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A hypothesis to explain division site selection in Escherichia coli by combining nucleoid occlusion and Min.

The positioning of the site of cell division in Escherichia coli results, it is generally believed, from the operation of nucleoid occlusion in combination with the Min system. Nucleoid occlusion prevents division over the nucleoids and directs it by default to the mid-cell region between segregating nucleoids or to polar regions while the Min system prevents division in polar regions. Unresolved questions include how these systems interact to control the earliest known event in division, the assembly at the membrane of the tubulin-like protein, FtsZ, and, more importantly, what exactly constitutes a division site. Evidence exists that (1) the coupled transcription, translation and insertion of proteins into membrane (transertion), can structure the cytoplasmic membrane into phospholipid domains, (2) the MinD protein can convert vesicles into tubes and (3) a variety of membranous structures can be observed at mid-cell. These data support a model in which transertion from the segregating daughter chromosomes leads to the formation of a distinct proteolipid domain between them at mid-cell; the composition of this domain allows phospholipid tubes to extend like fingers into the cytoplasm; these tubes then become the substrate for the dynamic assembly and disassembly of FtsZ which converts them into the invaginating fold responsible for division; the Min system inhibits division at unwanted sites and times by removing these tubes especially at the cell poles.

Adenosine Triphosphatases↗

"Fingering" the vertebrate limb.

A detailed and precise picture is being pieced together about how the pattern of digits develops in vertebrate limbs. What is particularly exciting is that it will soon be possible to trace the process all the way from establishment of a signalling centre in a small bud of undifferentiated cells right through to final limb anatomy. The development of the vertebrate limb is a traditional model in which to explore mechanisms involved in pattern formation, and there is accelerating knowledge about the genes involved. One reason why the limb is holding its place in the post-genomic age is that it is rich in pre-genomic embryology. Here, we will focus on recent findings about the aspect of vertebrate limb development concerned with digit pattern across the anteroposterior axis of the limb. This process is controlled by a signalling region in the early limb bud known as the polarizing region. Interactions between polarizing region cells and other cells in the limb bud ensure that a thumb develops at one edge of the hand (anterior) and a little finger at the other (posterior).

Animals↗

Redistribution of the nuclear mitotic apparatus protein (NuMA) during mitosis and nuclear assembly. Properties of purified NuMA protein.

Monoclonal antibodies and human autoimmune sera specific for the nuclear mitotic apparatus protein (NuMA protein) were applied to study the structure of this protein and its intracellular distribution. The NuMA protein was purified using immuno-affinity columns. Studies on this large (250 kD) nuclear protein indicated that it is a highly asymmetric phosphoprotein. It is present in all mammalian cells examined and in those of some non-mammals. Immunofluorescence studies on fixed cells demonstrated that its intracellular distribution is essentially the same in all species at all stages of the cell cycle. Immunoblot (western blot) analysis showed that the size of the NuMA protein varies slightly in different species. At the onset of mitosis the NuMA protein redistributes from the nucleus to two centrosomal structures that later will become part of the mitotic spindle pole. This occurs at the time of nuclear breakdown and eventually leads to an accumulation of the NuMA protein at the polar region of the mitotic spindle. After anaphase the protein redistributes from the spindle polar region into the reforming nucleus and concentrates initially at the site where nuclear lamins and perichomatin have been reported to assemble. Living cells microinjected with fluorescent anti-NuMA antibodies were studied to examine parameters that effect the redistribution of the NuMA protein in vivo. These experiments indicate that microtubule assembly is essential for the NuMA protein to accumulate in the polar region.

Anaphase↗

Distinct signaling molecules control Hoxa-11 and Hoxa-13 expression in the muscle precursor and mesenchyme of the chick limb bud.

