Novel porphyrin amino acids as building blocks for artificial photosynthetic reaction centers : photoinduced energy and electron transfer

This thesis reports on the synthesis and characterisation of trans-(M)AB2C meso-substituted porphyrin amino acid esters (PAr) (M = 2H or Zn) with tunable electron donating and electron withdrawing Ar substituents at B positions (Ar = 4-C6H4OnBu, 4-C6H4OMe, 2,4,6-C6H2Me3, 4-C6H4Me, C6H5, 4-C6H4F, 4-C6H4CF3, C6F5). These porphyrins were used as key building blocks for photosynthetic LHC (LHC = light-harvesting antenna complex) and RC (RC = reaction center) model compounds.rnBased on free-base or zinc(II) porphyrin amino acid esters and porphyrin acids several amide linked free-base bis(porphyrins) PAr1-PAr2 (Ar1 = 2,4,6-C6H2Me3, C6F5 and Ar2 = 2,4,6-C6H2Me3, 4-C6H4F, 4-C6H4CF3, C6F5), mono metallated bis(porphyrin) PAr1-(Zn)PAr2 (Ar1 = 2,4,6-C6H2Me3 and Ar2 =4-C6H4F) and its doubly zincated complexes (Zn)PAr1-(Zn)PAr2 were prepared. In the fluorescence spectra of free-base bis(porphyrins) the porphyrin with the strongest electron donating power of Ar substituents at B positions is the light emitting unity. The emission of mono metallated bis(porphyrin) occurs only from the free-base porphyrin building block. This phenomenon is caused by an efficient energy transfer likely via the Dexter through-bond mechanism.rnLinking of anthraquinone (Q) as electron acceptor (A) to the N-terminus of porphyrin amino acid esters ((M)PAr) and aminoferrocene (Fc) as electron donor (D) to the C-terminus of the porphyrin resulting in Q-(M)PAr-Fc triads (M = 2H or Zn, Ar = 4-C6H4OnBu, 4-C6H4OMe, 2,4,6-C6H2Me3, 4-C6H4Me, C6H5, 4-C6H4F, 4-C6H4CF3, C6F5) with tunable electron density at the porphyrin chromophore. In these triads initial oxidative PET (Q←(M)PAr) and reductive PET ((M)PAr→Fc) (PET = photoinduced electron transfer) are possible. Both processes leads to an emission quenching of (M)PAr. The efficiency of the PET pathways occurring in the Marcus normal region is controlled by the specific porphyrin electron density.rnAmide-linked conjugates PAr-Fc (Ar = 2,4,6-C6H2Me3, C6F5) and Fmoc-Fc-PAr1 (N-Fmoc-Fc = N-Fmoc protected 1,1’-ferrocene amino acid; Ar1 = C6H5, 4-C6H4F, 4-C6H4CF3, C6F5) as well as hinges PAr2-Fc-PAr1 (Ar1 = C6H5, 4-C6H4F and Ar2 = 2,4,6-C6H2Me3) were studied with respect to the reductive PET. The PET driving force (−GET) in dyads increases with the increasing electron withdrawing character of Ar substituents. Additionally, intramolecular energy transfer between porphyrins PAr1 and PAr2 is feasible in the hinges via the Förster mechanism.rn

Expanding the scope of poly(ethylene glycol)s for bioconjugation to squaric acid-mediated PEGylation and pH-sensitivity

