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Fire blight caused by Erwinia amylovorareached Kazakhstan in 2008. Here, the disease poses a threat not only to agricultural production of apples and pears, but also to the forests of wild Malus sieversii, the progenitor of most domesticated apple varieties worldwide. In the period 2019–2021, the spread of fire blight in the growth area of wild apples was limited by the weather conditions. In 2022, late spring and early summer were characterized by increased rainfall and moderate temperatures favorable for the disease. The goal of this study was to monitor the distribution of fire blight in private households and small orchards in the zones adjacent to wild apple distribution areas. A total of 91 samples with fire blight-compatible symptoms were collected from cultural apples (68), wild apple (10), pear (5), hawthorn (7), and quince (1) in south-eastern and eastern Kazakhstan, resulting in 21 isolates (one from pear, one from quince, and 19 from apple) of E. amylovora. All isolates belonged to the archetypal CRISPR genotype A. Considering the relative proximity of the infections to the forests of wild M. sieversii, additional measures for fire blight control and prevention will have to be implemented, including state monitoring of the wild apple forests for disease symptoms and awareness campaigns for specially protected natural territories that safeguard M. sieversii, as well as for local pomaceous-fruit growing communities.

Fire blight, caused by the bacterial pathogen Erwinia amylovora, is a severe bacterial disease of apple and pear that can quickly destroy whole plants. In the last decade, it was also detected in Central Asia, where wild pomaceous fruit plants represent the dominant species in mid-altitude forests and constitute a critical foundation for the entire ecosystem. Efficiently informing farmers, forestry services and private persons about the instances and dangers of fire blight, the correct way to recognize the symptoms, and the methods of disease control is thus of paramount importance in a vast and fragmented natural landscape like the one characterizing countries like Kyrgyzstan, Kazakhstan and Tajikistan. For that purpose, we have developed an app for smartphones and mobile devices that can inform stakeholders about fire blight, simultaneously allowing a citizen science approach for mapping the spread of the disease in Central Asia. The app is available in the three national languages as well as in Russian, English, and German, and can easily be adapted to new countries, languages or even diseases.

Vaccination is one of medicine’s greatest achievements; however, its full potential is hampered by considerable variation in efficacy across populations and geographical regions. For example, attenuated malaria vaccines in high-income countries confer almost 100% protection, whereas in low-income regions these same vaccines achieve only 20–50% protection. This trend is also observed for other vaccines, such as bacillus Calmette–Guérin (BCG), rotavirus and yellow fever vaccines, in terms of either immunogenicity or efficacy. Multiple environmental factors affect vaccine responses, including pathogen exposure, microbiota composition and dietary nutrients. However, there has been variable success with interventions that target these individual factors, highlighting the need for a better understanding of their downstream immunological mechanisms to develop new ways of modulating vaccine responses. Here, we review the immunological factors that underlie geographical variation in vaccine responses. Through the identification of causal pathways that link environmental influences to vaccine responsiveness, it might become possible to devise modulatory compounds that can complement vaccines for better outcomes in regions where they are needed most.

Heat shock proteins 90 (Hsp90) are promising therapeutic targets due to their involvement in stabilizing several aberrantly expressed oncoproteins. In cancerous cells, Hsp90 expression is elevated, thereby exerting antiapoptotic effects, which is essential for the malignant transformation and tumor progression. Most of the Hsp90 inhibitors (Hsp90i) under investigation target the ATP binding site in the N-terminal domain of Hsp90. However, adverse effects, including induction of the prosurvival resistance mechanism (heat shock response or HSR) and associated dose-limiting toxicity, have so far precluded their clinical approval. In contrast, modulators that interfere with the C-terminal domain (CTD) of Hsp90 do not inflict HSR. Since the CTD dimerization of Hsp90 is essential for its chaperone activity, interfering with the dimerization process by small-molecule protein–protein interaction inhibitors is a promising strategy for anticancer drug research. We have developed a first-in-class small-molecule inhibitor (5b) targeting the Hsp90 CTD dimerization interface, based on a tripyrimidonamide scaffold through structure-based molecular design, chemical synthesis, binding mode model prediction, assessment of the biochemical affinity, and efficacy against therapy-resistant leukemia cells. 5breduces xenotransplantation of leukemia cells in zebrafish models and induces apoptosis in BCR-ABL1+(T315I) tyrosine kinase inhibitor-resistant leukemia cells, without inducing HSR.

Systemic immune cell dynamics during coronavirus disease 2019 (COVID-19) are extensively documented, but these are less well studied in the (upper) respiratory tract, where severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) replicates1–6. Here, we characterized nasal and systemic immune cells in individuals with COVID-19 who were hospitalized or convalescent and compared the immune cells to those seen in healthy donors. We observed increased nasal granulocytes, monocytes, CD11c+natural killer (NK) cells and CD4+T effector cells during acute COVID-19. The mucosal proinflammatory populations positively associated with peripheral blood human leukocyte antigen (HLA)-DRlowmonocytes, CD38+PD1+CD4+T effector (Teff) cells and plasmablasts. However, there was no general lymphopenia in nasal mucosa, unlike in peripheral blood. Moreover, nasal neutrophils negatively associated with oxygen saturation levels in blood. Following convalescence, nasal immune cells mostly normalized, except for CD127+granulocytes and CD38+CD8+tissue-resident memory T cells (TRM). SARS-CoV-2-specific CD8+T cells persisted at least 2 months after viral clearance in the nasal mucosa, indicating that COVID-19 has both transient and long-term effects on upper respiratory tract immune responses.

Erwinia amylovoracan rapidly become highly resistant to applications of the antibiotic streptomycin through a single nucleotide mutation in the rpsLgene, which causes an amino acid substitution that prevents inhibitory binding of streptomycin while preserving the functionality of the ribosome. Several viable mutations are described in the literature, but screenings in the orchards consistently revealed that the one leading to the substitution of lysin by arginine at position 43 (K43R) is by far the most abundant. In this work, we tested the rate of emergence of the various spontaneous mutants, their tolerance to different antibiotic concentrations and their general fitness in competition experiments. Furthermore, we developed a molecular assay based on the SNaPshot™ Multiplex Kit from Thermo Fisher Scientific that allows swift determination of rpsLmutations using a single-base primer extension approach directly from colonies on plate. Or results indicate that the prevalence of variant K43R is largely due to fact that it retains the highest environmental fitness even in absence of antibiotic pressure. Sequence analysis of two resistant colonies that did not respond as expected in the SNaPshot™ assay revealed the existence of two yet undescribed rpsLmutations resulting in conditional-lethal streptomycin-dependent (SmD) phenotypes, which are unable to grow in absence of the antibiotic.

