Crystal structure of the bacterial cell division inhibitor MinC.

Bacterial cell division requires accurate selection of the middle of the cell, where the bacterial tubulin homologue FtsZ polymerizes into a ring structure. In Escherichia coli, site selection is dependent on MinC, MinD and MINE: MinC acts, with MinD, to inhibit division at sites other than the midcell by directly interacting with FTSZ: Here we report the crystal structure to 2.2 A of MinC from Thermotoga maritima. MinC consists of two domains separated by a short linker. The C-terminal domain is a right-handed beta-helix and is involved in dimer formation. The crystals contain two different MinC dimers, demonstrating flexibility in the linker region. The two-domain architecture and dimerization of MinC can be rationalized with a model of cell division inhibition. MinC does not act like SulA, which affects the GTPase activity of FtsZ, and the model can explain how MinC would select for the FtsZ polymer rather than the monomer.

Morphological and physiological adjustment to N and P fertilization in nutrient-limited Metrosideros polymorpha canopy trees in Hawaii.

Leaf-level studies of Metrosideros polymorpha Gaud. (Myrtaceae) canopy trees at both ends of a substrate age gradient in the Hawaiian Islands pointed to differential patterns of adjustment to both nutrient limitation and removal of this limitation by long-term (8-14 years) nitrogen (N), phosphorus (P) and N + P fertilizations. The two study sites were located at the same elevation, had similar annual precipitation, and supported forests dominated by M. polymorpha, but differed in the age of the underlying volcanic substrate, and in soil nutrient availability, with relatively low N at the young site (300 years, Thurston, Hawaii) and relatively low P at the oldest site (4,100,000 years, Kokee, Kauai). Within each site, responses to N and P fertilization were similar, regardless of the difference in soil N and P availability between sites. At the young substrate site, nutrient addition led to a larger mean leaf size (about 7.4 versus 4.8 cm2), resulting in a larger canopy leaf surface area. Differences in foliar N and P content, chlorophyll concentrations and carboxylation capacity between the fertilized and control plots were small. At the old substrate site, nutrient addition led to an increase in photosynthetic rate per unit leaf surface area from 4.5 to 7.6 micromol m(-2) s(-1), without a concomitant change in leaf size. At this site, leaves had substantially greater nutrient concentrations, chlorophyll content and carboxylation capacity in the fertilized plots than in the control plots. These contrasting acclimation responses to fertilization at the young and old sites led to significant increases in total carbon gain of M. polymorpha canopy trees at both sites. At the young substrate site, acclimation to fertilization was morphological, resulting in larger leaves, whereas at the old substrate site, physiological acclimation resulted in higher leaf carboxylation capacity and chlorophyll content.

Crystal structure of the bacterial cell division regulator MinD.

In bacterial cell division MinD plays a pivotal role, selecting the mid-cell over other sites. With MinC, MinD forms a non-specific inhibitor of division, that interacts with FtsZ. Specificity is provided by MinD's interaction with MinE at the mid-cell. We have solved the crystal structure of MinD-1 from Archaeoglobus fulgidus to 2.6 A by multiple anomalous dispersion. MinD is a classic nucleotide binding protein, related to nitrogenase iron proteins, which have a fold of a seven-stranded parallel beta-sheet, surrounded by alpha-helices. Although MinD, unlike the proteins it interacts with and those it is structurally related to, is a monomer, not a dimer.

Crystal structure of the SMC head domain: an ABC ATPase with 900 residues antiparallel coiled-coil inserted.

SMC (structural maintenance of chromosomes) proteins are large coiled-coil proteins involved in chromosome condensation, sister chromatid cohesion, and DNA double-strand break processing. They share a conserved five-domain architecture with three globular domains separated by two long coiled-coil segments. The coiled-coil segments are antiparallel, bringing the N and C-terminal globular domains together. We have expressed a fusion protein of the N and C-terminal globular domains of Thermotoga maritima SMC in Escherichia coli by replacing the approximately 900 residue coiled-coil and hinge segment with a short peptide linker. The SMC head domain (SMChd) binds and condenses DNA in an ATP-dependent manner. Using selenomethionine-substituted protein and multiple anomalous dispersion phasing, we have solved the crystal structure of the SMChd to 3.1 A resolution. In the monoclinic crystal form, six SMChd molecules form two turns of a helix. The fold of SMChd is closely related to the ATP-binding cassette (ABC) ATPase family of proteins and Rad50, a member of the SMC family involved in DNA double-strand break repair. In SMChd, the ABC ATPase fold is formed by the N and C-terminal domains with the 900 residue coiled-coil and hinge segment inserted in the middle of the fold. The crystal structure of an SMChd confirms that the coiled-coil segments in SMC proteins are anti-parallel and shows how the N and C-terminal domains come together to form an ABC ATPase. Comparison to the structure of the MukB N-terminal domain demonstrates the close relationship between MukB and SMC proteins, and indicates a helix to strand conversion when N and C-terminal parts come together.

