Laboratory investigation of crest height statistics in intermediate water depths.

This paper concerns the statistical distribution of the crest heights associated with surface waves in intermediate water depths. The results of a new laboratory study are presented in which data generated in different experimental facilities are used to establish departures from commonly applied statistical distributions. Specifically, the effects of varying sea-state steepness, effective water depth and directional spread are investigated. Following an extensive validation of the experimental data, including direct comparisons to available field data, it is shown that the nonlinear amplification of crest heights above second-order theory observed in steep deep water sea states is equally appropriate to intermediate water depths. These nonlinear amplifications increase with the sea-state steepness and reduce with the directional spread. While the latter effect is undoubtedly important, the present data confirm that significant amplifications above second order (5-10%) are observed for realistic directional spreads. This is consistent with available field data. With further increases in the sea-state steepness, the dissipative effects of wave breaking act to reduce these nonlinear amplifications. While the competing mechanisms of nonlinear amplification and wave breaking are relevant to a full range of water depths, the relative importance of wave breaking increases as the effective water depth reduces.

Osteogenesis imperfecta and the teeth, eyes, and ears-a study of non-skeletal phenotypes in adults.

Osteogenesis imperfecta (OI) is a disease causing bone fragility; however, it potentially affects all organs with a high content of collagen, including ears, teeth, and eyes. The study is cross-sectional and compares non-skeletal characteristics in adults with OI that clinicians should be aware of when caring for patients with OI. INTRODUCTION: Osteogenesis imperfecta (OI) is a hereditary connective tissue disorder. The skeletal fragility is pronounced; however, OI leads to a number of extra-skeletal symptoms related to the ubiquity of collagen type 1 throughout the human body. The vast majority of knowledge is derived from studies performed in the pediatric population. Thus, we aimed to investigate the nature and prevalence of ophthalmologic, odontologic, and otologic phenotypes in an adult population with OI. METHODS: The study population comprises 85 Danish OI patients (age 44.9 ± 15.9 years). Fifty-eight patients had OI type I, 12 OI type III, and 15 OI type IV according to the classification by Sillence. Audiometric evaluations and dental examinations were performed in 62 and 73 patients, respectively. Ophthalmologic investigations were performed in 64 patients, including measurements of the central corneal thickness. RESULTS: All patients, except two, had corneal thickness below the normal reference value. Patients with OI type I and patients with a quantitative collagen defect had thinner corneas compared to patients with OI type III and other patients with a qualitative collagen defect. One patient in this cohort was diagnosed with and treated for acute glaucoma. Dentinogenesis imperfecta was diagnosed in one fourth of the patients, based on clinical and radiographic findings. This condition was predominately seen in patients with moderate to severe OI. Hearing loss requiring treatment was found in 15 of 62 patients, of whom three were untreated. The most prevalent type of hearing loss (HL) was sensorineural hearing loss, whereas conductive HL was solely seen in patients with OI type III. The patients with the most severe degrees of HL were patients with mild forms of OI. Age was associated with increased HL. CONCLUSION: Although significant health problems outside the skeleton are frequent in adult patients with OI, the patients are not consistently monitored and treated for their symptoms. Clinicians treating adult patients with OI should be aware of non-skeletal health issues and consider including regular interdisciplinary check-ups in the management plan for adult OI patients.

Children with Crohn's Disease Frequently Consume Select Food Additives.

OBJECTIVE: Certain food additives may promote the pathogenesis of Crohn's disease (CD), but thus far the evaluation of food additive exposures in humans has been limited. The objective of this study was to quantify food additive exposures in children with CD. METHODS: In a trial for bone health in CD, children were followed over 24 months with evaluation of disease characteristics, dietary intake, and body composition. At baseline, participants completed three 24-h dietary recalls. Foods were categorized, and the ingredient list for each item was evaluated for the presence of select food additives: polysorbate-80, carboxymethylcellulose, xanthan gum, soy lecithin, titanium dioxide, carrageenan, maltodextrin, and aluminosilicates. The frequency of exposures to these food additives was described for study participants and for food categories. RESULTS: At study baseline, 138 participants, mean age 14.2 ± 2.8 years, 95% having inactive or mild disease, were enrolled and dietary recalls were collected. A total of 1325 unique foods were recorded. Mean exposures per day for xanthan gum was 0.96 ± 0.72, carrageenan 0.58 ± 0.63, maltodextrin 0.95 ± 0.77, and soy lecithin 0.90 ± 0.74. The other additives had less than 0.1 exposures per day. For the 8 examined food additives, participants were exposed to a mean (SD) of 3.6 ± 2.1 total additives per recall day and a mean (SD) of 2.4 ± 1.0 different additives per day. CONCLUSION: Children with CD frequently consume food additives, and the impact on disease course needs further study.

