Synthesis of oligonucleotides containing the oxidatively modified kappaB site: unique binding affinity to the NFkappaB p50 homodimer.

Synthesis of oligonucleotide 26-mers including single 5-formyl-2'-deoxyuridine (1) or 5-formyl-2'-O-methyluridine (2) in place of thymidine at the kappaB site has been accomplished. One of the 26-mers with 1 was critically discriminated by the NFkappaB p50 homodimer in binding.     

Production of interferon by an SV40-transformed macrophage line, BB-W-531-2

A simian virus 40-transformed mouse macrophage line, BB-W-531-2, was examined for its ability to produce interferon. BB-W-531-2 cells showed a phenotypic change between the macrophage and the nonmacrophage states. A viral inhibitor (interferon) was produced by the cells during the phenotypic change from the nonmacrophage to the macrophage state. Cells having macrophage properties were well capable of producing interferon when they were stimulated with ultraviolet-inactivated vaccinia virus, lipopolysaccharide, a streptococcal preparation (OK-432) or polyinosinate . polycytidylate. In contrast, cells that had lost their macrophage properties did not produce interferon even when they were given the same treatments as the cells having macrophage properties. The results suggest that the ability of BB-W-531-2 cells to produce interferon is associated with the expression of several macrophage properties.     

Inactivation of the Heme Degrading Enzyme IsdI by an Active Site Substitution That Diminishes Heme Ruffling

IsdG and IsdI are paralogous heme degrading enzymes from the bacterium Staphylococcus aureus. Heme bound by these enzymes is extensively ruffled such that the meso-carbons at the sites of oxidation are distorted toward bound oxygen. In contrast, the canonical heme oxygenase family degrades heme that is bound with minimal distortion. Trp-66 is a conserved heme pocket residue in IsdI implicated in heme ruffling. IsdI variants with Trp-66 replaced with residues having less bulky aromatic and alkyl side chains were characterized with respect to catalytic activity, heme ruffling, and electrochemical properties. The heme degradation activity of the W66Y and W66F variants was approximately half that of the wild-type enzyme, whereas the W66L and W66A variants were inactive. A crystal structure and NMR spectroscopic analysis of the W66Y variant reveals that heme binds to this enzyme with less heme ruffling than observed for wild-type IsdI. The reduction potential of this variant (−96 ± 7 mV versus standard hydrogen electrode) is similar to that of wild-type IsdI (−89 ± 7 mV), so we attribute the diminished activity of this variant to the diminished heme ruffling observed for heme bound to this enzyme and conclude that Trp-66 is required for optimal catalytic activity.     

Can Acupuncture Treatment Be Double-Blinded? An Evaluation of Double-Blind Acupuncture Treatment of Postoperative Pain

Blinding protects against bias but the success of blinding is seldom assessed and reported in clinical trials including studies of acupuncture where blinding represents a major challenge. Recently, needles with the potential for double-blinding were developed, so we tested if acupuncture can be double-blinded in a randomized study of sixty-seven patients with acute pain ≥ 3 (0-10 scale following third molar removal) who received active acupuncture with a penetrating needle or placebo acupuncture with a non-penetrating needle. To test if acupuncture was administered double-blind, patients and acupuncturists were asked about perceived treatment allocation at the end of the study. To test if there were clues which led to identification of the treatment, deep dull pain associated with needle application and rotation (termed “de qi” in East Asian medicine), and patients’ pain levels were assessed. Perceived treatment allocation depended on actual group allocation (p < 0.015) for both patients and acupuncturists, indicating that the needles were not successful in double-blinding. Up to 68% of patients and 83% of acupuncturists correctly identified the treatment, but for patients the distribution was not far from 50/50. Also, there was a significant interaction between actual or perceived treatment and the experience of de qi (p = 0.027), suggesting that the experience of de qi and possible non-verbal clues contributed to correct identification of the treatment. Yet, of the patients who perceived the treatment as active or placebo, 50% and 23%, respectively, reported de qi. Patients’ acute pain levels did not influence the perceived treatment. In conclusion, acupuncture treatment was not fully double-blinded which is similar to observations in pharmacological studies. Still, the non-penetrating needle is the only needle that allows some degree of practitioner blinding. The study raises questions about alternatives to double-blind randomized clinical trials in the assessment of acupuncture treatment.     

