Human cytomegalovirus deploys molecular mimicry to recruit VPS4A to sites of virus assembly.

The AAA-type ATPase VPS4 is recruited by proteins of the endosomal sorting complex required for transport III (ESCRT-III) to catalyse membrane constriction and membrane fission. VPS4A accumulates at the cytoplasmic viral assembly complex (cVAC) of cells infected with human cytomegalovirus (HCMV), the site where nascent virus particles obtain their membrane envelope. Here we show that VPS4A is recruited to the cVAC via interaction with pUL71. Sequence analysis, deep-learning structure prediction, molecular dynamics and mutagenic analysis identify a short peptide motif in the C-terminal region of pUL71 that is necessary and sufficient for the interaction with VPS4A. This motif is predicted to bind the same groove of the N-terminal VPS4A Microtubule-Interacting and Trafficking (MIT) domain as the Type 2 MIT-Interacting Motif (MIM2) of cellular ESCRT-III components, and this viral MIM2-like motif (vMIM2) is conserved across ß-herpesvirus pUL71 homologues. However, recruitment of VPS4A by pUL71 is dispensable for HCMV morphogenesis or replication and the function of the conserved vMIM2 during infection remains enigmatic. VPS4-recruitment via a vMIM2 represents a previously unknown mechanism of molecular mimicry in viruses, extending previous observations that herpesviruses encode proteins with structural and functional homology to cellular ESCRT-III components.

Gait Analysis in Walking and Trotting Dairy Cows on Different Flooring Types with Novel Mobile Pressure Sensors and Inertial Sensors.

Mechanical overburdening is a major risk factor that provokes non-infectious claw diseases. Moreover, lameness-causing lesions often remain undetected and untreated. Therefore, prevention of claw tissue overburdening is of interest, especially by analyzing harmful effects within dairy cows' housing environment. However, objective "on-cow" methods for bovine gait analysis are underdeveloped. The purpose of the study was to apply an innovative mobile pressure sensor system attached at the claws to perform pedobarometric gait analysis. A further goal was the supplementation with accelerative data, generated simultaneously by use of two inertial measurement units (IMUs), attached at metatarsal level. IMU data were analyzed with an automatic step detection algorithm. Gait analysis was performed in ten dairy cows, walking and trotting on concrete flooring and rubber mats. In addition to the basic applicability of the sensor systems and with the aid of the automatic step detection algorithm for gait analysis in cows, we were able to determine the impact of the gait and flooring type on kinematic and kinetic parameters. For pressure sensor output, concrete was associated with significantly (p < 0.001) higher maximum and average pressure values and a significantly smaller contact area, compared to rubber mats. In contrast to walking, trotting led to a significantly higher force, especially under the medial claw. Further, IMU-derived parameters were significantly influenced by the gait. The described sensor systems are useful tools for detailed gait analysis in dairy cows. They allow the investigation of factors which may affect claw health negatively.

[Curative claw trimming for mechanical relief of sole ulcers - an ex-vivo study]. / Kurative Klauenpflege zur mechanischen Entlastung von Sohlengeschwüren ­ eine Ex-vivo-Studie.

