Structural analysis of covalently labeled estrogen receptors by limited proteolysis and monoclonal antibody reactivity

We have used limited proteolysis of affinity-labeled estrogen receptors (ER), coupled with antireceptor antibody immunoreactivity, to assess structural features of ER and the relatedness of ER from MCF-7 human breast cancer and rat uterine cells. MCF-7 ER preparations covalently labeled with [3H]tamoxifen aziridine [( 3H]TAZ) were treated with trypsin (T), alpha-chymotrypsin (C), or Staphylococcus aureus V8 protease prior to electrophoresis on sodium dodecyl sulfate gels. Fluorography revealed a distinctive ladder of ER fragments containing TAZ for each protease generated from the Mr 66,000 ER: for T, fragments of 50K, 38K, 36K, 31K, 29K, and 28K that with longer exposure generated a 6K fragment; for C, fragments of 50K, 38K, 35K, 33K, 31K, 19K, and 18K that with longer exposure generated 14K and 6K fragments; and for V8, ca. 10 fragments between 62K and 28K. Two-dimensional gels revealed charge heterogeneity (two to three spots between pI 5.5 and 6.2) of the 66K ER and the T-generated 28K meroreceptor form. Immunoblot detection with the primate-specific antibody D75P3 gamma revealed that all immunoreactive fragments corresponded to TAZ-labeled fragments but that some small TAZ-labeled fragments (V8-generated forms less than 47K and T-generated forms less than 31K) were no longer immunoreactive. In contrast, use of the antibody H222Sp gamma revealed a correspondence between TAZ-labeled and immunoreactive fragments down to the smallest fragments generated, ca. 6K for T and C and 28K for V8. MCF-7 nuclear and cytosol ER showed very similar digest patterns, and there was a remarkable similarity in the TAZ-labeled and H222-immunoreactive fragments generated by proteolysis of both MCF-7 and rat uterine ER. These findings reveal great structural similarities between the human (breast cancer) and rat (uterine) ER and between nuclear and cytosol ER, indicate charge heterogeneity of ER, and allow a comparison of the immunoreactive and hormone attachment site domains of the ER. The observation that T and C generate a ca. 6K TAZ-labeled fragment that is also detectable with the H222 antibody should be of interest in studies determining the hormone binding domain of the ER and in amino acid sequencing of this region.     

Colorectal Cancers Mimic Structural Organization of Normal Colonic Crypts

Colonic crypts are stereotypical structures with distinct stem cell, proliferating, and differentiating compartments. Colorectal cancers derive from colonic crypt epithelia but, in contrast, form morphologically disarrayed glands. In this study, we investigated to which extent colorectal cancers phenocopy colonic crypt architecture and thus preserve structural organization of the normal intestinal epithelium. A subset of colon cancers showed crypt-like compartments with high WNT activity and nuclear β-Catenin at the leading tumor edge, adjacent proliferation, and enhanced Cytokeratin 20 expression in most differentiated tumor epithelia of the tumor center. This architecture strongly depended on growth conditions, and was fully reproducible in mouse xenografts of cultured and primary colon cancer cells. Full crypt-like organization was associated with low tumor grade and was an independent prognostic marker of better survival in a collection of 221 colorectal cancers. Our findings suggest that full activation of preserved intestinal morphogenetic programs in colon cancer requires in vivo growth environments. Furthermore, crypt-like architecture was linked with less aggressive tumor biology, and may be useful to improve current colon cancer grading schemes.     

On the non-existence of a tryptic peptide with biological activity derived from mouse nerve growth factor

Reverse-phase high-performance liquid chromatography was used to analyse the products of treatment of mouse nerve growth factor with cyanogen bromide followed by trypsin as described by Mercanti et al. All the biological activity was found to be due to incompletely cleaved starting material. Total digestion with trypsin led to complete loss of activity.     

