Bacteriophage resistance and flocculation deficiency of Flavobacterium sp. are phenotypically interrelated

Summary An analysis of Flavobacterium sp. strain 304 variants selected for flocculation deficiency and phage resistance has been carried out. The results show that flocculation is efficiently hindered by overexpressed protein in the outer membrane or carbohydrate on the cell surface. The floc-forming protein is suggested to be a minor component of the cell surface. The 12 flocculation-deficient and five phage-insensitive variants obtained were grouped into ten classes based on nine characteristics. In addition, bacteriophage 304 resistance and the pure culture flocculation deficiency of this strain are interrelated. Offsprint requests to: D. H. Bamford     

Blood Platelets Contain and Secrete Laminin-8 (α4β1γ1) and Adhere to Laminin-8 via α6β1 Integrin

Laminins, a family of heterotrimeric proteins with cell adhesive/signaling properties, are characteristic components of basement membranes of vasculature and tissues. In the present study, permeabilized platelets were found to react with a monoclonal antibody to laminin γ1 chain by immunofluorescence. In Western blot analysis of platelet lysates, several monoclonal antibodies to γ1 and β1 laminin chains recognized 220- to 230-kDa polypeptides, under reducing conditions, and a structure with much slower electrophoretic mobility under nonreducing conditions. Immunoaffinity purification on a laminin β1 antibody–Sepharose column yielded polypeptides of 230, 220, 200, and 180 kDa from platelet lysates. In the purified material, mAbs to β1 and γ1 reacted with the two larger polypeptides, while affinity-purified rabbit antibodies to laminin α4 chain recognized the smallest polypeptide. Identity of the polypeptides was confirmed by microsequencing. One million platelets contained on average 1 ng of laminin (approximately 700 molecules per cell), of which 20–35% was secreted within minutes after stimulation with either thrombin or phorbol ester. Platelets adhered to plastic surfaces coated with the purified platelet laminin, and this process was largely inhibited by antibodies to β1 and α6 integrin chains. We conclude that platelets contain and, following activation, secrete laminin-8 (α4β1γ1) and that the cells adhere to the protein by using α6β1 integrin.     

Identification and quantification of proteins differentially secreted by a pair of normal and malignant breast‐cancer cell lines

Secreted proteins play a pivotal role in cellular functions. To better understand malignant behavior, we adapted stable isotopic labeling with amino acids in cell culture technology to identify and quantify proteins differentially released into the extracellular media by a pair of normal and malignant breast‐cancer cell lines. Approximately 380 non‐redundant proteins were quantified in serum‐free media. Of the assigned proteins, 62% are classified secreted in protein databases and an additional 25% are designated secreted in the literature. A number of growth factors were found differentially regulated. Tumor necrosis factor, pigment epithelial‐differentiating factor and stem‐cell growth factor precursor showed decreased expression in breast‐cancer cell line, whereas Inhibin beta and macrophage migration inhibitory factor show increased expression. Interestingly, protease inhibitors, including plasma protease (C1) inhibitor, PZP precursor, and SerpinE2 were significantly down‐regulated in cancer cell line as were angiostatic factors from extracellular matrix (ECM) such as endorepillin. Further, the C‐terminal fragment of type XVIII collagen, endostatin, a potent angiostatic factor, was down‐regulated as well whereas extracellular collagens and osteoblast‐specific factor 2 (OSF‐2), were up‐regulated. Differential expression and secretion of SerpinE2 and OSF‐2 were confirmed using Western blotting. These results corroborate models of invasive tumors sustained by elaborate coordination of stromal cells via chemokines and growth factors, while protease inhibitors remodel the ECM to stimulate angiogenesis.     

Biochemical characterization of CA IX, one of the most active carbonic anhydrase isozymes

Carbonic anhydrase IX (CA IX) is an exceptional member of the CA protein family; in addition to its classical role in pH regulation, it has also been proposed to participate in cell proliferation, cell adhesion, and tumorigenic processes. To characterize the biochemical properties of this membrane protein, two soluble recombinant forms were produced using the baculovirus-insect cell expression system. The recombinant proteins consisted of either the CA IX catalytic domain only (CA form) or the extracellular domain, which included both the proteoglycan and catalytic domains (PG + CA form). The produced proteins lacked the small transmembrane and intracytoplasmic regions of CA IX. Stopped-flow spectrophotometry experiments on both proteins demonstrated that in the excess of certain metal ions the PG + CA form exhibited the highest catalytic activity ever measured for any CA isozyme. Investigations on the oligomerization and stability of the enzymes revealed that both recombinant proteins form dimers that are stabilized by intermolecular disulfide bond(s). Mass spectrometry experiments showed that CA IX contains an intramolecular disulfide bridge (Cys(119)-Cys(299)) and a unique N-linked glycosylation site (Asn(309)) that bears high mannose-type glycan structures. Parallel experiments on a recombinant protein obtained by a mammalian cell expression system demonstrated the occurrence of an additional O-linked glycosylation site (Thr(78)) and characterized the nature of the oligosaccharide structures. This study provides novel information on the biochemical properties of CA IX and may help characterize the various cellular and pathophysiological processes in which this unique enzyme is involved.     

