Large-Scale Conformational Transitions and Dimerization Are Encoded in the Amino-Acid Sequences of Hsp70 Chaperones

Hsp70s are a class of ubiquitous and highly conserved molecular chaperones playing a central role in the regulation of proteostasis in the cell. Hsp70s assist a myriad of cellular processes by binding unfolded or misfolded substrates during a complex biochemical cycle involving large-scale structural rearrangements. Here we show that an analysis of coevolution at the residue level fully captures the characteristic large-scale conformational transitions of this protein family, and predicts an evolutionary conserved–and thus functional–homo-dimeric arrangement. Furthermore, we highlight that the features encoding the Hsp70 dimer are more conserved in bacterial than in eukaryotic sequences, suggesting that the known Hsp70/Hsp110 hetero-dimer is a eukaryotic specialization built on a pre-existing template.     

Enzymatic evidence for involvement of the methylmalonyl-CoA pathway in propionate oxidation by Syntrophobacter wolinii

Enzyme measurements were carried out with crude cell-free extracts of the propionate oxidizing coculture of Syntrophobacter wolinii and Desulfovibrio G11. Using cell-free extracts of a pure culture of Desulfovibrio G11 as a blank, most of the enzymes involved in the methylmalonyl-CoA pathway for propionate oxidation, including a propionyl-CoA: oxaloacetate transcarboxylase, were demonstrated in S. wolinii.     

A phase II trial of murine monoclonal antibody 17-1A and interferon-γ: Clinical and immunological data

Summary A group of 15 patients with metastatic colorectal adenocarcinoma received a combination of interferon (0.1 mg/m², days 1–15) and the murine monoclonal antibody 17-1A (400 mg, days 5, 7, 9 and 12). The treatment was tolerated with minimal toxicity. Of the 14 evaluable patients, 13 developed human antibody to murine 17-1A, with 11 patients demonstrating antibody to the variable region of 17-1A (anti-idiotype). Antibody to the variable region was inhibited by 17-1A but not by mouse immunoglobulin. Sera from patients with substantial anti-idiotype reactivity were capable of inhibiting the binding of murine 17-1A to antigen expressing LS174-T cells thus indicating the presence of antibody directed against the 17-1A combining site (mirror-image anti-idiotype). The median survival of the whole group was 56 weeks and there was no correlation between clinical response/survival and the development of anti-idiotype antibody.Supported by the Veterans Administration Medical Center and by Public Health Services grant CA 45 232 from the National Cancer Institute, National Institutes of Health, Department of Health and Human Services     

Serial studies of autologous antibody reactivity to squamous cell carcinoma of the head and neck

Summary In previous studies we evaluated the incidence and specificity of autologous antibody reactivity against squamous cell carcinoma of the head and neck (SCCHN). We were able to demonstrate that autologous antibody reactivity is present in native sera but was usually of too low a titer to allow further analysis. Dissociation of immune complexes by acidification and ultrafiltration of serum augmented autologous antibody reactivity in nine out of nine autologous systems tested. Native antibody and antibody derived from immune complexes produced by the host and reactive with autologous tumor cells may be directed against physiologically relevant antigens. Therefore, correlations of antibody titers with clinical course may provide insight into the nature of the host response to cancer. In the present analysis, serological studies of six patients with SCCHN were performed with serum samples obtained over many months. Results of serial serological assays were correlated to tumor progression and clinical course. Fluctuations in autologous antibody reactivity were noted over time. In four cases, rises in autologous antibody titers preceded the clinical diagnosis of recurrence by several months. Drops in autologous antibody reactivity were noted in two cases following surgery or radiation therapy. In two cases of long-term survivors, no correlation between antibody reactivity and clinical course was noted. Specificity analysis of the six autologous systems demonstrated reactivity against autologous and allogeneic SCCHN as well as melanoma cell lines. These sera did not react with glioma, neuroblastoma, renal cell, breast, bladder and colon carcinoma cell lines nor with fetal calf serum, pooled lymphocytes, red blood cells and platelets. Autologous serial serological studies may provide a means by which to evaluate the host/tumor relationship in patients with SCCHN.     

On the effect of temperature and pH on the settling behaviour of a flocculent strain ofZymomonas mobilis

Summary In this paper the effect of temperature and pH on the settling behaviour of a flocculent strain ofZymomonas mobilis is studied by using the old fashioned batch settling technique. Plots are given to show the influence of the above mentioned parameters on the settling curve behaviour.     

Allelic variation at the frizzled locus of Drosophila

The epidermis of Drosophila has a tissue polarity that is manifested by a parallel array of polarized structures (primarily hairs and bristles). The production of normal tissue polarity requires the function of the frizzled (fz) locus. We have isolated a large number of alleles at this locus and have phenotypically characterized more than 25 of them. We have found extensive allelic variation that a previous study failed to detect. Most of the alleles fall into a hypomorphic to amorphic series. Two alleles, however, have unusual properties. These alleles, which in general are moderately strong alleles, fail to produce a rough eye phenotype that is characteristic of all the other moderate or strong fz alleles. Thus, these two alleles are tissue specific in effect. Furthermore, these two alleles also have a neomorphic or antimorphic effect on hair polarity in one region of the wing.     

Growth-associated modifications of low-molecular-weight thiols and protein sulfhydryls in human bronchial fibroblasts

The thiol redox status of cultured human bronchial fibroblasts has been characterized at various growth conditions using thiol-reactive monobromobimane, with or without the combination of dithiotreitol, a strong reducing agent. This procedure has enabled measurement of the cellular content of reduced glutathione (GSH), total glutathione equivalents, cysteine, total cysteine equivalents, protein sulfhydryls, protein disulfides, and mixed disulfides. Passage of cells with trypsin perturbs the cellular thiol homeostasis and causes a 50% decrease in the GSH content, whereas the total cysteine content is subsequently increased severalfold during cell attachment. During subsequent culture, transient severalfold increased levels of GSH, protein-bound thiols, and protein disulfides are reached, whereas the total cysteine content gradually declines. These changes in the redox balance of both low-molecular-weight thiols and protein-bound thiols correlate with cell proliferation and mostly precede the major growth phase. When the onset of proliferation is inhibited by maintenance of cells in medium containing decreased amounts of serum, the GSH content remains significantly increased. Subsequent stimulation of growth by addition of serum results in decreased GSH levels at the onset of proliferation. In thiol-depleted medium, proliferation is also inhibited, whereas GSH levels are increased to a lesser extent than in complete medium. Exposure to buthionine sulfoximine inhibits growth, prevents GSH synthesis, and results in accumulation of total cysteine, protein-bound cysteine, and protein disulfides. For extracellular cystine, variable rates of cellular uptake correlate with the initial increase in the total cysteine content observed following subculture and with the GSH peak that precedes active proliferation. The results strongly suggest that specific fluctuations in the cellular redox balance of both free low-molecular-weight thiols and protein sulfhydryls are involved in growth regulation of normal human fibroblasts.