Immune complexome analysis reveals the presence of immune complexes and identifies disease-specific immune complex antigens in saliva samples from patients with Sjögren's syndrome.

Sjögren's syndrome (SS) is a chronic autoimmune disease that mainly damages the salivary and lacrimal glands. Immune complex (IC) formation triggers local inflammation through IC deposition and decreased antigen function. Some ICs can leak from the lesion and into the saliva, but no salivary ICs have been reported to date. We used immune complexome analysis to comprehensively identify antigens incorporated into IC (IC-antigens) in saliva samples from patients with SS (n = 9) or with xerostomia (n = 7). Neutrophil defensin 1 (67%), small proline-rich protein 2D (67%), myeloperoxidase (44%), neutrophil elastase (44%), cathepsin G (33%), nuclear mitotic apparatus 1 (33%) and phosphatidylinositol 4-phosphate 3-kinase C2 domain-containing subunit gamma (33%) were identified as new IC-antigens specifically and frequently detected in the saliva of SS patients. Of these, neutrophil defensin 1, myeloperoxidase, neutrophil elastase and cathepsin G are neutrophil intracellular proteins, which suggests that repeated destruction of neutrophils due to abnormal autoimmunity may be involved in the pathogenesis of SS. We also analyzed serum samples from three SS patients. There was little overlap of IC-antigens between two of the samples (fewer than 30% of the IC-antigens in the saliva samples), suggesting that many ICs are formed locally and independently of the circulation. In addition, we found that four SS-specific salivary antigens show sequence homology with several proteins of oral microbiomes but no antigen has homology with Epstein-Barr virus proteins. The homology between some IC-antigens and oral microbiome proteins may indicate the impact of oral infection on local autoimmunity through molecular mimicry theory.

Picosecond rotation of a ring-shaped optical lattice by using a chirped vortex-pulse pair.

A novel method of ultrafast rotation of a ring-shaped optical lattice in the picosecond time region was proposed and demonstrated. Our ring-lattice generator was assembled by a pair of linearly chirped pulses with a time delay, a high-order birefringent retarder, and an axially symmetric polarization element. Using a mode-locked Ti:sapphire laser oscillator as a light source, stable two-, four-, and six-petaled ring-lattice rotations were demonstrated with the rotation periods of 1.6, 3.2, and 4.8 ps, respectively. Our method has the potential to open up a new technique to resonantly excite propagating quasi-particles together with their coherent enhancement.

Influenza A virus infection of vascular endothelial cells induces GSK-3ß-mediated ß-catenin degradation in adherens junctions, with a resultant increase in membrane permeability.

Multiorgan failure with vascular hyperpermeability is the final outcome in the progression of seasonal influenza virus pneumonia and influenza-associated encephalopathy, and it is also common in infection with highly pathogenic avian influenza virus. However, the precise molecular mechanism by which influenza virus infection causes vascular endothelial cell hyperpermeability remains poorly defined. We investigated the mechanisms of hyperpermeability of human umbilical vein endothelial cells infected with influenza A virus (IAV)/Puerto Rico/8/34 (PR8) (H1N1). The levels of ß-catenin, a key regulatory component of the vascular endothelial-cadherin cell adhesion complex, were markedly decreased during infection for 28 h, with increments of vascular hyperpermeability measured by transendothelial electrical resistance. Lactacystin (at 2 µM), a proteasome inhibitor, inhibited the decrease in ß-catenin levels. Since the N-terminal phosphorylation of ß-catenin by glycogen synthase kinase (GSK)-3ß is the initiation step of proteasome-dependent degradation, we examined the effects of GSK-3ß suppression by RNA interference in endothelial cells. IAV-infection-induced ß-catenin degradation was significantly inhibited in GSK-3ß-knockdown cells, and transfection of cells with recombinant ß-catenin significantly suppressed IAV-induced hyperpermeability. These findings suggest that IAV infection induces GSK-3ß-mediated ß-catenin degradation in the adherens junctional complexes and induces vascular hyperpermeability. The in vitro findings of ß-catenin degradation and activation of GSK-3ß after IAV infection were confirmed in lungs of mice infected with IAV PR8 during the course of infection from day 0 to day 6. These results suggest that GSK-3ß-mediated ß-catenin degradation in adherens junctions is one of the key mechanisms of vascular hyperpermeability in severe influenza.

