An immunocytochemical method for studying the kinetics of osteoclast nuclei on intact mouse parietal bone

Summary An immunocytochemical method using an antibody against 5-bromo-2-deoxyuridine has been applied to the study of the kinetics of osteoclast nuclei on intact mouse parietal bones. Osteoclasts containing tartrate-resistant acid phosphatase show nuclei that are positive for the thymidine analogue within 24 hours of injection into four-day old mice. Labelled osteoclast nuclei decline in number with a half-life of 1.3 days, compatible with a random mechanism of cell death rather than a fixed lifespan. This is shorter than has previously been reported and the possible reasons for this are suggested. The main advantages compared with autoradiography are the shortened processing time and the large number of osteoclasts that can be examined per parietal bone.     

High level of surface CD4 prevents stable human immunodeficiency virus infection of T-cell transfectants.

CD4 is the principal receptor for the human immunodeficiency virus (HIV). We have isolated and studied CD4-expressing tumor cell clones made by expressing CD4 in the T-cell tumor line HSB. Two clones, one designated HSBCD4, a clone expressing low levels of CD4, and the other, HSB10xCD4, a high-expresser CD4+ clone, were studied for their ability to bind and replicate HIV. In contrast to many other CD4+ cells that down-modulate CD4 following HIV infection, the HSB10xCD4 clones continued to express high levels of surface CD4 following infection with HIV. Unlike infection of HSBCD4 or many other human CD4+ cells, HIV infection of HSB10xCD4 clone was short lived: p24 antigen, provirus, or coculturable virus was present for less than 14 days following infection with several strains of HIV-1 or with HIV-2. When infection was initiated by transfection of proviral DNA, high and low CD4 expressers initially produced p24 antigen at approximately the same level. However, high CD4 expressers produced coculturable virus only during the first few days following transfection, whereas low CD4 expressers transfected with HIV continued to produce virus beyond 6 weeks. Monoclonal antibody-mediated down-modulation of CD4 surface expression on HSB10xCD4 clones permitted these formerly HIV-resistant cells to become persistently infected with HIV. Thus, high concentrations of CD4 on the surface of an HIV-infected cell prevent persistent HIV infection of CD4+ cells.     

Structural investigation of helices II, III, and IV of B. megaterium 5S ribosomal RNA by molecular dynamics calculations.

The structures of the helices II-III region and the helix IV region of B. megaterium 5S rRNA have been examined by means of energy minimization and molecular dynamics calculations. Calculated distances between neighboring hydrogen-bonded imino protons in helices II, III, and IV were between 3.5 and 4.5 A. The overall axis for the helices II-III region is warped rather than straight. Formation of additional Watson-Crick base pairs in loop B and loop C was not evident from the atomic positions calculated by molecular dynamics. Bases in loop C are well stacked, showing no significant change during dynamics. Bulge migration in helix III does not seem to be possible; the helices II-III region prefers one conformation. Helix II is more stable than helix III. Five base pairs in helix IV were sufficiently stable to establish that helix IV is terminated by a hairpin loop of three nucleotides. U87 protrudes from loop D. Structures of the helices II-III segment and the helix IV segment of B. megaterium 5S rRNA obtained by molecular dynamics were generally consistent with the solution structure inferred from high-field proton nmr spectroscopy.     

A hybrid protein of urokinase growth-factor domain and plasminogen-activator inhibitor type 2 inhibits urokinase activity and binds to the urokinase receptor.

The binding of urokinase-type plasminogen activator (uPA) to its specific cell-surface receptor (uPAR) localises the proteolytic cascade initiated by uPA to the pericellular environment. Inhibition of uPA activity or prevention of uPA binding to uPAR might have a beneficial effect on disease states wherein this activity is deregulated, e.g. cancer and some inflammatory diseases. To this end, a bifunctional hybrid molecule consisting of the uPAR-binding growth-factor domain of uPA (amino acids 1-47; GFuPA) at the N-terminus of plasminogen-activator inhibitor type 2 (PAI-2) was produced in Saccharomyces cerevisiae. The purified protein inhibited uPA with kinetics similar to placental or recombinant PAI-2 and was also found to bind to U937 cells and to FL amnion cells. GFuPA-PAI-2 competed with uPA, the N-terminal fragment of uPA and a proteolytic fragment of uPA (amino acids 4-43) in cell binding experiments, indicating that the molecule bound to the cells via uPAR. Hence, both the uPA-inhibitory and uPAR-binding domains of the hybrid molecule were functional, demonstrating the feasibility of the novel concept of introducing an unrelated, functional domain onto a member of the serine-protease-inhibitor superfamily.     

Reconstitution of interactions between the Src tyrosine kinases and Ras GTPase-activating protein using a baculovirus expression system.

Ras GTPase-activating protein (GAP) has been implicated in mitogenic signal transduction downstream of oncogenic and receptor tyrosine kinases. Previous studies have suggested that GAP is phosphorylated by oncogenic viral Src (v-Src) and that GAP is associated with a complex containing normal cellular Src (c-Src) in vertebrate fibroblasts. To investigate molecular interactions between the Src kinases and GAP, we developed an in vitro system for reconstituting Src-GAP complexes. For this purpose, we constructed recombinant baculovirus vectors that direct expression of Rous sarcoma virus v-Src, chicken c-Src, and bovine GAP in infected Sf9 insect cells. In vitro reconstitution experiments using baculovirus-expressed proteins demonstrate that both v-Src and c-Src associate in complexes with GAP. In addition, in vitro and in vivo phosphorylation analyses indicate that GAP serves as a substrate for both the v-Src and c-Src tyrosine kinases. To determine which structural features of GAP are involved in interactions with the Src kinases, we constructed recombinant baculoviruses that encode deletion mutants of bovine GAP. Deletion of the GAP amino-terminal portion containing Src homology 2 regions, which are highly conserved structural motifs postulated to mediate interactions among proteins, diminishes GAP phosphorylation and association with Src. This reconstitution system should facilitate further studies of molecular interactions between the Src kinases and GAP.     