Asthma is a complex disease that often starts in childhood. Genomic and environmental factors as well as aberrant immune maturation early in life can contribute to the onset of disease, with great disparity over time and geographical regions. Epidemiological studies have scrutinised environmental exposures and attempted to translate these exposures into prevention strategies. Some approaches for patients with asthma have been successful (eg, smoking ban, the Finnish Asthma Programme), and primary prevention of wheeze in pre-school children (age 0–5 years) by the supplementation of vitamin D or fish oil, or both, to pregnant women seems promising. Several recent prevention initiatives are based on strong asthma-protective environmental microbial exposures associated with traditional rural lifestyles. Preclinical studies with various bacterial lysates, bacterial and dietary metabolites, or helminthic compounds have yielded promising results that await translation into clinical practice. Given the immense societal and individual burden of asthma, there is an urgent need to further develop novel strategies to eradicate the disease.

During acute COVID-19, there is little correlation between the nose and blood in terms of antibodies or cytokines; instead, these factors are associated with nasal microbiota.

Knowledge of protein structures is essential to understand proteins’ functions, evolution, dynamics, stabilities, and interactions and for data-driven protein- or drug design. Yet, experimental structure determination rates are far exceeded by that of next-generation sequencing, resulting in less than 1/1000th of proteins having an experimentally known 3D structure. Computational structure prediction seeks to alleviate this problem, and the Critical Assessment of Protein Structure Prediction (CASP) has shown the value of consensus and meta-methods that utilize complementary algorithms. However, traditionally, such methods employ majority voting during template selection and model averaging during refinement, which can drive the model away from the native fold if it is underrepresented in the ensemble. Here, we present TopModel, a fully automated meta-method for protein structure prediction. In contrast to traditional consensus and meta-methods, TopModel uses top-down consensus and deep neural networks to select templates and identify and correct wrongly modeled regions. TopModel combines a broad range of state-of-the-art methods for threading, alignment, and model quality estimation and provides a versatile workflow and toolbox for template-based structure prediction. TopModel shows a superior template selection, alignment accuracy, and model quality for template-based structure prediction on the CASP10–12 datasets compared to 12 state-of-the-art stand-alone primary predictors. TopModel was validated by prospective predictions of the nisin resistance protein (NSR) protein from Streptococcus agalactiaeand LipoP from Clostridium difficile, showing far better agreement with experimental data than any of its constituent primary predictors. These results, in general, demonstrate the utility of TopModel for protein structure prediction and, in particular, show how combining computational structure prediction with sparse or low-resolution experimental data can improve the final model.

The current study compared the effect of hydroxychloride trace minerals (HTM) with the effect of inorganic trace minerals (ITM) on growth performance and carcass quality in grower-finisher pigs. The results of 6 studies conducted throughout Europe were combined into one meta-analysis. All included studies were performed using pigs from about 19 kg of body weight until slaughter. In all studies, 2 different mineral sources were compared, HTM and sulfates as ITM. Zn from either HTM or ITM was added at a level of 80 ppm to the diet, and Cu was added at a level of 15 ppm from the same source as Zn. In most studies, an additional treatment was included in which 20 ppm Zn was used from either source in combination with 15 ppm Cu from the same source. Diets were fed in 3 phases according to local commercial standards. The body weight, average daily gain, average daily feed intake, and gain:feed ratio were measured at the end of each phase. At the end of each study, the carcass yield, back fat thickness, and lean meat percentage were measured at commercial slaughterhouses. The meta-analysis was conducted using a MIXED model in SAS taking into account the within-study and between-study variation. The comparison was done only between HTM and ITM added at the same Zn level. No statistical differences were observed for growth performance or carcass characteristics between the mineral sources in pigs fed 20 ppm Zn. When 80 ppm Zn was used, a significant improvement in lean meat percentage was observed in pigs fed HTM compared with pigs fed ITM. In the overall study period, there was a tendency towards an increased gain:feed ratio in pigs fed 80 ppm Zn from HTM. In the last feeding phase, before slaughter gain:feed ratio and average daily gain were both significantly improved by 3.9%. In conclusion, HTM addition improved growth performance and lean meat percentage in grower-finisher pigs.

Gene knock outs (KOs) are efficiently engineered through CRISPR–Cas9-induced frameshift mutations. While the efficiency of DNA editing is readily verified by DNA sequencing, a systematic understanding of the efficiency of protein elimination has been lacking. Here we devised an experimental strategy combining RNA sequencing and triple-stage mass spectrometry to characterize 193 genetically verified deletions targeting 136 distinct genes generated by CRISPR-induced frameshifts in HAP1 cells. We observed residual protein expression for about one third of the quantified targets, at variable levels from low to original, and identified two causal mechanisms, translation reinitiation leading to N-terminally truncated target proteins or skipping of the edited exon leading to protein isoforms with internal sequence deletions. Detailed analysis of three truncated targets, BRD4, DNMT1 and NGLY1, revealed partial preservation of protein function. Our results imply that systematic characterization of residual protein expression or function in CRISPR–Cas9-generated KO lines is necessary for phenotype interpretation.

Tumors accumulate high levels of mutant p53 (mutp53), which contributes to mutp53 gain-of-function properties. The mechanisms that underlie such excessive accumulation are not fully understood. To discover regulators of mutp53 protein accumulation, we performed a large-scale RNA interference screen in a Burkitt lymphoma cell line model. We identified transformation/transcription domain-associated protein (TRRAP), a constituent of several histone acetyltransferase complexes, as a critical positive regulator of both mutp53 and wild-type p53 levels. TRRAP silencing attenuated p53 accumulation in lymphoma and colon cancer models, whereas TRRAP overexpression increased mutp53 levels, suggesting a role for TRRAP across cancer entities and p53 mutations. Through clustered regularly interspaced short palindromic repeats (CRISPR)–Cas9 screening, we identified a 109-amino-acid region in the N-terminal HEAT repeat region of TRRAP that was crucial for mutp53 stabilization and cell proliferation. Mass spectrometric analysis of the mutp53 interactome indicated that TRRAP silencing caused degradation of mutp53 via the MDM2-proteasome axis. This suggests that TRRAP is vital for maintaining mutp53 levels by shielding it against the natural p53 degradation machinery. To identify drugs that alleviated p53 accumulation similarly to TRRAP silencing, we performed a small-molecule drug screen and found that inhibition of histone deacetylases (HDACs), specifically HDAC1/2/3, decreased p53 levels to a comparable extent. In summary, here we identify TRRAP as a key regulator of p53 levels and link acetylation-modifying complexes to p53 protein stability. Our findings may provide clues for therapeutic targeting of mutp53 in lymphoma and other cancers.