Long term urinary platinum, palladium, and gold excretion of patients after insertion of noble metal dental alloys

The aim of this study was to investigate to which extent noble-metal dental alloys contribute to the total platinum (Pt), palladium (Pd), and gold (Au) body burden of the general population. The urinary Pt, Pd, and Au excretion was determined in three non-occupationally exposed volunteers before and up to 3 months after insertion of a highgold dentalalloy. The in-vitro release of Pt, Pd, and Au from four different types of dental alloys into either artificial saliva or 1% lactic acid solution was additionally investigated. The Pt, Pd, and Au concentrations were determined by sector field inductively coupled plasma mass spectrometry (SF-ICP-MS). Before insertion of the high-gold dental alloy, the Pt excretion of the patients ranged between 1.0 and 7.4 ng l-1 (0.6-3.3 ng g-1 creatinine). In the immediate post-insertion phase the Pt excretion rose to 10.5-59.6 ng l-1 (14.5-33.2 ng g-1 creatinine). This is a mean increase by a factor of 12 compared with the average Pt excretion before insertion. Three months after insertion, the Pt excretion was still elevated by a factor of 7. Contrary to Pt, the Au and Pd excretion in urine was not significantly increased after insertion of this type of high-gold dental alloy. Our in-vitro investigations confirm the assumption that Pt, Pd, and Au are released from noble metal containing dental alloys by corrosion. Under the applied conditions, the release was in the lower ng cm-2 range. It can be concluded that the Pt release from dental alloys can predominantly contribute to the Pt exposure of non-occupationally exposed persons. It can exceed the exposure from all other environmental sources including the Pt release from automobile exhaust catalysts.     

Decision Support through Mass and Energy Flow Management in the Vehicle-Refinishing Sector

In the past few decades, major advances in environmental protection within the coating application industry have been made. In spite of this technological progress, approximately 50% of industrial-solvent emissions still come from the paint-application sector. The advances made in reducing emissions for plants requiring licensing have unfortunately had no influence on the environmental efforts of smaller companies. Solvent-reduced painting systems, such as high-solid paints, water-based coating, and powder coating have not been able to achieve acceptance, nor have innovative application technologies. The principal arguments against a conversion to these ecologically more favorable alternatives were related to cost and quality.
Recently, the EU Solvent Directive (1999/13/EC) went into effect, aiming to significantly reduce industrial-solvent emissions. Up until this point, however, instruments enabling smaller companies to determine their solvent emissions and to simultaneously develop process-improvement potentials while keeping costs in mind have been missing.
Using the mass and energy flow-management approach, cost structures and environmental benefits can be made transparent to the entrepreneur. The primary result of the research projects presented here is the computer-based mass and energy flow model called the individual computer-aided mass and energy flow model for the vehicle-refinishing sector (IMPROVE). It can be used as a detailed business-consultancy tool. Based upon this, practical guidelines were developed for easy orientation and activity planning. They can be used by companies to help them fulfill the requirements of environmental legislation and to display the benefits that can be achieved by various emission-reduction measures.     

Monoclonal antibodies to CD2 and lymphocyte function-associated antigen 3 inhibit human thymic epithelial cell-dependent mature thymocyte activation

Recent study of human thymocyte-thymic epithelial (TE) cell interactions has demonstrated that thymocytes bind to TE cells, and a consequence of this binding is the provision of accessory cell signals by TE cells for phytohemagglutinin (PHA)-induced mature thymocyte activation. In this paper we report on studies of the molecules involved in TE cell-dependent mature thymocyte activation. TE-thymocyte interactions necessary for PHA-induced thymocyte activation were inhibited by monoclonal antibodies against the cluster of differentiation (CD)2 antigen on thymocytes and lymphocyte function-associated (LFA)-3 antigen on TE cells. Inhibition of TE accessory cell signals by antibodies against CD2 (alpha CD2) and LFA-3 (alpha LFA-3) antigens occurred early on during thymocyte activation and prevented thymocyte interleukin 2 receptor expression. Further, alpha CD2 and alpha LFA-3 inhibited PHA-induced thymocyte activation in whole thymic explant cultures suggesting a significant role of the CD2 and LFA-3 antigens in thymocyte activation when accessory cell signals for PHA-induced thymocyte triggering were delivered by cells within an intact thymic microenvironment.     

Genomic organization of the ribosomal DNA of sorghum and its close relatives

Summary The structure and organization of the ribosomal DNA (rDNA) of sorghum (Sorghum bicolor) and several closely related grasses were determined by gel blot hybridization to cloned maize rDNA. Monocots of the genus Sorghum (sorghum, shattercane, Sudangrass, and Johnsongrass) and the genus Saccharum (sugarcane species) were observed to organize their rDNA as direct tandem repeats of several thousand rDNA monomer units. For the eight restriction enzymes and 14 cleavage sites examined, no variations were seen within all of the S. bicolor races and other Sorghum species investigated. Sorghum, maize, and sugarcane were observed to have very similar rDNA monomer sizes and restriction maps, befitting their close common ancestry. The restriction site variability seen between these three genera demonstrated that sorghum and sugarcane are more closely related to each other than either is to maize. Variation in rDNA monomer lengths were observed frequently within the Sorghum genus. These size variations were localized to the intergenic spacer region of the rDNA monomer. Unlike many maize inbreds, all inbred Sorghum diploids were found to contain only one rDNA monomer size in an individual plant. These results are discussed in light of the comparative timing, rates, and modes of evolutionary events in Sorghum and other grasses. Spacer size variation was found to provide a highly sensitive assay for the genetic contribution of different S. bicolor races and other Sorghum species to a Sorghum population.     