Influx of extracellular calcium is required for the membrane translocation of 5-lipoxygenase and leukotriene synthesis.

Our studies assessed the effects of increases in intracellular calcium concentrations [( Ca2+]i) on leukotriene synthesis and membrane translocation of 5-lipoxygenase (5LO). The calcium ionophore ionomycin and the tumor promoter thapsigargin stimulated leukotriene production and translocation of 5-lipoxygenase to the membrane. Both agents elicited prolonged rises in [Ca2+]i. Leukotriene C4 production associated with [Ca2+]i in cells stimulated with various concentrations of ionomycin and thapsigargin suggests that a threshold [Ca2+]i level of approximately 300-400 nM is required. In the absence of extracellular Ca2+, both the ionomycin- and thapsigargin-induced rises in [Ca2+]i were transient, indicating that the prolonged [Ca2+]i elevation is due to an influx of extracellular Ca2+. Addition of EGTA to the external medium before, or at different times during, the treatment with ionomycin or thapsigargin instantaneously inhibited 5LO translocation and leukotriene synthesis, indicating that Ca2+ influx plays an essential role in 5LO membrane translocation and leukotriene synthesis. No leukotriene production was detected when cells were stimulated by a physiological stimulus of leukotriene D4. The addition of 100 nM leukotriene D4 triggered peak rises in [Ca2+]i that were comparable to those achieved by the ionomycin and thapsigargin. However, the leukotriene D4 induced rise was transient and rapidly declined to a lower but still elevated steady-state level, which was attributed to Ca2+ influx. Stimulation with 100 nM leukotriene D4 for 15 s increased the cellular levels of 1,4,5-inositol triphosphate (IP3), 1,3,4-IP3, and 1,3,4,5-inositol tetraphosphate (IP4).(ABSTRACT TRUNCATED AT 250 WORDS)     

Fluid production by in vitro lungs from fetal guinea pigs

Lungs from fetal guinea pigs (54-67 days of gestation) were supported in vitro, and lung liquid secretion rates were measured by a dye-dilution technique. The average secretion rate in the first hour was 2.14 +/- 0.08 (SE) mL x kg-1 body weight.h-1 (0.21 +/- 0.01 mL/h) (n = 450); this was comparable to intact preparations. In an independent study of 30 lungs, secretion continued unchanged for 3 h, with no significant change in fluid composition. Between 54 days and term, production appeared to fall in terms of millilitres per kilogram per hour. The following agents were placed in the supporting saline during the middle hour of incubation. (i) Sodium iodoacetate: at 10(-4) M this produced a fall in secretion (fall, succeeding hours; 55.4 +/- 23.0 and 64.9 +/- 17.5%; n = 6); at 10(-3) M it stopped secretion (fall, succeeding hours; 87.2 +/- 10.3 and 100%, n = 6). (ii) Ouabain: at 10(-5) M there was no change in production (n = 6); at 10(-4) M, four preparations were unaffected, two reduced production. (iii) Epinephrine (10(-7) M) produced a significant fall in production in all cases (n = 6); in four preparations secretion reduced (average fall, 64.4 +/- 10.8%); in two preparations there was reabsorption (average rate, -1.03 mL.kg-1.h-1). This extends the effect of epinephrine to the guinea pig, and suggests that the in vitro preparation is a useful model for studies of the fetal lung.     

INFLORESCENCE DEVELOPMENT IN TWO ANNUAL TEOSINTES: ZEA MAYS SUBSP. MEXICANA AND Z. MAYS SUBSP. PARVIGLUMIS

The ontogeny of tassels and ears in two annual Mexican teosintes, Zea mays subsp. mexicana and Z. mays subsp. parviglumis, was examined using scanning electron microscopy and light microscopy. Ear development in these annual teosintes follows a pattern previously described as leading to the bisexual mixed inflorescence in Z. diploperennis. Common bud primordia are initiated in the axils of distichously arranged bracts along the ear axis. These common primordia bifurcate to form paired sessile and pedicellate spikelet primordia. Development of pedicellate spikelets is arrested leaving the sessile spikelets, along with the adjoining rachis segment, to form solitary grains enclosed within cupulate fruitcases. Development of the central tassel spike is similar to that previously described in the Z. diploperennis tassel, except that the first formed axillary bud primordia form precocious tassel branches. The origin of these tassel branches suggests a possible mechanism for the transition from a distichous spike, characteristic of teosinte, to a polystichous spike, typical of maize.     

Genes Responsible for Size Reduction of Marine Vibrios during Starvation Are Located on the Chromosome

In a survey of 21 marine Vibrio spp., all responded to nutrient deprivation by undergoing a reduction in size (dwarfing). However, only 43% of these strains possessed one or more plasmids, suggesting that the genes responsible for dwarfing were located on the chromosome rather than on the plasmids. This conclusion was confirmed by the observation that fragmentation and subsequent size reduction occurred in three strains from which the plasmids had been removed by curing. The cured strains lost certain characteristics, such as resistance to some heavy metals and antibiotics, that were restored when the plasmids were reintroduced by either transformation or electroporation.     

The Sabinas syndrome

We have defined a new autosomal recessive disorder in patients stemming from a small community in northern Mexico. Diagnosable at birth, its major symptoms include brittle hair, mental retardation, and nail dysplasia. Structural hair abnormalities are seen by both light and electron microscopy. Hair cystine content is reduced while the copper/zinc ratio in hair is increased.