Functional proteometrics for cell migration.

BACKGROUND: Advances in living cellular fluorescence biosensors and computerized microscopy enable a vision of fully automated high-resolution measurements of the detailed intracellular molecular dynamics directly linked to cellular behaviors. Given the heterogeneity of cell populations, a statistically relevant study of molecular-cellular dynamics is a key motivation for improved automation. METHODS: We explored automating computerized, microscope-based data extraction and analyses that monitor cell locomotion, rates of mitoses, and spatiotemporal activities of intracellular proteins via ratiometric fluorescent biosensors in mouse fibroblasts. Novel image processing methods included K-means clustering segmentation preprocessing followed by modified discrete, normalized cross-correlational alignment of two-color images; ratiometric processing for fluorescence resonance energy transfer (FRET) measurements; and intracellular spatial distribution measurements of RhoA GTPase activity. RESULTS: The interdivision time was 19.4 h (mean) +/- 6.0 h (SD) (n = 7) for the GFP-histone cells in the two-by-two field that was scanned for 72 h. After registration and ratioing of the cells with the RhoA biosensor, increases in both cell protrusion and retraction were coincident with to increases in RhoA activity. CONCLUSIONS: These advances lay the foundation for extracting and correlating measurements characterizing the functional relationships of spatial localization and protein activation with features of cell migration such as velocity, polarization, protrusion, retraction, and mitosis.     

Behavioural and Growth Responses of a Territorial Fish (Atlantic Salmon, Salmo salar, L.) to Multiple Predatory Cues

Antipredatory behaviours are important fitness components. The probability of survival decreases if animals fail to respond to signs of danger, but in contrast, energetic costs increase if the response to the threat is exaggerated. We conducted a laboratory experiment designed to examine the behavioural and growth responses of a territorial fish (Atlantic salmon fry, Salmo salar, L.) to different predatory cues (no predatory cues, chemical cues alone, physical cues alone and combined chemical and physical cues). We evaluated the response of Atlantic salmon, focussing on behaviours linked to predator avoidance and to other fitness‐enhancing activities (territory defence and energy acquisition) both during the day and the night. The cost of such responses in terms of growth was assessed and we compared the relative contributions of behaviours in explaining individual growth rate, according to each predation treatment. We demonstrated that the magnitude and nature of behavioural modification varied according to the response variables we considered. An index of predator avoidance and the distance from the food source were affected in an additive fashion by predatory cues (interaction term, p = 0.469 and p = 0.888 for the index of predator avoidance and the distance from the food source respectively); the effect of physical cues was stronger than the effect of chemical cues and the effect of the combined cues was highest. An index of territoriality was affected in a threshold‐like fashion (interaction term, p = 0.040); chemical or physical cues alone had no effect but when both cues were combined, Atlantic salmon significantly reduced their territorial defence. An index of foraging activity was not significantly affected by predatory cues (alone or combined). We detected no effect on the growth rate of Atlantic salmon (p = 0.328). Finally, we found that the relative contribution of behaviours in explaining individual growth rate changed according to the treatments we considered. Overall, these results demonstrated that fish were able to accurately integrate multiple predatory cues and that this information was used to modulate their antipredatory response. Behaviours involved in the response were relatively independent of each other, allowing fish to adopt behavioural tactics that maximized the ratio of net energy gain to predator avoidance.     

AN EVALUATION OF METHODS USED TO ASSESS INTERGENERIC HYBRIDIZATION IN KELP USING PACIFIC LAMINARIALES (PHAEOPHYCEAE)1

Kelp intergeneric laminarialean hybridizations and hybridization protocol were assessed using seven northeast Pacific kelp species: Alaria marginata Postels and Ruprecht, Costaria costata (C. A. Agardh) Saunders, Eisenia arborea Areschoug, Laminaria saccharina (L) Lamouroux, Lessoniopsis littoralis (Tilden) Reinke, Macrocystis integrifolia Bory, and Nereocystis leutkeana (Mertens) Postels and Ruprecht. Survival and development of sporophyte morphologies derived from selfings, separate males and females, and reciprocal crosses were evaluated over 30 weeks of cultivation. All cultures were initiated from cloned gametophytes. Two closely related species, Laminaria angustata Kjellman and L. japonica Areschoug, demonstrated the efficacy of long‐term (up to 30 years) cloned gametophytes in hybridization studies. Sporophyte morphologies appeared in 34%–69% of control and hybridization trials, and 6%–16% of all trials produced sporophytes in control and hybridization conditions that persisted through 30 weeks of cultivation. Sporophytes in control and hybridization conditions could appear normal or abnormal. Usually, the morphology of sporophytes in hybridizations and female controls resembled the female parent, whereas the sporophytes in male controls often had an abbreviated morphology, lacking definitive generic features. Species‐specific rDNA internal transcribed spacer molecular primers were used to determine the parentage of five putative hybrids. Only the L. japonica♀/L. angustata♂ hybrid bore both parental genomes. That negative controls could produce persistent and normal‐appearing sporophytes negates their value and emphasizes the importance of molecular confirmation in hybridization studies. These findings were applied to critique the only known wild intergeneric hybrid, Pelagophycus/Macrocystis.     

