The mode of action of the lantibiotic lacticin 3147--a complex mechanism involving specific interaction of two peptides and the cell wall precursor lipid II

Lacticin 3147 is a two-peptide lantibiotic produced by Lactococcus lactis in which both peptides, LtnA1 and LtnA2, interact synergistically to produce antibiotic activities in the nanomolar concentration range; the individual peptides possess marginal (LtnA1) or no activity (LtnA2). We analysed the molecular basis for the synergism and found the cell wall precursor lipid II to play a crucial role as a target molecule. Tryptophan fluorescence measurements identified LtnA1, which is structurally similar to the lantibiotic mersacidin, as the lipid II binding component. However, LtnA1 on its own was not able to substantially inhibit cell wall biosynthesis in vitro; for full inhibition, LtnA2 was necessary. Both peptides together caused rapid K(+) leakage from intact cells; in model membranes supplemented with lipid II, the formation of defined pores with a diameter of 0.6 nm was observed. We propose a mode of action model in which LtnA1 first interacts specifically with lipid II in the outer leaflet of the bacterial cytoplasmic membrane. The resulting lipid II:LtnA1 complex is then able to recruit LtnA2 which leads to a high-affinity, three-component complex and subsequently inhibition of cell wall biosynthesis combined with pore formation.     

The systematic position of the Lower Cretaceous heteromorphic ammonite <Emphasis Type="Italic">Pictetia</Emphasis> Uhlig, 1883

The heteromorphic ammonite genus Pictetia has previously been included in the Lytoceratidae. A reassessment of literature data and a detailed analysis of the suture shows that Pictetia lacks a septal lobe as well as a bifid internal lobe and lateral lobe, all features indicative of the Lytoceratidae. This and the quadrilobate suture line, combined with the loose coiling of the shell, indicate that it should be included in the polyphyletic suborder “Ancyloceratina”. Any further systematic assessment is impossible at the moment.     

Overexpression of RAD51 suppresses recombination defects: a possible mechanism to reverse genomic instability

RAD51, a key protein in the homologous recombinational DNA repair (HRR) pathway, is the major strand-transferase required for mitotic recombination. An important early step in HRR is the formation of single-stranded DNA (ss-DNA) coated by RPA (a ss-DNA-binding protein). Displacement of RPA by RAD51 is highly regulated and facilitated by a number of different proteins known as the ‘recombination mediators’. To assist these recombination mediators, a second group of proteins also is required and we are defining these proteins here as ‘recombination co-mediators’. Defects in either recombination mediators or co-mediators, including BRCA1 and BRCA2, lead to impaired HRR that can genetically be complemented for (i.e. suppressed) by overexpression of RAD51. Defects in HRR have long been known to contribute to genomic instability leading to tumor development. Since genomic instability also slows cell growth, precancerous cells presumably require genomic re-stabilization to gain a growth advantage. RAD51 is overexpressed in many tumors, and therefore, we hypothesize that the complementing ability of elevated levels of RAD51 in tumors with initial HRR defects limits genomic instability during carcinogenic progression. Of particular interest, this model may also help explain the high frequency of TP53 mutations in human cancers, since wild-type p53 represses RAD51 expression.     

A proteomic approach towards the identification of the matrix protein content of the two types of microbodies in Neurospora crassa

Microbodies (peroxisomes) comprise a class of organelles with a similar biogenesis but remarkable biochemical heterogeneity. Here, we purified the two distinct microbody family members of filamentous fungi, glyoxysomes and Woronin bodies, from Neurospora crassa and analyzed their protein content by HPLC/ESI‐MS/MS. In the purified Woronin bodies, we unambiguously identified only hexagonal 1 (HEX1), suggesting that the matrix is probably exclusively filled with the HEX1 hexagonal crystal. The proteomic analysis of highly purified glyoxysomes allowed the identification of 191 proteins. Among them were 16 proteins with a peroxisomal targeting signal type 1 (PTS1) and three with a PTS2. The collection also contained the previously described N. crassa glyoxysomal matrix proteins FOX2 and ICL1 that lack a typical PTS. Three PTS1 proteins were identified that likely represent the long sought glyoxysomal acyl‐CoA dehydrogenases of filamentous fungi. Two of them were demonstrated by subcellular localization studies to be indeed glyoxysomal. Furthermore, two PTS proteins were identified that are suggested to be involved in the detoxification of nitroalkanes. Since the glyoxysomal localization was experimentally demonstrated for one of these enzymes, a new biochemical reaction is expected to be associated with microbody function.     

