Release of the cytokines colony-stimulating factor-1, granulocyte-macrophage colony-stimulating factor, and IL-6 by cloned murine vascular smooth muscle cells

Vascular smooth muscle cells were cultured from the mesenteric arteries of MRL lpr/lpr, MRL +/+, CBA/J, or C3H/HeJ mice and evaluated for their ability to synthesize a range of cytokines. Vascular smooth muscle cells of MRL +/+, MRL lpr/lpr, and CBA/J origin released biologically significant amounts of CSF-1 and IL-6 and relatively low but detectable amounts of granulocyte macrophage-CSF (GM-CSF) but not IL-2, IL-3, or IL-4. Vascular smooth muscle cells of C3H/HeJ origin produced lower amounts of CSF-1 and IL-6, and GM-CSF was barely detectable. Production of these cytokines did not require the exogenous growth factors present in FCS and occurred, although at lower levels, in serum-free medium supplemented with insulin, transferrin, and albumin. Cloned lines of MRL +/+ vascular smooth muscle cells, with electron microscopic and immunochemical properties of vascular smooth muscle cells, produced CSF-1, IL-6, and GM-CSF, establishing that vascular smooth muscle cells were a direct source of CSF-1, IL-6, and GM-CSF. These observations highlight the need for experiments to directly address the question of whether vascular smooth muscle cells constitutively produce these cytokines under physiologic conditions in vivo and suggest that vascular smooth muscle cells may participate actively in inflammation by releasing cytokines that are active on lympho-hemopoietic and other cells.     

Assessment of structural similarities in chick oviduct progesterone receptor subunits by partial proteolysis of photoaffinity-labeled proteins

Partial proteolytic fragmentation of the two chick oviduct progesterone receptor subunits was used to identify structural features shared by the two proteins. Both subunits can be photoaffinity labeled at their hormone-binding sites (Birnbaumer, M., Schrader, W. T., and O'Malley, B. W. (1983) J. Biol. Chem. 258, 1637-1644) using the radioactive steroid [methyl-3H] 17 alpha, 21-dimethyl-19-nor-pregn-4,9-diene-3,20-dione. Native subunits A (Mr = 79,000) and B (Mr = 108,000) were partially purified, photoaffinity-labeled, and then subjected to various mild proteolytic digestions. Labeled fragments were analyzed by fluorography after electrophoresis of the digests under denaturing conditions. Digestion patterns were characteristic for each protease tested. However, fragments from both A and B were indistinguishable for all peptides of less than Mr = 60,000. Time course studies demonstrated the sequential production of progressively smaller discrete fragments in a manner consistent with a precursor-product relationship among them and established the existence of similar structural domains resistant to proteolysis in both proteins. Autoradiographic peptide maps were obtained by 125I-labeling of pure A and B protein isolated by two-dimensional gel electrophoresis followed by exhaustive tryptic digestion and two-dimensional separation. These studies revealed that a significant proportion of the smaller A protein differs in its primary sequence from that of the B protein which excludes the possibility of their sharing a precursor-product relationship. We conclude that B and A subunits are separate proteins with common structural features in the native state, but with considerable amino acid sequence differences. The simplest hypothesis consistent with these findings is that B and A are the products of two separate genes which have diverged to give rise to two different but related proteins that fold in such a manner as to be almost indistinguishable by proteolytic attack of their native conformation.     

Identification of a Caldariomyces fumago Mutant Secreting an Inactive Form of Chloroperoxidase Lacking the Heme Group and N-Glycans

By mutant colony screening of Caldariomyces fumago a mutant was isolated which was slightly greenish on fructose minimal medium and grew slower in comparison to the wild type. The supernatant samples lacked the Soret band typical for the heme group of the CPO and nearly no CPO activity was detected. SDS-PAGE analysis of mutant culture supernatant samples showed production of a 38–40 kDa protein while wild type samples contain the 42 kDa CPO protein. Protein identification using nanoLC-ESI-MS/MS was performed and based on three peptides the protein in the mutant culture was identified as CPO. No differences in the CPO gene sequences of wild type and mutant were found indicating a post-translational defect in protein maturation. Deglycosylation experiments using CPO from wild type and mutant were carried out. After removing N-linked oligosaccharides from wild type CPO a protein band at 38–40 kDa was detected. Our results reveal that the mutant protein lacks the heme group as well as the N-glycans.     

Assessment of SLX4 Mutations in Hereditary Breast Cancers

Background

SLX4 encodes a DNA repair protein that regulates three structure-specific endonucleases and is necessary for resistance to DNA crosslinking agents, topoisomerase I and poly (ADP-ribose) polymerase (PARP) inhibitors. Recent studies have reported mutations in SLX4 in a new subtype of Fanconi anemia (FA), FA-P. Monoallelic defects in several FA genes are known to confer susceptibility to breast and ovarian cancers.

