Cytokine regulation of adult human osteoblast-like cell prostaglandin biosynthesis

Prostaglandin (PG) biosynthesis by cytokine stimulated normal adult human osteoblast-like (hOB) cells was evaluated by thin layer chromatography, high performance liquid chromatography, and specific immunoassays. PGE₂ was the predominant PG formed under all incubation conditions tested. Control samples produced measurable amounts of PGE₂, and the measured level of this metabolite increased by 22-fold (from 7 to 152 ng/ml) following a 20 h treatment with the combination of TGFβ and tumor necrosis factor-α(TNF). The production of 6-keto-PGF_1α (the stable metabolite of prostacyclin) and of PGF_2α were each increased by about five-fold (from about 0.5 to 2.5 ng/ml) in samples treated with the cytokines. Thus, TGFβ and TNF exerted a regulation of hOB cell PG biosynthesis that was principally directed towards an increased PGE₂ biosynthesis, with lesser effects on the production of other PG metabolites. COX-2 mRNA levels were increased within 2 h of cytokine stimulation, reached a maximum at 6–12 h, and levels had appreciably diminished by 24 h after treatment. Both TGFβ and TNF could independently increase COX-2 mRNA levels and PG biosynthesis. However, the increased production of PGE₂ resulting from TNF stimulation was blocked by the addition of an interleukin-1β (IL-1β) neutralizing antibody, suggesting that TNF regulation of hOB cell PG synthesis was secondary to its capacity to increase hOB cell IL-1β production. TGFβ regulation of PG production was not affected by the addition of the neutralizing antibody. These studies support the proposition that PGs can be important autocrine/paracrine mediators of bone biology, whose production by hOB cells is responsively regulated by osteotropic cytokines. J. Cell. Biochem. 64:618–631. © 1997 Wiley-Liss, Inc.     

1,25-Dihydroxyvitamin D3 or dexamethasone modulate arachidonic acid uptake and distribution into glycerophospholipids by normal adult human osteoblast-like cells

The effects of treatment with the osteotropic steroids 1,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃), 17β-estradiol, or dexamethasone on [1-¹⁴C]arachidonic acid (AA) uptake and distribution into glycerophospholipid classes by normal adult human osteoblast-like (hOB) cells were investigated. Total uptake of [1-¹⁴C]AA was decreased in cells treated with dexamethasone when assayed after a 24-, 48-, or 96-h exposure to the hormone. Specific radiolabel incorporation into phosphatidylcholine was reduced by a 48-h treatment with dexamethasone with a concurrent increase in the radiolabeling of phosphatidylethanolamine. However, these changes were transient, and by 96 h of dexamethasone treatment the distribution of the radiolabeled fatty acid had reequilibrated to resemble the pattern found for vehicle treated samples. Total uptake of [1-¹⁴C]AA was diminished by 96-h treatment with 1,25(OH)₂D₃ (79 ± 3% of control, P < 0.01); at that time point, a significant decrease in the proportional radiolabeling of the phosphatidylinositol pool was identified (92 ± 2% of control, P < 0.05). The 1,25(OH)₂D₃-dependent decrease in total uptake and in phosphatidylinositol incorporation of [1-¹⁴C]AA were found to be hormone dose dependent. Treatment with 24,25(OH)₂D₃ was without effect on either total [1-¹⁴C]AA uptake or the specific [1-¹⁴C]AA radiolabeling of the phosphatidylinositol pool. 1,25(OH)₂D₃ treatment decreased hOB cell uptake of [1-¹⁴C]oleic acid and decreased its proportional incorporation into the phosphatidylinositol pool. Gas chromatographic analyses revealed no 1,25(OH)₂D₃-dependent effects on total phosphatidylinositol lipid mass or on the mole percent of arachidonic acid within the phosphatidylinositol pool, leaving the mechanism of the effects of the secosteroid on hOB cell AA metabolism unexplained. 17β-Estradiol had no effects on the parameters of AA metabolism measured. As a consequence of their modulation of arachidonic acid uptake and its distribution into hOB cellular phospholipids, steroids might alter the biological effects of other hormones whose actions include the stimulated production of bioactive AA metabolites, such as prostaglandins or the various lipoxygenase products.     

