Nucleotide Binding by Lhs1p Is Essential for Its Nucleotide Exchange Activity and for Function in Vivo

Protein translocation and folding in the endoplasmic reticulum of Saccharomyces cerevisiae involves two distinct Hsp70 chaperones, Lhs1p and Kar2p. Both proteins have the characteristic domain structure of the Hsp70 family consisting of a conserved N-terminal nucleotide binding domain and a C-terminal substrate binding domain. Kar2p is a canonical Hsp70 whose substrate binding activity is regulated by cochaperones that promote either ATP hydrolysis or nucleotide exchange. Lhs1p is a member of the Grp170/Lhs1p subfamily of Hsp70s and was previously shown to function as a nucleotide exchange factor (NEF) for Kar2p. Here we show that in addition to this NEF activity, Lhs1p can function as a holdase that prevents protein aggregation in vitro. Analysis of the nucleotide requirement of these functions demonstrates that nucleotide binding to Lhs1p stimulates the interaction with Kar2p and is essential for NEF activity. In contrast, Lhs1p holdase activity is nucleotide-independent and unaffected by mutations that interfere with ATP binding and NEF activity. In vivo, these mutants show severe protein translocation defects and are unable to support growth despite the presence of a second Kar2p-specific NEF, Sil1p. Thus, Lhs1p-dependent nucleotide exchange activity is vital for ER protein biogenesis in vivo.     

Diversity,structure and dynamics of a gallery forest in central Brazil

The structure and dynamics of approximately 64 ha of undisturbed gallery forest were studied over six years. Trees from 31 cm gbh (c. 10 cm dbh) were measured every three years from 1985. They were in 151 (10×20 m) permanent plots in the Gama forest in the Federal District of Brazil. Natural regeneration (individuals under 31 cm gbh) was measured in subplots (of 2×2 m, 5×5 m and 10×10 m) within the 200 m² plots. The total tree flora (gbh31 cm) consisted of 93 species, 81 genera and 44 families in 1985. The Leguminosae, Myrtaceae and Rubiaceae were the families richest in number of species. Most individuals and species were under 45 cm diameter and 20 m high while the maximum diameter per species ranged from 30 to 95 cm. The density structure of trees and natural regeneration was similar, in which the densities of c. 80% of the species represented less than 1% of the total density. The periodic mean annual diameter increment for trees from 10 cm dbh, was c. 0.25 cm/year. Variability was high with coefficients of variation c. 100% or more. The Gama community may maintain tree diversity and structure in undisturbed conditions. Regeneration of c. 80% of the species was found in the establishing phase (poles); the diameter structure was typical of native forests with the number of individuals decreasing with increasing size classes and showing little change over the six years; recruitment compensated for the mortality of most of the abundant species. The soils in Gama gallery forest were dystrophic with high aluminium content. Multivariate analysis suggested the stream, natural gaps and edges as the main causes of floristic differentiation at the community level.     

Intermuscular adipose tissue-free skeletal muscle mass: estimation by dual-energy X-ray absorptiometry in adults.

Skeletal muscle (SM) is a large and physiologically important compartment. Adipose tissue is found interspersed between and within SM groups and is referred to as intermuscular adipose tissue (IMAT). The study objective was to develop prediction models linking appendicular lean soft tissue (ALST) estimates by dual-energy X-ray absorptiometry (DXA) with whole body IMAT-free SM quantified by magnetic resonance imaging. ALST and total-body IMAT-free SM were evaluated in 270 healthy adults [body mass index (BMI) of <35 kg/m(2)]. The SM prediction models were then validated by the leave-one-out method and by application in a new group of subjects who varied in SM mass [anorexia nervosa (AN), n = 23; recreational athletes, n = 16; patients with acromegaly, n = 7]. ALST alone was highly correlated with whole body IMAT-free SM [model 1: R(2) = 0.96, standard error (SE) = 1.46 kg, P < 0.001]; age (model 2: R(2) = 0.97, SE = 1.38 kg, P < 0.001) and sex and race (model 3: R(2) = 0.97, SE = 1.06 kg, both P < 0.001) added significantly to the prediction models. All three models validated in the athletes and patients with acromegaly but significantly (P < 0.01-0.001) over-predicted SM in the AN group as a whole. However, model 1 was validated in AN patients with BMIs in the model-development group range (n = 11; BMI of >16 kg/m(2)) but not in those with a BMI of <16 kg/m(2) (n = 12). The DXA-based models are accurate for predicting IMAT-free SM in selected populations and thus provide a new opportunity for quantifying SM in physiological and epidemiological investigations.     

Is there a PGM1 4 allele specific to Amerindian populations?

Summary Rare PGM₁ variants in Macushi and Wayampi Amerindian populations have been compared electrophoretically and by means of electrofocusing. They appear to be identical. The findings are discussed.     

