel serpin (NewBG-III) binds cortisol with a 33-fold lower affinity than CBG, but shares a similar ligand-binding profile and binding mode when probed with different steroid ligands and site-directed mutagenesis. An additional substitution, i.e. A349R, created NewBG-III-allo, which introduced an allosteric coupling between ligand binding and the serpin-like S-to-R transition in ACT. In NewBG-III-allo, the proteinase-triggered S-to-R transition leads to a greater than 200-fold reduction in ligand affinity, and crystal structures suggest that this is mediated by the L55V and A349R substitutions. This reduction significantly exceeds the 10-fold reduction in binding affinity observed in human CBG.

Is Alpha-1 Antichymotrypsin Gene Polymorphism a Risk Factor for Primary Intracerebral Hemorrhage? A Case-Control Study and Meta-Analysis.

BACKGROUND: Alpha-1 antichymotrypsin (ACT) signal peptide A/T polymorphism has been suggested to play a role in various brain diseases with arterial wall pathology. We conducted a case-control study and a meta-analysis to evaluate the association between this polymorphism and risk of primary intracerebral hemorrhage. MATERIAL AND METHODS: A total of 188 patients and 200 age- and sex-matched healthy controls were enrolled in our case-control study. The ACT polymorphism was genotyped by PCR-LDR. Further meta-analysis was conducted by searching literature from PUBMED, EMBASE, and Chinese National Knowledge Infrastructure databases until December 2014, then combining data using STATA10.0. RESULTS: Similar genotype distribution was detected between PICH patients and healthy controls (p=0.523). Further analysis based on hypertension and location of hemorrhage did not observe significant association. Multiple logistic regression analysis also failed to identify ACT polymorphism as an independent risk factor for PICH. With regard to meta-analysis, a total of 6 case-control studies including 932 PICH patients and 1140 controls were enrolled. Pooled ORs failed to detect a significant association of ACT signal peptide A/T polymorphism with PICH (dominant model: OR=1.03, 95%CI=0.72-1.46; recessive model: OR=1.08, 95%CI=0.88-1.32). Subgroup analysis based on hypertension revealed no association in hypertensive PICH or in normotensive PICH. CONCLUSIONS: Our case-control study in a Chinese population did not detect a significant association between ACT signal peptide A/T polymorphism and PICH. Moreover, meta-analysis combining data from relevant studies failed to provide evidence for the association. Further well-designed studies with larger sample sizes are warranted to verify our findings.

Nuclear α1-antichymotrypsin promotes chromatin condensation and inhibits proliferation of human hepatocellular carcinoma cells.

BACKGROUND & AIMS: α1-Antichymotrypsin (α1-ACT), a member of the serpin family (SERPINA3), is an acute-phase protein secreted by hepatocytes in response to cytokines such as oncostatin M. α1-ACT is a protease inhibitor thought to limit tissue damage produced by excessive inflammation-associated proteolysis. However, α1-ACT also is detected in the nuclei of cells, where its activities are unknown. Expression of α1-ACT is down-regulated in human hepatocellular carcinoma (HCC) tissues and cells; we examined its roles in liver regeneration and HCC proliferation. METHODS: We measured levels of α1-ACT messenger RNA in human HCC samples and healthy liver tissue. We reduced levels of α1-ACT using targeted RNA interference in human HCC (HepG2) and mouse hepatocyte (AML12) cell lines, and overexpressed α1-ACT from lentiviral vectors in Huh7 (HCC) cells and adeno-associated viral vectors in livers of mice. We assessed proliferation, differentiation, and chromatin compaction in cultured cells, and liver regeneration and tumor formation in mice. RESULTS: Reducing levels of α1-ACT promoted proliferation of HCC cells in vitro. Oncostatin M up-regulated α1-ACT expression and nuclear translocation, which inhibited HCC cell proliferation and activated differentiation of mouse hepatocytes. We identified amino acids required for α1-ACT nuclear localization, and found that α1-ACT inhibits cell-cycle progression and anchorage-independent proliferation of HCC cells. HCC cells that overexpressed α1-ACT formed smaller tumors in mice than HCC cells that did not express the protein. α1-ACT was observed to self-associate and polymerize in the nuclei of cells; nuclear α1-ACT strongly bound chromatin to promote a condensed state that could prevent cell proliferation. CONCLUSIONS: α1-ACT localizes to the nuclei of hepatic cells to control chromatin condensation and proliferation. Overexpression of α1-ACT slows the growth of HCC xenograft tumors in nude mice.

