中国蚋科新纪录,5

作者在整理采自云南和广东的蚋科标本中,发现蚋属3个国內新纪录。 1. 节蚋 Simulium nodosum Puri, 1933 模式产地为印度库尔格附近的考维里河。在云南省保山(道街,1983. V.17,属于峡谷地带,海拔600米)采到12♀♀,6♀♀,68蛹,95幼虫;昌宁(湾甸,1983. V. 20,峡谷地带,海拔700—800米),35♀♀,4♀♀,59蛹,87幼虫。为一常见种,雌成虫喜刺叮人血。幼虫与蛹孳生于小河中的树枝、庄稼     

5乙,5异戊巴比妥酸对胆碱脱氢酶的抑制

5乙,5异戊巴比妥酸(Amytal)对鼠肝线粒体胆碱脱氢酶的抑制为竞争性,K_i为0.80×10~(-3)M。当胆碱脱氢酶被Triton X-100从膜上增溶下来后Amytal不再抑制溶解的胆碱脱氢酶。Amytal对鼠肝线粒体胆碱-细胞色素c还原酶的抑制为反竞争性,K_i为1.80×10~(-4)M。Amytal的这种抑制不受线粒体老化的影响,也不被维生素K_3所解除,说明不是继发于胆碱的氧化产物三甲铵乙醛的氧化被抑制的结果。     

Mammalian genome,volume 5, number 7, 1994, pp 416–423

Erratum

Mammalian genome, volume 5, number 7, 1994, pp 416–423     

5α,6α- AND 5β,6β-dichloromethylene adducts of 3β-acetoxy-5-androsten-17-one

Both the 5α, 6α- and 5β, 6β-dichloromethylene adducts ($\text{2a}$ and $\text{2b}$) of 3β-acetoxy-5-androsten-17-one ($\text{1}$) are produced when the latter is exposed to dichlorocarbene generated from chloroform and base by Phase Transfer Catalysis using ultrasound as a means of agitation. The ¹H NMR substituent effects of 5α, 6α- and 5β, 6β-dichloromethylene on the angular methyl groups (Zürcher values) are given. The ¹³C NMR spectra for both compounds are presented and discussed.     

5′-Nucleotidase from Yeast,Having Special Affinity for 5′-AMP

Three kinds of diketopiperazines which have retarditive activity for the growth of plant seedlings and plant roots at concentrations ranging from 1 : 2,500 to 1 : 100,000, were isolated from the neutral fraction by extracting the cultured broth of Rosellinia necatrix. These three diketopiperazines have been proved to be l-prolyl-l-leucine anhydride, l-prolyl-l-valine anhydride and l-prolyl-l-phenylalanine anhydride respectively, and the last one seems to be a new diketo-piperazine.

Furthermore, a crystalline wax having m.p. 52°C, a physiologically inactive substance, was also isolated from the same neutral fraction and presumed to be the saturated hydrocarbon of n-pentacosane C₂₅H₅₂.     

Identification of Novel Regulatory Cholesterol Metabolite, 5-Cholesten, 3β,25-Diol,Disulfate

Oxysterol sulfation plays an important role in regulation of lipid metabolism and inflammatory responses. In the present study, we report the discovery of a novel regulatory sulfated oxysterol in nuclei of primary rat hepatocytes after overexpression of the gene encoding mitochondrial cholesterol delivery protein (StarD1). Forty-eight hours after infection of the hepatocytes with recombinant StarD1 adenovirus, a water-soluble oxysterol product was isolated and purified by chemical extraction and reverse-phase HPLC. Tandem mass spectrometry analysis identified the oxysterol as 5-cholesten-3β, 25-diol, disulfate (25HCDS), and confirmed the structure by comparing with a chemically synthesized compound. Administration of 25HCDS to human THP-1-derived macrophages or HepG2 cells significantly inhibited cholesterol synthesis and markedly decreased lipid levels in vivo in NAFLD mouse models. RT-PCR showed that 25HCDS significantly decreased SREBP-1/2 activities by suppressing expression of their responding genes, including ACC, FAS, and HMG-CoA reductase. Analysis of lipid profiles in the liver tissues showed that administration of 25HCDS significantly decreased cholesterol, free fatty acids, and triglycerides by 30, 25, and 20%, respectively. The results suggest that 25HCDS inhibits lipid biosynthesis via blocking SREBP signaling. We conclude that 25HCDS is a potent regulator of lipid metabolism and propose its biosynthetic pathway.     

