Hereditary hemochromatosis genetic testing of at-risk children: what is the appropriate age?

The objective of this study was to consider the objective evidence and ethical arguments for the appropriate age to test children at risk of developing hereditary hemochromatosis. A literature search for information on iron overload in children, onset of disease expression for hemochromatosis, and recommendations for age of cascade screening was undertaken. We examined the objective evidence and arguments for testing in early childhood and those for delaying testing until later teenage years. Cascade testing of offspring of people with hemochromatosis is widely advocated because it is an easily preventable disease. The ideal age to test those offspring is a matter of debate. Some authorities advocate testing at a very young age whereas others recommend delaying testing until late teenage years. To date there has been no published overview of the objective evidence and arguments central to this debate. In children who are C282Y homozygous, iron overload is rare in the first two decades of life and associated morbidity has only been documented in 1 patient. In the cascade setting, genetic testing for hemochromatosis need not be offered until late teenage years.     

Expression of LEKTI domains 6-9' in the baculovirus expression system: recombinant LEKTI domains 6-9' inhibit trypsin and subtilisin A

The precursor lympho-epithelial Kazal-type-related inhibitor (LEKTI), containing two Kazal-type and 13 nonKazal-type domains, is an efficient inhibitor of multiple serine proteinases, among them plasmin, subtilisin A, cathepsin G, elastase, and trypsin. To gain insight into the structure and function of some of these domains, a portion of the cDNA coding for LEKTI domains 6-9' was cloned and expressed in Sf9 cells using the baculovirus expression vector system (BEVS). Through a single purification step using a Co2+ column, 3-4 mg of purified recombinant LEKTI-domains 6-9' (rLEKTI6-9') with the predicted molecular mass of 34.6 kDa was obtained from the cell pellet of a 1-L culture. Unlike full-length LEKTI, rLEKTI6-9' inhibited trypsin and subtilisin A but not plasmin, cathepsin G, or elastase. The inhibition of trypsin and subtilisin A by rLEKTI6-9' occurred through a noncompetitive mechanism, with inhibitory constants (Ki) of 356 +/- 12 and 193 +/- 10 nM, respectively. On the basis of the Ki values, rLEKTI6-9' was determined to be a more potent trypsin inhibitor and a less potent subtilisin A inhibitor than the full-length LEKTI. In contrast to LEKTI domains 6-9', recombinant LEKTI domain 6 does not inhibit subtilisin A but competitively inhibited trypsin with a Ki of 200 +/- 10 nM. Taking LEKTI6-9' as an example, the BEVS should facilitate the structure-function analysis of naturally occurring processed LEKTI forms that have physiological relevance.     

Tumor necrosis factor-alpha induces functionally active hyaluronan-adhesive CD44 by activating sialidase through p38 mitogen-activated protein kinase in lipopolysaccharide-stimulated human monocytic cells

Interaction of CD44, an adhesion molecule, with its ligand, hyaluronan (HA), in monocytic cells plays a critical role in cell migration, inflammation, and immune responses. Most cell types express CD44 but do not bind HA. The biological functions of CD44 have been attributed to the generation of the functionally active, HA-adhesive form of this molecule. Although lipopolysaccharide (LPS) and cytokines induce HA-adhesive CD44, the molecular mechanism underlying this process remains unknown. In this study, we show that LPS-induced CD44-mediated HA (CD44-HA) binding in monocytes is regulated by endogenously produced tumor necrosis factor (TNF)-alpha and IL-10. Furthermore, p38 mitogen-activated protein kinase (MAPK) activation was required for LPS- and TNF-alpha-induced, but not IL-10-induced, CD44-HA-binding in normal monocytes. To dissect the signaling pathways regulating CD44-HA binding independently of cross-regulatory IL-10-mediated effects, IL-10-refractory promonocytic THP-1 cells were employed. LPS-induced CD44-HA binding in THP-1 cells was regulated by endogenously produced TNF-alpha. Our results also suggest that lysosomal sialidase activation may be required for the acquisition of the HA-binding form of CD44 in LPS- and TNF-alpha-stimulated monocytic cells. Studies conducted to understand the role of MAPKs in the induction of sialidase activity revealed that LPS-induced sialidase activity was dependent on p42/44 MAPK-mediated TNF-alpha production. Blocking TNF-alpha production by PD98059, a p42/44 inhibitor, significantly reduced the LPS-induced sialidase activity and CD44-HA binding. Subsequently, TNF-alpha-mediated p38 MAPK activation induced sialidase activity and CD44-HA binding. Taken together, our results suggest that TNF-alpha-induced p38 MAPK activation may regulate the induction of functionally active HA-binding form of CD44 by activating sialidase in LPS-stimulated human monocytic cells.     

Collection and culture techniques for gelatinous zooplankton

Gelatinous zooplankton are the least understood of all planktonic animal groups. This is partly due to their fragility, which typically precludes the capture of intact specimens with nets or trawls. Specialized tools and techniques have been developed that allow researchers and aquarists to collect intact gelatinous animals at sea and to maintain many of these alive in the laboratory. This paper summarizes the scientific literature on the capture, collection, and culture of gelatinous zooplankton and incorporates many unpublished methods developed at the Monterey Bay Aquarium in the past 15 years.     

