Using amplicon sequencing of rpoB for identification of inoculant rhizobia from peanut nodules.

To improve the nitrogen fixation, legume crops are often inoculated with selected effective rhizobia. However, there is large variation in how well the inoculant strains compete with the indigenous microflora in soil. To assess the success of the inoculant, it is necessary to distinguish it from other, closely related strains. Methods used until now have generally been based either on fingerprinting methods or on the use of reporter genes. Nevertheless, these methods have their shortcomings, either because they do not provide sufficiently specific information on the identity of the inoculant strain, or because they use genetically modified organisms that need prior authorization to be applied in the field or other uncontained environments. Another possibility is to target a gene that is naturally present in the bacterial genomes. Here we have developed a method that is based on amplicon sequencing of the bacterial housekeeping gene rpoB, encoding the beta-subunit of the RNA polymerase, which has been proposed as an alternative to the 16S rRNA gene to study the diversity of rhizobial populations in soils. We evaluated the method under laboratory and field conditions. Peanut seeds were inoculated with various Bradyrhizobium strains. After nodule development, DNA was extracted from selected nodules and the nodulating rhizobia were analysed by amplicon sequencing of the rpoB gene. The analyses of the sequence data showed that the method reliably identified bradyrhizobial strains in nodules, at least at the species level, and could be used to assess the competitiveness of the inoculant compared to other bradyrhizobia.

Antibody-Drug Conjugates with Indolinobenzodiazepine Dimer Payloads: DNA-Binding Mechanism of Indolinobenzodiazepine Dimer Catabolites in Target Cancer Cells.

DNA-targeting indolinobenzodiazepine dimer (IGN) payloads are used in several clinical-stage antibody-drug conjugates. IGN drugs alkylate DNA through the single imine moiety present in the dimer in contrast to the pyrrolobenzodiazepine dimer drugs, such as talirine and tesirine, which contain two imine moieties per dimer and cross-link DNA. This study explored the mechanism of binding of IGN to DNA in cells and to synthetic duplex and hairpin oligonucleotides. New, highly sensitive IGN-DNA binding enzyme-linked immunosorbent assay methods were developed using biotinylated IGN analogues (monoimine, diimine, and diamine IGNs) and digoxigenin-labeled duplex oligonucleotides, which allowed the measurement of drug-DNA adducts in viable cells at concentrations below IC50. Furthermore, the release of free drug from the IGN-DNA adduct upon treatment with nuclease ex vivo was tested under physiological conditions. The monoimine IGN drug formed a highly stable adduct with DNA in cells, with stability similar to that of the diimine drug analogue. Both monoimine and diimine IGN-DNA adducts released free drugs upon DNA cleavage by nuclease at 37 °C, although more free drug was released from the monoimine compared to the diimine adduct, which presumably was partly cross-linked. The strong binding of the monoimine IGN drug to duplex DNA results from both the noncovalent IGN-DNA interaction and the covalent bond formation between the 2-amino group of a guanine residue and the imine moiety in IGN.

Optimizing Lysosomal Activation of Antibody-Drug Conjugates (ADCs) by Incorporation of Novel Cleavable Dipeptide Linkers.

Although peptide linkers are used in multiple clinical-stage ADCs, there are only few reports on optimizing peptide linkers for efficient lysosomal proteolysis and for stability in circulation. We screened multiple dipeptide linkers for efficiency of proteolysis and compared them to the dipeptide linkers currently being evaluated in the clinic: Val-Cit, Val-Ala, and Ala-Ala. Lead dipeptide linkers selected from the initial screen were incorporated into ADCs with indolinobenzodiazepine dimer (IGN) payloads to evaluate cellular processing, in vitro cytotoxic activity, plasma stability, and in vivo efficacy. ADCs with several dipeptide linkers bearing l-amino acids showed faster lysosomal processing in target cancer cells compared to the l-Ala-l-Ala linked ADC. These variances in linker processing rates did not result in different in vitro and in vivo activities among peptide linker ADCs, presumably due to accumulation of threshold cytotoxic catabolite levels for ADCs of several peptide linkers in the cell lines and xenografts tested. ADCs with l-amino acid dipeptide linkers exhibited superior in vitro cytotoxic potencies in multiple cell lines compared to an ADC with a d-Ala-d-Ala dipeptide linker and an ADC with a noncleavable linker. This work adds to the toolbox of stable, lysosomally cleavable peptide linkers for ADCs.

