Control of Cell Proliferation,Organ Growth,and DNA Damage Response Operate Independently of Dephosphorylation of the Arabidopsis Cdk1 Homolog CDKA;1

Entry into mitosis is universally controlled by cyclin-dependent kinases (CDKs). A key regulatory event in metazoans and fission yeast is CDK activation by the removal of inhibitory phosphate groups in the ATP binding pocket catalyzed by Cdc25 phosphatases. In contrast with other multicellular organisms, we show here that in the flowering plant Arabidopsis thaliana, cell cycle control does not depend on sudden changes in the phosphorylation pattern of the PSTAIRE-containing Cdk1 homolog CDKA;1. Consistently, we found that neither mutants in a previously identified CDC25 candidate gene nor plants in which it is overexpressed display cell cycle defects. Inhibitory phosphorylation of CDKs is also the key event in metazoans to arrest cell cycle progression upon DNA damage. However, we show here that the DNA damage checkpoint in Arabidopsis can also operate independently of the phosphorylation of CDKA;1. These observations reveal a surprising degree of divergence in the circuitry of highly conserved core cell cycle regulators in multicellular organisms. Based on biomathematical simulations, we propose a plant-specific model of how progression through the cell cycle could be wired in Arabidopsis.     

Construction of a Gene Knockout System for Application in Paenibacillus alvei CCM 2051T,Exemplified by the S-Layer Glycan Biosynthesis Initiation Enzyme WsfP