Deletion of IκBα activates RelA to reduce acute pancreatitis in mice through up-regulation of Spi2A.

BACKGROUND & AIMS: The transcription factor nuclear factor-κB (NF-κB) (a heterodimer of NF-κB1p50 and RelA) is activated rapidly in acute pancreatitis (AP). However, it is not clear whether NF-κB promotes or protects against AP. We used the NF-κB inhibitor protein, inhibitor of κB (IκB)α, to study the roles of NF-κB in the development of AP in mice. METHODS: IκBα or the combination of IκBα and RelA selectively were deleted from pancreas of mice using the Cre/locus of cross-over P strategy; cerulein or L-arginine were used to induce AP. We performed microarray analyses of the IκBα- and RelA-deficient pancreata. DNA from healthy individuals and patients with acute or chronic pancreatitis were analyzed for variants in coding regions of alpha-1-antichymotrypsin. RESULTS: Mice with pancreas-specific deletion of IκBα had constitutive activation of RelA and a gene expression profile consistent with NF-κB activation; development of AP in these mice was attenuated and trypsin activation was impaired. However, AP was fully induced in mice with pancreas-specific deletion of IκBα and RelA. By using genome-wide expression analysis, we identified a cluster of NF-κB-regulated genes that might protect against the development of AP. The serine protease inhibitor 2A (Spi2a) was highly up-regulated in IκBα-deficient mice. Lentiviral-mediated expression of Spi2A reduced the development of AP in C57BL/6 and RelA-deficient mice. However, we did not correlate any variants of alpha-1-antichymotrypsin, the human homologue of Spi2a, with acute or chronic pancreatitis. CONCLUSIONS: Pancreas-specific deletion of IκBα results in nuclear translocation of RelA and reduces AP induction and trypsin activation in mice after administration of cerulein or L-arginine. Constitutive activation of RelA up-regulates Spi2A, which protects mice against the development of AP.

Tomato cystatin SlCYS8 as a stabilizing fusion partner for human serpin expression in plants.

Studies have reported the usefulness of fusion proteins to bolster recombinant protein yields in plants. Here, we assess the potential of tomato SlCYS8, a Cys protease inhibitor of the cystatin protein superfamily, as a stabilizing fusion partner for human alpha-1-antichymotrypsin (α1ACT) targeted to the plant cell secretory pathway. Using the model expression platform Nicotiana benthamiana, we show that the cystatin imparts a strong stabilizing effect when expressed as a translational fusion with α1ACT, allowing impressive accumulation yields of over 2 mg/g of fresh weight tissue for the human serpin, a 25-fold improvement on the yield of α1ACT expressed alone. Natural and synthetic peptide linkers inserted between SlCYS8 and α1ACT have differential effects on protease inhibitory potency of the two protein partners in vitro. They also have a differential impact on the yield of α1ACT, dependent on the extent to which the hybrid protein may remain intact in the plant cell environment. The stabilizing effect of SlCYS8 does not involve Cys protease inhibition and can be partly reproduced in the cytosol, where peptide linkers are less susceptible to degradation. The effect of SlCYS8 on α1ACT yields could be explained by: (i) an improved translation of the human protein coding sequence; and/or (ii) an overall stabilization of its tertiary structure preventing proteolytic degradation and/or polymerization. These findings suggest the potential of plant cystatins as stabilizing fusion partners for recombinant proteins in plant systems. They also underline the need for an empirical assessment of peptide linker functions in plant cell environments.

A locus for familial early-onset Alzheimer's disease on the long arm of chromosome 14, proximal to the alpha 1-antichymotrypsin gene.

Although mutations in the beta-amyloid precursor protein gene (APP) on chromosome 21 cause some cases of early-onset Alzheimer's disease (AD), most cases evidently do not have mutations in APP. We analysed ten early-onset families for linkage to APP and markers elsewhere in the genome. One family (F172) was consistent with linkage to chromosome 21 and was subsequently found to have an APP Val to Ile mutation. Of the others, all but one were consistent with linkage to markers in the middle long arm of chromosome 14. However, no family showed independent evidence of linkage with two point analysis and only one showed independent evidence of linkage on multipoint analysis. Therefore, we cannot rule out heterogeneity at these loci although tests for heterogeneity were not significant.