Tumors accumulate high levels of mutant p53 (mutp53), which contributes to mutp53 gain-of-function properties. The mechanisms that underlie such excessive accumulation are not fully understood. To discover regulators of mutp53 protein accumulation, we performed a large-scale RNA interference screen in a Burkitt lymphoma cell line model. We identified transformation/transcription domain-associated protein (TRRAP), a constituent of several histone acetyltransferase complexes, as a critical positive regulator of both mutp53 and wild-type p53 levels. TRRAP silencing attenuated p53 accumulation in lymphoma and colon cancer models, whereas TRRAP overexpression increased mutp53 levels, suggesting a role for TRRAP across cancer entities and p53 mutations. Through clustered regularly interspaced short palindromic repeats (CRISPR)–Cas9 screening, we identified a 109-amino-acid region in the N-terminal HEAT repeat region of TRRAP that was crucial for mutp53 stabilization and cell proliferation. Mass spectrometric analysis of the mutp53 interactome indicated that TRRAP silencing caused degradation of mutp53 via the MDM2-proteasome axis. This suggests that TRRAP is vital for maintaining mutp53 levels by shielding it against the natural p53 degradation machinery. To identify drugs that alleviated p53 accumulation similarly to TRRAP silencing, we performed a small-molecule drug screen and found that inhibition of histone deacetylases (HDACs), specifically HDAC1/2/3, decreased p53 levels to a comparable extent. In summary, here we identify TRRAP as a key regulator of p53 levels and link acetylation-modifying complexes to p53 protein stability. Our findings may provide clues for therapeutic targeting of mutp53 in lymphoma and other cancers.

Ubiquitin-binding proteins play an important role in eukaryotes by translating differently linked polyubiquitin chains into proper cellular responses. Current knowledge about ubiquitin-binding proteins and ubiquitin linkage-selective interactions is mostly based on case-by-case studies. We have recently reported a method called ubiquitin interactor affinity enrichment–mass spectrometry (UbIA-MS), which enables comprehensive identification of ubiquitin interactors for all ubiquitin linkages from crude cell lysates. One major strength of UbIA-MS is the fact that ubiquitin interactors are enriched from crude cell lysates, in which proteins are present at endogenous levels, contain biologically relevant post-translational modifications (PTMs) and are assembled in native protein complexes. In addition, UbIA-MS uses chemically synthesized nonhydrolyzable diubiquitin, which mimics native diubiquitin and is inert to cleavage by endogenous deubiquitinases (DUBs). Here, we present a detailed protocol for UbIA-MS that proceeds in five stages: (i) chemical synthesis of ubiquitin precursors and click chemistry for the generation of biotinylated nonhydrolyzable diubiquitin baits, (ii) in vitro affinity purification of ubiquitin interactors, (iii) on-bead interactor digestion, (iv) liquid chromatography (LC)–MS/MS analysis and (v) data analysis to identify differentially enriched proteins. The computational analysis tools are freely available as an open-source R software package, including a graphical interface. Typically, UbIA-MS allows the identification of dozens to hundreds of ubiquitin interactors from any type of cell lysate, and can be used to study cell type or stimulus-dependent ubiquitin interactions. The nonhydrolyzable diubiquitin synthesis can be completed in 3 weeks, followed by ubiquitin interactor enrichment and identification, which can be completed within another 2 weeks.

Nisin (NisA) is an antimicrobial peptide produced by Lactococcus lactisand belongs to the class of lanthipeptides, more specifically to the class of lantibiotics. They are ribosomally synthesized as a precursor peptide and are comprised of an N-terminal leader peptide and a C-terminal core peptide. The core peptide is post-translationally modified and contains dehydrated amino acids in addition to five (methyl)-lanthionine rings, which are crucial for its activity. The leader peptide serves as a signal sequence and ensures that NisA remains inactive but secretion-competent within the cell. After translocation into the extracellular space, the leader peptide is cleaved by the leader peptidase NisP, resulting in active nisin. NisP is an extracellular subtilisin-like serine protease, which recognizes the cleavage site GASPR|IT located at the C-terminal end of the leader peptide. Here, we present the biochemical characterization of secreted and purified NisP (NisPs) with its natural substrate, the fully modified NisA (mNisA). Furthermore, we determined the kinetic parameters of NisPsin the presence of NisA containing different modification states. Additionally, in vitrodata revealed that NisPscan efficiently cleave the leader peptide of mNisA. However, it is strictly dependent on the modification state of the core peptide. Thus, NisPshas a sequence-based cleavage activity, and the presence of at least one lanthionine ring is crucial for optimal substrate recognition and subsequent cleavage.

Innate mononuclear phagocytic system (MPS) cells preserve mucosal immune homeostasis. We investigated their role at nasal mucosa following allergen challenge with house dust mite. We combined single-cell proteome and transcriptome profiling on nasal immune cells from nasal biopsies cells from 30 allergic rhinitis and 27 non-allergic subjects before and after repeated nasal allergen challenge. Biopsies of patients showed infiltrating inflammatory HLA-DRhi/CD14+ and CD16+ monocytes and proallergic transcriptional changes in resident CD1C+/CD1A+ conventional dendritic cells (cDC)2 following challenge. In contrast, non-allergic individuals displayed distinct innate MPS responses to allergen challenge: predominant infiltration of myeloid-derived suppressor cells (MDSC: HLA-DRlow/CD14+ monocytes) and cDC2 expressing inhibitory/tolerogenic transcripts. These divergent patterns were confirmed in ex vivo stimulated MPS nasal biopsy cells. Thus, we identified not only MPS cell clusters involved in airway allergic inflammation but also highlight novel roles for non-inflammatory innate MPS responses by MDSC to allergens in non-allergic individuals. Future therapies should address MDSC activity as treatment for inflammatory airway diseases.

Epitope-tagging is an effective tool to facilitate protein enrichment from crude cell extracts. Traditionally, N- or C-terminal fused tags are employed, which, however, can perturb protein function. Unnatural amino acids (UAAs) harboring small reactive handles can be site-specifically incorporated into proteins, thus serving as a potential alternative for conventional protein tags. Here, we introduce Click-MS, which combines the power of site-specific UAA incorporation, bioorthogonal chemistry, and quantitative mass spectrometry-based proteomics to specifically enrich a single protein of interest from crude mammalian cell extracts. By genetic encoding of p-azido-l-phenylalanine, the protein of interest can be selectively captured using copper-free click chemistry. We use Click-MS to enrich proteins that function in different cellular compartments, and we identify protein–protein interactions, showing the great potential of Click-MS for interaction proteomics workflows.

