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Methylococcus capsulatus (Texas), when grown on methane, undergoes with age a progressive degeneration of internal membrane structure with a simultaneous accumulation of intracellular inclusions. When M. capsulatus is grown on methanol, virtually no internal membranes are present but, instead, cells contain many intracellular droplets morphologically similar to inclusions in old methane-grown cells. Membranes are regenerated by the cells when a methanol-grown culture is transferred back to methane. The oxidative ability of methane- and methanol-grown cells was compared.

The use of high-performance hydrophobic-interaction chromatography (HPHIC) on SynChropak 500 propyl columns has been evaluated for the first time in the analysis of estrogen receptors labeled with [125I]iodoestradiol-17 beta. These receptors were extracted from reproductive tissues with 500 mM phosphate buffer and applied to the stationary phase. Utilizing an inverse phosphate gradient (500 to 10 mM), elution resulted in rapidly excluded components in the void volume followed by a second radioactive peak at 400 mM phosphate. Both peaks appeared to contain specific estrogen-binding components in that steroid association was inhibited by diethylstilbestrol and free ligand was eluted with a different retention time. A great deal of [125I]iodoestradiol-17 beta was retained by the column. Inclusion of acetonitrile (20%) in the mobile phase resulted in the elution of [125I]iodoestradiol-receptor complexes at a different position from free ligand. Distribution of specific estrogen-binding components appeared to be tumor-dependent. These preliminary results indicate that HPHIC may be useful for isolating various isoforms of steroid hormone receptors so that detailed information regarding their intrinsic properties may be ascertained.

Evidence from a variety of target organs has shown that progesterone receptor (PR) is induced by estrogen receptor (ER) in normal and neoplastic tissues. However, approximately 12% of the normal human uterine samples exhibit only PR with no measurable ER, suggesting the expression of both inducible and constitutive receptor isoforms. We investigated several molecular properties of PR from tissues either exhibiting or lacking ER. All studies were conducted in potassium phosphate buffer containing 10 mM sodium molybdate with a synthetic progestin, [3H]R5020 as the ligand. Radioinert R5020 was used as competitor to assess nonspecific association. Competition analysis showed that PR from both sources exhibited similar ligand specificities and affinities. Relative affinities were ORG 2058 greater than R5020 greater than medroxy-progesterone acetate greater than progesterone much greater than testosterone (Kd values ranged from 10(-9) to 10(-10) M; testosterone showed no specific competition). We utilized high-performance liquid chromatography in the size-exclusion (HPSEC) and ion-exchange (HPIEC) modes to probe the size and ionic properties of PR. HPSEC profiles showed that the PR isoform from both sources was eluted as a single, sharp peak greater than 75 A. HPIEC elution profiles indicated no differences in the surface ionic properties in that PR from both tissue types eluted with ca. 100 mM phosphate. These experiments show no difference between the inducible and the putative constitutive form of PR. Thus, some PR species may not require estrogen for their formation.

Breast tumor biopsies required for steroid receptor determination are normally frozen in liquid nitrogen and stored until assay. However, some limitations of this type of storage exist. To try to both eliminate the need for liquid nitrogen and as part of a study of serial assays on a single tumor biopsy, alternative storage media were investigated. This study shows that storage of breast tumor biopsies at -20 degrees in sucrose buffer made 50% in glycerol prevented the tissues from freezing, yet retained the specific estrogen receptor content both quantitatively and in terms of molecular form (8S:4S ratio). Receptor was stable for up to 100 days, and individual samples could be successfully reassayed throughout this period. Forty-four biopsies from 40 patients were halved, and one section from each was stored in liquid nitrogen, while the other was stored in sucrose:glycerol. Overall, the correlation of receptor content between the two storage methods was good. Using a clinical cutoff value of 20 fmol/mg cytosol protein, only one sample of the 44 would have been classified differently after storage in the two media. Progesterone receptor in biopsies stored in sucrose:glycerol also appears to be stable for at least a limited period.