APOE*4-associated Alzheimer's disease risk is modified by alpha 1-antichymotrypsin polymorphism.

Genetic studies on Alzheimer's disease (AD), a devastating neurodegenerative disorder, have identified the apolipoprotein E (APOE) gene as a strong susceptibility marker for AD. The E*4 allele of APOE is a major risk factor for AD regardless of age of onset or family history. However, the observation that the APOE*4 allele is neither necessary nor sufficient for the expression of AD emphasizes the involvement of other environmental or genetic elements that, either in conjunction with APOE*4 or alone, increase an individual's risk of developing AD. Among the candidate genes that may affect the risk of this multifactorial disease is the gene coding for alpha 1-antichymotrypsin (ACT). Like APOE protein, ACT binds to beta-amyloid peptide (A beta P) with high affinity in the filamentous deposits found in the AD brain and serves as a strong stimulatory factor in the polymerization of A beta P into amyloid filaments. In AD brains, ACT expression is enhanced, particularly in areas that develop amyloid plaques, suggesting that ACT may play an important role in the pathogenesis of AD. Here we show that a common polymorphism in the signal peptide of ACT confers a significant risk for AD. Furthermore, the APOE*4 gene dosage effect associated with AD risk is significantly modified by the ACT polymorphism. We have also identified a unique combination of the ACT/AA and APOE 4/4 genotypes as a potential susceptibility marker for AD, as its frequency was 1/17 in the AD group compared to 1/313 in the general population control. Our data show that ACT behaves as a modifier gene that alters the AD risk conventionally associated with the APOE*4 allele.

Recurrent intracerebral hemorrhage in patients with hypertension is associated with APOE gene polymorphism: a preliminary study.

BACKGROUND: Recurrent intracerebral hemorrhage (ICH) in patients with hypertension has been reported in Asia and is attributed to poor control of blood pressure, but there may be a genetic basis. This study evaluates the roles of apolipoprotein E (APOE) and α-1 antichymotrypsin (ACT) genes in patients with recurrent hypertensive ICH and compares patients with nonrecurring hypertensive ICH and normal controls. METHODS: Thirty-three recurrent and 101 nonrecurrent patients with hypertension and ICH were included. The demographic, stroke risk factors, and computed tomographic or magnetic resonance imaging findings were recorded. Magnetic resonance angiography or digital subtraction angiography and vasculitic profile were done in recurrent group to exclude secondary causes of ICH. APOE and ACT gene polymorphisms were assessed with polymerase chain reaction studies in patients with ICH and 188 healthy controls. RESULTS: The demographic and clinical variables were similar in patients with recurrent and nonrecurrent ICH, but patients with recurrent ICH were older (61.1 vs 57.2 years). In the recurrent ICH group, only 7 (10%) out of 69 episodes were lobar; the remaining were deep-seated hematomas. In the nonrecurrent group, 7 (6.9%) patients had lobar ICH. The E2 (odds ratio 4.32; 95% confidence interval 1.65-11.28; P = .003) and E4 alleles of APOE (odds ratio 11.33; 95% confidence interval 5.37-23.02; P < .0001) were significantly related to recurrent ICH compared to healthy controls. The E4 allele was also independently related to recurrent compared to nonrecurrent ICH, even after adjustment for stroke risk factors (odds ratio 25.99; 95% confidence interval 11.65-57.97; P < .0001). ACT gene polymorphism, however, was not related to recurrent ICH compared to controls and nonrecurrent ICH. CONCLUSIONS: APOE polymorphism may contribute to the recurrence of hypertensive ICH.

Association between α1-antichymotrypsin signal peptide -15A/T polymorphism and the risk of Alzheimer's disease: a meta-analysis.

