600 ps Molecular dynamics reveals stable substructures and flexible hinge points in cAMP dependent protein kinase

Molecular dynamics simulations of the catalytic subunit of cAMP dependent protein kinase (cAPK) have been performed in an aqueous environment. The relations among the protein hydrogen‐bonding network, secondary structural elements, and the internal motions of rigid domains were examined. The values of fluctuations of protein dihedral angles during dynamics show quite distinct maxima in the regions of loops and minima in the regions of α‐helices and β‐strands. Analyses of conformation snapshots throughout the run show stable subdomains and indicate that these rigid domains are constrained during the dynamics by a stable network of hydrogen bonds. The most stable subdomain during the dynamics was in the small lobe including part of the carboxy‐terminal tail. The most significant flexible region was the highly conserved glycine‐rich loop between β strands 1 and 2 in the small lobe. Many of the main chain dihedral angle changes measured in a comparison of the crystallographic structures of “open” and “closed” conformations of cAPK correspond to the highly flexible residues found during dynamics. © 1999 John Wiley & Sons, Inc. Biopoly 50: 513–524, 1999     

Distinct thyroid hormone‐dependent expression of trkB and p75NGFR in nonneuronal cells during the critical TH‐dependent period of the cochlea

Analyzing the thyroid hromone (TH)‐dependent period of the inner ear, we observed that the presence of triiodothyronine (T3) between postnatal day 3 (P3) and P12 is sufficient for functional maturation of the auditory system. Within this short time period, an unusual transient TH‐dependent expression of nonneuronal neurotrophin receptors (NT‐R) trkB and p75^NGFR was observed in correlation with neuronal and morphogenetic processes. The availability of thyroid hormone was revealed to be invariably correlated with (a) a transient expression of full‐length trkB in TRα1‐, TRα2‐ and TRβ1‐expressing hair cells concomitant to the segregation of afferent fibers and the synaptogenesis of efferent fibers; and (b) a transient expression of p75^NGFR in TRα1‐ and TRβ1‐expressing great epithelia ridge cells in direct spatiotemporal correlation with the appearance of apoptotic cells and morphogenetic maturation of the organ. For the first time, these data suggest a TH dependency of the expression of neurotrophin receptors in nonneuronal cells. A potential role of these peculiar neurotrophin receptor expression for the conversion of the biological function of TH on innervation patterning and morphogenesis during the critical TH‐dependent period of the inner ear may be considered. © 1999 John Wiley & Sons, Inc. J Neurobiol 38: 338–356, 1999     

Life and death of <Emphasis Type="Italic">Trypanosoma cruzi</Emphasis> in presence of metals

Trypanosoma cruzi has many molecules that need metallic elements to work, allowing cell invasion and the establishment of infection, causing Chagas disease. Nonetheless, knowledge regarding how the parasites address metals and maintain homeostasis is lacking. To study this relationship, zinc, cadmium and mercury were chosen. Epimastigote, trypomastigote and intracellular forms of T. cruzi were incubated with these metals for different times and at different concentrations. In general, epimastigotes were the most sensitive and trypomastigotes the most resistant to metals. ZnCl₂ induced low toxic effects to all parasite forms. Although the parasites were very sensitive to the toxic effects of CdCl₂ and HgCl₂, pretreatment with ZnCl₂ decreased the death rate. The trypomastigotes pretreated with CdCl₂ were unable to infect the host cells, and the treated intracellular forms were damaged after 2 h of incubation, when the toxic effects were poorly reverted. New insights on metal toxicity mechanisms are provided, helping to understand how metallic ions influence the parasite’s biochemical and physiological processes.     

<Emphasis Type="Italic">Deinococcus knuensis</Emphasis> sp. nov., a bacterium isolated from river water

Strain 16F3H^T, a Gram-negative, aerobic, non-motile, and oval-shaped bacterium, was isolated from river water collected from the Han River in South Korea. Growth of strain 16F3H^T was observed at 10–42 °C (optimum at 25–30 °C), but no growth occurred at 4 °C. The strain is able to grow at pH 4–10 (optimum at pH 7–8) and tolerates up to 4% NaCl (w/v), with optimum growth at 0.5% NaCl. The isolate was found to be resistant to UV irradiation. Based on 16S rRNA gene sequence analysis, it is closely related to ‘Deinococcus seoulensis’ 16F1E (98.8%), Deinococcus aquaticus PB314^T (98.1%) and Deinococcus caeni Ho-08^T (98.0%). The level of DNA–DNA homology between the novel strain and the three related strains was 57.4, 41.2, and 35.8%, respectively. Chemotaxonomic data revealed that strain 16F3H^T possesses MK-8 as the predominant respiratory quinone, an unidentified phosphoglycolipid as the major polar lipid, and C_15:1 ω6c and C_16:1 ω7c as the major fatty acids. The DNA G + C content was determined to be 65.7 mol%. Based on polyphasic evidence, strain 16F3H^T (=KCTC 33794^T = JCM 31406^T) should be classified as the type strain of a novel Deinococcus species, for which the name Deinococcus knuensis sp. nov. is proposed.     