Poly(ethylene glycol) (PEG) is used in a broad range of applications due to its unique combination of properties and is approved use in formulations for body-care products, edibles and medicine. This thesis aims at the synthesis and characterization of novel heterofunctional PEG structures and the establishment of diethyl squarate as a suitable linker for the covalent attachment to proteins. Chapter 1 is an introduction on the properties and applications of PEG as well as the fascinating chemistry of squaric acid derivatives. In Chapter 1.1, the synthesis and properties of PEG are described, and the versatile applications of PEG derivatives in everyday products are emphasized with a focus on PEG-based pharmaceuticals and nonionic surfactants. This chapter is written in German, as it was published in the German Journal Chemie in unserer Zeit. Chapter 1.2 deals with PEGs major drawbacks, its non-biodegradability, which impedes parenteral administration of PEG conjugates with polyethers exceeding the renal excretion limit, although these would improve blood circulation times and passive tumor targeting. This section gives a comprehensive overview of the cleavable groups that have been implemented in the polyether backbone to tackle this issue as well as the synthetic strategies employed to accomplish this task. Chapter 1.3 briefly summarizes the chemical properties of alkyl squarates and the advantages in protein conjugation chemistry that can be taken from its use as a coupling agent. In Chapter 2, the application of diethyl squarate as a coupling agent in the PEGylation of proteins is illustrated. Chapter 2.1 describes the straightforward synthesis and characterization of squaric acid ethyl ester amido PEGs with terminal hydroxyl functions or methoxy groups. The reactivity and selectivity of theses activated PEGs are explored in kinetic studies on the reactions with different lysine and other amino acid derivatives, followed by 1H NMR spectroscopy. Further, the efficient attachment of the novel PEGs to a model protein, i.e., bovine serum albumin (BSA), demonstrates the usefulness of the new linker for the PEGylation with heterofunctional PEGs. In Chapter 2.3 initial studies on the biocompatibility of polyether/BSA conjugates synthesized by the squaric acid mediated PEGylation are presented. No cytotoxic effects on human umbilical vein endothelial cells exposed to various concentrations of the conjugates were observed in a WST-1 assay. A cell adhesion molecule - enzyme immunosorbent assay did not reveal the expression of E-selectin or ICAM-1, cell adhesion molecules involved in inflammation processes. The focus of Chapter 3 lies on the syntheses of novel heterofunctional PEG structures which are suitable candidates for the squaric acid mediated PEGylation and exhibit superior features compared to established PEGs applied in bioconjugation. Chapter 3.1 describes the synthetic route to well-defined, linear heterobifunctional PEGs carrying a single acid-sensitive moiety either at the initiation site or at a tunable position in the polyether backbone. A universal concept for the implementation of acetal moieties into initiators for the anionic ring-opening polymerization (AROP) of epoxides is presented and proven to grant access to the degradable PEG structures aimed at. The hydrolysis of the heterofunctional PEG with the acetal moiety at the initiating site is followed by 1H NMR spectroscopy in deuterium oxide at different pH. In an exploratory study, the same polymer is attached to BSA via the squarate acid coupling and subsequently cleaved from the conjugate under acidic conditions. Furthermore, the concept for the generation of acetal-modified AROP initiators is demonstrated to be suitable for cholesterol, and the respective amphiphilic cholesteryl-PEG is cleaved at lowered pH. In Chapter 3.2, the straightforward synthesis of α-amino ω2-dihydroxyl star-shaped three-arm PEGs is described. To assure a symmetric length of the hydroxyl-terminated PEG arms, a novel AROP initiator is presented, who’s primary and secondary hydroxyl groups are separated by an acetal moiety. Upon polymerization of ethylene oxide for these functionalities and subsequent cleavage of the acid-labile unit no difference in the degree of polymerization is seen for both polyether fragments.

Icatibant , the bradykinin B2 receptor antagonist with target to the interconnected kinin systems

INTRODUCTION: HOE-140/ Icatibant is a selective, competitive antagonist to bradykinin (BK) against its binding to the kinin B2 receptor. Substitution of five non-proteogeneic amino acid analogues makes icatibant resistant to degradation by metalloproteases of kinin catabolism. Icatibant has clinical applications in inflammatory and vascular leakage conditions caused by an acute (non-controlled) production of kinins and their accumulation at the endothelium B2 receptor. The clinical manifestation of vascular leakage, called angioedema (AE), is characterized by edematous attacks of subcutaneous and submucosal tissues, which can cause painful intestinal consequences, and life-threatening complications if affecting the larynx. Icatibant is registered for the treatment of acute attacks of the hereditary BK-mediated AE, i.e., AE due to C1 inhibitor deficiency. AREAS COVERED: This review discusses emerging knowledge on the kinin system: kinin pharmacological properties, biochemical characteristics of the contact phase and kinin catabolism proteases. It underlines the responsibility of the kinins in AE initiation and the potency of icatibant to inhibit AE formation by kinin-receptor interactions. EXPERT OPINION: Icatibant antagonist properties protect BK-mediated AE patients against severe attacks, and could be developed for use in inflammatory conditions. More studies are required to confirm whether or not prolonged and frequent applications of icatibant could result in the impairment of the cardioprotective effect of BK.