The limb muscles, originating from the ventrolateral portion of the somites, exhibit position-specific morphological development through successive splitting and growth/differentiation of the muscle masses in a region-specific manner by interacting with the limb mesenchyme and the cartilage elements. The molecular mechanisms that provide positional cues to the muscle precursors are still unknown. We have shown that the expression patterns of Hoxa-11 and Hoxa-13 are correlated with muscle patterning of the limb bud (Yamamoto et al., 1998) and demonstrated that muscular Hox genes are activated by signals from the limb mesenchyme. We dissected the regulatory mechanisms directing the unique expression patterns of Hoxa-11 and Hoxa-13 during limb muscle development. HOXA-11 protein was detected in both the myogenic cells and the zeugopodal mesenchymal cells of the limb bud. The earlier expression of HOXA-11 in both the myogenic precursor cells and the mesenchyme was dependent on the apical ectodermal ridge (AER), but later expression was independent of the AER. HOXA-11 expression in both myogenic precursor cells and mesenchyme was induced by fibroblast growth factor (FGF) signal, whereas hepatocyte growth factor/scatter factor (HGF/SF) maintained HOXA-11 expression in the myogenic precursor cells, but not in the mesenchyme. The distribution of HOXA-13 protein expression in the muscle masses was restricted to the posterior region. We found that HOXA-13 expression in the autopodal mesenchyme was dependent on the AER but not on the polarizing region, whereas expression of HOXA-13 in the posterior muscle masses was dependent on the polarizing region but not on the AER. Administration of BMP-2 at the anterior margin of the limb bud induced ectopic HOXA-13 expression in the anterior region of the muscle masses followed by ectopic muscle formation close to the source of exogenous BMP-2. In addition, NOGGIN/CHORDIN, antagonists of BMP-2 and BMP-4, downregulated the expression of HOXA-13 in the posterior region of the muscle masses and inhibited posterior muscle development. These results suggested that HOXA-13 expression in the posterior muscle masses is activated by the posteriorizing signal from the posterior mesenchyme via BMP-2. On the contrary, the expression of HOXA-13 in the autopodal mesenchyme was affected by neither BMP-2 nor NOGGIN/CHORDIN. Thus, mesenchymal HOXA-13 expression was independent of BMP-2 from polarizing region, but was under the control of as yet unidentified signals from the AER. These results showed that expression of Hox genes is regulated differently in the limb muscle precursor and mesenchymal cells.

Animals↗

Mercury (micro)biogeochemistry in polar environments.

The contamination of polar regions with mercury that is transported as inorganic mercury from lower latitudes has resulted in the accumulation of methylmercury in the food chain of polar environments, risking the health of humans and wildlife. This problem is likely to be particularly severe in coastal marine environments where active cycling occurs. Little is currently known about how mercury is methylated in polar environments. Relating observations on mercury deposition and transport through polar regions to knowledge of the microbiology of cold environments and considering the principles of mercury transformations as have been elucidated in temperate aquatic environments, we propose that in polar regions (1) variable pathways for mercury methylation may exist, (2) mercury bioavailability to microbial transformations may be enhanced, and (3) microbial niches within sea ice are sites where active microorganisms are localized in proximity to high concentrations of mercury. Thus, microbial transformations, and consequently mercury biogeochemistry, in the Arctic and Antarctic are both unique and common to these processes in lower latitudes, and understanding their dynamics is needed for the management of mercury-contaminated polar environments.

Antarctic Regions↗

Lateral diffusion of gramicidin S, M-13 coat protein and glycophorin in bilayers of saturated phospholipids. Mean field and Monte Carlo studies.

We have developed a general model that relates the lateral diffusion coefficient of one isolated large intrinsic molecule (mol. wt. greater than or approximately 1000) in a phosphatidylcholine bilayer to the static lipid hydrocarbon chain order. We have studied how protein lateral diffusion can depend upon protein-lipid interactions but have not investigated possible non-specific contributions from gel-state lattice defects. The model has been used in Monte Carlo simulations or in mean-field approximations to study the lateral diffusion coefficients of Gramicidin S, the M-13 coat protein and glycophorin in dimyristoyl- and dipalmitoylphosphatidylcholine (DMPC and DPPC) bilayers as functions of temperature. Our calculated lateral diffusion coefficients for Gramicidin S and the M-13 coat protein are in good agreement with what has been observed and suggest that Gramicidin S is in a dimeric form in DMPC bilayers. In the case of glycophorin we find that the 'ice breaker' effect can be understood as a consequence of perturbation of the lipid polar region around the protein. In order to understand this effect is necessary that the protein hydrophilic section perturb the polar regions of at least approx. 24 lipid molecules, in good agreement with the numbers of 29-30 measured using 31P-NMR. Because of lipid-lipid interactions this effect extends itself out to four or five lipid layers away from the protein so that the hydrocarbon chains of between approx. 74 and approx. 108 lipid molecules are more disordered in the gel phase, so contributing less to the transition enthalpy, in agreement with the numbers of 80-100 deduced from differential scanning calorimetry (DSC). An understanding of the abrupt change in the diffusion coefficient at a temperature below the main bilayer transition temperature requires an additional mechanism. We propose that this change may be a consequence of a 'coupling-uncoupling' transition involving the protein hydrophilic section and the lipid polar regions, which may be triggered by the lipid bilayer pretransition. Our calculation of the average number of gauche bonds per lipid chain as a function of temperature and distance away from an isolated polypeptide or integral protein shows the extent of statically disordered lipid around such molecules. The range of this disorder depends upon temperature, particularly near the main transition.