OBJECTIVE: Overloading or excessive mechanical stress to the claws may damage the sensitive claw tissues and subsequently lead to sole ulcers. Corrective treatment can prevent complications of these sole ulcers. The aim of this study was to investigate the effects of a wedge-shaped relief incision from the bulb to the tip of the outer claw of the pelvic limb on the pressure distribution beneath the pertaining claw. Furthermore, it aimed to clarify whether a change in the pressure distribution would lead to dermal pressure relief in the area of a horn defect. MATERIAL AND METHODS: Nineteen isolated bovine limbs from an abattoir were gradually trimmed resulting in following stages. A: initial condition; B: functional claw trimming, steps 1-3; C: extension of the model and drill of a circular lesion at the Rusterholz site (outer claw ulcer simulation); D: wedge shaped, weight-relieving incision from the bulb halfway towards the sole tip; E: further extension of the wedge-shaped incision towards the sole tip. The limbs were loaded with 200 kg following each of the procedures. A pressure sensor system was used in order to analyze the pressure distribution beneath the claws. Furthermore, positioning of the claws on a glass plate allowed for an evaluation of corium resp. fat cushion protrusion through the artificial lesion. RESULTS: In the untrimmed claws, an average of 63 % of the total force applied rested on the outer claw area. This value decreased to 30 % following step E. Maximum pressure values shifted from the bulb area towards the sole tip with increasing degree of claw trimming. The relative loading area of the inner claw increased to up to 18.4 % following the procedures. The described incision technique (step E) resulted in a significant reduction of corium protrusion through the artificial horn defect in comparison to the claw situation in step C. CONCLUSION: The wedge-shaped, weight-relieving incision (step E) led to pressure reduction in the area of the outer claw and the bulb region in this ex-vivo-study. Therefore, this trimming method may serve to relieve pressure from the vulnerable Rusterholz site as well as providing means for reducing corium compression. CLINICAL RELEVANCE: The described trimming method facilitates pressure relief on the outer claw area in live animals. In this, it may exert a positive effect on disease progression in Rusterholz sole ulcers.

Impact of DAA/water composition on PFSA ionomer conformation.

Performance of a proton exchange membrane fuel cell (PEMFC) is significantly determined by the structure and composition of the electrode layer. Electrode layers are formed from inks consisting of platinum-doped carbon black particles, perfluorosulfonic acid (PFSA) ionomer and a dispersing solvent. Interaction between these materials mainly influences suspension stability, ionomer conformation and therewith layer morphology. We characterize the interplay between a short sidechain (SSC) PFSA ionomer (Aquivion® D79-25BS) and a solvent mixture (diacetone alcohol (DAA) and water with different weight ratios) by using Hansen solubility/dispersibility parameters (HSP) and by experimental tests. It was found that HSPs are well suitable to describe the ionomer/solvent interactions. In particular, the HSP difference in terms of the hydrogen bonds is responsible for the poor affinity between ionomer and solvent at low DAA concentrations. With increasing DAA content the affinity between ionomer and solvent increases as indicated by better matching HSPs. For an ionomer concentration of 4 wt%, Aquivion always forms molecular solutions for all DAA-in-water mixing ratios. Self-organization of the ionomer molecules changes from densely packed/collapsed molecules with highly deprotonated sulfonic acid side groups at low DAA concentrations to unfolded Aquivion molecules with a low dissociation degree of the sulfonic acid groups at high DAA concentrations.

A Phase 1 Study of mTORC1/2 Inhibitor BI 860585 as a Single Agent or with Exemestane or Paclitaxel in Patients with Advanced Solid Tumors.

This phase 1 trial (NCT01938846) determined the maximum tolerated dose (MTD) of the mTOR serine/threonine kinase inhibitor, BI 860585, as monotherapy and with exemestane or paclitaxel in patients with advanced solid tumors. This 3+3 dose-escalation study assessed BI 860585 monotherapy (5-300 mg/day; Arm A), BI 860585 (40-220 mg/day; Arm B) with 25 mg/day exemestane, and BI 860585 (80-220 mg/day; Arm C) with 60-80 mg/m2/week paclitaxel, in 28-day cycles. Primary endpoints were the number of patients with dose-limiting toxicities (DLTs) in cycle 1 and the MTD. Forty-one, 25, and 24 patients were treated (Arms A, B, and C). DLTs were observed in four (rash (n = 2), elevated alanine aminotransferase/aspartate aminotransferase, diarrhea), four (rash (n = 3), stomatitis, and increased gamma-glutamyl transferase), and two (diarrhea, increased blood creatine phosphokinase) patients in cycle 1. The BI 860585 MTD was 220 mg/day (Arm A) and 160 mg/day (Arms B and C). Nine patients achieved an objective response (Arm B: Four partial responses (PRs); Arm C: Four PRs; one complete response). The disease control rate was 20%, 28%, and 58% (Arms A, B, and C). The most frequent treatment-related adverse events (AEs) were hyperglycemia (54%) and diarrhea (39%) (Arm A); diarrhea (40%) and stomatitis (40%) (Arm B); fatigue (58%) and diarrhea (58%) (Arm C). The MTD was determined in all arms. Antitumor activity was observed with BI 860585 monotherapy and in combination with exemestane or paclitaxel.

