Structural and evolutionary analysis of an orangutan foamy virus

The full-length proviral genome of a foamy virus infecting a Bornean orangutan was amplified, and its sequence was analyzed. Although the genome showed a clear resemblance to other published foamy virus genomes from apes and monkeys, phylogenetic analysis revealed that simian foamy virus SFVora was evolutionarily equidistant from foamy viruses from other hominoids and from those from Old World monkeys. This finding suggests an independent evolution within its host over a long period of time.     

Identification and characterization of NIF3L1 BP1, a novel cytoplasmic interaction partner of the NIF3L1 protein

The NIF3L1 protein is strongly conserved during evolution from bacteria to mammals and recently its function in neuronal differentiation has been demonstrated. In the present study we identified novel binding partners of human NIF3L1 by screening a HeLa cDNA-library using the yeast two-hybrid system. We could show that the NIF3L1 protein is interacting with itself and with the NIF3L1 binding protein 1 (NIF3L1 BP1), a novel protein of 23.67kDa bearing a putative leucine zipper domain. Furthermore, both interactions were confirmed using the mammalian two-hybrid system. Deletion analyses clearly demonstrated that a C-terminal region of 100 amino acids of the NIF3L1 BP1 is sufficient for the interaction with NIF3L1. The NIF3L1 BP1 is ubiquitously expressed and cotransfection experiments revealed that NIF3L1 and NIF3L1 BP1 interact in the cytoplasm of human LNCaP cells. This study provides novel insights into the cellular function of the NIF3L1 protein.     

Physical mapping of two Xp markers DXS16 and DXS143

Summary Lymphocyte karyotyping of an infant girl with the clinical features of microphthalmia, iridoschisis, goiter, hip joint dysplasia, labium synechia and craniotabes revealed an Xp deletion. The lymphocyte karyotypes of the parents were normal. Bromodeoxyuridine incorporation studies showed that, in 42 out of 43 metaphases, the deleted X chromosome was late replicating. In one metaphase, the normal X chromosome was observed to be allocyclic. Using DNA markers from the Xp22 region, the breakpoint was assigned distal to DXS16 (pXUT23) and proximal to DXS143 (dic56). Dosage intensity measurements confirmed that the STS gene and the DNA marker DXS31 were involved in the deleted area. Restriction fragment length polymorphism analysis revealed that the paternally derived X-chromosome was deleted.     

In vitro assembly of gap junctions.

Gap junction structures were assembled in vitro from octyl-beta-D-glucopyranoside-solubilized components of lens fiber cell membranes. Individual pore structures (connexons), short double-membrane structures, and other amorphous material were evident in the solubilized mixture. Following the removal of the detergent by dialysis, these connexons associated to form single- and double-layered, two-dimensional hexagonal arrays (unit cell size a = b = 8.5 nm). The formation of larger arrays was dependent on the lipid-to-protein ratio and the presence of Mg2+ ions. Crystallographic analysis of electron micrographs revealed that lens junctional connexons consisted of six subunits surrounding a stain-filled channel. Upon further detergent treatment, in vitro assembled gap junctions were insoluble and formed three-dimensional stacks while other components were solubilized. SDS-PAGE and mass data from scanning transmission electron microscopy strongly suggest that a 38-kDa polypeptide, which is a processed form of the lens specific gap junction protein MP70, is a major component of the arrays. The in vitro assembly of gap junctions opens new avenues for the structural analysis of gap junctions and for the study of the intermolecular interactions of connexons during junctional assembly.     

Performance of non-motile male gametes in the sea: analysis of paternity and fertilization success in a natural population of a red seaweed,Gracilaria gracilis

In haploid–diploid red seaweeds, the dispersal of male gametes is presumed limited due to their lack of flagella. It has been suggested that this group suffers from sperm limitation and, consequently, that fertilization is relatively inefficient. Fertilization in most floridean rhodophytes results in the formation a cystocarp, a swelling on the haploid female thallus housing the diploid zygote and its thousands of diploid daughter spores. To study the performance of non-motile male gametes in the sea, we evaluated both female and male fertilization success in a natural population of the red marine alga Gracilaria gracilis. Female fertilization success, estimated by cystocarp yield per unit female thallus, was evaluated with respect to the availability of male gametes. Male fertilization success, estimated by the individual contribution of different males to zygotes, was assessed by paternity analyses on 350 cystocarps produced in one reproductive season using two microsatellite loci. The results show that cystocarp yield is not sperm limited and that the large variation in male fertilization success cannot be solely explained by the distance travelled by the male gamete to find a mate. Taken together, the results suggest that, not only is fertilization efficient, but that male–male competition and/or female choice may play a role in shaping population mating patterns.     

Anisotropic propagation of Ca2+ waves in isolated cardiomyocytes.