Capsid↗

Positional signalling and the development of the humerus in the chick limb bud.

The positional signal model for specification of the cartilaginous elements in limb development has been tested by examining the effect on the humerus of grafting a polarizing region to different positions along the anteroposterior axis of the limb bud at stage 16. The humerus between the host and grafted polarizing region was largely normal though there were variations in width, particularly the distal epiphysis. The humerus often showed mirror-image symmetry along the anteroposterior axis. When the grafted polarizing region was in a very anterior position, there were a few cases where a second humerus developed. Anterior to the graft an additional humerus often developed. This was associated with the splitting of the bud into two domains. It is suggested that these results are not consistent with a positional signal model and that an additional mechanism involving an isomorphic prepattern may be involved in the specification of the cartilaginous elements.

Animals↗

Morphogen gradients in vertebrate limb development.

The developing limb is an excellent model for pattern formation in vertebrate embryos. Signalling by the polarizing region controls limb pattern across the antero-posterior axis of the chick limb. It was suggested first on theoretical grounds that signalling by the polarizing region could involve a morphogen gradient. Embryological manipulations provided evidence consistent with this model and, more recently, signalling molecules associated with the polarizing region have been identified and tested for their role as morphogens. It is still not clear whether any of the known molecules act directly as a morphogen. The extension of the morphogen model to patterning along the other axes of the limb has been proposed but this may not be applicable.

Animals↗

Lipopolysaccharide O antigen chains mask IcsA (VirG) in Shigella flexneri.

Shigella flexneri 2a strain 2457T lipopolysaccharide (LPS) has O antigen (Oag) chains with two modal lengths (S-type and VL-type), and has IcsA apparently located at one pole on its cell surface. Treatment of Y serotype derivatives of 2457T and RMA696 (2457T wzz(SF)) with Sf6 tailspike protein (TSP) resulted in hydrolysis of Oag chains, and an increase in detection of IcsA by indirect immunofluorescence staining on both the lateral and polar regions of the cell surface. Newly synthesised IcsA expressed from a pBAD promoter in a S. flexneri Y strain was also detected on both the lateral and polar regions of the cell when incubated with TSP prior to immunofluorescence staining. We conclude that IcsA is actually located on both lateral and polar regions of the S. flexneri cell surface, and that LPS Oag chains mask the presence of IcsA by hindering its detection with antibodies. These results have implications for the mechanism of IcsA export. They suggest that while IcsA export is predominantly targeted to the old cell pole, it can also occur on the lateral regions of the cell surface.

Bacterial Proteins↗

[Spectroscopic study of the structure and intramolecular mobility of yeast pyruvate decarboxylase].

Steady-state and time-resolved fluorimetry were used to study the properties of holo- and apopyruvate decarboxylase (EC 4.1.1.1, PDC) from Brewer's yeast after interaction with substrate (pyruvate), cofactor (thiamine diphosphate, ThDP) and Mg2+ ions. The analysis of the enzyme's intrinsic fluorescence as well as of its complex with the probe 2-(p-toluidinylnaphthalene)-6-sulphonate (TNS) revealed that ThDP was found at the polar region of the PDC active sites, inducing a decrease in the mobility of the protein's nearest surroundings. The fluorescent probe had three different sites of binding to the protein apoform, two of which being located at the catalytic site and having different rotation freedom. The study of the PDC complex with thiochrome pyrophosphate, a ThDP structural analogue, pointed to the occurrence of a non-polar region of the enzyme active site for pyruvate absorption besides the polar region. The binding of pyruvate to the protein does not depend upon the cofactor's binding. On the basis of the fluorescent studies a model of the ThDP and pyruvate arrangement at the PDC active site is suggested.