No consensus has been recently reached at the relationship between the α1-antichymotrypsin (ACT) signal peptide -15A/T polymorphism and Alzheimer's disease (AD) risk. Thus, our study aimed to better assess this association by performing a meta-analysis, including 4,212 cases and 4,039 controls from 29 studies. Odds ratios (ORs) with the 95% confidence interval (CI) were used to assess the strength of relationship between ACT -15A/T polymorphism and AD risk. Overall, a borderline statistically significant association was detected under recessive model comparison in all subjects (AA vs. AT+TT: OR 1.12, 95% CI 1.01-1.25, P = 0.04). But in subgroup analysis by ethnicity, no significant association was found in Caucasians, Asians, or Africans. Moreover, after exclusion of one study which affect the heterogeneity, the ACT A allele and AA genotype were statistically associated with late-onset AD (LOAD) risk (AA vs. TT: OR 1.25, 95% CI 1.06-1.48, P = 0.007, A vs. T: OR 1.12, 95% CI 1.03-1.21, P = 0.008), especially in Caucasians. In conclusion, our study suggests that the common α1-antichymotrypsin signal peptide -15A/T polymorphism may not be a major risk factor for AD. However, the polymorphism is capable of increasing LOAD risk.

A companion protease inhibitor for the protection of cytosol-targeted recombinant proteins in plants.

We reported earlier the potential of tomato cathepsin D inhibitor (SlCDI) as an in-built stabilizing agent for the protection of recombinant proteins in transgenic plant leaf crude extracts (Plant Biotechnol J.4, 359-368). Here we document the potential of SlCDI for the in situ protection of proteins in potato leaves. Total protein assays with control and SlCDI-expressing potato lines indicated a positive impact of slcdi transgene expression on leaf protein content, with a mean relative increase of 35%-40% depending on the light regime. Out of approximately 700 proteins detected on 2-D gels, only 20 exhibited a significantly altered level on a protein-specific basis, whereas most proteins were up-regulated on a leaf fresh weight basis, albeit at variable rates. Quantitative reverse trancriptase-PCR assays for rubisco activase showed similar transcript levels in leaves of test and control lines despite protein levels increased by two- to threefold in SlCDI-expressing lines. These observations, along with the unrelated biological functions assigned to MS-identified proteins up-regulated in leaves and protease assays showing slightly increased proteasome activity in protein extracts of SlCDI-expressing lines, suggest a general, proteasome-independent protein stabilizing effect of SlCDI in planta. Transient expression assays with human alpha(1)-antichymotrypsin also showed a stabilizing effect for SlCDI on heterologous proteins, leading to net levels of the human protein increased by approximately 2.5-fold in SlCDI-expressing plants. These data illustrate, overall, the potential of SlCDI as an in vivo protein-stabilizing agent in transgenic plant systems, useful to improve protein levels and recombinant protein accumulation.

[Microarray analysis of human primary astrocytes activated by beta amyloid peptide and alpha1-antichymotrypsin].

OBJECTIVE: To investigate gene expression of astrocytes under the actions of amyloid peptide Abeta(1-42) and alpha1-antichymotrypsin (ACT) and to explore the characteristics of inflammatory reactions occurring in brain of Alzheimer's patients. METHODS: Human primary astrocytes were cultured to the second passage and then treated with lipopolysaccharide (LPS), Abeta(1-42) (50 micromol/L) and Abeta(1-42)/ACT (50:5 micromol/L) respectively. At 24 h, cells were harvested for total RNA extraction. Gene expression profile was screened by microarray technique. And the function enrichment of differentially expressed genes and the signal transduction pathways involved were analyzed. RESULTS: In comparison with LPS, both Abeta(1-42) and Abeta(1-42)/ACT had demonstrated marked effects on altering the astrocyte gene expression. And the gene up-regulation was predominant. But the gene expression spectrum varied between different groups. Gene ontology analysis showed that Abeta(1-42) up-regulated genes modulated inflammation, oxidative stress and immune response. But Abeta(1-42)/ACT had significant effects on genes related with mitochondrial impairment, apoptosis, oxidative stress, epithelial differentiation and vasculogenesis. The analysis of up-regulated genes, such as interleukin 6 (IL-6) and tumor necrosis factor alpha (TNFalpha), showed that transcriptional factors and downstream genes of signal transduction pathways potentiated further the inflammatory response and cell apoptosis and increased the production of abnormal Abeta. CONCLUSION: Abeta(1-42) induces the inflammation of astrocytes. And the Abeta(1-42)/ACT complex has diverse effects on the gene expression of astrocytes. Thus both proteins play important roles in the activation of astrocytes. In addition, IL-6, TNFalpha and their signal pathways are important in the pathogenic process of Alzheimer's disease.

