Microcystin production by a freshwater spring cyanobacterium of the genus Fischerella

We investigated the production of a hepatotoxic, cyclic heptapeptide, microcystin, by a filamentous branched cyanobacterium belonging to the order Stigonematales, genus Fischerella. The freshwater Fischerella sp. strain CENA161 was isolated from spring water in a small concrete dam in Piracicaba, Sao Paulo State, Brazil, and identified by combining a morphological description with 16S rRNA gene sequencing and phylogenetic analysis. Microcystin (MCYST) analysis performed using an ELISA assay on cultured cells gave positive results. High performance liquid chromatography-mass spectrometry (HPLC-MS) analysis detected 33.6 mu g MCYST-LR per gram dry weight of cyanobacterial cells. Microcystin profile revealed by quadrupole time-of-flight tandem mass spectrometry (Q-TOF-MS/MS) analysis confirmed the production of MCYST-LR. Furthermore, genomic DNA was analyzed by PCR for sequences similar to the ketosynthase (KS) domain of the type I polyketide synthase gene, which is involved in microcystin biosynthesis. This revealed the presence of a KS nucleotide fragment similar to the mcyD and ndaD genes of the microcystin and nodularin synthetase complexes. Phylogenetic analysis grouped the Fischerella KS sequence together with mcyD sequences of the three known microcystin synthetase operon (Microcystis, Planktothrix and Anabaena) and ndaD of the nodularin synthetase operon, with 100% bootstrap support. Our findings demonstrate that Fischerella sp. CENA161 produces MYCST-LR and for the first time identify a nucleotide sequence putatively involved in microcystin synthesis in this genus. (C) 2009 Elsevier Ltd. All rights reserved.

Stabilization of native protein fold by intein-mediated covalent cyclization

A mutant version of the N-terminal domain of Escherichia coli DnaB helicase was used as a model system to assess the stabilization against unfolding gained by covalent cyclization. Cyclization was achieved in vivo by formation of an amide bond between the N and C termini with the help of a split mini-intein. Linear and circular proteins were constructed to be identical in amino acid sequence. Mutagenesis of Phe102 to Glu rendered the protein monomeric even at high concentration. A difference in free energy of unfolding, DeltaDeltaG, between circular and linear protein of 2.3(+/-0.5) kcal mol(-1) was measured at 10degreesC by circular dichroism. A theoretical estimate of the difference in conformational entropy of linear and circular random chains in a three-dimensional cubic lattice model predicted DeltaDeltaG = 2.3 kcal mol(-1), suggesting that stabilization by protein cyclization is driven by the reduced conformational entropy of the unfolded state. Amide-proton exchange rates measured by NMR spectroscopy and mass spectrometry showed a uniform, approximately tenfold decrease of the exchange rates of the most slowly exchanging amide protons, demonstrating that cyclization globally decreases the unfolding rate of the protein. The amide proton exchange was found to follow EX1 kinetics at near-neutral pH, in agreement with an unusually slow refolding I measured by stopped-flow circular dichroism. rate of less than 4 min(-1) The linear and circular proteins differed more in their unfolding than in their folding rates. Global unfolding of the N-terminal domain of E. coli DnaB is thus promoted strongly by spatial separation of the N and C termini, whereas their proximity is much less important for folding. (C) 2005 Elsevier Ltd. All rights reserved.

Identification of intracellular and extracellular domains mediating signal transduction in the inhibitory glycine receptor chloride channel

Fast synaptic neurotransmission is mediated by transmitter-activated conformational changes in ligand-gated ion channel receptors, culminating in opening of the integral ion channel pore. Human hereditary hyperekplexia, or startle disease, is caused by mutations in both the intracellular or extracellular loops flanking the pore-lining M2 domain of the glycine receptor alpha 1 subunit. These flanking domains are designated the M1-M2 loop and the M2-M3 loop respectively. We show that four startle disease mutations and six additional alanine substitution mutations distributed throughout both loops result in uncoupling of the ligand binding sites from the channel activation gate. We therefore conclude that the M1-M2 and M2-M3 loops act in parallel to activate the channel. Their locations strongly suggest that they act as hinges governing allosteric control of the M2 domain. As the members of the ligand-gated ion channel superfamily share a common structure, this signal transduction model may apply to all members of this superfamily.