Bacteria of the genus Pseudomonasoccupy diverse environments. The Pseudomonas fluorescensgroup is particularly well-known for its plant-beneficial properties including pathogen suppression. Recent observations that some strains of this group also cause lethal infections in insect larvae, however, point to a more versatile ecology of these bacteria. We show that 26 P. fluorescensgroup strains, isolated from three continents and covering three phylogenetically distinct sub-clades, exhibited different activities toward lepidopteran larvae, ranging from lethal to avirulent. All strains of sub-clade 1, which includes Pseudomonas chlororaphisand Pseudomonas protegens, were highly insecticidal regardless of their origin (animals, plants). Comparative genomics revealed that strains in this sub-clade possess specific traits allowing a switch between plant- and insect-associated lifestyles. We identified 90 genes unique to all highly insecticidal strains (sub-clade 1) and 117 genes common to all strains of sub-clade 1 and present in some moderately insecticidal strains of sub-clade 3. Mutational analysis of selected genes revealed the importance of chitinase C and phospholipase C in insect pathogenicity. The study provides insight into the genetic basis and phylogenetic distribution of traits defining insecticidal activity in plant-beneficial pseudomonads. Strains with potent dual activity against plant pathogens and herbivorous insects have great potential for use in integrated pest management for crops.

Bacteria of the genus Pseudomonas occupy diverse environments. The Pseudomonas fluorescens group is particularly well-known for its plant-beneficial properties including pathogen suppression. Recent observations that some strains of this group also cause lethal infections in insect larvae, however, point to a more versatile ecology of these bacteria. We show that 26 P. fluorescens group strains, isolated from three continents and covering three phylogenetically distinct sub-clades, exhibited different activities toward lepidopteran larvae, ranging from lethal to avirulent. All strains of sub-clade 1, which includes Pseudomonas chlororaphis and Pseudomonas protegens, were highly insecticidal regardless of their origin (animals, plants). Comparative genomics revealed that strains in this sub-clade possess specific traits allowing a switch between plant- and insect-associated lifestyles. We identified 90 genes unique to all highly insecticidal strains (sub-clade 1) and 117 genes common to all strains of sub-clade 1 and present in some moderately insecticidal strains of sub-clade 3. Mutational analysis of selected genes revealed the importance of chitinase C and phospholipase C in insect pathogenicity. The study provides insight into the genetic basis and phylogenetic distribution of traits defining insecticidal activity in plant-beneficial pseudomonads. Strains with potent dual activity against plant pathogens and herbivorous insects have great potential for use in integrated pest management for crops.

The vertebrate body plan and organs are shaped during a conserved embryonic phase called the phylotypic stage. However, the mechanisms that guide the epigenome through this transition and their evolutionary conservation remain elusive. Here we report widespread DNA demethylation of enhancers during the phylotypic period in zebrafish, Xenopus tropicalis and mouse. These enhancers are linked to developmental genes that display coordinated transcriptional and epigenomic changes in the diverse vertebrates during embryogenesis. Binding of Tet proteins to (hydroxy)methylated DNA and enrichment of 5-hydroxymethylcytosine in these regions implicated active DNA demethylation in this process. Furthermore, loss of function of Tet1, Tet2 and Tet3 in zebrafish reduced chromatin accessibility and increased methylation levels specifically at these enhancers, indicative of DNA methylation being an upstream regulator of phylotypic enhancer function. Overall, our study highlights a regulatory module associated with the most conserved phase of vertebrate embryogenesis and suggests an ancient developmental role for Tet dioxygenases.

Omega-1, a glycosylated T2 ribonuclease (RNase) secreted by Schistosoma mansoni eggs and abundantly present in soluble egg antigen, has recently been shown to condition dendritic cells (DCs) to prime Th2 responses. However, the molecular mechanisms underlying this effect remain unknown. We show in this study by site-directed mutagenesis of omega-1 that both the glycosylation and the RNase activity are essential to condition DCs for Th2 polarization. Mechanistically, we demonstrate that omega-1 is bound and internalized via its glycans by the mannose receptor (MR) and subsequently impairs protein synthesis by degrading both ribosomal and messenger RNA. These experiments reveal an unrecognized pathway involving MR and interference with protein synthesis that conditions DCs for Th2 priming.

The anaerobic degradation potential at a chloroethene‐contaminated site was investigated by operating two anoxic column aquifer microcosms enriched in iron(III). One column was fed with vinyl chloride (VC) only (column A) and one with VC and acetate (column B). In column A, after about 600 pore volume exchanges (PVEs), VC started to disappear and reached almost zero VC recovery in the effluent after 1000 PVEs. No formation of ethene was observed. In column B, effluent VC was almost always only a fraction of influent VC. Formation of ethene was observed after 800 PVEs and started to become an important degradation product after 1550 PVEs. However, ethene was never observed in stoichiometric amounts compared with disappeared VC. The average stable isotope enrichment factor for VC disappearance in column A was determined to be ‐4.3‰. In column B, the isotope enrichment factor shifted from ‐10.7 to ‐18.5‰ concurrent with an increase in ethene production. Batch microcosms inoculated with column material showed similar isotope enrichment factors as the column microcosms. These results indicated that two degradation processes occurred, one in column A and two in parallel in column B with increasing importance of reductive dechlorination with time. This study suggests that in addition to reductive dechlorination, other degradation processes such as anaerobic oxidation should be taken into account when evaluating natural attenuation of VC and that isotope analysis can help to differentiate between different pathways of VC removal.

Für viele Wissenschaftler sind Membranproteine ein faszinierendes Forschungsobjekt, für viele aber auch der schlimmste Alptraum, dem sie je begegnet sind. Letztere kämpfen mit intrinsischen Problemen: Expression, Reinigung, Stabilität, biochemische und biophysikalische Charakterisierung; die anderen begeistert die Funktion von Membranproteinen — Informationsaustausch über biologische Membranen, ohne die wahrscheinlich das Leben, wie wir es heute kennen, nicht existieren würde.

