Design of chimeric receptor mimics with different TcRVbeta isoforms. Type-specific inhibition of superantigen pathogenesis

The Staphylococcus aureus enterotoxins (S.E.) A-I, and toxic-shock syndrome toxin TSST-1 act as superantigens to cause overstimulation of the host immune system, leading to the onset of various diseases including food poisoning and toxic shock syndrome. SAgs bind as intact proteins to the DRalpha1 domain of the MHC class II receptor and the TcRVbeta domain from the T cell receptor and cause excessive release of cytokines such as IL-2, TNF-alpha, and IFN-gamma, and hyperproliferation of T cells. In addition, different SAgs bind and activate different TcRVbeta isoforms during pathogenesis of human immune cells. These two properties of SAgs prompted us to design several chimeric DRalpha1-linker-TcRVbeta proteins using different TcRVbeta isoforms to create chimeras that would specifically inhibit the pathogenesis of SAgs against which they were designed. In this study, we compare the design, interaction, and inhibitory properties of three different DRalpha1-linker-TcRVbeta chimeras targeted against three different SAgs, SEB, SEC3, and TSST-1. The inhibitory properties of the chimeras were tested by monitoring IL-2 release and T cell proliferation using a primary human cell model. We demonstrate that the three chimeras specifically inhibit the pathogenesis of their target superantigen. We performed molecular modeling to analyze the structural basis of the type specificity exhibited by different chimeras designed against their target SAgs, examine the role of the linker in determining binding and specificity, and suggest site-specific mutations in the chimera to enhance binding affinity. The fact that our strategy works equally well for SEB and TSST-1, two widely different phylogenic variants, suggests that the DRalpha1-linker-TcRVbeta chimeras may be developed as a general therapy against a broad spectrum of superantigens released during Staphylococcal infection.     

The 5'-AMP-activated protein kinase gamma3 isoform has a key role in carbohydrate and lipid metabolism in glycolytic skeletal muscle

5'-AMP-activated protein kinase (AMPK) is a metabolic stress sensor present in all eukaryotes. A dominant missense mutation (R225Q) in pig PRKAG3, encoding the muscle-specific gamma3 isoform, causes a marked increase in glycogen content. To determine the functional role of the AMPK gamma3 isoform, we generated transgenic mice with skeletal muscle-specific expression of wild type or mutant (225Q) mouse gamma3 as well as Prkag3 knockout mice. Glycogen resynthesis after exercise was impaired in AMPK gamma3 knock-out mice and markedly enhanced in transgenic mutant mice. An AMPK activator failed to increase skeletal muscle glucose uptake in AMPK gamma3 knock-out mice, whereas contraction effects were preserved. When placed on a high fat diet, transgenic mutant mice but not knock-out mice were protected against excessive triglyceride accumulation and insulin resistance in skeletal muscle. Transfection experiments reveal the R225Q mutation is associated with higher basal AMPK activity and diminished AMP dependence. Our results validate the muscle-specific AMPK gamma3 isoform as a therapeutic target for prevention and treatment of insulin resistance.     

Influence of a Fusarium culmorum inoculation of wheat on the progression of mycotoxin accumulation, ingredient concentrations and ruminal in sacco dry matter degradation of wheat residues

The Fusarium head blight (FHB)-susceptible winter wheat cv. Ritmo was inoculated with spores of Fusarium culmorum at the beginning of full blossom. Samples of whole wheat plants were taken once weekly from anthesis until harvest and subsequently fractionated into straw, glumes and spindles, which were examined for deoxynivalenol (DON) and zearalenone (ZON). Additionally, the content of crude protein (CP) and non-starch polysaccharides (NSP) was scrutinized. Synthesis of the Fusarium toxins DON and ZON generally differed in terms of date of formation and concentration. Final mean DON concentrations of 37.5, 28.1 and 5.0 mg/kg DM were measured in glumes, spindles and straw, respectively, at the time of harvest. At this time, maximal mean ZON concentrations of 587, 396 and 275 microg/kg DM in spindles, glumes and straw, respectively, were determined. Moreover, Fusarium infected wheat residues contained higher CP but lower NSP contents at the last three sampling dates. In addition, collective samples of wheat straw and chaff were taken to investigate the effect of the Fusarium contamination on their in sacco DM degradation in dairy cows. Samples were analysed for mycotoxins and selected quality parameters. The dried and milled collective samples of straw and chaff were weighed into nylon bags and subjected to ruminal incubation for 4, 8, 16, 24, 48, 72, 96 and 120 h in two dairy cows equipped with a permanent rumen cannula. Marked differences in level of mycotoxin contamination as well as in ingredient composition between the variants of straw and chaff were detected. Moreover, after 120 h rumen incubation the in sacco DM degradation of inoculated straw and chaff were lower compared to the accordant controls. The soluble fraction was increased in inoculated samples, whereas a diminishment in the potentially degradable but insoluble fraction was more pronounced. Thereby, a decrease in the potential degradability was obtained for inoculated straw and even if less pronounced for chaff compared to the non inoculated corresponding controls. In conclusion, infection with F. culmorum of wheat involves an increased risk of mycotoxin contamination in straw. Also, a Fusarium infection may have an impact on chemical composition and may result in Fusarium growth-related modifications of host cell wall components.     

