A Grp on the Hsp90 mechanism

In a recent issue of Molecular Cell, Dollins et al. (2007) present the crystal structure of Grp94, which highlights the similarity between Grp94 and Hsp90 and provides insight into the resting state of Grp94 and potentially other Hsp90 family members.     

The ATPase cycle of the endoplasmic chaperone Grp94

Grp94, the Hsp90 paralog of the endoplasmic reticulum, plays a crucial role in protein secretion. Like cytoplasmic Hsp90, Grp94 is regulated by nucleotide binding to its N-terminal domain. However, the question of whether Grp94 hydrolyzes ATP was controversial. This sets Grp94 apart from other members of the Hsp90 family where a slow but specific turnover of ATP has been unambiguously established. In this study we aimed at analyzing the nucleotide binding properties and the potential ATPase activity of Grp94. We show here that Grp94 has an ATPase activity comparable with that of yeast Hsp90 with a k(cat) of 0.36 min(-1) at 25 degrees C. Kinetic and equilibrium constants of the partial reactions of the ATPase cycle were determined using transient kinetic methods. Nucleotide binding appears to be tighter compared with other Hsp90s investigated, with dissociation constants (K(D)) of approximately 4 microm for ADP, ATP, and AMP-PCP. Interestingly, all nucleotides and inhibitors (radicicol, 5'-N-ethylcarboxamidoadenosine) studied here bind with similar rate constants for association (0.2-0.3 x 10(6) M(-1) s(-1)). Furthermore, there is a marked difference from cytosolic Hsp90s in that after binding, the ATP molecule does not seem to become trapped by conformational changes in Grp94. Grp94 stays predominantly in the open state concerning the nucleotide-binding pocket as evidenced by kinetic analyses. Thus, Grp94 shows mechanistically important differences in the interaction with adenosine nucleotides, but the basic hydrolysis reaction seems to be conserved between cytosolic and endoplasmic members of the Hsp90 family.     

Influence of the internal disulfide bridge on the folding pathway of the CL antibody domain

Disulfide bridges are one of the most important factors stabilizing the native structure of a protein. Whereas the basis for their stabilizing effect is well understood, their role in a protein folding reaction still seems to require further attention. We used the constant domain of the antibody light chain (C(L)), a representative of the ubiquitous immunoglobulin (Ig)-superfamily, to delineate the kinetic role of its single buried disulfide bridge. Independent of its redox state, the monomeric C(L) domain adopts a typical Ig-fold under native conditions and does not retain significant structural elements when unfolded. Interestingly, its folding pathway is strongly influenced by the disulfide bridge. The more stable oxidized protein folds via a highly structured on-pathway intermediate, whereas the destabilized reduced protein populates a misfolded off-pathway species on its way to the native state. In both cases, the formation of the intermediate species is shown to be independent of the isomerization state of the Tyr(141)-Pro(142) bond. Our results demonstrate that the internal disulfide bridge in an antibody domain restricts the folding pathway by bringing residues of the folding nucleus into proximity thus facilitating the way to the native state.     

Cooking and Season as Risk Factors for Acute Lower Respiratory Infections in African Children: A Cross-Sectional Multi-Country Analysis

Background

Acute lower respiratory infections (ALRI) are a leading cause of death among African children under five. A significant proportion of these are attributable to household air pollution from solid fuel use.

Methods

We assessed the relationship between cooking practices and ALRI in pooled datasets of Demographic and Health Surveys conducted between 2000 and 2011 in countries of sub-Saharan Africa. The impacts of main cooking fuel, cooking location and stove ventilation were examined in 18 (n = 56,437), 9 (n = 23,139) and 6 countries (n = 14,561) respectively. We used a causal diagram and multivariable logistic mixed models to assess the influence of covariates at individual, regional and national levels.