The gram-positive bacterium Paenibacillus alvei CCM 2051^T is covered by an oblique surface layer (S-layer) composed of glycoprotein subunits. The S-layer O-glycan is a polymer of [→3)-β-d-Galp-(1[α-d-Glcp-(1→6)]→4)-β-d-ManpNAc-(1→] repeating units that is linked by an adaptor of -[GroA-2→OPO₂→4-β-d-ManpNAc-(1→4)]→3)-α-l-Rhap-(1→3)-α-l-Rhap-(1→3)-α-l-Rhap-(1→3)-β-d-Galp-(1→ to specific tyrosine residues of the S-layer protein. For elucidation of the mechanism governing S-layer glycan biosynthesis, a gene knockout system using bacterial mobile group II intron-mediated gene disruption was developed. The system is further based on the sgsE S-layer gene promoter of Geobacillus stearothermophilus NRS 2004/3a and on the Geobacillus-Bacillus-Escherichia coli shuttle vector pNW33N. As a target gene, wsfP, encoding a putative UDP-Gal:phosphoryl-polyprenol Gal-1-phosphate transferase, representing the predicted initiation enzyme of S-layer glycan biosynthesis, was disrupted. S-layer protein glycosylation was completely abolished in the insertional P. alvei CCM 2051^T wsfP mutant, according to sodium dodecyl sulfate-polyacrylamide gel electrophoresis evidence and carbohydrate analysis. Glycosylation was fully restored by plasmid-based expression of wsfP in the glycan-deficient P. alvei mutant, confirming that WsfP initiates S-layer protein glycosylation. This is the first report on the successful genetic manipulation of bacterial S-layer protein glycosylation in vivo, including transformation of and heterologous gene expression and gene disruption in the model organism P. alvei CCM 2051^T.Bacterial cell surface layer (S-layer) glycoproteins provide a unique self-assembly matrix that has been optimized by nature for regular and periodic display of glycans with nanometer scale accuracy (21, 31). Exploitation of this self-assembly system for surface display of functional, tailor-made glycans is an attractive alternative to the use of common cell surface anchors (7), with potential areas of application relating to any biological phenomenon that is based on carbohydrate recognition, such as receptor-substrate interaction, signaling, or cell-cell communication. A prerequisite for this endeavor is the availability of an S-layer glycoprotein-covered bacterium that is amenable to genetic manipulation. Despite the high application potential offered by the S-layer glycan display system, there are so far only two reports in the literature dealing with the genetic manipulation of S-layer glycoprotein-carrying bacteria. Both reports concern the gram-negative periodontal pathogen Tannerella forsythia ATCC 43037, but neither of them affects S-layer protein glycosylation (12, 24). In archaea, in contrast, molecular studies of S-layer protein glycosylation are quite advanced (1), but with the archaeal system, S-layer glycoprotein self-assembly, which is a prerequisite for the desired glycan display, has not been manageable in vitro so far.Our model organisms and, hence, candidates for S-layer-mediated glycan display enabled by carbohydrate engineering techniques are members of the Bacillaceae family. Currently, the S-layer glycosylation system of the thermophilic bacterium Geobacillus stearothermophilus NRS 2004/3a is best understood (20, 23, 29, 33, 34). However, a drawback of this organism is its resistance to take up foreign DNA. Although described in the literature (13, 14, 37), transformation of thermophilic bacilli seems to be a strain-specific trait. Based on successful transformation experiments in our laboratory, the mesophilic bacterium Paenibacillus alvei CCM 2051^T (ATCC 6344; DSM 29) (formerly Bacillus alvei [4]) was chosen to set up a system for genetic manipulation. The bacterium is completely covered with an oblique S-layer lattice composed of glycoprotein species. Various aspects of its S-layer, including ultrastructural characterization (27), glycosylation analysis (2, 18), and glycan biosynthesis (11), have been investigated so far. The S-layer O-glycans are polymers of [→3)-β-d-Galp-(1[α-d-Glcp-(1→6)]→4)-β-d-ManpNAc-(1→] repeating units that are linked via the adaptor -[GroA-2→OPO₂→4-β-d-ManpNAc-(1→4)]→3)-α-l-Rhap-(1→3)-α-l-Rhap-(1→3)-α-l-Rhap-(1→3)-β-d- Galp-(1→ to specific tyrosine residues (2, 18) of the S-layer protein SpaA (GenBank accession number {"type":"entrez-nucleotide","attrs":{"text":"FJ751775","term_id":"225546017","term_text":"FJ751775"}}FJ751775).Due to the presence of an identical adaptor saccharide backbone in the S-layer glycan of G. stearothermophilus NRS 2004/3a (29), where its biosynthesis is initiated by the UDP-Gal:phosphoryl-polyprenol Gal-1-phosphate transferase WsaP (33), it was conceivable that a homologous enzyme would initiate S-layer glycosylation in P. alvei CCM 2051^T. Considering that the S-layer protein glycosylation machinery has been found to be encoded by S-layer glycosylation (slg) gene clusters (21), degenerate primers for the rml genes catalyzing the dTDP-l-Rha biosynthesis required for building up the adaptor saccharide of the P. alvei CCM 2051^T S-layer glycan were used to define a point of entry into the glycosylation locus (K. Zarschler, B. Janesch, P. Messner, and C. Schäffer, unpublished data). Chromosome walking revealed the existence of an slg gene cluster of about 24 kb, including an open reading frame (ORF) predicted to encode the initiation enzyme of S-layer protein glycosylation. The corresponding gene, named wsfP, served as a first target for the gene knockout system developed in the course of the present study. This target was chosen because loss of function would be easily screenable, resulting in an S-layer glycosylation-deficient mutant. The gene knockout system constructed for insertional inactivation of the chromosomal wsfP gene of P. alvei CCM 2051^T is based on the commercially available bacterial mobile group II intron Ll.LtrB of Lactococcus lactis, in combination with further components available in our laboratory, including the broad-host-range S-layer gene promoter of sgsE from G. stearothermophilus NRS 2004/3a (22) and the Geobacillus-Bacillus-Escherichia coli shuttle vector pNW33N. Bacterial mobile group II introns are retroelements inserted into specific DNA target sites at high frequency by use of the retrohoming mechanism, by which the excised intron lariat RNA is inserted directly into a DNA target site and is then reverse transcribed by the associated intron-encoded enzyme protein (6, 8, 17). Since the DNA target site is recognized primarily by base pairing of intron RNA, which can be modified, and a few intron-encoded-enzyme-protein recognition positions, these introns can be inserted efficiently into any specific DNA target (9, 15, 35, 40).The aim of this study is the development of a genetic tool for manipulation of S-layer protein glycosylation in P. alvei CCM 2051^T. For proof of concept, we specifically deal with (i) the construction of a broad-host-range gene knockout system based on the L. lactis Ll.LtrB intron; (ii) its modification for specific disruption of the wsfP gene on the P. alvei CCM 2051^T chromosome, encoding the putative initiation enzyme of S-layer glycan biosynthesis; and (iii) the reconstitution of enzyme activity by plasmid-based expression of wsfP and its predicted functional homologue wsaP from G. stearothermophilus NRS 2004/3a.     

Choice of Mouse Strain Influences the Outcome in a Mouse Model of Chemical-Induced Asthma

Background

The development of occupational asthma is the result of interactions between environmental factors and individual susceptibility. We assessed how our model of chemical-induced asthma is influenced by using different mouse strains.

