Is nectar reabsorption restricted by the stalk cells of floral and extrafloral nectary trichomes?

Reabsorption is a phase of nectar dynamics that occurs concurrently with secretion; it has been described in floral nectaries that exude nectar through stomata or unicellular trichomes, but has not yet been recorded in extrafloral glands. Apparently, nectar reabsorption does not occur in multicellular secretory trichomes (MST) due to the presence of lipophilic impregnations - which resemble Casparian strips - in the anticlinal walls of the stalk cells. It has been assumed that these impregnations restrict solute movement within MST to occur unidirectionally and exclusively by the symplast, thereby preventing nectar reflux toward the underlying nectary tissues. We hypothesised that reabsorption is absent in nectaries possessing MST. The fluorochrome lucifer yellow (LYCH) was applied to standing nectar of two floral and extrafloral glands of distantly related species, and then emission spectra from nectary sections were systematically analysed using confocal microscopy. Passive uptake of LYCH via the stalk cells to the nectary tissues occurred in all MST examined. Moreover, we present evidence of nectar reabsorption in extrafloral nectaries, demonstrating that LYCH passed the stalk cells of MST, although it did not reach the deepest nectary tissues. Identical (control) experiments performed with neutral red (NR) demonstrated no uptake of this stain by actively secreting MST, whereas diffusion of NR did occur in plasmolysed MST of floral nectaries at the post-secretory phase, indicating that nectar reabsorption by MST is governed by stalk cell physiology. Interestingly, non-secretory trichomes failed to reabsorb nectar. The role of various nectary components is discussed in relation to the control of nectar reabsorption by secretory trichomes.

Response of the hairy mussel Trichomya hirsuta to sediment-metal contamination in the presence of a bioturbator.

The accumulation of metals in tissue compartments of bivalve biomonitors is expected to reflect the phases in which metals are most bioavailable. In concurrent field and laboratory experiments we measured Zn, Cd and Pb concentrations in the gills and digestive glands of mussels exposed to sediments from Lake Macquarie in NSW, Australia. Mussels in the laboratory were also exposed to the bioturbating gastropod Batillaria australis. Zn, Cd and Pb concentrations in gills and digestive glands of mussels from both experiments were accumulated in proportion with levels of metal contamination in the sediments. An interaction in the field between site and tissue type was found for Cd and Pb suggesting variation in the phases in which metals are most bioavailable. No effect of bioturbation on metal accumulation in the bivalve was detected and we conclude that it is unlikely to be a significant factor in metal uptake when these species interact.

First Report of Podosphaera xanthii Race 1W Causing Powdery Mildew of Watermelon in California.