Investigation Of Leucine And Glutamic Acid Properties From The Nanoscale To The Macroscale

Aerosols are known to have important effects on climate, the atmosphere, and human health. The extent of those effects is unknown and largely depend on the interaction of aerosols with water in the atmosphere. Ambient aerosols are complex mixtures of both inorganic and organic compounds. The cloud condensation nuclei (CCN) activities, hygroscopic behavior and particle morphology of a monocarboxylic amino acid (leucine) and a dicarboxylic amino acid (glutamic acid) were investigated. Activation diameters at various supersaturation conditions were experimentally determined and compared with Köhler theoretical values. The theory accounts for both surface tension and the limited solubility of organic compounds. It was discovered that glutamic acid aerosols readily took on water both when relative humidity was less than 100% and when the supersaturation condition was reached, while leucine did not show any water activation at those conditions. Moreover, the study also suggests that Köhler theory describes CCN activity of organic compounds well when only surface tension of the compound is taken into account and complete solubility is assumed. Single parameter ¿ was also computed using both CCN data and hygroscopic growth factor (GF). The results of ¿ range from 0.17 to 0.53 using CCN data and 0.09 to 0.2 using GFs. Finally, the study suggests that during the water-evaporation/particle-nucleation process, crystallization from solution droplets takes place at different locations: for glutamic acid at the particles¿ center and leucine at the particles¿ boundary.

Mechanism of Ca(v)1.2 channel modulation by the amino terminus of cardiac beta2-subunits

L-type calcium channels are composed of a pore, alpha1c (Ca(V)1.2), and accessory beta- and alpha2delta-subunits. The beta-subunit core structure was recently resolved at high resolution, providing important information on many functional aspects of channel modulation. In this study we reveal differential novel effects of five beta2-subunits isoforms expressed in human heart (beta(2a-e)) on the single L-type calcium channel current. These splice variants differ only by amino-terminal length and amino acid composition. Single-channel modulation by beta2-subunit isoforms was investigated in HEK293 cells expressing the recombinant L-type ion conducting pore. All beta2-subunits increased open probability, availability, and peak current with a highly consistent rank order (beta2a approximately = beta2b > beta2e approximately = beta2c > beta2d). We show graded modulation of some transition rates within and between deep-closed and inactivated states. The extent of modulation correlates strongly with the length of amino-terminal domains. Two mutant beta2-subunits that imitate the natural span related to length confirm this conclusion. The data show that the length of amino termini is a relevant physiological mechanism for channel closure and inactivation, and that natural alternative splicing exploits this principle for modulation of the gating properties of calcium channels.

Dysphagia due to triple A syndrome: successful treatment of achalasia by balloon dilatation

Triple A syndrome is a rare autosomal recessive inherited disorder which is characterized by alacrima, adrenal insufficiency, and achalasia. We report on a 14-year old girl with dysphagia, regurgitation, and vomiting since 5 years. At the age of five years an Addison crisis was diagnosed and cortisone substitution was initiated. In addition, the patient had episodes of conjunctivitis. Severe esophagitis and candida infection were diagnosed by esophago-gastro-duodenoscopy and treated with omeprazole and fluconazole. The esophageal barium swallow was typical for achalasia. Medical treatment of achalasia with oral nifedipine resulted only in a partial and temporal improvement. But after seven balloon dilatations dysphagia and nocturnal coughing improved clearly and a remarkable gain of weight could be seen. Direct sequencing showed a homozygous nonsense mutation in exon 11 of the AAAS gene leading to truncation at position 342 of the 546 amino acid protein. CONCLUSION: Triple A syndrome has to be considered in patients with dysphagia. In our patient, the absence of tears since birth followed by adrenal insufficiency were early signs of the triple A syndrome. Balloon dilatation of the esophago-gastric junction is an effective treatment, which can avoid surgical interventions.

