Actin filaments are required for fibripositor-mediated collagen fibril alignment in tendon

Cells in tendon deposit parallel arrays of collagen fibrils to form a functional tissue, but how this is achieved is unknown. The cellular mechanism is thought to involve the formation of intracellular collagen fibrils within Golgi to plasma membrane carriers. This is facilitated by the intracellular processing of procollagen to collagen by members of the tolloid and ADAMTS families of enzymes. The carriers subsequently connect to the extracellular matrix via finger-like projections of the plasma membrane, known as fibripositors. In this study we have shown, using three-dimensional electron microscopy, the alignment of fibripositors with intracellular fibrils as well as an orientated cable of actin filaments lining the cytosolic face of a fibripositor. To demonstrate a specific role for the cytoskeleton in coordinating extracellular matrix assembly, cytochalasin was used to disassemble actin filaments and nocodazole or colchicine were used to disrupt microtubules. Microtubule disruption delayed procollagen transport through the secretory pathway, but fibripositor numbers were unaffected. Actin filament disassembly resulted in rapid loss of fibripositors and a subsequent disappearance of intracellular fibrils. Procollagen secretion or processing was not affected by cytochalasin treatment, but the parallelism of extracellular collagen fibrils was altered. In this case a significant proportion of collagen fibrils were found to no longer be orientated with the long axis of the tendon. The results suggest an important role for the actin cytoskeleton in the alignment and organization of the collagenous extracellular matrix in embryonic tendon.     

Effects of Handling or Immobilization on Plasma Levels of 3,4-Dihydroxyphenylalanine, Catecholamines, and Metabolites in Rats

In conscious animals, handling and immobilization increase plasma levels of the catecholamines norepinephrine (NE) and epinephrine (EPI). This study examined plasma concentrations of endogenous compounds related to catecholamine synthesis and metabolism during and after exposure to these stressors in conscious rats. Plasma levels of 3,4-dihydroxyphenylalanine (DOPA), NE, EPI, and dopamine (DA), the deaminated catechol metabolites 3,4-dihydroxyphenylglycol (DHPG), and 3,4-dihydroxyphenylacetic acid (DOPAC), and their O-methylated derivatives methoxyhydroxyphenylglycol (MHPG) and homovanillic acid (HVA) were measured using liquid chromatography with electrochemical detection at 1, 3, 5, 20, 60, and 120 min of immobilization. By 1 min of immobilization, plasma NE and EPI levels had already reached peak values, and plasma levels of DOPA, DHPG, DOPAC, and MHPG were increased significantly from baseline, whereas plasma DA and HVA levels were unchanged. During the remainder of the immobilization period, the increased levels of DOPA, NE, and EPI were maintained, whereas levels of the metabolites progressively increased. In animals immobilized briefly (5 min), elevated concentrations of the metabolites persisted after release from the restraint, whereas DOPA and catecholamine levels returned to baseline. Gentle handling for 1 min also significantly increased plasma levels of DOPA, NE, EPI, and the NE metabolites DHPG and MHPG, without increasing levels of DA or HVA. The results show that in conscious rats, immobilization or even gentle handling rapidly increases plasma levels of catecholamines, the catecholamine precursor DOPA, and metabolites of NE and DA, indicating rapid increases in the synthesis, release, reuptake, and metabolism of catecholamines.     