Methodology for high-performance hydrophobic interaction chromatography (HPHIC) of estrogen receptors (ER) was developed, utilizing a polyether-bonded stationary phase, which was non-ionic in nature. Using a descending salt gradient (2 M to 0 M ammonium sulphate in 40 min), ERs from human breast cancer separated into two isoforms, which retained ligand-binding domains. The same isoforms were observed with ER preparations from rat uterus. When sodium molybdate, a stabilizer of receptor structure, was incorporated into the mobile phase, it altered the ER characteristics, producing an earlier elution of one component, while the other one remained unchanged. Treatment of breast cancer cytosol with RNase A did not alter ER elution from either the hydrophobic or size-exclusion (TSK 3000 SW) columns. Modification of cysteine residues with N-ethylmaleimide led to a broad elution pattern of receptor from the hydrophobic column, implying the existence of multiple conformations of ER. Limited trypsin treatment of ER, which removes the DNA binding domain, led to the elution of only one receptor peak from the hydrophobic column. The receptor eluted at 24 min both in the presence and in the absence of sodium molybdate. Thus, at least one mechanism of the sodium molybdate effect must involve its direct interaction with ER to influence the sequence between the DNA-binding domain and the N-terminus. This also indicates that the most hydrophobic species of ER (sodium molybdate sensitive) may arise due to the interaction of the DNA-binding site with the stationary phase. Other possibilities, such as differential post-translational modifications of the receptor protein could also account for the two isoforms of ER, observed in HPHIC analysis.

The DNA regulatory enzyme topoisomerase I (TpI) from human breast cancer cells has been analyzed by high-performance liquid chromatography (HPLC) for the first time. Cells were homogenized in Tris buffer and TpI activity was extracted with 0.5 M sodium chloride. Negatively supercoiled plasmid pBR322 was used as the substrate to monitor TpI activity, as judged by relaxed products, analyzed on 1% agarose gels. HPLC in the anion-exchange mode (HPIEC) provided an approximately 6-fold purification of the enzyme. Enhanced purification was subsequently obtained by chromatography of a HPIEC eluate on size-exclusion columns (30- to 60-fold). Recovery of TpI from size-exclusion columns, whether used in multistep analysis or as the first step, was dependent on inclusion of organic solvent, 1-propanol (0.5%, v/v), in the mobile phase. Marked resolution of TpI activity was observed with HPIEC on a SynChrom CM-300 column. Enzyme activity was noted in the void volume, at 150-200 mM phosphate and at 250-350 mM phosphate. TpI purification was 10- and 120-fold in the latter two peaks, respectively. Silver-stained polyacrylamide gels of TpI-containing activity, eluted from a CM-300 column, showed considerable purification of all but the void volume fraction. A distinct protein band at approximately 88-90 kD was seen in the peak eluted from the CM-300 column with 250-350 mM phosphate. These results indicate that HPLC is useful for rapid purification of the labile enzyme, TpI, in the analysis of its structure-function relationship.

We have determined that high-performance hydrophobic-interaction chromatography (HPHIC) with weakly hydrophobic columns permit the rapid separation of the labile isoforms of estrogen receptor proteins. Previously we reported the use of the SynChrom propyl 500 column for HPHIC of steroid receptors. However, due to the strongly hydrophobic characteristics of the ligand, [125I]iodoestradiol-17 beta, and the receptor protein, organic solvent was required in the mobile phase for greater recovery of receptor proteins. Here, we report separation of steroid receptors from human breast tumors and rat uteri, using the Beckman CAA-HIC, a non-ionic polyether-bonded column, without the need for organic solvents and with virtually 100% recoveries. Receptors were extracted in 10 mM phosphate buffer (pH 7.4). Maximum resolution and separation were achieved when a descending salt gradient of ammonium sulfate in phosphate buffer (pH 7.4) was used (2-0 M in 30 min). Estrogen receptor (ER) was resolved into two isoforms with tR = 22 +/- 1 min (n = 16, designated as peak I) and 27.5 +/- 0.5 min (n = 14, designated as peak II) and a purification of five- to twenty-fold in a single pass. Free steroid was eluted at tR = 35 +/- 1 min (n = 4). Separation was dependent on adjusting the ionic strength of cytosol to 1.5 M ammonium sulfate. ER, purified by HPHIC, retained ligand binding capacity and exhibited protein kinase activity, which was dominant in the less hydrophobic peak I (tR = 22 min) when immunoprecipitated with the monoclonal antibody D547. This method of rapidly purifying ER with high retention of biological activity may now be applied to the study of the molecular interrelationships of steroid receptor isoforms.