Novel Polyomaviruses in Mammals from Multiple Orders and Reassessment of Polyomavirus Evolution and Taxonomy.

As the phylogenetic organization of mammalian polyomaviruses is complex and currently incompletely resolved, we aimed at a deeper insight into their evolution by identifying polyomaviruses in host orders and families that have either rarely or not been studied. Sixteen unknown and two known polyomaviruses were identified in animals that belong to 5 orders, 16 genera, and 16 species. From 11 novel polyomaviruses, full genomes could be determined. Splice sites were predicted for large and small T antigen (LTAg, STAg) coding sequences (CDS) and examined experimentally in transfected cell culture. In addition, splice sites of seven published polyomaviruses were analyzed. Based on these data, LTAg and STAg annotations were corrected for 10/86 and 74/86 published polyomaviruses, respectively. For 25 polyomaviruses, a spliced middle T CDS was observed or predicted. Splice sites that likely indicate expression of additional, alternative T antigens, were experimentally detected for six polyomaviruses. In contrast to all other mammalian polyomaviruses, three closely related cetartiodactyl polyomaviruses display two introns within their LTAg CDS. In addition, the VP2 of Glis glis (edible dormouse) polyomavirus 1 was observed to be encoded by a spliced transcript, a unique experimental finding within the Polyomaviridae family. Co-phylogenetic analyses based on LTAg CDS revealed a measurable signal of codivergence when considering all mammalian polyomaviruses, most likely driven by relatively recent codivergence events. Lineage duplication was the only other process whose influence on polyomavirus evolution was unambiguous. Finally, our analyses suggest that an update of the taxonomy of the family is required, including the creation of novel genera of mammalian and non-mammalian polyomaviruses.

Chemical (C, N, S, black carbon, soot and char) and stable carbon isotope composition of street dusts from a major West African metropolis: Implications for source apportionment and exposure.

Street dust is a major source of pollution and exposure of residents of West Africa to toxic chemicals. There is however, limited knowledge about the chemical composition and sources of street dust in urban areas of sub-Saharan Africa. The total carbon (TC), nitrogen (TN), sulfur (TS) and the stable carbon isotope ratios (δ13C) contents of street dust sampled from 25 sites distributed across Kumasi (a metropolis in Ghana with a population of ca. 2 million) were determined. In addition, black carbon (BC) and their subunits (soot and char) in these samples were also determined. The concentrations of TC, TN and TS in the dusts were 5-71 mg g-1, 0.3-4.3 mg g-1 and 0.2-1.4 mg g-1, respectively. The concentrations of TC, TN and TS were higher than at the background site of the metropolis by a factor of 5.1 (range: 1.7-12), 3.9 (1.1-13) and 2.8 (0.7-5), respectively. The BC, char and soot concentrations in these samples averaged 1.6 mg g-1 (0.13-4.4), 1.2 mg g-1 (0.08-3.7) and 0.36 mg g-1 (0.05-1.5), respectively. The concentrations of BC, char and soot in the street dust were higher than in the background location by factors of 5 (range: 0.8-13), 6 (0.7-17) and 3 (0.5-12), respectively. The TC, TN, TS, BC, soot and char concentrations were positively correlated with each other and with polycyclic aromatic compounds (PAHs, oxygenated PAHs and azaarenes from a previous study), indicating their common origin and fate. The δ13C values ranged from -27 to -24 [‰], with more polluted sites being more depleted in 13C. Based on the chemical composition of the street dusts, the 25 sites could be clustered into four groups by hierarchical cluster analysis which reflect areas of varying anthropogenic influence and, accordingly, exposure to hazardous chemicals.

