Search

Background Oral squamous cell carcinomas (OSCC) secrete exosomes into the surrounding extracellular environment to promote the horizontal transfer of bioactive molecules including microRNA (miRNA). The primary objective of this study was to explore potential differences in miRNA content between OSCC and OSCC stem cells. Methods The OSCC cell lines SCC4, SCC15, SCC25 and CAL27 were used in these studies. The corresponding OSCC stem cells that demonstrated phenotypic adhesion independent tumor spheres (AiTS) were manually isolated. All cells were cultured in DMEM containing 10% exosome-free fetal bovine serum. Exosomes were isolated using Total Exosome Isolation reagent (Invitrogen) and RNA was purified using Total Exosome RNA isolation kit (Invitrogen). Exosome miRNA content was evaluated using miRNA Advanced Taqman Assays for miR-21, − 155, − 133, − 34, − 31, − 32, and − 365. The fold change of miRNA content was calculated using the comparative CT (ΔΔCT) method using miR-16 as an endogenous control. Results After successful cell cultures were established, AiTS (cancer stem cells) were manually separated and confirmed using CD133 and Sox-2 biomarkers. Exosomes and extracellular vesicles were successfully isolated from all cell lines and AiTS isolates for miRNA screening. All isolates exhibited miR-16 expression (positive control), but none contained mir-31, − 32, or 133a. Differential expression of miR-21, miR-34 and miR-155 were observed with patterns observed among the cancer cell lines which were distinct from the corresponding AiTS isolates. Conclusions Exosomes isolated from these different OSCC stem cell populations displayed nearly consistent downregulation/loss of miR-21 and miR-34 suggesting the possibility of a unique miRNA profile characteristic of oral cancer stem cells. These findings highlight the need to investigate the comprehensive functions of miR-21 and miR-34 in tumor progression and continued research to refine a miRNA profile that could aid in distinguishing tumors with poor prognosis.

First estimates of sex allocation patterns and body size-at-sexual maturity and at protogynous sex change are presented for the five major (including one endemic) species of parrotfishes of Hawaii. Median body size at initial maturation as a female (LM50) and at protogynous sex change from adult female to adult male (LΔ50) varied greatly among the five species. Estimates of LM50 were about 14, 17, 24, 34 and 35 cm fork length (LF) in palenose Scarus psittacus, Pacific bullethead Chlorurus spilurus, stareye Calotomus carolinus, spectacled Chlorurus perspicillatus and redlip parrotfish Scarus rubroviolaceus. Values of LΔ50 were c. 23, 27, 37, 46 and 47 cm LF in the respective species. Length at female maturation was proportional to maximum body size (Lmax) of the respective species, ranging from 50 to 72% and averaging 62% of Lmax across species. LΔ50 was also proportional to Lmax, ranging from 82 to 97% and averaging 92%. Males of both pairs of Scarus and Chlorurus spp. reported here are diandric. Only one of the five major species (C. carolinus) is functionally monandric, with either all or nearly all males secondarily derived from adult females. The broadly differing absolute body sizes at sexual maturation and at sex change among the five species have important implications for improving regulatory size limits for parrotfishes in the State of Hawaii, where parrotfish species have historically been managed based on a single minimum size limit of 30·5 cm LF. This study provides a model demonstration of why catch data for parrotfishes, and other size-structured reef-fish populations, should be recorded either by species or by functional size-groups of species that allow setting more meaningful minimum size limits.