The Biogeochemistry of Carbon at Hubbard Brook

The biogeochemical behavior of carbon in the forested watersheds of the Hubbard Brook Experimental Forest (HBEF) was analyzed in long-term studies. The largest pools of C in the reference watershed (W6) reside in mineral soil organic matter (43% of total ecosystem C) and living biomass (40.5%), with the remainder in surface detritus (14.5%). Repeated sampling indicated that none of these pools was changing significantly in the late-1990s, although high spatial variability precluded the detection of small changes in the soil organic matter pools, which are large; hence, net ecosystem productivity (NEP) in this 2nd growth forest was near zero (± about 20 g C/m²-yr) and probably similar in magnitude to fluvial export of organic C. Aboveground net primary productivity (ANPP) of the forest declined by 24% between the late-1950s (462 g C/m²-yr) and the late-1990s (354 g C/m²-yr), illustrating age-related decline in forest NPP, effects of multiple stresses and unusual tree mortality, or both. Application of the simulation model PnET-II predicted 14% higher ANPP than was observed for 1996–1997, probably reflecting some unknown stresses. Fine litterfall flux (171 g C/m²-yr) has not changed much since the late-1960s. Because of high annual variation, C flux in woody litterfall (including tree mortality) was not tightly constrained but averaged about 90 g C/m²-yr. Carbon flux to soil organic matter in root turnover (128 g C/m²-yr) was only about half as large as aboveground detritus. Balancing the soil C budget requires that large amounts of C (80 g C/m²-yr) were transported from roots to rhizosphere carbon flux. Total soil respiration (TSR) ranged from 540 to 800 g C/m²-yr across eight stands and decreased with increasing elevation within the northern hardwood forest near W6. The watershed-wide TSR was estimated as 660 g C/m²-yr. Empirical measurements indicated that 58% of TSR occurred in the surface organic horizons and that root respiration comprised about 40% of TSR, most of the rest being microbial. Carbon flux directly associated with other heterotrophs in the HBEF was minor; for example, we estimated respiration of soil microarthropods, rodents, birds and moose at about 3, 5, 1 and 0.8 g C/m²-yr, respectively, or in total less than 2% of NPP. Hence, the effects of other heterotrophs on C flux were primarily indirect, with the exception of occasional irruptions of folivorous insects. Hydrologic fluxes of C were significant in the watershed C budget, especially in comparison with NEP. Although atmospheric inputs (1.7 g C/m²-yr) and streamflow outputs (2.7 g C/m²-yr) were small, larger quantities of C were transported within the ecosystem and a more substantial fraction of dissolved C was transported from the soil as inorganic C and evaded from the stream as CO₂ (4.0 g C/m²-yr). Carbon pools and fluxes change rapidly in response to catastrophic disturbances such as forest harvest or major windthrow events. These changes are dominated by living vegetation and dead wood pools, including roots. If biomass removal does not accompany large-scale disturbance, the ecosystem is a large net source of C to the atmosphere (500–1200 g C/m²-yr) for about a decade following disturbance and becomes a net sink about 15–20 years after disturbance; it remains a net sink of about 200–300 g C/m²-yr for about 40 years before rapidly approaching steady state. Shifts in NPP and NEP associated with common small-scale or diffuse forest disturbances (e.g., forest declines, pathogen irruptions, ice storms) are brief and much less dramatic. Spatial and temporal patterns in C pools and fluxes in the mature forest at the HBEF reflect variation in environmental factors. Temperature and growing-season length undoubtedly constrain C fluxes at the HBEF; however, temperature effects on leaf respiration may largely offset the effects of growing season length on photosynthesis. Occasional severe droughts also affect C flux by reducing both photosynthesis and soil respiration. In younger stands nutrient availability strongly limits NPP, but the role of soil nutrient availability in limiting C flux in the mature forest is not known. A portion of the elevational variation of ANPP within the HBEF probably is associated with soil resource limitation; moreover, sites on more fertile soils exhibit 20–25% higher biomass and ANPP than the forest-wide average. Several prominent biotic influences on C pools and fluxes also are clear. Biomass and NPP of both the young and mature forest depend upon tree species composition as well as environment. Similarly, litter decay differs among tree species and forest types, and forest floor C accumulation is twice as great in the spruce–fir–birch forests at higher elevations than in the northern hardwood forests, partly because of inherently slow litter decay and partly because of cold temperatures. This contributes to spatial patterns in soil solution and streamwater dissolved organic carbon across the Hubbard Brook Valley. Wood decay varies markedly both among species and within species because of biochemical differences and probably differences in the decay fungi colonizing wood. Although C biogeochemistry at the HBEF is representative of mountainous terrain in the region, other sites will depart from the patterns described at the HBEF, due to differences in site history, especially agricultural use and fires during earlier logging periods. Our understanding of the C cycle in northern hardwood forests is most limited in the area of soil pool size changes, woody litter deposition and rhizosphere C flux processes.     

Kinetic investigation of Escherichia coli RNA polymerase mutants that influence nucleotide discrimination and transcription fidelity

Recent RNA polymerase (RNAP) structures led to a proposed three-step model of nucleoside triphosphate (NTP) binding, discrimination, and incorporation. NTPs are thought to enter through the secondary channel, bind to an E site, rotate into a pre-insertion (PS) site, and ultimately align in the catalytic (A) site. We characterized the kinetics of correct and incorrect incorporation for several Escherichia coli RNAPs with substitutions in the proposed NTP entry pore (secondary channel). Substitutions of the semi-conserved residue betaAsp(675), which is >10A away from these sites, significantly reduce fidelity; however, substitutions of the totally conserved residues betaArg(678) and betaAsp(814) do not significantly alter the correct or incorrect incorporation kinetics, even though the corresponding residues in RNAPII crystal structures appear to be interacting with the NTP phosphate groups and coordinating the second magnesium ion in the active site, respectively. Structural analysis suggests that the lower fidelity of the betaAsp(675) mutants most likely results from reduction of the negative potential of a small pore between the E and PS sites and elimination of several structural interactions around the pore. We suggest a mechanism of nucleotide discrimination that is governed both by rotation of the NTP through this pore and subsequent rearrangement or closure of RNAP to align the NTP in the A site.     