Quantitative utilization of L-cysteate (2-amino-3-sulphopropionate) as the sole source of carbon and energy for growth of the aerobic, marine bacterium Silicibacter pomeroyi DSS-3T was observed. The sulphonate moiety was recovered in the medium largely as sulphite, and the appropriate amount of the ammonium ion was also observed. Genes [suyAB (3-sulpholactate sulpho-lyase)] encoding the known desulphonation reaction in cysteate degradation were absent from the genome, but a homologue of a putative sulphate exporter gene (suyZ) was found, and its neighbour, annotated as a D-cysteine desulphhydrase, was postulated to encode pyridoxal 5′-phosphate-coupled L-cysteate sulpho-lyase (CuyA), a novel enzyme. Inducible CuyA was detected in cysteate-grown cells. The enzyme released equimolar pyruvate, sulphite and the ammonium ion from L-cysteate and was purified to homogeneity by anion-exchange, hydrophobic-interaction and gel-filtration chromatography. The N-terminal amino acid sequence of this 39-kDa subunit confirmed the identification of the cuyA gene. The native enzyme was soluble and homomultimeric. The Km-value for L-cysteate was high (11.7 mM) and the enzyme also catalysed the D-cysteine desulphhydrase reaction. The gene cuyZ, encoding the putative sulphite exporter, was co-transcribed with cuyA. Sulphite was exported despite the presence of a ferricyanide-coupled sulphite dehydrogenase. CuyA was found in many bacteria that utilize cysteate.

ABSTRACTInfection with human immunodeficiency virus (HIV) and the resulting immunosuppression are associated with an increased risk for human papillomavirus (HPV) persistence and related malignancies. In the present study we investigated the prevalence of HPV in urine samples from 104 HIV-infected men with low CD4+cell counts (<100 per mm3) and 115 urine samples from HIV-negative men. A high prevalence of HPV DNA (39.4%) was found in the HIV patients. Most of the HPV types were high risk (81.4%), with HPV 52 as the most prevalent type (12.5%), followed by HPV 18 (6.7%), HPV 35 (5.8%), and HPV 70 (4.8%). Multiple HPV genotypes were observed in 17 (41%) of the 41 HPV- and HIV-positive men. In contrast, only 11 (9.6%) HPV DNA-positive cases were observed among the 115 HIV-uninfected men, and 3 (27.3%) contained multiple genotypes. Quantitative analyses indicated that the HPV viral load, as measured in urine samples, is significantly higher in HIV-positive men compared to HIV-negative men. In the present study we show that urine samples are useful for detecting HPV DNA, there is a high prevalence of HPV in HIV-positive men, and the HPV viral load is substantially higher in HIV-positive than in HIV-negative men. More studies are needed to evaluate the risk and natural development of HPV-related malignancies in HIV-positive men.

The objective of the present experiment was to investigate the effects of transportation, lairage, and preslaughter stressor treatment on glycolytic potential and pork quality of the glycolytic longissimus and the oxidative supraspinatus (SSP) or serratus ventralis (SV) muscles. In a 2 × 2 × 2 factorial design, 384 pigs were assigned randomly either to short (50 min) and smooth or long (3 h) and rough transport, long (3 h) or short (< 45 min) lairage, and minimal or high preslaughter stress. Muscle samples were taken from the LM at 135 min and from the SSP at 160 min postmortem for determination of the glycolytic potential and rate of glycolysis. At 23 h postmortem, pork quality was assessed in the LM and the SV. Effects of transport and lairage conditions were similar in both muscle types. Long transport increased (P< 0.01) the glycolytic potential and muscle lactate concentrations compared with short transport. Both long transportation and short lairage decreased (P< 0.01) redness (a* values) and yellowness (b* values) of the LM and SV. In combination with short lairage, long transport decreased (P< 0.05) pork lightness (lower L* values), and electrical conductivity was increased (P< 0.05) after long transport. Several interactions between stress level and muscle type (P< 0.001) were observed. High preslaughter stress decreased (P< 0.001) muscle glycogen in both the LM and SSP, but this decrease was greater in the LM. Lactate concentrations were increased (P< 0.001) only in the LM by high preslaughter stress. Increases in ultimate pH (P< 0.001) and decreases in a* values (P< 0.01) were greatest in the SV, whereas increases in electrical conductivity (P< 0.001) were greatest in the LM. The lack of interactions among transportation, lairage, and muscle type was attributed to the relatively minor differences in stress among treatments. It was concluded that, in glycolytic muscle types such as the LM, the high physical and psychological stress levels associated with stress in the immediate preslaughter period have a greater effect on the water-holding capacity of the meat and may promote PSE development. Conversely, oxidative muscle types tend to have higher ultimate pH values and produce DFD pork in response to intense physical activity and/or high psychological stress levels preslaughter.

The objectives of the present experiment were 1) to study the effects of transport conditions and lairage duration on stress level, muscle glycolytic potential, and pork quality; and 2) to investigate whether the negative effects of high stress immediately preslaughter are affected by preceding handling factors (transport and lairage). In a 2 × 2 × 2 factorial design, halothane-free pigs (n = 384) were assigned to either short (50 min) and smooth or long (3 h) and rough transport; long (3 h) or short (<45 min) lairage; and minimal or high preslaughter stress. Pigs were processed in eight groups (48 pigs per group) on various days at a commercial plant. Blood samples were taken at exsanguination to measure plasma cortisol and lactate concentrations. Muscle pH and temperature were measured at 30 and 40 min, respectively, and both were measured at 3 h, postmortem. A LM sample was taken 135 min postmortem to estimate glycogen content and rate of glycolysis. Pork quality attributes were assessed 23 h postmortem. Short transport increased cortisol when followed by short lairage (transport × lairage; P< 0.01). Long transport, but not lairage (P> 0.30), tended to increase (P= 0.06) muscle glycolytic potential. Long transport tended to increase (P= 0.08) electrical conductivity, and decreased a* (P< 0.01) and b* (P< 0.02) values. Decreasing lairage from 3 h to <45 min decreased (P< 0.05) the L* value, but it did not (P> 0.10) affect other pork quality traits. High stress decreased (P< 0.001) muscle glycolytic potential, and increased (P< 0.001) plasma lactate, cortisol, muscle temperature, rate of pH decline, and ultimate pH. Except for decreased (P< 0.001) b* values, pork color was not (P> 0.40) affected by high stress, but water-holding properties (measured by electrical conductivity, filter paper moisture, and drip loss) were impaired (P< 0.001) by high stress. Fiber optic-measured light scattering and Warner-Bratzler shear force were not (P> 0.12) affected by any treatment. Comparisons with the “optimal” handling (short transport, long lairage, and minimal stress) revealed that, with regard to water-holding properties, the negative effects of high stress were aggravated by suboptimal transport and lairage conditions. High stress alone increased electrical conductivity by 56%, whereas high stress in combination with short lairage led to an 88% increase. However, high preslaughter stress contributed most and was the major factor responsible for reductions in pork quality.