Regulation of leaf life-span and nutrient-use efficiency of Metrosideros polymorpha trees at two extremes of a long chronosequence in Hawaii.

Leaf traits related to life-span and nutrient-use efficiency were studied in the dominant Hawaiian tree species, Metrosideros polymorpha, at both ends of a natural fertility gradient, from young, nitrogen-poor soils to older, phosphorus-poor soils. The main objective of this study was to understand how nutrient limitations affect leaf-level attributes that ultimately play a mechanistic role in regulating whole-ecosystem function. Different types of adjustments to removal of nutrient limitation by long-term fertilization (9-15 years) with nitrogen (N), phosphorus (P), and a combined treatment of N plus P were observed at each site. Nitrogen fertilization at the young, mostly N-limited site did not significantly affect net CO2 assimilation (A), foliar N content, or N resorption. The primary response to N fertilization was a decrease in average leaf life span to approximately 553 days compared with 898 days in the control plot. Significantly shorter average leaf life-span coupled with constant A and foliar N content resulted in reduced integrated photosynthetic nitrogen-use efficiency (PNUE: A summed over the life-span of a leaf divided by foliar N) in the fertilized plots. In contrast, removal of nutrient limitations at the old, mostly P-limited site resulted in increased A, and increased foliar P concentration which also resulted in reduced integrated photosynthetic phosphorus-use efficiency (PPUE). P resorption was also reduced at this site, yet leaf life-span remained constant. When results from both sites and all treatments were combined, statistically significant relationships between leaf life-span, and A, leaf mass per area (LMA), and the cost of leaf construction per unit carbon gain (cost of construction determined by combustion of leaf samples divided by A) were found. As leaf life-span increased, A decreased asymptotically, and LMA and the carbon cost per carbon gain increased linearly. It appears that the balance between leaf carbon cost and carbon uptake is a major determinant of leaf longevity in M. polymorpha despite contrasting responses to removal of N and P limitations by long-term fertilization. Removal of the main nutrient limitations at both sites also resulted in reduced integrated nutrient use efficiency. However, the regulatory mechanisms were different depending on the site limitations: a shorter leaf life-span in the young, N-limited site and substantially higher foliar P concentration in the P-fertilized plots at the old, P-limited site.

Supercooling Capacity Increases from Sea Level to Tree Line in the Hawaiian Tree Species Metrosideros polymorpha.

Population-specific differences in the freezing resistance of Metrosideros polymorpha leaves were studied along an elevational gradient from sea level to tree line (located at ca. 2500 m above sea level) on the east flank of the Mauna Loa volcano in Hawaii. In addition, we also studied 8-yr-old saplings grown in a common garden from seeds collected from the same field populations. Leaves of low-elevation field plants exhibited damage at -2 degrees C, before the onset of ice formation, which occurred at -5.7 degrees C. Leaves of high-elevation plants exhibited damage at ca. -8.5 degrees C, concurrent with ice formation in the leaf tissue, which is typical of plants that avoid freezing in their natural environment by supercooling. Nuclear magnetic resonance studies revealed that water molecules of both extra- and intracellular leaf water fractions from high-elevation plants had restricted mobility, which is consistent with their low water content and their high levels of osmotically active solutes. Decreased mobility of water molecules may delay ice nucleation and/or ice growth and may therefore enhance the ability of plant tissues to supercool. Leaf traits that correlated with specific differences in supercooling capacity were in part genetically determined and in part environmentally induced. Evidence indicated that lower apoplastic water content and smaller intercellular spaces were associated with the larger supercooling capacity of the plant's foliage at tree line. The irreversible tissue-damage temperature decreased by ca. 7 degrees C from sea level to tree line in leaves of field populations. However, this decrease appears to be only large enough to allow M. polymorpha trees to avoid leaf tissue damage from freezing up to a level of ca. 2500 m elevation, which is also the current tree line location on the east flank of Mauna Loa. The limited freezing resistance of M. polymorpha leaves may be partially responsible for the occurrence of tree line at a relatively low elevation in Hawaii compared with continental tree lines, which can be up to 1500 m higher. If the elevation of tree line is influenced by the inability of M. polymorpha leaves to supercool to lower subzero temperatures, then it will be the first example that freezing damage resulting from limited supercooling capacity can be a factor in tree line formation.