In September and October 2012, powdery mildew was detected on watermelon (Citrullus lanatus var. lanatus) plants of various breeding lines growing in field plots in Davis, California. Plants had partially necrotic leaves, yellowing to brown in color, with white surface mycelium and faint sporulation. No teleomorph was observed. Infected leaves were collected for examination and a spore suspension of the field isolate was made in water with 0.01% Tween 20 to spray inoculate watermelon seedlings of cultivar Dixie Lee with two true leaves. Plants were incubated in a growth chamber (22 to 26°C, 12-h photoperiod) for approximately 10 days, until sporulation was apparent. Microscopic observation of conidial chains showed that they had clearly crenate edges indicative of Podosphaera xanthii (4). To confirm the identity of the pathogen, we used Podosphaera-specific primers PFITS-F (5'-CCAACTCGTGCTGAGTGT-3') and PF5.8-R (5'-TGTTGGTTTCTTTTCCTCCG-3') to amplify and sequence the internal transcribed spacer regions of the nuclear rDNA. The 326-bp sequence had 98% homology to the GenBank sequence (accessions JQ340082.1 and AB774158.1) for P. xanthii. Infected 'Dixie Lee' leaves were used to make a spore suspension (approximately 5 × 104 conidia/ml) as described above to inoculate watermelon, melon, and squash seedlings (2 to 3 plants per cultivar) in a greenhouse. It caused severe symptoms on all watermelon plants cv. Charleston 76, P8, and Sugar Baby in the form of a powdery mildew with surface mycelium and chains of conidia, with leaves becoming gradually more necrotic and eventually dying, with the appearance of a melting down. Non-inoculated plants did not develop symptoms. The isolate also infected all squash plants 'Zucchini Elite' and melon powdery mildew differentials Iran H and 'Védrantais.' On these plants, the pathogen produced a powdery mildew (white surface mycelium with sporulation) but did not cause extensive necrosis. All other melon powdery mildew differentials ('PMR5,' 'PMR45,' WMR29, MR1, PI 124112, and PI 313970) did not develop any powdery mildew. A follow-up test in a growth chamber (22 to 26°C, 12-h photoperiod) with the same set of species and cultivars gave the same results. Based on these results, we conclude that this isolate belongs to race 1W (1,2). The presence of race 1W could have implications in disease management for this crop in the Central Valley of California as most cultivars are not resistant to it and the disease has been shown to cause severe damage in other states (1,3). References: (1) A. R. Davis et al. J. Am. Soc. Hort. Sci. 132:790, 2007. (2) J. D. McCreight. Amer. Soc. Hort. Sci. 131:59, 2006. (3) A. Y. Tetteh et al. Crop Sci. 50:933, 2010. (4) T. A. Zitter. Page 28 in: Compendium of Cucurbit Diseases, The American Phytopathological Society, St. Paul, MN, 1996.

Sponges as sentinels: patterns of spatial and intra-individual variation in trace metal concentration.

If sponges are to be effective biomonitors we require a better understanding of the spatial scales over which metals vary in these organisms. We determined how concentration of Cd, Zn, Cu, Pb, Hg and Se varied over four spatial scales for two common estuarine sponge species in the Sydney region. We examined variability with a fully nested sampling design; between coastal lakes, within coastal lakes, between sponges and within sponges. Calculation of variance components confirmed that 'within-sponge' variation in Cd, Zn, Cu, Pb and Se concentrations were low (1-14%) relative to the two largest spatial scales (49-98%) examined. In contrast, Hg concentrations exhibited marked variability 'between-sponges' and were below detection at one location. There was little evidence that sponge size was a good predictor of metal concentration. Taken together, these outcomes confirm that fragments of these sponges could be successfully transplanted and therefore show promise as biomonitors of metal contamination.

Phylogeny of the families Pyuridae and Styelidae (Stolidobranchiata, Ascidiacea) inferred from mitochondrial and nuclear DNA sequences.

The Order Stolidobranchiata comprises the families Pyuridae, Styelidae and Molgulidae. Early molecular data was consistent with monophyly of the Stolidobranchiata and also the Molgulidae. Internal phylogeny and relationships between Styelidae and Pyuridae were inconclusive however. In order to clarify these points we used mitochondrial and nuclear sequences from 31 species of Styelidae and 25 of Pyuridae. Phylogenetic trees recovered the Pyuridae as a monophyletic clade, and their genera appeared as monophyletic with the exception of Pyura. The Styelidae, on the other hand, appeared as a paraphyletic group split into several clades. One of them was formed by solitary oviparous species, of which the Pyuridae were a sister group. A second clade included the colonial genera Botryllus, Botrylloides and Symplegma. The remaining colonial and solitary genera formed several poorly resolved clades. One of the more species genus, Polycarpa, was shown to be polyphyletic, and the species Styela plicata grouped into two genetically distant clades suggesting the existence of two cryptic species. The internal phylogeny of Styelidae has bearings on the origin of coloniality in this family. We suggest to abandon the traditional division of colonial forms into social and compound species and use instead the categories of aggregated colonies that do not have common vascular systems, and integrated colonies, that do possess such systems. Our molecular results indicate that there have been several independent acquisitions of coloniality in the Styelidae, and that viviparity may be a pre-adaptation for a colonial life-style.