Significant disparities in allergy prevalence and microbiota between the young people in Finnish and Russian Karelia.

BACKGROUND: Atopic allergy has been more common among schoolchildren in Finland, as compared to Russian Karelia. These adjacent regions show one of the most contrasting socio-economical differences in the world. OBJECTIVE: We explored changes in allergy from school age to young adulthood from 2003 to 2010/2012 in these two areas. The skin and nasal microbiota were also compared. METHODS: Randomly selected children from Finnish (n = 98) and Russian Karelia (n = 82) were examined in 2003, when the children were 7-11 years of age, and again in 2010 (Finnish Karelia) and 2012 (Russian Karelia). We analysed self-reported allergy symptoms and sensitization to common allergens by serum sIgE values. The skin (volar forearm) and nasal mucosa microbiota, collected in 2012 (aged 15-20 years), identified from DNA samples, were compared with multivariate methods. RESULTS: Asthma, hay fever, atopic eczema, self-reported rhinitis, as well as atopic sensitization, were threefold to 10-fold more common in Finland, as compared to Russian Karelia. Hay fever and peanut sensitization were almost non-existent in Russia. These patterns remained throughout the 10-year follow-up. Skin microbiota, as well as bacterial and fungal communities in nasal mucosa, was contrastingly different between the populations, best characterized by the diversity and abundance of genus Acinetobacter; more abundant and diverse in Russia. Overall, diversity was significantly higher among Russian subjects (Pskin < 0.0001, Pnasal-bacteria < 0.0001 and Pnasal-fungi < 0.01). Allergic diseases were not associated with microbial diversity in Finnish subjects. CONCLUSIONS AND CLINICAL RELEVANCE: Differences in allergic phenotype, developed in early life, remain between populations. A parallel difference in the composition of skin and nasal microbiota suggests a potential underlying mechanism. Our results also suggest that high abundance and diversity of Acinetobacter might contribute to the low allergy prevalence in Russia. Implications of early-life exposure to Acinetobacter should be further investigated.

Clonal Analysis of Regulatory T Cell Defect in Patients with Autoimmune Polyendocrine Syndrome Type 1 Suggests Intrathymic Impairment.

Mutations in the autoimmune regulator gene disrupt thymic T cell development and negative selection, leading to the recessively inherited polyendocrine autoimmune disease autoimmune polyendocrine syndrome type 1 (APS-1). The patients also have a functional defect in the FOXP3+ regulatory T cell population, but its origin is unclear. Here, we have used T cell receptor sequencing to analyse the clonal relationship of major CD4+ T cell subsets in three patients and three healthy controls. The naive regulatory T cells showed little overlap with helper T cell subsets, supporting divergence in the thymus. The activated/memory regulatory T cell subset displayed more sharing with helper T cells, but was mainly recruited from the naive regulatory T cell population. These clonal patterns were very similar in both patients and controls. However, naive regulatory T cells isolated from the patients had a significantly longer T cell receptor complementarity-determining region 3 than any other population, suggesting failure of thymic selection. These data indicate that the peripheral differentiation of regulatory T cells in APS-1 patients is not different from that in healthy controls. Rather, the patients' naive regulatory T cells may have an intrinsic defect imprinted already in the thymus.

More than constipation - bowel symptoms in Parkinson's disease and their connection to gut microbiota.

