Purification and characterization of acyl carrier protein from two cyanobacteria species

The acyl carrier protein (ACP), an essential protein cofactor for fatty acid synthesis, has been isolated from two cyanobacteria: the filamentous, heterocystous, Anabaena variabilis (ATCC 29211) and the unicellular Synechocystis 6803 (ATCC 27184). Both ACPs have been purified to homogeneity utilizing a three-column procedure. Synechocystis 6803 ACP was purified 1800-fold with 67% yield, while A. variabilis ACP was purified 1040-fold with 50% yield. Yields of 13.0 micrograms ACP/g Synechocystis 6803 and 9.0 micrograms ACP/g A. variabilis were achieved. Amino acid analysis indicated that these ACPs were highly charged acidic proteins similar to other known ACPs. Sequence analysis revealed that both cyanobacterial ACPs were highly conserved with both spinach and Escherichia coli ACP at the phosphopantetheine prosthetic group region. Examining the probability of alpha-helix and beta-turn regions in various ACPs, showed that cyanobacterial ACPs were more closely related to E. coli ACP than spinach ACP I. Immunoblot analysis and a competitive binding assay for ACP illustrated that both ACPs bound poorly to spinach ACP I antibody. SDS/PAGE and native PAGE of Synechocystis 6803 ACP and A. variabilis ACP showed that cyanobacteria ACPs co-migrated with E. coli ACP and had relative molecular masses of 18,100 and 17,900 respectively. Both native and urea gel analysis of acyl-ACP products from fatty acid synthase reactions demonstrated that bacterial ACPs and plant ACP gave essentially the same metabolic products when assayed using either bacterial or plant fatty acid synthase. A. variabilis and Synechocystis 6803 ACP could be acylated using E. coli acyl ACP synthetase.     

Matrix Metalloproteinase 8 (Collagenase 2) Induces the Expression of Interleukins 6 and 8 in Breast Cancer Cells

Matrix metalloproteinase 8 (MMP-8) is a tumor-suppressive protease that cleaves numerous substrates, including matrix proteins and chemokines. In particular, MMP-8 proteolytically activates IL-8 and, thereby, regulates neutrophil chemotaxis in vivo. We explored the effects of expression of either a WT or catalytically inactive (E198A) mutant version of MMP-8 in human breast cancer cell lines. Analysis of serum-free conditioned media from three breast cancer cell lines (MCF-7, SK-BR-3, and MDA-MB-231) expressing WT MMP-8 revealed elevated levels of IL-6 and IL-8. This increase was mirrored at the mRNA level and was dependent on MMP-8 catalytic activity. However, sustained expression of WT MMP-8 by breast cancer cells was non-permissive for long-term growth, as shown by reduced colony formation compared with cells expressing either control vector or E198A mutant MMP-8. In long-term culture of transfected MDA-MB-231 cells, expression of WT but not E198A mutant MMP-8 was lost, with IL-6 and IL-8 levels returning to base line. Rare clonal isolates of MDA-MB-231 cells expressing WT MMP-8 were generated, and these showed constitutively high levels of IL-6 and IL-8, although production of the interleukins was no longer dependent upon MMP-8 activity. These studies support a causal connection between MMP-8 activity and the IL-6/IL-8 network, with an acute response to MMP-8 involving induction of the proinflammatory mediators, which may in part serve to compensate for the deleterious effects of MMP-8 on breast cancer cell growth. This axis may be relevant to the recognized ability of MMP-8 to orchestrate the innate immune system in inflammation in vivo.     

Phylodynamic Inference of Bacterial Outbreak Parameters Using Nanopore Sequencing