A laboratory study of nonlinear changes in the directionality of extreme seas.

This paper concerns the description of surface water waves, specifically nonlinear changes in the directionality. Supporting calculations are provided to establish the best method of directional wave generation, the preferred method of directional analysis and the inputs on which such a method should be based. These calculations show that a random directional method, in which the phasing, amplitude and direction of propagation of individual wave components are chosen randomly, has benefits in achieving the required ergodicity. In terms of analysis procedures, the extended maximum entropy principle, with inputs based upon vector quantities, produces the best description of directionality. With laboratory data describing the water surface elevation and the two horizontal velocity components at a single point, several steep sea states are considered. The results confirm that, as the steepness of a sea state increases, the overall directionality of the sea state reduces. More importantly, it is also shown that the largest waves become less spread or more unidirectional than the sea state as a whole. This provides an important link to earlier descriptions of deterministic wave groups produced by frequency focusing, helps to explain recent field observations and has important practical implications for the design of marine structures and vessels.

Pre- and post-operative dizziness, tinnitus, and taste disturbances among cochlear implant recipients.

OBJECTIVE: To determine the pre- and post-operative prevalence of dizziness, tinnitus and taste disturbances in adult cochlear implant recipients. METHODS: A questionnaire regarding pre- and post-operative dizziness, tinnitus and taste disturbances was sent to 170 cochlear implant recipients implanted between January 2003 and March 2009. Seventy-seven patients (41 per cent) responded. RESULTS: Pre-operatively, 20 per cent of the participants experienced dizziness, 52 per cent experienced tinnitus and 3 per cent experienced taste disturbances. Post-operative dizziness developed in 46 per cent of patients and resolved in the majority of these; however, 15 per cent reported dizziness more than six months after implantation. Tinnitus worsened in 25 per cent of patients, whereas 73 per cent reported attenuation or termination of tinnitus. Post-operatively, tinnitus developed in 12 per cent and taste disturbances developed in 17 per cent of the patients. CONCLUSION: The high prevalence of dizziness, tinnitus and taste disturbances reported by cochlear implant recipients necessitates that assessment of symptoms related to inner ear and chorda tympani damage are included when evaluating operative results.

The interaction between steep waves and a surface-piercing column.

Experimental observations are presented of a single surface-piercing column subject to a wide range of surface gravity waves. With the column diameter, D, chosen such that the flow lies within the drag-inertia regime, two types of high-frequency wave scattering are identified. The first is driven by the run-up and wash-down on the surface of the column in the vicinity of the upstream and downstream stagnation points. The second concerns the circulation of fluid around the column, leading to the scattering of a pair of non-concentric wavefronts. The phasing of the wave cycle at which this second mode evolves is dependent upon the time taken for fluid to move around the column. This introduces an additional time-scale, explaining why existing diffraction solutions, based upon a harmonic analysis of the incident waves, cannot describe this scattered component. The interaction between the scattered waves and the next (steep) incident wave can produce a large amplification of the scattered waves, particularly the second type. Evidence is provided to show that these interactions can produce highly localized free-surface effects, including vertical jetting, with important implications for the setting of deck elevations, the occurrence of wave slamming and the development of large run-up velocities.

Dendritic network structure constrains metacommunity properties in riverine ecosystems.

