Comparative analysis of Dp427-deficient mdx tissues shows that the milder dystrophic phenotype of extraocular and toe muscle fibres is associated with a persistent expression of beta-dystroglycan

The cell biological hypothesis of Duchenne muscular dystrophy assumes that deficiency in the membrane cytoskeletal element dystrophin triggers a loss in surface glycoproteins, such as beta-dystroglycan, thereby rendering the sarcolemmal membrane more susceptible to micro-rupturing. Secondary changes in ion homeostasis, such as increased cytosolic Ca2+ levels and impaired luminal Ca2+ buffering, eventually lead to Ca2+-induced myonecrosis. However, individual muscle groups exhibit a graded pathological response during the natural time course of x-linked muscular dystrophy. The absence of the dystrophin isofom Dp427 does not necessarily result in a severe dystrophic phenotype in all muscle groups. In the dystrophic mdx animal model, extraocular and toe muscles are not as severely affected as limb muscles. Here, we show that the relative expression and sarcolemmal localization of the central trans-sarcolemmal linker of the dystrophin-glycoprotein complex, beta-dystroglycan, is preserved in mdx extraocular and toe fibres by means of two-dimensional immunoblotting and immunofluorescence microscopy. Thus, with respect to improving myology diagnostics, the relative expression levels of beta-dystroglycan appear to represent reliable markers for the severity of secondary changes in dystrophin-deficient fibres. Immunoblotting and enzyme assays revealed that mdx toe muscle fibres exhibit an increased expression and activity of the sarcoplasmic reticulum Ca2+-ATPase. Chemical crosslinking studies demonstrated impaired calsequestrin oligomerization in mdx gastrocnemius muscle indicating that abnormal calsequestrin clustering is involved in reduced Ca2+ buffering of the dystrophic sarcoplasmic reticulum. Previous studies have mostly attributed the sparing of certain mdx fibres to the special protective properties of small-diameter fibres. Our study suggests that the rescue of dystrophin-associated glycoproteins, and possibly the increased removal of cytosolic Ca2+ ions, might also play an important role in protecting muscle cells from necrotic changes.     

Impact on Arbuscular Mycorrhiza Formation of Pseudomonas Strains Used as Inoculants for Biocontrol of Soil-Borne Fungal Plant Pathogens

The arbuscular mycorrhizal symbiosis, a key component of agroecosystems, was assayed as a rhizosphere biosensor for evaluation of the impact of certain antifungal Pseudomonas inoculants used to control soil-borne plant pathogens. The following three Pseudomonas strains were tested: wild-type strain F113, which produces the antifungal compound 2,4-diacetylphloroglucinol (DAPG); strain F113G22, a DAPG-negative mutant of F113; and strain F113(pCU203), a DAPG overproducer. Wild-type strain F113 and mutant strain F113G22 stimulated both mycelial development from Glomus mosseae spores germinating in soil and tomato root colonization. Strain F113(pCU203) did not adversely affect G. mosseae performance. Mycelial development, but not spore germination, is sensitive to 10 μM DAPG, a concentration that might be present in the rhizosphere. The results of scanning electron and confocal microscopy demonstrated that strain F113 and its derivatives adhered to G. mosseae spores independent of the ability to produce DAPG.     

Effects of Hormone Therapy on Cognition and Mood in Recently Postmenopausal Women: Findings from the Randomized,Controlled KEEPS–Cognitive and Affective Study

Background

Menopausal hormone therapy (MHT) reportedly increases the risk of cognitive decline in women over age 65 y. It is unknown whether similar risks exist for recently postmenopausal women, and whether MHT affects mood in younger women. The ancillary Cognitive and Affective Study (KEEPS-Cog) of the Kronos Early Estrogen Prevention Study (KEEPS) examined the effects of up to 4 y of MHT on cognition and mood in recently postmenopausal women.