BACKGROUND AND PURPOSE: The majority of Parkinson's disease (PD) patients suffer from gastrointestinal symptoms of which constipation is considered the most prominent. Recently, in addition to constipation, a diagnosis of irritable bowel syndrome (IBS) was also found to be associated with increased PD risk. Gut microbiota alterations have been reported in IBS and recently also in PD. IBS-like bowel symptoms in PD and their possible connection to other non-motor symptoms and faecal microbiota were assessed. METHODS: This case-control study compared 74 PD patients with 75 controls without any signs of parkinsonism or potential premotor symptoms. IBS-like symptoms were assessed using the Rome III questionnaire. The non-motor symptoms were assessed using the Non-Motor Symptoms Questionnaire and Non-Motor Symptom Scale. Faecal microbiota were assessed by pyrosequencing of the V1-V3 regions of the bacterial 16S ribosomal RNA gene. RESULTS: Symptoms that were IBS-like were significantly more prevalent in PD patients than in controls (24.3% vs. 5.3%; P = 0.001). Criteria for functional constipation were met by 12.2% of PD patients and 6.7% of controls (P = 0.072). PD patients with IBS-like symptoms had more non-motor symptoms and a lower faecal abundance of Prevotella bacteria than those without IBS-like symptoms. CONCLUSION: Our results indicate that PD patients may suffer from colonic dysfunction beyond pure constipation. Therefore, a more comprehensive assessment of bowel symptoms could provide valuable information. The lower abundance of Prevotella bacteria in PD patients with IBS-like symptoms suggests that the microbiota-gut-brain axis may be implicated in the gastrointestinal dysfunction of PD patients.

Performance of microbiological, serological, molecular, and modified seminal plasma methods in the diagnosis of Brucella abortus in semen and serum of bovine bulls.

Brucellosis remains as a major infectious disease of domestic animals and is considered a re-emerging zoonosis in several countries. B. abortus infections in bulls are related to reproductive tract infections, although infected animals show transient serological titers or nonreactor status. Thus, diagnosis of bovine brucellosis based exclusively on serological tests probably underestimates B. abortus infections in bulls. In this scenario, three hundred thirty-five serum samples from reproductively mature bovine bulls were subjected simultaneously to standard serodiagnosis using the rose Bengal test (RBT), 2-mercaptoethanol (2-ME), complement fixation (CFT), and fluorescence polarization assay (FPA). Furthermore, conventional semen plasma agglutination (SPA) and modified 2-ME, FC and, FPA were carried out in all bulls replaing serum by seminal plasma. Semen from all bulls was also analyzed for sperm viability, microbiological culture in Farrell media, and polymerase chain reaction (PCR). Only eight (2.38%) semen samples were considered improper for reproduction services (necrospermia and azoospermia), although none of these animals was positive in any of the diagnosis methods used. Five bulls (1.49%) were simultaneously positive in conventional RBT, 2-ME, SPA, modified 2-ME, microbiological culture in Farrell media, and in PCR for B. abortus strain 19. Two (1.67%) bulls were positive in PCR for B. abortus field strains and negative in all other tests, although semen was considered viable to reproduction service. The identification of B. abortus B19 strain in serum and semen of bulls occurred probably due to improper vaccination of males or infection by B19 strain shedding by vaccinated females that could to contaminated environment of farms. In addition, detection of B. abortus field strains only using PCR in bulls without sperm viability abnormalities indicate the need for including molecular methods to improve diagnosis of the disease in bovine bulls.

Temperature- and sex-related effects of serine protease alleles on larval development in the Glanville fritillary butterfly.

The body reserves of adult Lepidoptera are accumulated during larval development. In the Glanville fritillary butterfly, larger body size increases female fecundity, but in males fast larval development and early eclosion, rather than large body size, increase mating success and hence fitness. Larval growth rate is highly heritable, but genetic variation associated with larval development is largely unknown. By comparing the Glanville fritillary population living in the Åland Islands in northern Europe with a population in Nantaizi in China, within the source of the post-glacial range expansion, we identified candidate genes with reduced variation in Åland, potentially affected by selection under cooler climatic conditions than in Nantaizi. We conducted an association study of larval growth traits by genotyping the extremes of phenotypic trait distributions for 23 SNPs in 10 genes. Three genes in clip-domain serine protease family were associated with larval growth rate, development time and pupal weight. Additive effects of two SNPs in the prophenoloxidase-activating proteinase-3 (ProPO3) gene, related to melanization, showed elevated growth rate in high temperature but reduced growth rate in moderate temperature. The allelic effects of the vitellin-degrading protease precursor gene on development time were opposite in the two sexes, one genotype being associated with long development time and heavy larvae in females but short development time in males. Sexually antagonistic selection is here evident in spite of sexual size dimorphism.

Sensitive ELISA Method for the Measurement of Catabolites of Antibody-Drug Conjugates (ADCs) in Target Cancer Cells.