1. Increasingly, ecologists conceptualize local communities as connected to a regional species pool rather than as isolated entities. By this paradigm, community structure is determined through the relative strengths of dispersal-driven regional effects and local environmental factors. However, despite explicit incorporation of dispersal, metacommunity models and frameworks often fail to capture the realities of natural systems by not accounting for the configuration of space within which organisms disperse. This shortcoming may be of particular consequence in riverine networks which consist of linearly -arranged, hierarchical, branching habitat elements. Our goal was to understand how constraints of network connectivity in riverine systems change the relative importance of local vs. regional factors in structuring communities. 2. We hypothesized that communities in more isolated headwaters of riverine networks would be structured by local forces, while mainstem sections would be structured by both local and regional processes. We examined these hypotheses using a spatially explicit regional analysis of riverine macroinvertebrate communities, focusing on change in community similarity with distance between local communities [i.e., distance-decay relationships; (DDRs)], and the change in environmental similarity with distance. Strong DDRs frequently indicate dispersal-driven dynamics. 3. There was no evidence of a DDR in headwater communities, supporting our hypothesis that dispersal is a weak structuring force. Furthermore, a positive relationship between community similarity and environmental similarity supported dynamics driven by local environmental factors (i.e., species sorting). In mainstem habitats, significant DDRs and community x environment similarity relationships suggested both dispersal-driven and environmental constraints on local community structure (i.e., mass effects). 4. We used species traits to compare communities characterized by low vs. high dispersal taxa. In headwaters, neither strength nor mode (in-network vs. out of network) of dispersal changed our results. However, outcomes in mainstems changed substantially with both dispersal mode and strength, further supporting the hypothesis that regional forces drive community dynamics in mainstems. 5. Our findings demonstrate that the balance of local and regional effects changes depending on location within riverine network with local (environmental) factors dictating community structure in headwaters, and regional (dispersal driven) forces dominating in mainstems.

Leaf litter species evenness influences nonadditive breakdown in a headwater stream.

Species loss directly affects the magnitude and stability of various ecosystem processes, and species composition can drive this phenomenon. Much of the evidence that species loss affects ecosystem processes comes from experiments where species richness was manipulated while holding abundance/biomass of individual species constant. Given that species rarely coexist in equal proportions, neglecting evenness might under/overestimate the role of important species combinations. We examined leaf litter breakdown in a small stream based on species-specific input rates of leaf litter from the four dominant species (Liriodendron tulipifera, Fraxinus pennsylvanica, Fagus grandifolia, Quercus prinus), comprising 71% of inputs over peak leaf fall, for a full-factorial litter mixture study. Our experimental approach departed from previous mixture studies in that while we created all two-, three-, and four-species combinations holding species-specific mass constant, we also created a complementary set of mixtures that reflected the natural proportion we estimated from the survey. We found that species richness and evenness alone did not explain variation in breakdown rate, but an interaction between the two did, and mixtures reflecting ambient evenness lost mass nearly 33% faster than single species treatments. Analysis of individual treatments revealed that the emergent effect of mixing species was nearly twice as common in uneven vs. even mixtures. The compositional effects of litter diversity on breakdown uncovered in previous studies might be more pronounced if evenness, and not just richness, is considered when evaluating the role of species loss in these ecosystems.

Identification of a localized mucosal immune response in rainbow trout, Oncorhynchus mykiss (Walbaum), following immunization with a protein-hapten antigen.

The ability of rainbow trout, Oncorhynchus mykiss (RBT), to produce a localized mucosal immune response was investigated following intraperitoneal (i.p.) or peranal (p.a.) immunization with a protein-hapten carrier, fluorescein isothiocyanate conjugated to keyhole limpet haemocyanin (FITC/KLH). Antibody levels in serum, mucus, tissue culture supernatant from blood and spleen leucocytes, and excised skin, intestine and gill tissues were determined by ELISA. Significantly, elevated antigen-specific antibodies were elicited in both serum and mucus of fish immunized i.p. Mucosal antibody responses, in general, paralleled serum responses over time. Leucocytes isolated from spleen and blood of i.p. immunized fish at week 10 produced significantly elevated antibody levels against FITC when cultured in vitro. Excised skin, intestine and gill tissues from these fish also exhibited significantly elevated antibody responses indicating localized production in the mucosa from tissue-specific B cells. A localized mucosal immune response was elicited only after i.p. and not p.a. immunization, suggesting that systemically stimulated B cells migrate to mucosal tissues where they produce antibodies locally.