The achievement of mass balance by simultaneous quantification of floxuridine prodrug, floxuridine, 5-fluorouracil, 5-dihydrouracil, α-fluoro-β-ureidopropionate, α-fluoro-β-alanine using LC-MS

5-Fluoro-2'-deoxyuridine (floxuridine, 5-FdUrd) and 5-fluorouracil (5-FU) are widely used for the treatment of colorectal cancers. The mechanisms of action of 5-FdUrd and 5-FU, as well as the biochemical pathway responsible for their metabolism, are well understood. Identification of every metabolite and achieving mass balance by conventional UV absorption-based HPLC analysis are not feasible because the metabolites beyond 5-FU in the 5-FdUrd metabolic pathway are undetectable by UV light. We therefore established a mass spectrometry method, designed for fast and convenient analysis, for simultaneously measuring 5-FdUrd, 5-FU, and their metabolites. Linearity, precision and accuracy were validated in the concentration ranges studied for each compound. Hydrolysis studies of 5-FdUrd and amino acid mono ester prodrugs of 5-FdUrd in Capan-2 cell homogenates were carried out and the achievement of mass balance was established with this method (recovery of 5'-O-l-leucyl-FdUrd was 96.6-108.2% and that of 5-FdUrd was 79.4-117.4%). This simple LC-MS method achieves reliable quantitation and mass balance of 5-FdUrd, 5-FU, and their metabolites and can be effectively utilized for further kinetic studies.     

Pavement Life Cycle Assessment Workshop,May 5–7, 2010 in Davis,California, USA

Purpose  

A workshop was convened on life cycle assessment (LCA) applied to pavement. The workshop’s primary goals were to establish common practices for conducting LCAs for pavements. In general, pavement LCA has been implemented without clear guidelines for modeling assumptions and reporting. This shortcoming has led to challenges in interpreting and comparing pavement LCA outcomes.     

Synthesis, SAR, and preliminary mechanistic evaluation of novel antiproliferative N⁶,5'-bis-ureido- and 5'-carbamoyl-N⁶-ureidoadenosine derivatives

We have developed efficient methods for the preparation of N(6),5'-bis-ureidoadenosine derivatives and their 5'-carbamoyl-N(6)-ureido congeners. Treatment of 5'-azido-5'-deoxy-N(6)-(N-alkyl or -arylurea)adenosine derivatives (6a-d) with H(2)/Pd-C or Ph(3)P/H(2)O, followed by N-methyl-p-nitrophenylcarbamate gave N(6),5'-bis-ureido products 7a-d in 49-78% yield. Analogous derivatives in the 5'-carbamoyl-N(6)-ureido series were prepared by treatment of 2',3'-bis-O-TBS-adenosine (11) with N-methyl-p-nitrophenylcarbamate followed by acylation with appropriate isocyanates which gave 13a-d in 45-69% yield. A more versatile route for obtaining potentially vast libraries of compounds from both series was achieved by treatment of 5'-N-methylureido- or 5'-N-methylcarbamoyladenosine derivatives with ethylchlorformate to give N(6)-ethoxycarbonyl derivatives (9 and 14) in 55-63% yields, respectively. Simple heating of 9 or 14 in the presence of primary alkyl- or arylamines gave the corresponding N(6),5'-bis-ureido- or 5'-carbamoyl-N(6)-ureidoadenosine derivatives in good yields (33-72% and 39-83%; 10a-e and 15a-e, respectively). Significant antiproliferative activities (IC(50)≈4-10 μg/mL) were observed for a majority of the N(6),5'-bis-ureido derivatives, whereas the 5'-carbamoyl-N(6)-ureido derivatives were generally less active (IC(50) >100 μg/mL). A 2',3'-O-desilylated derivative (5'-amino-5'-deoxy-5'-N-methylureido-N(6)-(N-phenylcarbamoyl)adenosine, 16) was shown to inhibit binding of 16 of 441 protein kinases to immobilized ATP-binding site ligands by 30-40% in a competitive binding assay at 10 μM. Compound 16 was also shown to bind to bone morphogenetic protein receptor 1b (BMPR1b) with a Kd=11.5 ± 0.7 μM.     