The human gastric pathogen Helicobacter pylori spontaneously switches lipopolysaccharide (LPS) Lewis (Le) antigens on and off (phase-variable expression), but the biological significance of this is unclear. Here, we report that Le+ H. pylori variants are able to bind to the C-type lectin DC-SIGN and present on gastric dendritic cells (DCs), and demonstrate that this interaction blocks T helper cell (Th)1 development. In contrast, Le− variants escape binding to DCs and induce a strong Th1 cell response. In addition, in gastric biopsies challenged ex vivo with Le+ variants that bind DC-SIGN, interleukin 6 production is decreased, indicative of increased immune suppression. Our data indicate a role for LPS phase variation and Le antigen expression by H. pylori in suppressing immune responses through DC-SIGN.

The objective of the present experiment was to study physiological changes elicited in response to stress in the immediate preslaughter period and to link them to pork quality characteristics. Crossbred, halothane-free pigs (n = 192) were processed in eight groups (24 pigs per group) on various days at one of two commercial processing plants operating different stunning systems (electrical and CO2stunning in Plants A and B, respectively). In each group, half the pigs were exposed to either minimal or high preslaughter stress. Blood samples were taken at exsanguination, and lactate, cortisol, and catecholamines, as well as blood pH and temperature, were assessed and linked to various longissimus muscle quality attributes. Additionally, muscle pH and temperature were measured 30 min postmortem, and muscle glycolytic potential was determined 22 h postmortem. At both processing plants, high preslaughter stress resulted in higher (P< 0.05) blood cortisol and lactate; however, the effects of preslaughter stress on catecholamines and blood pH were believed to be biased by the different stunning methods employed at the plants. High preslaughter stress increased (P< 0.05) blood temperature at Plant A but not at Plant B. At both plants, high stress increased (P< 0.05) 30-min muscle temperature and decreased (P< 0.05) 30-min muscle pH. Ultimate pH was increased (P< 0.05) and muscle glycolytic potential was decreased (P< 0.05) by high preslaughter stress. At both plants, high stress resulted in inferior pork quality attributes (P< 0.05), including reflectance, electrical conductivity, filter paper moisture, drip loss, and L* value. The effect of stress was greater on water-holding capacity than on pork color, with drip losses increased by 56%. Of all stress indicators measured at exsanguination, only blood lactate was strongly correlated with pork quality attributes. Regression analyses revealed that blood lactate and glycolytic potential accounted for 52 and 48% of the variation in drip loss and L* value, respectively. In combination with high preslaughter stress, high glycolytic potentials were related to increased drip losses. We conclude that high preslaughter stress leads to impaired pork quality, with high muscle energy levels aggravating the negative effects of preslaughter stress. Monitoring stress level by blood lactate measurement in combination with strategies to control muscle energy present at slaughter may help to improve meat quality.

The present experiment investigated whether increasing chilling rate could improve meat quality in pigs exposed to either minimal or high stress immediately preslaughter. Pigs (n = 192) were offspring of halothane-free lines. On various days, four groups of 48 pigs were processed at a commercial plant. Within each group, half the pigs were exposed to either minimal or high preslaughter stress. Before entering the cooler at 45 min postmortem, carcasses of both minimal and high preslaughter stress treatments were allocated randomly to either conventional (+4°C for 22 h) or rapid (three-phase chilling tunnel: −15, −10, and −1°C for 15, 38, and 38 min, respectively, followed by storage at 4°C until 22 h postmortem) chilling. Temperature and pH were measured in the blood at exsanguination and in the longissimus lumborum (LL) and semimembranosus (SM) muscle at 0.5, 2.5, 4.5, 6.5, and 22 h postmortem. Meat quality attributes (water-holding capacity and objective color measurements) were assessed on the LL. Preslaughter stress level affected pH and temperature in both blood and muscle, with lower (P< 0.001) pH values and higher (P< 0.001) temperatures for pigs exposed to high vs. minimal stress. Rapid chilling led to a faster (P< 0.001) temperature decline regardless of preslaughter stress level. Rapid chilling did not (P> 0.05) influence the rate of pH decline in the LL muscle, but reduced (P= 0.061) pH decline in the SM. Rapid chilling, as opposed to conventional chilling, decreased (P< 0.05) electrical conductivity in the LL, regardless of preslaughter stress; however, it could not compensate for the detrimental effect (P< 0.05) of stress on drip loss, filter paper moisture absorption, and meat color (L* value). Results from the present study indicated that increasing chilling rate is not a suitable method to resolve pork quality problems caused by inadequate preslaughter handling.

Percutaneous thrombolysis has become an accepted treatment of thrombosed haemodialysis grafts. Several devices have been developed for mechanical thrombolysis, which macerate the clot using different mechanisms such as aspiration and fragmentation. The aim of our study was to compare the efficacy of three devices for mechanical thrombolysis in removing the thrombus from thrombosed haemodialysis access grafts and to determine the initial technical and clinical success, complication rates of each device, and graft patency after the procedure.

Interleukin 12 (IL-12) is a potent enhancer of interferon γ production by activated T cells. The high-affinity IL-12 receptor (IL-12R) is a heterodimer of a β1 and a β2 subunit. Expression of the signaling IL-12Rβ2 chain is usually low, as compared with the more abundant β1 chain, and may be rate-limiting for IL-12 sensitivity. Little is known about the mechanisms controllingIL-12Rβ2gene expression. Reporter gene assays in IL-12Rβ2-expressing Jurkat cells showed that truncation of the region from −151 to −61 abrogated promoter activity. The proximal promoter region does not contain a typical TATA box, suggesting a role for SP-1. Indeed, mutagenesis of the −63 SP-1 consensus site decreased transcription by 50%. Electrophoretic mobility shift experiments confirmed the binding of SP-1 and SP-3 at this site. In contrast, truncation of −252 to −192 increased promoter activity. Likewise, mutagenesis of the consensus nuclear factor of activated T cells site at −206 increased promoter activity by 70%, suggesting silencer activity of this element. Electrophoretic mobility shift experiments with primary Th (T helper) cells showed the formation of a specific, T-cell receptor-inducible complex at this site that is sensitive to cyclosporin A and supershifted with anti-NFATc2 in both Th1 and Th2 cells. Accordingly, cyclosporin A dose-dependently increased IL-12Rβ2 mRNA expression. These first data onIL-12Rβ2gene regulation indicate a TATA-less promoter, depending on SP-1/SP-3 transcription factors, and a negative regulatory NFAT element at −206. This element may contribute to the overall low level of IL-12Rβ2 expression on Th cells.

