Periodic fluctuations in proliferation of SV-40 transformed human skin fibroblast lines with prolonged lifespan

A human fibroblastic cell line transformed by the SV40-T antigen sequence and continuously cultured for 7 months displayed large periodic variations in cell proliferation. This contrasted with other characteristics of this cell line that remained constant: mosaic cell shape, absence of cell contact inhibition, and predominance of a hypodiploid population. Similar fluctuations in proliferative capacity were also found during the long-term growth of a transformed but nonimmortalized human fibroblastic line prior to senescence, and in the established hamster fibroblastic Nil cell line. This growth pattern suggests a recurrent stimulation of growth in these three transformed cell lines. The proliferation pattern from cultured transformed cells may thus be complex and requires further investigation. These variations presumably influence major cell functions. This observation has important implications for the analysis of data from such cell lines.Abbreviations I-SF immortalized human skin fibroblasts - T-SF transformed human skin fibroblasts - FBS fetal bovine serum     

Effects of omnivorous shrimp in a montane tropical stream: sediment removal,disturbance of sessile invertebrates and enhancement of understory algal biomass

Freshwater shrimp dominate the faunal biomass of many headwater tropical streams: however, their role in community organization is unclear. Enclosure/exclosure experiments in a montane Puerto Rican stream examined direct and indirect effects of two dominant taxa of atyid (Atyidae) shrimp, Atya lanipes Holthuis and Xiphocaris elongata Guerin-Meneville. Both shrimp taxa caused significant reductions in sediment cover on rock substrata, reducing sedimentation and enhancing algal biovolume on clay tiles in cages. When tiles incubated in shrimp exclosures for 2 wks were placed outside of cages, atyid shrimp removed 100% of the sediment cover within a 30 min observation period. Atyid shrimp appear to play an important role in stream recovery after high discharge events by rapidly removing sediments and detritus deposited on benthic substrata in pools. We evaluated the mechanism by which A. lanipes influences algae and benthic insects by comparing patterns of algal biomass, taxonomic composition, and insect abundance between shrimp-exclusion and shrimp-presence treatments both with and without manual sediment removal. The shrimp exclusion treatment without manual sediment removal bad significantly lower algal biomass and greater sedimentation than all other treatments. The treatment in which shrimp were excluded but sediment was manually removed, however, accrued almost the same algal biovolume as the shrimp enclosure treatment, supporting the hypothesis that sediment removal enhances the biovolume of understory algal taxa. Algal community composition was similar between stream bottom bedrock exposed to natural densities of shrimp and all experimental treatments for both Atya and Xiphocaris: a diatom community strongly dominated (78–95%) by the adnate taxon, Achnanthes lanceolata Breb ex. Kutz. Atyid shrimp are important in determining the distribution and abundance of benthic insects through both direct and indirect effects. Sessile, retreat-building chironomid larvae (Chironomidae: Diptera) are negatively affected by both A. lanipes and X. elongata, through direct removal by foraging activities and/or indirectly through depression of sediment resources available to larvae for the construction of retreats. In constrast, the mobile grazer, Cloeodes maculipes (Baetidae: Ephemeroptera) was not adversely affected and atyid shrimp have the potential to exert positive indirect effects on this taxon by facilitating its exploitation of algal resources and/or through enhancement of understory algal food resources through sediment removal.     

Citrate metabolism in 16 Leuconostoc mesenteroides subsp. mesenteroides and subsp. dextranicum strains

Citrate metabolism was studied in non-growing cells of Leuconostoc mesenteroides subsp. mesenteroides and subsp. dextranicum with respect to energetics, formation of degradation products and stoichiometry. The use of selective ionophores and uncoupler showed that citrate utilization was coupled to the proton motive force generated by ATP hydrolysis. Differences in citrate metabolism observed in 20 Leuconostoc strains were related to strains but not to the species or subspecies studied. Citrate metabolism was stimulated by glucose up to a concentration of 25 mmol 1^-1 and decreased at higher concentrations. The main degradation products resulting from the co-metabolism of citrate (10 mmol 1^-1) and glucose (2 mmol 1^-1) were acetate, lactate and pyruvate. Only four Leuconostoc strains produced low levels of acetoin and diacetyl. No strains produced ethanol or acetaldehyde. Citrate degradation ability was stable for at least 130 generations in 81% of the Leuconostoc strains.     