Methods and Findings

KEEPS, a randomized, double-blinded, placebo-controlled clinical trial, was conducted at nine US academic centers. Of the 727 women enrolled in KEEPS, 693 (95.3%) participated in the ancillary KEEPS-Cog, with 220 women randomized to receive 4 y of 0.45 mg/d oral conjugated equine estrogens (o-CEE) plus 200 mg/d micronized progesterone (m-P) for the first 12 d of each month, 211 women randomized to receive 50 μg/d transdermal estradiol (t-E2) plus 200 mg/d m-P for the first 12 d of each month, and 262 women randomized to receive placebo pills and patches. Primary outcomes included the Modified Mini-Mental State examination; four cognitive factors: verbal learning/memory, auditory attention/working memory, visual attention/executive function, and speeded language/mental flexibility; and a mood measure, the Profile of Mood States (POMS). MHT effects were analyzed using linear mixed-effects (LME) models, which make full use of all available data from each participant, including those with missing data. Data from those with and without full data were compared to assess for potential biases resulting from missing observations. For statistically significant results, we calculated effect sizes (ESs) to evaluate the magnitude of changes.On average, participants were 52.6 y old, and 1.4 y past their last menstrual period. By month 48, 169 (24.4%) and 158 (22.8%) of the 693 women who consented for ancillary KEEPS-Cog were lost to follow-up for cognitive assessment (3MS and cognitive factors) and mood evaluations (POMS), respectively. However, because LME models make full use all available data, including data from women with missing data, 95.5% of participants were included in the final analysis (n = 662 in cognitive analyses, and n = 661 in mood analyses). To be included in analyses, women must have provided baseline data, and data from at least one post-baseline visit. The mean length of follow-up was 2.85 y (standard deviation [SD] = 0.49) for cognitive outcomes and 2.76 (SD = 0.57) for mood outcomes. No treatment-related benefits were found on cognitive outcomes. For mood, model estimates indicated that women treated with o-CEE showed improvements in depression and anxiety symptoms over the 48 mo of treatment, compared to women on placebo. The model estimate for the depression subscale was −5.36 × 10⁻² (95% CI, −8.27 × 10⁻² to −2.44 × 10^−2; ES = 0.49, p < 0.001) and for the anxiety subscale was −3.01 × 10⁻² (95% CI, −5.09 × 10⁻² to −9.34 × 10⁻³; ES = 0.26, p < 0.001). Mood outcomes for women randomized to t-E2 were similar to those for women on placebo. Importantly, the KEEPS-Cog results cannot be extrapolated to treatment longer than 4 y.

Conclusions

The KEEPS-Cog findings suggest that for recently postmenopausal women, MHT did not alter cognition as hypothesized. However, beneficial mood effects with small to medium ESs were noted with 4 y of o-CEE, but not with 4 y of t-E2. The generalizability of these findings is limited to recently postmenopausal women with low cardiovascular risk profiles.

Trial Registration

ClinicalTrials.gov NCT00154180 and NCT00623311     

Biomechanics of single chondrocytes under direct shear

Articular chondrocytes experience a variety of mechanical stimuli during daily activity. One such stimulus, direct shear, is known to affect chondrocyte homeostasis and induce catabolic or anabolic pathways. Understanding how single chondrocytes respond biomechanically and morphologically to various levels of applied shear is an important first step toward elucidating tissue level responses and disease etiology. To this end, a novel videocapture method was developed in this study to examine the effect of direct shear on single chondrocytes, applied via the controlled lateral displacement of a shearing probe. Through this approach, precise force and deformation measurements could be obtained during the shear event, as well as clear pictures of the initial cell-to-probe contact configuration. To further study the non-uniform shear characteristics of single chondrocytes, the probe was positioned in three different placement ranges along the cell height. It was observed that the apparent shear modulus of single chondrocytes decreased as the probe transitioned from being close to the cell base (4.1 ± 1.3 kPa), to the middle of the cell (2.6 ± 1.1 kPa), and then near its top (1.7 ± 0.8 kPa). In addition, cells experienced the greatest peak forward displacement (~30% of their initial diameter) when the probe was placed low, near the base. Forward cell movement during shear, regardless of its magnitude, continued until it reached a plateau at ~35% shear strain for all probe positions, suggesting that focal adhesions become activated at this shear level to firmly adhere the cell to its substrate. Based on intracellular staining, the observed height-specific variation in cell shear stiffness and plateau in forward cell movement appeared to be due to a rearrangement of focal adhesions and actin at higher shear strains. Understanding the fundamental mechanisms at play during shear of single cells will help elucidate potential treatments for chondrocyte pathology and loading regimens related to cartilage health and disease.     