Rapid method for total carotenoid detection in canary yellow-fleshed watermelon.

Yellow-fleshed watermelons (Citrullus lanatus[Thunb.] Matsum. and Nakai) contain many different carotenoids, all in low to trace amounts. Since there is not 1 predominant carotenoid in yellow-fleshed watermelon, testing the total carotenoid content among watermelon lines is important in determining the antioxidant potential and thus potential health benefits of different varieties. Unfortunately, current methods to assay total carotenoid content are time consuming and require organic solvents. This report describes a rapid and reliable light absorption method to assay total carotenoid content for yellow-fleshed watermelon that does not require organic solvents. Light absorption of 78 watermelon flesh purees was measured with a diode array xenon flash spectrophotometer that can measure actual light absorption from opaque samples; results were compared with a hexane extraction method. The puree absorbance method gave a linear relationship (R(2)= 0.88) to total carotenoid content and was independent of watermelon variety within the total carotenoid concentration range measured (0 to 7 mug/g fresh weight).

Residual symptomatic superior oblique palsy.

PURPOSE: To determine the outcome of patients treated for residual symptomatic hyperdeviations, in a tertiary referral centre, following a previous weakening procedure of the ipsilateral Inferior Oblique (IO) muscle in Superior Oblique (SO) palsy. METHODS: A retrospective review of 37 patients seen over 6 years at one institution who had remained symptomatic from a SO palsy despite having had an initial weakening procedure to their ipsilateral IO (myectomy or recession). Median age was 19 years (range 3 to 56 years). Information recorded included pre- and postoperative deviation and ocular motility findings, preoperative symptoms, findings at the time of surgery, and outcome. RESULTS: Nine patients underwent repeat weakening surgery (disinsertion) on the ipsilateral IO only. Thirteen patients underwent strengthening surgery on the ipsilateral SO only. Nine patients had surgery on both the ipsilateral IO and SO. Six patients had surgery on the ipsilateral IO with either horizontal or vertical rectus surgery. Nine (24%) patients remained symptomatic after their initial procedure and are regarded as initial failures. Four of these patients had masked bilateral IO weakness. Five patients required additional surgery. At final outcome, 84% were discharged with resolution of their symptoms. CONCLUSIONS: In the light of these findings we suggest an approach for the management of these patients. This should always include exploring a previously operated ipsilateral IO. Despite this, patients should be warned that they have a 1 in 4 chance of needing further surgery to achieve adequate ocular motility.

Efficient adenoviral-mediated gene delivery into porcine mesenchymal stem cells.

Mesenchymal stem cell (MSC) mediated gene therapy research has been conducted predominantly on rodents. Appropriate large animal models may provide additional safety and efficacy information prior to human clinical trials. The objectives of this study were: (a) to optimize adenoviral transduction efficiency of porcine bone marrow MSCs using a commercial polyamine-based transfection reagent (GeneJammer, Stratagene, La Jolla, CA), and (b) to determine whether transduced MSCs retain the ability to differentiate into mesodermal lineages. Porcine MSCs (pMSCs) were infected under varying conditions, with replication-defective adenoviral vectors carrying the GFP gene and GFP expression analyzed. Transduced cells were induced to differentiate in vitro into adipogenic, chondrogenic, and osteogenic lineages. We observed a 5.5-fold increase in the percentage of GFP-expressing pMSCs when adenovirus type 5 carrying the adenovirus type 35 fiber (Ad5F35eGFP) was used in conjunction with GeneJammer. Transduction of pMSCs at 10.3-13.8 MOI (1,500-2,000 vp/cell) in the presence of Gene Jammer yielded the highest percentage of GFP-expressing cells ( approximately 90%) without affecting cell viability. A similar positive effect was detected when pMSCs were infected with an Ad5eGFP vector. Presence of fetal bovine serum (FBS) during adenoviral transduction enhanced vector-encoded transgene expression in both GeneJammer-treated and control groups. pMSCs transduced with adenovirus vector in the presence of GeneJammer underwent lipogenic, chondrogenic, and osteogenic differentiation. Addition of GeneJammer during adenoviral infection of pMSCs can revert the poor transduction efficiency of pMSCs while retaining their pluripotent differentiation capacity. GeneJammer-enhanced transduction will facilitate the use of adenoviral vectors in MSC-mediated gene therapy models and therapies.