"Eu sou quem então?": o trabalho docente na educação infantil e os impactos da organização do trabalho na dinâmica do reconhecimento / "Then who am I?": the teaching work in childhood education and the impacts of the work organization in the dynamic of recognition

Este estudo teve como intuito compreender como o reconhecimento do trabalho docente na educação infantil interfere na saúde das professoras. Para isso, buscou entender as modificações no contexto da educação infantil e os fatores produtores de prazer e sofrimento no ambiente laboral. A pesquisa foi realizada em um município do interior do Rio Grande do Sul, com oito docentes da primeira etapa da educação da rede pública, por meio de entrevistas semiestruturadas. A metodologia utilizada foi uma adaptação da psicodinâmica do trabalho strictu sensu. O trabalho docente na educação infantil tem passado por modificações, inclusive em sua organização. Algumas professoras precisaram assumir a responsabilidade pedagógica de mais de uma turma, o que acarretou o aumento da carga de trabalho. Além disso, possuem pouco tempo com as crianças e a principal atividade se refere a atribuições de tarefas. Nesse sentido, a dinâmica da contribuição e do reconhecimento das profissionais é afetada. O reconhecimento é fundamental para que o trabalho tenha sentido para o sujeito. Para as professoras pesquisadas ele provém das crianças com as quais trabalham e é manifestado por meio do afeto.

This study had as a general objective to understand how the recognition of the teaching work in the childhood education interferes with the health of the teachers. For this purpose, it sought to understand the changes in the context of childhood education and the factors that produce pleasure and suffering due to the work environment. The research was carried out in a municipality in the countryside of Rio Grande do Sul, with eight teachers from the first stage of public education, through semi-structured interviews. The methodology used was an adaptation of the Psychodynamics of Work Stricto Sensu. Teaching work in childhood education has undergone changes, its organization included. Some teachers had to assume the pedagogical responsibility of more than one class, which has led to an increase in the workload. In addition, they have little time with the children and the main activity refers to tasks distribution. In this sense, the dynamic of contribution and recognition of the professionals is affected. The acknowledgment is essential so that the work has a meaning for the subject. For the researched teachers, it comes from the children with whom they work, manifested through affection.

Efficacy of methyl nonyl ketone as an in-soil repellent for common voles (Microtus arvalis).

BACKGROUND: Common voles (Microtus arvalis) can cause enormous damage in agriculture. Tests were conducted using an alternative approach to rodenticide-based vole management by developing an in-soil odour repellent based on the secondary plant metabolite methyl nonyl ketone (MNK). Replicated 25 m(2) plots were established in forage grassland to test efficacy and application schemes using a foam application that included MNK. RESULTS: MNK significantly reduced the number of reopened burrow entrances in all field trials compared with untreated control plots, with repeated application for 6 days reducing vole activity by up to 90%. The addition of black pepper oil (BPO) did not enhance the efficacy of the MNK foam. Voles tended to avoid burrows where MNK was applied and rather opened new burrows instead of reopening the treated burrow entrances. The foam application scheme led to a repellent effect that lasted for about 4-5 days. A single additional application of MNK after 4 days can extend the treatment effects for a further 2 days. CONCLUSION: The results indicate that MNK has the potential to repel common voles in the field. However, more studies are needed to enhance the duration of a one-off treatment effect to yield a user-friendly field application to manage vole populations.

The repelling effect of plant secondary metabolites on water voles, Arvicola amphibius.

