The force exerted by a single kinesin molecule against a viscous load.

Kinesin is a motor protein that uses the energy derived from the hydrolysis of ATP to power the transport of organelles along microtubules. To probe the mechanism of this chemical-to-mechanical energy transduction reaction, the movement of microtubules across glass surfaces coated with kinesin was perturbed by raising the viscosity of the buffer solution. When the viscosity of the solution used in the low density motility assay was increased approximately 100-fold through addition of polysaccharides and polypeptides, the longer microtubules, which experienced a larger drag force from the fluid, moved more slowly than the shorter ones. The speed of movement of a microtubule depended linearly on the drag force loading the motor. At the lowest kinesin density, where dilution experiments indicated that the movement was caused by a single kinesin molecule, extrapolation of the linear relationship yielded a maximum time-averaged drag force of 4.2 +/- 0.5 pN per motor (mean +/- experimental SE). The magnitude of the force argues against one type of "ratchet" model in which the motor is hypothesized to rectify the diffusion of the microtubule; at high viscosity, diffusion is too slow to account for the observed speeds. On the other hand, our data are consistent with models in which force is a consequence of strain developed in an elastic element within the motor; these models include a different "ratchet" model (of the type proposed by A. F. Huxley in 1957) as well as "power-stroke" models.     

Linkage analysis of sex determination in the honey bee (Apis mellifera)

A colony-level phenotype was used to map the major sex determination locus (designatedX) in the honey bee (Apis mellifera). Individual queen bees (reproductive females) were mated to single drones (fertile males) by instrumental insemination. Haploid drone progeny of an F1 queen were each backcrossed to daughter queens from one of the parental lines. Ninety-eight of the resulting colonies containing backcross progeny were evaluated for the trait low brood-viability resulting from the production of diploid drones that were homozygous atX. DNA samples from the haploid drone fathers of these colonies were used individually in polymerase chain reactions (PCR) with 10-base primers. These reactions generated random amplified polymorphic DNA (RAPD) markers that were analyzed for cosegregation with the colony-level phenotype. One RAPD marker allele was shared by 22 of 25 drones that fathered low brood-viability colonies. The RAPD marker fragment was cloned and partially sequenced. Two primers were designed that define a sequence-tagged site (STS) for this locus. The primers amplified DNA marker fragments that cosegregated with the original RAPD marker. In order to more precisely estimate the linkage betweenX and the STS locus, another group of bees consisting of progeny from one of the low-brood viability colonies was used in segregation analysis. Four diploid drones and 181 of their diploid sisters (workers, nonfertile females) were tested for segregation of the RAPD and STS markers. The cosegregating RAPD and STS markers were codominant due to the occurrence of fragment-length alleles. The four diploid drones were homozygous for these markers but only three of the 181 workers were homozygotes (recombinants). Therefore the distance betweenX and the STS locus was estimated at 1.6 cM. An additional linked marker was found that was 6.6 cM from the STS locus.     

Role of Oxygen in the Limitation and Inhibition of Nitrogenase Activity and Respiration Rate in Individual Soybean Nodules

Although infected cell O2 concentration (Oi) is known to limit respiration and nitrogenase activity in legume nodules, techniques have not been available to measure both processes simultaneously in an individual legume nodule. Consequently, details of the relationship between nitrogenase activity and Oi are not fully appreciated. For the present study, a probe was designed that allowed open circuit measurements of H2 evolution (nitrogenase activity) and CO2 evolution (respiration rate) in a single attached soybean nodule while simultaneously monitoring fractional oxygenation of leghemoglobin (and thereby Oi) with a nodule oximeter. Compared to measurements of whole nodulated roots, use of the probe led to inhibition of nitrogenase activity in the single nodules. During oximetry measurements, total nitrogenase activity (TNA; peak H2 evolution in Ar/O2) in the single nodules was 16% of that in whole nodulated roots and 48% of nodulated root activity when Oi was not being measured simultaneously. This inhibition did not affect the nodules' ability to regulate Oi, because exposure to Ar/O2 (80:20, v/v) caused nitrogenase activity and respiration rate to decline, and this decline was linearly correlated with a concurrent decrease in Oi. When the nodules were subsequently exposed to a linear increase in external pO2 from 20 to 100% O2 at 2.7% O2/min, fractional leghemoglobin oxygenation first increased gradually and then more rapidly, reaching saturation at a pO2 between 76 and 100% O2. Plots of nitrogenase activity and respiration rate against Oi showed that rates increased with Oi up to a value of 57 nM, with half-maximal rates being attained at Oi values between 10 and 14 nM O2. The maximum nitrogenase activity achieved during the increase in pO2 (potential nitrogenase activity) was 30 to 57% of that measured in intact nodulated roots, showing that O2 limitation of nitrogenase activity could account for a significant proportion of the inhibition of TNA associated with the use of the probe. However, some factor(s) in addition to O2 must have limited the activity of single nodules at both subsaturating and saturating Oi. At Oi values greater than about 57 nM, nitrogenase activity and nodule respiration were inhibited, but, because this inhibition has been shown previously to be readily reversible when the Oi was lowered, it was not attributed to direct O2 inactivation of the nitrogenase protein. These results indicate that maximum nitrogenase activity in legume nodules is supported by a narrow range of Oi values. Possible biochemical mechanisms are discussed for both O2 limitation of nitrogenase activity at low Oi and inhibition of nitrogenase activity at high Oi.     