Human papillomavirus (HPV) can be detected by DNA amplification from clinical samples. The aim of the present study was to compare the HPV status in both cervical scrape and biopsy specimens obtained from 174 patients, using the recently developed broad spectrum SPF10PCR‐LiPA method. The detection rate of HPV in these materials was determined and the spectrum of HPV genotypes was compared. Cervical scrapes and biopsy specimens were obtained, either on the same day (group I), or with an interval of up to almost 2 years (group II, mean interval 97 days, range 1–469 days). HPV DNA was amplified by SPF10PCR and detected in a microtitre plate hybridization assay. Of the HPV‐positive cases, the genotype was determined by reverse hybridization of the same SPF10amplimer on a line probe assay (LiPA), discriminating between HPV genotypes 6, 11, 16, 18, 31, 33–35, 39, 40, 42–45, 51–54, 56, 58, 59, 66, 68, 70, and 74. The results showed that the detection rate and the spectrum of HPV genotypes in cervical scrapes and the corresponding biopsy specimens were highly comparable in both patient groups, even when multiple genotypes were present. In both groups, multiple HPV genotypes were more frequently detected in cervical scrapes than in the corresponding biopsy specimens. In conclusion, HPV infection can be diagnosed in cervical scrapes and biopsy specimens using the SPF10PCR‐LiPA system. Analysis of cervical scrapes accurately reflects the spectrum of HPV genotypes in the patient's cervical region, even with a sampling interval between the cervical scrape and the biopsy specimen. Copyright © 2001 John Wiley & Sons, Ltd.

IL-12 is a potent inducer of IFN-γ production and drives the development of Th1 cells. Human polarized Th2 cells do not express the signaling β2-subunit of the IL-12R and, therefore, do not signal in response to IL-12. The question was raised as to what extent the loss of the IL-12Rβ2 chain in Th2 cells has bearing on the stability of the human Th2 phenotype. In the present report, we show that restimulation of human fully polarized Th2 cells in the presence of IL-12 primes for a shift towards Th0/Th1 phenotypes, accompanied by suppression of GATA-3 expression and induction of T-bet expression. These reversed cells are further characterized by a marked IL-12Rβ2 chain expression and fully restored IL-12-inducible STAT4 activation. The IL-12-induced phenotypic shift proved to be stable as a subsequent restimulation in the presence of IL-4 and in the absence of IL-12 could not undo the accomplished changes. Identical results were obtained with cells from atopic patients, both with polyclonal Th2 cell lines and allergen-specific Th2 cell clones. These findings suggest the possibility of restoring IL-12 responsiveness in established Th2 cells of atopic patients by stimulation in the presence of IL-12, and that IL-12-promoting immunotherapy can be beneficial for Th2-mediated immune disorders, targeting both naive and memory effector T cells.

The host‐searching behaviour of Heterorhabditis megidisstrain NLH‐E 87.3 in the presence of insect hosts and plant roots, offered individually and in combination, was studied using a newly developed Y‐tube olfactometer filled with sand. Within a period of 24 hours infective juveniles (IJs) were significantly attracted to living G. mellonellalarvae and caused 100% larval mortality. Otiorhynchus sulcatuslarvae, however, did not elicit host‐oriented movement of IJs and no larval mortality was observed. Roots of strawberry plants induced a negative response in IJs. The combination of strawberry roots and O. sulcatuslarvae, however, strongly attracted IJs leading to 37% host mortality. It was shown that this type of Y‐tube choice arena is a useful tool in studying the searching behaviour of entomopathogenic nematodes in a semi‐natural habitat.

Genetic lack of interleukin 12 receptor β1 (IL-12Rβ1) surface expression predisposes to severe infections by poorly pathogenic mycobacteria or Salmonella and causes strongly decreased, but not completely abrogated, interferon (IFN)-γ production. To study IL-12Rβ1–independent residual IFN-γ production, we have generated mycobacterium–specific T cell clones (TCCs) from IL-12Rβ1–deficient individuals. All TCCs displayed a T helper type 1 phenotype and the majority responded to IL-12 by increased IFN-γ production and proliferative responses upon activation. This response to IL-12 could be further augmented by exogenous IL-18. IL-12Rβ2 was found to be normally expressed in the absence of IL-12Rβ1, and could be upregulated by IFN-α. Expression of IL-12Rβ2 alone, however, was insufficient to induce signal transducer and activator of transcription (Stat)4 activation in response to IL-12, whereas IFN-α/IFN-αR ligation resulted in Stat4 activation in both control and IL-12Rβ1–deficient cells. IL-12 failed to upregulate cell surface expression of IL-18R, integrin α6, and IL-12Rβ2 on IL-12Rβ1–deficient cells, whereas this was normal on control cells. IL-12–induced IFN-γ production in IL-12Rβ1–deficient T cells could be inhibited by the p38 mitogen-activated protein kinase (MAP) kinase inhibitor SB203580 and the MAP kinase kinase (MEK) 1/2 inhibitor U0126, suggesting involvement of MAP kinases in this alternative, Stat4-independent, IL-12 signaling pathway. Collectively, these results indicate that IL-12 acts as a partial agonist in the absence of IL-12Rβ1. Moreover, the results reveal the presence of a novel IL-12Rβ1/Stat4–independent pathway of IL-12 responsiveness in activated human T cells involving MAP kinases. This pathway is likely to play a role in the residual type 1 immunity in IL-12Rβ1 deficiency.

Virulence and speed of action, as related to dose, are important effectiveness-determining properties of insect-pathogenic biocontrol agents. We used the droplet-feeding bioassay to compare dose responses between two wild-type baculoviruses, Autographa californicamulticapsid nucleopolyhedrovirus (AcMNPV) and Spodoptera exiguaMNPV (SeMNPV), and three deletion mutants of AcMNPV in S. exigualarvae. In each mutant one gene was deleted by genetic engineering: pp34,coding for the polyhedral membrane; egt,coding for ecdysteroid UDP–glucosyltransferase; or p10,coding for fibrillar structures in infected insect cells. SeMNPV had the lowest median lethal dose (LD50) as well as the highest speed of action (LT50) of all viruses investigated. In our comparative bioassays the only significant effect of gene deletions in AcMNPV was a slightly lower speed of action for the p10deletion mutant. Otherwise, wild-type and recombinant AcMNPVs had similar biological activities. Our results suggest, in contrast to what is generally assumed, that gene deletions in AcMNPV for improved insecticidal activity should be critically assessed in each host system prior to further implementation as a control agent. Insertion of foreign genes coding for entomotoxins is less questionable and more promising in this respect.