Optimization of Low-Cost Culture Media for the Production of Biomass and Bacteriocin by a Urogenital Lactobacillus salivarius Strain

The aim of this work was to formulate a culture medium of lower cost than conventional laboratory media, in order to simultaneously obtain high amounts of both biomass and bacteriocin of vaginal Lactobacillus salivarius CRL 1328. The growth assays under different culture conditions were performed by using a 2^8?2 central composite experimental design, with a central point and sixteen additional points. The factors taken into consideration were glucose, lactose, yeast extract, tryptone, ammonium citrate, sodium acetate, MgSO₄ and MnSO₄. The simultaneous presence of a carbon source (mainly glucose), a nitrogen source (mainly yeast extract) and salts (mainly MnSO₄, MgSO₄ and sodium acetate) allowed the highest cell biomass and bacteriocin levels to be reached in the experimental design. Through the application of the desirability function, several optimal medium compositions to achieve efficient production of biomass and bacteriocin were predicted. The optimized growth media allow a cost reduction of around 25 to 40% compared with conventional broths. The results obtained represent an advance in the search of the most suitable strategies for the production of bioactive compounds for pharmaceutical products to prevent or treat female urogenital infections.     

Intracellular Trafficking and Synaptic Function of APL-1 in Caenorhabditis elegans

Background

Alzheimer''s disease (AD) is a neurodegenerative disorder primarily characterized by the deposition of β-amyloid plaques in the brain. Plaques are composed of the amyloid-β peptide derived from cleavage of the amyloid precursor protein (APP). Mutations in APP lead to the development of Familial Alzheimer''s Disease (FAD), however, the normal function of this protein has proven elusive. The organism Caenorhabditis elegans is an attractive model as the amyloid precursor-like protein (APL-1) is the single ortholog of APP, and loss of apl-1 leads to a severe molting defect and early larval lethality.

Methodology/Principal Findings

We report here that lethality and molting can be rescued by full length APL-1, C-terminal mutations as well as a C-terminal truncation, suggesting that the extracellular region of the protein is essential for viability. RNAi knock-down of apl-1 followed by drug testing on the acetylcholinesterase inhibitor aldicarb showed that loss of apl-1 leads to aldicarb hypersensitivity, indicating a defect in synaptic function. The aldicarb hypersensitivity can be rescued by full length APL-1 in a dose dependent fashion. At the cellular level, kinesins UNC-104/KIF-1A and UNC-116/kinesin-1 are positive regulators of APL-1 expression in the neurons. Knock-down of the small GTPase rab-5 also leads to a dramatic decrease in the amount of apl-1 expression in neurons, suggesting that trafficking from the plasma membrane to the early endosome is important for apl-1 function. Loss of function of a different small GTPase, UNC-108, on the contrary, leads to the retention of APL-1 in the cell body.

Conclusions/Significance

Our results reveal novel insights into the intracellular trafficking of APL-1 and we report a functional role for APL-1 in synaptic transmission.     

Cloning, nucleotide sequence and expression of a new L-N-carbamoylase gene from Arthrobacter aurescens DSM 3747 in E. coli

An L-N-carbamoyl amino acid amidohydrolase (L-N-carbamoylase) from Arthrobacter aurescens DSM 3747 was cloned in E. coli and the nucleotide sequence was determined. After expression of the gene in E. coli the enzyme was purified to homogeneity and characterized. The enzyme was shown to be strictly L-specific and exhibited the highest activity in the hydrolysis of beta-aryl substituted N alpha-carbamoyl-alanines as e.g. N-carbamoyl-tryptophan. Carbamoyl derivatives of beta-alanine and charged aliphatic amino acids were not accepted as substrates. The N-carbamoylase of A. aurescens DSM 3747 differs from all known enzymes with respect to its substrate specificity although amino acid sequence identity scores of 35-38% to other N-carbamoylases have been detected. The enzyme consists of two subunits of 44,000 Da, and has an isoelectric point of 4.3. The optima of temperature and pH were determined to be 50 degrees C and pH 8.5 respectively. At 37 degrees C the enzyme was completely stable for several days.     