CFTR mutations in Turkish and North African cystic fibrosis patients in Europe: implications for screening

AIMS: To obtain more insight into the variability of the CFTR mutations found in immigrant cystic fibrosis (CF) patients who are living in Europe now, and to estimate the test sensitivity of different frequently used methods of DNA analysis to detect CF carriers or patients among these Turkish or North African immigrants. METHODS: A survey among 373 European CF centers asking which CFTR mutations had been found in Turkish and North African CF patients. RESULTS: 31 and 26 different mutations were reported in Turkish and North African patients, identifying 64.2% (113/176) and 87.4% (118/135) alleles, respectively (p < 0.001). The mean sensitivity (detection rate) of three most common CFTR mutation panels to detect these mutations differed between Turkish and North African people, 44.9% (79/176) versus 69.6% (94/135) (p < 0.001), and can be increased to 57.4% (101/176) and 79.3% (107/135) (p < 0.001), respectively, by expanding these panels with 13 mutations which have been found on two or more alleles. CONCLUSION: 35.8% and 12.6%, respectively, of CF alleles in Turkish and North African patients living in Europe now had not been identified. Among these populations, the test sensitivity of common CFTR mutation panels is insufficient for use in screening programs in Europe, even after expansion with frequent Turkish and North African mutations. This raises questions about whether and how to implement CF carrier and neonatal screening in a multiethnic society.     

Recent insights into the biological roles of mucin-type O-glycosylation

In this special issue of the Glycoconjugate Journal focusing on glycosciences and development, we summarize recent advances in our understanding of the role of mucin-type O-glycans in development and disease. The presence of this widespread protein modification has been known for decades, yet identification of its biological functions has been hampered by the redundancy and complexity of the enzyme family controlling the initiation of O-glycosylation, as well as the diversity of extensions of the core sugar. Recent studies in organisms as diverse as mammals and Drosophila have yielded insights into the function of this highly abundant and evolutionarily-conserved protein modification. Gaining an understanding of mucin-type O-glycans in these diverse systems will elucidate crucial conserved processes underlying many aspects of development and homeostasis.     

Role of CLASP2 in microtubule stabilization and the regulation of persistent motility

In motile fibroblasts, stable microtubules (MTs) are oriented toward the leading edge of cells. How these polarized MT arrays are established and maintained, and the cellular processes they control, have been the subject of many investigations. Several MT "plus-end-tracking proteins," or +TIPs, have been proposed to regulate selective MT stabilization, including the CLASPs, a complex of CLIP-170, IQGAP1, activated Cdc42 or Rac1, a complex of APC, EB1, and mDia1, and the actin-MT crosslinking factor ACF7. By using mouse embryonic fibroblasts (MEFs) in a wound-healing assay, we show here that CLASP2 is required for the formation of a stable, polarized MT array but that CLIP-170 and an APC-EB1 interaction are not essential. Persistent motility is also hampered in CLASP2-deficient MEFs. We find that ACF7 regulates cortical CLASP localization in HeLa cells, indicating it acts upstream of CLASP2. Fluorescence-based approaches show that GFP-CLASP2 is immobilized in a bimodal manner in regions near cell edges. Our results suggest that the regional immobilization of CLASP2 allows MT stabilization and promotes directionally persistent motility in fibroblasts.     

Importance of the N-distal AP-2 binding element in Nef for simian immunodeficiency virus replication and pathogenicity in rhesus macaques

Point mutations in SIVmac239 Nef disrupting CD4 downmodulation and enhancement of virion infectivity attenuate viral replication in acutely infected rhesus macaques, but changes selected later in infection fully restore Nef function (A. J. Iafrate et al., J. Virol. 74:9836-9844, 2000). To further evaluate the relevance of these Nef functions for viral persistence and disease progression, we analyzed an SIVmac239 Nef mutant containing a deletion of amino acids Q64 to N67 (delta64-67Nef). This mutation inactivates the N-distal AP-2 clathrin adaptor binding element and disrupts the abilities of Nef to downregulate CD4, CD28 and CXCR4 and to stimulate viral replication in vitro. However, it does not impair the downmodulation of CD3 and class I major histocompatibility complex (MHC-I) or MHC-II and the upregulation of the MHC-II-associated invariant chain, and it has only a moderate effect on the enhancement of virion infectivity. Replication of the delta64-67Nef variant in acutely infected macaques was intermediate between grossly nef-deleted and wild-type SIVmac239. Subsequently, three of six macaques developed moderate to high viral loads and developed disease, whereas the remaining animals efficiently controlled SIV replication and showed a more attenuated clinical course of infection. Sequence analysis revealed that the deletion in nef was not repaired in any of these animals. However, some changes that slightly enhanced the ability of Nef to downmodulate CD4 and moderately increased Nef-mediated enhancement of viral replication and infectivity in vitro were observed in macaques developing high viral loads. Our results imply that both the Nef functions that were disrupted by the delta64-67 mutation and the activities that remained intact contribute to viral pathogenicity.