Insights into base selectivity from the 1.8 Å resolution structure of an RB69 DNA polymerase ternary complex

Bacteriophage RB69 DNA polymerase (RB69 pol) has served as a model for investigating how B family polymerases achieve a high level of fidelity during DNA replication. We report here the structure of an RB69 pol ternary complex at 1.8 ? resolution, extending the resolution from our previously reported structure at 2.6 ? [Franklin, M. C., et al. (2001) Cell 105, 657-667]. In the structure presented here, a network of five highly ordered, buried water molecules can be seen to interact with the N3 and O2 atoms in the minor groove of the DNA duplex. This structure reveals how the formation of the closed ternary complex eliminates two ordered water molecules, which are responsible for a kink in helix P in the apo structure. In addition, three pairs of polar-nonpolar interactions have been observed between (i) the Cα hydrogen of G568 and the N3 atom of the dG templating base, (ii) the O5' and C5 atoms of the incoming dCTP, and (iii) the OH group of S565 and the aromatic face of the dG templating base. These interactions are optimized in the dehydrated environment that envelops Watson-Crick nascent base pairs and serve to enhance base selectivity in wild-type RB69 pol.     

Limb venous distension evokes sympathetic activation via stimulation of the limb afferents in humans

We have recently shown that a saline infusion in the veins of an arterially occluded human forearm evokes a systemic response with increases in muscle sympathetic nerve activity (MSNA) and blood pressure. In this report, we examined whether this response was a reflex that was due to venous distension. Blood pressure (Finometer), heart rate, and MSNA (microneurography) were assessed in 14 young healthy subjects. In the saline trial (n = 14), 5% forearm volume normal saline was infused in an arterially occluded arm. To block afferents in the limb, 90 mg of lidocaine were added to the same volume of saline in six subjects during a separate visit. To examine whether interstitial perfusion of normal saline alone induced the responses, the same volume of albumin solution (5% concentration) was infused in 11 subjects in separate studies. Lidocaine abolished the MSNA and blood pressure responses seen with saline infusion. Moreover, compared with the saline infusion, an albumin infusion induced a larger (MSNA: Δ14.3 ± 2.7 vs. Δ8.5 ± 1.3 bursts/min, P < 0.01) and more sustained MSNA and blood pressure responses. These data suggest that venous distension activates afferent nerves and evokes a powerful systemic sympathoexcitatory reflex. We posit that the venous distension plays an important role in evoking the autonomic adjustments seen with postural stress in human subjects.     

Cortical bone viscoelasticity and fixation strength of press-fit femoral stems: finite element model

Many cementless implant designs rely upon a diaphyseal press-fit in conjunction with a porous coated implant surface to achieve primary or short term fixation, thereby constraining interface micromotion to such a level that bone ingrowth and consequent secondary or long-term fixation, i.e., osseointegration, can occur. Bone viscoelasticity, however, has been found to affect stem primary stability by reducing push-out load. In this investigation, an axisymmetric finite element model of a cylindrical stem and diaphyseal cortical bone section was created in order to parametrically evaluate the effect of bone viscoelasticity on stem push-out while controlling coefficient of friction (mu = 0.15, 0.40, and 1.00) and stem-bone diametral interference (delta = 0.01, 0.05, 0.10, and 0.50 mm). Based on results from a previous study, it was hypothesized that stem-bone interference (i.e., press-fit) would elicit a bone viscoelastic response which would reduce the initial fixation of the stem as measured by push-out load. Results indicate that for all examined combinations of mu and delta, bone viscoelastic behavior reduced the push-out load by a range of 2.6-82.6% due to stress relaxation of the bone. It was found that the push-out load increased with mu for each value of delta, but minimal increases in the push-out load (2.9-4.9%) were observed as delta was increased beyond 0.10 mm. Within the range of variables reported for this study, it was concluded that bone viscoelastic behavior, namely stress relaxation, has an asymptotic affect on stem contact pressure, which reduces stem push-out load. It was also found that higher levels of coefficient of friction are beneficial to primary fixation, and that an interference "threshold" exists beyond which no additional gains in push-out load are achieved.     

