Analysis of Chaperone Function and Formation of Hetero-oligomeric Complexes of Hsp18.1 and Hsp17.7, Representing Two Different Cytoplasmic sHSP Classes in Pisum sativum

Small heat shock proteins (sHSPs) are the most abundant stress proteins in plants. Usually not expressed under permissive conditions, they can accumulate to more than 2% of the total cellular protein content during heat stress. At present several points of evidence indicate that these proteins act as molecular chaperones by keeping partially denatured proteins in a folding-competent state. In plants sHSPs are encoded by a multigene family, which can be segregated into several classes according to their subcellular position and/or sequence homology. Curiously, two different classes appear in the cytoplasm. Their specific role during heat shock remains elusive. Here we present some evidence that both classes of sHSPs enhance recovery of reporter protein activity in the presence of HSP70. Applying peptide arrays prepared by SPOT synthesis and in situ analysis by confocal laser scanning microscopy, we could further show that the two classes of sHSP are attached to each other and are able to interact with non-native proteins both in vivo and in vitro. Although both of the sHSPs act similarly as molecular chaperones, immunohistochemistry experiments support the hypothesis that the two have different cellular functions in the development of heat-induced cytoplasmic heat shock granules under elevated temperatures. Daniela Wagner Deceased 24 Feburary 2004.     

Mesozoic calcareous nannofossils — state of the art

Calcareous nannofossils originated in the Triassic, radiated in the Jurassic and became a dominant component of the marine biosphere from the earliest Jurassic onward. They can be considered as one of the most important “innovations” of the Mesozoic oceans. Their basic morphology allows the differentiation of three different groups: coccoliths, nannoliths and calcispheres (= calcareous dinocysts). Only coccoliths and nannoliths are discussed in this article in some detail. Coccoliths and nannoliths have contributed greatly in the Interpretation of Mesozoic marine Systems through biostratigraphy and palaeoecology/palaeoceanography. Ever since the late 1960s both coccoliths and nannoliths have proven to be useful and reliable zonal markers for biostratigraphic schemes, allowing detailed zonations for the Jurassic and Cretaceous. Though affected by palaeobiogeographic provincialism, coccoliths and nannoliths have supplied many cosmopolitan biostratigraphic markers. These allow a global correlation of marine sedimentary units both from onshore sections in the classical European and North American areas and pelagic sequences recovered in the course of the DSDP/ODP drilling from the world’s oceans. Thus research on calcareous nannofossils Covers both, regional and global aspects. Research in the last 15 years concentrated on palaeoecological aspects. Apart from dinoflagellates, coccolithophores were the most important primary producers in Mesozoic oceans. As such they heavily relied on autecological factors such as light, nutrients and temperature. Variations in the assemblage composition of these groups may thus be viewed as a key for understanding palaeoecological, palaeoceanographic and palaeoclimatic changes of the past.      

Facies and faunal assemblage changes in response to the Holocene transgression in the Lagoon of Mayotte (Comoro Archipelago, SW Indian Ocean)

This paper documents the facies change in response to the Holocene transgression within five sediment cores taken in the lagoon of Mayotte, which contain a Type-1 depositional sequence (lowstand, transgressive and highstand deposits underlain by an erosive sequence boundary). Quantitative compositional analysis and visual examination of the bioclasts were used to document the facies changes. The distribution of the skeletal and non-skeletal grains in the lagoon of Mayotte is clearly controlled by (1) the rate and amplitude of the Holocene sea-level rise, (2) the pre-Holocene basement topography and (3) the growth-potential of the barrier reef during sea-level rise, and the changes in bathymetry and continuity during this period. The sequence boundary consists of the glacial karst surface. The change-over from the glacial lowstand is marked by the occurrence of mangrove deposits. Terrigenous and/or mixed terrigenous-carbonate muds to sandy muds with a mollusc or mollusc-ostracod assemblage dominate the transgressive deposits. Mixed carbonate-siliciclastic or carbonate sand to gravel with a mollusc-foraminifer or mollusc-coral-foraminifer assemblage characterize the early highstand deposits on the inner lagoonal plains. The early highstand deposits in the outer lagoonal plains consist of carbonate muds with a mollusc-foraminifer assemblage. Late highstand deposits consist of terrigenous muds in the nearshore bays, mixed terrigenous-carbonate sandy muds to sands with a mollusc-foraminifer assemblage on the inner lagoonal plains and mixed muds with a mollusc-foraminifer assemblage on the outer deep lagoonal plains. The present development stage of the individual lagoons comprises semi-enclosed to open lagoons with fair or good water exchange with the open ocean.     