High variability and non-neutral evolution of the mammalian avpr1a gene

BACKGROUND: The arginine-vasopressin 1a receptor has been identified as a key determinant for social behaviour in Microtus voles, humans and other mammals. Nevertheless, the genetic bases of complex phenotypic traits like differences in social and mating behaviour among species and individuals remain largely unknown. Contrary to previous studies focusing on differences in the promotor region of the gene, we investigate here the level of functional variation in the coding region (exon 1) of this locus. RESULTS: We detected high sequence diversity between higher mammalian taxa as well as between species of the genus Microtus. This includes length variation and radical amino acid changes, as well as the presence of distinct protein variants within individuals. Additionally, negative selection prevails on most parts of the first exon of the arginine-vasopressin receptor 1a (avpr1a) gene but it contains regions with higher rates of change that harbour positively selected sites. Synonymous and non-synonymous substitution rates in the avpr1a gene are not exceptional compared to other genes, but they exceed those found in related hormone receptors with similar functions. DISCUSSION: These results stress the importance of considering variation in the coding sequence of avpr1a in regards to associations with life history traits (e.g. social behaviour, mating system, habitat requirements) of voles, other mammals and humans in particular.

Autosomal dominant neurohypophyseal diabetes insipidus in a Swiss family, caused by a novel mutation (C59Delta/A60W) in the neurophysin moiety of prepro-vasopressin-neurophysin II (AVP-NP II).

OBJECTIVE To study clinical, morphological and molecular characteristics in a Swiss family with autosomal dominant familial neurohypophyseal diabetes insipidus (adFNDI). PARTICIPANTS AND METHODS A 15-month-old girl presenting with symptoms of polydipsia and polyuria was investigated by water deprivation test. Evaluation of the family revealed three further family members with symptomatic vasopressin-deficient diabetes insipidus. T1-weighted magnetic resonance images of the posterior pituitary were taken in two affected adult family members and molecular genetic analysis was performed in all affected individuals. RESULTS The water deprivation test in the 15-month-old child confirmed the diagnosis of vasopressin-deficient diabetes insipidus and the pedigree was consistent with autosomal dominant inheritance. The characteristic bright spot of the normal vasopressin-containing neurophypophysis was absent in both adults with adFNDI. Direct sequence analysis revealed a new deletion (177-179DeltaCGC) in exon 2 of the AVP-NP II gene in all affected individuals. At the amino acid level, this deletion eliminates cysteine 59 (C59Delta) and substitutes alanine 60 by tryptophan (A60W) in the AVP-NP II precursor; interestingly, the remainder of the reading frame remains unchanged. According to the three-dimensional structure of neurophysin, C59 is involved in a disulphide bond with C65. CONCLUSIONS Deletion of C59 and substitution of A60W in the AVP-NP II precursor is predicted to disrupt one of the seven disulphide bridges required for correct folding of the neurophysin moiety and thus disturb the function of neurophysin as the vasopressin transport protein. These data are in line with the clinical and morphological findings in the reported family with adFNDI.

Whole Proteome Analysis of the Protozoan Parasite Trypanosoma brucei using Stable Isotope Labeling by Amino Acids in Cell Culture and Mass Spectrometry

The single-celled protozoan Trypanosoma brucei spp. is the causative agent of human African trypanosomiasis and nagana in cattle. Quantitative proteomics for the first time allowed for the characterization of the proteome from several different life stages of the parasite (1-3). To achieve this, stable isotope labeling by amino acids in cell culture (SILAC; (4)) was adapted to T. brucei spp. cultures. T. brucei cells grown in standard media with dialyzed fetal calf serum containing heavy isotope-labeled amino acids (arginine and lysine) show efficient incorporation of the labeled amino acids into the whole cell proteome (8-12 divisions) and no detectable amino acid conversions. The method can be applied to both of the major life stages of the parasite and in combination with RNAi or gene knock-out approaches.