Myocarditis following adeno-associated viral gene expression of human soluble TNF receptor (TNFRII-Fc) in baboon hearts.

Sequestration of tumor necrosis factor-alpha (TNFalpha) by TNF-receptor immunoglobulin G (IgG)-Fc fusion proteins can limit heart failure progression in rodent models. In this study we directly injected an adeno-associated viruses (AAV)-2 construct encoding a human TNF receptor II IgG-Fc fusion protein (AAV-TNFRII-Fc) into healthy baboon hearts and assessed virally encoded gene expression and clinical response. Adult baboons received direct cardiac injections of AAV-TNFRII-Fc ( approximately 5 x 10(12) viral/genomes/baboon) or an equivalent dose of AAV-2 empty capsids, and were analyzed after 5 or 12 weeks. Viral genomes were restricted to the myocardium, and routine analyses (blood cell counts, clinical chemistries) remained unremarkable. Echocardiograms were unchanged but electrocardiograms revealed marked ST- and T-wave changes consistent with myocarditis only in baboons receiving AAV-TNFRII-Fc. TNFRII serum levels peaked at approximately 3 times the baseline levels at 1-2 weeks postinjection and subsequently declined to baseline levels. TNFRII-Fc protein and transcripts were detected in the heart at harvest. After AAV injection, anti-AAV-2 antibody levels increased in all baboons, while anti-TNFRII-Fc could not be detected. Baboons that received AAV-TNFRII-Fc developed myocardial infiltrates including CD8+ cells. Thus, a cellular immune response to cardiac delivery of AAV encoding foreign proteins may be an important consideration for AAV-based cardiac gene therapy.

Can gene delivery close the door to HIV-1 entry after escape?

BACKGROUND: Research efforts to prevent viral entry by developing small molecule inhibitors against HIV-1 chemokine coreceptors have yielded promising clinical results. However, resistance to some chemokine receptor inhibitors has been recently documented, and therefore, alternative methods of HIV-1 coreceptor disruption are needed. CONCLUSION: We will describe current HIV-1 vector-delivered genetic disruption mechanisms that target HIV-1 chemokine coreceptors, such as RNA interference, ribozymes, zinc fingers, intrakines, and intrabodies, and frame the use of these gene delivery chemokine receptor disruption mechanisms in the context of current small molecule blocker/antagonists of CCR5 and CXCR4. In addition, we will discuss the importance of evaluating HIV-1 vector-delivered viral entry prevention mechanisms in the rhesus macaque SIV non-human primate model in regard to pathogenesis and therapeutic efficacy.

T-cell protection and enrichment through lentiviral CCR5 intrabody gene delivery.

CCR5 is the chemokine co-receptor for R5-tropic human immunodeficiency virus type 1 (HIV-1) isolates most often associated with primary infection. We have developed an HIV-1 self-inactivating vector, CAD-R5, containing a CCR5 single-chain antibody (intrabody) gene, which when expressed in T-cell lines and primary CD4+ T cells disrupts CCR5 cell surface expression and provides protection from R5-tropic isolate exposure. Furthermore, CAD-R5 intrabody expression in primary CD4+ T cells supports significant growth and enrichment over time during HIV-1-pulsed dendritic cell-T-cell interactions. These results indicate that CCR5 intrabody-expressing CD4+ T cells are refractory against this highly efficient primary route of infection. CD34+ cells transduced with the CAD-R5 vector gave rise to CD4+ and CD8+ thymocytes in non-obese diabetic (NOD)/ severely combined-immunodeficient (SCID)-human thymus/liver (hu thy/liv) mice, suggesting that CCR5 intrabody expression can be maintained throughout differentiation without obvious cellular effects. CD4+ T cells isolated from NOD/SCID-hu thy/liv mice were resistant to R5-tropic HIV-1 challenge demonstrating the maintenance of protection. Our findings demonstrate delivery of anti-HIV-1 activity through CCR5 intrabodies in primary CD4+ T cells and CD34+ cell-derived T-cell progeny. Thus, gene delivery strategies that provide a selective survival and growth advantage for T effector cells may provide a therapeutic benefit for HIV-1-infected individuals who have failed conventional therapies.