Physiology and Phylogeny of Green Sulfur Bacteria Forming a Monospecific Phototrophic Assemblage at a Depth of 100 Meters in the Black Sea

The biomass, phylogenetic composition, and photoautotrophic metabolism of green sulfur bacteria in the Black Sea was assessed in situ and in laboratory enrichments. In the center of the western basin, bacteriochlorophyll e (BChl e) was detected between depths of 90 and 120 m and reached maxima of 54 and 68 ng liter⁻¹. High-pressure liquid chromatography analysis revealed a dominance of farnesyl esters and the presence of four unusual geranyl ester homologs of BChl e. Only traces of BChl e (8 ng liter⁻¹) were found at the northwestern slope of the Black Sea basin, where the chemocline was positioned at a significantly greater depth of 140 m. Stable carbon isotope fractionation values of farnesol indicated an autotrophic growth mode of the green sulfur bacteria. For the first time, light intensities in the Black Sea chemocline were determined employing an integrating quantum meter, which yielded maximum values between 0.0022 and 0.00075 μmol quanta m⁻² s⁻¹ at the top of the green sulfur bacterial layer around solar noon in December. These values represent by far the lowest values reported for any habitat of photosynthetic organisms. Only one 16S rRNA gene sequence type was detected in the chemocline using PCR primers specific for green sulfur bacteria. This previously unknown phylotype groups with the marine cluster of the Chlorobiaceae and was successfully enriched in a mineral medium containing sulfide, dithionite, and freshly prepared yeast extract. Under precisely controlled laboratory conditions, the enriched green sulfur bacterium proved to be capable of exploiting light intensities as low as 0.015 μmol quanta m⁻² s⁻¹ for photosynthetic ¹⁴CO₂ fixation. Calculated in situ doubling times of the green sulfur bacterium range between 3.1 and 26 years depending on the season, and anoxygenic photosynthesis contributes only 0.002 to 0.01% to total sulfide oxidation in the chemocline. The stable population of green sulfur bacteria in the Black Sea chemocline thus represents the most extremely low-light-adapted and slowest-growing type of phototroph known to date.     

Efficacy and safety of insulin glulisine in patients with type 1 diabetes.

Insulin glulisine (glulisine), a human insulin analogue with a rapid-acting time-action profile, has been developed to fulfil the mealtime (bolus) insulin requirement in patients with diabetes. The aim of this multinational, multi-centre, controlled, open-label, randomized, parallel-group study was to compare the efficacy and safety of insulin glulisine (glulisine) to that of insulin lispro (lispro) in adults diagnosed with Type 1 diabetes. Of the 683 patients randomized, 672 received treatment (339 patients received glulisine, 333 patients received lispro). Over the 26-week study, a similar reduction in mean HbA1c occurred in both groups (adjusted mean change from baseline -0.14% in both groups). The basal insulin dose was relatively unchanged from baseline in the glulisine group but increased in the lispro group (glulisine: 0.12 IU vs. lispro: 1.82 IU; p = 0.0001). As a consequence, total daily insulin dose decreased in the glulisine group but increased in the lispro group (glulisine: -0.86 IU vs. lispro: 1.01 IU; p = 0.0123). There was no relevant difference between the two groups in the reporting of symptomatic hypoglycaemia (overall, nocturnal and severe). This study demonstrates that glulisine provides equivalent glycaemic control to lispro. The clinical relevance of any difference in total daily insulin dose remains to be established.     