The expression profile of the toll-like receptor family in scleroderma dermal fibroblasts.

OBJECTIVES: The toll-like receptor (TLR) family is thought to be expressed in many cell types in the skin and play a role in various diseases. The expression pattern and role of TLRs in systemic sclerosis (SSc) is to be clarified. We investigated the expression profiles of TLR-related genes in SSc fibroblasts, and tried to clarify their roles in the pathogenesis of this disease. METHODS: The expression profile of TLR-related genes was assessed by gene array. Real-time PCR was used to confirm the array result. The protein expression of TLRs and type I collagen was determined by immunoblotting and immunohistochemistry. RESULTS: PCR array revealed that several genes were up- or down-regulated in SSc fibroblasts compared to normal cells. Among them, both mRNA and protein levels of TLR5 and TLR10 were up-regulated in SSc fibroblasts. The transfection of Smad3 siRNA into SSc fibroblasts resulted in the down-regulation of TLR proteins. There was no significant difference in mRNA half-lives of TLR5 and TLR10 between normal and SSc fibroblasts. Immunohistochemical staining revealed that TLRs expression was strongly detected in SSc fibroblasts in vivo. The stimulation of TLR5 signal with flagellin reduced the expression of type I collagen in SSc fibroblasts, but not in normal fibroblasts. CONCLUSIONS: TLR5 and TLR10 expression is increased in SSc fibroblasts in vitro and in vivo, probably at transcript level via the TGF-ß/Smad3 activation. Furthermore, TLR5 itself may have suppressive effects on collagen expression, and its overexpression in SSc fibroblasts may be the negative feedback against tissue fibrosis.

Pathogenicity of exopolysaccharide-producing Actinomyces oris isolated from an apical abscess lesion.

AIM: To demonstrate a capacity for producing exopolysaccharides (EPSs) and an ability to form biofilm on abiotic materials of Actinomyces oris strain K20. METHODOLOGY: The productivity of EPSs and the ability to form biofilm of strain K20 were evaluated by measuring viscosity of spent culture media and by scanning electron microscopy (SEM) and the biofilm assay on microtitre plates, respectively. High-performance liquid chromatography was used to determine the chemical composition of the viscous materials. To examine the role of the viscous materials attributable to the pathogenicity in this organism, the ability of strain K20 to induce abscess formation was compared in mice to that of ATCC 27044. RESULTS: The viscosity of the spent culture media of K20 was significantly higher than that of ATCC 27044. Strain K20 showed dense meshwork structures around the cells and formed biofilms on microtitre plates, whereas ATCC 27044 did not. Chemical analysis of the viscous materials revealed that they were mainly composed of neutral sugars with mannose constituting 77.5% of the polysaccharides. Strain K20 induced persistent abscesses in mice lasting at least 5 days at a concentration of 10(8) cells mL(-1), whereas abscesses induced by ATCC 27044 healed and disappeared or decreased in size at day 5. CONCLUSIONS: Strain K20 produced EPSs, mainly consisting of mannose, and formed biofilms. This phenotype might play an important role for A. oris to express virulence through the progression of apical periodontitis.

Prevalence of and risk factors for depressive symptoms in non-tuberculous mycobacterial pulmonary disease.

BACKGROUND: The presence of depressive symptoms in patients with non-tuberculous mycobacterial pulmonary disease (NTM-PD) is an important research topic; however, the prevalence of depressive symptoms and the factors that influence their development are unclear.OBJECTIVE: To analyse the association between CES-D (Center for Epidemiological Studies Depression Scale) scores and clinical parameters such as age, disease duration, pulmonary function, imaging findings, blood data, physical functions, sleep disturbances, respiratory symptoms and health-related quality of life (HRQOL).METHODS: We conducted a cross-sectional retrospective study of 114 patients with NTM-PD at a single centre from March 2016 to January 2021 to evaluate the relationship between CES-D scores and clinical parameters.RESULTS: Participants had a median age of 64 years; 32.5% of them had depressive symptoms. Disease duration, albumin, C-reactive protein, pulmonary function, dyspnoea, exercise capacity, respiratory symptoms, cough-related HRQOL and sleep disturbances were associated with depressive symptoms. Binomial logistic regression analyses indicated that the CES-D score was significantly associated with cough-related HRQOL and sleep disturbances.CONCLUSION: A high percentage of NTM-PD patients in this study experienced depressive symptoms, and these patients had abnormalities of various clinical parameters. Cough-related HRQOL and sleep disturbance had a strong influence on the development of depressive symptoms.

microRNA-mediated keratinocyte hyperproliferation in psoriasis vulgaris.

