Proteins encoded by the mre gene cluster in Streptomyces coelicolor A3(2) cooperate in spore wall synthesis

It is still an open question how an intracellular cytoskeleton directs the synthesis of the peptidoglycan exoskeleton. In contrast to MreB of rod-shaped bacteria, which is essential for lateral cell wall synthesis, MreB of Streptomyces coelicolor has a role in sporulation. To study the function of the S. coelicolor mre gene cluster consisting of mreB, mreC, mreD, pbp2 and sfr, we generated non-polar replacement mutants. The individual mutants were viable and growth of substrate mycelium was not affected. However, all mutants produced enlarged spores, which frequently germinated prematurely and were sensitive to heat, high osmolarity and cell wall damaging agents. Protein-protein interaction assays by bacterial two-hybrid analyses indicated that the S. coelicolor Mre proteins form a spore wall synthesizing complex, which closely resembles the lateral wall synthesizing complex of rod-shaped bacteria. Screening of a genomic library identified several novel putative components of this complex. One of them (sco2097) was deleted. The Δsco2097 mutant formed sensitive spores with an aberrant morphology, demonstrating that SCO2097 is a new player in cell morphogenesis of Streptomyces. Our results suggest that all Mre proteins cooperate with the newly identified proteins in the synthesis of the thickened spore wall required to resist detrimental environmental conditions.     

Ex situ conservation genetics: a review of molecular studies on the genetic consequences of captive breeding programmes for endangered animal species

Captive breeding has become an important tool in species conservation programmes. Current management strategies for ex situ populations are based on theoretical models, which have mainly been tested in model species or assessed using studbook data. During recent years an increasing number of molecular genetic studies have been published on captive populations of several endangered species. However, a comprehensive analysis of these studies is still outstanding. Here, we present a review of the published literature on ex situ conservation genetics with a focus on molecular studies. We analysed 188 publications which either presented empirical studies using molecular markers (105), studbook analyses (26), theoretical work (38), or tested the genetic effects of management strategies using model species (19). The results show that inbreeding can be minimized by a thorough management of captive populations. There seems to be a minimum number of founders (15) and a minimum size of a captive population (100) necessary in order to minimize a loss of genetic diversity. Optimally, the founders should be unrelated and new founders should be integrated into the captive population successively. We recommend that genetic analyses should generally precede and accompany ex situ conservation projects in order to avoid inbreeding and outbreeding depression. Furthermore, many of the published studies do not provide all the relevant parameters (founder size, captive population size, H_o, H_e, inbreeding coefficients). We, therefore, propose that a general standard for the presentation of genetic studies should be established, which would allow integration of the data into a global database.     

TSH compensates thyroid-specific IGF-I receptor knockout and causes papillary thyroid hyperplasia

Although TSH stimulates all aspects of thyroid physiology IGF-I signaling through a tyrosine kinase-containing transmembrane receptor exhibits a permissive impact on TSH action. To better understand the importance of the IGF-I receptor in the thyroid in vivo, we inactivated the Igf1r with a Tg promoter-driven Cre-lox system in mice. We studied male and female mice with thyroidal wild-type, Igf1r(+/-), and Igf1r(-/-) genotypes. Targeted Igf1r inactivation did transiently reduce thyroid hormone levels and significantly increased TSH levels in both heterozygous and homozygous mice without affecting thyroid weight. Histological analysis of thyroid tissue with Igf1r inactivation revealed hyperplasia and heterogeneous follicle structure. From 4 months of age, we detected papillary thyroid architecture in heterozygous and homozygous mice. We also noted increased body weight of male mice with a homozygous thyroidal null mutation in the Igf1r locus, compared with wild-type mice, respectively. A decrease of mRNA and protein for thyroid peroxidase and increased mRNA and protein for IGF-II receptor but no significant mRNA changes for the insulin receptor, the TSH receptor, and the sodium-iodide-symporter in both Igf1r(+/-) and Igf1r(-/-) mice were detected. Our results suggest that the strong increase of TSH benefits papillary thyroid hyperplasia and completely compensates the loss of IGF-I receptor signaling at the level of thyroid hormones without significant increase in thyroid weight. This could indicate that the IGF-I receptor signaling is less essential for thyroid hormone synthesis but maintains homeostasis and normal thyroid morphogenesis.     