A new, sensitive ELISA method has been developed which measures catabolites in cells and media upon processing of antibody-drug conjugates (ADCs) by target cancer cells. This ELISA method, exemplified for maytansinoid ADCs, uses competitive inhibition by a maytansinoid analyte of the binding of biotinylated antimaytansine antibody to an immobilized BSA-maytansinoid conjugate. Synthetic standards of several maytansinoid catabolites derived from ADCs with different linkers were tested and showed similar inhibition curves, with an EC50 of about 0.1 nM (0.03 pmol in an assay volume of 0.25 mL). This high sensitivity allowed quantification of catabolites from a methanolic cell extract and from the medium, generated from an ADC in 1 day using only about 1 million cells. The processing of anti-EpCAM and anti-CanAg ADCs with noncleavable linker (SMCC-DM1), disulfide linker (SPDB-DM4), and charged sulfonate-bearing disulfide linker (sulfo-SPDB-DM4), each containing an average of about four maytansinoid molecules per antibody, were compared in colon cancer cell lines (COLO 205 and HT-29). An 8-10-fold higher total level of catabolite was observed for anti-CanAg ADCs than for anti-EpCAM ADCs upon processing by COLO 205 cells, consistent with a higher cell-surface expression of CanAg. In a multidrug resistant HCT-15 colon cancer cell line, the anti-EpCAM-SPDB-DM4 linker conjugate was not cytotoxic and showed a significantly lower level of catabolite within cells compared to that in medium, presumably due to Pgp-mediated efflux of the nonpolar DM4 catabolite. In contrast, sulfo-SPDB-DM4 and SMCC-DM1 linker conjugates were cytotoxic, which correlated with higher amounts of catabolites found within the HCT-15 cells relative to amounts in medium. In a nonmultidrug resistant HT-29 cell line, the anti-EpCAM-SPDB-DM4 linker conjugate was cytotoxic, with most of the catabolite found in cells and little in the medium. In conclusion, this highly sensitive ELISA method for measurement of ADC catabolite is convenient for screening multiple ADC parameters such as linkers and antibodies in a number of cell lines, does not require concentration of sample or extraction of media, and is complementary to other reported methods such as radiolabeling of ADCs or mass spectrometry.

Home interventions are effective at decreasing indoor nitrogen dioxide concentrations.

UNLABELLED: Nitrogen dioxide (NO2 ), a by-product of combustion produced by indoor gas appliances such as cooking stoves, is associated with respiratory symptoms in those with obstructive airways disease. We conducted a three-armed randomized trial to evaluate the efficacy of interventions aimed at reducing indoor NO2 concentrations in homes with unvented gas stoves: (i) replacement of existing gas stove with electric stove; (ii) installation of ventilation hood over existing gas stove; and (iii) placement of air purifiers with high-efficiency particulate air (HEPA) and carbon filters. Home inspection and NO2 monitoring were conducted at 1 week pre-intervention and at 1 week and 3 months post-intervention. Stove replacement resulted in a 51% and 42% decrease in median NO2 concentration at 3 months of follow-up in the kitchen and bedroom, respectively (P = 0.01, P = 0.01); air purifier placement resulted in an immediate decrease in median NO2 concentration in the kitchen (27%, P < 0.01) and bedroom (22%, P = 0.02), but at 3 months, a significant reduction was seen only in the kitchen (20%, P = 0.05). NO2 concentrations in the kitchen and bedroom did not significantly change following ventilation hood installation. Replacing unvented gas stoves with electric stoves or placement of air purifiers with HEPA and carbon filters can decrease indoor NO2 concentrations in urban homes. PRACTICAL IMPLICATIONS: Several combustion sources unique to the residential indoor environment, including gas stoves, produce nitrogen dioxide (NO2), and higher NO2 concentrations, are associated with worse respiratory morbidity in people with obstructive lung disease. A handful of studies have modified the indoor environment by replacing unvented gas heaters; this study, to our knowledge, is the first randomized study to target unvented gas stoves. The results of this study show that simple home interventions, including replacement of an unvented gas stove with an electric stove or placement of HEPA air purifiers with carbon filters, can significantly decrease indoor NO2 concentrations.