BACKGROUND: Psoriasis is a chronic inflammatory skin disease characterized by intense proliferation and abnormal differentiation of keratinocytes, although the pathogenesis is still not completely clarified. OBJECTIVES: We investigated the mechanism of keratinocyte proliferation seen in psoriasis, focusing on microRNA (miRNA). MATERIALS AND METHODS: miRNAs were extracted from tissues and sera of psoriasis, atopic dermatitis and healthy control. To determine pathogenic miRNAs, we performed miRNA polymerase chain reaction (PCR) array analysis. The results were confirmed with quantitative real-time PCR, in situ hybridization, immunohistochemistry, transient transfection of siRNA and inhibitor in cultured keratinocytes and Western blotting. RESULTS: PCR array analysis using tissue miRNA demonstrated miR-424 level was markedly decreased in psoriasis skin in vivo. Protein expression of mitogen-activated protein kinase kinase 1 (MEK1) or cyclin E1, predicted target genes of miR-424, was increased in psoriatic skin, although their mRNA levels were not. The transfection of specific inhibitor of miR-424 in normal human keratinocytes led to upregulation of MEK1 or cyclin E1 protein, and resulted in increased cell proliferation. On the other hand, cell number was significantly decreased when cells were transfected with siRNA for MEK1 or cyclin E1. Furthermore, we first investigated serum miRNA levels in psoriasis. Although not significant, serum miR-424 concentration tended to be decreased in patients with psoriasis compared with healthy controls. CONCLUSIONS: Decreased miR-424 expression and subsequently increased MEK1 or cyclin E1 may play a key role in the pathogenesis of psoriasis. Investigation of the regulatory mechanisms of keratinocyte proliferation by miRNA may lead to new treatments and a disease activity marker.

Low serum levels of total and high-molecular-weight adiponectin predict the development of metabolic syndrome in Japanese-Americans.

BACKGROUND: Adiponectin is thought to play a significant role in the development of both insulin resistance and metabolic syndrome. Yet, there is very few evidence about the association plasma adiponectin and metabolic syndrome in the prospective study. Adiponectin exists as multimers in serum, and high-molecular-weight (HMW) adiponectin is particularly considered to be the active form of the protein. AIM: We investigated whether serum HMW adiponectin as well as total adiponectin is associated with the development of metabolic syndrome in a longitudinal study. SUBJECTS AND METHODS: We enrolled 224 men and 312 women of Japanese- Americans without metabolic syndrome at baseline who were followed for an average of 3.2 yr. The association of plasma total and HMW adiponectin with a progression to metabolic syndrome was examined. RESULTS: Subjects who developed metabolic syndrome had significantly lower plasma total and HMW adiponectin levels at baseline than those who did not develop metabolic syndrome. In a Cox proportional hazards model, lower total and HMW adiponectin levels were independent risk factors for the development of metabolic syndrome after adjusting for age, body mass index, classification of 75-g glucose tolerance test, and homeostasis model assessment (hazards ratio: total, 0.684, p=0.017, in men; 0.606, p=0.003, in women; HMW, 0.687, p=0.014, in men; 0.704, p=0.029, in women, respectively). CONCLUSIONS: Low circulating levels of total and HMW adiponectin may be a possible predictor for the development of metabolic syndrome.

Health-related quality of life associates with clinical parameters in patients with NTM pulmonary disease.

BACKGROUND: Previous studies have shown a reduction in health-related quality of life (HRQoL) in patients with non-tuberculous mycobacterial pulmonary disease (NTM-PD). However, the causes of this decline and the factors that contribute to it are unknown. This study was conducted to analyse the association between the St George´s Respiratory Questionnaire (SGRQ) and clinical parameters, including age, disease duration, body composition, pulmonary function, chest X-ray findings, blood data and physical function.METHODS: We performed a single-centre, cross-sectional, retrospective study of 101 patients with NTM-PD from December 2016 to October 2019. The relationship between the SGRQ scores and clinical parameters was evaluated.RESULTS: The median patient age was 67.0 years. Pulmonary function, radiological score, albumin levels, C-reactive protein levels and incremental shuttle walk test distance (ISWD) were significantly correlated with the total and component scores on the SGRQ. Multiple regression analysis showed that the SGRQ score was significantly associated with radiological score, pulmonary function and ISWD.CONCLUSION: This study was the first to assess the effect of clinical parameters on the SGRQ in patients with NTM-PD. HRQoL as determined using the SGRQ was associated with the radiological score, pulmonary function and ISWD in patients with NTM-PD.