The role of growth factors in gastrointestinal cell proliferation.

The epithelial lining of the gastrointestinal (GI) tract is in a state of continuous cell renewal, and the proliferating and differentiating/differentiated cell populations are spatially clearly demarcated. Members of the epidermal growth factor (EGF) family of peptides, the trefoil peptides and enteroglucagon appear to be the most important enterotrophic molecules for both normal cell renewal and healing after cell damage. Transforming growth factor-a (TGF-a) appears to be the primary physiological ligand for the EGF receptor (EGFR), promoting normal cell renewal, and TGF-a/EGFR are part of an autocrine loop in many intestinal cancers. In response to damage, a differentiating cell lineage arises from adjacent epithelium secreting EGF, TGF-a and trefoil peptides; this may be viewed as part of a ‘repair kit’ in damaged endodermally-derived tissue.     

Alkaline phosphatase and peptidase activities in Caco-2 cells: Differential response to triiodothyronine

Summary Caco-2 cell human colon adenocarcinoma cell line was used to study the hormonal regulation of small intestinal epithelial cell differentiation. We had previously shown that insulin-transferrin-selenium and triiodothyronine (5 × 10⁻⁸ M)-supplemented medium can best replace serum after 2 days of culture for both the maintenance and differentiation of Caco-2 cells. The present study demonstrates that precoating petri dishes with complete serum allows the growth and differentiation of Caco-2 cells seeded directly in serum-free medium. On the other hand, precoating with dialyzed serum inhibits alkaline phosphatase and dipeptidyl-dipeptidase IV activities by more than 50%. The results obtained with complete serum-precoated culture plates indicate that there is no synergy between insulin and triiodothyronine because cells maintained in transferrin-selenium and triiodothyronine-supplemented medium, with or without insulin, express comparable enzyme activities. Moreover, large increases in alkaline phosphatase and dipeptidyl-dipeptidase IV activities were observed when triiodothyronine was added to the culture medium by the time confluency was reached. In contrast, γ-glutamyltransferase was lowered to a greater extent when triiodothyronine was present from the beginning of culture. These findings show that triiodothyronine preferentially stimulates alkaline phosphatase and dipeptidyl-dipeptidase IV activities during the differentiation period whereas it selectively inhibits γ-glutamyltransferase during the proliferation phase. Triiodothyronine acts in a dose-dependent manner.     

A Hydrogen-Donating Monohydroxamate Scavenges Ferryl Myoglobin Radicals

The addition of 25μM hydrogen peroxide to 20μM metmyoglobin produces ferryl (Fe^IV = O) myoglobin. Optical spectroscopy shows that the ferryl species reaches a maximum concentration (60-70% of total haem) after 10 minutes and decays slowly (hours). Low temperature EPR spectroscopy of the high spin metmyoglobin (g = 6) signal is consistent with these findings. At this low peroxide concentration there is no evidence for iron release from the haem. At least two free radicals are detectable by EPR immediately after H₂O₂ addition, but decay completely after ten minutes. However, a longer-lived radical is observed at lower concentrations that is still present after 90 minutes. The monohydroxamate N-methylbutyro-hydroxamic acid (NMBH) increases the rate of decay of the fenyl species. In the presence of NMBH, none of the protein-bound free radicals are detectable; instead nitroxide radicals produced by oxidation of the hydroxamate group are observed. Similar results are observed with the trihydroxamate, desferoxamine. “Ferryl myoglobin” is still able to initiate lipid peroxidation even after the short-lived protein free radicals are no longer detectable (E.S.R. Newman, C.A. Rice-Evans and M.J. Davies (1991) Biochemical and Biophysical Research Communications 179, 1414-1419). It is suggested that the longer-lived protein radicals described here may be partly responsible for this effect. The mechanism of inhibition of initiation of lipid peroxidation by hydroxamate drugs, such as NMBH, may therefore be due to reduction of the protein-derived radicals, rather than reduction of ferryl haem.     