Demographic feedback between clonal growth and fragmentation in an invasive seaweed.

Many abundant plants, invertebrates, and seaweed are clonal, and this allows the formation of high-density aggregations, foraging, and the placement of modules into new space, and rapid rates of expansion. For these species, population density and rates of expansion are functions of recruitment of asexual modules and post-recruitment vegetative growth and survivorship. In this study, we provide the first experimental test of the relative importance of these two processes in determining the abundance of a clonal seaweed using Caulerpa taxifolia, an invasive green alga that spreads rapidly and reaches very high abundance. We asked two main questions: What is the relative importance to abundance (biomass) of vegetative stolon growth and fragment recruitment during expansion of established patches? Does greater fragment recruitment result in greater abundance in established patches? Vegetative growth of stolons underpinned patch expansion. Plots with stolons growing into them always had a greater abundance than plots where stolons were removed, even when fragment recruitment was increased. Greater recruitment only resulted in greater abundance when stolons were absent, a situation analogous to the establishment of new populations. Although post-recruitment processes were more important in determining abundance during patch expansion, there was greater ambient fragment recruitment when stolons were present compared to when they were absent, and as the abundance of C. taxifolia increased, demonstrating an important feedback between stolon growth, abundance, and fragment recruitment. In established patches, greater fragment recruitment over six months (six levels ranging from 0 to 480 recruits x m(-2) x mo(-1)) had no effect on biomass. Our experiments demonstrate that the rapid expansion and high abundance of invasive C. taxifolia are underpinned by post-recruitment vegetative growth and, during expansion, by a feedback between vegetative growth and asexual fragmentation.

UV-induced mortality in encapsulated intertidal embryos: are mycosporine-like amino acids an effective sunscreen?

Mycosporine-like amino acids (MAAs) are believed to protect a variety of marine organisms against the negative effects of ultraviolet radiation (UVR). However, their role in protecting developing intertidal encapsulated embryos remains untested. In the present study, we focused on the UV protective role of natural concentrations of MAAs for two intertidal gastropod species, Bembicium nanum and Siphonaria denticulata, which lay egg masses in habitats exposed to direct sunlight. We predicted that in both species, a higher concentration of MAAs within the egg mass would increase the likelihood of embryonic survivorship in the presence of UVR. Egg masses from both species were collected along the rocky shores of southeastern New South Wales, and a portion from each was subjected to one of three separate spectral treatments: full spectrum, UV-B block, and UV block. Proportions of surviving embryos were recorded following 72 hr exposure to spectral treatment. In addition, MAAs in each egg mass were quantified. Levels of variation in MAA concentration were striking, with S. denticulata egg masses showing more intraspecific variation than those of B. nanum. Surprisingly, survivorship under all spectral treatments was extremely high for both species, irrespective of MAA concentration. Under full spectrum treatments, B. nanum survivorship and total MAA concentration were significantly and positively correlated; however, MAA accounted for just 23.6% (R = 0.486) of the variation in survivorship. In contrast, survivorship in S. denticulata was not correlated with MAA concentration under full spectrum light. We conclude that the dependence on MAAs as photoprotection may be species-specific; however, it is likely that both species possess alternative mechanisms that minimize the negative effects of UVR.

A quantitative survey of mycosporine-like amino acids (MAAS) in intertidal egg masses from temperate rocky shores.