Photorhabdus luminescensisolates were exchanged between four entomopathogenic nematode strains; two isolates ofHeterorhabditis megidisfrom The Netherlands and two isolates ofH. bacteriophora, one from Australia and one from Moldavia. When cultured onH. megidissymbionts only fewH. bacteriophorainfective juveniles retained the symbiotic bacterium. These infective juveniles, without symbiotic bacteria, were not pathogenic to insects. WhenP. luminescenswas injected into insect larvae it could killGalleria mellonella(Lepidoptera: Galleridae) larvae but it was not pathogenic toTipula oleracea(Diptera: Tipulidae). The combination of nematode and bacterium killedT. oleracea, showing that the nematode is more than just a vector for the bacterium. Small differences in pathogenicity between the combinations could only be observed inT. oleracea, not in the highly susceptibleG. mellonella. The pathogenicity of a combination depends on the pathogenicity of the bacterium, the pathogenicity of the nematode and the interaction between them. The pathogenicity ofH. bacteriophorastrains againstT. oleraceawas low, partly because of the low penetration rate of these strains.

The insect pathogenic nematode Steinernema feltiae (Nematoda: Steinernematidae) was shown to offer an alternative to the use of diflubenzuron for the control of the mushroom fly Lycoriella auripila (Diptera: Sciaridae). The influence of diflubenzuron and S. feltiae treatments on the yield and numbers of the mushroom, Agaricus bisporus , was studied. Applications of diflubenzuron after casing significantly reduced total mushroom yields by 10-13%. The greatest yield reductions were observed in the first flush. In the second flush, high yields were found despite significantly lower numbers of mushrooms. These high yields, however, could not compensate for the yield loss of the first flush. Some of the treatments with S. feltiae significantly increased yields, but these were not associated with a particular time of application. As nematodes did not reduce mushroom yields they were able to compete with diflubenzuron. Although the purchase costs of nematodes were high, their use was more economical than that of diflubenzuron when the yield losses of 10% due to casing treatments were taken into account.

The nematode Steinernema feltiae(Nematoda: Steinemematidae) was tested for its ability to control two main mushroom pests i.e. the sciarid Lycoriella auripila(Diptera: Sciaridae) and the phorid Megaselia halterata(Diptera: Phoridae) in growing-rooms filled with spawned compost. A clear difference between female and male sciarid control was observed. A nematode application 1 day after casing preceded by an application 1 day before casing on the compost caused an almost complete control (97) of the F1-generation of female sciarids. The F2-generation of females was similarly controlled (95) by an application 7 days after casing. A dosage of 1 × 106nematodes m-2was found to be equally effective as higher dosages. Diflubenzuron remained active throughout entire the cropping period with high sciarid mortality rates varying from 72 to 99.

The efficacy of different species of entomopathogenic nematodes was tested against larvae of the mushroom phorid Megaselia halterata (Diptera: Phoridae) and the mushroom sciarid Lycoriella auripila (Diptera: Sciaridae). Sciarid larvae originating from infestations in casing soil during colonization by Agaricus bisporus were almost completely controlled by applications of Steinernema feltiae to the casing soil. When larvae originated from infestations in freshly spawned compost, they could be controlled by compost applications halfway through spawnrunning and by very early casing treatments. The control of phorids in compost was maximally 31% when nematodes were mixed within the infested compost at a concentration of 3 106 nematodes/m2. Only slightly higher reduction rates were obtained at higher concentrations. The control of phorids was more promising in the infested casing layer, in which S. carpocapsae was most successful. At concentrations of 6 and 15 106 nematodes/m2 this species obtained reduction rates of 65 and 73% respectively when it was applied 3 days after the end of the infestation period. These concentrations are, however, too high for practical application.

Experiments were conducted to examine the location of oviposition by the phorid fly Megaselia halterata(Wood) (Diptera: Phoridae) in uncased and cased compost. Clearly, a majority of the gravid females choose oviposition sites directly after entering the top layer of the compost. In uncased compost, 60% of all adults emerged from the top of four compost layers of equal thickness. When the compost was covered by a casing layer which was still uncolonized by Agaricus bisporus, oviposition was further concentrated in the top compost layer. In this situation, 91% of all adults emerged from the top compost layer whereas only 1.5% emerged from the casing. When the casing layer was colonized by mushroom mycelium, 45% of all adults emerged from the casing layer and 53% emerged from the top compost layer. Further concentration in the top compost layer and the casing layer occurred as a result of upward migration of larvae. When compost was cased after oviposition, up to 43% of all adults emerged from the casing layer. We concluded that in the control of phorid infestations with insect pathogenic nematodes, applications in uncased compost can be restricted to the upper compost layer. When compost and casing are filled simultaneously, nematode applications in the casing layer only could be considered.

In a comparative investigation of Critical Fusion Frequency (C. F. F.) values of 10 squirrels and 12 hedgehogs, it was shown that the C. F. F. values of a diurnal retina (squirrel) lie between about 30 and higher than 103 per sec., whereas these for a nocturnal retina (hedgehog) are about 30 per sec. or lower.Electroretinographic Critical Fusion Frequency determinations (C. F. F.) can be used to distinguish between « rod » and « cone » systems.

The existence of a fibre connection from the deep layers of the dorsal lateral geniculate body to the lateral posterior thalamic nucleus has been confirmed with histological methods in the cat. A fibre tract from the lateral posterior thalamic nucles to the ventral anterior thalamic nucleus and to the central lateral thalamic neucleus has been demonstrated.Electrical stimulation of the lateral posterior thalamic nucleus resulted in ipsilateral head movements, loss of posture, jerking and fully developed seizures with loss of bladder control, and with spikewave activity in the EEG in the freely moving cat.Electrical stimulation of anterior thalamic regions at the ventral anterior thalamic nucleus and at the central lateral thalamic nucleus produced only some jerking of the legs, which stopped on termination of the stimulus and was never combined with loss of bladder control.Electrical stimulation of the dorsal lateral geniculate body and of the rostral colliculus and of the nucleus cuneiformus confirmed earlier results from the literature.The lateral posterior thalamic nucleus is thought to be a major part of the centrencephalic epileptic system with a direct connection to the occipital cortex and with an indirect connection, through the anterior thalamic nuclei, to the frontal cortex.

Early Xenopusdevelopment is characterized by a poor correlation between global mRNA and protein abundances due to maternal mRNA and protein loading. Therefore, proteome profiling is necessary to study gene expression dynamics during early Xenopusdevelopment. In contrast to mammals, single Xenopuseggs and embryos contain enough protein to allow identification and quantification of thousands of proteins using mass spectrometry-based proteomics. In addition to investigating developmental processes, single egg or blastomere proteomes can be used to study cell-to-cell variability at an unprecedented depth. In this protocol, we describe a mass spectrometry-based proteomics approach for the identification and absolute quantification of Xenopus laevisegg or embryo proteomes, including sample preparation, peptide fractionation and separation, and data analysis.