The effect of age at introduction and number of milk-portions on the behaviour of group housed dairy calves fed by a computer controlled milk feeder

This study is an investigation of the effect of age at introduction (6 days versus 14 days) and number of milk-portions (four milk-portions a day versus eight milk-portions a day) on integration into a large dynamic group of calves, fed by a computer controlled milk feeder. Forty calves (Jerseys, Danish Reds and Holstein-Friesians) were allocated equally to the two age conditions (A6 and A14) and the two milk-portion conditions (M4 and M8) in a 2 × 2 factorial design, according to sex and breed, and introduced into the group in pairs (one A6 and one A14). The behaviour of each pair was video-recorded for 8 h from 8 a.m. to 4 p.m. and 1 h from 1 to 2 a.m. on days 1, 8 and 15 after introduction.

The A6 calves performed less licking and sniffing, changed posture more often and tended to spend less time standing than the A14 calves. In the course of time A14 calves lay closer to other calves than the A6 calves.

The M8 calves, which were offered the same daily milk allowance as the M4 calves stood for a longer time in the milk feeding station and the M8 calves also sucked the empty teat more frequently than the M4 calves. Finally, the M8 calves initiated more social play behaviour than the M4 calves. On the first day after introduction the M8 calves lay closer to other calves than the M4 calves.

The results suggest that calves integrate better into a group when introduced at the age of 14 days than at the age of 6 days. Distributing the milk into eight daily milk-portions, rather than four milk-portions in the first period after introduction, increased milk feeder occupancy, which may facilitate learning to use the milk feeder. Surprisingly, more milk-portions also stimulated play behaviour, which is suggested to be due to M8 calves encountering more calves in the milk feeder area.     

Body composition, weight status, body image and weight control practices among female adolescents from eastern Austria

The prevalence rates of obesity, but also of altered eating behaviour up to clinically manifested eating disorders have increased dramatically during the last years. In the present study the weight status, body composition and eating behaviour as well as weight controlling strategies of 1752 female adolescents ageing between 12 and 18 years (x=14.6) from Eastern Austria were documented. Furthermore, data regarding socioeconomic status and educational level of the probands were collected. It could be shown, that a higher educational level or a higher socioeconomic status was associated with a significantly lower fat percentage and a significantly lower prevalence of overweight and obesity. Underweight and severe underweight was predominantly found among girls ageing between 15 and 18 years of higher educational level. Weight controlling behaviour such as stepping on scale and altered or restricted eating behaviour was widespread among the probands, independent of weight status and socioeconomic parameters. Body weight and shape concerns were also found very frequently independent of educational level. The results of the present study corroborate the findings of former investigations, that disturbed eating behaviour and concerns regarding body weight and shape are found among all socioeconomic groups.     

Association of Bcl-2 with misfolded prion protein is linked to the toxic potential of cytosolic PrP

Protein misfolding is linked to different neurodegenerative disorders like Alzheimer's disease, polyglutamine, and prion diseases. We investigated the cytotoxic effects of aberrant conformers of the prion protein (PrP) and show that toxicity is specifically linked to misfolding of PrP in the cytosolic compartment and involves binding of PrP to the anti-apoptotic protein Bcl-2. PrP targeted to different cellular compartments, including the cytosol, nucleus, and mitochondria, adopted a misfolded and partially proteinase K-resistant conformation. However, only in the cytosol did the accumulation of misfolded PrP induce apoptosis. Apoptotic cell death was also induced by two pathogenic mutants of PrP, which are partially localized in the cytosol. A mechanistic analysis revealed that the toxic potential is linked to an internal domain of PrP (amino acids 115-156) and involves coaggregation of cytosolic PrP with Bcl-2. Increased expression of the chaperones Hsp70 and Hsp40 prevented the formation of PrP/Bcl-2 coaggregates and interfered with PrP-induced apoptosis. Our study reveals a compartment-specific toxicity of PrP misfolding that involves coaggregation of Bcl-2 and indicates a protective role of molecular chaperones.     