Conformational properties of Eu(III) complexes of 3,3′; 5,3″-polymethylene bridged derivatives of 2,2′; 6,2″-terpyridine

The complex [Eu(tpy)₃](ClO₄)₃ where TPY=2,2′; 6,2″-terpyridine, has been prepared and reexamined. The complex appears to be stable in acetonitrile solution with respect to decomplexation of the ligands but the addition of water does cause partial replacement of tpy. Analogous complexes have been prepared with 3,3′; 5,3″-polymethylene bridged derivatives of tpy having two or three carbons in the bridge. The bridging enforces a cisoid geometry of the ligand and prohibits its replacement by added water. An X-ray determination was carried out for [Eu(3b)₃](ClO₄)₃, where 3b=3,3′; 5,3″-dimethylene tpy, which crystallizes in the monoclinic space group P2₁/c with a=11.908(4), b=15.768(5), c=29.513(9) Å, β=93.60(2)°, μ=13.5 cm⁻¹ and Z=4. The complex forms a tricapped trigonal prism with each of the ligands adopting the same dl conformation. Variable temperature NMR analysis of the bridged ligand complexes indicates that conformational inversion of the bound ligand is not a concerted process and barriers for inversion of individual methylene units can be estimated from coalescence of the signals from the geminal methylene protons. The luminescence properties of the bridged tpy complexes are similar to the parent unbridged system.     

Allosterism and Na++-d-glucose cotransport kinetics in rabbit jejunal vesicles: Compatibility with mixed positive and negative cooperativities in a homo- dimeric or tetrameric structure and experimental evidence for only one transport protein involved

Summary We first present two simple dimeric models of cotransport that may account for all of the kinetics of Na⁺⁺-d-glucose cotransport published so far in the small intestine. Both the sigmoidicity in the Na⁺⁺ activation of transport (positive cooperativity) and the upward deviations from linearity in the Eadie-Hofstee plots relative to glucose concentrations (negative cooperativity) can be rationalized within the concept of allosteric kinetic mechanisms corresponding to either of two models involving sequential or mixed concerted and sequential conformational changes. Such models also allow for 2 Na⁺⁺ 1 S and 1 Na⁺⁺ 1 S stoichiometries of cotransport at low and high substrate concentrations, respectively, and for partial inhibition by inhibitors or substrate analogues. Moreover, it is shown that the dimeric models may present physiological advantages over the seemingly admitted hypothesis of two different cotransporters in that tissue. We next address the reevaluation of Na⁺⁺-d-glucose cotransport kinetics in rabbit intestinal brush border membrane vesicles using stable membrane preparations, a dynamic approach with the Fast Sampling Rapid Filtration Apparatus (FSRFA), and both nonlinear regression and statistical analyses. Under different conditions of temperatures, Na⁺⁺ concentrations, and membrane potentials clamped using two different techniques, we demonstrate that our data can be fully accounted for by the presence of only one carrier in rabbit jejunal brush border membranes since transport kinetics relative to glucose concentrations satisfy simple Michaelis-Menten kinetics. Although supporting a monomeric structure of the cotransporter, such a conclusion would conflict with previous kinetic data and more recent studies implying a polymeric structure of the carrier protein. We thus consider a number of alternatives trying to reconcile the observation of Michaelis-Menten kinetics with allosteric mechanisms of cotransport associated with both positive and negative cooperativities for Na⁺⁺ and glucose binding, respectively. Such models, implying energy storage and release steps through conformational changes associated with ligand binding to an allosteric protein, provide a rational hypothesis to understand the long-time debated question of energy transduction from the Na⁺⁺ electrochemical gradient to the transporter.This research was supported by grant MT-7607 from the Medical Research Council of Canada. One of the authors (A.B.) was supported by a scholarship from the Fonds de la Recherche en Santé du Québec and C. C. was supported by a fellowship from the GRTM. The technical assistance of Mrs. C. Leroy has been greatly appreciated. The authors also thank D.D. Maenz and C. Malo for insightful discussions and C. Gauthier for the art work.     