A loss-of-function variant of PTPN22 is associated with reduced risk of systemic lupus erythematosus.

A gain-of-function R620W polymorphism in the PTPN22 gene, encoding the lymphoid tyrosine phosphatase LYP, has recently emerged as an important risk factor for human autoimmunity. Here we report that another missense substitution (R263Q) within the catalytic domain of LYP leads to reduced phosphatase activity. High-resolution structural analysis revealed the molecular basis for this loss of function. Furthermore, the Q263 variant conferred protection against human systemic lupus erythematosus, reinforcing the proposal that inhibition of LYP activity could be beneficial in human autoimmunity.

The molecular mechanism of retinoic acid action: Regulation of tissue transglutaminase gene expression

Retinoic acid is a small lipophilic molecule that exerts profound effects on the growth and differentiation of both normal and transformed cells. It is also a natural morphogen that is critical in the development of embryonic structures. The molecular effects of retinoic acid involve alterations in the expression of several proteins and these changes are presumably mediated in part by alterations in gene expression. For instance, retinoic acid causes a rapid induction of tissue transglutaminase, an enzyme involved in protein cross-linking. The molecular mechanisms responsible for the effects of retinoic acid on gene expression have not been characterized. To approach this question, I have isolated and characterized tissue transglutaminase of cDNA clones. The deduced amino acid sequences of tissue transglutaminase and of factor XIIIa showed a relatively high degree of homology in their putative calcium binding domains.^ To explore the mechanism of induction of this enzyme, both primary (macrophages) and cultured cells (Swiss 3T3-C2 and CHO fibroblasts) were used. I found that retinoic acid is a general inducer of tissue transglutaminase mRNA in these cells. In murine peritoneal macrophages retinoic acid causes a rapid accumulation of this mRNA and this effect is independent of concurrent protein synthesis. The retinoic acid effect is not mediated by a post-transcriptional increase in the stability of the tissue transglutaminase mRNA, but appears to involve an increase in the transcription rate of the tissue transglutaminase gene. This provides the first example of regulation by retinoic acid of a specific gene, supporting the hypothesis that these molecules act by directly regulating the transcriptional activity of specific genes. A molecular model for the effects of retinoic acid on the expression of genes linked to cellular proliferation and differentiation is proposed. ^

Phenotypic and genotypic analysis of mouse hepatitis virus (JHM) complementation group A temperature-sensitive mutants

Temperature sensitive (ts) mutant viruses have helped elucidate replication processes in many viral systems. Several panels of replication-defective ts mutants in which viral RNA synthesis is abolished at the nonpermissive temperature (RNA$\sp{-})$ have been isolated for Mouse Hepatitis Virus, MHV (Robb et al., 1979; Koolen et al., 1983; Martin et al., 1988; Schaad et al., 1990). However, no one had investigated genetic or phenotypic relationships between these different mutant panels. In order to determine how the panel of MHV-JHM RNA$\sp{-}$ ts mutants (Robb et al., 1979) were genetically related to other described MHV RNA$\sp{-}$ ts mutants, the MHV-JHM mutants were tested for complementation with representatives from two different sets of MHV-A59 ts mutants (Koolen et al., 1983; Schaad et al., 1990). The three ts mutant panels together were found to comprise eight genetically distinct complementation groups. Of these eight complementation groups, three complementation classes are unique to their particular mutant panel; genetically equivalent mutants were not observed within the other two mutant panels. Two complementation groups were common to all three mutant panels. The three remaining complementation groups overlapped two of the three mutant sets. Mutants MHV-JHM tsA204 and MHV-A59 ts261 were shown to be within one of these overlapping complementation groups. The phenotype of the MHV-JHM mutants within this complementation class has been previously characterized (Leibowitz et al., 1982; Leibowitz et al, 1990). When these mutants were grown at the permissive temperature, then shifted up to the nonpermissive temperature at the start of RNA synthesis, genome-length RNA and leader RNA fragments accumulated, but no subgenomic mRNA was synthesized. MHV-A59 ts261 produced leader RNA fragments identical to those observed with MHV-JHM tsA204. Thus, these two MHV RNA$\sp{-}$ ts mutants that were genetically equivalent by complementation testing were phenotypically similar as well. Recombination frequencies obtained from crosses of MHV-A59 ts261 with several of the gene 1 MHV-A59 mutants indicated that the causal mutation(s) of MHV-A59 ts261 was located near the overlapping junction of ORF1a and ORF1b, in the 3$\sp\prime$ end of ORF1a, or the 5$\sp\prime$ end of ORF1b. Sequence analysis of this junction and 1400 nucleotides into the 5$\sp\prime$ end of ORF1b of MHV-A59 ts261 revealed one nucleotide change from the wildtype MHV-A59. This substitution at nucleotide 13,598 (A to G) was a silent mutation in the ORF1a reading frame, but resulted in an amino acid change in ORF1b gene product (I to V). This amino acid change would be expressed only in the readthrough translation product produced upon successful ribosome frameshifting. A revertant of MHV-A59 ts261 (R2) also retained this guanidine residue, but had a second substitution at nucleotide 14,475 in ORF1b. This mutation results in the substitution of valine for an isoleucine.^ The data presented here suggest that the mutation in MHV-A59 ts261 (nucleotide 13,598) would be responsible for the MHV-JHM complementation group A phenotype. A second-site reversion at nucleotide 14,475 may correct this defect in the revertant. Sequencing of gene 1 immediately upstream of nucleotide 13,296 and downstream of nucleotide 15,010 must be conducted to test this hypothesis. ^