Regulation of carbohydrate and fatty acid utilization by L-carnitine during cardiac development and hypoxia

This study is designed to investigate whether substrate preference in the myocardium during the neonatal period and hypoxia-induced stress is controlled intracellularly or by extracellular substrate availability. To determine this, the effect of exogenous L-carnitine on the regulation of carbohydrate and fatty acid metabolism was determined during cardiac stress (hypoxia) and during the postnatal period. The effect of L-carnitine on long chain (palmitate) and medium chain (octonoate) fatty acid oxidation was studied in cardiac myocytes isolated from less than 24 h old (new born; NB), 2 week old (2 week) and hypoxic 4 week old (HY) piglets. Palmitate oxidation was severely decreased in NB cells compared to those from 2 week animals (0.456 ± 0.04 vs. 1.207 ± 0.52 nmol/mg protein/30 min); surprisingly, cells from even older hypoxic animals appeared shifted toward the new born state (0.695 ± 0.038 nmol/mg protein/30 min). Addition of L-carnitine to the incubation medium, which stimulates carnitine palmitoyl-transferase I (CPTI) accelerated palmitate oxidation 3 fold in NB and approximately 2 fold in HY and 2 week cells. In contrast, octanoate oxidation which was greater in new born myocytes than in 2 week cells, was decreased by L-carnitine suggesting a compensatory response. Furthermore, oxidation of carbohydrates (glucose, pyruvate, and lactate) was greatly increased in new born myocytes compared to 2 week and HY cells and was accompanied by a parallel increase in pyruvate dehydrogenase (PDH) activity. The concentration of malonyl-CoA, a potent inhibitor of CPTI was significantly higher in new born heart than at 2 weeks. These metabolic data taken together suggest that intracellular metabolic signals interact to shift from carbohydrate to fatty acid utilization during development of the myocardium. The decreased oxidation of palmitate in NB hearts probably reflects decreased intracellular L-carnitine and increased malonyl-CoA concentrations. Interestingly, these data further suggest that the cells remain compliant so that under stressful conditions, such as hypoxia, they can revert toward the neonatal state of increased glucose utilization.     

Identification and activation of mitogen-activated protein (MAP) kinase in normal human osteoblastic and bone marrow stromal cells: Attenuation of MAP kinase activation by cAMP,parathyroid hormone and forskolin

The mitogen-activated protein (MAP) kinases (p44^mapk and p42^mapk), also known as extracellular signal-regulated kinases 1 and 2 (ERK1 and ERK2), are activated in response to a variety of extracellular signals, including growth factors, hormones and, neurotransmitters. We have investigated MAP kinase signal transduction pathways in normal human osteoblastic cells. Normal human bone marrow stromal (HBMS), osteoblastic (HOB), and human (TE85, MG-63, SaOS-2), rat (ROS 17/2.8, UMR-106) and mouse (MC3T3-E1) osteoblastic cell lines contained immunodetectable p44^mapk/ERK1 and p42^mapk/ERK2. MAP kinase activity was measured by 'in-gel' assay myelin basic protein as the substrate. Mainly ERK2 was rapidly activated (within 10 min) by bFGF, IGF-I and PDGF-BB in normal HOB, HBMS and human osteosarcoma cells, whereas both ERK1 and ERK2 were activated by growth factors in rat osteoblast-like cell lines, ROS 17/2.8 and UMR-106. The ERK1 activation was greater than the ERK2 in ROS 17/2.8 cells. Furthermore, ERK2 was also activated by bFGF and PDGF-BB in the mouse osteoblastic cell line, MC3T3-E1. This is the first demonstration of inter-species differences in the activation of MAP kinases in osteoblastic cells. Cyclic AMP derivatives or cAMP generating agents such as PTH and forskolin inhibited ERK2 activation by bFGF and PDGF-BB suggesting a 'cross-talk' between the two different signalling pathways activated by receptor tyrosine kinases and cAMP-dependent protein kinase. The accumulated results also suggest that the MAP kinases may be involved in mediating mitogenic and other biological actions of bFGF, IGF-I and PDGF-BB in normal human osteoblastic and bone marrow stromal cells.     