BACKGROUND: Water voles (Arvicola amphibius Linnaeus 1758) are abundant in most parts of Germany and other European countries. They are known to cause serious damage in fruit and horticulture as well as in agriculture. Currently available repellents, scaring devices and household remedies are mostly inefficient. Tests were conducted to establish whether water voles can be repelled using plant secondary metabolites. These compounds are produced by many plant species as part of their defence against herbivores and pathogens. RESULTS: In this study, 12 volatile substances were tested in T-maze trials. The voles could choose between a test box including a test substance and a control box without odour. The extracts were considered to be repellent if the test box was avoided. Five potential repellents were identified: the essential oils of black pepper oil, Chinese geranium oil and onion, as well as the pure substances methyl nonyl ketone and n-valeric acid. Application of a combination of black pepper oil, Chinese geranium oil and methyl nonyl ketone did not increase efficacy. CONCLUSION: The identification of an effective water vole repellent could help to reduce damage to crops. It may also minimise the use of kill traps and of rodenticides, which will be of benefit for non-target organisms.

The ergonomic process of an automotive company in Brazil: a study case.

The goal of this paper is to present the ergonomic process of an automotive company, whose focus is on the adaptation of the work conditions to the psychophysiological characteristics of its employees. The planning and the development of the ergonomic actions took place in three distinctive stages: ergonomic analysis of the work post (stage 1), ergonomic adaptations (stage 2) and Ergonomic Committee (stage 3). The activities started in June 2006 and have lasted to the current date, keeping a permanent improving process. The procedure adopted was based on the ergonomic analysis methodology proposed by Wisner (1994:1997) and the stages of the Ergonomic Analysis of Work presented in the 17 NR Regulatory Manual (MET, 2002). The paper's approach focused on the voluntary participation of workers from different areas and different hierarchical levels of the organization throughout all the stages of the process. The methodological procedures included descriptive research techniques, exploratory and qualitative research criteria, background and guidelines available in literature and legislation, as well as company information. Among the main results it can be mentioned the satisfaction of the employees regarding the appropriate work conditions, cultural and organizational changes and the creation of an Ergonomic Committee in the company.

The tegument protein UL71 of human cytomegalovirus is involved in late envelopment and affects multivesicular bodies.

Morphogenesis of human cytomegalovirus (HCMV) is still only partially understood. We have characterized the role of HCMV tegument protein pUL71 in viral replication and morphogenesis. By using a rabbit antibody raised against the C terminus of pUL71, we could detect the protein in infected cells, as well as in virions showing a molecular mass of approximately 48 kDa. The expression of pUL71, detected as early as 48 h postinfection, was not blocked by the antiviral drug foscarnet, indicating an early expression. The role of pUL71 during virus replication was investigated by construction and analysis of a UL71 stop mutant (TBstop71). The mutant could be reconstituted on noncomplementing cells proving that pUL71 is nonessential for virus replication in human fibroblasts. However, the inhibition of pUL71 expression resulted in a severe growth defect, as reflected by an up to 16-fold reduced extracellular virus yield after a high-multiplicity infection and a small-plaque phenotype. Ultrastructural analysis of cells infected with TBstop71 virus revealed an increased number of nonenveloped nucleocapsids in the cytoplasm, many of them at different stages of envelopment, indicating that final envelopment of nucleocapsids in the cytoplasm was affected. In addition, enlarged multivesicular bodies (MVBs) were found in close proximity to the viral assembly compartment, suggesting that pUL71 affects MVBs during virus infection. The observation of numerous TBstop71 virus particles attached to MVB membranes and budding processes into MVBs indicated that these membranes can be used for final envelopment of HCMV.

Conformational changes specific for pseudophosphorylation at serine 262 selectively impair binding of tau to microtubules.

