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.     

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.     

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.     

Oral [13C]bicarbonate measurement of CO2 stores and dynamics in children and adults

During exercise, less additional CO2 is stored per kilogram body weight in children than in adults, suggesting that children have a smaller capacity to store metabolically produced CO2. To examine this, tracer doses of [13C]bicarbonate were administered orally to 10 children (8-12 yr) and 12 adults (25-40 yr) at rest. Washout of 13CO2 in breath was analyzed to estimate recovery of tracer, mean residence time (MRT), and size of CO2 stores. CO2 production (VCO2) was also measured breath by breath using gas exchange techniques. Recovery did not differ significantly between children [73 +/- 13% (SD)] and adults (71 +/- 9%). MRT was shorter in children (42 +/- 7 min) compared with adults (66 +/- 15 min, P less than 0.001). VCO2 per kilogram was higher in the children (5.4 +/- 0.9 ml.min-1.kg-1) compared with adults (3.1 +/- 0.5, P less than 0.0001). Tracer estimate of CO2 production was correlated to VCO2 (r = 0.86, P less than 0.0001) and when corrected for mean recovery accurately predicted the VCO2 to within 3 +/- 14%. There was no difference in the estimate of resting CO2 stores between children (222 +/- 52 ml CO2/kg) and adults (203 +/- 42 ml CO2/kg). We conclude that orally administered [13C]bicarbonate can be used to assess CO2 transport dynamics. The data do not support the hypothesis of lower CO2 stores under resting conditions in children.     

Pathway of rhinovirus disruption by soluble intercellular adhesion molecule 1 (ICAM-1): an intermediate in which ICAM-1 is bound and RNA is released.

We have examined the pathway of rhinovirus interaction with soluble intercellular adhesion molecule 1 (sICAM-1). Binding of sICAM-1 to rhinovirus serotypes 3 and 14 gives particles with sedimentation coefficients from 145 to 120S, depending on the amount of sICAM-1 bound. The formation of 120S particles is faster and more extensive at a neutral pH than at an acidic pH. A large number of receptors (> 30) can bind to human rhinovirus 3 without disruption. Disruption by sICAM-1 of rhinovirus that yields 80S particles is strongly temperature dependent and is antagonized by a low pH. Interestingly, sICAM-1 remains bound to the viral capsid after RNA is released, although in smaller amounts than those observed for the native virus. We have found heterogeneity both between and within 80S particle preparations in the VP4 content and number of bound receptors. The ability of the virus to remain bound to its receptor during the uncoating process may facilitate the transport of the viral genome into the cytoplasm in vivo.     

Efficient neutralization and disruption of rhinovirus by chimeric ICAM-1/immunoglobulin molecules.

The intercellular adhesion molecule 1 (ICAM-1) is used as a cellular receptor by 90% of human rhinoviruses (HRVs). Chimeric immunoadhesin molecules containing extracellular domains of ICAM-1 and constant regions of immunoglobulins (Igs) were designed in order to determine the effect of increased valency, Ig isotype, and number of ICAM-1 domains on neutralization and disruption of rhinovirus structure. These immunoadhesins include ICAM-1 amino-terminal domains 1 and 2 fused to the hinge and constant domains of the heavy chains of IgA1, IgM, and IgG1 (IC1-2D/IgA, -/IgM, and -/IgG). In addition, all five extracellular domains were fused to IgA1 (IC1-5D/IgA). Immunoadhesins were compared with soluble forms of ICAM-1 containing five and two domains (sICAM-1 and ICI-2D, respectively) in assays of HRV binding, infectivity, and conformation. In prevention of HRV plaque formation, IC1-5D/IgA was 200 times and IC1-2D/IgM and IC1-2D/IgA were 25 and 10 times more effective, respectively, than ICAM-1. The same chimeras were highly effective in inhibiting binding of rhinovirus to cells and disrupting the conformation of the virus capsid, as demonstrated by generation of approximately 65S particles. The results show that the number of ICAM-1 domains and a flexible Ig hinge are important factors contributing to the efficacy of neutralization. The higher efficiency of chimeras that bound bivalently in disrupting HRV was attributed to higher binding avidity. The IC1-5D/IgA immunoadhesin was effective at nanomolar concentrations, making it feasible therapy for rhinovirus infection.     

Molecular characterization of the Salmonella typhimurium parE gene.

The DNA sequence of the wild type S. typhimurium parE gene was determined. The predicted protein has 96.7% amino acid identity with the ParE protein of E.coli, but is 29 amino acids longer, due to an additional basepair in the 3' end of the S. typhimurium gene. Subclones of the S. typhimurium parE gene localized the sites of four heat sensitive mutations within parE. The parE206 and parE374 mutations are identical (Val67-Met) and lie in a highly conserved region corresponding to the ATP binding pocket of GyrB. Two additional heat sensitive mutations were sequenced and predict the following amino acid substitutions: parE377 (Gly399-Ser) and parE493 (Thr583-Pro). All of the heat sensitive mutations lie in regions with strong amino acid homology to GyrB.     