During 1994 and 1995, tests of ultra-low-volume applications of insecticides conducted on cotton, Gossypium hirsutum L., at Stoneville, MS, evaluated the effectiveness of malathion applied with a new technology in ground application, the air-assisted ultra-low-volume ground sprayer. Two tests conducted in 1994 determined the effectiveness of ultra-low-volume applications of malathion (91%) with and without cottonseed oil and established effective malathion rates against boll weevils , Anthonomus grandis grandis Boheman. An emulsifiable concentrate (EC) formulation of azinphosmethyl was used as a standard. In 1995, ultra-low-volume malathion (95%) was applied at 1.36 and 1.02 kg (AI)/ha plus cottonseed oil in a total volume of 1.17 liter/ha. These treatments were compared with a 1.12kg (AI)/ha rate of an EC formulation of malathion applied with water in a total volume of 46.8 liter/ha. In the 1994 insecticide comparison test, ultra-low-volume malathion treatments resulted in 70% mortality throughout the 48-h test period, whereas mortality from the azinphosmethyl standard steadily decreased from 71% at 4 h after treatment to 12% at 48 h after treatment. In the 1994 malathion rate test, 1.30 and 0.98 kg (AI)/ha produced mortalities comparable to those of azinphosmethyl at 0.28 kg (AI)/ha. In 1995, 48-h mortalities at 24 and 48 h after treatment were highest for the rate at 1.36 kg (AI)/ha (99 and 90%, respectively), followed by the rate at 1.02 kg (AI)/ha (78 and 54%, respectively), and lowest in the EC formulation treatment (28 and 21%, respectively). There were no differences in mortality at 72 h after treatment. The air-assisted ground sprayer permitted application of insecticides at considerably lower volumes than a conventional hydraulic ground sprayer and also permitted a reduction in the current rate of ultra-low-volume malathion required for effective boll weevil control.

Biological herbicides will assume greater importance for weed control as the number of chemicals registered for use decreases. Two promising fungi pathogenic on Datura stramonium and Cassia obtusifolia are, respectively, Alternaria crassa and Alternaria cassiae. In order to develop technologies for the efficient production of spore inocula in submerged liquid culture, we studied the effect of volatile factors which influenced sporulation on solid media. Sporulation was quantified on Richard's V8 agar under individual and joint variation of carbon dioxide (CO2) and relative humidity (RH). Sporulation of both fungi was suppressed when the two factors increased but not when CO2 was absorbed and RH remained high. Incubation of cultures in a CO2 controlled cabinet with RH constant indicated that CO2 was the suppressant.

Activation of endothelin (ET) receptors in the liver causes vasoconstriction, glucose production, and lipid and peptide mediator synthesis. In the intact rat, a bolus infusion of endotoxin into a mesenteric vein served as an acute exposure model of endotoxemia. In response to this challenge, a ninefold increase in hepatic ET-1 mRNA occurred within 3 h. The plasma level of immunoreactive ET-1 (irET-1) increased correspondingly by 8.5-fold within 6 h. ET-1 mRNA levels in liver endothelial cells (EC) isolated from livers of endotoxin-treated rats at various times after endotoxin challenge showed a more gradual increase. Northern blot analyses of the major liver cell types demonstrated that ET-1 mRNA was most abundant in the EC. The present results document a significant increase in the circulating level of irET-1 during episodes of endotoxemia. The increased hepatic ET-1 production in response to endotoxin infusion suggests that ET-1 produced in the liver could make a significant contribution to the plasma irET-1 and may be an important component in the hepatic responses to systemic trauma.

During development, the precursors for eggs and sperm — the germ cells — migrate to the gonads to mature. But what attracts them there? Results from a fruitfly screen indicate that one component in this pathway is a gene called columbus, which encodes HMG-CoA reductase. In humans, this enzyme is involved in cholesterol biosynthesis, raising the intriguing possibility that the attractant may be a lipid.

