Fractionation of aqueous ethanolic extracts from bread

Column chromatography of an aqueous ethanolic extract from bread crust on alkylated Sephadex (LH-20) partly separated it into five coloured fractions. Further partial separation of two of these fractions was achieved by chromatography on Sephadex G-15 and silica gel columns, and by thin-layer chromatography. Maltose, glucose and fructose were determined in crust and crumb extracts by quantitative paper chromatography.     

Immortalization of osteoclast precursors by targeting Bcl -XL and Simian virus 40 large T antigen to the osteoclast lineage in transgenic mice

Cellular and molecular characterization of osteoclasts (OCL) has been extremely difficult since OCL are rare cells, and are difficult to isolate in large numbers. We used the tartrate-resistant acid phosphatase promoter to target the bcl-XL and/or Simian Virus 40 large T antigen (Tag) genes to cells in the OCL lineage in transgenic mice as a means of immortalizing OCL precursors. Immunocytochemical studies confirmed that we had targeted Bcl-XL and/or Tag to OCL, and transformed and mitotic OCL were readily apparent in bones from both Tag and bcl-XL/Tag mice. OCL formation in primary bone marrow cultures from bcl-XL, Tag, or bcl-XL/Tag mice was twofold greater compared with that of nontransgenic littermates. Bone marrow cells from bcl-XL/Tag mice, but not from singly transgenic bcl-XL or Tag mice, have survived in continuous culture for more than a year. These cells form high numbers of bone-resorbing OCL when cultured using standard conditions for inducing OCL formation, with approximately 50% of the mononuclear cells incorporated into OCL. The OCL that form express calcitonin receptors and contract in response to calcitonin. Studies examining the proliferative capacity and the resistance of OCL precursors from these transgenic mice to apoptosis demonstrated that the increased numbers of OCL precursors in marrow from bcl-XL/Tag mice was due to their increased survival rather than an increased proliferative capacity compared with Tag, bcl-XL, or normal mice. Histomorphometric studies of bones from bcl-XL/Tag mice also confirmed that there were increased numbers of OCL precursors (TRAP + mononuclear cells) present in vivo. These data demonstrate that by targeting both bcl-XL and Tag to cells in the OCL lineage, we have immortalized OCL precursors that form bone-resorbing OCL with an efficiency that is 300-500 times greater than that of normal marrow.     

Inhibition of DNA cytosine methyltransferase by chemopreventive selenium compounds, determined by an improved assay for DNA cytosine methyltransferase and DNA cytosine methylation

The organoselenium compounds benzyl selenocyanate (BSC) and 1,4-phenylenebis(methylene)selenocyanate (p-XSC), as well as sodium selenite, are effective chemopreventive agents for various chemically induced tumors in animal models at both the initiation and postinitiation stages. The mechanisms involved at the postinitiation stage are not clear. Because several lines of evidence indicate that inhibition of excess DNA (cytosine-5)-methyltransferase (Mtase) may be a sufficient factor for the suppression or reversion of carcinogenesis, we examined the effects of sodium selenite, BSC, p-XSC and benzyl thiocyanate (BTC), the sulfur analog of BSC, on Mtase activity in nuclear extracts of human colon carcinomas, and of p-XSC on the Mtase activity of HCT116 human colon carcinoma cells in culture. For this purpose, we developed an improved Mtase assay, in which the incorporation of the methyl-[3H] group from S-adenosyl[methyl-3H]methionine into deoxycytidine of poly(dI-dC)-poly(dI-dC), is specifically determined by HPLC with radioflow detection after enzymatic hydrolysis, enhancing specificity and reliability. In a variation, using SssI methyltransferase and labeled S-adenosylmethionine, the overall methylation status of DNA in various tissues can also be compared. Selenite, BSC and p-XSC inhibited Mtase extracted from a human colon carcinoma with IC50s of 3.8, 8.1 and 5.2 microM, respectively; BTC had no effect. p-XSC also inhibited the Mtase activity and growth of human colon carcinoma HCT116 cells, with an IC50 of approximately 20 microM. The improved Mtase assay should prove to be a reliable method for screening potential Mtase inhibitors, especially using cells in culture. We suggest that inhibition of Mtase may be a major mechanism of chemoprevention by selenium compounds at the postinitiation stage of carcinogenesis.     