Ultrastructure and lipid composition of etioplasts in developing dark-grown wheat leaves

Changes in the ultrastructure and lipid composition of etioplasts have been evaluated in three regions from the base to the tip of 8-day-old darkgrown wheat leaves and in the upper-2/3 region of etiolated leaves of different ages. In developing darkgrown tissues, the main morphological changes that etioplasts undergo consist of an increase in the amount of thylakoïds which, in the most mature etioplasts, align in parallel arrays. Concomitantly, galactolipids and sulfolipid form an increasing proportion of the total lipids. Trans-3-hexadecenoic acid was not detectable in phosphatidylglycerol (PG) of etioplasts showing appressed thylakoïds isolated from 5-day-old leaves, but was present in significant amounts in etioplasts in the basal part of 8-day-ols leaves in which membrane appression was barely visible. The proportions of this acid increase as etioplasts develop, reaching 25% of the PG fatty acids (1.2% of the total fatty acids) in the most differentiated etioplasts. In wheat etioplasts, it appears that trans-3-hexadecenoic acid may accumulate in considerable amounts in darkgrown tissues and that its accumulation is not directly involved in membrane appression.Abbreviations AP phosphatidic acid - DGDG digalactosyldiacylglycerol - MGDG monogalactosyldiacylglycerol - PC phosphatidylcholine - PE phosphatidylethanolamine - PG phosphatidylglycerol - PI phosphatidylinositol - PS phosphatidylserine - SL sulfolipid     

Single acetylcholine-activated channel currents in developing muscle cells

The properties of single acetylcholine-activated ion channels in developing rat myoblasts and myotubes in tissue culture have been investigated using the gigaohm seal patch clamp technique. Two classes of ACh-activated channels were identified. The major class of channels (accounting for >95% of all channel openings) has a conductance of 35 pS and a mean open time of 15 msec (at room temperature and ?80 mV). The minor class of channels has a larger conductance (55 pS) and a briefer mean open time (2–3 msec). Functional ACh-activated channels are present in undifferentiated mononucleated myoblasts 1–2 days in culture, although the channel density on such cells is low. Over the next week in culture, as the myoblasts fuse to form multinucleate myotubes, there is a marked increase in channel density and an increase in the proportion of large conductance channels. No significant change, however, occurs in channel conductance or open time (within a given class of channels) during this period. At high concentrations of ACh, channels desensitize and channel openings occur in groups, similar to what has been previously described in adult muscle. The rate of channel opening within a group of openings increases with increasing agonist concentration while mean open time is independent of agonist concentration, as expected from simple models of drug action. During a group of openings, the channel is open for half the time (i.e., channel opening rate is equal to channel closing rate) at a concentration of approximately 6 μm ACh.     

Empowering marine science through genomics

Marine scientists in Europe summarize their successes with genome technologies in the marine sciences and make a plea for a concerted international effort to raise greater public education for support.     

Combining evidence for association from transmission disequilibrium and case-control studies using single-nucleotide polymorphisms

The aim of the present analysis is to combine evidence for association from the two most commonly used designs in genetic association analysis, the case-control design and the transmission disequilibrium test (TDT) design. The cases here are affected offspring from nuclear families and are used in both the case-control and TDT designs. As a result, inference from these designs is not independent. We applied a simple logistic regression method for combining evidence for association from case-control and TDT designs to single-nucleotide polymorphism data purchased on a region on chromosome 3, replicate 1 of the Aipotu population. Combining the evidence from the case-control and TDT designs yielded a 5-10% reduction in the standard errors of the relative risk estimates. The authors did not know the results before the analyses were conducted.     

Expressed sequence tags from heat-shocked seagrass Zostera noltii (Hornemann) from its southern distribution range

Predicted global climate change threatens the distributional ranges of species worldwide. We identified genes expressed in the intertidal seagrass Zostera noltii during recovery from a simulated low tide heat-shock exposure. Five Expressed Sequence Tag (EST) libraries were compared, corresponding to four recovery times following sub-lethal temperature stress, and a non-stressed control. We sequenced and analyzed 7009 sequence reads from 30 min, 2 h, 4 h and 24 h after the beginning of the heat-shock (AHS), and 1585 from the control library, for a total of 8594 sequence reads. Among 51 Tentative UniGenes (TUGs) exhibiting significantly different expression between libraries, 19 (37.3%) were identified as ‘molecular chaperones’ and were over-expressed following heat-shock, while 12 (23.5%) were ‘photosynthesis TUGs’ generally under-expressed in heat-shocked plants. A time course analysis of expression showed a rapid increase in expression of the molecular chaperone class, most of which were heat-shock proteins; which increased from 2 sequence reads in the control library to almost 230 in the 30 min AHS library, followed by a slow decrease during further recovery. In contrast, ‘photosynthesis TUGs’ were under-expressed 30 min AHS compared with the control library, and declined progressively with recovery time in the stress libraries, with a total of 29 sequence reads 24 h AHS, compared with 125 in the control. A total of 4734 TUGs were screened for EST-Single Sequence Repeats (EST-SSRs) and 86 microsatellites were identified.