Methodology/Principal Findings

On days 1 and 8, male mice of 7 different strains (BALB/c, BP/2, A/J, C57Bl/6, DBA/2, CBA and AKR) were dermally treated with toluene-2,4-diisocyanate (TDI) (0.3%) or vehicle (acetone/olive oil, AOO, 2∶3) on each ear (20 µl). On day 15, they received an oropharyngeal instillation of TDI (0.01%) or AOO (1∶4). Airway reactivity to methacholine, total and differential cell counts in bronchoalveolar lavage (BAL) and total serum IgE and IgG_2a levels were measured. Lymphocyte subpopulations in auricular lymph nodes and in vitro release of cytokines by ConA stimulated lymphocytes were assessed. In TDI-sensitized and challenged mice, airway hyper-reactivity was only observed in BALB/c, BP/2, A/J and AKR mice; airway inflammation was most pronounced in BALB/c mice; numbers of T-helper (CD4⁺), T-activated (CD4⁺CD25⁺), T-cytotoxic (CD8⁺) and B- lymphocytes (CD19⁺) were increased in the auricular lymph nodes of BALB/c, BP/2, A/J and CBA mice; elevated concentrations of IL-4, IL-10, IL-13 and IFN-γ were detected in supernatant of lymphocytes from BALB/c, BP/2, A/J, C57Bl/6 and CBA mice cultured with concanavaline A, along with an increase in total serum IgE.

Conclusion

The used mouse strain has considerable and variable impacts on different aspects of the asthma phenotype. The human phenotypical characteristics of chemically-induced occupational asthma were best reproduced in Th2-biased mice and in particular in BALB/c mice.     

Immunological determinants of ventilatory changes induced in mice by dermal sensitization and respiratory challenge with toluene diisocyanate

The objective of the study was to characterize better the immunologic mechanisms underlying a previously developed animal model of chemical-induced asthma. BALB/c and severe combined immunodeficiency disease (SCID) mice received toluene diisocyanate (TDI) or vehicle on each ear on day 1 and/or day 7. On day 10, they were intranasally challenged with TDI or vehicle. Ventilatory function was monitored by whole body plethysmography for 40 min after challenge. Reactivity to methacholine was measured 23 h later: enhanced pause and actual resistance measurements. Pulmonary inflammation was assessed 1, 6, and 24 h after challenge by bronchoalveolar lavage (BAL). Tumor necrosis factor-alpha and macrophage inflammatory protein (MIP)-2 levels were measured in BAL. Immunological parameters included total IgE, IgG1, and IgG2a in serum, lymphocyte populations in auricular and cervical lymph nodes, and IL-4 and IFN-gamma levels in supernatants of lymph node cells, cultured with or without concanavalin A. Ventilatory changes suggestive of airway obstruction and increased methacholine reactivity were observed in all TDI-sensitized and TDI intranasally instilled mice, except in SCID mice. A neutrophil influx, accompanied by an increase in MIP-2 levels, was found in BAL of all responding groups 6 and 24 h after intranasal challenge. In BALB/c mice an increased level of CD19+ B cells was found in the auricular lymph nodes. IL-4 and IFN-gamma levels were increased in supernatants of concanavalin A-stimulated auricular lymph node cells from BALB/c mice completely treated with TDI. These results indicate that our model is dependent on the presence of lymphocytes, but it is not characterized by a preferential stimulation of Th1 or Th2 lymphocytes.     

Longevity and life history of cave bears – a review and novel data from tooth cementum and relative emergence of permanent dentition

Abstract

Longevity and other life history variables are key to understanding evolutionary processes and the biology of extinct animals. For the past 20 years, the lifespan of cave bears received an increased interest. Studies focusing on incremental lines of tooth cementum resulted in detailed mortality patterns from different localities. In this review, we summarise literature on age estimation as well as mortality of different European cave bear localities and present novel data on longevity from 94 teeth originating from 20 European localities. Additionally, the relative tooth emergence pattern of the permanent dentition is investigated under the Schultz’s rule framework of possible life history implications. For this, the known sequences of extant bear species are compared with the one of cave bears. Our results suggest that the typical duration of the life of cave bears was 19 years but data from literature show that in rare cases ages of up to 30–32 years were achieved. Additionally, we present the oldest known age for the Middle Pleistocene cave bear Ursus deningeri, 29 years. The tooth eruption pattern of cave bears exhibits a heterochronic shift that implies, under the assumption of Schulz’ rule, a slightly faster life history than closely related species.     