Ecological significance of bacterial polyphosphate metabolism in sediments

The purpose of this study was to find theoretical evidence that bacteria, in particular those capable of polyphosphate (polyP) metabolism, are directly implicated in sediment phosphorus (P) dynamics and control P metabolism of freshwater ecosystems. The specific attributes and functional role of such bacteria were investigated on successive levels of ecological organization: individual microorganism, microbial community, freshwater ecosystem. The results of this systematic approach have been formulated as a number of hypotheses.

1. PolyP metabolism is the mechanism which enables individual polyP bacteria to survive and grow under the fluctuating redox conditions characteristic of their habitat at the sediment-water interface.
2. PolyP metabolism together with anaerobic Mn and/or Fe respiration is the mechanism that confers upon polyP bacteria the advantage required to fill a unique ecological niche within the microbial community to which they belong.
3. To the freshwater ecosystem as a whole bacterial polyP metabolism is a homeostatic mechanism which limits P availability and makes ecosystem productivity self-correcting as a function of oxygen availability. Bacterial polyP pools in the sediment are vital components of the P cycle. It was suggested that the impact of this bacterial mechanism should be tested with regard to the eutrophication issue.

     

Mature Tissue and Crop Canopy Respiratory Characteristics of Rye, Triticale and Wheat

Crop dry matter and its chemical composition, together withcanopy and mature tissue respiration rates were measure at equivalentgrowth stages and temperatures for spring and winter rye, triticaleand wheat crops grown under irrigated field conditions. Canopyrespiration was partitioned into growth and maintenance respirationusing information from the chemical composition analysis ofthe crop biomass. Rates of dry matter accumulation early inthe growing season were significantly greater for rye cropsin comparison to triticale and wheat. However, when dry matterwas measured at similar ontogenetic stages, the productivityadvantage of the rye crop was no longer evident. Nevertheless,canopy respiration rates per unit ground area were significantlylower for rye than wheat over all temperatures and growth stages.Intergeneric differences in the respiration rates of matureleaf and stem tissues were consistent with those measured atcanopy scales. Differences in the chemical composition of thebiomass among genera were minimal, and insufficient to accountfor differences in canopy respiration due to synthesis respirationrequirement. Estimates of biomass maintenance requirements appearto be significantly lower for rye than wheat when calculatedat similar temperatures and ontogenetic stages. The maintenancecoefficient (m) depended on stage of development, suggestingthat m will decline earlier chronologically for rye than wheat,which implies that greater carbon retention is another aspectcontributing to the higher early-season crop growth rates ofspring and winter rye. Considering the lower respiration ratesof mature stems relative to leaves, the dependence of m on stem:leafratio was suggested as a useful approach to modelling ontogeneticeffects on maintenance respiration.Copyright 1993, 1999 AcademicPress Rye, triticale, wheat, dry matter, growth and maintenance respiration     

Developing Regenerate: A circular economy engagement tool for the assessment of new and existing buildings

The transition toward a circular economy (CE) is key in decarbonizing the built environment. Despite this, knowledge of—and engagement with—CE philosophies remains limited within the construction industry. Discussion with practitioners reveals this to be contributed to by a lack of clarity regarding CE principles, with numerous organizations recommending implementation of differing and sometimes conflicting principles. In addition, a systematic assessment of how building designs consider CE is made difficult by the multiple design areas required to be considered and the large amount of design data required to do so. The absence of a systematic CE assessment causes a lack of comparability across designs, preventing benchmarking of CE practices in building design at present. This paper details the development of Regenerate, a CE engagement tool for the assessment of new and existing buildings, established in an effort to overcome the aforementioned barriers to the adoption of CE within the construction sector. A CE design workflow for the built environment is proposed, comprising four overarching circularity principles (Design for Adaptability; Design for Deconstructability; Circular Material Selection; Resource Efficiency) and contributing design actions. In addition to engaging stakeholders by enabling the assessment of building designs, the tool retrieves key data for further research. Information on completed design actions as well as recycling and waste metrics is collected to facilitate future CE benchmarking. “Bill of materials” data (i.e., material quantities) is also compiled, with this being key in material stock modeling research and embodied carbon benchmarking.     

In vitro fusion of endocytic vesicles is inhibited by cyclin A-cdc2 kinase.

Receptor-mediated endocytosis and recycling are inhibited in mitotic mammalian cells, and previous studies have shown that inhibition of endocytic vesicle fusion in vitro occurs via cyclin B-cdc2 kinase. To test for the ability of cyclin A-cdc2 kinase to inhibit endocytic vesicle fusion, we employed recombinant cyclin A proteins. Addition of cyclin A to interphase extracts activated a histone kinase and markedly reduced the efficiency of endocytic vesicle fusion. By a number of criteria, inhibition of fusion was shown to be due to the action of cyclin A, via the mitosis-specific cdc2 kinase, and not an indirect effect through cyclin B. Two-stage incubations were used to demonstrate that at least one target of cyclin A-cdc2 kinase is a cytosolic component of the fusion apparatus. Reconstitution experiments showed that this component was also modified in mitotic cytosols and was unaffected by N-ethyl maleimide treatment.