Dynamic rearrangement of the filamentous actin network occurs during zoosporogenesis and encystment in the oomycete phytophthora cinnamomi

The organization of filamentous actin (F-actin) in living cells of the oomycete Phytophthora cinnamomi was determined during zoosporogenesis and zoospore encystment by microinjecting sporangia with fluorescently labeled phalloidin and observing resultant fluorescence by confocal microscopy. In multinucleate sporangia prior to the induction of cleavage, phalloidin labeling took the form of plaques which occurred mainly in the periphery of the sporangia. After induction of cleavage, phalloidin labeling showed that the plaques disappeared and that F-actin began to accumulate along the developing cleavage planes and around nuclei and water expulsion vacuoles. F-actin labeling was also observed near the plasma membrane in zoospores and young cysts but reverted to the plaque form in older cysts. Localization of F-actin close to the developing cleavage planes is consistent with the idea that actin microfilaments function in the positioning and expansion of the cleavage membranes. Observations of plaques of actin in living sporangia provide evidence that plaques are not aldehyde-induced fixation artifacts. Copyright 1998 Academic Press.     

Serglycin expression during monocytic differentiation of U937-1 cells

Serglycin is the major proteoglycan in most hematopoietic cells, including monocytes and macrophages. The monoblastic cell line U937-1 was used to study the expression of serglycin during proliferation and differentiation. In unstimulated proliferating U937-1 cells serglycin mRNA is nonconstitutively expressed. The level of serglycin mRNA was found to correlate with the synthesis of chondroitin sulfate proteoglycan (CSPG). The U937-1 cells were induced to differentiate into different types of macrophage-like cells by exposing the cells to PMA, RA, or VitD3. These inducers of differentiation affected the expression of serglycin mRNA in three different ways. The initial upregulation seen in the normally proliferating cells was not observed in PMA treated cells. In contrast, RA increased the initial upregulation, giving a reproducible six times increase in serglycin mRNA level from 4 to 24 h of incubation, compared to a four times increase in the control cells. VitD3 had no effect on the expression of serglycin mRNA. The incorporation of (35S)sulfate into CSPG decreased approximately 50% in all three differentiated cell types. Further, the (35S)CSPGs expressed were of larger size in PMA treated cells than controls, but smaller after RA treatment. This was due to the expression of CSPGs, with CS-chains of 25 and 5 kDa in PMA and RA treated cells, respectively, compared to 11 kDa in the controls. VitD3 had no significant effect on the size of CSPG produced. PMA treated cells secreted 75% of the (35S)PGs expressed, but the major portion was retained in cells treated with VitD3 or RA. The differences seen in serglycin mRNA levels, the macromolecular properties of serglycin and in the PG secretion patterns, suggest that serglycin may have different functions in different types of macrophages.      

Abrupt permafrost thaw drives spatially heterogeneous soil moisture and carbon dioxide fluxes in upland tundra

Permafrost thaw causes the seasonally thawed active layer to deepen, causing the Arctic to shift toward carbon release as soil organic matter becomes susceptible to decomposition. Ground subsidence initiated by ice loss can cause these soils to collapse abruptly, rapidly shifting soil moisture as microtopography changes and also accelerating carbon and nutrient mobilization. The uncertainty of soil moisture trajectories during thaw makes it difficult to predict the role of abrupt thaw in suppressing or exacerbating carbon losses. In this study, we investigated the role of shifting soil moisture conditions on carbon dioxide fluxes during a 13-year permafrost warming experiment that exhibited abrupt thaw. Warming deepened the active layer differentially across treatments, leading to variable rates of subsidence and formation of thermokarst depressions. In turn, differential subsidence caused a gradient of moisture conditions, with some plots becoming consistently inundated with water within thermokarst depressions and others exhibiting generally dry, but more variable soil moisture conditions outside of thermokarst depressions. Experimentally induced permafrost thaw initially drove increasing rates of growing season gross primary productivity (GPP), ecosystem respiration (R_eco), and net ecosystem exchange (NEE) (higher carbon uptake), but the formation of thermokarst depressions began to reverse this trend with a high level of spatial heterogeneity. Plots that subsided at the slowest rate stayed relatively dry and supported higher CO₂ fluxes throughout the 13-year experiment, while plots that subsided very rapidly into the center of a thermokarst feature became consistently wet and experienced a rapid decline in growing season GPP, R_eco, and NEE (lower carbon uptake or carbon release). These findings indicate that Earth system models, which do not simulate subsidence and often predict drier active layer conditions, likely overestimate net growing season carbon uptake in abruptly thawing landscapes.     