Requirements for p53 and the ATM gene product in the regulation of G1/S and S phase checkpoints

We investigated the requirements for protein p53 and the ATM gene product in radiation-induced inhibition of DNA synthesis and regulation of the cyclin E/ and cyclin A/cyclin dependent kinases (Cdks). Wild type (WT) mouse lung fibroblasts (MLFs), p53(-/-) knock-out MLFs, normal human skin fibroblasts (HSF-55), and human AT skin fibroblasts (GM02052) were used in the investigations. The absence of p53 had no significant effect on the inhibition or recovery of DNA synthesis throughout the S phase, as determined from BrdU labeling and flow cytometry, or the rapid inhibition of cyclin A/Cdks. Gamma radiation (8 Gy) inhibited DNA synthesis and progression into G2 during the first 3 h after irradiation, and the recovery of these processes occurred at similar rates in both WT and p53(-/-) MLFs. The cyclin A/Cdks were inhibited 55-70% at 1 h after irradiation in both cell types, but p21WAF1/Cip1 levels or p21 interaction with Cdk2 did not increase in the irradiated p53(-/-) MLFs. Although p53(-/-) MLFs do not exhibit prolonged arrest at a G1 checkpoint, radiation did induce a rapid 20% reduction and small super-recovery of cyclin E/Cdk2 within 1-2 h after irradiation. Similar inhibition and recovery of cyclin E/Cdk2 previously had been associated with regulation of transient G1 delay and the inhibition of initiation at an apparent G1/S checkpoint in Chinese hamster cells. In contrast, loss of the ATM gene product abrogated transient cyclin E/Cdk2 inhibition, most inhibition of DNA synthesis and all, but a 10-15% inhibition, of the cyclin A/Cdks. The results indicate that neither p53 nor p21 is required for transient inhibition of cyclin E/Cdk2 associated with the G1/S checkpoint or for inhibition of DNA synthesis at 'checkpoints' within the S phase. Conversely, the ATM gene product appears to be essential for regulation of the G1/S checkpoint and for inhibition of DNA replication associated with the inhibition of cyclin A/Cdk2. Differential aspects of DNA synthesis inhibition among cell types are presented and discussed in the context of S phase checkpoints.     

Selective radiation coatings. Preparation and high temperature stability

Selective radiation coatings suitable for operation at high temperatures with focused sunlight are described. Coatings of CuO and Co₃O₄ on polished nickel, silver, and platinum have high absorptivity for solar radiation and low emissivity in the infrared when heated. They are stable at at high temperatures.     

Ontogeny of vesicular monoamine transporter mRNAs VMAT1 and VMAT2. I. The developing rat central nervous system

We used in situ hybridization histochemistry to study the expression of the mRNA of the two vesicular monoamine transporters (VMAT1 and VMAT2) during embryonic and postnatal development of the central nervous system (CNS) in the rat. In the adult rat, VMAT2 mRNA is present exclusively in monoaminergic cell groups of the CNS and VMAT1 mRNA was reported to be present in the adrenal medulla and certain intestinal epithelial cells. In contrast to the above, the expression of VMAT1 mRNA has previously never been detected in the central nervous system. This study shows the first evidence that both transporter molecules are expressed in CNS during ontogenesis. We here demonstrate four main expression patterns detected during development: 1. VMAT2 mRNA expression in monoaminergic neurons of the brainstem beginning as early as embryonic day E13. 2. Expression of VMAT2 mRNA in all major sensory relay nuclei of central nervous system. 3. Co-expression of VMAT1 and VMAT2 mRNA in most limbic structures, basal ganglia, as well as in some hypothalamic nuclei. 4. Exclusive expression of VMAT1 mRNA in the neocortical subventricular zone, in the amygdala at early (E15-18) and late (P1-P28) timepoints, the granular cell layer of cerebellum, and in several brainstem motor nuclei. Based on their distribution during development we suggest that monoamines, released in a controlled fashion, might affect wiring of sensory and also motor circuits. VMAT1 mRNA expression may reflect a specific effect of monoamines in glial differentiation and cerebellar granule cell migration and/or differentiation.     

