Behavior of the yolk nuclei during embryogenesis, and development of the midgut diverticulum in the horseshoe crab Limulus polyphemus

Abstract. Classical studies of horseshoe crab development have provided relatively little information about the earliest stages, and the contribution of yolk cells and yolk nuclei—a deficiency due in large part to the difficulty of preparing the eggs and embryos for sectioning. Using newly developed histological resins, we show that the yolk nuclei undergo a series of changes during embryogenesis, before cellularizing and forming the midgut epithelium during the first larval stage. The digestive diverticulum forms in a 2-step process. A mesodermally derived lamina divides the yolk mass into distinct lobes, defining the boundaries of the digestive caeca. The yolk nuclei then cellularize to form the midgut epithelium.     

Tryptase inhibition blocks airway inflammation in a mouse asthma model

Release of human lung mast cell tryptase may be important in the pathophysiology of asthma. We examined the effect of the reversible, nonelectrophilic tryptase inhibitor MOL 6131 on airway inflammation and hyper-reactivity in a murine model of asthma. MOL 6131 is a potent selective nonpeptide inhibitor of human lung mast cell tryptase based upon a beta-strand template (K(i) = 45 nM) that does not inhibit trypsin (K(i) = 1,061 nM), thrombin (K(i) = 23, 640 nM), or other serine proteases. BALB/c mice after i.p. OVA sensitization (day 0) were challenged intratracheally with OVA on days 8, 15, 18, and 21. MOL 6131, administered days 18-21, blocked the airway inflammatory response to OVA assessed 24 h after the last OVA challenge on day 22; intranasal delivery (10 mg/kg) had a greater anti-inflammatory effect than oral delivery (10 or 25 mg/kg) of MOL 6131. MOL 6131 reduced total cells and eosinophils in bronchoalveolar lavage fluid, airway tissue eosinophilia, goblet cell hyperplasia, mucus secretion, and peribronchial edema and also inhibited the release of IL-4 and IL-13 in bronchoalveolar lavage fluid. However, tryptase inhibition did not alter airway hyper-reactivity to methacholine in vivo. These results support tryptase as a therapeutic target in asthma and indicate that selective tryptase inhibitors can reduce allergic airway inflammation.     

Sialylated core 1 O-glycans influence the sorting of Pmel17/gp100 and determine its capacity to form fibrils

Pmel17 is a melanocyte/melanoma-specific protein that is essential for the maturation of melanosomes to form mature, fibrillar, and pigmented organelles. Recently, we reported that the less glycosylated form of Pmel17 (termed iPmel17) is sorted via the plasma membrane in a manner distinct from mature Pmel17 (termed mPmel17), which is sorted directly to melanosomes. To clarify the mechanism(s) underlying the distinct processing and sorting of Pmel17, we generated a highly specific antibody (termed alphaPEP25h) against an epitope within the repeat domain of Pmel17 that is sensitive to changes in O-glycosylation. alphaPEP25h recognizes only iPmel17 and allows analysis of the processing and sorting of iPmel17 when compared with alphaPEP13h, an antibody that recognizes both iPmel17 and mPmel17. Our novel findings using alphaPEP25h demonstrate that iPmel17 differs from mPmel17 not only in its sensitivity to endoglycosidase H, but also in the content of core 1 O-glycans modified with sialic acid. This evidence reveals that iPmel17 is glycosylated differently in the Golgi and that it is sorted through the secretory pathway. Analysis of Pmel17 processing in glycosylation-deficient mutant cells reveals that Pmel17 lacking the correct addition of sialic acid and galactose loses the ability to form fibrils. Furthermore, we show that addition of sialic acid affects the stability and sorting of Pmel17 and reduces pigmentation. Alterations in sialyltransferase activity and substrates differ between normal and transformed melanocytes and may represent a critical change during malignant transformation.     

Durable Suppression of HIV-1 after Virologic Monitoring-Based Antiretroviral Adherence Counseling in Rakai,Uganda

ObjectivesHIV viral load is recommended for monitoring antiretroviral treatment and identifying treatment failure. We assessed the durability of viral suppression after viral load-triggered adherence counseling among patients with HIV viremia 6 months after ART initiation.DesignObservational cohort enrolled in an antiretroviral treatment program in rural Uganda.MethodsParticipants who underwent routine viral load determination every 24 weeks and had at least 48 weeks of follow-up were included in this analysis. Patients with viral loads >400 copies/ml at 24 weeks of treatment were given additional adherence counseling, and all patients were followed to assess the duration of viral suppression and development of virologic failure.Results1,841 participants initiating antiretroviral therapy were enrolled in the Rakai Health Sciences Program between June 2005 and June 2011 and were followed with viral load monitoring every 24 weeks. 148 (8%) of patients did not achieve viral suppression at 24 weeks and were given additional adherence counseling. 85 (60%) of these patients had undetectable viral loads at 48 weeks, with a median duration of viral suppression of 240 weeks (IQR 193-288 weeks). Failure to achieve an undetectable viral load at 48 weeks was associated with age <30 years and 24 week viral load >2,000 copies/ml in multivariate logistic regression analysis.ConclusionsThe majority of patients with persistent viremia who were provided adherence counseling achieved robust viral suppression for a median 4.6 years. Access to virologic monitoring and adherence counseling is a priority in resource-limited settings.     

Potential evolutionary influences on overlapping reading frames in the bovine leukemia virus pXBL region

Bovine leukemia virus contains a pXBL region encoding the 3' parts of four regulatory proteins (Tax, Rex, G4, R3) in overlapping reading frames. Here we report the pXBL polymorphisms of 30 isolates from four countries. Rates of overall and synonymous substitutions were consistently lower, and nucleotide/amino acid composition bias and codon bias higher, in more-overlapped than in less-overlapped regions. Ratios of nonsynonymous/synonymous substitutions were lowest in the tax gene and its subregions. The 5' parts of the four genes showed selection patterns corresponding to their genomic context outside of the pXBL region. Longer G4 variants due to a natural stop codon mutation had additional triple overlap with reduced sequence variability. These data support the concept that a higher level of overlapping in coding regions correlates with greater evolutionary constraint. Tax, the most conserved among the four regulatory proteins, showed purifying selection consistent with its importance in the viral life cycle.     

The vas deferens of the spider crab,Libinia emarginata

Sperm enter the anterior vas deferens individually in the spider crab male. There they become surrounded by secretion products from the cells of the vas deferens, and are compartmentalized into spermatophores of varying size. The anterior vas deferens can be divided into three regions. The epithelium of the anterior vas deferens varies regionally from low to high columnar. The cytoplasm contains vast arrays of rough endoplasmic reticulum and Golgi complexes but few mitochondria. Intercellular spaces contain septate junctions, gap junctions and vesicles. Once the spermatophores have been formed in the anterior vas deferens, they are moved posteriorly to the middle vas deferens where they are stored and surrounded by seminal fluids. The epithelial cells of the middle vas deferens contain large amounts of rough endoplasmic reticulum and Golgi complexes. Numerous micropinocytotic vesicles appear, forming at the cell surface and within the apical cytoplasm. Their suggested function is the resorption of secretion products of the anterior vas deferens which initiated compartmentalization of the spermatozoa into spermatophores. The posterior vas deferens functions primarily as a storage center for spermatophores until they are released at the time of copulation. Seminal fluid surrounding the spermatophores is produced in this region as well as in the middle vas deferens. The cells of this region contain vast arrays of vesicular rough endoplasmic reticulum and Golgi complexes. The cells are multinucleate. Microtubules are numerous throughout the length of the cells and appear to insert on the plasma membrane.