Purification and characterization of squid brain myosin.

Myosin was extracted from frozen squid brain and purified by a modification of the procedure of Pollard et al. (Pollard, T.D., Thomas, S.M., and Niederman, R. (1974) Anal. Biochem. 60, 258-266). Myosin was eluted from Bio-Gel A-15m column as a single peak of (K+-EDTA)-activated ATPase ((K+-EDTA)-ATPase) activity with an average partition coefficient (Kav) of 0.22. In sodium dodecyl sulfate-acrylamide gel electrophoresis, the purified myosin showed a predominant band with similar electrophoretic mobility as the heavy chain of rabbit skeletal muscle myosin, and two less intense bands near the bottom of the gel. No actin band was seen. The properties of the (K+-EDTA)-ATPase activity were: (a) the time course of the reaction was biphasic at 25 degrees but linear at 32 degrees; (b) the optimum rate of reaction was obtained between 0.3 and 0.8 M KCl; (c) the pH optimum was between 8.0 and 9.0; (d) the reaction was specific for ATP with an apparent Km of 0.19 mM. ATPase activity in 0.06 M KCl and 5 mM MgCl2 was increased about 1.5 times by a 10-fold excess of rabbit skeletal muscle F-actin and about 5 times by a 40-fold excess. The actin activation was inhibited slightly by the addition of 0.2 mM CaCl2 and completely by the addition of 10 mM CaCl2. Myosin formed arrowhead patterns with rabbit skeletal muscle F-actin as observed by electron microscopy of negatively stained samples. It also aggregated in bipolar filaments which attached to decorated actin filaments at different angles, as well as formed cross-connections and ladder-like patterns between actin filaments. These two forms of interactions between myosin and actin were abolished by treatment with MgATP.     

Catalytic degradation of vitamin D up-regulated protein 1 mRNA enhances cardiomyocyte survival and prevents left ventricular remodeling after myocardial ischemia

Vitamin D3 up-regulated protein 1 (VDUP1) is a key mediator of oxidative stress on various cellular processes via downstream effects on apoptosis signaling kinase 1 (ASK1) and p38 mitogen-activated protein kinase (MAPK). Here, we report that VDUP1 expression is significantly increased in rat hearts following acute myocardial ischemia, suggesting it may have important regulatory effects on cardiac physiological processes during periods of oxidative stress. Transfection of H9C2 cardiomyoblasts with a sequence-specific VDUP1 DNA enzyme to down-regulate VDUP1 mRNA expression significantly reduced apoptosis and enhanced cell survival under conditions of H(2)O(2) stress, and these effects involved inhibition of ASK1 activity. Direct intracardiac injection of the DNA enzyme at the time of acute myocardial infarction reduced myocardial VDUP1 mRNA expression and resulted in prolonged reduction in cardiomyocyte apoptosis and ASK1 activity. Moreover, down-regulation of VDUP1 was accompanied by significant reduction in cardiac expression of pro-collagen type I alpha2 mRNA level, as well as marked reduction in myocardial scar formation. These features were accompanied by significant improvement in cardiac function. Together, these results suggest a direct role for VDUP1 in the adverse effects of ischemia and oxidative stress on cardiomyocyte survival, left ventricular collagen deposition, and cardiac function. Strategies to inhibit VDUP1 expression and/or function during acute ischemic events may be beneficial to cardiac functional recovery and prevention of left ventricular remodeling.     