Methods and Results

To determine if SLX4 is involved in breast cancer susceptibility, we sequenced the entire SLX4 coding region in 738 (270 Jewish and 468 non-Jewish) breast cancer patients with 2 or more family members affected by breast cancer and no known BRCA1 or BRCA2 mutations. We found a novel nonsense (c.2469G>A, p.W823*) mutation in one patient. In addition, we also found 51 missense variants [13 novel, 23 rare (MAF<0.1%), and 15 common (MAF>1%)], of which 22 (5 novel and 17 rare) were predicted to be damaging by Polyphen2 (score = 0.65–1). We performed functional complementation studies using p.W823* and 5 SLX4 variants (4 novel and 1 rare) cDNAs in a human SLX4-null fibroblast cell line, RA3331. While wild type SLX4 and all the other variants fully rescued the sensitivity to mitomycin C (MMC), campthothecin (CPT), and PARP inhibitor (Olaparib) the p.W823* SLX4 mutant failed to do so.

Conclusion

Loss-of-function mutations in SLX4 may contribute to the development of breast cancer in very rare cases.     

Semi-automated 3D Leaf Reconstruction and Analysis of Trichome Patterning from Light Microscopic Images

Trichomes are leaf hairs that are formed by single cells on the leaf surface. They are known to be involved in pathogen resistance. Their patterning is considered to emerge from a field of initially equivalent cells through the action of a gene regulatory network involving trichome fate promoting and inhibiting factors. For a quantitative analysis of single and double mutants or the phenotypic variation of patterns in different ecotypes, it is imperative to statistically evaluate the pattern reliably on a large number of leaves. Here we present a method that enables the analysis of trichome patterns at early developmental leaf stages and the automatic analysis of various spatial parameters. We focus on the most challenging young leaf stages that require the analysis in three dimensions, as the leaves are typically not flat. Our software TrichEratops reconstructs 3D surface models from 2D stacks of conventional light-microscope pictures. It allows the GUI-based annotation of different stages of trichome development, which can be analyzed with respect to their spatial distribution to capture trichome patterning events. We show that 3D modeling removes biases of simpler 2D models and that novel trichome patterning features increase the sensitivity for inter-accession comparisons.     

Using neutron crystallography to elucidate the basis of selective inhibition of carbonic anhydrase by saccharin and a derivative

Up-regulation of carbonic anhydrase IX (CA IX) expression is an indicator of metastasis and associated with poor cancer patient prognosis. CA IX has emerged as a cancer drug target but development of isoform-specific inhibitors is challenging due to other highly conserved CA isoforms. In this study, a CA IX_mimic construct was used (CA II with seven point mutations introduced, to mimic CA IX active site) while maintaining CA II solubility that make it amenable to crystallography. The structures of CA IX_mimic unbound and in complex with saccharin (SAC) and a saccharin-glucose conjugate (SGC) were determined using joint X-ray and neutron protein crystallography. Previously, SAC and SGC have been shown to display CA isoform inhibitor selectivity in assays and X-ray crystal structures failed to reveal the basis of this selectivity. Joint X-ray and neutron crystallographic studies have shown active site residues, solvent, and H-bonding re-organization upon SAC and SGC binding. These observations highlighted the importance of residues 67 (Asn in CA II, Gln in CA IX) and 130 (Asp in CA II, Arg in CA IX) in selective CA inhibitor targeting.     

Differential sensitivity of chicken progesterone receptor forms to sulfhydryl reactive reagents

DNA binding of chick progesterone receptor B form (PRB) has been examined and compared to that of the A form (PRA). We found that the elution profiles of the two receptors overlap on DNA-cellulose columns. Both PRA or PRB could bind to plasmid DNA equivalently as assayed by sedimentation velocity studies. However, DNA-binding activity of the two receptor forms showed differential sensitivity to reducing agents and to sulfhydryl (SH) reactive reagents. Reducing agents stabilized DNA-binding activity of PRA more efficiently than they stabilized PRB. Moreover, removal of reducing agents from receptor preparations caused preferential loss of DNA binding by PRB compared to the PRA. DNA-binding activity of PRA was readily destroyed by sulfhydryl modifying reagents such as N-ethylmaleimide and iodoacetamide while PRB was 3-4 times less sensitive to these reagents. We conclude the DNA-binding activity of PRB is less stable due to altered accessibility of SH groups despite the amino acid sequence identity of the DNA-binding domains of PRA and PRB.