Identification of a Gialpha binding site on type V adenylyl cyclase

The stimulatory G protein alpha subunit Gsalpha binds within a cleft in adenylyl cyclase formed by the alpha1-alpha2 and alpha3-beta4 loops of the C2 domain. The pseudosymmetry of the C1 and C2 domains of adenylyl cyclase suggests that the homologous inhibitory alpha subunit Gialpha could bind to the analogous cleft within C1. We demonstrate that myristoylated guanosine 5'-3-O-(thio)triphosphate-Gialpha1 forms a stable complex with the C1 (but not the C2) domain of type V adenylyl cyclase. Mutagenesis of the membrane-bound enzyme identified residues whose alteration either increased or substantially decreased the IC50 for inhibition by Gialpha1. These mutations suggest binding of Gialpha within the cleft formed by the alpha2 and alpha3 helices of C1, analogous to the Gsalpha binding site in C2. Adenylyl cyclase activity reconstituted by mixture of the C1 and C2 domains of type V adenylyl cyclase was also inhibited by Gialpha. The C1b domain of the type V enzyme contributed to affinity for Gialpha, but the source of C2 had little effect. Mutations in this soluble system faithfully reflected the phenotypes observed with the membrane-bound enzyme. The pseudosymmetrical structure of adenylyl cyclase permits bidirectional regulation of activity by homologous G protein alpha subunits.     

Transfectable and transplantable postmitotic human neurons: a potential "platform" for gene therapy of nervous system diseases

OBJECTIVE: To evaluate F-18 fluorodeoxyglucose positron emission tomography (PET) in terms of its sensitivity and specificity in diagnosing malignant pulmonary nodules and staging bronchogenic carcinoma. METHODS: A retrospective review of any patient that presented to the VA Palo Alto Health Care System with a pulmonary nodule between 9/94 and 3/96 revealed 49 patients (four female, 45 male) age 37-85 (mean 63) with 54 pulmonary nodules who had: chest CT scan, PET scan; and tissue characterization of the nodule. Characterization of each nodule was achieved by histopathologic (N = 44) or cytopathologic (N = 10) analysis. Of the 49 patients, 18 had bronchogenic carcinoma which was adequately staged. Mediastinal PET and CT findings in these 18 patients were compared with the surgical pathology results. N2 disease was defined as mediastinal lymph node involvement by the American Thoracic Society's classification system. Mediastinal lymph nodes were interpreted as positive by CT if they were larger that 1.0 cm in the short-axis diameter. RESULTS: Sensitivity and specificity for the diagnosis of malignant pulmonary nodules using PET was 93 and 70%, respectively. All nodules (N = 3) that were falsely positive by PET scan were infectious in origin. All nodules (N = 4) that were falsely negative by PET were technically limited studies (outdated scanner, no attenuation correction, hyperglycemia) except for one case of metastatic adenocarcinoma. The sensitivity and specificity of PET in diagnosing N2 disease was 67 and 100%, compared with 56% and 100% for CT scan (not statistically significant). However, one more patient with N2 disease was correctly diagnosed by PET than by CT scan. CONCLUSION: PET is a valuable tool in the diagnosis and management of pulmonary nodules and may more accurately stage patients with bronchogenic carcinoma than CT scanning alone.     

Specific and efficient peptide substrates for assaying the proteolytic activity of prostate-specific antigen

Prostate-specific antigen (PSA) is a serine protease secreted by both normal prostate glandular cells and prostate cancer cells. The major proteolytic substrates for PSA are the gel-forming proteins in semen, semenogelin (Sg) I and II. On the basis of the PSA cleavage map for Sg I and II, a series of small peptides (i.e., < or = 7 amino acids) was synthesized and coupled at the COOH terminus to 7-amino-4-methyl coumarin. Using these fluorescently tagged substrates, K(m)s and k(cat)s were determined for PSA hydrolysis, and the substrates were also tested for activity against a panel of purified proteases. Previously, a variety of chymotrypsin substrates have been used to assay the enzymatic activity of PSA. The present studies have identified a peptide sequence with a high degree of specificity for PSA (ie., no detectable hydrolysis by chymotrypsin) and improved K(m)s and k(cat)s over previously used substrates. On the basis of these parameters, the best peptide substrate for PSA has the amino acid sequence HSSKLQ. Using PC-82 human prostate cancer xenografts and human prostate tissues, this PSA substrate was used to document that prostate cancer cells secrete enzymatically active PSA into the extracellular fluid but that once in the blood, PSA is not enzymatically active. On the basis of this information, it should be possible to use the HSSKLQ peptide as a carrier to target peptide-coupled prodrugs for selective activation within sites of PSA-secreting, metastatic prostate cancer cells and not within the blood or other nonprostatic normal tissues.