Functional interaction between human histocompatibility leukocyte antigen (HLA) class II and mouse CD4 molecule in antigen recognition by T cells in HLA-DR and DQ transgenic mice.

Studies in vitro have suggested that a species barrier exists in functional interaction between human histocompatibility leukocyte antigen (HLA) class II and mouse CD4 molecules. However, whether mouse CD4+ T cells restricted by HLA class II molecules are generated in HLA class II transgenic mice and respond to peptide antigens across this barrier has remained unclear. In an analysis of T cell responses to synthetic peptides in mice transgenic for HLA-DR51 and -DQ6, we found that DR51 and DQ6 transgenic mice acquired significant T cell response to influenza hemagglutinin-derived peptide 307-319 (HA 307) and Streptococcus pyogenes M12 protein-derived peptide 347-397 (M6C2), respectively. Inhibition studies with several monoclonal antibodies showed that transgenic HLA class II molecules presented these peptides to mouse CD4+ T cells. Furthermore, T cell lines specific for HA 307 or M6C2 obtained from the transgenic mice could respond to the peptide in the context of relevant HLA class II molecules expressed on mouse L cell transfectants that lack the expression of mouse MHC class II. These findings indicate that interaction between HLA class II and mouse CD4 molecules is sufficient for provoking peptide-specific HLA class II-restricted T cell responses in HLA class II transgenic mice.

Analysis of DNA sequence polymorphism at the cMWG699 locus reveals phylogenetic relationships and allopolyploidy within Hordeum murinum subspecies.

Hordeum murinum L. is one of the most widely distributed species in the genus Hordeum. This species is composed of three subspecies with three ploidy levels, namely subsp. glaucum (2x=14), subsp. murinum (4x=28) and subsp. leporinum (4x=28, 6x=42). These three subspecies are morphologically similar and are frequently referred to as the 'murinum complex'. Although many cytological studies suggest that the murinum complex is allopolyploid, one inter-specific hybridization study suggested that it is autopolyploid. The goals of the present study are to identify nucleotide variation in the cMWG699 locus in the polyploid genomes of the murinum complex, to conduct molecular phylogenetic analysis of this locus, and to clarify the allo- versus auto-polyploidy status of the murinum complex. For this purpose, PCR-RFLP analysis was conducted with HhaI and SspI restriction enzymes on 80 H. murinum accessions. Single enzyme digestion data revealed polymorphism between diploid and polyploids, and double-digestion revealed polymorphism between tetra- and hexaploids. The nucleotide sequences of clones clearly show that polyploid murinum species are allopolyploid. In addition, DNA sequence analysis indicated that one donor of the tetraploid was subsp. glaucum (2x), as has been suggested previously by cytological studies. The other diploid donors were not identified, but at least one group of sequences common to 4x and 6x genomes (namely clonetype B) was highly diverged from 2x subsp. glaucum. The two tetraploid subspecies, 4x subsp. murinum and 4x subsp. leporinum, had identical DNA sequences, suggesting that these two subspecies are not differentiated at the cMWG699 locus.

Angularly-dispersed optical parametric amplification of optical pulses with one-octave bandwidth toward monocycle regime.

We demonstrated experimentally the generation of 65-microJ, 5.8-fs optical pulses with an ultrabroad bandwidth (540-1000 nm) by the use of a double-pass angularly-dispersed non-collinear optical parametric amplifier. We also confirmed up to the 95-microJ output from the amplifier when seed pulses were not pre-compensated for. Furthermore, we confirmed that the broadband pump pulses brought in the broader gain bandwidth (from 520 to 1080 nm) than numerical estimation based on CW-pump approximation. To the best of our knowledge, this is the system with the broadest gain bandwidth.

[Left atrial myxoma surgically resected in acute phase of hemorrhagic cerebral infarction; report of a case].