Study of retinoic acid signaling in cancer cells: Cloning and characterization of retinoic acid responsive genes

Retinoids such as all-trans-retinoic acid (ATRA) are promising agents for cancer chemoprevention and therapy. ATRA can cause growth inhibition, induction of differentiation and apoptosis of a variety of cancer cells. These effects are thought to be mediated by nuclear retinoids receptors which are involved in ligand-dependent transcriptional activation of downstream target genes. Using differential display, we identified several retinoic acid responsive genes in the head and neck squamous carcinoma cells and lung cancer cells, including tissue type transglutaminase, cytochrome P450-related retinoic acid hydroxylase, and a novel gene, designated RAIG1. RAIG1 has two transcripts of 2.4 and 6.8 kbp, respectively, that are generated by alternative selection of polyadenylation sites. Both transcripts have the same open reading frame that encodes a protein comprised of 357 amino acid residues. The deduced RAIG1 protein sequence contains seven transmembrane domains, a signature structure of G protein-coupled receptors. RAIG1 mRNA is expressed at high level in fetal and adult lung tissues. Induction of RAIG1 expression by ATRA is rapid and dose-dependent. A fusion protein of RAIG1 and the green fluorescent protein was localized in the cell surface membrane and perinuclear vesicles in transiently transfected cells. The locus for RAIG1 gene was mapped to a region between D12S358 and D12S847 on chromosome 12p12.3-p13. Our study of the novel retinoic acid induced gene RAIG1 provide evidence for a possible interaction between retinoid and G protein signaling pathways.^ We further examined RAIG1 expression pattern in a panel of 84 cancer cell lines of different origin. The expression level varies greatly from very high to non-detectable. We selected a panel of different cancer cells to study the effects of retinoids and other differentiation agents. We observed: (1) In most cases, retinoids (including all-trans retinoic acid, 4HPR, CD437) could induce the expression of RAIG-1 in cells from cancers of the breast, colon, head and neck, lung, ovarian and prostate. (2) Compare to retinoids, butyrate is often a more potent inducer of RAIG-1 expression in many cancer cells. (3) Butyrate, Phenylacetate butyrate, (R)P-Butyrate and (S)P-Butyrate have different impact on RAIG1 expression which varies among different cell lines. Our results indicate that retinoids could restore RAIG1 expression that is down-regulated in many cancer cells.^ A mouse homologous gene, mRAIG1, was cloned by 5$\sp\prime$ RACE reaction. mRAIG1 cDNA has 2105 bp and shares 63% identity with RAIG1 cDNA. mRAIG1 encodes a polypeptide of 356 amino acid which is 76% identity with RAIG1 protein. mRAIG1 protein also has seven transmembrane domains which are structurally identical to those of RAIG1 protein. Only one 2.2 kbp mRAIG1 transcript could be detected. The mRAIG1 mRNA is also highly expressed in lung tissue. The expression of mRAIG1 gene could be induced by ATRA in several mouse embryonal carcinoma cells. The induction of mRAIG1 expression is associated with retinoic acid-induced neuroectoderm differentiation of P19 cells. Similarity in cDNA and protein sequence, secondary structure, tissue distribution and inducible expression by retinoic acid strongly suggest that the mouse gene is the homologue of the human RAIG1 gene. ^