Linker substitution influences succinimide ring hydrolysis equilibrium impacting the stability of attachment to antibody-drug conjugates.

Maleimide chemistry is widely used in antibody-drug conjugate (ADC) generation to connect drugs to antibodies through a succinimide linker. The resulting ADC is prone to payload loss via a reverse Michael reaction, leading to premature drug release in vivo. Complete succinimide hydrolysis is an effective strategy to overcome the instability of ADC. However, we discovered through previous work that hydrolysed succinimide rings can close again in a liquid formulation during storage and under thermal stress conditions. In this work, a set of maleimide linkers with hydrolysis-prone groups were designed. The corresponding ADCs were prepared and subjected to thermal stress conditions. The extent of succinimide hydrolysis and drug release was measured, and ADC properties such as SEC, DAR, pI and clog P of linkers were calculated. Our results demonstrated that even though all these groups increased the hydrolysis rate, they have different impacts on maintaining the hydrolysed succinimide ring in an open conformation and ADC stability in a liquid formulation.

Identification of allele-specific KIV-2 repeats and impact on Lp(a) measurements for cardiovascular disease risk.

The abundance of Lp(a) protein holds significant implications for the risk of cardiovascular disease (CVD), which is directly impacted by the copy number (CN) of KIV-2, a 5.5 kbp sub-region. KIV-2 is highly polymorphic in the population and accurate analysis is challenging. In this study, we present the DRAGEN KIV-2 CN caller, which utilizes short reads. Data across 166 WGS show that the caller has high accuracy, compared to optical mapping and can further phase ~50% of the samples. We compared KIV-2 CN numbers to 24 previously postulated KIV-2 relevant SNVs, revealing that many are ineffective predictors of KIV-2 copy number. Population studies, including USA-based cohorts, showed distinct KIV-2 CN, distributions for European-, African-, and Hispanic-American populations and further underscored the limitations of SNV predictors. We demonstrate that the CN estimates correlate significantly with the available Lp(a) protein levels and that phasing is highly important.

Mechanisms behind the varying severity of Aleutian mink disease virus: Comparison of three farms with a different disease status.

Aleutian mink disease virus (AMDV) is distributed widely among mink farms and wild mustelids despite ongoing attempts to stop the spread. The severity of Aleutian disease (AD) varies from subclinical to fatal but the reasons for its varying severity are complex and unclear. Recently, breeding of tolerant mink has drawn attention as the possible solution to reduce the effects of AD in farms. The aim of this study was to gather information on the effects of breeding based on overall health, production traits, and antibody titer on AD severity by comparing a positive farm (farm 1) that has been breeding for tolerance in mink to an infected farm without tolerance selection, and an AMDV-free farm. During the 2.5-year follow-up, the mink in farm 1 remained mostly free of clinical AD, had normal pelt quality and litter size, and had low virus copy numbers in tissues and low antibody titers in ELISA. In histopathological studies, most of the farm 1 mink had no/mild lesions in their kidneys. 29-43% of the mink were ELISA negative but PCR positive throughout the follow-up and frequent changes in virus strains and coinfections were observed. Several differences in gene expression between animals from different farms were also detected. These results indicate that the disease burden of AMDV can be reduced, with seemingly normal health and production rates, despite continual circulation of ADMV in cases where eradication attempts are unsuccessful.

Determination of fungal succession during municipal solid waste composting using a cloning-based analysis.

AIMS: The microbiota at industrial full-scale composting plants has earlier been fragmentarily studied with molecular methods. Here, fungal communities from different stages of a full-scale and a pilot-scale composting reactors were studied before and after wood ash amendment. METHODS AND RESULT: The portion of fungal biomass, determined using phospholipid fatty acid analysis, varied between 6.3% and 38.5% in different composting phases. The fungal internal transcribed spacer (ITS) area was cloned and sequenced from 19 samples representing different stages of the composting processes. Altogether 2986 sequenced clones were grouped into 166 phylotypes from which 35% had a close match in the sequence databases. The fungal communities of the samples were related with the measured environmental variables in order to identify phylotypes typical of certain composting conditions. The fungal phylotypes could be grouped into those that dominated the mesophilic low pH initial phases (sequences similar to genera Candida, Pichia and Dipodascaceae) and those found mostly or exclusively in the thermophilic phase (sequences clustering to Thermomyces, Candida and Rhizomucor), but a few were also present throughout the whole process. CONCLUSIONS: The community composition was found to vary between suboptimally and optimally operating processes. In addition, there were differences in fungal communities between processes of industrial and pilot scale. SIGNIFICANCE AND IMPACT OF THE STUDY: The results of this study reveal the fungal diversity with molecular methods in industrial composting process. This is also one of the first studies conducted with samples from an industrial biowaste composting process.