Enzymatic preparation of 5-hydroxy-L-proline, N-Cbz-5-hydroxy-L-proline, and N-Boc-5-hydroxy-L-proline from (α-N-protected)-L-ornithine using a transaminase or an amine oxidase

N-Cbz-4,5-dehydro-L-prolineamide or N-Boc-4,5-dehydro-L-prolineamide are alternative key intermediates for the synthesis of saxagliptin, a dipeptidyl peptidase IV (DPP4) inhibitor recently approved for treatment of type 2 diabetes mellitus. An efficient biocatalytic method was developed for conversion of L-ornithine, N-α-benzyloxycarbonyl (Cbz)-L-ornthine, and N-α-tert-butoxycarbonyl (Boc)-L-ornithine to 5-hydroxy-L-proline, N-Cbz-5-hydroxy-L-proline, and N-Boc-5-hydroxy-L-proline, respectively. Rec. Escherichia coli expressing lysine-ε-aminotransferase and rec Pichia pastoris expressing L-ornithine oxidase were used for these conversions. N-Cbz-5-hydroxy-L-proline, and N-Boc-5-hydroxy-L-proline were chemically converted to key intermediates N-Cbz-4,5-dehydro-L-prolineamide and N-Boc-4,5-dehydro-L-prolineamide, respectively.     

5th Annual Monoclonal Antibodies Conference: March 24–25, 2009, London,UK

The conference, which was organized by Visiongain and held at the BSG Conference Center in London, provided an excellent opportunity for participants to exchange views on the development, production and marketing of therapeutic antibodies, and discuss the current business environment. The conference included numerous interactive panel and group discussions on topics such as isotyping for therapeutic antibodies (panel chair: Nick Pullen, Pfizer), prospects for fully human monoclonal antibodies (chair: Christian Rohlff, Oxford BioTherapeutics), perspectives on antibody manufacturing and development (chair: Bo Kara, Avecia), market impact and post-marketing issues (chair: Keith Rodgers, Bodiam Consulting) and angiogenesis inhibitors (chair: David Blakey, AstraZeneca).

• MAbs. 2009 Jul-Aug; 1(4): 308.
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• March 24, 2009 Day 1