Resonance raman investigations of site-directed mutants of myoglobin: effects of distal histidine replacement

The resonance Raman spectra of met-, deoxy-, and (carbonmonoxy)myoglobin (MbCO) are studied as a function of amino acid replacement at the distal histidine-E7 position. The synthetic wild type is found to be spectroscopically identical with the native material. The methionine and glycine replacements do not affect the met or deoxy spectra but do lead to distinct changes in the nu Fe-CO region of the MbCO spectrum. The native MbCO displays a pH-dependent population redistribution of the nu Fe-CO modes, while the analogous population in the mutant systems is found to be pH independent. This indicates that histidine-E7 is the titratable group in native MbCO. Moreover, the pH dependence of the population dynamics is found to be inconsistent with a simple two-state Henderson-Hasselbalch analysis. Instead, we suggest a four-state model involving the coupling of histidine protonation and conformational change. Within this model, the pK of the distal histidine is found to be 6.0 in the "open" configuration and 3.8 in the "closed" conformation. This corresponds to a 3 kcal/mol destabilization of the positively charged distal histidine within the hydrophobic pocket and suggests how protonation can lead to a larger population of the "open" conformation. At pH 7, the pocket is found to be "open" approximately 3% of the time. Further work, involving both IR and Raman measurements, allows the electron-nuclear coupling strengths of the various nu Fe-CO and nu C-O Raman modes to be determined. The slowly rebinding conformational state, corresponding to nu Fe-CO = 518 cm-1 (nu C-O = 1932 cm-1), displays unusually weak coupling of the Fe-CO mode to the Soret transition. Studies of the nu Fe-CO region as a function of temperature reveal that the equilibria between the conformational states are quenched in both the native and glycine mutant below the freezing point of the solvent. Unusual line narrowing of the nu Fe-CO modes at the phase transition is also observed in all samples studied. This line narrowing stands in marked contrast to the other heme Raman modes and suggests that Fe-CO librational motion and/or distal pocket vibrational (or conformational) excitations are involved in the line broadening at room temperature.     

Microbial communities in the saturated groundwater environment II: Diversity of bacterial communities in a Pleistocene sand aquifer and their in vitro activities

Bacterial cell numbers obtained from 103 water and sediment samples from a Pleistocene sandy aquifer in the Lower Rhine region (Bocholt, FRG) were determinated on P-agar and by direct count. Below 5 m under the surface, colony-forming unit (cfu) numbers in water samples were less than 100/ml, and in many cases less than 50/ml. In sediment samples, they were 10- to 100-fold higher (10²–10⁴ cfu/g dry wt), but changing markedly between different depths. Direct cell counts yielded numbers two to three orders of magnitude higher.About 2,700 strains of bacteria from 60 samples were isolated randomly and characterized by morphological and physiological properties. Of all the isolates, 71.6% were gram-negative, and 52.2% were gram-negative straight rods. Water communities, with one exception, had low proportions of gram-positive bacteria (<11%), whereas in all but one of the sediment communities percentages of gram-positive isolates were three- to sevenfold higher (35–43%). Water and sediment communities, as well as communities from different sampling sites and communities from different depths of the same sampling site, differed in their qualitative and quantitative morphotype composition and physiological capabilities.The in vitro activities of strains within a single community were quite different, indicating that each community is composed of many diverse bacteria, several having extremely different capabilities. Thus, each community has its own specific activity pattern. Gram-positive bacteria showed on an average lower total activities than did gram-negative bacteria. Grampositive bacteria as well as gram-negative bacteria from sediment had higher values of in vitro activities than the corresponding groups isolated from water. Many water and sediment bacteria preferred the same substrates which were utilized at high rates. However, there were differences in the degradation of the various other substrates present, and each community showed preferences for particular substrates, which they degraded best.The results of cell morphology and physiology studies indicated that all eight characterized communities were very different from one another and very diversely structured.