Tuberous sclerosis genes regulate cellular 14-3-3 protein levels

The genes TSC1, encoding hamartin, and TSC2, encoding tuberin are responsible for tuberous sclerosis. This autosomal dominant tumor suppressor gene syndrome affects about 1 in 6000 individuals. A variety of tumors characteristically occur in different organs of tuberous sclerosis patients and are believed to result from defects in cell cycle/cell size control. We performed a proteomics approach of two-dimensional gel electrophoresis with subsequent mass spectrometrical identification of protein spots after ectopic overexpression of human TSC1 or TSC2. We found the cellular levels of four isoforms of the 14-3-3 protein family, 14-3-3 gamma, 14-3-3, 14-3-3 sigma, and 14-3-3 zeta, to be regulated by the two tuberous sclerosis gene products. In the same experiments the protein levels of keratin 7, capZ alpha-1 subunit, ezrin, and nedasin were not affected by ectopic TSC1 or TSC2. Western blot analyses confirmed the deregulation of 14-3-3 proteins upon ectopic overexpression of TSC1 and TSC2. A TSC1 mutant not encoding the transmembrane domain and the tuberin-binding domain but harbouring most of the coiled-coil region and the ERM protein interaction domain of hamartin did not affect 14-3-3 protein levels. The here presented findings suggest that deregulation of 14-3-3 protein amounts might contribute to the development of tumors in tuberous sclerosis patients. These data provide important new insights into the molecular development of this disease especially since both, the TSC genes and the 14-3-3 proteins, are known to be involved in mammalian cell cycle control.     

Reproductive factors and serum uric acid levels in females from the general population: the KORA F4 study

Objective

Hyperuricemia is associated with an increased risk of metabolic and cardiovascular diseases. There are pronounced sex differences in the levels of uric acid. It is largely unknown whether or not reproductive parameters which induce hormonal changes are responsible for this. We examined if there are associations between reproductive parameters and uric acid levels in a female population-based sample.

Methods

In this cross-sectional analysis, data of 1530 women aged 32 to 81 years participating in the KORA F4 study, conducted between 2006 and 2008 in Southern Germany were used. Reproductive parameters were obtained by standardized interviews. Uric acid levels were tested by the uricase method. The whole study sample and stratified in pre- and postmenopausal women was analyzed.

Results

Menopausal status and earlier age at menarche were associated with higher serum uric acid levels (age-adjusted: p-values 0.003, <0.001 respectively; after multivariable adjustment, including BMI: p-values 0.002, 0.036). A history of oral contraceptive use showed an association with uric acid levels only after multivariable adjustment (p-value 0.009). Hot flushes showed an association with uric acid levels only after age-adjustment (p-value 0.038), but lost significance after adding other confounders. Other reproductive factors, including parity, current or ever use of hormone replacement therapy, current use of oral contraceptives, hysterectomy, bilateral oophorectomy, or depressive mood related to menopausal transition were not associated with uric acid levels.

Conclusions

Postmenopausal status, earlier age at menarche and a history of oral contraceptive use were independently associated with higher serum uric acid concentrations in women from the general population. Further studies, especially longitudinal population-based studies investigating the relationship of female reproductive parameters with uric acid levels are necessary to confirm our findings.     

lemmingA encodes the Apc11 subunit of the APC/C in Drosophila melanogaster that forms a ternary complex with the E2-C type ubiquitin conjugating enzyme, Vihar and Morula/Apc2

Background

The spindle assembly checkpoint (SAC) inhibits anaphase progression in the presence of insufficient kinetochore-microtubule attachments, but cells can eventually override mitotic arrest by a process known as mitotic slippage or adaptation. This is a problem for cancer chemotherapy using microtubule poisons.

Results

Here we describe mitotic slippage in yeast bub2?? mutant cells that are defective in the repression of precocious telophase onset (mitotic exit). Precocious activation of anaphase promoting complex/cyclosome (APC/C)-Cdh1 caused mitotic slippage in the presence of nocodazole, while the SAC was still active. APC/C-Cdh1, but not APC/C-Cdc20, triggered anaphase progression (securin degradation, separase-mediated cohesin cleavage, sister-chromatid separation and chromosome missegregation), in addition to telophase onset (mitotic exit), during mitotic slippage. This demonstrates that an inhibitory system not only of APC/C-Cdc20 but also of APC/C-Cdh1 is critical for accurate chromosome segregation in the presence of insufficient kinetochore-microtubule attachments.

Conclusions

The sequential activation of APC/C-Cdc20 to APC/C-Cdh1 during mitosis is central to accurate mitosis. Precocious activation of APC/C-Cdh1 in metaphase (pre-anaphase) causes mitotic slippage in SAC-activated cells. For the prevention of mitotic slippage, concomitant inhibition of APC/C-Cdh1 may be effective for tumor therapy with mitotic spindle poisons in humans.