Total synthesis of heterocyclic steroids,part V. Ionic hydrogenation of heteroaromatic steroidal 8, 14-dienes

Some heteroaromatic steroidal 8, 14-dienes were found to undergo 1, 4-addition of the hydrogen molecule during ionic hydrogenation.     

Convenient preparation of A-ring fused pyridines from steroidal enamides

A facile strategy for the preparation of A-ring fused pyridosteroids has been accomplished in high yields by the reaction of Vilsmeier reagent (chloromethyleneiminium salt) with steroidal A-ring enamides (2- and 3-ene) under thermal conditions. The structure of 6'-chloro-5alpha-cholest [3,2-b]pyridine was determined by X-ray analysis.     

Biochemical properties of A-homovitamin D derivatives: 1,4-dihydroxy-3-deoxy-A-homo-19-nor-9, 10-secocholesta-5,7-dienes

A study has been made in the chick of the stereostructural requirements of A-ring-functionalized vitamin D analogs which elicit vitamin D₃ and 1,25-(OH)₂D₃-dependent biological responses of intestinal calcium absorption (ICA) and bone calcium mobilization (BCM). Ring expansion of vitamin D₃ to produce (1S,4S), (1S,4R), or (1R,4S)-(7E)-1,4-dihydroxy-3-deoxy-A-homo-19-nor-9,10-secocholesta-5,7-dienes resulted in the loss of both ICA and BCM biological activity at dose levels of steroid of up to 650 nmol/0.1 kg birds. Accordingly the three A-homo analogs of vitamin D₃ were assessed for their ability to inhibit or increase the ICA or BCM responses of D₃ and 1,25-(OH)₂D₃. Only (1R,4S)-(7E)-diol-C, maintaining a cis-β,β-hydroxyl orientation showed antagonistic biological activity. Intraperitoneal doses (65–325 nmol) of diol-C administered in conjunction with D₃ (0.8–3.25 nmol) inhibited the BCM responses selectively and had no effect on the ICA response. Doses of analog-C (16.3-3.25 nmol) injected before and after the active hormone 1,25-(OH)₂D₃ (0.13–01.30 nmol) stimulated the ICA response of the latter above its normal levels (a synergistic response) when administered alone.     

Efficient preparation of plant metaphase spreads

A simple and efficient method for the preparation of a high number of plant metaphase spreads from a broad range of plant species suitable for light microscopy and high-resolution electron microscopy is described.     

High-throughput laser-mediated in situ cell purification with high purity and yield.

BACKGROUND: Technologies for purification of living cells have significantly advanced basic and applied research in many settings. Nevertheless, certain challenges remain, including the robust and efficient purification (e.g., high purity, yield, and sterility) of adherent and/or fragile cells and small cell samples, efficient cell cloning, and safe purification of biohazardous cells. In addition, existing purification methods are generally open loop and exhibit an inverse relation between cell purity and yield. METHODS: An automated closed-loop (i.e., employing feedback control) cell purification technology was developed by building upon medical laser applications and laser-based semiconductor manufacturing equipment. Laser-enabled analysis and processing has combined high-throughput in situ cell imaging with laser-mediated cell manipulation via large field-of-view optics and galvanometer steering. Laser parameters were determined for cell purification using three mechanisms (photothermal, photochemical, and photomechanical), followed by demonstration of system performance and utility. RESULTS: Photothermal purification required approximately 10(8) W/cm(2) at 523 nm in the presence of Allura Red, resulting in immediate protein coagulation and cell necrosis. Photochemical purification required approximately 10(9) W/cm(2) at 355 nm, resulting in apoptosis induction over 4 to 24 h. Photomechanical purification required more than 10(10) W/cm(2) independent of wavelength, resulting in immediate cell lysis. Each approach resulted in high efficiency purification (>99%) after a single operation, as demonstrated with eight cell types. An automated closed-loop process to re-image and irradiate remaining targets in situ was implemented, resulting in improved purification (99.5-100%) without decreasing cell yield or affecting sterility in this closed system. Efficient purification was demonstrated with B- and T-cell mixtures over a wide range of contaminating cell percentages (0.1-99%) and cell densities (10(4)-10(6)/cm(2)). Efficient cloning of 293T cells based on fluorescence with green fluorescent protein after plasmid transfection was also demonstrated. CONCLUSIONS: In situ laser-mediated purification was achieved with nonadherent and adherent cells on the automated laser-enabled analysis and processing platform. Closed-loop processing routinely enabled greater than 99.5% purity with a greater than 90% cell yield in sample sizes ranging from 10(1) to 10(8) cells. Throughput ranged from approximately 10(3) to 10(5) total cells/s for contaminating percentages ranging from 99% to 0.1%, respectively.     

