Effect of hepatocyte growth factor/scatter factor and other growth factors on motility and morphology of non-tumorigenic and tumor cells

Summary Using an automated cell analyzer system, the effect of hepatocyte growth factor/scatter factor (HGF/SF), epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), endothelial acidic fibroblast growth factor (a-FGF), platelet derived growth factor (PDGF), and recombinant human insulinlike growth factor (IGF) on the motility and morphology of Madin-Darby canine kidney (MDCK), rat hepatomas, C₂, and H_5–6 and murine mammary carcinoma (EMT-6) cells was investigated. Treatment of MDCK cells with HGF/SF, bFGF, EGF, and a-FGF resulted in an increase in average cell velocity and in the fraction of moving cells. Cells treated with the PDGF and IGF did not show significant alterations in velocity. MDCK cells treated with each growth factor were classified into groups of “fast” and “slow” moving cells based on their average velocities, and the average morphologic features of the two groups were quantitated. Fast-moving cells had larger average area, circularity, and flatness as compared to slow-moving cells. Factors that stimulated cell movement also induced alterations in cell morphologic parameters including spreading, flatness, area, and circularity. HGF/SF also scattered and stimulated motility of C₂ and H_5–6 hepatoma cells. In contrast to MDCK cells, there was no significant difference between the morphology of the fast moving and slow moving C₂ and H_5–6 cells. These studies suggest that growth factor cytokines have specific effects on motility of normal and tumor cells.     

Synthesis of lipopolysaccharide O side chains by Pseudomonas aeruginosa PAO1 requires the enzyme phosphomannomutase.

We have cloned a lipopolysaccharide (LPS) biosynthetic gene from Pseudomonas aeruginosa PAO1 that complements the defect in the production and incorporation of LPS O side chains in the LPS-rough strain AK1012. This gene was characterized by pulsed-field gel electrophoresis, deletion and restriction mapping of the cloned DNA, and biochemical analysis of the protein product. The cloned DNA was found to map to the 7-to-11-min region of the P. aeruginosa chromosome, and the gene needed for complementation of the LPS-rough phenotype was contained on a 2.6-kb HindIII-SacI fragment. This same size restriction fragment contains the alginate gene algC, which encodes the enzyme phosphomannomutase (PMM) and also maps to this region of the P. aeruginosa chromosome. The LPS-rough strain AK1012 was deficient in PMM activity, and this activity was restored to parental levels when the cloned gene was transferred to strain AK1012. In addition, the cloned gene could complement the PMM deficiency in the algC mutant strain 8858, and the cloned algC gene could restore the LPS-smooth phenotype to strain AK1012. These results indicate that the gene we have cloned is equivalent to the alginate gene algC. We designate this gene pmm to emphasize that it encodes the enzyme PMM, which has been shown to be essential for alginate production, and we demonstrate that PMM activity is required for the LPS-smooth phenotype in P. aeruginosa PAO1.     

A Radioimmunoassay-Based Method for Measuring the True Affinity of a Monoclonal Antibody with Trace Amounts of Radioactive Antigen: Illustration with the Products of a Cell-Free Protein Synthesis System

This communication describes a novel highly sensitive method for measuring the affinity of a monoclonal antibody for its antigen. It is based on a radioimmunoassay in which the antigen is labeled with radioactivity. It is therefore particularly well adapted to the study of trace amounts of radiolabeled polypeptide chains produced either in vivo, or in vitro by a cell free protein synthesis system or by chemical radiolabeling. It offers several advantages over previously described methods. Though making use of insolubilized antibody, it does measure the true affinity constant of the monoclonal antibody in solution for the antigen. It can be used even when the antigen is present at concentrations far below the dissociation constant of the antibody/antigen complex. It does not require the antigen or the antibody to be purified. In most cases, it requires no sophisticated equipment. This method could be easily adapted to the determination of the equilibrium constant of any type of protein/ligand system.     

Framework for validation and implementation of in vitro toxicity tests

Summary The development and application of in vitro alternatives designed to reduce or replace the use of animals, or to lessen the distress and discomfort of laboratory animals, is a rapidly developing trend in toxicology. However, at present there is no formal administrative process to organize, coordinate, or evaluate validation activities. A framework capable of fostering the validation of new methods is essential for the effective transfer of new technologic developments from the research laboratory into practical use. This committee has identified four essential validation resources: chemical bank(s), cell and tissue banks, a data bank, and reference laboratories. The creation of a Scientific Advisory Board composed of experts in the various aspects and endpoints of toxicity testing, and representing the academic, industrial, and regulatory communities, is recommended. Test validation acceptance is contingent on broad buy-in by disparate groups in the scientific community—academics, industry, and government. This is best achieved by early and frequent communication among parties and agreement on common goals. It is hoped that the creation of a validation infrastructure composed of the elements described in this report will facilitate scientific acceptance and utilization of alternative methodologies and speed implementation of replacement, reduction, and refinement alternatives in toxicity testing.     