A 61-year-old man who had been suffered from several episodes of cerebral infarction for 3 years complained of left-sided paresthesia and was pointed out hemorrhagic infarction in the right frontal area of the brain by computed tomography (CT) and magnetic resonance imaging (MRI). The echocardiography showed left atrial mass 9 cm in length attaching to the atrial septum, which was diagnosed as left atrial myxoma causing cerebral embolism. As serial MRI showed frequent episode of cerebral infarction, we performed surgical resection of the cardiac tumor on the 10th day after the onset of the neurological symptom. Anticoagulation during cardiopulmonary bypass was maintained with 1.5 mg/kg of heparin sodium and 80 mg/hour of nafamostat mesilate (FUT). Postoperative course was uneventful without neurological deterioration.

Conserved BRCT regions of TopBP1 and of the tumor suppressor BRCA1 bind strand breaks and termini of DNA.

The BRCT region, the carboxyl-terminus of BRCA1 (the breast cancer susceptibility gene 1 product), is a ubiquitous region homologous to regions in DNA repair enzymes and cell cycle regulators. We showed that the BRCT regions bound DNA fragments, using the TopBP1 protein (topoisomerase II binding protein 1), with eight BRCTs as a model protein. The bindings were independent of DNA sequences, forms of DNA termini and energy. The BRCT-DNA complex showed resistance to an exonuclease, indicating that BRCT bound DNA breaks. The BRCTs also bound DNA nicks, suggesting that BRCTs play an important role in detection of both single- and double-strand DNA breaks or ends. On the other hand, BRCTs did not bind circular intact DNA. BRCTs of BRCA1 also bound DNA termini. Since some BRCTs are unique general elements in some tumor suppressions, these findings will reveal novel aspects of the tumor suppression mechanism.

p53 contains a DNA break-binding motif similar to the functional part of BRCT-related region of Rb.

The BRCT regions are two repeating structures at BRCA1 carboxyl-terminus and are ubiquitous in some proteins involved in DNA repair and cell cycle checkpoints. It was shown that BRCTs of TopBP1, BRCA1, and BRCT-Ws of Rb bound DNA ends and breaks. We indicate here that the C-terminus of p53 tumor suppressor contains a DNA binding motif (residues 327-333 in human), whose features are similar to those of the part of BRCT-W in Rb with DNA binding activity. The short motif was required for the gel retardation activity of DNA fragments, since residues 311-333 showed the activity while residues 311-326 showed no activity. Significant numbers of total p53 and its fragments with the motif formed multimerizing complexes and associated with DNA ends and breaks. These results suggest the common presence of DNA binding motifs that can recognize DNA ends or damages in major tumor suppressors, Rb, BRCA1 and p53. The oncogenic activity of p53 C-terminus (residues 311-393) required both the DNA damage recognition motif and the repair enzyme-associating domain.

Retinoblastoma susceptibility protein, Rb, possesses multiple BRCT-Ws, BRCA1 carboxyl-terminus-related W regions with DNA break-binding activity.

The BRCT region, the carboxyl-terminus of BRCA1 (the breast cancer susceptibility gene 1 product), is ubiquitous in several proteins that participate in cell cycle checkpoints and DNA repair. We have previously shown that the BRCT regions of TopBP1 (DNA topoisomerase II binding protein 1) and BRCA1 bound DNA breaks. A BRCT-related region, BRCT-W1, in the retinoblastoma susceptibility gene product (Rb) also could bind DNA fragments, independently of DNA sequences. Five BRCT-W regions were found in the Rb family. All BRCT-Ws of Rb bound DNA fragments. Electron microscopy and treatment with an exonuclease showed that BRCT-Ws bound double-strand DNA breaks. Since some BRCTs are exceptional common relating elements in tumor suppression, our findings reveal novel aspects of the tumor suppression mechanism.

Hybrid alpha-amylases produced by the transformants of Bacillus subtilis. III. A possible mechanism of formation of hybird alpha-amylases.