[A systemic disease mimicking a rheumatoid arthritis]. / Une maladie systémique mimant une polyarthrite rhumatoïde.

INTRODUCTION: Rare systemic diseases such as amyloidosis can mimic inflammatory rheumatic diseases. Because of their poor prognosis, physicians should rule them out at the onset of inflammatory rheumatism. We report a case of AL amyloidosis misdiagnosed as rheumatoid arthritis. CASE REPORT: A 71-year-old woman was referred for seronegative rheumatoid arthritis, resistant to three biologic therapies. She had an IgA lambda monoclonal gammopathy of undetermined significance (MGUS). The patient subsequently developed glomerular proteinuria. Abdominal fat and accessory salivary glands biopsies revealed amyloid light-chain (AL) amyloidosis. Treatment with bortezomib-cyclophosphamide-dexamethasone, led to complete hematologic, renal and rheumatologic remission. Ten months after treatment interruption, the patient had an articular and hematologic relapse. CONCLUSION: Amyloid light-chain amyloidosis arthropathy is probably underdiagnosed. A review of amyloid arthropathy associated with multiple myeloma found that 33% of patients had been misdiagnosed with rheumatoid arthritis.

Characterization of Russian rabies virus vaccine strain RV-97.

The RV-97 rabies virus vaccine strain is widely used in Russia as a component of the live attenuated oral anti-rabies vaccine "Sinrab". This vaccine has also been used in some other countries, such as Kazakhstan, Belarus, and Ukraine. Entire genome sequencing is an effective tool for studying the genetic properties of virus strains. In this study, a simple technique for obtaining the entire genome sequence of the rabies virus was used. The entire genome sequence and the deduced amino acid sequences of the major viral proteins were compared with those of other rabies vaccine virus strains. The RV-97 strain forms a separate phylogenetic branch and seems to be phylogenetically more related to the group of Japanese vaccine strains. It also contains several unique amino acid changes in known immunodominant sites of G and P proteins.

Analysis of fungal flora in indoor dust by ribosomal DNA sequence analysis, quantitative PCR, and culture.

In recent years increasing attention has been given to the potential health effects of fungal exposure in indoor environments. We used large-scale sequencing of the fungal internal transcribed spacer region (ITS) of nuclear ribosomal DNA to describe the mycoflora of two office buildings over the four seasons. DNA sequencing was complemented by cultivation, ergosterol determination, and quantitative PCR analyses. Sequences of 1,339 clones were clustered into 394 nonredundant fungal operational taxonomical units containing sequences from 18 fungal subclasses. The observed flora differed markedly from that recovered by cultivation, the major differences being the near absence of several typical indoor mold genera such as Penicillium and Aspergillus spp. and a high prevalence of basidiomycetes in clone libraries. A total of 55% of the total diversity constituted of unidentifiable ITS sequences, some of which may represent novel fungal species. Dominant species were Cladosporium cladosporioides and C. herbarum, Cryptococcus victoriae, Leptosphaerulina americana and L. chartarum, Aureobasidium pullulans, Thekopsora areolata, Phaeococcomyces nigricans, Macrophoma sp., and several Malassezia species. Seasonal differences were observed for community composition, with ascomycetous molds and basidiomycetous yeasts predominating in the winter and spring and Agaricomycetidae basidiomycetes predominating in the fall. The comparison of methods suggested that the cloning, cultivation, and quantitative PCR methods complemented each other, generating a more comprehensive picture of fungal flora than any of the methods would give alone. The current restrictions of the methods are discussed.

Structural basis for the interaction between the cell polarity proteins Par3 and Par6.