Alternaria crassa (Sacc.) Rands. and Alternaria cassiae Jurair & Khan are important fungal pathogens of jimsonweed (Datura stramonium) and sicklepod (Cassia obtusifolia), respectively (Walker, 1982; Boyette, 1986). Commercial production of these Alternaria spp. depends on numerous factors including cost-effective sporulation techniques. Efforts to elicit sporulation of Alternaria spp. in submerged culture have largely been unsuccessful. Other fungi produce far more new conidia than were initially present in the inoculum after growth in a bioreactor. However, in Alternaria spp. there is no morphological difference between newly formed conidia and old conidia and the inability to distinguish the two has hampered efforts to increase or improve conditions for conidia production. Consequently, a staining procedure was sought to identify newly formed spores in fungi, such as A. crassa and A. cassiae, where spore production may be limited by numerous factors. Studies were conducted in column bioreactors containing a minimal medium. Conidial inoculum was stained with Nile red fluorescent dye and added to the column bioreactors yielding final concentrations of 1 x 103 to 1 x 104 conidia/ml. Between 430 and 3.5 x 103 conidia/ ml were recovered from each fungus after 96 h in minimal media and approximately 20% of the total spores were identified as new conidia. Nile red, 9-diethylamino-5H-benzo(a)phenoxazine-5one (Sigma Co.), is a hydrophobic fluorescent dye which is highly selective for intracellular lipid droplets (Greenspan et al., 1985). It is nontoxic to fungi at concentrations up to 100 Ag/ml, stable, intensely fluorescent only in a hydrophobic environment and very soluble in lipids (Butt et al.

Utilization of soybean oil (SBO) in swine starter diets and the effect of an adaptation period to SBO on performance was studied in two 4-wk experiments with pigs initially 4 to 5 wk of age. One trial was extended an additional 4 wk and apparent DE was measured during the 4th wk postweaning. Corn-soybean meal-based diets were used in which SBO was substituted for cornstarch at 2, 4 and 6% of the diet in Exp. 1 and at 3 and 6% of the diet in Exp. 2. In Exp. 2, SBO was added either initially (adaptation) or after 2 wk (no adaptation). In Exp. 1 daily gain and efficiency of feed utilization were improved (P less than .05) by adding SBO during wk 3 and 4, but not earlier. Feed intake was not affected by treatment. In Exp. 2, for the initial 4-wk postweaning period, there was no effect of SBO addition on growth rate; feed intake was depressed (P less than .05) when SBO was added at wk 3, and efficiency of feed utilization was improved (P less than .05) for pigs fed SBO without an adaptation period. The primary improvements in performance occurred in the 3rd and 4th wk postweaning, with a tendency for pigs to perform better without an adaptation period. During wk 4, pigs adapted to SBO consumed more (P less than .05) feed but tended to be less efficient (P less than .10) than nonadapted pigs. With or without adaptation, apparent DE was unaffected by SBO additions. Regardless of adaptation period, SBO additions depressed feed intake (P less than .05) and improved efficiency of feed utilization (P less than .01) for the 5- to 8-wk growing phase. In conclusion, adding SBO the first 2 wk postweaning did not improve performance; an adaptation period was not necessary for a performance response from added SBO.

The genetic loci fushi tarazu (ftz), hairy (h), and engrailed (en) must be expressed during embryogenesis for the correct segmental organization of the Drosophila embryo to be established. Transcripts from these genes accumulate in periodic patterns at the blastoderm stage of development. We demonstrate that generation of the normal pattern of ftz RNA requires the h+ function whereas formation of the h pattern does not require the ftz gene. In addition, we show that the en pattern is altered in both h and ftz mutant embryos. The nature of these changes and the cuticular phenotype of h mutant larvae suggest that both h and ftz phenotypes are a consequence of incorrect compartmentalization and that the effect of h is mediated via ftz and other genes.

We conducted a study of the relationship between changes in photosynthetic pigment content and water depth in Great Harbor near Woods Hole, Massachusetts, USA, on the green algae Ulva lactuca and Codium fragile and the red algae Porphyra umbilicalis and Chondrus crispus. A calibrated underwater photometer equipped with spectral band filters measured light attenuation by the water column. The depth required for a 10-fold diminution of photon flux was 3.6, 5.3, 6.0 and 6.0 m for red, blue, yellow and green light, respectively. Seaweeds were attached to vertically buoyed lines and left to adapt for 7 days; then, with their positions reversed, they were allowed to readapt for 7 days. All species showed greater photosynthetic pigment content with increased depth. Further, the ratio of phycobiliproteins and chlorophyll b to chlorophyll a increased with depth. Changes in pigment content were reversible and occurred in the absence of cell division. There was a net loss of pigments near the surface (high irradiance), and subsequent synthesis when seaweeds were transferred to a position deep in the water column (low irradiance). In contrast, seaweeds which were found in intertidal habitats changed only their pigment concentration, and not pigment ratio, a phenomena analogous to higher plant sun and shade adaptation. Therefore, seaweeds modify their photon-gathering photosynthetic antennae to ambient light fields in the water column by both intensity adaptation and complementary chromatic adaptation.