The epidermal growth factor receptor ligand amphiregulin is a negative regulator of hepatic acute-phase gene expression.

BACKGROUND/AIMS: The modulation of the hepatic acute-phase reaction (APR) that occurs during inflammation and liver regeneration is important for allowing normal hepatocellular proliferation and the restoration of homeostasis. Activation of acute-phase protein (APP) gene expression by interleukin-6 (IL-6)-type cytokines is thought to be counteracted by growth factors released during hepatic inflammation and regeneration. The epidermal growth factor receptor (EGFR) ligand amphiregulin (AR) is readily induced by inflammatory signals and plays a nonredundant protective role during liver injury. In this paper, we investigated the role of AR as a modulator of liver APP gene expression. METHODS: Expression of APP genes was measured in the livers of AR(+/+) and AR(-/-)mice during inflammation and regeneration and in cultured liver cells treated with AR and oncostatin M (OSM). Crosstalk between AR and OSM signalling was studied. RESULTS: APP genes were overexpressed in the livers of AR(-/-) mice during inflammation and hepatocellular regeneration. In cultured AR-null hepatocytes and human hepatocellular carcinoma (HCC) cells after AR knockdown, APP gene expression is enhanced. AR counteracts OSM-triggered signal transducer and activator of transcription 3 signalling in hepatocytes and attenuates APP gene transcription. CONCLUSIONS: Our data support the relevance of EGFR-mediated signalling in the modulation of cytokine-activated pathways. We have identified AR as a key regulator of hepatic APP gene expression during inflammation and liver regeneration.

Effect of reactive center loop structure of antichymotrypsin on inhibition of duodenase activity.

Interaction between duodenase (a granase family member) from bovine duodenal mucosa and recombinant antichymotrypsin (rACT) and its P1 variants has been studied. Association rate constants (k(a)) were 11, 6.8, and 17 mM(-1).sec(-1) for rACT, ACT L358M, and ACT L358R, respectively. Natural antitrypsin (AT) compared to ACT was a 20 times more effective duodenase inhibitor (in terms of k(a)). Duodenase interacted with P1 variants of ACT via a suicide mechanism with stoichiometry of the process SI = 1.2. The nature of the P1 residue of the inhibitor did not influence the interaction if other residues did not meet conformational requirements of the duodenase substrate-binding pocket. Also, interaction of duodenase with ACT variants containing residues from AT reaction center loop (rACT P2-P3', rACT P3-P4', rACT P4-P3', and rACT P6-P4') was studied. The inhibition type ([E](0) = 1.10(-7) M, 25 degrees C) was revealed to be reversible-like, and efficacy of inhibition decreased with increase in the substituted part of the reactive center loop. Constants of inhibition (K(i)) were measured. Efficacy of interaction between the enzyme (duodenase) and inhibitor depends on topochemical correspondence between a substrate-binding pocket of the enzyme and substrate structure.

Pharmacogenetics in pancreatic cancer. Highlights from the 45th ASCO annual meeting. Orlando, FL, USA. May 29-June 2, 2009.

Cancer pharmacogenetics is a popular and evolving field in medicine with applications in various types of tumours helping clinicians to apply a more personalized medicine by providing information of prognostic, predictive and therapeutic value. Such evidence of pharmacogenetic applications is been already available in colon cancer (e.g. KRAS status, mismatch repair genes status, UGT1A1 polymorphisms), lung cancer (EGFR mutations, ERCC1 mutations), breast cancer (HER2/neu overexpression) and many others. In all these tumors, the genetic information is rendering the management of the involved patients safer and more effective. Interesting abstracts and announcements from the perspective of pharmacogenomics in pancreatic cancer included Abstract #4611 which suggested the use of a novel genomic study able to detect specific single nucleotide polymorphisms (SNPs) with prognostic value, Abstract #4615 which showed that the known proteins alpha1-antitrypsin and alpha1-antichymotrypsin may be predictive of response to gemcitabine and survival, and Abstract #11097 which suggested that human R protein (HuR) expression may be a useful predictive biomarker of gemcitabine treatment. The authors also present here a few other abstracts of pharmacogenomic interest which had negative findings, but believed to be of clinical importance.