Increased puroindoline levels slow ruminal digestion of wheat (Triticum aestivum L.) starch by cattle.

Starch is the primary nutrient in ruminant diets used to promote high levels of performance. The site of starch digestion alters the nature of digestive end products (VFA in the rumen vs. glucose in the small intestine) and the efficiency of use. Cereal grain endosperm texture plays a major role in the rate and extent of starch degradation in ruminants. Wheat grain texture is regulated by the starch surface protein complex friabilin that consists primarily of puroindoline (PIN) A and B. Soft kernel texture in wheat is a result of both PIN genes being in the wild type active form and bound to starch. The objective of this study was to investigate the effect of varying PIN content in wheat on the rate of starch digestion in the rumen of beef cattle. In Exp. 1, 6 transgenic soft pin a/b isolines created in a hard wheat background, and 2 hard wheat controls were milled to yield a wide range of mean particle sizes across all lines. Milled samples were incubated in situ for 3 h. Increased expression of both PINA and PINB decreased DM digestibility (DMD) by 29.2% (P < 0.05) and decreased starch digestibility by 30.8% (P < 0.05). Experiment 2 separated the effects of particle size and total PIN content on digestion by milling the hardest and softest lines such that the mean particle size was nearly identical. Increased PIN decreased DMD by 21.7% (P < 0.05) and starch digestibility by 19.9% (P < 0.05) across particle sizes smaller than whole kernel. Experiment 3 addressed the time course of PIN effects in the rumen by observing ground samples of the hardest and softest lines over a 12-h in situ period. Increased PIN decreased DMD by 10.4% (P < 0.05) and starch digestibility by 11.0% (P < 0.05) across all time points. Dry matter and starch digestibility results demonstrated that increased expression of PIN was associated with a decreased rate of ruminal digestion independent of particle size. Puroindolines seem to aid in the protection of starch molecules from microbial digestion in the rumen, potentially increasing the amount of starch entering the small intestine.

Modeling deformation-induced fluid flow in cortical bone's canalicular-lacunar system.

To explore the potential role that load-induced fluid flow plays as a mechano-transduction mechanism in bone adaptation, a lacunar-canalicular scale bone poroelasticity model is developed and implemented. The model uses micromechanics to homogenize the pericanalicular bone matrix, a system of straight circular cylinders in the bone matrix through which bone fluids can flow, as a locally anisotropic poroelastic medium. In this work, a simplified two-dimensional model of a periodic array of lacunae and their surrounding systems of canaliculi is used to quantify local fluid flow characteristics in the vicinity of a single lacuna. When the cortical bone model is loaded, microscale stress, and strain concentrations occur in the vicinity of individual lacunae and give rise to microscale spatial variations in the pore fluid pressure field. Furthermore, loading of the bone matrix containing canaliculi generates fluid pressures in the contained fluids. Consequently, loading of cortical bone induces fluid flow in the canaliculi and exchange of fluid between canaliculi and lacunae. For realistic bone morphology parameters, and a range of loading frequencies, fluid pressures and fluid-solid drag forces in the canalicular bone are computed and the associated energy dissipation in the models compared to that measured in physical in vitro experiments on human cortical bone. The proposed model indicates that deformation-induced fluid pressures in the lacunar-canalicular system have relaxation times on the order of milliseconds as opposed to the much shorter times (hundredths of milliseconds) associated with deformation-induced pressures in the Haversian system.

Patient-led variable dosing with balsalazide as long-term therapy for maintenance in ulcerative colitis: a 3-year prospective observational study.

BACKGROUND: The patient-centred approach is new to the management of ulcerative colitis. To date, it has only been shown to be successful in a short-term study. AIM: To assess the feasibility, safety and efficacy of patient-led dosing using balsalazide in the long-term treatment of ulcerative colitis. METHODS: This was a 3-year, two-cohort, multi-centre study: one cohort was in stable remission (52 patients) and the other was newly in remission (76 patients) from ulcerative colitis. Two 750-mg balsalazide capsules were given twice daily for maintenance, increased by 750-mg increments to a maximum of 6 g for up to 7 days depending on symptom severity. Clinical assessments were made every 12-14 weeks; laboratory assessments were made every 6 months. RESULTS: The average median daily dose of balsalazide was 3 g (range, 1.5-6 g). In the cohort with stable remission, 23 patients (44%) had relapsed by 3 years [median time to relapse, > 1095 days (36 months)]. In the cohort newly in remission, these values were 45 patients (59%) and 656 days (22 months), respectively. In the cohort with stable remission, the time since last relapse was significantly associated with relapse during the first year of treatment (P < 0.033). CONCLUSIONS: Long-term, patient-led, maintenance treatment with balsalazide is well tolerated with a good safety profile and is effective for patients with ulcerative colitis.