Cortical bone viscoelasticity and fixation strength of press-fit femoral stems: an in-vitro model

Cementless total hip femoral components rely on press-fit for initial stability and bone healing and remodeling for secondary fixation. However, the determinants of satisfactory press-fit are not well understood. In previous studies, human cortical bone loaded circumferentially to simulate press-fit exhibited viscoelastic, or time dependent, behavior. The effect of bone viscoelastic behavior on the initial stability of press-fit stems is not known. Therefore, in the current study, push-out loads of cylindrical stems press-fit into reamed cadaver diaphyseal femoral specimens were measured immediately after assembly and 24 h with stem-bone diametral interference and stem surface treatment as independent variables. It was hypothesized that stem-bone interference would result in a viscoelastic response of bone that would decrease push-out load thereby impairing initial press-fit stability. Results showed that push-out load significantly decreased over a 24 h period due to bone viscoelasticity. It was also found that high and low push-out loads occurred at relatively small amounts of stem-bone interference, but a relationship between stem-bone interference and push-out load could not be determined due to variability among specimens. On the basis of this model, it was concluded that press-fit fixation can occur at relatively low levels of diametral interference and that stem-bone interference elicits viscoelastic response that reduces stem stability over time. From a clinical perspective, these results suggest that there could be large variations in initial press-fit fixation among patients.     

Characterization of link protein(s) from human intervertebral-disc tissues.

Proteoglycan aggregates (A1) were prepared from the anulus fibrosus, nucleus pulposus and cartilage-endplate tissues of postnatal (0-6-month-old)-and young-adult (20-30-year-old)-human intervertebral discs. The A1 fractions from young-adult disc contained a greater proportion of non-aggregating proteoglycans than did postnatal tissues. After dissociative CsCl-density-gradient fractionation of the A1, more than 90% of the uronic acid was found in the postnatal A1D1, whereas only 60-80% of the hexuronate was present in the A1D1 isolated from young-adult disc tissues. These results indicated that more lower-buoyant-density proteoglycans occur in the young-adult disc. Link-protein-rich fractions (A1D3) were subjected to SDS/polyacrylamide-gel electrophoresis and immunolocation analyses using monoclonal antibodies specific for epitopes on link protein or proteoglycan. Under non-reducing conditions, the major link protein present in postnatal disc tissues was link protein 1. By contrast, all three link proteins (1, 2 and 3) were detected in young-adult tissues, with the smaller link protein 3 predominating. Analyses of the A1D3 fractions under reducing conditions also indicated the presence of link-protein-degradation peptides (Mr approx. 26,000) from young-adult disc tissues, but not from postnatal tissues. Sequential Sepharose CL-6B and Sephacryl S-300 chromatography in 4 M-guanidinium chloride was employed to separate the link proteins of the A1D3 fraction from protein-rich proteoglycan. Immunolocation analyses indicated that postnatal samples contained no detectable contaminating proteoglycan fragments. However, young-adult link-protein preparations could not be separated from hyaluronic acid-binding region and other proteoglycan fragments by means of these chromatographic procedures. The studies indicate that, compared with hyaline articular cartilage, degraded link protein and proteoglycan accumulate at an early age in young-adult disc tissues. These partially degraded proteoglycan aggregate components may significantly alter the biomechanical properties of disc tissues.     

Molecular population genetics of the alcohol dehydrogenase locus in the Hawaiian drosophilid D. mimica

Sequence variation among 10 alleles of the alcohol dehydrogenase (Adh) gene of the Hawaiian drosophilid D. mimica was analyzed with reference to the evolutionary history of the Hawaiian subgroup as well as to levels and patterns of polymorphism of the Adh gene in continental drosophilid species. The Adh gene of D. mimica is less polymorphic than that of other drosophilid species, and no replacement substitutions were found. Statistical analyses of the Adh alleles suggested the action of balancing selection and revealed significant linkage disequilibrium among three of the variable sites. The effective population size was estimated to be only slightly smaller than that of continental species and, surprisingly, on the same order of magnitude as the actual size.      

Studies on the role of the S4 substrate binding site of HIV proteinases

Kinetic analysis of the hydrolysis of the peptide H-Val-Ser-Gln-Asn-Tyr*Pro-Ile-Val-Gln-NH2 and its analogs obtained by varying the length and introducing substitutions at the P4 site was carried out with both HIV-1 and HIV-2 proteinases. Deletion of the terminal Val and Gln had only moderate effect on the substrate hydrolysis, while the deletion of the P4. Ser as well as P'3 Val greatly reduced the substrate hydrolysis. This is predicted to be due to the loss of interactions between main chains of the enzyme and the substrate. Substitution of the P4 Ser by amino acids having high frequency of occurrence in beta turns resulted in good substrates, while large amino acids were unfavorable in this position. The two proteinases acted similarly, except for substrates having Thr, Val and Leu substitutions, which were better accommodated in the HIV-2 substrate binding pocket.