Treatment-resistant depression: definition,prevalence, detection,management, and investigational interventions

Treatment-resistant depression (TRD) is common and associated with multiple serious public health implications. A consensus definition of TRD with demonstrated predictive utility in terms of clinical decision-making and health outcomes does not currently exist. Instead, a plethora of definitions have been proposed, which vary significantly in their conceptual framework. The absence of a consensus definition hampers precise estimates of the prevalence of TRD, and also belies efforts to identify risk factors, prevention opportunities, and effective interventions. In addition, it results in heterogeneity in clinical practice decision-making, adversely affecting quality of care. The US Food and Drug Administration (FDA) and the European Medicines Agency (EMA) have adopted the most used definition of TRD (i.e., inadequate response to a minimum of two antidepressants despite adequacy of the treatment trial and adherence to treatment). It is currently estimated that at least 30% of persons with depression meet this definition. A significant percentage of persons with TRD are actually pseudo-resistant (e.g., due to inadequacy of treatment trials or non-adherence to treatment). Although multiple sociodemographic, clinical, treatment and contextual factors are known to negatively moderate response in persons with depression, very few factors are regarded as predictive of non-response across multiple modalities of treatment. Intravenous ketamine and intranasal esketamine (co-administered with an antidepressant) are established as efficacious in the management of TRD. Some second-generation antipsychotics (e.g., aripiprazole, brexpiprazole, cariprazine, quetiapine XR) are proven effective as adjunctive treatments to antidepressants in partial responders, but only the olanzapine-fluoxetine combination has been studied in FDA-defined TRD. Repetitive transcranial magnetic stimulation (TMS) is established as effective and FDA-approved for individuals with TRD, with accelerated theta-burst TMS also recently showing efficacy. Electroconvulsive therapy is regarded as an effective acute and maintenance intervention in TRD, with preliminary evidence suggesting non-inferiority to acute intravenous ketamine. Evidence for extending antidepressant trial, medication switching and combining antidepressants is mixed. Manual-based psychotherapies are not established as efficacious on their own in TRD, but offer significant symptomatic relief when added to conventional antidepressants. Digital therapeutics are under study and represent a potential future clinical vista in this population.     

Intracellular Ionic Variations in the Apoptotic Death of L Cells by Inhibitors of Cell Cycle Progression

Treatment with VP-16 (1-50 μM) or excess thymidine (5 mM) caused a block of L cells at different steps in their progression through the replicative cycle. The arrest was followed by an asynchronous process of cell death that conformed to criteria for apoptosis. Careful monitoring of this process in the whole cell population by flow cytometry showed a virtual absence of necrosis, an increase in side light scattering, followed by the occurrence of a population with subdiploid DNA fluorescence as well as reduced forward and side light seattering. The development of apoptosis required sufficient time and adequate ion gradients in the cells. By the combined use of flow cytometry and fluorescence microscopy data were obtained suggesting that (i) intracellular free Ca²⁺ and pH and/or their drug-induced alterations had to be adequately controlled for the apoptotic process to evolve; (ii) mitochondria were compromised earlier than the plasma membrane or lysosomes; and (iii) K⁺ extrusion possibly played a role in the final loss of cell volume. Interfering with the control of ion gradients and/or their changes in drug-treated cells resulted in cell death by necrosis.     

Genetical and physical assignments of equine microsatellites—first integration of anchored markers in horse genome mapping

Twenty equine microsatellites were isolated from a genomic phage library, and their genetical and physical localization was sought by linkage mapping and fluorescent in situ hybridization (FISH). Nineteen of the markers were found to be polymorphic with, in most cases, heterozygosities exceeding 50%. The markers were mapped in a Swedish reference family for gene mapping, comprising eight half-sib families from Standardbred and Icelandic horse sires. Segregation was analyzed against a set of 35 other markers typed in the pedigree. Thirteen of the microsatellites showed linkage to at least one other marker, with a total of 21 markers being involved in these linkages. In parallel, 18 of the microsatellites could be assigned to their chromosomal region by FISH. These assignments involved eight equine autosomes: ECA1, 2, 4, 6, 9, 10, 15, and 16. The genetical and physical mappings revealed by this study represent a significant extension of the current knowledge of the equine genome map. Received: 24 September 1996 / Accepted: 1 December 1996     

Interaction of workplace demands and cardiovascular reactivity in progression of carotid atherosclerosis: population based study.

OBJECTIVE: To examine the combined influence of workplace demands and changes in blood pressure induced by stress on the progression of carotid atherosclerosis. DESIGN: Population based follow up study of unestablished as well as traditional risk factors for carotid atherosclerosis, ischaemic heart disease, and other outcomes. SETTING: Eastern Finland. SUBJECTS: 591 men aged 42-60 who were fully employed at baseline and had complete data on the measures of carotid atherosclerosis, job demands, blood pressure reactivity, and covariates. MAIN OUTCOME MEASURES: Change in ultrasonographically assessed intima-media thickness of the right and left common carotid arteries from baseline to 4 year follow up. RESULTS: Significant interactions between workplace demands and stress induced reactivity were observed for all measures of progression (P < 0.04). Men with large changes in systolic blood pressure (20 mm Hg or greater) in anticipation of a maximal exercise test and with high job demands had 10-40% greater progression of mean (0.138 v 0.123 mm) and maximum (0.320 v 0.261 mm) intima-media thickness and plaque height (0.347 v 0.264) than men who were less reactive and had fewer job demands. Similar results were obtained after excluding men with prevalent ischaemic heart disease at baseline. Findings were strongest among men with at least 20% stenosis or non-stenotic plaque at baseline. In this subgroup reactive men with high job demands had more than 46% greater atherosclerotic progression than the others. Adjustment for atherosclerotic risk factors did not alter the results. CONCLUSIONS: Men who showed stress induced blood pressure reactivity and who reported high job demands experienced the greatest atherosclerotic progression, showing the association between dispositional risk characteristics and contextual determinants of disease and suggesting that behaviourally evoked cardiovascular reactivity may have a role in atherogenesis.