Micromechanically based poroelastic modeling of fluid flow in Haversian bone.

To explore the hypothesis that load-induced fluid flow in bone is a mechano-transduction mechanism in bone adaptation, unit cell micro-mechanical techniques are used to relate the microstructure of Haversian cortical bone to its effective poroelastic properties. Computational poroelastic models are then applied to compute in vitro Haversian fluid flows in a prismatic specimen of cortical bone during harmonic bending excitations over the frequency range of 10(0) to 10(6) Hz. At each frequency considered, the steady state harmonic response of the poroelastic bone specimen is computed using complex frequency-domain finite element analysis. At the higher frequencies considered, the breakdown of Poisueille flow in Haversian canals is modeled by introduction of a complex fluid viscosity. Peak bone fluid pressures are found to increase linearly with loading frequency in proportion to peak bone stress up to frequencies of approximately 10 kHz. Haversian fluid shear stresses are found to increase linearly with excitation frequency and loading magnitude up until the breakdown of Poisueille flow. Tan delta values associated with the energy dissipated by load-induced fluid flow are also compared with values measured experimentally in a concurrent broadband spectral analysis of bone. The computational models indicate that fluid shear stresses and fluid pressures in the Haversian system could, under physiologically realistic loading, easily reach the level of a few Pascals, which have been shown in other works to elicit cell responses in vitro.

Effects of progestins on progesterone synthesis in a stable porcine granulosa cell line: control of transcriptional activity of the cytochrome p450 side-chain cleavage gene.

The purpose of the present study was to examine the effects of progestins on progesterone synthesis and expression of the cytochrome P450 cholesterol side-chain cleavage gene (P450(scc)) in a stable porcine granulosa cell line, the JC-410. Cells were incubated for 48 h with the synthetic progestogen-levornorgestrel with or without RU486 (progesterone and glucocorticoid receptor antagonist) or RWJ26819 (progesterone agonist without affinity to glucocorticoid receptors). Both levonorgestrel and RU486 enhanced progesterone accumulation in a dose-dependent manner. RU486 did not antagonize the effects of levonorgestrel, and RWJ26819 had no effect on progesterone production in cultured JC-410 cells. Progesterone and levonorgestrel increased steady state P450(scc) mRNA levels after 3-6 h of treatment. Progesterone and RU486 at 0.1, 1, and 10 microM increased the transcription rate of P450(scc) transiently expressed in JC-410 cells after 18 h of incubation; 30 microM had no effect, and 100 microM suppressed transcription. Levonorgestrel did not affect transcription of the P450(scc) gene, and RWJ26819 reduced its transcription. Progesterone and RU486 significantly decreased the number of cells and total protein content after 72 and 24 h of incubation, respectively. Levonorgestrel had no effect, whereas RWJ26819 increased (24 h) but subsequently reduced (72 h) cell number and protein content. The present results indicate that progestins are capable of directly modulating progesterone biosynthesis in porcine JC-410 granulosa cells. These effects may be exerted in part through the regulation of P450(scc) gene expression. Ostensible differences exist between progesterone and its synthetic analogues in the control of progesterone secretion in the stable porcine granulosa cell line in vitro.

A broadband viscoelastic spectroscopic study of bovine bone: implications for fluid flow.