2009 Jul-Aug; 1(4): 308.

March 24, 2009 Day 1

Mari HerigstadAuthor information Copyright and License information DisclaimerVisiongain; London, UKCorresponding author.Correspondence to: Mari Herigstad; Visiongain; BSG House; 226-236 City Road; London EC1V 2QU UK; Email: moc.liamg@datsgireh.iramCopyright © 2009 Landes BioscienceThe first day was dedicated to discussion of antibody development and engineering, as well as debate on use of various types of antibodies. The session was chaired by David Blakey (AstraZeneca). The day opened with an overview on global trends in the antibody development and probabilities of approval success for human and humanized monoclonal antibodies (mAbs). The speakers then provided insights into the engineering and development of new therapeutic antibodies. Prospects for novel antibody formats, and assessment of immunogenicity, stability and aggregation risks in the development of therapeutic antibodies through use of in vivo and in silico methods were reviewed.Global trends in antibody development were discussed by Janice Reichert (Tufts Center for the Study of Drug Development and Editor-in-Chief, mAbs). Dr. Reichert emphasized the increased focus on mAbs as therapeutic agents. Of the therapeutic proteins entering clinical study each year, the majority are mAbs. Major pharmaceutical firms are acquiring biotechnology companies to enter this market and new solutions to problems of immunogenicity, stability, affinity, specificity and production are being developed. The research on clinical pipelines undertaken at Tufts CSDD allows calculation of metrics such as clinical development and approval times and probabilities of approval success. Insights gained from these results are important for strategic planning.The cumulative approval success rate for humanized mAbs was 16% for candidates entering clinical study during 1988 and 2008, and 29% for candidates entering clinical study during 1988 and 1997.¹ A conservative estimate of the success rate for humanized monoclonal antibodies would be somewhere in between, at approximately 20%. The trend, however, is toward fully human monoclonal antibodies. There are currently two marketed human mAbs, with another four in regulatory review. The cumulative US approval success rate for human antibodies is currently low, but will rise to 18% if the four in regulatory review are approved.In terms of therapeutic categories, oncology mAbs comprises approximately 50% of the total. Of 228 oncology mAbs that have entered clinical study since 1988, 56% are currently in clinical development. By comparison, 125 immunological mAb therapeutics have entered clinical study since 1990, of which 54% are currently in clinical development. The cumulative success rate for humanized oncology and immunological mAbs is 15% and 20%, respectively. Other therapeutic categories are being considered, including infectious disease. Sixteen anti-infective mAbs are currently in clinical study and one anti-infective mAb (palivizumab) has been approved to date. Oncology and immunology mAbs exhibit similar patterns for phase lengths and transition probabilities. The phase transition probability for phase 1 to 2 is high, followed by a lower phase 2–3 transition probability due to a proof-of-concept barrier. The transition probability for phase 3 to approval is comparable to that of phase 1 to 2.¹Other interesting trends include an increasing emphasis on antibody fragments.² Fragments may be easier and less costly to produce, but have shorter circulating half-life compared to full size antibodies and no effector functions unless this is added. Also worth noting is the growing prevalence of modified versions of mAbs (glycosylation and Fc region engineering) and improvements on circulation half-life through PEGylation.^3,4 Production methods as well as development and approval pathways for mAbs are well established and marketing approvals are set to increase if success rates are consistent with previous rates. This, together with competitive R&D times and potentially large markets, makes mAbs attractive for development as therapeutics.Julian Burke (Genetix) presented a clinical update on the selection of cell lines for antibody expression and protein production. A hybridoma is a hybrid cell that has been engineered to produce a desired antibody in large amounts. ClonePix FL is an antigen based system for in vitro detection and selection of hybridomas. The system incorporates plating hybridomas into a 3D cell matrix-a method which was first described 25 years ago.⁵ Whilst this method is not new, the novel aspect of the ClonePix system lies in the screening and collection of only those clones secreting a specific antibody. There are two options for screening hybridomas: immunoglobin G (IgG) secretion assays and antigenspecific assays. Unlike IgG secretion assays, antigen-specific assays isolate only antigen-specific clones with the desired IgG isotype. The system can also optimize production through detection of the highest producing cell lines. This approach allows the production of 10,000 clones in three weeks compared to the conventional approach which produces approximately 1,000 clones in two months. After a few days growth post-selection, isolated clones can be rapidly re-screened for cell-line stability. This stability test can be run in parallel with the scaling up of clones, thus making the process highly time efficient. To summarize, ClonePix minimizes the labor requirement, shortens the process timeline and permits parallel interrogation of multiple antigens.Masa Fujiwara (Chiome Bioscience) described the generation of antibodies using a novel antibody-generation technology