Anilino Naphthalenesulfonates↗

Bilayer thickness and lipid interface area in unilamellar extruded 1,2-diacylphosphatidylcholine liposomes: a small-angle neutron scattering study.

Small-angle neutron scattering (SANS) experiments have been performed on large unilamellar liposomes prepared from 1,2-dilauroylphosphatidylcholine (DLPC), 1,2-dimyristoyl-phosphatidylcholine (DMPC) and 1,2-distearoylphosphatidylcholine (DSPC) in heavy water by extrusion through polycarbonate filters with 500 A pores. The neutron scattering intensity I(Q) in the region of scattering vectors Q corresponding to 0.0015 A(-2) < or = Q(2) < or = 0.0115 A(-2) was fitted using a step function model of bilayer neutron scattering length density and supposing that the liposomes are spherical and have a Gaussian distribution of radii. Using the lipid volumetric data, and supposing that the thickness of bilayer polar region equals to d(H) = 9+/-1 A and the water molecular volume intercalated in the bilayer polar region is the same as in the aqueous bulk aqueous phase, the steric bilayer thickness d(L), the lipid surface area A(L) and the number of water molecules per lipid molecule N intercalated in the bilayer polar region were obtained: d(L) = 41.58+/-1.93 A, A(L) = 57.18+/-1.00 A(2) and N = 6.53+/-1.93 in DLPC at 20 degrees C, d(L) = 44.26+/-1.42 A, A(L) = 60.01+/-0.75 A(2) and N = 7.37+/-1.94 in DMPC at 36 degrees C, and d(L) = 49.77+/-1.52 A, A(L) = 64.78+/-0.46 A(2) and N = 8.67+/-1.97 in DSPC at 60 degrees C. After correcting for area thermal expansivity alpha approximately 0.00417 K(-1), the lipid surface area shows a decrease with the lipid acyl chain length at 60 degrees C: A(L) = 67.56+/-1.18 A(2) in DLPC, A(L) = 66.33+/-0.83 A(2) in DMPC and A(L) = 64.78+/-0.46 A(2) in DSPC. It is also shown that a joint evaluation of SANS and small-angle X-ray scattering on unilamellar liposomes can be used to obtain the value of d(H) and the distance of the lipid phosphate group from the bilayer hydrocarbon region d(H1).

1,2-Dipalmitoylphosphatidylcholine↗

Retinoic acid and pattern formation in the developing chick wing: SEM and quantitative studies of early effects on the apical ectodermal ridge and bud outgrowth.

When retinoic acid is locally applied to the anterior margin of developing chick wing buds on ion-exchange beads, dose-dependent changes in the skeletal pattern result. At low doses, additional digits develop. At high doses, there is thinning of the symmetrical wing. Local application of retinoic acid to the apex of the bud also leads to pattern changes, but in contrast normal wing patterns are almost always obtained following application posteriorly. These effects are manifest at 6-7 days after the operation although only a brief exposure (14-20 h) to retinoic acid is required. Therefore the morphology of wing buds was studied at shorter times after the start of treatment. The local application of retinoic acid to the wing bud margin leads to changes in extent of the apical ridge that can be detected at 24 h after application. The behaviour of the apical ridge with varying doses and positions of retinoic acid application has been analysed quantitatively and dose response curves obtained. At low doses of retinoic acid, the length of the apical ridge increases or remains constant, but then progressively decreases with higher doses. The progressive obliteration of the ridge starts first near the bead and then involves more distant parts of the bud. Thus the region of the ridge affected depends on the position at which the retinoic acid is applied. We propose that these effects on the apical ridge reflect dose-dependent responses to the local concentration of retinoic acid that varies with distance from the source. At high doses, the apical ridge disappears but at low doses it is maintained. Since grafts of polarizing region tissue also have a graded effect on ridge morphology, a possible interpretation of the retinoic acid effects is that tissue adjacent to the source is converted into polarizing region tissue. Alternatively, retinoic acid may act directly on the ridge cells. The changes in the extent of the apical ridge produced by retinoic acid lead to different forms of bud outgrowth. The form of the outgrowth depends on the dose of retinoic acid, the position of application and the interaction between the effects of the local source of retinoic acid and those of the polarizing region of the host bud. These considerations give some insights into why anterior application of retinoic acid leads to the development of additional digits whereas posterior application generally gives normal wings.

