Diagnosis of Hunter's syndrome carriers; Radioactive sulphate incorporation into fibroblasts in the presence of fructose 1-phosphate

Summary Mutual correction of co-cultivated fibroblasts from patients with Hunter's and Hurler's syndrome could be inhibited by either fructose 1-phosphate or mannose 6-phosphate. In the presence of fructose 1-phosphate a 50% mixture of fibroblasts from a patient with Hunter's syndrome and a normal homozygous individual showed an increased³⁵S-sulphate incorporation into acid mucopolysaccharides. When fibroblast cultures from one obligate and two possible carriers of Hunter's syndrome were tested for³⁵S-sulphate incorporation, the cultures showed either twice the normal³⁵S-sulphate incorporation into acid mucopolysaccharides in the presence of fructose 1-phosphate or an abnormally high incorporation in the presence as well as in the absence of the sugar phosphate.     

Characterizing the functional significance of the neonatal rat vibrissae prior to the onset of whisking

The present series of experiments assessed how information from the whiskers controls and modulates infant rat behavior during early learning and attachment. Passive vibrissal stimulation can elicit behavioral activity in pups throughout the first two postnatal weeks, although orienting to the source of stimulation is evident only after ontogenetic emergence of whisking. In addition, while pups were capable of demonstrating learning in a classical conditioning paradigm pairing vibrissa stimulation with electric shock, no corresponding changes were detected in the anatomy of the barrel cortex as determined by cytochrome oxidase (CO) staining. Finally, the role of whiskers in a more naturalistic setting was determined in postnatal day (PN)3-5 and PN11-12 pups. Our results showed that both nipple attachment and huddling were disrupted in whisker-clipped PN3-5 pups but only marginally altered in PN11-12 pups. Together, these results suggest that the neonatal whisker system is behaviorally functional and relevant for normal mother-infant interactions, though it lacks the sophistication of a mature whisker system that evokes very specific and directed responses.     

Interaction network of the ribosome assembly machinery from a eukaryotic thermophile

Ribosome biogenesis in eukaryotic cells is a highly dynamic and complex process innately linked to cell proliferation. The assembly of ribosomes is driven by a myriad of biogenesis factors that shape pre‐ribosomal particles by processing and folding the ribosomal RNA and incorporating ribosomal proteins. Biochemical approaches allowed the isolation and characterization of pre‐ribosomal particles from Saccharomyces cerevisiae, which lead to a spatiotemporal map of biogenesis intermediates along the path from the nucleolus to the cytoplasm. Here, we cloned almost the entire set (～180) of ribosome biogenesis factors from the thermophilic fungus Chaetomium thermophilum in order to perform an in‐depth analysis of their protein–protein interaction network as well as exploring the suitability of these thermostable proteins for structural studies. First, we performed a systematic screen, testing about 80 factors for crystallization and structure determination. Next, we performed a yeast 2‐hybrid analysis and tested about 32,000 binary combinations, which identified more than 1000 protein–protein contacts between the thermophilic ribosome assembly factors. To exemplary verify several of these interactions, we performed biochemical reconstitution with the focus on the interaction network between 90S pre‐ribosome factors forming the ctUTP‐A and ctUTP‐B modules, and the Brix‐domain containing assembly factors of the pre‐60S subunit. Our work provides a rich resource for biochemical reconstitution and structural analyses of the conserved ribosome assembly machinery from a eukaryotic thermophile.     

A hidden Markov movement model for rapidly identifying behavioral states from animal tracks

Electronic telemetry is frequently used to document animal movement through time. Methods that can identify underlying behaviors driving specific movement patterns can help us understand how and why animals use available space, thereby aiding conservation and management efforts. For aquatic animal tracking data with significant measurement error, a Bayesian state‐space model called the first‐Difference Correlated Random Walk with Switching (DCRWS) has often been used for this purpose. However, for aquatic animals, highly accurate tracking data are now becoming more common. We developed a new hidden Markov model (HMM) for identifying behavioral states from animal tracks with negligible error, called the hidden Markov movement model (HMMM). We implemented as the basis for the HMMM the process equation of the DCRWS, but we used the method of maximum likelihood and the R package TMB for rapid model fitting. The HMMM was compared to a modified version of the DCRWS for highly accurate tracks, the DCRWS, and to a common HMM for animal tracks fitted with the R package moveHMM. We show that the HMMM is both accurate and suitable for multiple species by fitting it to real tracks from a grey seal, lake trout, and blue shark, as well as to simulated data. The HMMM is a fast and reliable tool for making meaningful inference from animal movement data that is ideally suited for ecologists who want to use the popular DCRWS implementation and have highly accurate tracking data. It additionally provides a groundwork for development of more complex modeling of animal movement with TMB. To facilitate its uptake, we make it available through the R package swim.     