The role of the amino terminus of Bacillus subtilis sigmaK in transcription initiation

The sigma (σ) subunit of eubacterial RNA polymerase (RNAP) is required for specific recognition of promoter DNA sequences and transcription initiation. Regulation of bacterial gene expression can be achieved by modulating a factor activity. The Bacillus subtilis sporulation a σ factor, σ K, controls gene expression of the late sporulation regulon. σ K is synthesized as an inactive precursor protein, pro-σ K, with a 20 amino acid pro sequence. Proteolytic processing of the pro sequence produces the active form, σK, which is able to bind to the core subunits of RNAP to direct gene expression. Thus, the pro sequence renders σK inactive in vivo. After processing, the amino terminus of σK consists of region 1.2, which is conserved among various σ factors. To understand the role of the amino terminus of σK, namely the pro sequence and region 1.2, mutagenesis of both regions was pursued. NH 2-terminal truncations of pro-σK were constructed to address how the pro sequence silences σK activity. The work described here shows that the pro sequence inhibits the ability of σ K to associate with the core subunits and that a deletion of only six amino acids of the pro sequence is sufficient to activate pro-σ K for DNA binding and transcription initiation to levels similar to σ K. Additionally, site directed mutagenesis was used to obtain single amino acid substitutions in region 1.2 to address the role of region 1.2 in σ K transcriptional activity. Two mutations were isolated, converting a lysine (K) to an alanine (A) at position three, and an asparagine (N) to a tyrosine (Y) at position five, both of which alter the efficiency of transcription initiation by RNAP containing the mutant σKs. Surprisingly, σ KK3A increased transcript production when compared to wild type. This increase is due to improvement in DNA affinity and increased stability of RNAP-DNA promoter open complexes. σKN5Y showed a decrease in transcription activity that is related to defects in the ability of RNAP to make the transition from the closed to open RNAP-DNA complex. Results of both the pro sequence and region 1.2 analyses indicate that the amino terminus of σK is important for transcription activity and this work adds to the increasing body of evidence that the amino termini of many σ factors modulate transcription initiation by RNAP. ^

Trypanosoma brucei eflornithine transporter AAT6 is a low-affinity low-selective transporter for neutral amino acids

Amino acid transporters are crucial for parasite survival since the cellular metabolism of parasitic protozoa depends on the uptake of exogenous amino acids. Amino acid transporters are also of high pharmacological relevance because they may mediate uptake of toxic amino acid analogues. In the present study we show that the eflornithine transporter AAT6 from Trypanosoma brucei (TbAAT6) mediates growth on neutral amino acids when expressed in Saccharomyces cerevisiae mutants. The transport was electrogenic and further analysed in Xenopus laevis oocytes. Neutral amino acids, proline analogues, eflornithine and acivicin induced inward currents. For proline, glycine and tryptophan the apparent affinities and maximal transport rates increased with more negative membrane potentials. Proline-induced currents were dependent on pH, but not on sodium. Although proline represents the primary energy source of T. brucei in the tsetse fly, down-regulation of TbAAT6-expression by RNAi showed that in culture TbAAT6 is not essential for growth of procyclic form trypanosomes in the presence of glucose or proline as energy source. TbAAT6-RNAi lines of both bloodstream and procyclic form trypanosomes showed reduced susceptibility to eflornithine, whereas the sensitivity to acivicin remained unchanged, indicating that acivicin enters the cell by more than one transporter