Purification and enzymological characterization of murine neurotrypsin

An increasing number of studies indicate that serine proteases play an important role in structural plasticity associated with learning and memory formation. Neurotrypsin is a multidomain serine protease located at the presynaptic terminal of neurons. It is thought to be crucial for cognitive brain functions. A deletion in the neurotrypsin gene causes severe mental retardation in humans. For a biochemical characterization, we produced murine neurotrypsin recombinantly in a eukaryotic expression system using myeloma cells. From the culture medium we purified neurotrypsin using heparin-, hydrophobic interaction- and immobilized metal affinity chromatography. For an enzymological characterization two fragments of agrin containing the natural cleavages sites of neurotrypsin were used as substrates. The highest catalytic activity of neurotrypsin was observed in the pH range between 7.0 and 8.5. Calcium ions were required for neurotrypsin activity and an ionic strength exceeding 500 mM decreased substrate cleavage. Site-specific mutations of the amino acids flanking the scissile bonds showed that cleavage is highly specific and requires a basic amino acid preceded by a glutamate residue on the N-terminal side of the scissile bond. This sequence requirement argues for a unique substrate binding pocket of neurotrypsin. This observation was further substantiated by the fact that almost all tested serine protease inhibitors except dichloroisocoumarin and PMSF did not affect neurotrypsin activity.     

Neurochemical approaches of cerebrospinal fluid diagnostics in neurodegenerative diseases

Alzheimer's disease (AD), Parkinson's disease dementia (PDD)/Lewy-body disease (DLB), and frontotemporal dementia (FTD) are the major causes of memory impairment and dementia. As new therapeutic agents are visible for the different diseases, there is an ultimate need for an early and an early differential diagnosis. Since cerebrospinal fluid (CSF) is in direct contact with the central nervous system (CNS), potentially promising biomarkers might be seen there first. In principle, two research approaches can be considered for the laboratory diagnosis of dementias: (i) the direct detection of disease specific protein like Abeta-peptide-oligomers in AD or alpha-synuclein-aggregates in DLB and (ii) the detection of surrogate markers that show an altered pattern of expression in early stages of the disease or are used in the differential diagnosis of other dementias and thus enable an exclusion diagnosis. Especially Abeta-peptides and tau-protein measurements seem to employ a combination of these approaches. Until now it was shown that a combined determination of just these few markers (tau-proteins and Abeta-peptides) is already sufficient to achieve a high degree of diagnostic certainty in the diagnosis of AD. However although these markers seem to correlate with neuropathological changes and memory disturbances, these markers are not specific for a single form of dementia and further research is necessary to improve especially the early differential diagnosis of dementias.     

Phylogenetic relationships, character evolution and biogeography of southern African members of Zygophyllum (Zygophyllaceae) based on three plastid regions

The plastid coding rbcL and non-coding trnLF regions of 53 of 55 southern African Zygophyllum species were sequenced and used to evaluate the phylogenetic relationships within the southern African representatives of the genus. Published sequences of the same gene regions of Australian, Asian and North African Zygophyllum species were included to assess the relationships of the species from these regions to the southern African species. The addition of Z. stapffii from Namibia, found to be conspecific with Z. orbiculatum from Angola, lead to a greatly resolved tree. The molecular results were largely congruent with a recent sectional classification of the southern African species and supported their subdivision into subgenera Agrophyllum and Zygophyllum. Reconstruction of the character evolution of capsule dehiscence, seed attachment and seed mucilage showed that these characters allowed a division of southern African species into the two subgenera but that this could not be applied to species occurring elsewhere. Other morphological characters were found to vary and unique character combinations, rather than unique characters, were found to be of systematic value in sectional delimitation. The study suggests that repeated radiations from the horn of Africa to southern Africa and Asia and back lead to the present distribution of the taxa in the subfamily Zygophylloideae. Although this study supports some of the recent taxonomic changes in the group, the unresolved relationships between the proposed genera Tetraena and Roepera and those retained as Zygophyllum species suggest that changes to the taxonomy may have been premature.