Polarity is a fundamental property of most cell types. The Par protein complex is a major driving force in generating asymmetrically localized protein networks and consists of atypical protein kinase C (aPKC), Par3, and Par6. Dysfunction of this complex causes developmental abnormalities and diseases such as cancer. We identified a PDZ domain-binding motif in Par6 that was essential for its interaction with Par3 in vitro and for Par3-mediated membrane localization of Par6 in cultured cells. In fly embryos, we observed that the PDZ domain-binding motif was functionally redundant with the PDZ domain in targeting Par6 to the cortex of epithelial cells. Our structural analyses by x-ray crystallography and NMR spectroscopy showed that both the PDZ1 and PDZ3 domains but not the PDZ2 domain in Par3 engaged in a canonical interaction with the PDZ domain-binding motif in Par6. Par3 thus has the potential to recruit two Par6 proteins simultaneously, which may facilitate the assembly of polarity protein networks through multivalent PDZ domain interactions.

Morphometric Differentiation Among Anastrepha fraterculus (Diptera: Tephritidae) Exploiting Sympatric Alternate Hosts.

Anastrepha fraterculus (Wiedemann) is currently considered a complex of cryptic species infesting fruits from Mexico to Argentina and represents an interesting biological model for evolutionary studies. Moreover, detecting and quantifying behavioral, morphological, and genetic differentiation among populations is also relevant to the application of environment-friendly control programs. Here, phenotypic differentiation among individuals coexisting in the wild in a Northern region of Argentina was unveiled and associated with host choice. Six morphometric traits were measured in sympatric flies exploiting three different host species. Phenotypic variation was shown to be host-dependent regardless of geographical or temporal overlap. Flies collected from synchronous alternate hosts (peach and walnut) differed from each other despite the lack of geographical isolation. By contrast, flies emerging from guavas that ripen about two months later than peach and walnut showed no significant differentiation in comparison to flies collected from walnuts, but they differ significantly from flies originating from peaches. This result is consistent with the hypothesis that the same population of flies shifts from walnuts to guavas throughout the year, whereas the population of flies that uses peaches as a host is probably exploiting other alternate hosts when peach availability decreases. Further research is needed to study the underlying mechanism. Results are consistent with previous molecular markers (inter-simple sequence repeat-ISSR) research on flies stemming from the same hosts and the same area, suggesting that differentiation among flies emerging from alternative hosts occurs at both genetic and phenotypic levels. The contribution of host preference in long-term genetic differentiation is discussed.

Ornithine decarboxylase activity from an extremely thermophilic bacterium, Clostridium thermohydrosulfuricum. Effect of GTP analogues on enzyme activity.

The activity of ornithine decarboxylase has been detected for the first time in extracts of a thermophilic bacterium, Clostridium thermohydrosulfuricum. The temperature optimum of the thermoresistant ornithine decarboxylase was 55 degrees C and the pH optimum was 7.5. It required pyridoxal phosphate and a thiol (dithiothreitol) for activity. The activity of the enzyme was closely connected to the growth of the thermophilic bacteria, since the activity was highest during the logarithmic growth. The enzyme was not inhibited (in contrast to the enzyme from Escherichia coli) by putrescine, spermidine or other naturally occurring polyamines. When the effect of GTP and a number of GTP analogues was tested on the activity of the enzyme, it was observed that GTP or dGTP was necessary for the full activity. The modification of either the purine base or 5'-phosphate chain of GTP leads to a stimulation smaller than that caused by GTP. Modification of the 3'-carbon of the ribose part of GTP (magic spot I and II of Cashel and Gallant, Nature 221 (1969) 838-841) caused a distinct inhibition of the enzyme activity, indicating that ornithine decarboxylase contains at least two domains for binding of GTP. The enzyme was inhibited irreversibly by high concentrations (50 mM) of difluoromethylornithine. Extracts of the bacterium contained also arginine decarboxylase, but its activity was always very much lower than that of ornithine decarboxylase. The activity of arginine decarboxylase was inhibited irreversibly by difluoromethylarginine, which is an irreversible suicide inhibitor of bacterial arginine decarboxylase (Kallio, A., McCann, P.P. and Bey, P. (1981) Biochemistry 20, 3163-3166).