A SYMMETRICAL, EXTRACELLULAR FIBRIL

Symmetrical, extracellular fibrils, which are related to the "special fibrils" of the dermis described by Palade and Farquhar, have been found along the outer surface of the basement membrane covering the notochord in the tail of Rana catesbeiana (bullfrog) tadpoles. The fibrils are ~7,500 A long and occur singly or in clusters. The single fibrils are characterized by a symmetrical transverse band pattern and by attachment at both ends to the basement membrane. The clusters are various complex configurations which seemingly represent symmetrical fibrils in different states of aggregation. Symmetrical fibrils also occur in the skin of the tadpole tail and in the skin of the toad, Bufo marinus. It is proposed that a narrow, symmetrical fibril is the fundamental "special fibril."     

STUDIES ON BLOOD CAPILLARIES : II. Transport of Ferritin Molecules across the Wall of Muscle Capillaries

The pathway by which intravenously injected ferritin molecules move from the blood plasma across the capillary wall has been investigated in the muscle of the rat diaphragm. At 2 min after administration, the ferritin molecules are evenly distributed in high concentration in the blood plasma of capillaries and occur within vesicles along the blood front of the endothelium. At the 10-min time point, a small number of molecules appear in the adventitia, and by 60 min they are relatively numerous in the adventitia and in phagocytic vesicles and vacuoles of adventitial macrophages. Thereafter, the amount of ferritin in the adventitia and pericapillary regions gradually increases so that at 1 day the concentration in the extracellular spaces approaches that in the blood plasma. Macrophages and, to a lesser extent, fibroblasts contain large amounts of ferritin. 4 days after administration, ferritin appears to be cleared from the blood and from the capillary walls, but it still persists in the adventitial macrophages and fibroblasts. At all time points examined, ferritin molecules within the endothelial tunic were restricted to vesicles or to occasional multivesicular or dense bodies; they were not found in intercellular junctions or within the cytoplasmic matrix. Ferritin molecules did not accumulate within or against the basement membranes. Over the time period studied, the concentration of ferritin in the blood decreased, first rapidly, then slowly, in two apparently exponential phases. Liver and spleen removed large amounts of ferritin from the blood. Diaphragms fixed at time points from 10 min to 1 day, stained for iron by the Prussian Blue method, and prepared as cleared whole mounts, showed a progressive and even accumulation of ferritin in adventitial macrophages along the entire capillary network. These findings indicate: (1) that endothelial cell vesicles are the structural equivalent of the large pore system postulated in the pore theory of capillary permeability; (2) that the basement membrane is not a structural restraint in the movement of ferritin molecules across the capillary wall; (3) that transport of ferritin occurs uniformly along the entire length of the capillary; and (4) that the adventitial macrophages monitor the capillary filtrate and partially clear it of the tracer.     

Characteristics of a Hydrated, Alginate-Based Delivery System for Cultivation of the Button Mushroom

The production of the button mushroom Agaricus bisporus with mycelium-colonized alginate pellets as an inoculant of the growing medium was investigated. Pellets having an irregular surface and porous internal structure were prepared by complexing a mixture of 1% sodium alginate, 2 to 6% vermiculite, 2% hygramer, and various concentrations of Nutrisoy (soy protein) with calcium chloride. The porous structure allowed the pellets to be formed septically and then inoculated and colonized with the fungus following sterilization. By using an enzyme-linked immunosorbent assay (ELISA) to estimate fungal biomass, the matrix components of the pellet were found to be of no nutritive value to A. bisporus. Pellets amended with Nutrisoy at a concentration of 0.5 to 8% supported extensive mycelial growth, as determined by significantly increased ELISA values, with a concentration of 4% being optimal and higher concentrations proving inhibitory. The addition of hydrated, mycelium-invaded pellets to the compost or casing layer supported the thorough colonization of the growing substrate and culminated in the formation of mushrooms that showed normal development and typical morphology. Yields and sizes of mushrooms were comparable from composts seeded with either colonized pellets or cereal grain spawn. Similarly, amending the casing layer with pelletized-mycelium-colonized compost resulted in a 2- to 3-day-earlier and more-synchronous emergence of mushrooms than with untreated casing. This technology shows the greatest potential as a pathogen-free inoculant of the casing layer in the commercial cultivation of mushrooms.     