Biologically active conformer of the effector region of human C5a and modulatory effects of N-terminal receptor binding determinants on activity

A conformationally biased decapeptide agonist of human C5a (C5a(65-74)Y65,F67,P69,P71,D-Ala73 or YSFKPMPLaR) was used as a functional probe of the C5a receptor (C5aR) in order to understand the conformational features in the C-terminal effector region of C5a that are important for C5aR binding and signal transduction. YSFKPMPLaR was a potent, full agonist of C5a, but at higher concentrations had a superefficacious effect compared to the natural factor. The maximal efficacy of this analogue was 216 +/- 56% that of C5a in stimulating the release of beta-glucuronidase from human neutrophils. C5aR activation and binding curves both occurred in the same concentration range with YSFKPMPLaR, characteristics not observed with natural C5a or more conformationally flexible C-terminal agonists. YSFKPMPLaR was then used as a C-terminal effector template onto which was synthesized various C5aR binding determinants from the N-terminal core domain of the natural factor. In general, the presence of N-terminal binding determinants had little effect on either potency or binding affinity when the C-terminal effector region was presented to the C5aR in this biologically active conformation. However, one peptide, C5a(12-20)-Ahx-YSFKPMPLaR, expressed a 100-fold increase in affinity for the neutrophil C5aR and a 6-fold increase in potency relative to YSFKPMPLaR. These analyses showed that the peptides used in this study have up to 25% of the potency of C5a in human fetal artery and up to 5% of the activity of C5a in the PMN enzyme release assay.

Conformational dynamics of thyroid hormones by variable temperature nuclear magnetic resonance: The role of side chain rotations and cisoid/transoid interconversions

H-1 NMR spectra of the thyroid hormone thyroxine recorded at low temperature and high field show splitting into two peaks of the resonance due to the H2,6 protons of the inner (tyrosyl) ring. A single resonance is observed in 600 MHz spectra at temperatures above 185 K. An analysis of the line shape as a function of temperature shows that the coalescence phenomenon is due to an exchange process with a barrier of 37 kJ mol(-1). This is identical to the barrier for coalescence of the H2',6' protons of the outer (phenolic) ring reported previously for the thyroid hormones and their analogues. It is proposed that the separate peaks at low temperature are due to resonances for H2,6 in cisoid and transoid conformers which are populated in approximately equal populations. These two peaks are averaged resonances for the individual H2 and H6 protons. Conversion of cisoid to transoid forms can occur via rotation of either the alanyl side chain or the outer ring, from one face of the inner ring to the other. It is proposed that the latter process is the one responsible for the observed coalescence phenomenon. The barrier to rotation of the alanyl side chain is greater than or equal to 37 kJ mol(-1), which is significantly larger than has previously been reported for Csp(2)-Csp(3) bonds in other Ph-CH2-X systems. The recent crystal structure of a hormone agonist bound to the ligand-binding domain of the rat thyroid hormone receptor (Wagner et al. Nature 1995, 378, 690-697) shows the transoid form to be the bound conformation. The significant energy barrier to cisoid/transoid interconversion determined in the current study combined with the tight fit of the hormone to its receptor suggests that interconversion between the forms cannot occur at the receptor site but that selection for the preferred bound form occurs from the 50% population of the transoid form in solution.

Expression of activated mutants of the human interleukin-3/interleukin-5 granulocyte-macrophage colony-stimulating factor receptor common beta subunit in primary hematopoietic cells induces factor-independent proliferation and differentiation

To date, several activating mutations have been discovered in the common signal-transducing subunit (h beta c) of the receptors for human granulocyte-macrophage colony-stimulating factor, interleukin-3, and interleukin-5. Two of these, Fl Delta and 1374N, result in a 37 amino acid duplication and a single amino acid substitution in the extracellular domain of h beta c, respectively. A third, V449E, results in a single amino acid substitution in the transmembrane domain, Previous studies comparing the activity of these mutants in different hematopoietic cell lines imply that the transmembrane and extracellular mutations act by different mechanisms and suggest the requirement for cell type-specific molecules in signalling. To characterize the ability of these mutant hpc subunits to mediate growth and differentiation of primary cells and hence investigate their oncogenic potential, we have expressed all three mutants in primary murine hematopoietic cells using retroviral transduction. It is shown that, whereas expression of either extracellular hpc mutant confers factor-independent proliferation and differentiation on cells of the neutrophil and monocyte lineages only, expression of the transmembrane mutant does so on these lineages as well as the eosinophil, basophil, megakaryocyte, and erythroid lineages, Factor-independent myeloid precursors expressing the transmembrane mutant display extended proliferation in liquid culture and in some cases yielded immortalized cell lines. (C) 1997 by The American Society of Hematology.