Digital imaging microscopy of fluor-3 fluorescence was used to study the propagation of intracellular Ca2+ waves in isolated adult rat cardiomyocytes from 17 to 37 degrees C. Ca2+ waves spread in both transverse and longitudinal direction of a myocyte. Transverse propagation was pronounced in waves starting from a focus at the edge of a myocyte and in waves following an irregular, curved path (spiral waves). For the former type of waves, propagation velocities were determined. Both transverse and longitudinal wave components propagated at constant velocity ranging from 30 to 125 micron/s. Myocytes were anisotropic with respect to wave propagation: waves propagated faster in the longitudinal than in the transverse direction. The ratio between longitudinal and transverse velocity increased from 1.30 at 17 degrees C to 1.55 at 37 degrees C. Apparent activation energies for transverse and longitudinal wave propagation were estimated to be -20 kJ/mol, suggesting that these processes are limited by diffusion of Ca2+. Direction-dependent propagation velocities are interpreted to result from the highly ordered structure of the myocytes, especially from the anisotropic arrangement of diffusion obstacles such as myofilaments and mitochondria.     

Cutting edge: long-term B cell memory in humans after smallpox vaccination

Memory B cells are a central component of humoral immunity, and yet little is known about their longevity in humans. Immune memory after smallpox vaccination (DryVax) is a valuable benchmark for understanding the longevity of B cell memory in the absence of re-exposure to Ag. In this study, we demonstrate that smallpox vaccine-specific memory B cells last for >50 years in immunized individuals. Virus-specific memory B cells initially declined postimmunization, but then reached a plateau approximately 10-fold lower than peak and were stably maintained for >50 years after vaccination at a frequency of approximately 0.1% of total circulating IgG(+) B cells. These persisting memory B cells were functional and able to mount a robust anamnestic Ab response upon revaccination. Additionally, virus-specific CD4(+) T cells were detected decades after vaccination. These data show that immunological memory to DryVax vaccine is long-lived and may contribute to protection against smallpox.     

Binding and calcium-induced aggregation of laminin onto lipid bilayers

Direct binding of laminin in the form of its complex with nidogen to planar lipid bilayers was demonstrated with total internal reflection fluorescence microscopy. Binding occurred equally well to zwitterionic (phosphatidylcholine) and negatively charged (phosphatidylglycerol) lipids and was enhanced by sulfatides but only at nonphysiological molar ratios higher than 30 mol %. Strong interactions with lipid bilayers were also observed for bovine serum albumin. This explains a strong inhibition of laminin binding by this protein. However, binding of laminin to sulfatide-rich bilayers was not completely inhibited. Observable by the microscopic technique was the formation of laminin clusters on the surface of the bilayer which occurred concomitantly with binding. Both processes were strongly enhanced by the presence of calcium. These results show that calcium-induced laminin self-assembly is enhanced at lipid surfaces.     

Pressure-diameter relationships of the upper airway in awake supine subjects

In awake supine normal subjects, dimensional changes of the oropharyngeal airway were measured during exposure to negative intraluminal pressures. The pressure was generated 1) "actively" by subjects inspiring against an externally occluded airway or 2) "passively" by external suction at the mouth during voluntary glottic closure with no inspiratory effort. Airway dimensions were imaged with X-ray fluoroscopy and anteroposterior diameters measured at levels corresponding to cervical vertebra 3 and 4 (C3 and C4). Cephalad axial displacement of the hyoid bone (CDHY) was also measured. During the "active" maneuver, airway diameters and position were maintained at resting levels despite airway pressure up to -15 cmH2O. In contrast, during the passive maneuver at -15 cmH2O, C3 was only 15 +/- 9% and C4 only 47 +/- 8% of control; CDHY was 5.6 +/- 1.8 mm. In three subjects airway wall apposition occurred and persisted until an active inspiratory effort. We conclude that, in the absence of inspiratory effort, negative oropharyngeal airway pressures result in marked narrowing and cephalad displacement of the upper airway, even during wakefulness. Therefore, our data suggest that the complex interaction of upper airway and thoracic muscle activity is critical in determining the effective compliance and patency of the upper airway, which is readily collapsible even in normal subjects.     

Radiological evaluation of bone status in the jaw and in the vertebral column in a group of women

Vertebral bone mineral content was determined in a group of 56 women, ages 30–62. These measurements were compared with the status of supporting bone in the jaws (alveolar, molar and bicuspid) and with gingival health. There was a significant decline in vertebral bone mineral content from the pre- to post-menopausal group. Molar and bicuspid measurements were highly correlated. There was some association between lumbar bone mineral content and molar bone status for postmenopausal women. For postmenopausal women, the cases of greatest percent bone loss in alveolar crest were associated with lower lumbar bone mineral content. Gingival health did not confound the bone status measurements. The 56 subjects did not exhibit the degree of reduction in bone density that is observed in the general population. Further investigation using these radiographic techniques may reveal a link between substantial bone loss in the jaw and moderate to severe bone loss in the lumbar vertebrae.