Aggregation of the microtubule-associated protein tau into neurofibrillary tangles is the pathological hallmark of a variety of dementias. For reasons not yet known, tau becomes excessively phosphorylated in Alzheimer's brains and as a result no longer binds properly to microtubules. Here we studied the impact of phosphorylation on the conformational and binding properties of the repeat region of tau (K18) that is necessary for microtubule assembly and forms the core of paired helical filaments. To mimic phosphorylation, we introduced four mutations of serine to glutamate residues at positions 262, 293, 324, and 356. NMR spectroscopy demonstrates that pseudophosphorylation at these sites modifies the structural properties in repeats 1 and 2, in particular for Gln265-Lys267. Gln265-Lys267 are in close proximity to Ser262, the phosphorylation site that most strongly attenuates binding to microtubules. In contrast, the pseudophosphorylation mimic of tau efficiently interacts with the polyanion heparin. Thus, phosphorylation of the repeat region of natively unfolded tau induces specific conformational changes that have a strong impact on its biological function and involvement in disease.

Opposed steric constraints in human DNA polymerase beta and E. coli DNA polymerase I.

DNA polymerase selectivity is crucial for the survival of any living species, yet varies significantly among different DNA polymerases. Errors within DNA polymerase-catalyzed DNA synthesis result from the insertion of noncanonical nucleotides and extension of misaligned DNA substrates. The substrate binding characteristics among DNA polymerases are believed to vary in properties such as shape and tightness of the binding pocket, which might account for the observed differences in fidelity. Here, we employed 4'-alkylated nucleotides and primer strands bearing 4'-alkylated nucleotides at the 3'-terminal position as steric probes to investigate differential active site properties of human DNA polymerase beta (Pol beta) and the 3'-->5'-exonuclease-deficient Klenow fragment of E. coli DNA polymerase I (KF(exo-)). Transient kinetic measurements indicate that both enzymes vary significantly in active site tightness at both positions. While small 4'-methyl and -ethyl modifications of the nucleoside triphosphate perturb Pol beta catalysis, extension of modified primer strands is only marginally affected. Just the opposite was observed for KF(exo-). Here, incorporation of the modified nucleotides is only slightly reduced, whereas size augmentation of the 3'-terminal nucleotide in the primer reduces the catalytic efficiency by more than 7000- and 260,000-fold, respectively. NMR studies support the notion that the observed effects derive from enzyme substrate interactions rather than inherent properties of the modified substrates. These findings are consistent with the observed differential capability of the investigated DNA polymerases in fidelity such as processing misaligned DNA substrates. The results presented provide direct evidence for the involvement of varied steric effects among different DNA polymerases on their fidelity.

Fully automated quantification of hepatitis C virus (HCV) RNA in human plasma and human serum by the COBAS AmpliPrep/COBAS TaqMan system.

BACKGROUND: HCV RNA is commonly recognized as key parameter for reliable diagnosis and treatment monitoring of HCV infection. Determination of blood HCV RNA concentrations reduces the pre-seroconversion period in the diagnosis of HCV infection and supports management of interferon alpha-based therapies of chronic HCV infection. OBJECTIVES AND STUDY DESIGN: The COBAS AmpliPrep/COBAS TaqMan HCV Test combines automated extraction of nucleic acids on the COBAS AmpliPrep Instrument with real-time PCR on the COBAS TaqMan Analyzer, thus greatly reducing hands-on time during sample preparation and amplification/detection. The test, which is calibrated to the 1st International HCV WHO Standard, was evaluated for sensitivity, dynamic range, precision, matrix equivalence, genotype inclusivity, interfering substances, diagnostic and analytical specificity, as well as for correlation with two other commercial tests for HCV RNA quantification. RESULTS: The COBAS AmpliPrep/COBAS TaqMan HCV Test demonstrated a >6-log dynamic range of 43-6.90 E+7 IU/mL, a sensitivity (95% hit rate) of at least 15 IU/mL for HCV WHO Standard and a comparable quantification of genotypes 1-6. HCV quantification results were in good correlation with those obtained by the COBAS AMPLICOR HCV MONITOR Test v2.0 and the VERSANT HCV RNA 3.0 test. CONCLUSIONS: The fully automated COBAS AmpliPrep/COBAS TaqMan HCV Test excellently accomplishes the requirements for highly sensitive detection and reliable quantification of HCV in clinical samples and thus improves therapy monitoring and management of HCV infection.