called the ADLib (Autonomously Diversifying Library) System. This is a selection technology system based on cell-cell interactions and surface-displayed antigens in their native conformations. The system provides high-affinity antibody generation against difficult antigens such as self/human/homologous antigens, GPCRs, sugars/lipids, haptens and pathogens.Dr. Fujiwara explained how the ADLib system can be used to generate specific monoclonal antibodies using a chicken B-cell line (DT40) that undergoes gene conversion at immunoglobulin loci. This gene conversion is enhanced by treatment of the cells with trichostatin A, a histone deacetylase inhibitor. DT40 cells that are specific to the target antigen are obtained through ‘fishing’ the ADLib library with antigens conjugated with magnetic beads. This selection process and the subsequent screening for specificity can be completed in approximately one week.^6,7 As such, one of ADLib''s attractive features is the system''s ability to develop diverse monoclonal antibodies within weeks, not months.Optimization of antibodies, with a focus on structure-function relationships, was discussed by Bryan Edwards (MedImmune). Complementary determining regions (CDRs) are found in the variable domains of antibodies and confer the antigen specificity of the molecule. The CDR regions show high levels of natural sequence variability and are targeted during somatic hypermutation to generate higher affinity antibodies to the antigen. When considering strategies for in vitro optimization of antibodies, the CDR regions are typically targeted for mutagenesis. Three CDRs (CDR1, CDR2 and CDR3) are found on both the heavy and light chain regions of an antibody, and the highest level of natural sequence variability is found in the heavy chain CDR3 domain.Dr. Edwards described the optimization of two lead antibody candidates, where the heavy and light chain CDR3 domains were randomized at all amino acid positions and higher affinity variants isolated by both phage and ribosome display. Further gains in antibody potency were then obtained by combining the beneficial amino changes introduced into the heavy and light chain CDR3 domains. Additional sequence space was also explored outside of the CDR3 regions by the generation of error-prone libraries, and subsequent selection by ribosome display. Lead antibody candidates were improved several thousand-fold in potency through a combination of these approaches.By studying the solved crystal structures of Fab:antigen complexes, Dr. Edwards explained that not all CDR regions make direct contact with the target antigen and that amino acids in the framework regions can also contribute to antibody specificity. Moreover, beneficial changes introduced during optimization are not always due to the introduction of new contacts with the target antigen. Several amino acid changes can indirectly improve the potency of the antibody despite being some distance from the antigen binding site. It has been postulated that these amino acid changes improved affinity by reducing the free energy of the antibody:antigen complex, for example by stabilizing CDR loop conformations or by improving the stability of the VH-VL interface.Pavel Bondarenko (Amgen) presented data on the structure and function of disulfide isoforms of the human IgG2 subclass. There are five different known human antibody isotypes; IgA, IgD, IgE, IgM and IgG, of which IgG is the isotype that provides the majority of immune responses against pathogens. IgG antibodies have predictable properties, controlled function and long circulation half-life. Due to these properties, IgG antibodies are the most common therapeutic modalities. There are four human IgG subclasses, IgG1, IgG2, IgG3 and IgG4, of which IgG1 is the most abundant.One therapeutic function of monoclonal IgG antibodies involves binding Fab regions to target receptors, which blocks ligand-receptor interaction. Additional functions include initiating cell destruction through the attraction of immune complexes by the Fc and hinge region on the antibody. Due to their lower affinity for Fc receptors than IgG1s, IgG2s show reduced propensity for activating immune responses. This may be beneficial in some therapeutic aspects.Scientists at Amgen have recently discovered that structural heterogeneity is a naturally occurring feature of human IgG2 antibodies.^8,9 These distinct IgG2 forms are due to differences in the disulfide connectivity at the hinge region. There are three human IgG2 isoforms; IgG2-A, IgG2-B and IgG2-A/B. IgG2-A is defined by structurally independent Fab domains and hinge region. In IgG2-B, on the other hand, both Fab regions are covalently linked to the hinge. IgG2-A/B is an arrangement in which only one Fab arm is covalently linked to the hinge through disulfide bonds.The disulfide isoforms may show differences in potency. Against a cell-surface receptor, IgG1 and IgG2-A forms of a mAb were shown to have approximately similar potency and both had greater potency that IgG2-B. The difference between the IgG2 isotopes was attributed to the greater flexibility of IgG2-A and its ability to bind with both Fab regions. IgG2 disulfide exchange is facilitated by the close proximity of cysteine residues at the hinge region of IgG2. The mutation of a single cysteine residue in the IgG2 hinge region resulted in a loss of disulfide heterogeneity.¹⁰ Also, redox treatments with a cysteine/cystamine mixture have been shown to cause enrichment of both IgG2-A and IgG2-B.⁹ Human IgG2 isoforms are dynamic and exhibit disulfide rearrangement in both blood and cell culture.