Involvement of Calcium Channels in Depolarization-Evoked Release of Adenosine from Spinal Cord Synaptosomes

Abstract: The potential involvement of L- and N-type voltage-sensitive calcium (Ca²⁺) channels and a voltage-independent receptor-operated Ca²⁺ channel in the release of adenosine from dorsal spinal cord synaptosomes induced by depolarization with K⁺ and capsaicin was examined. Bay K 8644 (10 n M ) augmented release of adenosine in the presence of a partial depolarization with K⁺ (addition of 6 m M ) but not capsaicin (1 and 10 μ M ). This augmentation was dose dependent from 1 to 10 n M and was followed by inhibition of release from 30 to 100 n M . Nifedipine and nitrendipine inhibited the augmenting effect of Bay K 8644 in a dose-dependent manner, but neither antagonist had any effect on release of adenosine produced by K⁺ (24 m M ) or capsaicin (1 and 10 μ M ) ω-Conotoxin inhibited K⁺-evoked release of adenosine in a dose-dependent manner but had no effect on capsaicin-evoked release. Ruthenium red blocked capsaicin-induced release of adenosine but had no effect on K⁺-evoked release. Although L-type voltage-sensitive Ca²⁺ channels can modulate release of adenosine when synaptosomes are partially depolarized with K⁺, N-type voltage-sensitive Ca²⁺ channels are primarily involved in K⁺-evoked release of adenosine. Capsaicin-evoked release of adenosine does not involve either L- or N-type Ca²⁺ channels, but is dependent on a mechanism that is sensitive to ruthenium red.     

Purification and Properties of an S-Adenosylmethionine: 2,4-Disubstituted Phenol O-Methyltransferase from Phanerochaete chrysosporium

An enzyme catalyzing the O-methylation of acetovanillone (3-methoxy-4-hydroxyacetophenone) by S-adeno-sylmethionine was isolated from Phanerochaete chrysosporium and purified 270-fold by ultrafiltration, anion-exchange chromatography, and gel filtration. The enzyme exhibited a pH optimum between 7 and 9 and was rapidly denatured at temperatures above 55°C. The K_m values for acetovanillone and S-adenosylmethionine were 34 and 99 μM, respectively. S-Adenosylhomocysteine acted as a powerful competitive inhibitor of S-adenosylmethionine, with a K_i of 41 μM. The enzyme was also susceptible to inhibition by thiol reagents and low concentrations of heavy metal ions. Gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that the enzyme was monomeric and had a molecular weight of approximately 53,000. Substrate specificity studies showed that 3-methoxy- and 3,5-dimethoxy-substituted 4-hydroxy-benzaldehydes, -benzoic acids, and -acetophenones were the preferred substrates for the enzyme. The corresponding 3,4-dihydroxy compounds were methylated relatively slowly, while the 3-hydroxy-4-methoxy compounds were almost inactive as substrates. Substituents in both the 2 and 4 positions relative to the hydroxyl group appeared to be essential for significant enzyme attack of a substrate. Provided that certain steric criteria were satisfied, the nature of the substituent was not critical. Hence, xenobiotic compounds such as 2,4-dichlorophenol and 2,4-dibromophenol were methylated almost as readily as acetovanillone. However, an extended side chain in the 4 position was not compatible with activity as a substrate, and neither homovanillic, caffeic, nor ferulic acid was methylated. The substrate range of the O-methyltransferase tends to imply a role in the catabolism or detoxification of lignin degradation products such as vanillic and syringic acids.