Combined prenatal and postnatal protein restriction influences adult kidney structure, function, and arterial pressure

The effects of prenatal protein restriction on adult renal and cardiovascular function have been studied in considerable detail. However, little is known about the effects of life-long protein restriction, a common condition in the developing world. Therefore, we determined in rats the effects of combined pre- and postnatal protein restriction on adult arterial pressure and renal function and responses to increased dietary sodium. Nephron number was also determined. Male Sprague-Dawley rats were born to mothers fed a low [8% (wt/wt), LP] or normal [20% (wt/wt), NP] isocaloric protein diet throughout pregnancy and maintained on these diets after birth. At postnatal day 135, nephron number, mean arterial pressure (MAP), and renal function were determined. A high-NaCl [8.0% (wt/wt), high-salt] diet was fed to a subset of rats from weaning. MAP was less in LP than in NP rats (120 +/- 2 vs. 128 +/- 2 mmHg, P < 0.05) and was not significantly altered by increased salt intake. Nephron number was 31% less in LP than in NP rats (P < 0.001). The volume of individual glomeruli was also less in LP than in NP rats, as were calculated effective renal plasma flow and glomerular filtration rate. Glomerular filtration rate, but not effective renal plasma flow, appeared to be increased by high salt intake, particularly in LP rats. In conclusion, protein restriction induced a severe nephron deficit, but MAP was lower, rather than higher, in protein-restricted than in control rats in adulthood. These findings indicate that the postnatal environment plays a key role in determining the outcomes of developmental programming.     

Nicotine inhibits cytokine production by placenta cells via NFkappaB: potential role in pregnancy-induced hypertension

Pregnancy-induced hypertension (PIH), also known as preeclampsia, is one of the major causes of maternal and fetal death. While the precise cause of PIH is not known, aberrant cytokine production and placenta participation are considered to be important factors. Gestational cigarette smoking, which is widely accepted to be harmful to both the mother and fetus, is protective against PIH. Based on the antiinflammatory activity of nicotine, the major component of cigarettes, we examined the effect of nicotine and other cholinergic agonists on placental inflammatory responses ex vivo. We observed that nicotine and other cholinergic agonists significantly suppress placenta cytokine production following stimulation. Placenta cells express the alpha7 nicotinic acetylcholine receptor (alpha7nAChR), and using cholinergic antagonists, we demonstrated that the antiinflammatory effect of nicotine and other cholinergic agonists is, in part, mediated through the nAChR pathway. By contrast, cholinergic stimulation had no effect on the expression of soluble fms-like tyrosine kinase (sFlt), an antiangiogenic substance implicated in maternal vascular dysfunction during PIH. Mechanistic studies reveal that cholinergic agonists exert their antiinflammatory effects through the NFkappaB pathway. Taken together, our results suggest that cholinergic agonists, including nicotine, may reduce cytokine production by placenta cells via NFkappaB to protect against PIH.     

The Mcf1 toxin induces apoptosis via the mitochondrial pathway and apoptosis is attenuated by mutation of the BH3-like domain

Photorhabdus are Gram-negative, nematode-vectored bacteria that produce toxins to kill their insect hosts. The expression of one of these, Makes caterpillars floppy 1 (Mcf1), is sufficient to allow Escherichia coli to survive within, and kill, caterpillars which are otherwise able to clear E. coli infection. Mcf1 treated caterpillars show rapid loss of body turgor (the 'floppy' phenotype) and death is associated with massive apoptosis of both the midgut epithelium and insect phagocytes. Mammalian tissue culture cells treated with Mcf1 also display key features of apoptosis including zeiosis, apoptotic nuclear morphology, DNA laddering, activation of the effector caspase-3 and PARP cleavage. As Mcf1 carries a single BH3-like domain, here we investigate the hypothesis that this toxin promotes apoptosis via the mitochondrial pathway by mimicking a BH3 domain-only protein. Consistent with this hypothesis, a double mutant within the BH3-like domain causes a dramatic decline in apoptosis. Mcf1 also alters mitochondrial membrane potential and triggers the release of cytochrome c. Cells overexpressing Bcl-x(L), an anti-apoptotic Bcl-2 family member, are resistant to Mcf1-mediated apoptosis, as are cells deficient in Bax. In addition, translocation of Bax to the mitochondrion is observed in response to Mcf1 treatment. Together, these results show that Mcf1 mediates apoptosis via the mitochondrial pathway, and are consistent with the hypothesis that the BH3-like domain in Mcf1 is a functional requirement for the pro-apoptotic activity of Mcf1.