Analysis of the mouse mutant Cloth-ears shows a role for the voltage-gated sodium channel Scn8a in peripheral neural hearing loss

Deafness is the most common sensory disorder in humans and the aetiology of genetic deafness is complex. Mouse mutants have been crucial in identifying genes involved in hearing. However, many deafness genes remain unidentified. Using N -ethyl N −nitrosourea (ENU) mutagenesis to generate new mouse models of deafness, we identified a novel semi-dominant mouse mutant, Cloth-ears ( Clth ). Cloth-ears mice show reduced acoustic startle response and mild hearing loss from ∼30 days old. Auditory-evoked brainstem response (ABR) and distortion product otoacoustic emission (DPOAE) analyses indicate that the peripheral neural auditory pathway is impaired in Cloth-ears mice, but that cochlear function is normal. In addition, both Clth/Clth and Clth/+ mice display paroxysmal tremor episodes with behavioural arrest. Clth/Clth mice also show a milder continuous tremor during movement and rest. Longitudinal phenotypic analysis showed that Clth/+ and Clth/Clth mice also have complex defects in behaviour, growth, neurological and motor function. Positional cloning of Cloth-ears identified a point mutation in the neuronal voltage-gated sodium channel α-subunit gene, Scn8a , causing an aspartic acid to valine (D981V) change six amino acids downstream of the sixth transmembrane segment of the second domain (D2S6). Complementation testing with a known Scn8a mouse mutant confirmed that this mutation is responsible for the Cloth-ears phenotype. Our findings suggest a novel role for Scn8a in peripheral neural hearing loss and paroxysmal motor dysfunction.     

Ostracods and the Holocene palaeolimnology of Lake Qarun, with special reference to past human–environment interactions in the Faiyum (Egypt)

We present an ostracod record covering the past two millennia from an 8.25-m core taken from Lake Qarun, in the Faiyum Depression of Egypt. The occurrence of ostracod species in the lake is controlled primarily by variations in solute composition, which are in turn related to shifts in catchment land use. At times when the Faiyum Depression supported thriving agriculture, lake water contained Na⁺–Cl^? brine, and Cyprideis torosa dominated the ostracod assemblage. When the Faiyum Depression experienced periods of environmental and economic decline, lake water contained Na⁺–HCO₃ ^? brine, and Limnocythere inopinata dominated. The relative abundance of other ostracod species provides additional information about past conditions in Lake Qarun including salinity and lake level changes. Overall, the ostracod assemblages provide evidence for human influences in the Faiyum, which extend back before instrumental or detailed observational records began.     

Elaboration of cellulose fibrils by Agrobacterium tumefaciens during attachment to carrot cells.

The attachment of virulent strains of Agrobacterium tumefaciens to plant cells is the first step in the bacterial induction of tumors. Binding of A. tumefaciens to carrot tissue culture cells occurred as a two-step process. The initial step was the attachment of the bacteria to the plant cell wall. Living plant cells were not required. Bacterial attachment to heat-killed or glutaraldehyde-fixed carrot cells proceeded with only slightly altered kinetics and unaltered bacterial strain specificity. After the bacteria bound to the carrot cell surface, scanning electron microscopy showed that fibrils developed, surrounded the bacteria, and anchored them to the plant cell surface. These fibrils were synthesized by the bacteria and not by the plant cell since they were also made after the attachment of A. tumefaciens to dead carrot cells and since under some conditions the bacteria synthesized fibrils in the absence of plant cells. Calcofluor staining, acid hydrolysis, enzymatic digestion studies, and infrared spectroscopy showed that the fibrils were composed of cellulose. The formation of these cellulose fibrils occurred during the attachment of virulent strains of A. tumefaciens to plant cells in vitro. The fibrils anchored the bacteria to the plant cell surface and entrapped additional bacteria. The multiplication of entrapped and attached bacteria resulted in the formation of large clusters of bacteria held close to the plant cell wall and plasma membrane by cellulose fibrils. This high concentration of bacteria may facilitate transfer of Ti plasmid deoxyribonucleic acid to the plant cell resulting in the formation of tumors.