Results

Main cooking fuel had a statistically significant impact on ALRI risk (p<0.0001), with season acting as an effect modifier (p = 0.034). During the rainy season, relative to clean fuels, the odds of suffering from ALRI were raised for kerosene (OR 1.64; CI: 0.99, 2.71), coal and charcoal (OR 1.54; CI: 1.21, 1.97), wood (OR 1.20; CI: 0.95, 1.51) and lower-grade biomass fuels (OR 1.49; CI: 0.93, 2.35). In contrast, during the dry season the corresponding odds were reduced for kerosene (OR 1.23; CI: 0.77, 1.95), coal and charcoal (OR 1.35; CI: 1.06, 1.72) and lower-grade biomass fuels (OR 1.07; CI: 0.69, 1.66) but increased for wood (OR 1.32; CI: 1.04, 1.66). Cooking location also emerged as a season-dependent statistically significant (p = 0.0070) determinant of ALRI, in particular cooking indoors without a separate kitchen during the rainy season (OR 1.80; CI: 1.30, 2.50). Due to infrequent use in Africa we could, however, not demonstrate an effect of stove ventilation.

Conclusions

We found differential and season-dependent risks for different types of solid fuels and kerosene as well as cooking location on child ALRI. Future household air pollution studies should consider potential effect modification of cooking fuel by season.     

GTP-binding proteins in bovine brain nuclear membranes.

Nuclear membranes and other subcellular fractions derived from bovine brain cortex were investigated for the existence of GTP-binding proteins. By using photolytic labeling with [alpha-32P]GTP a 29 kDa GTP-binding protein was shown to be present in nuclear membranes which was not present in the plasma membranes nor in microsomal or cytosolic fractions. Two-dimensional gel electrophoresis revealed that this protein is rather acidic with a pI lower than 4.5. Members of the heterotrimeric Gi/o family are not present in the nuclear envelope: a 39 kDa protein, ADP ribosylated by pertussis toxin, was shown to originate from plasma membrane contamination.     

Cytokine gene activation in synovial membrane, regional lymph nodes, and spleen during the course of rat adjuvant arthritis.

Cytokine gene activation was assessed during rat adjuvant arthritis (AA) in synovial membrane (SM), popliteal lymph node (popl-LN), and spleen, using semiquantitative, competitive RT-PCR. Changes in the popl-LN were considerably higher than in spleen or SM. In the preclinical phase (day 6), cytokine mRNA elevations occurred exclusively in the popl-LN and included IFN-gamma, IL-1beta, IL-5, IL-6, and IL-10. In the acute phase (days 13-16) all three organs became involved: (i) in the SM, significant elevations were limited to IL-1beta and IL-6, which, notably, correlated positively with the degree of arthritis; (ii) in the popl-LN, IFN-gamma, IL-1beta, IL-6, and IL-10 (but not IL-5) were still elevated, while IL-2 rose significantly; (iii) in the spleen, TNF-alpha peaked simultaneously with the arthritis score (day 16) and dramatically dropped thereafter. Upon transition into the chronic phase (day 20) the following phenomena were observed: (i) IL-1beta and IL-6 were still significantly increased in the SM; (ii) IFN-gamma, IL-1beta, IL-2, IL-6, and IL-10 were still elevated in the popl-LN; and (iii) there was a progressive rise of IL-5 mRNA in the spleen, positively correlated with the arthritis score. In conclusion, cytokines with pro- and anti-inflammatory functions overlap throughout disease, but in different organ-related patterns. Local (SM) and regional (popl-LN) IL-1beta and IL-6, elevated throughout the entire course of AA, may directly contribute to disease severity. While in AA spleen TNF-alpha appears to be a systemic marker of acute disease, spleen IL-5 may be involved in disease resolution.     