Analysis of candidate genes and hypertension in African American adults.

OBJECTIVE: We investigated the associations between hypertension status and the genotypes of four single nucleotide polymorphism (SNP) sites in four hypertension-related genes (Angiotensinogen [AGT], Angiotensin I Converting Enzyme [ACE], Angiotensinogen II receptor, subtype 1 [AGTR1], and Alpha 1-Antichymotrypsin [ACT or SERPINA3]), in an African American sample. METHODS: DNA from 628 participants of the Carolina African American Twin Study of Aging project, a population-based study of African American adult twins, was genotyped using SNPs shown to be associated with hypertension in other studies. RESULTS: The ACE SNP (ACE4 or A-240T) was associated with hypertension (P = .047 in a generalized estimating equations alternating logistics regression model that included age, body mass index, sex, and education. The analysis indicated a protective effect of the TT genotype (odds ratio [OR] 1.59, 95% confidence interval [CI] 1.03-2.48, P = .04) and of the AT genotype (OR 1.91, 95% CI 1.01-3.62, P = .047) compared with the AA genotype. DISCUSSION: These results extend previous findings of associations of various polymorphisms of ACE to hypertension and support the association of hypertension to the A allele of ACE4. The potential for this polymorphism to alter expression by its position in the gene's promoter region suggests that future studies of altered ACE protein activity are warranted.

[Biochemical and molecular diagnosis of alpha 1 antitrypsin deficiency in a Tunisian family]. / Diagnostic biochimique et moléculaire du déficit en alpha 1 antitrypsine dans une famille tunisienne.

Our study investigated alpha 1 antitrypsin deficiency (AATD) diagnosis in a family originated from central Tunisia and showing a familial history of asthma. Biochemical and genetic diagnosis for AATD was performed according to current diagnostic standards. AAT level quantification in affected individuals showed plasma AAT levels consistent with intermediate AATD (ranged from 0.91 to 1.04 g/L). The molecular analysis was assessed using the genotyping of the most prevalent PI*S and PI*Z SERPINA1 mutations and the sequencing of AAT coding exons for rare AATD variants detection. No PI*S or PI*Z deficient variants were seen in this family. Sequencing results showed the inheritance of the deficient rare variant PI*M(wurzburg) (P369S) at the heterozygous state in the mother and two affected siblings. However, AATD status remains unexplained in the third affected case, with no mutations detected in the AAT coding exons.

An original SERPINA3 gene cluster: elucidation of genomic organization and gene expression in the Bos taurus 21q24 region.

BACKGROUND: The superfamily of serine proteinase inhibitors (serpins) is involved in numerous fundamental biological processes as inflammation, blood coagulation and apoptosis. Our interest is focused on the SERPINA3 sub-family. The major human plasma protease inhibitor, alpha1-antichymotrypsin, encoded by the SERPINA3 gene, is homologous to genes organized in clusters in several mammalian species. However, although there is a similar genic organization with a high degree of sequence conservation, the reactive-centre-loop domains, which are responsible for the protease specificity, show significant divergences. RESULTS: We provide additional information by analyzing the situation of SERPINA3 in the bovine genome. A cluster of eight genes and one pseudogene sharing a high degree of identity and the same structural organization was characterized. Bovine SERPINA3 genes were localized by radiation hybrid mapping on 21q24 and only spanned over 235 Kilobases. For all these genes, we propose a new nomenclature from SERPINA3-1 to SERPINA3-8. They share approximately 70% of identity with the human SERPINA3 homologue. In the cluster, we described an original sub-group of six members with an unexpected high degree of conservation for the reactive-centre-loop domain, suggesting a similar peptidase inhibitory pattern. Preliminary expression analyses of these bovSERPINA3s showed different tissue-specific patterns and diverse states of glycosylation and phosphorylation. Finally, in the context of phylogenetic analyses, we improved our knowledge on mammalian SERPINAs evolution. CONCLUSION: Our experimental results update data of the bovine genome sequencing, substantially increase the bovSERPINA3 sub-family and enrich the phylogenetic tree of serpins. We provide new opportunities for future investigations to approach the biological functions of this unusual subset of serine proteinase inhibitors.