Malaria and urbanization in sub-Saharan Africa

There are already 40 cities in Africa with over 1 million inhabitants and the United Nations Environmental Programme estimates that by 2025 over 800 million people will live in urban areas. Recognizing that malaria control can improve the health of the vulnerable and remove a major obstacle to their economic development, the Malaria Knowledge Programme of the Liverpool School of Tropical Medicine and the Systemwide Initiative on Malaria and Agriculture convened a multi-sectoral technical consultation on urban malaria in Pretoria, South Africa from 2nd to 4th December, 2004. The aim of the meeting was to identify strategies for the assessment and control of urban malaria. This commentary reflects the discussions held during the meeting and aims to inform researchers and policy makers of the potential for containing and reversing the emerging problem of urban malaria.     

Assessment of interleukin 1 and interleukin 2 effects on cycling and noncycling murine thymocytes

When mouse thymocytes are stimulated with PHA, the proliferative response is very low, unless the culture medium is enriched with interleukin 1 (IL-1)- or interleukin 2 (IL-2)-containing supernatants. Cytofluorometric analyses show, however, that PHA stimulation generates a significant number of cells with increased RNA content (transition from the G₀ to G₁ phase of the cell cycle). If IL-2 is added to such cultures, the activated cells complete their process of RNA synthesis and then enter the S phase. The use of IL-2-containing culture medium thus permits one to obtain a high correlation between the number of g₁ cells and [³H]thymidine incorporation (r = 0.97). Enrichment with IL-1-containing supernatants also results in a statistically significant correlation (r = 0.68), but the regression lines are markedly different for the two interleukins (s = 20.3 for IL-2 and s = 9.2 for IL-1), when analyzed after 48 hr of incubation. These observations suggest that the G₁ phase must be divided into two subcompartments, G_1a and G_1b, the G_1a-G_1b transition being an IL-2-dependent event. If the number of G_1b cells is used to establish correlations with [³H]thymidine incorporation, all values fall on the same regression line, regardless of culture conditions and of the addition of interleukins. It is concluded that IL-2 regulates lymphocyte proliferation at the level of RNA synthesis (G_1a-G_1b transition) rather than that of DNA synthesis (G₁-S transition).     

Protein kinase CK2 phosphorylates the Fas-associated factor FAF1 in vivo and influences its transport into the nucleus

We previously identified the Fas-associated factor FAF1 as an in vitro substrate of protein kinase CK2 and determined Ser289 and Ser291 as phosphorylation sites. Here we demonstrate that these two serine residues are the only sites phosphorylated by CK2 in vitro, and that at least one site is phosphorylated in vivo. Furthermore, we analyzed putative physiological functions of FAF1 phosphorylation. The ability of FAF1 to potentiate Fas-induced apoptosis is not influenced by the FAF1 phosphorylation status; however, the nuclear import of a phosphorylation-deficient FAF1 mutant was delayed in comparison to wild-type FAF1.     

Planar cell polarity proteins differentially regulate extracellular matrix organization and assembly during zebrafish gastrulation

Zebrafish gastrulation cell movements occur in the context of dynamic changes in extracellular matrix (ECM) organization and require the concerted action of planar cell polarity (PCP) proteins that regulate cell elongation and mediolateral alignment. Data obtained using Xenopus laevis gastrulae have shown that integrin–fibronectin interactions underlie the formation of polarized cell protrusions necessary for PCP and have implicated PCP proteins themselves as regulators of ECM. By contrast, the relationship between establishment of PCP and ECM assembly/remodeling during zebrafish gastrulation is unclear. We previously showed that zebrafish embryos carrying a null mutation in the four-pass transmembrane PCP protein vang-like 2 (vangl2) exhibit increased matrix metalloproteinase activity and decreased immunolabeling of fibronectin. These data implicated for the first time a core PCP protein in the regulation of pericellular proteolysis of ECM substrates and raised the question of whether other zebrafish PCP proteins also impact ECM organization. In Drosophila melanogaster, the cytoplasmic PCP protein Prickle binds Van Gogh and regulates its function. Here we report that similar to vangl2, loss of zebrafish prickle1a decreases fibronectin protein levels in gastrula embryos. We further show that Prickle1a physically binds Vangl2 and regulates both the subcellular distribution and total protein level of Vangl2. These data suggest that the ability of Prickle1a to impact fibronectin organization is at least partly due to effects on Vangl2. In contrast to loss of either Vangl2 or Prickle1a function, we find that glypican4 (a Wnt co-receptor) and frizzled7 mutant gastrula embryos with disrupted non-canonical Wnt signaling exhibit the opposite phenotype, namely increased fibronectin assembly. Our data show that glypican4 mutants do not have decreased proteolysis of ECM substrates, but instead have increased cell surface cadherin protein expression and increased intercellular adhesion. These data indicate that Wnt/Glypican4/Frizzled signaling regulates ECM assembly through effects on cadherin-mediated cell cohesion. Together, our results demonstrate that zebrafish Vangl2/Prickle1a and non-canonical Wnt/Frizzled signaling have opposing effects on ECM organization underlying PCP and gastrulation cell movements.