Nanopore sequencing and phylodynamic modeling have been used to reconstruct the transmission dynamics of viral epidemics, but their application to bacterial pathogens has remained challenging. Cost-effective bacterial genome sequencing and variant calling on nanopore platforms would greatly enhance surveillance and outbreak response in communities without access to sequencing infrastructure. Here, we adapt random forest models for single nucleotide polymorphism (SNP) polishing developed by Sanderson and colleagues (2020. High precision Neisseria gonorrhoeae variant and antimicrobial resistance calling from metagenomic nanopore sequencing. Genome Res. 30(9):1354–1363) to estimate divergence and effective reproduction numbers (R_e) of two methicillin-resistant Staphylococcus aureus (MRSA) outbreaks from remote communities in Far North Queensland and Papua New Guinea (PNG; n = 159). Successive barcoded panels of S. aureus isolates (2 × 12 per MinION) sequenced at low coverage (>5× to 10×) provided sufficient data to accurately infer genotypes with high recall when compared with Illumina references. Random forest models achieved high resolution on ST93 outbreak sequence types (>90% accuracy and precision) and enabled phylodynamic inference of epidemiological parameters using birth–death skyline models. Our method reproduced phylogenetic topology, origin of the outbreaks, and indications of epidemic growth (R_e > 1). Nextflow pipelines implement SNP polisher training, evaluation, and outbreak alignments, enabling reconstruction of within-lineage transmission dynamics for infection control of bacterial disease outbreaks on portable nanopore platforms. Our study shows that nanopore technology can be used for bacterial outbreak reconstruction at competitive costs, providing opportunities for infection control in hospitals and communities without access to sequencing infrastructure, such as in remote northern Australia and PNG.     

Induced fit activation mechanism of the exceptionally specific serine protease, complement factor D

We have investigated the mechanism by which the complement protease, Factor D, achieves its high specificity for the cleavage of Factor B in complex with C3(H2O). Kinetic experiments showed that Factor B and C3(H2O) associate with a KD of >/=2.5 microM and that Factor D acts on this complex with a second-order rate constant of kcat/KM >/= 2 x 10(6) M-1 s-1, close to the rate of a diffusion-controlled reaction for proteins of this size. In contrast, Factor D, which is a member of the trypsin family of serine proteases, was 10(3)-10(4)-fold less active than trypsin toward both thioester and p-nitroanilide substrates containing an arginine at P1. Furthermore, peptides spanning the Factor B cleavage site were not detectably cleaved by Factor D (kcat/KM /=9 kcal/mol of binding energy to stabilize the transition state for reaction. In support of this, we demonstrate that chemical modification of Factor D at a single lysine residue that is distant from the active site abolishes the activity of the enzyme toward Factor B while not affecting activity toward small synthetic substrates. We propose that Factor D may exemplify a special case of the induced fit mechanism in which the requirement for conformational activation of the enzyme results in a substantial increase in substrate specificity.     

Binding of the basic fibroblast growth factor (bFGF) by soluble components of human umbilical cord

Pre-eclampsia, the most common pregnancy associated syndrome, is connected with remodelling of extracellular matrix of the umbilical cord tissues. Since the fibroblast growth factor (FGF) is known to be a stimulator of collagen and glycosaminoglycan biosynthesis, one may expect that it plays an important role in such a remodelling. Studies performed on the umbilical cords of 10 control and 10 pre-eclamptic newborns demonstrated that both the umbilical cord arterial wall and Wharton's jelly contain FGF mainly in complexes with the components of different molecular mass. Pre-eclampsia is associated with a decrease of endogenous FGF-binding by soluble high molecular mass components of the umbilical cord. It is suggested that FGF released from these complexes may be actively bound by fibroblasts of the umbilical cord, stimulating them to produce collagen and sulphated glycosaminoglycans.     

The effect of pre-eclamptic umbilical cord serum on fibroblast division in culture

Edema, proteinuria, hypertension (EPH-gestosis), most commonly termed as pre-eclampsia, is the most common pregnancy-associated pathological syndrome. It is accompanied by a thorough remodelling of extracellular matrix in the umbilical cord tissues. It is commonly known that the presence of serum in culture medium strongly stimulates many functions of cells cultured in vitro. It was decided to check how the pre-eclamptic serum affects the fibroblast division in culture. Ki-67 is a protein present in proliferating cells and can be detected during all phases of the cell cycle (G1, S, G2/M) but not in resting (G0) cells. PCNA (proliferating cell nuclear antigen) is an intranuclear polypeptide whose synthesis rate is at its maximum during the S-phase of the cell cycle. The expression of Ki-67 and PCNA was measured by immunocytochemical methods and biosynthesis of DNA was evaluated by [14C]-thymidine incorporation. The activity of pre-eclamptic umbilical cord serum (UC-serum) was found to be distinctly lower in comparison to control one. The expression of Ki and PCNA in fibroblast cultures treated with pre-eclamptic serum was also distinctly lower. Also the incorporation of [14C]-thymidine to DNA was lower than in the cultures treated with control UC-serum. It may by concluded that pre-eclampsia reduces the mitogenic activity of the umbilical cord serum.