Structural and microtubule binding properties of tau mutants of frontotemporal dementias.

Several mutations in the gene encoding the microtubule-associated protein tau are responsible for the formation of neurofibrillary inclusions in frontotemporal dementia with Parkinsonism linked to chromosome 17 (FTDP-17). Here we present the high-resolution characterization of the conformational properties of two FTDP-17 mutants of the four-repeat domain of tau, P301L and DeltaK280, and their properties for binding to polyanions and microtubules. Multidimensional NMR spectroscopy shows that the mutations do no lead to a significant increase in the level of beta-structure in their monomeric state, even though the mutations strongly promote beta-structure during aggregation. However, local structural changes are induced in the second repeat. These changes only weakly affect the binding to the polyanion heparin, which promotes paired helical filament formation. The extent of binding to microtubules, however, is strongly decreased. Our results demonstrate that the reversible binding of tau to microtubules involves specific interactions, which are not essential for binding to polyanions.

The "jaws" of the tau-microtubule interaction.

Tau is the major microtubule-associated protein in neuronal axons. It aggregates into "neurofibrillary tangles" during the course of Alzheimer disease. Binding to microtubules and microtubule assembly requires the "repeat domain" in the C-terminal half of Tau, as well as the two regions flanking the repeats. Here we report the NMR characterization of a 198-residue Tau fragment composed of the four tandem repeats and the flanking domains and containing the full microtubule binding and assembly activity of Tau. NMR secondary chemical shifts and dipolar couplings detect the highest propensity for beta-structure within the four-repeat region, whereas the flanking domains are largely random coil, with an increased rigidity in the proline-rich region. Chemical shift perturbation experiments identify two motifs in the upstream flanking domain, (225)KVAVVRT(231) and (243)LQTA(246), and one downstream of the repeats, (370)KIETHKTFREN(380), which strongly contribute to the binding to the acidic outside of microtubules, as well as to the binding of other polyanions such as heparin. This is consistent with the "jaws" model of Tau-microtubule interactions and highlights the importance of the regions flanking the repeats for both microtubule binding and pathological Tau aggregation.

Occurrence of glucosylsucrose [alpha-D-glucopyranosyl- (1-->2)-alpha-D-glucopyranosyl-(1-->2)-beta-D-fructofuranoside] and glucosylated homologues in cyanobacteria. Structural properties, cellular contents and possible function as thermoprotectants.

Little is known about the structure and function of oligosaccharides in cyanobacteria. In this study, a new class of saccharides from Nostoc was identified by MS and NMR techniques, consisting of alpha-D-glucopyranosyl-(1-->2)-[alpha-D-glucopyranosyl-(1-->2)]n-beta-D-fructofuranosides ranging from the trisaccharide (n = 1) to decasaccharide (n = 8). In Nostoc ellipsosporum the cell content of saccharides increased 10-20-fold after heat stress (1 day, 40 degrees C) or during prolonged cultivation. Under these conditions the abundance of homologues of higher molecular mass (> pentasaccharide) increased and finally exceeded that of homologues of lower molecular mass including sucrose. Total intracellular content of the saccharides after heat stress was 5-10 mg x (g dry weight)(-1) corresponding to intracellular concentrations of 0.25-0.5% (w/v). A possible role of the oligosaccharides identified is in the protection of enzymes against heat inactivation. Whereas amylase from Nostoc was only weakly protected by the decasaccharide, alpha-amylase from porcine pancreas was more efficiently stabilized by the octasaccharide and decasaccharide. Evidence is presented for the widespread occurrence of the newly identified saccharides in cyanobacteria. The results are discussed including previous reports on cyanobacterial oligosaccharides and with respect to possible functions of these compounds in the living cell.