Leaf rosette closure in the alpine rock species Saxifraga paniculata Mill.: significance for survival of drought and heat under high irradiation

The significance of leaf rosette closure for survival of drought and heat under high irradiation on alpine rock sites was investigated in the cushion forming rosette plant, Saxifraga paniculata Mill. With decreasing water content the leaves fold over the rosette centre reducing reversibly the evaporative leaf surface area by 80%. Internal water redistribution driven by an osmotic gradient from older to younger leaves occurs. The oldest leaves dry out to promote the survival of the individual. Leaf temperatures above 45 °C (which match heat tolerance limits 45–57 °C; LT₅₀) co-occurred with low substrate water potentials (less than – 0·5 MPa) on 11·3% of summer days. Shading by leaves can be crucial to surviving high temperatures as it keeps the rosette centre up to 10 °C colder. Mutual shading prevented sustained drought-induced photoinhibition in upper leaf surfaces at relative water contents below 60%. In exposed lower leaf surfaces restoration of photosystem II took several days. Leaf temperatures above 40 °C (21·3% of summer days) induced photoinhibition in situ. Periods with sufficient water supply can be fully utilized as rehydration is fast ( < 12 h) and exposes the upper leaf surfaces that showed only minor photoinhibition. By reversible leaf rosette closure environmental extremes that otherwise could exceed tolerance are efficiently avoided.     

Elementary movement detectors in an insect visual system

In the theoretical part of the present work the input-output relation for a multi-input system is developed into a functional power series. This is formally equivalent to a decomposition of the system into a sum of all possible combinations of 1-, 2-, 3-... input subsystems. The average response of the system to a uniformly moving patern is known to be a Fourier series with respect to spatial frequency. The coefficients of the series are linear combinations of the weights by which different subsystems contribute to the total reaction. If a system can be shown to have essential nonlinearities of no higher than second order it is possible to calculate, from a Fourier analysis of the average movement response, the weight by which the nonlinear interaction between any two input elements contributes to the total reaction. This interaction is termed elementary movement detector. By the analysis presented here the arrangement of the elementary movement detectors may be determined for a two-dimensional array of input elements and the strength of their contributions to the total movement reaction may be calculated. Special experimental methods have been developed which allow one to apply this analysis to the visual system of the fruitfly Drosophila. The preliminary data presented show that the direction sensitive optomotor response of Drosophila can be attributed predominantly to the contributions from two elementary movement detectors which interconnect neighbouring visual elements. The detectors are oriented in the hexagonal array of the compound eye at +30° and at-30° with respect to the horizontal line of symmetry. A weak contribution from a detector between neighbouring elements along the horizontal line of symmetry is suggested by the present data. In the course of the analysis the contrast transfer properties of the compound eye are characterized.     

Evidence for one-way movement detection in the visual system of Drosophila

Optomotor control of course and altitude in the fruitfly, Drosophila melanogaster, requires dense networks of elementary movement detectors (EMD's) which cover most if not all of the visual field. The predominant types of EMD's in these networks represent interactions between neighbouring visual elements along the three main directions of the hexagonal array in the compound eye. — Course control in the walking fly is achieved mainly by pairs of equivalent EMD's which occupy 2 o'clock and 4 o'clock positions with respect to the right eye (Buchner, 1976). Comparison of the turning response and the torque response in the present account confirms the particular properties of this network, and proves the presumed bidirectional sensitivity of its EMD's for the course control responses of legs and wings in the corresponding modes of locomotion. — Altitude control during flight is achieved by a less homogeneous network of EMD's which modifies lift and thrust simultaneously by the appropriate control of the wing beat amplitudes. The predominant types of EMD's in the lateral eye regions occupy 12 o'clock and 2 o'clock positions with respect to the right eye (Buchner et al., 1978). The present evaluation of the optomotor responses of thrust and wing beat confirms the preferred orientation of these EMD's and discloses a pecularity of their internal structure. The movement detectors of this network lack the bidirectional sensitivity of the EMD's in the course control system. At least the fronto-lateral network of the altitude control system seems to consist mainly of pairs of equivalent unidirectional EMD's. The detectors in 12 o'clock position increase wing beat in response to movement of the visual surroundings from inferior to superior. The opposite effect is produced by the detectors in 2 o'clock position which respond to movement from anterior-superior to posterior-inferior. These properties qualify unidirectional EMD's as the functional units of the optomotor control system in the fruitfly. Pairs of unidirectional antagonists would be sufficient to establish the bidirectional sensitivity found in the movement detectors of the course control system.