Caveolar endocytosis is critical for BK virus infection of human renal proximal tubular epithelial cells

In recent years, BK virus (BKV) nephritis after renal transplantation has become a severe problem. The exact mechanisms of BKV cell entry and subsequent intracellular trafficking remain unknown. Since human renal proximal tubular epithelial cells (HRPTEC) represent a main natural target of BKV nephritis, analysis of BKV infection of HRPTEC is necessary to obtain additional insights into BKV biology and to develop novel strategies for the treatment of BKV nephritis. We coincubated HRPTEC with BKV and the cholesterol-depleting agents methyl beta cyclodextrin (MBCD) and nystatin (Nys), drugs inhibiting caveolar endocytosis. The percentage of infected cells (detected by immunofluorescence) and the cellular levels of BKV large T antigen expression (detected by Western blot analysis) were significantly decreased in both MBCD- and Nys-treated HPRTEC compared to the level in HRPTEC incubated with BKV alone. HRPTEC infection by BKV was also tested after small interfering RNA (siRNA)-dependent depletion of either the caveolar structural protein caveolin-1 (Cav-1) or clathrin, the major structural protein of clathrin-coated pits. BKV infection was inhibited in HRPTEC transfected with Cav-1 siRNA but not in HRPTEC transfected with clathrin siRNA. The colocalization of labeled BKV particles with either Cav-1 or clathrin was investigated by using fluorescent microscopy and image cross-correlation spectroscopy. The rate of colocalization of BKV with Cav-1 peaked at 4 h after incubation. Colocalization with clathrin was insignificant at all time points. These results suggest that BKV entered into HRPTEC via caveolae, not clathrin-coated pits, and that BKV is maximally associated with caveolae at 4 h after infection, prior to relocation to a different intracellular compartment.     

Structure and absolute configuration of pyrophen, a novel pryrone derivative of L-phenylalanine from Aspergillus niger

Pyrophen, a novel 4-methoxy-2-pyrone derivative of L-phenylalanine isolated from cultures of Aspergillus niger, crystallizes in the orthorhombic space group P2(1)2(1)2(1), with a = 8.918(1), b = 9.199(3), and c = 18.092(4) A. The structure was solved by direct methods and the absolute configuration assigned by the Bijvoet method. Refinement gave R = 0.045 and S = 1.78 for 1677 reflections with I greater than 3 sigma (I). Though numerous other pyrone natural products are known, including other products of A. niger, this is the first report of an amino acid-pyrone derivative.     

CycloSal-2'-ara(ribo)-fluoro-2',3'-dideoxyadenosine monophosphates--an effort to solve the structure-activity relationship of 2'-fluoro-ddA.

Novel lipophilic cycloSal-triesters 3 and 4 from the ara- and ribo-configurated 2'-fluorinated ddAs 1 and 2, respectively, were prepared. The title compounds 3 and 4 delivered the corresponding monophosphates and thus, increasing the bioactivity or convert a formerly inactive compound into a RT inhibitor.     

Southern elephant seals: IgM concentration in milk of cows and serum of their pups

Serum and milk Immunoglobulin M (IgM) concentrations in 11 mother-pup pairs were measured in southern elephant seals (Mirounga leonina) throughout lactation during 2 breeding seasons at King George Island. Samples were obtained sequentially throughout the suckling period (approximately 23 days). The IgM concentration was measured by single radial immunodiffusion on agarose plates. Milk IgM concentrations showed significant differences throughout lactation, with the highest concentrations on the 1st day (x=989.7 mg/dL skimmed milk; SD=433.2) followed by a sharp fall during the next 3–6 days of the suckling period. The ratio of milk IgM/serum IgM concentrations from mothers ranged from 0.21 to 21.92, with highest values in the 1st day of lactation (x=8.25, SD=5.4) and a decrease in concentration as lactation progressed. This was due to the fact that, throughout lactation, milk IgM concentrations fell while serum IgM values showed an increasing trend. Pups showed the lowest serum IgM values in the 1st day of the suckling period (x=13.0 mg/dL, SD=4.3) with an increasing trend as lactation progressed. Despite the high IgM concentrations of milk at day 1 of lactation, by 1 week of age pups had serum IgM concentrations only slightly greater than at birth. This suggests that much of this Ig was newly formed and little or no milk IgM was absorbed during the 1st week. Possibly, the function of ingested IgM is to provide local immunity in the pup's gut, during the first few days of postnatal life. Accepted: 26 March 2000     

Lineage identification of Galápagos tortoises in captivity worldwide

Ex situ conservation strategies may be substantially informed by genetic data, and yet only recently have such approaches been used to facilitate captive population management of endangered species. The Galápagos tortoise Geochelone nigra is an endangered species that has benefited greatly from the application of molecular and population genetic data, but remains vulnerable throughout its range. The geographic and evolutionary origins of 98 tortoises in private collections and zoos on three continents were identified using mitochondrial DNA (mtDNA) control region sequences and multi-locus microsatellite genotype data relative to a large database of representative samplings from all extant populations, including historical population allele frequency data for the Geochelone nigra abingdoni taxon on Pinta by way of museum specimens. All but six individuals had mtDNA haplotypes previously sampled, with the novel haplotypes identified as most closely related to robust populations on the islands of Santa Cruz and Isabela. Multi-locus genotypic assignments corroborated the results obtained from the mtDNA analyses, with 83.7% of individuals consistently assigned to the same locality by both datasets. Overall, the majority of captive unknowns sampled were assigned to the La Caseta Geochelone nigra porteri population, with no fewer than six individuals of hybrid origin detected. Although a purported Pinta individual was revealed to be of Pinzón ancestry, the two females currently housed with Lonesome George exhibited haplotypic and genotypic signatures that indicate that they are among the most appropriate matches for captive breeding. More generally, molecular approaches continue to represent important tools for assessing conservation value, minimizing hybridization and guiding management programs for preserving the distinctiveness of G. nigra taxa in captivity.     