Dau c 1.01 and Dau c 1.02-silenced transgenic carrot plants show reduced allergenicity to patients with carrot allergy

Pathogenesis-related protein-10 (PR10) is a ubiquitous small plant protein induced by microbial pathogens and abiotic stress that adversely contributes to the allergenic potency of many fruits and vegetables, including carrot. In this plant, two highly similar genes encoding PR10 isoforms have been isolated and designated as allergen Dau c 1.01 and Dau c 1.02. The aim of the study was to generate PR10-reduced hypoallergenic carrots by silencing either one of these genes in transgenic carrots by means of RNA interference (RNAi). The efficiency of gene silencing by stably expressed hairpin RNA (hnRNA) was documented by means of quantitative RT-PCR (qPCR) and immunoblotting. Quantification of the residual protein revealed that PR10 accumulation was strongly decreased compared with untransformed controls. Treatment of carrot plants with the PR protein-inducing chemical salicylic acid resulted in an increase of PR10 isoforms only in wild-type but not in Dau c 1-silenced mutants. The decrease of the allergenic potential in Dau c 1-silenced plants was sufficient to cause a reduced allergenic reactivity in patients with carrot allergy, as determined with skin prick tests (SPT). However, simultaneous silencing of multiple allergens will be required to design hypoallergenic carrots for the market. Our findings demonstrate the feasibility of creating low-allergenic food by using RNAi. This constitutes a reasonable approach to allergen avoidance.     

Purification, cDNA structure and biological significance of a single insulin-like growth factor-binding domain protein (SIBD-1) identified in the hemocytes of the spider Cupiennius salei

Cupiennius salei single insulin-like growth factor-binding domain protein (SIBD-1), which exhibits an IGFBP N-terminal domain-like profile, was identified in the hemocytes of the spider C. salei. SIBD-1 was purified by RP-HPLC and the sequence determined by a combination of Edman degradation and 5′–3′- RACE PCR. The peptide (8676.08 Da) is composed of 78 amino acids, contains six intrachain disulphide bridges and carries a modified Thr residue at position 2. SIBD-1 mRNA expression was detected by quantitative real-time PCR mainly in hemocytes, but also in the subesophageal nerve mass and muscle. After infection, the SIBD-1 content in the hemocytes decreases and, simultaneously, the temporal SIBD-1 expression seems to be down-regulated. Two further peptides, SIBD-2 and IGFBP-rP1, also exhibiting IGFBP N-terminal domain variants with unknown functions, were identified on cDNA level in spider hemocytes and venom glands. We conclude that SIBD-1 may play an important role in the immune system of spiders.     

Biofilm formation by Salmonella enterica serovar Typhimurium colonizing solid tumours

Systemic administration of Salmonella enterica serovar Typhimurium to tumour bearing mice results in preferential colonization of the tumours and retardation of tumour growth. Although the bacteria are able to invade the tumour cells in vitro, in tumours they were never detected intracellularly. Ultrastructural analysis of Salmonella-colonized tumours revealed that the bacteria had formed biofilms. Interestingly, depletion of neutrophilic granulocytes drastically reduced biofilm formation. Obviously, bacteria form biofilms in response to the immune reactions of the host. Importantly, we tested Salmonella mutants that were no longer able to form biofilms by deleting central regulators of biofilm formation. Such bacteria could be observed intracellularly in immune cells of the host or in tumour cells. Thus, tumour colonizing S. typhimurium might form biofilms as protection against phagocytosis. Since other bacteria are behaving similarly, solid murine tumours might represent a unique model to study biofilm formation in vivo.     

Fc-glycosylation of IgG1 is modulated by B-cell stimuli

We have recently shown that IgG1 directed against antigens thought to be involved in the pathogenesis of rheumatoid arthritis harbor different glycan moieties on their Fc-tail, as compared with total sera IgG1. Given the crucial roles of Fc-linked N-glycans for the structure and biological activity of IgG, Fc-glycosylation of antibodies is receiving considerable interest. However, so far little is known about the signals and factors that could influence the composition of these carbohydrate structures on secreted IgG produced by B lymphocytes. Here we show that both "environmental" factors, such as all-trans retinoic acid (a natural metabolite of vitamin A), as well as factors stimulating the innate immune system (i.e. CpG oligodeoxynucleotide, a ligand for toll-like receptor 9) or coming from the adaptive immune system (i.e. interleukin-21, a T-cell derived cytokine) can modulate IgG1 Fc-glycosylation. These factors affect Fc-glycan profiles in different ways. CpG oligodeoxynucleotide and interleukin-21 increase Fc-linked galactosylation and reduce bisecting N-acetylglucosamine levels, whereas all-trans retinoic acid significantly decreases galactosylation and sialylation levels. Moreover, these effects appeared to be stable and specific for secreted IgG1 as no parallel changes of the corresponding glycans in the cellular glycan pool were observed. Interestingly, several other cytokines and molecules known to affect B-cell biology and antibody production did not have an impact on IgG1 Fc-coupled glycan profiles. Together, these data indicate that different stimuli received by B cells during their activation and differentiation can modulate the Fc-linked glycosylation of secreted IgG1 without affecting the general cellular glycosylation machinery. Our study, therefore, furthers our understanding of the regulation of IgG1 glycosylation at the cellular level.     