Animals↗

The state and future of Mars polar science and exploration.

As the planet's principal cold traps, the martian polar regions have accumulated extensive mantles of ice and dust that cover individual areas of approximately 10(6) km2 and total as much as 3-4 km thick. From the scarcity of superposed craters on their surface, these layered deposits are thought to be comparatively young--preserving a record of the seasonal and climatic cycling of atmospheric CO2, H2O, and dust over the past approximately 10(5)-10(8) years. For this reason, the martian polar deposits may serve as a Rosetta Stone for understanding the geologic and climatic history of the planet--documenting variations in insolation (due to quasiperiodic oscillations in the planet's obliquity and orbital elements), volatile mass balance, atmospheric composition, dust storm activity, volcanic eruptions, large impacts, catastrophic floods, solar luminosity, supernovae, and perhaps even a record of microbial life. Beyond their scientific value, the polar regions may soon prove important for another reason--providing a valuable and accessible reservoir of water to support the long-term human exploration of Mars. In this paper we assess the current state of Mars polar research, identify the key questions that motivate the exploration of the polar regions, discuss the extent to which current missions will address these questions, and speculate about what additional capabilities and investigations may be required to address the issues that remain outstanding.

Atmosphere↗

Ultrastructural study of a blood-muscle spindle barrier after systemic administration of horseradish peroxidase.

The permeability of the normal muscle spindle capsule to the entrance of an exogenous protein tracer was assessed by the use of horseradish peroxidase (HRP). Adult mice were injected intravenously with a solution of HRP (MW 40,000). After varying intervals, ranging from two to 240 minutes, animals were perfused aortically with fixative, and anterior tibialis muscles were removed, processed, and examined. The results were evaluated by electron microscopic cytochemical techniques, and attention was directed to the movement of tracer and its relationship to the outer and inner capsule of the muscle spindle. HRP was first demonstrated in cytoplasmic vesicles crossing the continuous capillary endothelium supplying the muscle spindle, and was then detected in the interstitial space surrounding the outer capsule. In polar regions, HRP had entered the periaxial space by two minutes, and it was seen in cytoplasmic vesicles of the attenuated outer capsule. Later, HRP abutted the sarcolemma of intrafusal fibers, and by 12.5 minutes there was evidence of tracer in T-tubules of these muscle cells. Whereas at no time was tracer observed traversing any of the numerous intracellular junctions of the capsule, it appeared that polar regions were leaky and open-ended at their distal portions. The sensory equatorial zone was considerably less permeable to the entrance of tracer. Whereas HRP was visible intially in cytoplasmic vesicles of the subjacent capillary endothelium, it was not until 12.5 minutes that racer could be demonstrated within some vesicles of equatorial outer capsule cells. At later times, a small amount of HRP was observed in the equatorial periaxial space, where it was phagocytosed and finally sequestered by cells of the inner capsule as membrane-bound lysosomal deposits. Consequently, at no time was tracer incorported into either intrafusal fibers or their sensory nerve terminals. Thus, in contract to polar regions, the muscle spindle capsule in the equatorial zone appears to be effective in preventing the indiscriminate penetration of HRP from the bloodstream. This suggests dissimilar paths of tracer movement from the microvasculature into sensory and non-sensory regions of the muscle spindle.

Animals↗

Synaptonemal complex karyotyping in Melanoplus differentialis.

The chromosomal axes of the spermatocytes of the grasshopper Melanoplus differentialis have been studied with a modification of the microspreading procedure used previously. The whole complement of synaptonemal complexes (SCs) and the axis of the X chromosome have been described and measured. The relative length of each SC is characteristic and constant and permits the construction of an idiogram. Relative lengths of SCs are almost equal to the relative lengths of mitotic chromosomes of spermatogonia (with the exception of the X chromosome), thus extending to an invertebrate the relationship between SCs and mitotic chromosomes that has been demonstrated in mammals. All the SCs except the 3 smallest (which are apparently telocentric) show a small short arm beyond the kinetochore. The progression of changes in the chromosomal axes during meiotic prophase has been staged by centriolar behaviour. During leptotene, axes are first formed near the nuclear envelope at a special (polar) region. SCs also begin to appear in the polar region and extend towards the nuclear interior. The beginning and completion of synapsis is not synchronous among bivalents. The X-axis is formed in midzygotene and shows a characteristic sequence of changes in shape during pachytene. Cells in post-synaptic stages show whole chromosome complements with characteristic chiasmatic configurations. Kinetochores are prominent and bipartite during diplotene-diakinesis.

