Nuclear envelope: nuclear pore complexity

A new study shows that the filamentous fungus, Aspergillus nidulans, which has a closed mitosis, does not maintain a continuous permeability barrier during mitosis. This work challenges current views of the differences between closed and open mitosis and has implications for understanding mitotic specific changes in the nuclear pore complex and Ran GTPase system in lower eukaryotes.     

Directional selectivity is formed at multiple levels by laterally offset inhibition in the rabbit retina

The excitatory and inhibitory inputs to directionally selective (DS) ganglion cells are themselves directionally selective. Directionality is achieved because excitation is reduced during null-direction movement along a GABAergic pathway. Inhibition is reduced during preferred-direction movement along a pathway that includes cholinergic synapses. Both excitation and inhibition are made directional by laterally offset inhibitory signals similar to the spatial offset of the direct inhibitory input to the DS cell dendrites. Thus, spatially offset lateral inhibition generates directionality at three different levels in the DS circuitry. We also found that for stimuli falling within the dendritic field, cholinergic input is delivered to the OFF but not the ON dendrites. Cholinergic pathways from outside the dendritic field reach both ON and OFF dendrites, but both of these pathways are normally inactivated by GABAergic synapses.     

Global-scale genetic identification of hammerhead sharks: Application to assessment of the international fin trade and law enforcement

The future status of sharks is an issue of widespread conservation concern due to declines in many species in the face of high levels of exploitation to satisfy market demands for products, especially fins. Substantial declines in the large-bodied hammerhead sharks, Sphyrna lewini, S. mokarran and S. zygaena, even in regions where some management occurs, indicate that informed conservation measures are warranted for these circumglobally distributed species. Despite the importance of assessing shark catch and trade on a species-specific basis to detect potential overexploitation of individual species, achieving this goal for hammerheads has proven elusive due to difficulties in identification of their products. Here, we present the development and application of a diagnostic, streamlined, five-primer multiplex polymerase chain reaction assay utilizing species-specific primers based on nuclear ribosomal ITS2 for the three hammerhead species throughout their global distribution. Application of this assay to investigations of the fin market confirmed the presence of hammerhead fins in the international trade. A study of the world’s largest fin market in Hong Kong revealed a high concordance between specific Chinese-name trade categories and fins from these three species (“Bai Chun” with S. lewini, “Gui Chun” with S. zygaena and “Gu Pian” with S.␣mokarran), and clear species preferences. This concordance information allows the use of market records for monitoring species-specific trends in trade and exploitation rates. The assay is also proving useful for identification of shark body parts in U.S. fisheries law-enforcement activities. Screening of morphologically identified “ S. lewini” from globally distributed areas using this assay with subsequent whole ITS2 sequencing suggests a cryptic species closely related to S. lewini occurs off the SE USA coast.     

Shigella dysenteriae ShuS promotes utilization of heme as an iron source and protects against heme toxicity

Shigella dysenteriae serotype 1, a major cause of bacillary dysentery in humans, can use heme as a source of iron. Genes for the transport of heme into the bacterial cell have been identified, but little is known about proteins that control the fate of the heme molecule after it has entered the cell. The shuS gene is located within the heme transport locus, downstream of the heme receptor gene shuA. ShuS is a heme binding protein, but its role in heme utilization is poorly understood. In this work, we report the construction of a chromosomal shuS mutant. The shuS mutant was defective in utilizing heme as an iron source. At low heme concentrations, the shuS mutant grew slowly and its growth was stimulated by either increasing the heme concentration or by providing extra copies of the heme receptor shuA on a plasmid. At intermediate heme concentrations, the growth of the shuS mutant was moderately impaired, and at high heme concentrations, shuS was required for growth on heme. The shuS mutant did not show increased sensitivity to hydrogen peroxide, even at high heme concentrations. ShuS was also required for optimal utilization of heme under microaerobic and anaerobic conditions. These data are consistent with the model in which ShuS binds heme in a soluble, nontoxic form and potentially transfers the heme from the transport proteins in the membrane to either heme-containing or heme-degrading proteins. ShuS did not appear to store heme for future use.     