A -436C>A polymorphism in the human FAS gene promoter associated with severe childhood malaria

Human genetics and immune responses are considered to critically influence the outcome of malaria infections including life-threatening syndromes caused by Plasmodium falciparum. An important role in immune regulation is assigned to the apoptosis-signaling cell surface receptor CD95 (Fas, APO-1), encoded by the gene FAS. Here, a candidate-gene association study including variant discovery at the FAS gene locus was carried out in a case-control group comprising 1,195 pediatric cases of severe falciparum malaria and 769 unaffected controls from a region highly endemic for malaria in Ghana, West Africa. We found the A allele of c.−436C>A (rs9658676) located in the promoter region of FAS to be significantly associated with protection from severe childhood malaria (odds ratio 0.71, 95% confidence interval 0.58–0.88, p_empirical = 0.02) and confirmed this finding in a replication group of 1,412 additional severe malaria cases and 2,659 community controls from the same geographic area. The combined analysis resulted in an odds ratio of 0.71 (95% confidence interval 0.62–0.80, p = 1.8×10⁻⁷, n = 6035). The association applied to c.−436AA homozygotes (odds ratio 0.47, 95% confidence interval 0.36–0.60) and to a lesser extent to c.−436AC heterozygotes (odds ratio 0.73, 95% confidence interval 0.63–0.84), and also to all phenotypic subgroups studied, including severe malaria anemia, cerebral malaria, and other malaria complications. Quantitative FACS analyses assessing CD95 surface expression of peripheral blood mononuclear cells of naïve donors showed a significantly higher proportion of CD69⁺CD95⁺ cells among persons homozygous for the protective A allele compared to AC heterozygotes and CC homozygotes, indicating a functional role of the associated CD95 variant, possibly in supporting lymphocyte apoptosis.     

Cutting edge: Chk1 directs senescence and mitotic catastrophe in recovery from G2 checkpoint arrest

Besides the well‐understood DNA damage response via establishment of G₂ checkpoint arrest, novel studies focus on the recovery from arrest by checkpoint override to monitor cell cycle re‐entry. The aim of this study was to investigate the role of Chk1 in the recovery from G₂ checkpoint arrest in HCT116 (human colorectal cancer) wt, p53^–/– and p21^–/– cell lines following H₂O₂ treatment. Firstly, DNA damage caused G₂ checkpoint activation via Chk1. Secondly, overriding G₂ checkpoint led to (i) mitotic slippage, cell cycle re‐entry in G₁ and subsequent G₁ arrest associated with senescence or (ii) premature mitotic entry in the absence of p53/p21^WAF1 causing mitotic catastrophe. We revealed subtle differences in the initial Chk1‐involved G₂ arrest with respect to p53/p21^WAF1: absence of either protein led to late G₂ arrest instead of the classic G₂ arrest during checkpoint initiation, and this impacted the release back into the cell cycle. Thus, G₂ arrest correlated with downstream senescence, but late G₂ arrest led to mitotic catastrophe, although both cell cycle re‐entries were linked to upstream Chk1 signalling. Chk1 knockdown deciphered that Chk1 defines long‐term DNA damage responses causing cell cycle re‐entry. We propose that recovery from oxidative DNA damage‐induced G₂ arrest requires Chk1. It works as cutting edge and navigates cells to senescence or mitotic catastrophe. The decision, however, seems to depend on p53/p21^WAF1. The general relevance of Chk1 as an important determinant of recovery from G₂ checkpoint arrest was verified in HT29 colorectal cancer cells.     

Characterization and Engineering of a Novel Pyrroloquinoline Quinone Dependent Glucose Dehydrogenase from <Emphasis Type="Italic">Sorangium cellulosum</Emphasis> So ce56

A novel pyrroloquinoline quinone dependent glucose dehydrogenase like enzyme (PQQ GDH) was isolated from Sorangium cellulosum So ce56. The putative coding region was cloned, over expressed in E. coli and the resulting enzyme was characterized. The recombinant protein has a relative molecular mass of 63 kDa and shows 43% homology to PQQ GDH-B from Acinetobacter calcoaceticus. In the presence of PQQ and CaCl₂ the enzyme has dehydrogenase activity with the substrate glucose as well as with other mono- and disaccharides. The thermal stability and its pH activity profile mark the enzyme as a potential glucose biosensor enzyme. In order to decrease the activity on maltose, which is unwanted for a potential application in biosensors, the protein was rationally modified at three specified positions. The best variant showed a 59% reduction in activity on maltose compared to the wild type enzyme. The catalytic efficiency (k _cat/K _M) was reduced fivefold but the specific activity still amounted to 63% of the wild type activity.