Molecular structures of two new anti-HIV nucleoside analogs: 9-(2,3-dideoxy-2-fluoro-beta-D-threo-pentofuranosyl)adenine and 9-(2,3-dideoxy-2-fluoro-beta-D-threo-pentofuranosyl)hypoxanthine.

The x-ray crystal structures of two new anti-HIV compounds, 9-(2,3-dideoxy-2-fluoro-beta-D-threo-pentofuranosyl)adenine (2'-F-dd-araA) and 9-(2,3-dideoxy-2-fluoro-beta-D-threo- pentofuranosyl)hypoxanthine (2'-F-dd-aral), have been determined at two temperatures. Both crystals are in the space group P2(1)2(1)2(1), and their structures were solved by direct methods. Least-squares refinement produced final R-factors of 0.027 for the 2'-F-dd-araA structure and of 0.044 for the 2'-F-dd-aral structure, respectively. The latter structure contains a two-fold disordered conformation of the sugar moiety. All three conformers (one for 2'-F-dd-araA and two for 2'-F-dd-aral) adopt an anti chi CN glycosyl torsion angle. The sugar in the 2'-F-dd-araA structure has a C2'-endo pucker conformation, whereas the sugar in the 2'-F-dd-aral structure has a mixture of C2'-endo and C3'-endo pucker conformations. When the sugar adopts the C2'-endo conformation, the torsion angle about the C4'-C5' bond is in a transgauche+ conformation. In contrast, when the sugar adopts the C3'-endo conformation, the torsion angle about the C4'-C5' bond is in a gauche(+)-gauche- conformation. The C2'-F bond distance is 1.406(3) A, similar to that found in other aliphatic C-F bonds. The results suggest that the 2'-fluoro-2',3'-dideoxyarabinosyl nucleosides do not have a strong preference for either C2'-endo or C3'-endo sugar pucker.     

Advertisement calls of the midwife toads Alytes (Amphibia, Anura, Discoglossidae) in continental Spain

The advertisement calls of six Spanish populations of midwife toads (genus Alytes) are described presenting characteristic audiospectrograms and oscillograms of the calls. Numerical data concerning the spectral and temporal features of the calls in relation to caller size (SVL) and temperature are also compared. The regression temperature vs. call duration groups the different populations according to the extant recognized phylogeny of the genus. Based on this character, the montane population of A. obstetricans from Penalara (Madrid) appears to be more closely related to the Pyrenean populations of A. o. almogavarü than to the more western populations of A. oboscai in Central Spain, which are closer geographically.     

Molecular analysis of barley mutants deficient in chloroplast glutamine synthetase

A barley leaf cDNA library has been screened with two oligonucleotide probes designed to hybridize to conserved sequences in glutamine synthetase (GS) genes from higher plants. Two GS cDNA clones were identified as hybridizing strongly to one or both probes. The larger clone (pcHvGS6) contained a 1.6 kb insert which was shown by primer extension analysis to be an almost full-length cDNA. Both clones were more closely related to cDNAs for the chloroplast form of GS (GS₂) from pea and Phaseolus vulgaris than to cDNAs for the cytosolic form (GS₁). A sequence identicalto an N-terminal sequence determined from a purified preparation of the mature GS₂ polypeptide (NH₂-XLGPETTGVIQRMQQ) was found in the pcHvGS6-encoded polypeptide at residues 46–61, indicating a pre-sequence of at least 45 amino acids. The pre-sequence has only limited sequence homology to the pre-sequences of pea and P. vulgaris GS₂ subunits, but is similarly rich in basic residues and possesses some of the structural features common to the targeting sequences of other chloroplast proteins. The molecular lesions responsible for the GS₂-deficient phenotypes of eight photorespiratory mutants of barley were investigated using a gene-specific probe from pcHvGS6 to assay for GS₂ mRNA, and an anti-GS antiserum to assay for GS₂ protein. Three classes of mutants were identified: class I, in which absence of cross-reacting material was correlated with low or undetectable levels of GS₂ mRNA; class II, which had normal or increased levels of GS₂ mRNA but very little GS₂ protein; and class III, which had significant amounts of GS₂ protein but little or no GS₂ activity.     

Phosphorylation of RhoB by CK1 impedes actin stress fiber organization and epidermal growth factor receptor stabilization

RhoB is a small GTPase implicated in cytoskeletal organization, EGF receptor trafficking and cell transformation. It is an immediate-early gene, regulated at many levels of its biosynthetic pathway. Herein we show that the serine/threonine protein kinase CK1 phosphorylates RhoB in vitro but not RhoA or RhoC. With the use of specific CK1 inhibitors, IC261 and D4476, we show that the kinase phosphorylates also RhoB in HeLa cells. Mass spectrometry analysis demonstrates that RhoB is monophosphorylated by CK1, in its C-terminal end, on serine 185. The substitution of Ser185 by Ala dramatically inhibited the phosphorylation of RhoB in cultured cells. Lastly we show that the inhibition of CK1 activates RhoB and promotes RhoB dependent actin fiber formation and EGF-R level. Our data provide the first demonstration of RhoB phosphorylation and indicate that this post-translational maturation would be a novel critical mechanism to control the RhoB functions.