To explore the hypothesis that mechanical excitation-induced fluid flow and/or fluid pressure are potential mechanical transduction mechanisms in bone adaptation, a complementary experimental and analytical modeling effort has been undertaken. Experimentally, viscoelastic tan delta properties of saturated cortical bovine bone were measured in both torsion and bending, and significant tan delta values in the 10(0)-10(5) Hz range were observed, although the nature of the damping is not consistent with a fluid pressure hypothesis. Analytically, micromechanically based poroelasticity models were exercised to quantify energy dissipation associated with load-induced fluid flow in large scale channels. The modeling results indicate that significant damping due to fluid flow occurs only above 1 MHz frequencies. Together, the experimental and analytical results indicate that at excitation frequencies presumed to be physiological (1-100 Hz), mechanical loading of bone generates extremely small pore fluid pressures, making the hypothesized fluid-pressure transduction mechanism upon osteocytes untenable.

Functional analysis of cis-regulatory elements within the promoter of the tobacco late pollen gene g10.

The tobacco gene g10 is preferentially and maximally expressed in mature pollen, shows homology to pectate lyases, and is the putative homologue of the tomato gene lat56. Analysis of regulatory elements within the g10 promoter was carried out to verify the importance of putative regulatory sequence motifs. Analysis of transgenic plants showed that 1190 bp of g10 5' sequence directed preferential expression of GUS in pollen, with bimodal peaks of expression just before and during pollen mitosis I, and in mature anthers. This was confirmed by northern analysis of native g10 transcripts in isolated spores. Transient expression analysis defined the minimal g10 promoter region capable of directing expression in pollen as -86 to +217. Three upstream regions within -427 bp modulate the expression from g10. Gain-of-function analyses showed that the region from -106 to -53 could enhance pollen-specific expression of a minimal CaMV 35S promoter. These analyses further showed that sequences upstream of -86 modulate expression in pollen, but are not essential for preferential pollen expression. The function of a conserved GTGA motif shared between the tobacco g10 and tomato lat56 promoters was demonstrated in g10. Thus, further functional evidence is provided for the conservation of mechanisms for the regulation of late pollen genes across species.

Inhibition of basal and stimulated progesterone synthesis by dichlorodiphenyldichloroethylene and methoxychlor in a stable pig granulosa cell line.

The effects of the insecticide dichlorodiphenyldichloroethylene (DDE) and methoxychlor in a stable pig granulosa cell line, JC-410, were investigated. The studies of DDE and methoxychlor were conducted in combination with studies of cholera toxin, the protein kinase A activator that stimulates cAMP and progesterone synthesis and gene expression of P450 cholesterol side chain cleavage (P450scc), which converts cholesterol to pregnenolone. Administration of DDE at 3000 and 10 000 ng ml (-1) was found to decrease progesterone synthesis 0.49- and 0.25-fold, respectively, and to block the stimulatory effect of 100 ng cholera toxin ml (-1), after 24 h incubation. At 1-100 ng ml (-1), methoxychlor did not affect progesterone synthesis after 48 h incubation. However, 1000 ng methoxychlor ml (-1) decreased progesterone synthesis 0.32-fold, and both 100 and 1000 ng methoxychlor ml (-1) blocked the stimulatory effect of cholera toxin. At 3000 and 10 000 ng ml(-1), DDE decreased cAMP synthesis 0.66-and 0.36-fold, respectively. At 300, 3000 and 10 000 ng ml (-1), DDE also decreased cholera toxin-stimulated cAMP synthesis 0.84-, 0.68-, and 0.52-fold, respectively. Administration of 1-100 ng methoxychlor ml (-1) did not affect basal or cholera toxin-stimulated cAMP synthesis. Cholera toxin increased P450scc mRNA 1.4-fold after 24 h incubation, while 3000 and 10 000 ng DDE ml (-1) led to 0.39- and 0.18-fold reductions, respectively. The stimulatory effect of cholera toxin on P450scc mRNA was blocked by 3000 and 10 000 ng DDE ml(-1). Cholera toxin increased P450scc mRNA 3.48-fold after 48 h incubation, while 100 and 1000 ng methoxychlor ml (-1) increased P450scc mRNA 1.79- and 3.0-fold, respectively, and further increased the stimulatory effect of cholera toxin 6.47- and 5.44-fold, respectively. The results of the present study indicate that DDE inhibits granulosa cell steroidogenesis by affecting cAMP production and P450scc gene expression. However, methoxychlor appears to inhibit steroidogenesis by a mechanism occurring before the conversion of cholesterol into pregnenolone.