Interstitial cells of Cajal: mediators of communication between circular and longitudinal muscle layers of canine colon

The network of interstitial cells of Cajal associated with Auerbach’s (myenteric) plexus in the canine colon was investigated to determine its role in facilitating communication between circular and longitudinal muscle layers. Electrical coupling between the muscle layers was demonstrated by propagating extracellularly evoked electrotonic pulses from circular muscle cells to nearby longitudinal muscle cells. The likelihood of cytoplasmic continuity across Auerbach’s plexus was further demonstrated by the ability of neurobiotin to spread between the interstitial cells and the circular and longitudinal muscle cells. Importantly, direct neurobiotin spread between circular and longitudinal muscle cells was not observed even when they were in close proximity as determined by confocal microscopy. When neurobiotin did spread across the two muscle layers, the intervening interstitial cells were always neurobiotin-positive. In regions where circular and longitudinal muscle cells approach each other closely, electron microscopy revealed the presence of close appositions between interstitial cells and smooth muscle cells. Gap junctions between interstitial cells and smooth muscle cells of both layers, as judged by electron microscopy, were extremely rare. Neither gap junctions nor close appositions were observed between longitudinal and circular muscle cells. The special arrangement for electrotonic coupling across Auerbach’s plexus through interstitial cells of Cajal suggests controlled coupling between the two muscle layers, explaining the preservation of their distinct electrical activities. Received: 21 July 1995 / Accepted: 22 April 1998     

Concentration and Purification of Poliovirus by Ultrafiltration and Isopycnic Centrifugation

A method is described by which poliovirus can be rapidly and simply concentrated by the use of a Diaflo XM-50 ultrafilter membrane. Freon-extracted ultrafilter concentrates banded in CsCl provided a 1,724-fold volumetric concentration of poliovirus. During concentration, trypsin-digested cellular material can pass through the ultrafilter membrane, thus providing a versatile means of degrading and eliminating extraneous contaminating proteins. The ultrafilter concentration system is compared with the CsCl cushion system of poliovirus concentration, and both systems are further compared by banding virus and virus capsid material in CsCl by use of isopycnic centrifugation.     

Purification and properties of the ribonucleic acid-dependent ribonucleic acid polymerase from Halobacterium cutirubrum

1. The RNA-dependent RNA polymerase from Halobacterium cutirubrum was purified to electrophoretic homogeneity. 2. It requires a single-stranded molecule of RNA or polyribonucleotide as template. 3. Nearest-neighbour analyses of the products formed on random poly(A,U) or alternating poly(A-U) templates and base analysis of the product of synthesis directed by wheat-germ RNA prove that the template is copied accurately. 4. The enzyme initiates new chains with purine ribonucleoside triphosphates. 5. Sucrose-density-gradient analysis of the product indicates that it has a size distribution similar to that of the template. 6. Preliminary amino acid analysis of the RNA-dependent polymerase shows that it contains much less serine than either of the subunits of H. cutirubrum DNA-dependent RNA polymerase. 7. The RNA-dependent enzyme is unable to substitute for either subunit of the DNA-dependent polymerase, and both the latter are devoid of RNA-dependent activity.     

Treatment of renal failure from diabetic nephropathy with cadaveric homograft

We report two patients with terminal renal failure secondary to diabetic nephropathy treated with cadaveric kidney transplantation. Neither of these patients had peripheral vascular disease or peripheral neuropathy. There was a proliferative diabetic retinopathy with hemorrhages and exudates in one patient and only background diabetic changes in the ocular fundi of the other; there have been no significant changes in visual acuity or retinopathy in either patient following the transplantation. Both have good kidney function after 8 and 15 months and are completely rehabilitated.The requirement for insulin decreased in both patients during the period of renal insufficiency and increased following transplantation; this seemed to be related to the large dose of steroids given because now that a maintenance level of steroids has been established, both patients require the same dosage of insulin as they did before the onset of renal insufficiency.