Suppression of gray leaf spot (blast) of perennial ryegrass turf by Pseudomonas aeruginosa from spent mushroom substrate

Bacteria isolated from spent mushroom substrate (SMS) were evaluated for the suppression of Pyricularia grisea, the causal agent of gray leaf spot of perennial ryegrass (Lolium perenne) turf. Thirty-two of 849 bacterial isolates (3.8%) from SMS significantly inhibited the mycelial growth of P. grisea in vitro. Six bacterial isolates that afforded maximum inhibition of P. grisea were also refractory to Rhizoctonia solani, Rhizoctonia cerealis, Sclerotinia homoeocarpa, and Fusarium culmorum. Each of the six isolates was identified as Pseudomonas aeruginosa by fatty acid profile analysis. The biocontrol activity of the bacterial isolates was not compromised by a prolonged exposure to UV radiation in vitro. In controlled-environment chamber experiments, all 32 bacterial isolates were tested for suppression of gray leaf spot on Pennfine perennial ryegrass when applied as seed treatment or foliar sprays. Foliar applications of the bacteria (10⁸ cfu/ml 0.1% carboxymethylcellulose), but not the seed treatment, significantly reduced disease severity and incidence. The three most efficient isolates from foliar application treatments, which were among the six bacterial isolates identified as P. aeruginosa, were further evaluated for suppression of gray leaf spot as a function of timing of application. The three isolates of P. aeruginosa suppressed gray leaf spot in perennial ryegrass in Cone-tainers when applied at 1, 3, and 7 days prior to inoculation with P. grisea both in controlled-environment chamber experiments, and in potted ryegrass plants maintained in the field. All application intervals, regardless of the bacterial isolate, provided significant reduction of gray leaf spot severity. Suppression of gray leaf spot by isolates of P. aeruginosa under controlled-environment chamber conditions was not different from that observed in potted ryegrass plants maintained in the field. In field experiments, an isolate of P. aeruginosa provided significant suppression of gray leaf spot when applied at 1, 7, and 14 days prior to inoculation with P. grisea. The bacterium proved effective against gray leaf spot of perennial ryegrass maintained as fairway and rough heights. These results indicate that P. aeruginosa may be a potential biocontrol agent for gray leaf spot of perennial ryegrass turf.     

SPERM WHALE DIVES TRACKED BY RADIO TAG TELEMETRY

Dives of a 12-m sperm whale ( Physeter catodon Linnaeus, 1758) were tracked in the southeast Caribbean by long range, 30 MHz radio tag with dive-profile telemetry over 4.6 d, 26 April-1 May 1995. Over the 295-km track, average speed was 0.7 m/sec (2.6 km/h). Of 158 dives (defined as submergences longer than 3 min), 65 were shallow (<200 m). The 93 deep dives averaged 990 m (range 420–1,330 m) in depth, and 44.4 min in duration (range 18.2–65.3 min). Water depth was at least 200 m deeper than the whale dive depth. The whale was engaged in activities at or near the surface, shallow dives, and deep dives during 22.6%, 23.4%, and 54% of the time, respectively. Depth and duration of dives were correlated, but there was little relationship between the length or depth of dives with the duration of surfacings either before or after dives. Deep dives occurred day and night. In 44.4% of the deep dives, the vertical movement of descents and ascents was interrupted at intermediate depths, lengthening these dives by an average of 10.8 min. During dives without stops at intermediate depths, descents averaged 11 min at 1.52 m/sec, and ascents averaged 11.8 min at 1.4 m/sec.     