PTCH1 gene mutations in exon 17 and loss of heterozygosity on D9S180 microsatellite in sporadic and inherited human basal cell carcinomas

Background Basal cell carcinomas (BCCs) are the most frequent human cancer that results from malignant transformation of basal cells in the epidermis. Gorlin syndrome is a rare inherited autosomal dominant disease that predisposes with multiple BCCs and other birth defects. Both sporadic and inherited BCCs are associated with mutations in the tumor suppressor gene PTCH1, but there is still uncertainty on the role of its homolog PTCH2. Objectives To search for mutations and genomic instability in sporadic and inherited BCCs. Methods DNA obtained from leukocytes and tumor cells was amplified by polymerase chain reaction regarding five exons of PTCH1 and PTCH2 and neighboring microsatellites. Exons were sequenced and compared with the GenBank database. Results Only D9S180, of six microsatellites, showed loss of heterozygosity in three BCCs (two sporadic and one inherited). One sporadic BCC presented the mutation g. 2885G>C in exon 17 of PTCH1, which predicts the substitution p.R962T in an external domain of the protein. In addition, the leukocytes and tumor cells of one patient with Gorlin syndrome showed the mutation g. 2839T>G in the same exon and gene, which predicts a p.E947stop and truncated protein. All control and tumor samples presented IVS9 + 217T in intron 9 of PTCH1. Conclusion Mutations found in the PTCH1 gene and neighboring repetitive sequences may have contributed to the development of the studied BCCs.

Inactivation of a c-Myb/estrogen receptor fusion protein in transformed primary cells leads to granulocyte/macrophage differentiation and down regulation of c-kit but not c-myc or cdc2

Primary murine fetal hemopoietic cells were transformed with a fusion protein consisting of the ligand-binding domain of the estrogen receptor and a carboxyl-terminally truncated c-Myb protein (ERMYB), The ERMYB-transformed hemopoietic cells exhibit an immature myeloid phenotype when grown in the presence of beta-estradiol. Upon removal of beta-estradiol, the ERMYB cells display increased adherence, decreased clonogenicity and differentiate to cells exhibiting granulocyte or macrophage morphology, The expression of the c-myc, c-kit, cdc2 and bcl-2 genes, which are putatively regulated by Myb, was investigated in ERMYB cells grown in the presence or absence of beta-estradiol. Neither c-myc nor cdc2 expression was down-regulated after removal of beta-estradiol demonstrating that differentiation is not a consequence of decreased transactivation of these genes by ERMYB. While bcl-2 expression was reduced by 50% in ERMYB cells grown in the absence of beta-estradiol, there was no increase in DNA laddering, suggesting that Myb was not protecting ERMYB cells from apoptosis, In contrast, a substantial (200-fold) decrease in c-kit mRNA level was observed following differentiation of ERMYB cells, and c-kit mRNA could be partially re-induced by the re-addition of beta-estradiol. Furthermore, a reporter construct containing the c-kit promoter was activated when cotransfected with a Myb expression vector, providing further evidence of a role for Myb in the regulation of c-kit.

Nonsense Mutations in FGF8 Gene Causing Different Degrees of Human Gonadotropin-Releasing Deficiency

Context: FGFR1 mutations cause isolated hypogonadotropic hypogonadism (IHH) with or without olfactory abnormalities, Kallmann syndrome, and normosmic IHH respectively. Recently, missense mutations in FGF8, a key ligand for fibroblast growth factor receptor (FGFR) 1 in the ontogenesis of GnRH, were identified in IHH patients, thus establishing FGF8 as a novel locus for human GnRH deficiency. Objective: Our objective was to analyze the clinical, hormonal, and molecular findings of two familial IHH patients due to FGF8 gene mutations. Methods and Patients: The entire coding region of the FGF8 gene was amplified and sequenced in two well-phenotyped IHH probands and their relatives. Results: Two unique heterozygous nonsense mutations in FGF8(p.R127X and p.R129X) were identified in two unrelated IHH probands, which were absent in 150 control individuals. These two mutations, mapped to the core domain of FGF8, impact all four human FGF8 isoforms, and lead to the deletion of a large portion of the protein, generating nonfunctional FGF8 ligands. The p.R127X mutation was identified in an 18-yr-old Kallmann syndrome female. Her four affected siblings with normosmic IHH or delayed puberty also carried the p.R127X mutation. Additional developmental anomalies, including cleft lip and palate and neurosensorial deafness, were also present in this family. The p.R129X mutation was identified in a 30-yr-old man with familial normosmic IHH and severe GnRH deficiency. Conclusions: We identified the first nonsense mutations in the FGF8 gene in familial IHH with variable degrees of GnRH deficiency and olfactory phenotypes, confirming that loss-of-function mutations in FGF8 cause human GnRH deficiency. (J Clin Endocrinol Metab 95: 3491-3496, 2010)