Mycosporine-like amino acids (MAAs) have been reported as functional chemical sunscreens in a variety of marine organisms, but their role in development of marine embryos and larvae remains largely unexplored. In this study, we quantified MAAs from intertidal egg masses of 46 species of mollusks, two species of polychaetes, and one species of fish from southeastern Australia. We aimed to elucidate potential patterns of occurrence and variation based on egg mass maturity, adult diet, spawning habitat, phylogeny, and viability. Our analyses revealed that maturity and spawning habitat did not affect MAA composition within egg masses. In contrast, adult diet, phylogeny, and viability affected MAA composition. Herbivores had higher levels of certain MAAs than carnivores; similarly, viable egg masses had higher levels of some MAAs than inviable ones. MAA composition varied according to the taxonomic group, with nudibranchs and anaspids showing different MAA composition compared to that of neogastropods, sacoglossans, and polychaetes. Basommatophoran egg masses had more porphyra-334 than the other taxa, and anaspids had more mycosporine-2-glycine than the other groups. MAAs occurred in relatively high concentrations in intertidal molluskan egg masses when compared to adult mollusks and other common intertidal organisms. Despite the complexity of factors affecting MAA composition, the prevalence of MAAs in some species is consistent with protection afforded to offspring against negative effects of UV radiation.

Passive flow through an unstalked intertidal ascidian: orientation and morphology enhance suspension feeding in Pyura stolonifera.

Passive flow is believed to increase the gains and reduce the costs of active suspension feeding. We used a mixture of field and laboratory experiments to evaluate whether the unstalked intertidal ascidian Pyura stolonifera exploits passive flow. We predicted that its orientation to prevailing currents and the arrangement of its siphons would induce passive flow due to dynamic pressure at the inhalant siphon, as well as by the Bernoulli effect or viscous entrainment associated with different fluid velocities at each siphon, or by both mechanisms. The orientation of P. stolonifera at several locations along the Sydney-Illawarra coast (Australia) covering a wide range of wave exposures was nonrandom and revealed that the ascidians were consistently oriented with their inhalant siphons directed into the waves or backwash. Flume experiments using wax models demonstrated that the arrangement of the siphons could induce passive flow and that passive flow was greatest when the inhalant siphon was oriented into the flow. Field experiments using transplanted animals confirmed that such an orientation resulted in ascidians gaining food at greater rates, as measured by fecal production, than when oriented perpendicular to the wave direction. We conclude that P. stolonifera enhances suspension feeding by inducing passive flow and is, therefore, a facultatively active suspension feeder. Furthermore, we argue that it is likely that many other active suspension feeders utilize passive flow and, therefore, measurements of their clearance rates should be made under appropriate conditions of flow to gain ecologically relevant results.

Bleeding patterns after vaginal misoprostol for treatment of early pregnancy failure.

BACKGROUND: Dilatation and curettage (D&C) has been the usual treatment for early pregnancy failure (EPF). Medical management with misoprostol may be an effective alternative. Bleeding patterns during and after medical management of EPF are unknown. METHODS: A prospective cohort study was conducted at University-based clinics and physician offices. Eighty women <11 weeks estimated gestational age with a diagnosis of missed abortion or fetal demise were enrolled. Treatment consisted of either 800 micro g of moistened (2 ml of saline) or dry vaginal misoprostol. Self-reported bleeding and sanitary product usage were recorded in a daily 2 week diary. Haemoglobin was assessed at enrollment and 2 weeks later. RESULTS: After misoprostol treatment, patients reported bleeding or spotting every day for the 14 days observed. Self-assessed heavy bleeding days were few (median 3) and usually occurred immediately after treatment. Sanitary pad use was highly variable (mean 30.5, range 2-125 pads over the 2 week period) and not related to changes in haemoglobin. The mean decrease in haemoglobin was 0.5 g/dl (SD 1.2). Complete expulsion without D&C occurred in 85% of subjects. CONCLUSIONS: Bleeding for at least 2 weeks after vaginal misoprostol for EPF is common. Heavy bleeding is usually limited to a few days after treatment. Clinically important changes in haemoglobin are rare.