¹¹ Initially, IgG2 exists as IgG2-A, and is then rapidly converted to the asymmetric IgG2-A/B, followed by a slower conversion to IgG2-B. The biological relevance of IgG2 isoforms and in vivo conversion is currently being studied.⁹Andrew Popplewell (UCB New Medicines) provided an introduction to the prospects for therapeutic antibody fragments. Out of the 22 currently FDA approved monoclonal antibody therapeutics, three are antibody fragment therapeutics. Antibody fragment formats include Fab regions, single chain variable domains (scFv) and variable loops on the heavy and light chain (dAbs).Antibody fragments are highly flexible formats that may be combined to form new multivalent or multi-specific structures. Compared to full-length IgGs, fragments may have improved biodistribution, tissue penetration,^12,13 target access, or potentially better safety profiles due to the lack of Fc regions. Antibody fragments can also be expressed in microbial systems. They also show shorter serum persistence, which, depending on use, can be either disadvantageous or advantageous. However, antibody fragment circulation time can be modified either through PEGylation,^3,4,14 or through use of serum proteins as carriers.¹⁵ Either strategy may alter the pharmacokinetic properties of fragments, allowing the infrequent therapeutic dosing commonly used for full-length IgG therapy.Stability is a major factor for the successful commercialization of antibody-based drugs. Fabs are generally more stable to thermal or physical stress compared to IgGs, and this stability is not affected by PEGylation or by antibody species origin (e.g. mouse, rat, human). ScFvs and dAbs typically exhibit reduced stability compared to Fabs, however advances in technology may contribute to improve stability in these fragments.Dr. Popplewell emphasized that a more complete understanding of biophysical properties and improved stability engineering are required before antibody fragments can reach their full potential. Full-length IgG1s offer active immune cell recruitment, and may thus be better suited for certain therapeutic uses, such as oncology treatments. The development of products with enhanced Fc functionality, the option of using inactive Fcs, and improvements in yields from mammalian cell expression systems are providing further options for the full length IgG format. Dr. Popplewell summarized by pointing out that the intended mechanism of action should guide the choice of format.The challenge of predicting immunogenicity in a potential drug was discussed by Phillipe Stas (Algonomics). This is a difficult, yet important, process because early and precise immunogenicity assessments can reduce the number of drugs that fail to demonstrate efficacy in clinical trials. In order to generate an accurate risk profile of the potential immunogenicity for a given drug, two questions in particular must be addressed. First, the probability of observing an immunogenic response must be analyzed. Second, the severity of the observed immunogenicity needs to be considered.Neutralizing antibody responses can neutralize not only the therapeutic protein, but also its endogenous counterpart; the latter may induce severe side-effects in the patient.¹⁶ Risk factors for immunogenicity include the degree of ‘non-self’ of the antibody, the dosing of the drug (acute versus repeated), route of administration (intravenous versus subcutaneous) and other drug characteristics such as clearance rate of the drug. The patient''s immune status and the properties of the disease (i.e., severity and availability of concomitant immunosuppressants) should be included in a risk analysis.The different drug development stages offer opportunities to use different strategies for immunogenicity assessment. In the clinical phase, the general approach is to conduct antidrug antibodies (ADA) screening on individuals exposed to the drug. However, efforts are now geared toward assessing immunogenicity at earlier stages. The generation of ADA is dependent on the presence of T-cell epitopes. These can be measured in the preclinical setting using in-vitro T-cell assays. Prior to this stage, in silico methods may be used to identify T-cell epitopes. One such T-cell epitope screening tool is Algonomics'' platform Epibase®, which can rapidly analyze and predict the potential immunogenicity of therapeutic protein leads. An in silico approach to T-cell identification can offer relatively inexpensive mapping of epitopes from a wide genetic background.¹⁷ Also, perhaps more importantly, the combined use of in vitro and in silico tools allows for a much more accurate and less time-consuming assessment of expected immunogenicity in a drug.In silico methods in the development of therapeutic antibodies were reviewed by Jesús Zurdo (Lonza Biologics). Dr. Zurdo focused on assessment of stability and aggregation risks. In addition to increased production costs, aggregation reduces product stability, increases immunogenicity and may also elevate the toxicity. AggreSolve is an in silico protein analysis platform that can be applied to predict and overcome protein stability and aggregation issues. The Aggresolve platform assesses protein aggregation propensity and identifies aggregation ‘hot-spots.’ The platform can also predict sequence changes that are likely to reduce aggregation propensity. This library of potential substitutions can then be used to re-engineer antibodies with elevated stability and fewer aggregation problems. Compared to wild-type, selected re-engineered molecules achieve significantly reduced aggregation levels whilst retaining biological activity. Furthermore, protein stabilisation using re-engineering methods can also translate into elevated antibody productivity.     