Antitumor effects of an antibody-carboxypeptidase g2 conjugate in combination with a benzoic acid mustard prodrug

The F(ab’)₂ fragment of the antitumor monoclonal antibody, A5B7, was covalently linked to the bacterial enzyme carboxypeptidase G2 (CPG2). The resulting conjugate was used in combination with a prodrug of a benzoic acid mustard alkylating agent to treat human colon tumor xenografts in a two-step targeting strategy, antibody-directed enzyme produrug therapy (ADEPT). The prodrug, 4-[(2-chloroethyl) (2-mesyloxyethyl) amino]-benzoyl-l-glutamic acid is rapidly converted by CPG2 to a drug that is at least 15x more toxic in vitro against LS174T colorectal tumor cells than the prodrug. Optimal tumor/ blood ratios of the A5B7-CPG2 were achieved 72 h after administration of the conjugate to athymic mice bearing established LS174T tumor xenografts. Significant antitumor activity was seen in LS174T tumor-bearing mice treated with the conjugate followed 3 d later by the prodrug. In contrast, prodrug, conjugate, or active drug alone did not result in any antitumor activity in this tumor model. These studies demonstrate the advantage of a two-step ADEPT system for the treatment of colorectal cancer.     

Physiological effects of translation initiation factor IF3 and ribosomal protein L20 limitation inEscherichia coli

To investigate the physiological roles of translation initiation factor IF3 and ribosomal protein L20 inEscherichia coli, theinfC, rpmI andrpIT genes encoding IF3, L35 and L20, respectively, were placed under the control oflac promoter/operator sequences. Thus, their expression is dependent upon the amount of inducer isopropyl thiogalactoside (IPTG) in the medium. Lysogenic strains were constructed with recombinant lambda phages that express eitherrpmI andrplT orinfC andrpmI in trans, thereby allowing depletion of only IF3 or L20 at low IPTG concentrations. At low IPTG concentrations in the IF3-limited strain, the cellular concentration of IF3, but not L20, decreases and the growth rate slows. Furthermore, ribosomes run off polysomes, indicating that IF3 functions during the initiation phase of protein synthesis in vivo. During slow growth, the ratio of RNA to protein increases rather than decreases as occurs with control strains, indicating that IF3 limitation disrupts feedback inhibition of rRNA synthesis. As IF3 levels drop, expression from an AUU-infC-lacZ fusion increases, whereas expression decreases from an AUG-infC-lacZ fusion, thereby confirming the model of autogenous regulation ofinfC. The effects of L20 limitation are similar; cells grown in low concentrations of IPTG exhibited a decrease in the rate of growth, a decrease in cellular L20 concentration, no change in IF3 concentration, and a small increase in the ratio of RNA to protein. In addition, a decrease in 50S subunits and the appearance of an aberrant ribosome peak at approximately 41–43S is seen. Previous studies have shown that the L20 protein negatively controls its own gene expression. Reduction of the cellular concentration of L20 derepresses the expression of anrplT-lacZ gene fusion, thus confirming autogenous regulation by L20.     

Molecular clocks and the incompleteness of the fossil record

Molecular clocks can be evaluated by comparing absolute rates of evolution and by performing relative-rate tests. Typically, calculations of absolute rates are based on earliest observed occurrences in the fossil record. Relative-rate tests, on the other hand, merely require an unambiguous outgroup. A major disadvantage of relative-rate tests is their insensitivity to concomitant and equal rate changes in all lineages. Apparent differences in absolute rates, in turn, may be artifacts that are attributable to an incomplete fossil record.Recently developed methods in quantitative biostratigraphy recognize the incompleteness of the fossil record and allow us to place confidence intervals on the endpoints of taxon ranges. These methods are applicable to taxa whose fossil records are of markedly different quality. When we extend these methods and integrate molecular and paleontologic data, we can test the null hypothesis that seemingly disparate rates of molecular evolution are in fact equal under the simplifying assumption that fossils are randomly and independently distributed over their temporal ranges and that fossils can be accurately placed in a phylogenetic context. We can also estimate the range of ticking rates, if any, that are compatible with known fossil data. Ultimately, more accurate rate estimates for widely divergent taxa should allow for more meaningful comparisons of evolutionary rates.DNA hybridization data for monotremes and marsupials suggest a 17-fold difference for 14 different rate calculations with a mean value of approximately 1% divergence per million years. Variation among marsupials is sevenfold. However, when we apply appropriate statistical tests and make additional allowances for fossils of uncertain taxonomic assignment, etc., all 14 rates are compatible with a molecular clock ticking at approximately 0.4% divergence per million years. In addition, this analysis brings relative- and absolute-rate tests into accord.