A polymerase chain reaction-based test for Verticillium fungicola causing dry bubble disease on the cultivated mushroom,Agaricus bisporus

A polymerase chain reaction (PCR)-based test is described for the specific detection of Verticillium fungicola var. aleophilum (Vfa), the fungal pathogen causing dry bubble disease on the cultivated button mushroom, Agaricus bisporus. PCR primers were tailored to target a 162-bp arbitrary sequence in the Vfa genome. In PCR amplifications using the primer pair, all of 20 isolates of Vfa that had been collected during a 29-year period at commercial mushroom operations located primarily in North America were found to generate the diagnostic 162-bp DNA product. Conversely, the primers failed to produce the specific amplicon with DNA from isolates representing 5 other species of Verticillium, the pathogenic subspecies V. fungicola var. fungicola from Europe, and 12 other fungal species commonly inhabiting mushroom compost. A protocol was designed enabling a confirmed diagnosis of dry bubble disease in less than 3 h. The PCR-based test should find application for the rapid diagnosis and detection of the fungal pathogen in disease management programs and, potentially, in screening for on-the-farm sources of infection.     

CLIP-170 interacts with dynactin complex and the APC-binding protein EB1 by different mechanisms

CLIP-170 is a "cytoplasmic linker protein" implicated in endosome-microtubule interactions and in control of microtubule dynamics. CLIP-170 localizes dynamically to growing microtubule plus ends, colocalizing with the dynein activator dynactin and the APC-binding protein EB1. This shared "plus-end tracking" behavior suggests that CLIP-170 might interact with dynactin and/or EB1. We have used site-specific mutagenesis of CLIP-170 and a transfection/colocalization assay to address this question in mammalian tissue culture cells. Our results indicate that CLIP-170 interacts, directly or indirectly, with both dynactin and EB1. We find that the CLIP-170/dynactin interaction is mediated by the second metal binding motif of the CLIP-170 tail. In contrast, the CLIP-170/EB1 interaction requires neither metal binding motif. In addition, our experiments suggest that the CLIP-170/dynactin interaction occurs via the shoulder/sidearm subcomplex of dynactin and can occur in the cytosol (i.e., it does not require microtubule binding). These results have implications for the targeting of both dynactin and EB1 to microtubule plus ends. Our data suggest that the CLIP-170/dynactin interaction can target dynactin complex to microtubule plus ends, although dynactin likely also targets MT plus ends directly via the microtubule binding motif of the p150(Glued) subunit. We find that CLIP-170 mutants alter p150(Glued) localization without affecting EB1, indicating that EB1 can target microtubule plus ends independently of dynactin.     

Placental expression of estrogen receptor beta and its hormone binding variant – comparison with estrogen receptor alpha and a role for estrogen receptors in asymmetric division and differentiation of estrogen-dependent cells

During human pregnancy, the production of 17-beta-estradiol (E2) rises steadily to eighty fold at term, and placenta has been found to specifically bind estrogens. We have recently demonstrated the expression of estrogen receptor alpha (ER-alpha) protein in human placenta and its localization in villous cytotrophoblast (CT), vascular pericytes, and amniotic fibroblasts. In vitro, E2 stimulated development of large syncytiotrophoblast (ST) aggregates. In the present study we utilized ER-beta affinity purified polyclonal (N19:sc6820) and ER-alpha monoclonal (clone h-151) antibodies. Western blot analysis revealed a single ~52 kDa ER-beta band in chorionic villi (CV) protein extracts. In CV, strong cytoplasmic ER-beta immunoreactivity was confined to ST. Dual color immunohistochemistry revealed asymmetric segregation of ER-alpha in dividing villous CT cells. Prior to separation, the cell nuclei more distant from ST exhibited high ER-alpha, while cell nuclei associated with ST showed diminution of ER-alpha and appearance of ER-beta. In trophoblast cultures, development of ST aggregates was associated with diminution of ER-alpha and appearance of ER-beta immunoreactivity. ER-beta was also detected in endothelial cells, amniotic epithelial cells and fibroblasts, extravillous trophoblast (nuclear and cytoplasmic) and decidual cells (cytoplasmic only). In addition, CFK-E12 (E12) and CWK-F12 (F12) monoclonal antibodies, which recognize ~64 kDa ER-beta with hormone binding domain, showed nuclear-specific reactivity with villous ST, extravillous trophoblast, and amniotic epithelium and fibroblasts. Western blot analysis indicated abundant expression of a ~64 kDa ER-beta variant in trophoblast cultures, significantly higher when compared to the chorionic villi and freshly isolated trophoblast cell protein extracts. This is the first report on ER-beta expression in human placenta and cultured trophoblast. Our data indicate that during trophoblast differentiation, the ER-alpha is associated with a less, and ER-beta with the more differentiated state. Enhanced expression of ~64 kDa ER-beta variant in trophoblast cultures suggests a unique role of ER-beta hormone binding domain in the regulation of trophoblast differentiation. Our data also indicate that asymmetric segregation of ER-alpha may play a role in asymmetric division of estrogen-dependent cells.     