Liposomal enhancement of the antitumor activity of conditionally replication-competent adenoviral plasmids.

Many human tumors have a functional deficiency in p53. Numerous studies have taken advantage of this phenomenon to use a conditionally replication-competent adenovirus (Ad dl1520) that will grow in and lyse tumor cells while sparing normal tissues. However, success has been limited, in part due to difficulties in reaching a sufficiently high proportion of tumor cells. Preexisting or developing immune responses directed toward viral proteins further decrease the efficacy of the approach. We have developed a liposome-encapsulated conditionally replication-competent plasmid based on the dl1520 virus. Like the parent virus, this plasmid generates infectious particles following transfection of p53-defective, but not p53-wild-type tumor cells, but unlike the parent virus it is able to infect CAR-negative tumor cells. The antitumor efficacy of this infectious plasmid was demonstrated in mice with xenografted human tumors, in which it was active after both local and intravenous administration for subcutaneous tumors and following intravenous administration for disseminated malignancy. Activity was retained systemically, even in the presence of neutralizing antibody. Such liposomally encapsulated conditionally replication-competent plasmids may complement the use of conventional viral particles, particularly in settings in which liver uptake of adenoviral vector is undesirable or there are problematic inhibitory effects from humoral immune responses.

Osteoinduction by ex vivo adenovirus-mediated BMP2 delivery is independent of cell type.

The objective of the study was to analyze and compare the abilities of various human cell types with inherently dissimilar osteogenic potentials to induce heterotopic bone formation following ex vivo transduction with two distinct adenoviral vectors encoding bone morphogenetic protein type 2 (BMP2). The cells comprised primary human bone marrow mesenchymal stem cells (BM-MSCs), primary human skin fibroblasts (SFs), and a human diploid fetal lung cell line (MRC-5). The vectors included adenovirus type 5 or a chimeric adenovirus type 5 with the fiber gene of adenovirus type 35 (Ad5F35-BMP2), both demonstrating significantly different expression of BMP2 in vitro. The experimental groups consisted of the three human cell types transduced with each of the two adenoviral vectors. Using nonobese diabetic severe combined immunodeficiency (NOD/SCID) mice, the transduced cells were injected intramuscularly following ex vivo adenoviral transduction. The nature and extent of heterotopic bone formation were analyzed radiographically and histologically. At 14 days postinjection, abundant, highly mineralized bone was formed in mice injected with Ad5F35-BMP2-transduced cells irrespective of the cell type. There was no statistically significant difference in the amount of bone formed between BM-MSCs, SFs, and MRC-5 cells transduced with Ad5F35-BMP2, as assessed from bone surface area on biplanar plain radiography. Substantially lesser amounts or no bone could be detected in mice injected with cells transduced with Ad5-BMP2. Immunohistochemical analysis confirmed the presence of human cells in muscle as early as 2 days postdelivery; however, at 6-7 days after injection, the transduced cells could not be detected in surrounding muscle, or in the heterotopic bone, indicating the host origin of the newly formed bone. The results of the study demonstrate no significant difference in osteoinductive properties between BM-MSCs, SFs, and MRC-5 cells transduced ex vivo with the same type of adenovirus encoding BMP2. The level of BMP2 expression appears to be a crucial factor determining the extent of heterotopic bone formation and was significantly affected by the type of adenovirus used. In the cell types studied, Ad5F35-BMP2 was more efficacious than Ad5-BMP2 in providing adequate levels of BMP2 for efficient osteoinduction.

The influence of vaginal bleeding on the sexual behavior of urban, Hispanic women and men.