Physiological and morphological variation in Metrosideros polymorpha, a dominant Hawaiian tree species, along an altitudinal gradient: the role of phenotypic plasticity.

Metrosideros polymorpha, a dominant tree species in Hawaiian ecosystems, occupies a wide range of habitats. Complementary field and common-garden studies of M. polymorpha populations were conducted across an altitudinal gradient at two different substrate ages to ascertain if the large phenotypic variation of this species is determined by genetic differences or by phenotypic modifications resulting from environmental conditions. Several characteristics, including ecophysiological behavior and anatomical features, were largely induced by the environment. However, other characteristics, particularly leaf morphology, appeared to be mainly determined by genetic background. Common garden plants exhibited higher average rates of net assimilation (5.8 µmol CO2 m-2 s-1) and higher average stomatal conductance (0.18 mol H2O m-2 s-1) than their field counterparts (3.0 µmol CO2 m-2 s-1, and 0.13 mol H2O m-2 s-1 respectively). Foliar δ13C of most common-garden plants was similar among sites of origin with an average value of -26.9‰. In contrast, mean values of foliar δ13C in field plants increased substantially from -29.5‰ at low elevation to -24.8‰ at high elevation. Leaf mass per unit area increased significantly as a function of elevation in both field and common garden plants; however, the range of values was much narrower in common garden plants (211-308 g m-2 for common garden versus 107-407 g m-2 for field plants). Nitrogen content measured on a leaf area basis in common garden plants ranged from 1.4 g m-2 to 2.4 g m-2 and from 0.8 g m-2 to 2.5 g m-2 in field plants. Photosynthetic nitrogen use efficiency (PNUE) decreased 50% with increasing elevation in field plants and only 20% in plants from young substrates in the common garden. This was a result of higher rates of net CO2 assimilation in the common garden plants. Leaf tissue and cell layer thickness, and degree of leaf pubescence increased significantly with elevation in field plants, whereas in common garden plants, variation with elevation of origin was much narrower, or was entirely absent. Morphological characteristics such as leaf size, petiole length, and internode length decreased with increasing elevation in the field and were retained when grown in the common garden, suggesting a potential genetic basis for these traits. The combination of environmentally induced variability in physiological and anatomical characteristics and genetically determined variation in morphological traits allows Hawaiian M. polymorpha to attain and dominate an extremely wide ecological distribution not observed in other tree species.

Acute and subacute effects of injury on the canine alveolar septum.

The effect of papain on the prevalence and distribution of alveolar macrophages, alveolar septal interstitial tissue gaps and epithelial cells in normal canine pulmonary alveoli was studied by light and electron microscopy. Serial sections of whole alveoli from control animals and from animals sacrificed 4 h, two weeks and one month after the instillation into one lung of crude papain in saline solution containing India ink as a marker were compared. In dogs, as in humans, there is normally a zonal distribution of alveolar macrophages and type 2 cells at alveolar junctional sites. We hypothesize that early alveolar septal injury takes place at these junctional sites, judging from concentration of alveolar macrophages and interstitial septal gaps at these sites following papain exposure, and also that septal repair activities are greatest at these sites, in view of the preponderance and high prevalence of type 2 cells occupying interstitial septal gaps in junctional zones. Consequently, the type 2 cell may play a role beyond that of merely replacing type 1 epithelial cells following alveolar septal injury.

In vitro epidermal cell proliferation in rat skin plugs.

Full-thickness skin plugs from immature and adult rats have been shown to incorporate -3H-thymidine in vitro in a semisynchronous fashion for up to 54 hr. DNA synthesis is minimal at 16 hr post sacrifice but increases again at 24 and 48 hr, and cells in S phase at 48 hr have passed through at least one mitosis in vitro (between 32 and 39 hr). Advantage can be taken of this semisynchronous burst of DNA synthetic activity to test the effects of potential inhibitors of skin cell proliferation, and to determine at which phase of the cell cyclic these inhibitors act. High concentrations of epinephrine or isoproterenol (10--5 M) are required to cause inhibition but the effect is specific for the G2 phase of the cell cycle. Propranolol does not demonstrate beta-antagonism in this system, since it is a very potent inhibitor acting in G1 phase. Dibutyryl cycle AMP and theophylline have two effects: one is a specific inhibitory action on cells in the G2 phase and the second is a short-term action limiting thymidine uptake by cells in S phase without affecting the transition of cells from G1 to S.