Geomicrobiology of the ocean crust: a role for chemoautotrophic Fe-bacteria

The delicate balance of the major global biogeochemical cycles greatly depends on the transformation of Earth materials at or near its surface. The formation and degradation of rocks, minerals, and organic matter are pivotal for the balance, maintenance, and future of many of these cycles. Microorganisms also play a crucial role, determining the transformation rates, pathways, and end products of these processes. While most of Earth's crust is oceanic rather than terrestrial, few studies have been conducted on ocean crust transformations, particularly those mediated by endolithic (rock-hosted) microbial communities. The biology and geochemistry of deep-sea and sub-seafloor environments are generally more complicated to study than in terrestrial or near-coastal regimes. As a result, fewer, and more targeted, studies usually homing in on specific sites, are most common. We are studying the role of endolithic microorganisms in weathering seafloor crustal materials, including basaltic glass and sulfide minerals, both in the vicinity of seafloor hydrothermal vents and off-axis at unsedimented (young) ridge flanks. We are using molecular phylogenetic surveys and laboratory culture studies to define the size, diversity, physiology, and distribution of microorganisms in the shallow ocean crust. Our data show that an unexpected diversity of microorganisms directly participate in rock weathering at the seafloor, and imply that endolithic microbial communities contribute to rock, mineral, and carbon transformations.     

Adaptation of rainbow fish to lake and stream habitats

Fish occupy a range of hydrological habitats that exert different demands on locomotor performance. We examined replicate natural populations of the rainbow fishes Melanotaenia eachamensis and M. duboulayi to determine if colonization of low-velocity (lake) habitats by fish from high-velocity (stream) habitats resulted in adaptation of locomotor morphology and performance. Relative to stream conspecifics, lake fish had more posteriorly positioned first dorsal and pelvic fins, and shorter second dorsal fin bases. Habitat dimorphism observed between wild-caught fish was determined to be heritable as it was retained in M. eachamensis offspring raised in a common garden. Repeated evolution of the same heritable phenotype in independently derived populations indicated body shape divergence was a consequence of natural selection. Morphological divergence between hydrological habitats did not support a priori expectations of deeper bodies and caudal peduncles in lake fish. However, observed divergence in fin positioning was consistent with a family-wide association between habitat and morphology, and with empirical studies on other fish species. As predicted, decreased demand for sustained swimming in lakes resulted in a reduction in caudal red muscle area of lake fish relative to their stream counterparts. Melanotaenia duboulayi lake fish also had slower sustained swimming speeds (Ucrit) than stream conspecifics. In M. eachamensis, habitat affected Ucrit of males and females differently. Specifically, females exhibited the pattern observed in M. duboulayi (lake fish had faster Ucrit than stream fish), but the opposite association was observed in males (stream males had slower Ucrit than lake males). Stream M. eachamensis also exhibited a reversed pattern of sexual dimorphism in Ucrit (males slower than females) relative to all other groups (males faster than females). We suggest that M. eachamensis males from streams responded to factors other than water velocity. Although replication of muscle and Ucrit phenotypes across same habitat populations within and/or among species was suggestive of adaptation, the common garden experiment did not confirm a genetic basis to these associations. Kinematic studies should consider the effect of the position and base length of dorsal fins.     

Saccharomyces cerevisiae RRM3, a 5' to 3' DNA helicase,physically interacts with proliferating cell nuclear antigen

Proliferating cell nuclear antigen (PCNA) plays an essential role in eukaryotic DNA replication, and numerous DNA replication proteins have been found to interact with PCNA through a conserved eight-amino acid motif called the PIP-box. We have searched the genome of the yeast Saccharomyces cerevisiae for open reading frames that encode proteins with putative PIP-boxes and initiated testing of 135 novel candidates for their ability to interact with PCNA-conjugated agarose beads. The first new PCNA-binding protein identified in this manner is the 5' to 3' DNA helicase RRM3. Yeast two-hybrid tests show that N-terminal deletions of RRM3, which remove the PIP-box but leave the helicase motifs intact, abolish the interaction with PCNA. In addition, mutating the two phenylalanine residues in the PIP-box to alanine or aspartic acid reduces binding to PCNA, confirming that the PIP-box in RRM3 is responsible for interaction with PCNA. The results presented here suggest that the RRM3 helicase functions at the replication fork.     

Antigen-driven oligoclonal expansion of tumor-infiltrating B cells in infiltrating ductal carcinoma of the breast

The objective of this study was to determine whether tumor-infiltrating B cells (TIL-B) of infiltrating ductal carcinoma (IDC) of the breast represent a tumor-specific humoral immune response. Immunohistochemical analysis of three Her-2/neu-negative IDC tumors from geriatric patients showed that TIL-B cluster in structures similar to germinal centers containing CD20(+) B lymphocyte and CD3(+) T lymphocyte zones with interdigitating CD21(+) follicular dendritic cells, suggesting an in situ immune response. A total of 29, 31, and 58 IgG1 H chain clones was sequenced from the three IDC tumors, respectively. Intratumoral oligoclonal expansion of TIL-B was demonstrated by a preponderance (45-68%) of clonal B cells. In contrast, only 7% of tumor-draining lymph node and 0% of healthy donor PBL IgG H chains were clonal, consistent with the larger repertoires of node and peripheral populations. Patterns and levels of TIL-B IgG H chain somatic hypermutation suggested affinity maturation in intratumoral germinal centers. To examine the specificity of TIL-B Ig, a phage-displayed Fab library was generated from the TIL-B of one IDC tumor. Panning with an allogeneic breast cancer cell line enriched Fab binding to breast cancer cells, but not nonmalignant cell lines tested. However, panning with autologous tumor tissue lysate increased binding of Fab to both tumor tissue lysate and healthy breast tissue lysate. These data suggest an in situ Ag-driven oligoclonal B cell response to a variety of tumor- and breast-associated Ags.