Atypical PKCzeta is involved in RhoA-dependent mitogenic signaling by the P2Y(12) receptor in C6 cells

When nucleotide hydrolysis is prevented, agonists of the P2Y(12) receptor enhance the proliferation of C6 glioma cells by RhoA-dependent, protein kinase C (PKC)-dependent activation of the extracellular signal-regulated kinase (ERK) pathway [Claes P, Grobben B, Van Kolen K, Roymans D & Slegers H (2001) Br J Pharmacol134, 402-408; Grobben B, Claes P, Van Kolen K, Roymans D, Fransen P, Sys SU & Slegers H (2001) J Neurochem78, 1325-1338]. In this study, we show that ERK1/2 phosphorylation was not affected by transfection of the cells with the Gbetagamma-subunit-scavenging adrenergic receptor kinase peptide [betaARK1-(495-689)] or with Rap1GAPII, indicating that P2Y(12) receptor stimulation enhances ERK1/2 phosphorylation by G(i)alpha subunit-mediated signaling independently of Rap1 activation. Inhibition of the RhoA downstream effector Rho-associated coiled-coil-containing kinase (ROCK) with Y-27632 did not affect the P2Y(12) receptor-induced increase in ERK1/2 phosphorylation but abrogated the mitogenic response. Involvement of growth factor receptor transactivation in the signaling towards ERK phosphorylation could be ruled out by the lack of an effect of PP2, AG1024, AG1296 or SU1498, inhibitors of Src, insulin-like growth factor receptor, platelet-derived growth factor receptor and vascular endothelial growth factor receptor kinase activity, respectively. Experiments with bisindolylmaleimide I and IX indicated the requirement of PKC activity. Classical and novel PKC isoforms could be excluded by treatment of the cells with G?6976 and calphostin C, whereas addition of a myristoylated PKCzeta pseudosubstrate inhibitor completely abolished P2Y(12) receptor-induced ERK1/2 activation. Moreover, coimmunoprecipitation experiments revealed PKCzeta/Raf1 and PKCzeta/ERK association, indicating the involvement of PKCzeta. From the data presented, we can conclude that the P2Y(12) receptor enhances cell proliferation by a G(i)alpha-dependent, RhoA-dependent PKCzeta/Raf1/MEK/ERK pathway that requires activation of ROCK, which is not involved in ERK1/2 signaling.     

Role of quorum sensing and antimicrobial component production by Serratia plymuthica in formation of biofilms, including mixed biofilms with Escherichia coli

We have previously characterized the N-acyl-l-homoserine lactone-based quorum-sensing system of the biofilm isolate Serratia plymuthica RVH1. Here we investigated the role of quorum sensing and of quorum-sensing-dependent production of an antimicrobial compound (AC) on biofilm formation by RVH1 and on the cocultivation of RVH1 and Escherichia coli in planktonic cultures or in biofilms. Biofilm formation of S. plymuthica was not affected by the knockout of splI or splR, the S. plymuthica homologs of the luxI or luxR quorum-sensing gene, respectively, or by the knockout of AC production. E. coli grew well in mixed broth culture with RVH1 until the latter reached 8.5 to 9.5 log CFU/ml, after which the E. coli colony counts steeply declined. In comparison, only a very small decline occurred in cocultures with the S. plymuthica AC-deficient and splI mutants. Complementation with exogenous N-hexanoyl-l-homoserine lactone rescued the wild-type phenotype of the splI mutant. The splR knockout mutant also induced a steep decline of E. coli, consistent with its proposed function as a repressor of quorum-sensing-regulated genes. The numbers of E. coli in 3-day-old mixed biofilms followed a similar pattern, being higher with S. plymuthica deficient in SplI or AC production than with wild-type S. plymuthica, the splR mutant, or the splI mutant in the presence of N-hexanoyl-l-homoserine lactone. Confocal laser scanning microscopic analysis of mixed biofilms established with strains producing different fluorescent proteins showed that E. coli microcolonies were less developed in the presence of RVH1 than in the presence of the AC-deficient mutant.     

Assessment of embryonic growth in chicken eggs by means of visible transmission spectroscopy

During this work, it was investigated whether spectral measurements can be used to monitor embryonic growth. An experiment was conducted in which both the transmission spectra and embryonic weight were determined on 240 eggs (Cobb, 37 weeks) between Day 5 and Day 10 of incubation. The spectral data were linked to embryonic weight by means of a partial least squares analysis. Different preprocessing procedures were compared during this work, that is, smoothing, multiplicative scatter correction (MSC), and first‐ and second‐order derivative. Compared to the remainder of the preprocessing procedures, MSC leads to a considerable improvement of the prediction capability of the embryonic weight. The ratio of performance to deviation obtained for the MSC spectra equaled 4.5 indicating that a very accurate prediction of embryonic weight is feasible based on the VIS/NIR transmission measurements. Important regions for the prediction are situated around 685–740 nm. It is suggested that the spectral changes in these spectral regions result from the displacement of carotenoids from the yolk into the blood circuitry. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010