Mutations of the hairy locus in Drosophila may affect both adult chaeta differentiation and embryonic segmentation. In an effort to understand this phenotypic complexity, we have analyzed 30 mutant alleles of the locus. We find that the alleles fall into four groups according to their complementation properties, suggesting a structurally complex locus in which two distinct functions share a common coding region.

A series of plasmids encoding various Klebsiella pneumoniae nif (nitrogen fixation) genes were constructed to determine which were required to produce active iron (Fe) protein in Escherichia coli, a species which does not normally fix nitrogen. The greatest success was achieved with binary plasmid systems that produced nifA regulatory protein under the control of a tac promoter on one plasmid, which then induced synthesis of nifH and nifM proteins from their native promoter sites on a second plasmid. nifH protein, the monomeric subunit of Fe protein, produced in the presence of nifM constituted nearly 10% of the whole cell protein and exhibited the corresponding amount of C2H2-reducing activity in nitrogenase assays conducted in vitro. nifH protein formed in the absence of nifM constituted 4.7% of the whole cell protein and exhibited no detectable activity in assays of whole cell extracts. The plasmid-encoded Fe protein was purified to homogeneity and was found to be indistinguishable from that isolated from derepressed wild type K. pneumoniae, having a similar specific activity, approximately 4 Fe/dimer of 68 kDa, and similar epr features. Although these experiments do not exclude the participation of other E. coli gene products in the maturation of nifH protein, they limit the nif-specific genes required for active Fe protein production to nifA, nifH, and nifM. Since nifA is thought to be the required activator protein involved in nif operon transcription, the simplest explanation for these observations is that nifH codes for the peptide of the Fe protein, while nifM acts to convert this nifH peptide to the functioning Fe protein of nitrogenase. In the absence of nifM, only an inactive nifH polypeptide is produced.

The distribution of cyanobacteria in the surface waters of the North Sea was measured during July 1987. Numbers of cyanobacteria ranged from 2.5x106 to 1.7x108 cells 1-1. In the majority of stations, cyanobacterial numbers were highest in the near-surface water and a subsurface maximum was found at only one station. The distribution of 14C among the end-products of photosynthesis was determined for picoplankton ( 1 μm throughout the North Sea. The majority of label was found in the protein fraction of both picoplankton and >1 μm phytoplankton; incorporation into lipids and polysaccharides plus nucleic acids was much lower. We interpret the large incorporation into protein to be a consequence of nutrient limitation of these natural assemblages. Photosynthetic parameters of the two size fractions were also determined. Assimilation number (PmB) and initial slope αβ were greater for the picoplankton fraction than for phytoplankton >1 μm but there was no evidence of significant photoinhibition of either fraction at irradiances up to 1 000 μE m-2 s-1.

Segmentation of the Drosophila embryo requires expression of the pair-rule genes, mutations of which cause reiterated deletions in alternate segments along the antero-posterior body axis. We find that transcripts of one such gene, hairy, accumulate in eight distinct regions of the early embryo. This pattern of expression is compared with that of another pair-rule gene, fushi tarazu, and its dependence on maternally expressed genes is described.