Irregular menstrual bleeding associated with use of hormonal contraceptives is a leading cause of contraception discontinuation. If bleeding disrupts sexual activity, this could contribute to contraceptive discontinuation. The impact of bleeding on sexual activities other than intercourse is unknown.A self-administered questionnaire in Spanish or English was used to collect information on demographics and the practice of various sexual behaviors during times of vaginal bleeding or spotting. The questionnaire was offered consecutively to 86 men and 75 women attending urban family-planning and young men's clinics. The men and women were approached individually, not as couples. Women reported a mean age of 25 years, a wide range of educational attainment, described themselves as mostly Hispanic (89%), and most chose the Spanish version of the questionnaire (67%). Men also described themselves as mostly Hispanic (80%), and reported a similar range of educational attainment, but were younger (mean age of 21, p < 0.001), and were less likely to choose the Spanish version of the questionnaire (23%, p < 0.001).Kissing, breast touching, and receiving non-genital touching did not change during bleeding or spotting. Forty-five percent of women reported usually receiving genital touching, which declined to 6% during bleeding and to 10% during spotting (p < 0.001). Similar proportions of women reported usually giving genital touching to their partner (50%) during bleeding (41%) or spotting (44%). Men also reported usually giving less genital touching to their partner, but no change in receiving genital touching during bleeding or spotting. Forty percent of women reported usually receiving oral sex, which declined to 4% during spotting or to 0% bleeding (p < 0.001). Forty-two percent of women reported usually giving oral sex, which declined to 23% during bleeding and to 35% during spotting (p = 0.06). Fifty percent of women and 68% of men reported engaging in vaginal intercourse. Among women, reported intercourse declined to 7% during bleeding and to 26% during spotting (p < 0.001). Among men, reported intercourse declined to 19% during bleeding and to 23% during spotting (p < 0.001). The presence of vaginal bleeding or spotting is associated with decreases in genital sexual behaviors among urban, Hispanic women and men. Such disruptions in sexual behavior could contribute to contraceptive discontinuation.

Cross-reactivity between hepatitis C virus and Influenza A virus determinant-specific cytotoxic T cells.

The cellular immune response contributes to viral clearance as well as to liver injury in acute and chronic hepatitis C virus (HCV) infection. An immunodominant determinant frequently recognized by liver-infiltrating and circulating CD8(+) T cells of HCV-infected patients is the HCV(NS3-1073) peptide CVNGVCWTV. Using a sensitive in vitro technique with HCV peptides and multiple cytokines, we were able to expand cytotoxic T cells specific for this determinant not only from the blood of 11 of 20 HCV-infected patients (55%) but also from the blood of 9 of 15 HCV-negative blood donors (60%), while a second HCV NS3 determinant was recognized only by HCV-infected patients and not by seronegative controls. The T-cell response of these healthy blood donors was mediated by memory T cells, which cross-reacted with a novel T-cell determinant of the A/PR/8/34 influenza A virus (IV) that is endogenously processed from the neuraminidase (NA) protein. Both the HCV NS3 and the IV NA peptide displayed a high degree of sequence homology, bound to the HLA-A2 molecule with high affinity, and were recognized by cytotoxic T lymphocytes with similar affinity (10(-8) M). Using the HLA-A2-transgenic mouse model, we then demonstrated directly that HCV-specific T cells could be induced in vivo by IV infection. Splenocytes harvested from IV-infected mice at the peak of the primary response (day 7 effector cells) or following complete recovery (day 21 memory cells) recognized the HCV NS3 peptide, lysed peptide-pulsed target cells, and produced gamma interferon. These results exemplify that host responses to an infectious agent are influenced by cross-reactive memory cells induced by past exposure to heterologous viruses, which could have important consequences for vaccine development.

Construction of adenoviral vectors.