Post-craniotomy headache: characteristics, behaviour and effect on quality of life in patients operated for treatment of supratentorial intracranial aneurysms

We prospectively studied headache characteristics during 6 months after craniotomy performed for treatment of cerebral aneurysms in 79 patients. Semistructured interviews, headache diaries, the Hospital Anxiety and Depression Scale and the Epworth Sleepiness Scales, the Short Form-36 Health Survey (SF-36) and McGill Pain Questionnaire were used. Seventy-two patients had headaches, half before the fifth day after surgery. Changes were observed in headache diagnosis, side and site in the postoperative period. Headache frequency increased immediately after surgery and then decreased over time. Headache frequency was associated with depressive and anxiety symptoms. Pain intensity was higher in women and in patients with more anxiety symptoms. An incidence of post-craniotomy headache of 40% was observed according to International Headache Society classification criteria, 10.7% of the acute and 29.3% of the chronic type. The bodily pain domain of the SF-36 was worse in patients with more anxiety symptoms. Greater frequencies of headache were associated with lower scores on bodily pain and social functioning.

Reliability and validity of the Brazilian-Portuguese version of the Burns Specific Pain Anxiety Scale (BSPAS)

Background: Pain and anxiety are a common problem in all recovery phases after a burn. The Burns Specific Pain Anxiety Scale (BSPAS) was proposed to assess anxiety in burn patients related to painful procedures. Objectives: To assess internal consistency, discriminative construct validity, dimensionality and convergent construct validity of the Brazilian-Portuguese version of the Burns Specific Pain Anxiety Scale. Design: In this cross-sectional study, the original version of the BSPAS, adapted into Brazilian Portuguese, was tested for internal consistency (Cronbach`s Alpha), discriminative validity (related to total body surface area burned and sex), dimensionality (through factor analysis), and convergent construct validity (applying the Visual Analogue Scale for pain and State-Anxiety-STAI) in a group of 91 adult burn patients. Results: The adapted version of the BSPAS displayed a moderate and positive correlation with pain assessments: immediately before baths and dressings (r = 0.32; p < 0.001), immediately after baths and dressings (r = 0.31; p < 0.001) and during the relaxation period (r= 0.31; p < 0.001) and with anxiety assessments (r = 0.34; p < 0.001). No statistically significant differences were observed when comparing the mean of the adapted version of the BSPAS scores with sex (p = 0.194) and total body surface area burned (p = 0.162) (discriminative validity). The principal components analysis applied to our sample seems to confirm anxiety as one single domain of the Brazilian-Portuguese version of the BSPAS. Cronbach`s Alpha showed high internal consistency of the adapted version of the scale (0.90). Conclusion: The Brazilian-Portuguese version of the BSPAS 9-items has shown statically acceptable levels of reliability and validity for pain-related anxiety evaluation in burn patients. This scale can be used to assess nursing interventions aimed at decreasing pain and anxiety related to the performance of painful procedures. (c) 2010 Elsevier Ltd. All rights reserved.

Patterns of community integration 2-5 years post-discharge from brain injury rehabilitation

Outcome after traumatic brain injury (TBI) is characterized by a high degree of variability which has often been difficult to capture in traditional outcome studies. The purpose of this study was to describe patterns of community integration 2-5 years after TBI. Participants were 208 patients admitted to a Brain Injury Rehabilitation Unit between 1991-1995 in Brisbane, Australia. The design comprised retrospective data collection and questionnaire follow-up by mail. Mean follow-up was 3.5 years. Demographic, injury severity and functional status variables were retrieved from hospital records. Community integration was assessed using the Community Integration Questionnaire (CIQ), and vocational status measured by a self administered questionnaire. Data was analysed using cluster analysis which divided the data into meaningful subsets. Based on the CIQ subscale scores of home, social and productive integration, a three cluster solution was selected, with groups labelled as working (n = 78), balanced (n = 46) and poorly integrated (n = 84). Although 38% of the sample returned to a high level of productive activity and 22% achieved a balanced lifestyle, overall community integration was poor for the remainder. This poorly integrated group had more severe injury characterized by longer periods of acute care and post-traumatic amnesia (PTA) and greater functional disability on discharge. These findings have implications for service delivery prior to and during the process of reintegration after brain injury.

