A 3'-5' exonuclease in human leukemia cells: implications for resistance to 1-beta -D-arabinofuranosylcytosine and 9-beta -D-arabinofuranosyl-2-fluoroadenine 5'-monophosphate

A 3'-5' exonuclease that excises the nucleotide analogs 1-beta-d-arabinofuranosylcytosine monophosphate and 9-beta-d-arabinofuranosyl-2-fluoroadenine 5'-monophosphate incorporated at 3' ends of DNA was purified from the nuclei of: 1) primary human chronic lymphocytic leukemia cells, 2) primary and established human acute myeloblastic leukemia cells, and 3) lymphocytes obtained from healthy individuals. The activity of this nuclear exonuclease (exoN) is elevated approximately 6-fold in 1-beta-d-arabinofuranosylcytosine-resistant leukemia cells as compared with drug-sensitive cells, and it differs between two healthy individuals and among three leukemia patients. exoN is a 46-kDa monomer, requires 50 mm KCl and 1 mm magnesium for optimal activity, and shows a preference for single-stranded over duplex DNA. Its physical and enzymatic properties indicate that exoN is a previously uncharacterized enzyme whose activity may confer resistance to clinical nucleoside analogs in leukemia cells.     

Differential expression of transforming growth factor-beta 1, -beta 2, and -beta 3 by glioblastoma cells, astrocytes, and microglia.

The type beta transforming growth factors (TGF) are potent regulators of the growth and functions of lymphocytes and macrophages. Recently the human glioblastoma cell line 308 was shown to produce TGF-beta 2. The relevance of this finding was evaluated further by comparing human glioblastoma cells with their nontransformed animal counterpart, astrocytes, with regard to the production of the three TGF-beta isoforms observed so far in mammals. In this report astrocytes are demonstrated to secrete also TGF-beta 2 and to express TGF-beta 1, -beta 2, and -beta 3 mRNA in vitro. In contrast, cultured murine brain macrophages release TGF-beta 1 and are positive for TGF-beta 1 mRNA only. Glia cell-derived TGF-beta 1 and -beta 2 are detected in latent form whereas both latent and active TGF-beta are identified in the supernatant of three human glioblastoma cell lines tested. These cell lines, however, show heterogeneity in regard to the isoform of TGF-beta expressed but share with astrocytes the inability to release TGF-beta 3. Provided production and activation of latent TGF-beta occur in vivo, astrocytes and microglia may then be expected to exert regulatory influences on immune mediated diseases of the central nervous system.     

Characterization of the binding of transforming growth factor-beta 1, -beta 2, and -beta 3 to recombinant beta 1-latency-associated peptide.

Preprotransforming growth factor-beta 1 (TGF beta 1) is a 390-amino acid precursor polypeptide that undergoes a number of processing steps to yield mature TGF beta 1 (amino acid residues 279-390) and a pro portion (residues 30-278) termed beta 1-latency-associated peptide (beta 1LAP). The dimeric form of beta 1LAP has been shown to associate noncovalently with the mature growth factor, resulting in inactivation of biological activity. To further characterize this interaction, the mature TGF beta 1 was radioiodinated and used to determine dissociation constants. A cross-linking method using the bifunctional covalent cross-linker bis-(sulfosuccinimidyl)suberate was found to be the best approach for measuring the amount of bound growth factor. The efficiency of cross-linking was constant within each experiment and varied between 45-55%. Saturation plots and their associated Scatchard analyses indicate apparent Kd values between 1.1-1.8 nM. Competition of TGF beta 1 binding to beta 1LAP by TGF beta 2 and TGF beta 3 (two closely related growth factors) revealed that the latter also bind beta 1LAP tightly, with apparent Kd values of 1.9 and 0.4 nM, respectively.     

Conversion of 20alpha-hydroxy-4-pregnen-3-one to 20alpha-hydroxy-5alpha-pregnan-3-one and 5alpha-pregnane-3alpha, 20alpha-diol by rat anterior pituitary

³H-20α-hydroxy-4-pregnen-3-one was incubated with anterior pituitaries from proestrous rats. The $\text{in vitro}$ metabolic products, identified by reverse isotopic dilution and purification to constant specific activity, were 20α-hydroxy-5α-pregnan-3-one (23.0%) and 5α-pregnane-3α,20α-diol (11.4%). These are qualitatively the same metabolites which result from $\text{in vitro}$ incubation of 20α-hydroxy-4-pregnen-3-one with medial basal hypothalamus. 68.8% of the recovered radioactivity remained as 20α-hydroxy-4-pregnen-3-one. These three compounds accounted for all of the recovered radioactivity.     

Hypocholesterolemic activity of 17-alpha-methyl-5-alpha-androstane-3-beta, 17-beta-diol, a metabolite of 17-alpha-methyltestosterone

The hypocholesterolemic activities of 17alpha-methyltestosterone and its major identified fecal metabolite, 17alpha-methyl-5alpha-androstane-3,17-diol, were compared in dogs. The dose-response curves indicated that the two compounds had similar effects, although at doses below 1 mg/kg per day the diol appeared to be more active than the parent compound.     

Transforming growth factor-beta 1, -beta 2 and -beta 3 in cartilage and bone cells during endochondral ossification in the chick.

The localization of TGF-beta 1, -beta 2 and -beta 3 was studied in the growth plate, epiphysis and metaphysis of the tibiotarsus of three-week-old chicks. The different TGF-beta isoforms were localized to hypertrophic chondrocytes, chondroclasts, osteoblasts and osteoclasts using immunohistochemical staining analysis with specific TGF-beta antibodies. TGF-betas in osteoclasts and chondroclasts were restricted to those cells located on the respective matrices. TGF-beta 3 localization was mainly cytoplasmic in the transitional (early hypertrophic) chondrocytes, but nuclear staining was also detected in some proliferating chondrocytes. The cell-specific localization of these TGF-beta isoforms supports the hypothesis that TGF-beta has a role in the coupling of new bone formation to bone and cartilage matrix resorption during osteochondral development and suggests that TGF-beta may be a marker of chondrocyte differentiation. TGF-beta localization preceded a marked increase in type II collagen mRNA expression in transitional chondrocytes, suggesting a role for TGF-beta in the induction of synthesis of extracellular matrix.     

Improved preparation of pregn-5-ene-3β, 16α, 20-triols and 5α-Pregnane-3α, 16α, 20-triols

The compounds named in the title were prepared by routes which included the reduction of suitable 16α,17-epoxypregnan-20-ones with aluminium amalgam to give 16α-hydroxypregnan-20-ones, and reduction of the 20-oxo function either with sodium borohydride to obtain the 3, 16α, 20β-triols or with lithium-liquid ammonia to obtain the 3, 16α, 20α-triols.     

Glucosylations of pregn-5-ene-3 beta,20R-diol

The four possible monoglucosides of the pregn-5-ene-3 beta,20R-diol were synthesized along with a mixture of the four possible 3,20-diglucosides. The glucosides were characterized by HPLC and mass spectrometry.     

A simple synthesis of 3beta-acetoxy-20-oxomethylpregn-5-ene and 3beta-acetoxy-20-hydroxymethylpregn-5-ene

3beta-Acetoxy-20-oxomethylpregn-5-ene and 3beta-acetoxy-20-hydroxymethylpregn-5-ene were synthesized from (22R,23R)-sitost-5-ene-3beta,22,23-triol in 66% overall yields.