Biomass allocation, needle structural characteristics and nutrient composition in Scots pine seedlings exposed to elevated CO2 and O3 concentrations

Three-year-old Scots pine (Pinus sylvestris L.) seedlings were exposed to ambient or elevated ozone (O₃) (1.52ambient) and carbon dioxide (CO₂) (590 µmol mol^-1) concentrations during two growing seasons in open-top field chambers (OTCs). Five different treatments were applied in the chambers: filtered air, ambient air, elevated O₃, elevated CO₂, and elevated O₃ and CO₂ combined. Ambient plots outside the OTCs were also included, but the chamber ambient was used as a control in O₃ and CO₂ treatments due to a significant chamber effect. Increases in yellowing and chlorotic mottling of previous-year (C+1) needles and in the amount of cytoplasmic ribosomes and electron density of the chloroplast stroma in current-year (C) and C+1 needle mesophyll cells were observed in elevated O₃ at both CO₂ concentrations. Elevated O₃ alone caused a non-significant 10.9% decrease in plant total dry mass and a significant decrease in manganese (Mn) content of C needles. CO₂ enrichment caused a significant increase in needle cross-sectional width after the first year of exposure, and an accumulation of starch and slight curling and swelling of the chloroplast thylakoids in the mesophyll tissue of C needles after the second year of exposure. Calcium and Mn contents were increased and copper and nitrogen contents were decreased, significantly, in CO₂-exposed needles. A non-significant 19.1% increase in plant total dry mass was measured in elevated CO₂ alone, whereas a 14.8% reduction in total dry mass, together with a significant reduction in current-year main shoot length, was found in the combined treatment. Overall, in spite of decreases in O₃-induced visible injuries by CO₂, elevated CO₂ levels were not able to counteract the impact of O₃ in this experiment.     

Effects of strength training on muscle strength characteristics, functional capabilities, and balance in middle-aged and older women

Progressive strength training can lead to substantial increases in maximal strength and mass of trained muscles, even in older women and men, but little information is available about the effects of strength training on functional capabilities and balance. Thus, the effects of 21 weeks of heavy resistance training--including lower loads performed with high movement velocities--twice a week on isometric maximal force (ISOmax) and force-time curve (force produced in 500 milliseconds, F0-500) and dynamic 1 repetition maximum (1RM) strength of the leg extensors, 10-m walking time (10WALK) and dynamic balance test (DYN.D) were investigated in 26 middle-aged (MI; 52.8 +/- 2.4 years) and 22 older women (O; 63.8 +/- 3.8 years). 1RM, ISOmax, and F0-500 increased significantly in MI by 28 +/- 10%, 20 +/- 19%, 31 +/- 34%, and in O by 27 +/- 8%, 20 +/- 16%, 18 +/- 45%, respectively. 10WALK (MI and O, p < 0.001) shortened and DYN.D improved (MI and O, p < 0.001). The present strength-training protocol led to large increases in maximal and explosive strength characteristics of leg extensors and in walking speed, as well to an improvement in the present dynamic balance test performance in both age groups. Although training-induced increase in explosive strength is an important factor for aging women, there are other factors that contribute to improvements in dynamic balance capacity. This study indicates that total body heavy resistance training, including explosive dynamic training, may be applied in rehabilitation or preventive exercise protocols in aging women to improve dynamic balance capabilities.     

Homogeneous time-resolved fluorescence quenching assay (LANCE) for caspase-3

In addition to kinases and G protein-coupled receptors, proteases are one of the main targets in modern drug discovery. Caspases and viral proteases, for instance, are potential targets for new drugs. To satisfy the current need for fast and sensitive high-throughput screening for inhibitors, new homogeneous protease assays are needed. We used a caspase-3 assay as a model to develop a homogeneous time-resolved fluorescence quenching assay technology. The assay utilizes a peptide labeled with both a luminescent europium chelate and a quencher. Cleavage of the peptide by caspase-3 separates the quencher from the chelate and thus recovers europium fluorescence. The sensitivity of the assay was 1 pg/microl for active caspase-3 and 200 pM for the substrate. We evaluated the assay for high-throughput usage by screening 9600 small-molecule compounds. We also evaluated this format for absorption/distribution/metabolism/excretion assays with cell lysates. Additionally, the assay was compared to a commercial fluorescence caspase-3 assay.