Dynamic changes in C-Raf phosphorylation and 14-3-3 protein binding in response to growth factor stimulation: differential roles of 14-3-3 protein binding sites

Phosphorylation events play a crucial role in Raf activation. Phosphorylation of serines 259 and 621 in C-Raf and serines 364 and 728 in B-Raf has been suggested to be critical for association with 14-3-3 proteins. To study the functional consequences of Raf phosphorylations at these positions, we developed and characterized phosphospecific antibodies directed against 14-3-3 binding epitopes: a monoclonal phosphospecific antibody (6B4) directed against pS621 and a polyclonal antibody specific for B-Raf-pS364 epitope. Although 6B4 detected both C- and B-Raf in Western blots, it specifically recognizes the native form of C-Raf but not B-Raf. Contrary to B-Raf, a kinase-dead mutant of C-Raf was found to be only poorly phosphorylated in the Ser-621 position. Moreover, serine 259 to alanine mutation prevented the Ser-621 phosphorylation suggesting an interdependence between these two 14-3-3 binding domains. Direct C-Raf.14-3-3 binding studies with purified proteins combined with competition assays revealed that the 14-3-3 binding domain surrounding pS621 represents the high affinity binding site, whereas the pS259 epitope mediates lower affinity binding. Raf isozymes differ in their 14-3-3 association rates. The time course of endogenous C-Raf activation in mammalian cells by nerve growth factor (NGF) has been examined using both phosphospecific antibodies directed against 14-3-3 binding sites (6B4 and anti-pS259) as well as phosphospecific antibodies directed against the activation domain (anti-pS338 and anti-pY340/pY341). Time course of Ser-621 phosphorylation, in contrast to Ser-259 phosphorylation, exhibited unexpected pattern reaching maximal phosphorylation within 30 s of NGF stimulation. Phosphorylation of tyrosine 340/341 reached maximal levels subsequent to Ser-621 phosphorylation and was coincident with emergence of kinase activity. Taken together, we found substantial differences between C-Raf.14-3-3 binding epitopes pS259 and pS621 and visualized for the first time the sequence of the essential C-Raf phosphorylation events in mammalian cells in response to growth factor stimulation.     

Identification of mitogen-activated protein kinase homologues from Leishmania mexicana

Mitogen-activated protein kinases are key-regulatory elements in the differentiation, proliferation, apoptosis and stress response of eukaryotic cells. Our recent identification of a mitogen-activated protein kinase homologue in Leishmania mexicana which is essential for the proliferation of the amastigote stage of the parasite living in the parasitophorous vacuole of the infected macrophage prompted us to screen the genome of L. mexicana for additional mitogen-activated protein kinase homologues using degenerate oligonucleotide primers in a polymerase chain reaction amplification approach. We cloned and sequenced the genes for eight new mitogen-activated protein kinase homologues which were subsequently shown to be present in one copy per haploid genome. The mRNA levels of the kinases varied significantly in pro- and amastigote life stages of the parasite. We used the structural information of the p38 stress-activated protein kinase, which belongs to the family of mitogen-activated protein kinases, for the alignment of the deduced proteins and the verification of the predicted secondary structure elements. All new mitogen-activated protein kinases reveal the typical 12 subdomain primary structure, the conserved residues characterising serine/threonine protein kinases and the characteristic TXY motif in the phosphorylation lip. Typical features of some of the molecules are amino acid insertions between the subdomains and long carboxy-terminal amino acid extensions carrying putative src-homology 3-binding motifs.     

Protein kinase involved in flagellar-length control

During its life cycle, the parasitic protozoon Leishmania mexicana differentiates from a flagellated form, the promastigote, to an amastigote form carrying a rudimentary flagellum. Besides biochemical changes, this process involves a change in overall cell morphology including flagellar shortening. A mitogen-activated protein kinase kinase homologue designated LmxMKK was identified in a homology screening and found to be critically involved in the regulation of flagellar assembly and cell size. LmxMKK is exclusively expressed in the promastigote stage and is likely to be regulated by posttranslational mechanisms such as phosphorylation. A deletion mutant for the single-copy gene revealed motile flagella dramatically reduced in length and lacking the paraflagellar rod, a structure adjacent to the axoneme in kinetoplastid flagella. Moreover, a fraction of the cells showed perturbance of the axonemal structure. Complementation of the deletion mutant with the wild-type gene restored typical promastigote morphology. We propose that LmxMKK influences anterograde intraflagellar transport to maintain flagellar length in Leishmania promastigotes; as such, it is the first protein kinase known to be involved in organellar assembly.