Synthesis of steroidal cyclophosphamides.

The Reformatsky product of estrone methyl ether and ethyl bromo-acetate was transformed by two separate routes to 21-amino-3-methoxy-17alpha-pregna-1,3,5(10)-trien-17beta-ol (9). Cyclization with bis- (2-chloroethyl) phosphoramide dichloride produced the steroidal cyclophosphamide 10. Analogous syntheses transformed androstenolone into steroidal cyclophosphamide 20 and androstenedione into steroidal cyclophosphamide 28.     

Isolation of intact nuclei of high purity from mouse liver

Current methods of nuclear isolation from liver disrupt the plasmalemmae via homogenization and separation of the nuclei by high centrifugal force (HCF) through gradients of sucrose or other substances for up to 80 min. The use of HCF for such a long time increases the potential for nuclear damage and degradation by endogenous proteases. We compared four combinations of alterations to classical nuclear isolation methods as follows. Mouse liver was gently crushed through a fine mesh with and without in vivo perfusion with collagenase. The cell suspension was centrifuged at 600g to remove gross debris and then at moderate centrifugal force (MCF, 16,000g) or high centrifugal force (HCF, 70,000g) through sucrose gradients for 30 min. The purity of the isolated nuclei was assessed biologically and morphologically, including analyses of representative marker proteins for nuclei and cytoplasm. The results indicate that MCF and no collagenase provided the highest nuclear integrity and purity, whereas MCF with collagenase is a viable option if priority is given to yield. The method is especially suited for small samples and so should facilitate studies with human liver biopsies and livers from mice, the most widely used species for gene targeting.     

Efficient preparation of L-cysteic acid and its esters

Summary. L-Cysteic acid and its esters were prepared in good yields from the oxidation of L-cystine by chlorine in water and in alcohols. When the reaction was carried out in alcohols the corresponding esters were produced.     

An optimized protocol for the production of high purity maltose

An economical protocol, which is simple, rapid and reproducible for the production of maltose by enzymatic hydrolysis of tapioca starch, has been optimized. The protocol involves liquefaction of 35% (w/w) tapioca starch by bacterial -amylase at 78±2°C to 3 to 5% (w/w) reducing sugars, followed by maximal (85±3% w/w maltose equivalent) saccharification with barley -amylase and pullulanase at 50°C for 24 to 30 h. The post-saccharification recovery protocol comprised decolourization by charcoal, de-dextrinization by denatured spirit precipitation, de-ionization by passage through cation and anion exchangers and dehydration by vacuum drying. A white crystalline maltose powder was obtained with specifications comparable to commercial high purity maltose. The protocol yields at least 60% (w/w) recovery of maltose and is suitable for use by the pharmaceutical industry. The protocol is unique in that it utilizes cheap and easily hydrolysed tapioca starch, leaves no mother liquor, enabling higher recovery of maltose, and allows almost quantitative recovery of limit maltodextrins, a value-added marketable by-product.     

Rapid extraction of high purity chromosomal DNA from Serratia marcescens

Rapid non-specific degradation of Serratia marcescens DNA extracted with guanidium thiocyanate, occurred within 10 min of incubation with restriction endo-nuclease enzymes. The described modified method based on chemical and enzymatic deproteinization produced preparations of Ser. marcescens DNA of high yield and quality which did not autodegrade when incubated with restriction endonucleases.     

Preparation of distinct ubiquitin chain reagents of high purity and yield

The complexity of protein ubiquitination signals derives largely from the variety of polyubiquitin linkage types that can modify a target protein, each imparting distinct functional consequences. Free ubiquitin chains of uniform linkages and length are important tools in understanding how ubiquitin-binding proteins specifically recognize these different polyubiquitin modifications. While some free ubiquitin chain species are commercially available, mutational analyses and labeling schemes are limited to select, marketed stocks. Furthermore, the multimilligram quantities of material required for detailed biophysical and/or structural studies often makes these reagents cost prohibitive. To address these limitations, we have optimized known methods for the synthesis and purification of linear, K11-, K48-, and K63-linked ubiquitin dimers, trimers, and tetramers on a preparative scale. The high purity and relatively high yield of these proteins readily enables material-intensive experiments and provides flexibility for engineering specialized ubiquitin chain reagents, such as fluorescently labeled chains of discrete lengths.