Identification of the regulatory subunit of Arabidopsis thaliana acetohydroxyacid synthase and reconstitution with its catalytic subunit

Acetohydroxyacid synthase (EC 4.1.3.18; AHAS) catalyzes the initial step in the formation of the branched-chain amino acids. The enzyme from most bacteria is composed of a catalytic subunit, and a smaller regulatory subunit that is required for full activity and for sensitivity to feedback regulation by valine. A similar arrangement was demonstrated recently for yeast AHAS, and a putative regulatory subunit of tobacco AHAS has also been reported. In this latter case, the enzyme reconstituted from its purified subunits remained insensitive to feedback inhibition, unlike the enzyme extracted from native plant sources. Here we have cloned, expressed in Escherichia coil, and purified the AHAS regulatory subunit of Ambidopsis thaliana. Combining the protein with the purified A. thaliana catalytic subunit results in an activity stimulation that is sensitive to inhibition by valine, leucine, and isoleucine. Moreover, there is a strong synergy between the effects of leucine and valine, which closely mimics the properties of the native enzyme. The regulatory subunit contains a sequence repeat of approximately 180 residues, and we suggest that one repeat binds leucine while the second binds valine or isoleucine. This proposal is supported by reconstitution studies of the individual repeats, which were also cloned, expressed, and purified. The structure and properties of the regulatory subunit are reminiscent of the regulatory domain of threonine deaminase (EC 4.2.1.16), and it is suggested that the two proteins are evolutionarily related.

X-linked neonatal diabetes mellitus, enteropathy and endocrinopathy syndrome is the human equivalent of mouse scurfy

To determine whether human X-linked neonatal diabetes mellitus, enteropathy and endocrinopathy syndrome (IPEX; MIM 304930) is the genetic equivalent of the scurfy (sf) mouse, we sequenced the human ortholog (FOXP3) of the gene mutated in scurfy mice (Foxp3), in IPEX patients. We found four non-polymorphic mutations. Each mutation affects the forkhead/winged-helix domain of the scurfin protein, indicating that the mutations may disrupt critical DNA interactions.

Gulliver, a long terminal repeat retrotransposon from the genome of the oriental blood fluke Schistosoma japonicum

We characterized the consensus sequence and structure of a long terminal repeat (LTR) retrotransposon from the genome of the human blood fluke, Schistosoma japonicum, and have earned this element, Gulliver. The full length, consensus Gulliver LTR retrotransposon was 4788 bp, and it was flanked at its 5'- and 3'-ends by LTRs of 259 bp. Each LTR included RNA polymerase II promoter sequences, a CAAT signal and a TATA box, Gulliver exhibited features characteristic of a functional LTR retrotransposon including two read through (termination) ORFs encoding retroviral gag and pol proteins of 312 and 1071 amino acid residues, respectively. The gag ORF encoded motifs conserved in nucleic acid binding proteins, while the pol ORF encoded conserved domains of aspartic protease, reverse transcriptase (RT), RNaseH and integrase, in that order, a pol pattern conserved in the gypsy lineage of LTR retrotransposons. Whereas the sequence and structure of Gulliver was similar to that of gypsy, phylogenetic analysis revealed that Gulliver did not group particularly closely with the gypsy family. Rather, its closest relatives were a LTR retrotransposon from Caenorhabditis elegans, mag from Bombyx mori and, to a lesser extent, easel from the salmon Oncorhynchus keta. Dot blot hybridizations indicated that Gulliver was present at between 100 and several thousand copies in the S. japonicum genome, and Southern hybridization analysis suggested its probable presence in the genome of Schistosoma mansoni. Transcripts encoding the RT domain of Gulliver were detected by RT-PCR in larval and adult stages of S. japonicum, indicating that (at least) the RT domain of Gulliver is transcribed. This is the first report of the sequence and structure of an LTR retrotransposon from any schistosome or indeed from any species belonging to the phylum Platyhelminthes. (C) 2001 Elsevier Science B.V. All rights reserved.