Characterization and cellular localization of human 5-lipoxygenase and its protein isoforms 5-LOΔ13, 5-LOΔ4 and 5-LOp12

Human 5-lipoxygenase (5-LO-WT) initiates the leukotriene (LT) biosynthesis. LTs play an important role in diseases like asthma, atherosclerosis and in many types of cancer. In this study, we investigated the 5-LO isoforms 5-LO∆13, 5-LO∆4 and 5-LOp12, lacking the exons 13, 4 or a part of exon 12, respectively. We were able to detect the mRNA of the isoforms 5-LO∆13 and 5-LOp12 in B and T cell lines as well as in primary B and T cells and monocytes. Furthermore, we found that expression of 5-LO and particularly of the 5-LO∆13 and 5-LOp12 isoforms is increased in monocytes from patients with rheumatoid arthritis and sepsis. Confocal microscopy of HEK293T cells stably transfected with tagged 5-LO-WT and/or the isoforms revealed that 5-LO-WT is localized in the nucleus whereas all isoforms are located in the cytosol. Additionally, all isoforms are catalytically inactive and do not seem to influence the specific activity of 5-LO-WT. S271A mutation in 5-LO-WT and treatment of the cells with sorbitol or KN-93/SB203580 changes the localization of the WT enzyme to the cytosol. Despite colocalization with the S271A mutant, the isoforms did not affect LT biosynthesis. Analysis of the phosphorylation pattern of 5-LO-WT and all the isoforms revealed that 5-LOp12 and 5-LO∆13 are highly phosphorylated at Ser271 and 5-LOp12 at Ser523. Furthermore, coexpression of the isoforms inhibited or stimulated 5-LO-WT expression in transiently and stably transfected HEK293T cells suggesting that the isoforms have other functions than canonical LT biosynthesis.     

Synthesis of 5α-Androstane-3α,16α,17β-triol from 3β-hydroxy-5-androsten-17-one

5α-Androstane-3α, 16α 17β-triol was synthesized from 3β-hy-droxy-5-androsten-17-one. The procedure Involved catalytic hydrogenation of 3β-hydroxy-5-androsten-17-one to 3β-hydroxy-5α-androstan-17-one. This was followed by conversion of the 3β-hydroxy group to 3α-benzoyloxy group by the Mitsunobu reaction. Further treatment with isopropenyl acetate yielded 5α-androsten-16-ene-3α, 17-diol 3-benzoate 17-acetate. This was then converted to 3α, 17-dihydroxy-5α-androstan-16-one 3-benzoate 17-acetate via the unstable epoxide intermediate after treatment with $\text{m}$-cloroperoxybenzoic acid. LiAlH₄ reduction of this compound formed 5α-androstane-3α, 16α, 17β-trlol. ¹H and ¹³C NMR of various steroids are presented to confirm the structure of this compound.