ADAMTS1 proteinase is up-regulated in wounded skin and regulates migration of fibroblasts and endothelial cells

The metalloproteinase ADAMTS1 (a disintegrin and metalloproteinase with thrombospondin motifs) is induced under inflammatory conditions, and it is also a potent inhibitor of angiogenesis. Due to these properties, we speculated about the role of ADAMTS1 in cutaneous wound repair. Here we have shown up-regulation of ADAMTS1 expression in wounds of normal and particularly of healing-impaired genetically diabetic mice. Immunofluorescence staining identified macrophages as the source of ADAMTS1 in early wounds, whereas keratinocytes and fibroblasts produce this protein at later stages of wound healing. The distribution of ADAMTS1 in the normal and wounded epidermis, its regulation in cultured keratinocytes, as well as the skin phenotype of ADAMTS1 knock-out mice suggests a role of this metalloproteinase in keratinocyte differentiation. Furthermore, we provide evidence for a novel dual function of ADAMTS1 in fibroblast migration; although low concentrations of this protein stimulate fibroblast migration via its proteolytic activity, high concentrations inhibit this process because of binding to fibroblast growth factor-2 and subsequent inhibition of its promotogenic activity. Similar effects were also observed with endothelial cells. Taken together, our results suggest a role of ADAMTS1 in keratinocyte differentiation and migration of fibroblasts and endothelial cells in healing skin wounds.     

Functional tissue engineering of chondral and osteochondral constructs

Due to the prevalence of osteoarthritis (OA) and damage to articular cartilage, coupled with the poor intrinsic healing capacity of this avascular connective tissue, there is a great demand for an articular cartilage substitute. As the bearing material of diarthrodial joints, articular cartilage has remarkable functional properties that have been difficult to reproduce in tissue-engineered constructs. We have previously demonstrated that by using a functional tissue engineering approach that incorporates mechanical loading into the long-term culture environment, one can enhance the development of mechanical properties in chondrocyte-seeded agarose constructs. As these gel constructs begin to achieve material properties similar to that of the native tissue, however, new challenges arise, including integration of the construct with the underlying native bone. To address this issue, we have developed a technique for producing gel constructs integrated into an underlying bony substrate. These osteochondral constructs develop cartilage-like extracellular matrix and material properties over time in free swelling culture. In this study, as a preliminary to loading such osteochondral constructs, finite element modeling (FEM) was used to predict the spatial and temporal stress, strain, and fluid flow fields within constructs subjected to dynamic deformational loading. The results of these models suggest that while chondral ("gel alone") constructs see a largely homogenous field of mechanical signals, osteochondral ("gel bone") constructs see a largely inhomogeneous distribution of mechanical signals. Such inhomogeneity in the mechanical environment may aid in the development of inhomogeneity in the engineered osteochondral constructs. Together with experimental observations, we anticipate that such modeling efforts will provide direction for our efforts aimed at the optimization of applied physical forces for the functional tissue engineering of an osteochondral articular cartilage substitute.     

Identification of genes involved in apoptosis and dominant follicle development during follicular waves in cattle

We hypothesize that granulosa and theca cells from growing dominant follicles, with relatively high intrafollicular concentrations of estradiol, have a greater expression of genes involved in inhibiting apoptosis pathways and lower expression of genes involved in apoptosis pathways than growing subordinate follicles with lower estradiol concentrations. Using the well-characterized bovine dominant follicle model, we collected granulosa and theca cells from individual dominant and the largest subordinate follicle 3 days after initiation of a follicular wave in four animals. Based on ultrasound analysis, both follicle types were in the growth phase at the time of ovariectomy. However, dominant follicles were larger (9.8 +/- 1.0 versus 7.6 +/- 0.6 mm in diameter, P < 0.05) and had greater intrafollicular concentrations of estradiol (132.2 +/-3 8.5 versus 24.1 +/- 12.1 ng/ml, P < 0.05), compared with the largest subordinate follicles. We used bovine cDNA microarrays, which contained a total of 1400 genes, including a subset of 53 genes known to be involved in apoptosis pathways, to determine which apoptosis and marker genes from each of the four dominant versus subordinate follicles were potentially differentially expressed. Using a low stringency-screening criterion, 22 genes were identified. Quantitative real-time polymerase chain reaction confirmed that 16 of these genes were differentially expressed. Our novel results demonstrate that the high intrafollicular concentrations of estradiol in growing dominant follicles were positively associated with enhanced expression of mRNAs in granulosa cells for aromatase, LH receptor, estradiol receptor beta, DICE-1, and MCL-1, compared with granulosa cells from subordinate follicles (all survival-associated genes). In contrast, the relatively low intrafollicular concentrations of estradiol in growing subordinate follicles were positively associated with enhanced expression of mRNAs in granulosa cells for beta glycan, cyclo-oxygenase-1, tumor necrosis factor alpha, caspase-activated DNase, and DRAK-2, and in theca cells for beta glycan, caspase 13, P58(IPK), Apaf-1, BTG-3, and TS-BCLL, compared with granulosa or theca cells from dominant follicles (genes that are all associated with cell death and/or apoptosis). We suggest that that these genes may be candidate estradiol target genes and that they may be early markers for the final stages of follicle differentiation or initiation of apoptosis and thus selection of dominant follicles during follicular waves.