Estradiol abrogates apoptosis in breast cancer cells through inactivation of BAD: Ras-dependent nongenomic pathways requiring signaling through ERK and Akt

Estrogens such as 17-beta estradiol (E(2)) play a critical role in sporadic breast cancer progression and decrease apoptosis in breast cancer cells. Our studies using estrogen receptor-positive MCF7 cells show that E(2) abrogates apoptosis possibly through phosphorylation/inactivation of the proapoptotic protein BAD, which was rapidly phosphorylated at S112 and S136. Inhibition of BAD protein expression with specific antisense oligonucleotides reduced the effectiveness of tumor necrosis factor-alpha, H(2)O(2), and serum starvation in causing apoptosis. Furthermore, the ability of E(2) to prevent tumor necrosis factor-alpha-induced apoptosis was blocked by overexpression of the BAD S112A/S136A mutant but not the wild-type BAD. BAD S112A/S136A, which lacks phosphorylation sites for p90(RSK1) and Akt, was not phosphorylated in response to E(2) in vitro(.) E(2) treatment rapidly activated phosphatidylinositol 3-kinase (PI-3K)/Akt and p90(RSK1) to an extent similar to insulin-like growth factor-1 treatment. In agreement with p90(RSK1) activation, E(2) also rapidly activated extracellular signal-regulated kinase, and this activity was down-regulated by chemical and biological inhibition of PI-3K suggestive of cross talk between signaling pathways responding to E(2). Dominant negative Ras blocked E(2)-induced BAD phosphorylation and the Raf-activator RasV12T35S induced BAD phosphorylation as well as enhanced E(2)-induced phosphorylation at S112. Chemical inhibition of PI-3K and mitogen-activated protein kinase kinase 1 inhibited E(2)-induced BAD phosphorylation at S112 and S136 and expression of dominant negative Ras-induced apoptosis in proliferating cells. Together, these data demonstrate a new nongenomic mechanism by which E(2) prevents apoptosis.     

Allosteric antagonism of insect odorant receptor ion channels

Background

At a molecular level, insects utilize members of several highly divergent and unrelated families of cell-surface chemosensory receptors for detection of volatile odorants. Most odors are detected via a family of odorant receptors (ORs), which form heteromeric complexes consisting of a well-conserved OR co-receptor (Orco) ion channel and a non-conserved tuning OR that provides coding specificity to each complex. Orco functions as a non-selective cation channel and is expressed in the majority of olfactory receptor neurons (ORNs). As the destructive behaviors of many insects are principally driven by olfaction, Orco represents a novel target for behavior-based control strategies. While many natural and synthetic odorants have been shown to agonize Orco/Or complexes, only a single direct Orco modulator, VUAA1, has been described. In an effort to identify additional Orco modulators, we have investigated the structure/activity relationships around VUAA1.

Results

A search of our compound library identified several VUAA1 analogs that were selected for evaluation against HEK cells expressing Orco from the malaria vector Anopheles gambiae (AgOrco). While the majority of compounds displayed no activity, many of these analogs possess no intrinsic efficacy, but instead, act as competitive VUAA1 antagonists. Using calcium mobilization assays, patch clamp electrophysiology, and single sensillum in vivo recording, we demonstrate that one such candidate, VU0183254, is a specific allosteric modulator of OR signaling, capable of broadly inhibiting odor-mediated OR complex activation.

Conclusions

We have described and characterized the first Orco antagonist, that is capable of non-competitively inhibiting odorant-evoked activation of OR complexes, thereby providing additional insight into the structure/function of this unique family of ligand-gated ion channels. While Orco antagonists are likely to have limited utility in insect control programs, they represent important pharmacological tools that will facilitate the investigation of the molecular mechanisms underlying insect olfactory signal transduction.