Rhodopseudomonas viridis ATCC 19567 grows by means of nitrogen fixation in yeast extract-N2 or nitrogen-free medium when sparged with 5% CO2 and 95% N2 in the light at 30 degrees C. Acetylene reduction assays for nitrogenase activity revealed an initially high level of activity during early-logarithmic growth phase, a lower plateau during mid- to late-logarithmic phase, and a dramatic reduction of activity at the beginning of the stationary phase. When viewed by electron microscopy, nitrogen-fixing R. viridis cells appeared to be morphologically and ultrastructurally similar to cells grown on nitrogen-rich media. Whole cells prepared under reducing conditions in the dark for electron spin resonance spectroscopy yielded g4.26 and g3.66 signals characteristic of the molybdenum-iron protein of nitrogenase. During growth on N2 in the absence of fixed-nitrogen sources, the nitrogenase activity of R. viridis measured by acetylene reduction stopped rapidly in response to the addition of NH4Cl as has been observed in other Rhodospirillaceae. However, unlike the nitrogenase of Rhodopseudomonas palustris or Rhodospirillum rubrum, which recover from this treatment within 40 min, the nitrogenase activity of R. viridis was not detectable for nearly 4 h.

The first hint of segmentation in the Drosophila embryo is the appearance of shallow transverse grooves that divide the main part of the germ band into 14 uniform domains1. These grooves are conspicuous in the 5–7-h-old embryo when viewed in the scanning electron microscope2 and can also be seen in longitudinal sections. Since their discovery in 1950, the domains delineated by the grooves haye been thought to be segments1,2, but now several lines of evidence suggest that they are ‘parasegments’—units consisting of a posterior compartment3 of one segment and an anterior compartment of the next4,5. If this is the case, the posterior compartments would lie in the anterior parts, of the domains delineated by the grooves. By using the engrailed gene product as a marker, we have now mapped the posterior compartments and report here that they are indeed located in the anterior quarter of the domains and that their anterior borders coincide with the grooves, establishing that the domains are parasegments.

Mutations of the segmentation gene Krüppel (Kr) cause deletions of contiguous sets of body segments from the middle region of the Drosophila embryo. We have monitored expression in situ of three other genes implicated in the establishment of the body plan, namely hairy (h), fushi tarazu (ftz) and engrailed (en), in mutant Kr embryos. Our results show that the pattern of expression of all three genes depends upon Kr+ activity and are consistent with a hierarchical model of segmentation gene activity. In addition, we find that the initial expression of the homoeotic selector gene Ultrabithorax(Ubx) follows a novel pattern in Kr- embroys indicating a close integration of the spatial control of homoeotic and segmantation gene expression.

The whole-body flux and protein-synthesis rates were determined in two groups of adult males aged 20-30 years who were matched for height and weight. [15N]Glycine was used as a tracer. Flux and synthesis rates were calculated from the urinary ammonia 15N excretion enrichment. Group I consisted of international competition level oarsmen, while Group II engaged in no exercise program. Measurement of the urinary nitrogen excretion rate showed that the oarsmen excreted more than twice as much nitrogen as the control group (P less than 0.05).flux and synthesis were 21.3 +/- 3.2 gN/9 hr and 188.7 +/- 59.9 g protein/day for the oarsmen and 16.4 +/- 2.1 g N/9 hr and 201.7 +/- 33.0 g protein/day for the controls. Data are +/- 1 SD for the eight oarsmen and eight controls. The flux values were different at the P less than 0.05 level. We concluded that (i) an exercise program does not lead to an increase in the basal protein turnover rate in adequately nourished individuals.

The ultrastructure of Neurospora crassa hyphae has been characterized primarily by the thin section technique. Since the original work of Shatkin and Tatum1, however, the quality of thin sections has been hindered by difficulty in fixing, embedding, and staining. A recent study of N. crassa ascospores compared the ultrastructure as revealed by freeze-etch to that revealed by thin sections, demonstrating the utility of the freeze-etch method when applied to organisms that have been difficult to prepare for EM observation2. The present study applies freeze-fracture to II. crassa in an attempt to confirm and increase the ultrastructural information presently available.Neurospora crassa strains 73a and 74A were grown in Vogel's minimal citrate medium for 18 to 24 hours. The resultant hyphae were treated with 3% glutaraldehyde in 0.1 M phosphate buffer for 2 hours followed by immersion in 30% glycerol for 1 hour or transferred directly to 30% glycerol from growth medium without glutaraldehyde fixation.