Alpha-Amylases (NA64 and NA20) produced by the representative transformants Bacillus subtilis NA64 and NA20 were hybrid enzymes between the two parental alpha-amylases (NAT and MAR) produced by the DNA donor strain of Bacillus natto IAM 1212 and the DNA recipient strain of B. subtilis 6160, a derivative of B. subtilis 168. In order to elucidate a possible mechanism of formation of the hybrid alpha-amylases, 14C-labeled alpha-amylase (SAC) produced by B. subtilis var. amylosarcchariticus, [3H]lysine- and [3H]arginine-labeled alpha-amylases (MAR, NA64, NA20, NAT and SAC), [3H]lysine-labeled alpha-amylase (SAC) and [3H]glucosamine-labeled alpha-amylase (NA64) were purified through ammonium sulfate precipitation, carboxy-methylcellulose and DEAE-Sephadex A-50 column chromatography and immunoprecipitation with rabbit antiserum against alpha-amylase (SAC). Peptide compositions of the tryptic digests from the labeled alpha-amylases were analyzed by double-label AG 50W-X2 column chromatography. On the other hand, amino- and carboxy-terminal amino acid residues of unlabeled alpha-amylases (MAR, NA64, NA20 and NAT) were analyzed. Based on these results, the possibility of DNA recombination events in the alpha-amylase structure gene and on the previous results, we attempted to estimate possible peptide arrangements for the four alpha-amylases (MAR, NA64, NA20 and NAT) and possible recombination regions to form the hybrid enzymes introduced by the DNA-mediated transformation of B. subtilis 6160.

Site-directed mutagenesis of active site residues in Bacillus subtilis alpha-amylase.

Site-directed mutagenesis of Bacillus subtilis N7 alpha-amylase has been performed to evaluate the roles of the active site residues in catalysis and to prepare an inactive catalytic-site mutant that can form a stable complex with natural substrates. Mutation of Asp-176, Glu-208, and Asp-269 to their amide forms resulted in over a 15,000-fold reduction of its specific activity, but all the mutants retained considerable substrate-binding abilities as estimated by gel electrophoresis in the presence of soluble starch. Conversion of His-180 to Asn resulted in a 20-fold reduction of kcat with a 5-fold increase in Km for a maltopentaose derivative. The relative affinities for acarbose vs. maltopentaose were also compared between the mutants and wild-type enzyme. The results are consistent with the roles previously proposed in Taka-amylase A and porcine pancreatic alpha-amylase based on their X-ray crystallographic analyses, although different pairs had been assigned as catalytic residues for each enzyme. Analysis of the residual activity of the catalytic-site mutants by gel electrophoresis has suggested that it derived from the wild-type enzyme contaminating the mutant preparations, which could be removed by use of an acarbose affinity column; thus, these mutants are completely devoid of activity. The affinity-purified mutant proteins should be useful for elucidating the complete picture of the interaction of this enzyme with starch.

Crystallization and preliminary X-ray studies of cyclodextrin glucanotransferase from alkalophilic Bacillus sp. 1011.

Large crystals of cyclodextrin glucanotransferase (CGTase) from alkalophilic Bacillus sp. 1011, a typical alkalophilic enzyme, have been obtained at room temperature using polyethylene glycol 3000 and 2-propanol as precipitant. They belong to the triclinic space group P1 with the following unit cell constants: a = 64.93 A, b = 74.45 A, c = 79.12 A, alpha = 85.2 degrees, beta = 105.0 degrees and gamma = 101.0 degrees. The crystallographic asymmetric unit seems to contain two molecules of CGTase, with crystal volume per protein mass (Vm) of 2.41 A3/Da and solvent content of 49% by volume. The crystals diffract to at least 2.0 A resolution and they are suitable for X-ray analysis.

DNA polymorphism at the cytosolic phosphoglucose isomerase (PgiC) locus of the wild plant Arabidopsis thaliana.

DNA variation in a 4.7-kb region of the cytosolic phosphoglucose isomerase (PgiC) locus was investigated for 21 ecotypes of Arabidopsis thaliana. The estimated nucleotide diversity was 0.0038, which was one-third of those in previously investigated loci. Since most of the nucleotide variations (93%) were singleton and doubleton, Tajima's test statistic was significantly negative. About 50% of nucleotide polymorphisms in exons were replacement, which caused significance in McDonald and Kreitman's test when compared with Arabis gemmifera and Cardaminopsis petraea. These results indicated that DNA polymorphism at the PgiC locus was not under neutrality. There were two divergent sequence types in the PgiC region, which were associated with allozyme variation. The Fast allozyme was shown to have originated from the Slow allozyme, since two outgroup species had the Slow form. A phylogenetic tree of ecotypes with the Fast allozyme had the shape of a star phylogeny. Mismatch distribution of the Fast allozyme ecotypes resembled that expected under an expanding population model. These results suggest positive selection for the Fast allozyme of the PGIC in A. thaliana.