An integrated map of human 6q22.3-q24 including a 3-Mb high-resolution BAC/PAC contig encompassing a QTL for fetal hemoglobin

Genetic studies have previously assigned a quantitative trait locus (QTL) for hemoglobin F and F cells to a region of approximately 4 Mb between the markers D6S408 and D6S292 on chromosome 6q23. An initial yeast artificial chromosome contig of 13 clones spanning this region was generated. Further linkage analysis of an extended kindred refined the candidate interval to 1-2 cM, and key recombination events now place the QTL within a region of <800 kb. We describe a high-resolution bacterial clone contig spanning 3 Mb covering this critical region. The map consists of 223 bacterial artificial chromosome (BAC) and 100 P1 artificial chromosome (PAC) clones ordered by sequence-tagged site (STS) content and restriction fragment fingerprinting with a minimum tiling path of 22 BACs and 1 PAC. A total of 194 STSs map to this interval of 3 Mb, giving an average marker resolution of approximately one per 15 kb. About half of the markers were novel and were isolated in the present study, including three CA repeats and 13 single nucleotide polymorphisms. Altogether 24 expressed sequence tags, 6 of which are unique genes, have been mapped to the contig.     

Urinary excretion of collagen degradation markers by sows during postpartum uterine involution

Incomplete uterine involution is the putative cause of the increased embryo mortality and reproductive failure often exhibited by sows that lactate for less than 21 days. Since such short lactation lengths are common in American swine production, an effective technique to monitor the postpartum involution process and test this hypothesis might be valuable. Rapid and extensive catabolism of uterine collagen is essential for normal postpartum involution. The objective of this study was to characterize postpartum excretion of two biochemical markers of collagen degradation. In experiment I, urine samples were collected from five sows every other day from the day before parturition (day -1), through a 21-day lactation, to day 8 postweaning. The collagen crosslinks hydroxylysyl pyridinoline (HP), which is present in many tissues, and lysyl pyridinoline (LP), which is primarily concentrated in bone, were assayed by both ELISA and HPLC. Urinary levels of both free (ELISA) and total (HPLC) HP and LP increased (P < 0.001) approximately two-fold during lactation. The mean molar ratio of total HP:LP increased (P < 0.001) from 6.6 +/- 1.6 at day 1 to a maximum of 10.2 +/- 1.5 at day 7 postpartum and averaged 9.1 +/- 0.3 for the entire sampling period. These data are consistent with a postpartum increase of soft tissue collagen catabolism since bone has a low HP:LP ratio of 4 and soft tissues like the uterus have a high HP:LP ratio of >/=20 because they contain only trace amounts of LP. Since HPLC (total) and ELISA (free) crosslinks estimates were highly correlated (r = 0.85-0.91, P < 0.001) in experiment I, only the less technical ELISA technique was used in experiment II. Urine samples were collected from 21 sows every third day from day 1 to 19 of lactation. Sows from this second group exhibited one of four distinct crosslinks excretion patterns: peak on day 1 (n = 3), peak on day 7 (n = 4), peak on day 10, 13 or 16 (n = 7), or no peak (n = 7). This variation of postpartum crosslinks excretion among sows was not related to parity, body weight, lactation body weight change, litter size, or litter birth weight. Overall, data from experiments I and II indicate that urinary HP does increase postpartum in a pattern temporally consistent with uterine involution. However, significant variation among sows in the magnitude and timing of peak HP excretion was evident.     

Localisation of the Vacuolar Proton Pump (V-H+-ATPase) and Carbonic Anhydrase II in the Human Eccrine Sweat Gland

The localisation of the vacuolar proton pump (V-H+ -ATPase) and the enzyme carbonic anhydrase II (CAII) was investigated in the human eccrine sweat gland employing standard immunohistochemical techniques after antigen retrieval using microwave heat treatment and high pressure. The high-pressure antigen retrieval unmasked the presence of V-H+ -ATPase in the clear cells of the secretory coil, with a distribution similar to that previously observed for CAII. However, the dark cells were unreactive to both antibodies. In addition, heat and high-pressure antigen retrieval demonstrated the presence of CAII in the apical zone of luminal cells of the reabsorptive duct, a location not previously reported. The localisation of V-H+ -ATPase and CAII in the secretory coil clear cells suggests that the formation of HCO3- and H+ by carbonic anhydrase II and the transport of H+ by V-H+ -ATPase may play an role in sweat fluid secretion. Their presence at the apex of the duct cells indicates involvement in ductal ion reabsorption.