RT1.P, rat class Ib genes related to mouse TL: evidence that CD1 molecules but not authentic TL antigens are expressed by rat thymus

 CD1 and TL were once thought to be genetic homologues because of their thymus-specific expression. We investigated their equivalents in the rat to clarify whether their structure and pattern of expression are conserved in rodents. Two rat class Ib genes, containing 3′ sequences very similar to mouse TL, were identified and designated RT1.P. Neither of them, however, can encode ordinary class I molecules due to the accumulation of harmful mutations in the 5′ regions that are unique to RT1.P, while the 3′TL-like regions still retain protein-coding capacity. Comparison of the structural organization of three types of TL family genes, which include mouse T3/T18-encoding TL antigens, mouse T1/T16, and rat RT1.P1/P2 pseudogenes, revealed the presence of a clear demarcation between the type-specific and TL-specific sequences at intron 3. This finding suggests that recombination plays an important role in creating the TL family genes in rodents. Characteristic features of TL, such as a low level of polymorphism and linkage to the major histocompatibility complex, were also observed in the rat. On the other hand, rat CD1 molecules were expressed at a high level on the surface of thymocytes. Absence of authentic TL antigens and thymic expression of CD1d molecules in the rat suggest the plasticity and conservation of class Ib genes in rodent evolution. Functions of TL may be substituted with CD1 or other class Ib molecules expressed by rat thymus. Received: 16 December 1996 / Revised: 11 March 1997     

Development of Artificial-Sebum-Containing Leeming and Notman Agar Medium to Enhance the Growth of Malassezia

Mycopathologia - Modified Leeming and Notman agar medium (mLNA) has been widely utilized to grow lipophilic fungi belonging to the genus Malassezia. We developed a new artificial-sebum-containing...     

A method for incorporating labeled lacithin into serum low density lipoproteins in vitro.

A sonicated dispersion of [14C]lecithin was incubated with high density lipoproteins (HDL) coupled to Sepharose. After washing the gels thoroughly with a buffer, the gels were incubated with low density lipoproteins (LDL); [14C]lecithin was transferred from the sonicated dispersion via HDL-Sepharose to the LDL. The LDL fraction thus prepared showed no contamination with lecithin dispersion or HDL. The lecithin:cholesterol acyltransferase (LCAT) reaction could be completely inhibited during preparation, and the net recovery of radioactivity in LDL was 16% of that of the original lecithin dispersion. The [14C]lecithin in the washed HDL-Sepharose was shown to be a substrate of the LCAT reaction in vitro.     

Preparation and characterization of biologically active 6'-O-(6-aminocaproyl)-4'-O-monophosphoryl lipid A and its conjugated derivative.

N-tert-butyloxycarbonyl (t-Boc) protected 6-aminocaproic (Cap) anhydride was reacted with unprotected hexaacyl-4'-O-monophosphoryl lipid A (MLA) obtained from the lipopolysaccharide of Escherichia coli J5 to yield t-Boc-Cap-MLA. After a column purification step, the t-Boc group was removed by incubating the sample at low temperature in the presence of acid to yield Cap-MLA. This product was analyzed by californium plasma desorption mass spectrometry (PDMS). Purified t-Boc-Cap-MLA was further fractionated by reverse-phase high-performance liquid chromatography as its methyl ester and characterized by laser desorption mass spectrometry, PDMS, and proton nuclear magnetic resonance spectroscopy. These analyses revealed that the Cap group was selectively introduced into the 6'-position of MLA. To demonstrate that Cap-MLA can be conjugated to other compounds, it was reacted with biotin-Cap N-hydroxysuccinimide ester to yield biotin-(Cap)2-MLA. Analysis of this product by PDMS confirmed its expected molecular weight of 2171 and showed the presence of fragments containing the biotin and Cap groups. Monoclonal antibodies and streptavidin were used to show the presence of both lipid A and biotin in this conjugated product. These two novel lipid A derivatives were then tested for their bioactivities. Although both Cap-MLA and biotin-(Cap)2-MLA showed mitogenic activity using murine splenocytes, they were about 4-8 times less active than MLA at 20 micrograms/mL or less and only one-half as active at 100 micrograms/mL.(ABSTRACT TRUNCATED AT 250 WORDS)