Recombinant adenovirus vectors have proven to be useful tools in facilitating gene transfer. Construction of such vectors requires a knowledge of the adenovirus genome structure and its life cycle. A commonly used recombinant adenovirus involves deletion of the E1 region; such a recombinant is traditionally produced by overlap recombination after cotransfection of 293 cells with a plasmid shuttle vector and a large right-end restriction fragment of viral DNA. The shuttle vector contains a cassette for a transgene placed in region E1 and flanking sequences from adenovirus for recombination. Normally, a high background of parental virus results because of the difficulty in separating right-end restriction fragment length DNA from uncut DNA. This paper describes a negative selection based on the traditional cotransfection method using viral DNA from an E1-deleted adenoviral recombinant that expresses green fluorescent protein (GFP). In situ fluorescent microscopy is used to distinguish the recombinant plaques (white or nonfluorescent) from the parental virus plaques (green or fluorescent). In addition, this system allows for the detection of contaminating parental virus at later stages when production lots of the recombinant vector are being made.

Adenovirus-mediated BMP2 expression in human bone marrow stromal cells.

Recombinant adenoviral vectors have been shown to be potential new tools for a variety of musculoskeletal defects. Much emphasis in the field of orthopedic research has been placed on developing systems for the production of bone. This study aims to determine the necessary conditions for sustained production of high levels of active bone morphogenetic protein 2 (BMP2) using a recombinant adenovirus type 5 (Ad5BMP2) capable of eliciting BMP2 synthesis upon infection and to evaluate the consequences for osteoprogenitor cells. The results indicate that high levels (144 ng/ml) of BMP2 can be produced in non-osteoprogenitor cells (A549 cell line) by this method and the resultant protein appears to be three times more biologically active than the recombinant protein. Surprisingly, similar levels of BMP2 expression could not be achieved after transduction with Ad5BMP2 of either human bone marrow stromal cells or the mouse bone marrow stromal cell line W20-17. However, human bone marrow stromal cells cultured with 1 microM dexamethasone for four days, or further stimulated to become osteoblast-like cells with 50 microg/ml ascorbic acid, produced high levels of BMP2 upon Ad5BMP2 infection as compared to the undifferentiated cells. The increased production of BMP2 in adenovirus transduced cells following exposure to 1 microM dexamethasone was reduced if the cells were not given 50 microg/ml ascorbic acid. When bone marrow stromal cells were allowed to become confluent in culture prior to differentiation, BMP2 production in response to Ad5BMP2 infection was lost entirely. Furthermore, the increase in BMP2 synthesis seen during differentiation was greatly decreased when Ad5BMP2 was administered prior to dexamethasone treatment. In short, the efficiency of adenovirus mediated expression of BMP2 in bone marrow stromal cells appears to be dependent on the differentiation state of these cells.

Primary dysmenorrhea in adolescent girls and treatment with oral contraceptives.

This review examines the prevalence, associated morbidity, and treatment of primary dysmenorrhea in adolescent girls. Relevant literature was examined by systematic, evidence-based review using MEDLINE and Cochrane Collaboration databases. Dysmenorrhea is highly prevalent during adolescence. Despite differences in measurement methods, 20%-90% of adolescent girls report dysmenorrhea and about 15% of adolescents describe their dysmenorrhea as severe. During adolescence, dysmenorrhea leads to high rates of school absence and activity nonparticipation. Most adolescents with dysmenorrhea self-medicate with over-the-counter preparations; few consult healthcare providers. Combined oral contraceptives (COC) are an accepted treatment for dysmenorrhea in nonadolescent women. However, data supporting the efficacy of COC is limited. Very small studies show decreased prostaglandin in menstrual fluid associated with high-dose COC use. Larger studies are limited to cross-sectional comparisons showing lower prevalence of dysmenorrhea in low-dose COC users compared to non-COC users. One small, randomized controlled trial including some adolescents demonstrated an improvement in dysmenorrhea with high-dose COC treatment compared to placebo. The efficacy of low-dose COC in the treatment of adolescent dysmenorrhea has yet to be determined. If effective, well-established safety and noncontraceptive health benefits may make COC an ideal treatment for dysmenorrhea in adolescent girls.