Animals↗

New insights on the behavior of PRODAN in homogeneous media and in large unilamellar vesicles.

The behavior of 6-propionyl-2-dimethylaminonaphthalene (PRODAN) was studied in homogeneous media and in large unilamellar vesicles (LUVs) of the phospholipid 1,2-di-oleoyl-sn-glycero-3-phosphatidylcholine (DOPC), using absorption, emission, depolarization, and time-resolved spectroscopies. In homogeneous media, the Kamlet and Taft solvatochromic comparison method quantified solute-solvent interactions from the absorption and emission PRODAN bands. These studies demonstrate that the absorption band is sensitive to the polarity-polarizability (pi) and the hydrogen bond donor ability (alpha) parameters of the media. PRODAN in the excited state is even more sensitive to these parameters and to the hydrogen bond acceptor ability (beta) of the media. The transition energy (expressed in kcal/mol) for both absorption and emission bands gives a linear correlation with the well-known polarity parameter E(T30). The results from the absorption and emission bands also reveal that PRODAN aggregates in water. The monomer has two fluorescence lifetimes, 2.27 and 0.65 ns, while the aggregate has a lifetime of 14.6 ns. Using steady-state anisotropy measurements, the calculated volumes of the aggregate and the monomer are 5590 and 222 mL mol(-1), respectively. In DOPC LUVs, PRODAN undergoes a partition process between the water bulk and the DOPC bilayer. We show that the partition constant (K(p)) value is large enough that only at [DOPC] below 0.15 mg/mL PRODAN in water can be detected. PRODAN dissolved in LUVs at [DOPC] > 1 mg/mL exists completely incorporated in its monomer form and senses two different microenvironments within the bilayer: a polar region in the interface near the water and a less polar and also less viscous environment, between the phospholipid tails. These environments were characterized by their fluorescence lifetimes (tau), showing that PRODAN in the polar microenvironment has a tau value of approximately 4 ns while in the less polar region gives a value of 1.2 ns. Moreover, this probe also senses the micropolarity of these two different regions of the bilayer and yields values similar to that of methanol and tetrahydrofuran.

2-Naphthylamine↗

Effects of biochemical inhibitors on positional signalling in the chick limb bud.

In 3- to 4-day embryonic chick limb buds, a region at the posterior margin of the limb, the zone of polarizing activity, appears to be responsible for signalling positional information along the anteroposterior axis. Our experiments were designed to test which biosynthetic processes are required for polarizing activity. We have treated polarizing regions with biochemical inhibitors, and then assayed their abilities to induce limb reduplications when grafted into anterior sites on host limb buds and also measured their capacities for protein, RNA, and DNA synthesis. DNA synthesis, and possibly oxidative phosphorylation, do not seem to be required for polarizing activity. But, glycolysis and protein and RNA synthesis are necessary, although not sufficient, for polarizing region activity. Activity seems particularly sensitive to inhibitors (actinomycin D and alpha-amanitin) of RNA synthesis.

Animals↗

People in high latitudes: the "Big Five" personality characteristics of the circumpolar sojourner.

The personality of the polar sojourner has been of interest to psychologists for a number of years. Using the NEO Five Factor Inventory, this study examined the general personality factors of the polar worker compared to a normative population, and how these factors differ according to the worker's occupational classification and the polar region in which he or she is working. It was found that polar workers scored higher than a normative group on all factors except Neuroticism. Comparisons across occupational groups showed that scientists were lower than military personnel on Extraversion and lower than technical/support staff on Agreeableness and Conscientiousness. The analysis by polar region indicated that Antarctic workers were higher than Arctic personnel on Extraversion, Agreeableness, and Conscientiousness. A group of Italian Antarctic personnel, completing a translated form of the NEO-FFI, scored lower than the rest of the polar groups on all factors. These findings are discussed in light of various features of the polar environment and Gunderson's 3-predictor model of polar adaptability.

Adaptation, Psychological↗