Two new prototype probes, BICAP and Heater, were assessed to determine their effectiveness in controlling post-biopsy bleeding during laparoscopy. A total of 88 patients with a wide spectrum of liver diseases were studied. Both probes were equally effective in stopping bleeding in all patients. We recommend the use of either of these devices in controlling post-biopsy bleeding.

High-performance hydrophobic interaction chromatography (HPHIC) was used to separate and characterize two molecular forms of estrogen receptor with a SynChropak propyl hydrophobic column (300 A pore size). The linear gradient utilized earlier with a polyether-bonded column (2 to 0 M) ammonium sulfate in 40 min, gave poor resolution with the propyl column. However, resolution was maximized with either an initial ammonium sulfate concentration of 1 M (40-min gradient) or with a two-phase gradient (2 to 0.5 M in 10 min, 0.5 to 0 M in 30 min). This indicated that the propyl column was more hydrophobic than the polyether column. Estrogen receptor separated into two isoforms, either in the presence [MI, retention time (tR) = 13-14 min; MII, tR = 20-21 min] or absence (I, tR = 21-23 min; II, tR = 31-33 min) of the estrogen receptor stabilizing reagent, sodium molybdate. Similar isoforms were observed in cytosols from human breast tumors, uterus, and MCF-7 breast cancer cells. Unlike others, MCF-7 estrogen receptor did not show MI. Since MCF-7 cells contain 90,000 dalton heat shock proteins (HSP-90), HSP-90 is probably not directly involved in MI formation. Sodium molybdate selectively interacted with isoform II and converted it to MI. All isoforms appeared to be high-molecular-weight proteins (greater than 60 A) when subsequently analyzed by high-performance size-exclusion chromatography. Interestingly, when estrogen receptor was immobilized on the stationary phase, no change was detected in either hydrophobicity or steroid-binding capacity. After 16-18 h, immobilized receptor was eluted with a slightly longer tR. During incubation on the column, component MI was converted into I and/or II. HPHIC appears to be a rapid, yet gentle procedure for isolating large receptor complexes in significant quantities with high recoveries. This allows one to discern the complicated structure-function relationships of estrogen receptor and associated non-receptor proteins and provides information about the on-column behavior of complex proteins.

STUDIES ON ERYTHROMYCIN RESISTANCE IN STRAINS OF GROUP A STREPTOCOCCI INDICATED THAT THEY WERE COMPRISED OF TWO TYPES: (i) an inducible, resistant type (IR strains) was seen, which manifested immediate logarithmic growth in media containing high concentrations of the drug only after brief previous exposure (induction period) of the organisms to subinhibitory concentrations of erythromycin, and (ii) a constitutive, resistant type (CR strains) which demonstrated, without prior drug exposure, continued logarithmic growth in media containing high concentrations of erythromycin. Subinhibitory concentrations of either chloramphenicol or puromycin, when added to IR strains prior to induction, interfered with their induction by erythromycin. Exposure of CR strains to chloramphenicol did not visibly affect the subsequent growth curve of these strains in media containing high concentrations of erythromycin. In IR strains, resistance to other macrolide antibiotics (oleandomycin, spiramycin, carbomycin, magnamycin) and to lincomycin also was inducible in nature. There was cross-inducibility between erythromycin, other macrolide antibiotics, and lincomycin. CR strains were constitutively resistant to these antibiotics.