Depletion of intracellular putrescine and spermidine by alpha-difluromethylornithine does not inhibit proliferation of 9L rat brain tumor cells.

Incubation of 9L rat brain tumor cells with 25 mM DL-α-difluoromethylornithine inhibits cell proliferation, while treatment with 10 mM and 1 mM do not. All three concentrations cause equal degrees of depletion of intracellular putrescine and spermidine content, but have no effect on spermine content. These observations show that 9L cells can continue to proliferate in spite of significant polyamine depletion and leads one to question the role of polyamines in 9L cell replication. These observations also suggest that inhibition of 9L cell proliferation by 25 mM DL-α-difluormethylornithine is probably not due to its effect on ornithine decarboxylase or on intracellular polyamine content.     

Depletion of donor Ia+ cells before transplantation does not prolong islet allograft survival

Culture of pancreatic islets reduces their immunogenicity and results in prolonged graft survival after allotransplantation. The mechanism by which immunogenicity is reduced by culture is not known, but it has been suggested that prolonged graft survival is the result of the depletion of Ia+ cells from the graft. We studied the effect of eliminating Ia+ cells from islets before allotransplantation. Freshly isolated islets were incubated with anti-Ia serum plus complement or with monoclonal antibody to IAk plus complement or were left untreated before transplantation beneath the renal capsule of diabetic recipients. Incubation with anti-Ia serum plus complement eliminated intra-islet IA+ cells as demonstrated by indirect immunofluorescence staining. Incubation with monoclonal antibody to IAk plus complement significantly reduced but did not eliminate IA+ cells. Neither pretreatment regimen influenced survival of islet allografts placed beneath the renal capsule. However, untreated islets injected into the portal circulation were rejected in a low percentage of cases. We conclude that decreased immunogenicity observed after culture is not due solely to the depletion of Ia+ cells and that the site of engraftment has an important impact on graft survival.     

Age does not alter protein kinase C isozymes mRNA expression in rat brain

Calcium and phospholipid dependent Protein kinase C (PKC) may play a role in memory function and pathogenesis of many neurodegenerative disorders such as Alzheimer's disease (AD). Abnormal phosphorylation by PKC as well as reduced levels of PKC has been implicated in the neurodegeneration associated with AD and aging. Recently, many subtypes of PKC isozymes have been identified by molecular biology techniques which are expressed differentially in various regions of the brain. The reduction and alterations in the activities and distribution of these subtypes of PKC isozymes may be accountable for the decline of selective neurons during aging. In order to investigate the role of PKC isozymes during aging, we examined the distribution of PKC-, , and mRNA, expressions between young (4 months) and old (25 months) rat brains using in situ hybridization histochemistry. Our studies showed that signals of three isoforms of PKC mRNA vary in cortical and hippocampal regions. However, no change was detected in any of the PKC isoforms mRNA expressions in aged animals.     

Dietary tryptophan does not alter the function of brain serotonin neurons

The hypothesis that alterations in dietary tryptophan modify the functional activity of brain serotonin-containing neurons was tested by recording the electrophysiological activity of single serotonergic cells in awake, behaving cats after meal ingestion of diets containing varying proportions of tryptophan and the neutral amino acids that compete with tryptophan for uptake into the brain. The data revealed that while the various diets produced significant changes in brain serotonin and its major metabolite, 5-hydroxyindoleacetic acid, there was no change in the activity of serotonin-containing dorsal raphe cells following meal ingestion. Furthermore, a pulse injection of tritiated labeled tryptophan following the various diets produced no significant change in the release of tritiated serotonin into the lateral ventricles, while tritiated 5-hydroxyindoleacetic acid was significantly increased. These data suggest that dietary tryptophan does not alter the functional activity of central serotonergic neurons, in contrast with current popular beliefs that such dietary manipulations alter brain function.     

Copper does not alter the intracellular distribution of ATP7B, a copper-transporting ATPase

Wilson's disease is a genetic disorder characterized by the accumulation of copper in the body due to a defect of biliary copper excretion. However, the mechanism of biliary copper excretion has not been fully clarified. We examined the effect of copper on the intracellular localization of the Wilson disease gene product (ATP7B) and green fluorescent protein (GFP)-tagged ATP7B in a human hepatoma cell line (Huh7). The intracellular organelles were visualized by fluorescence microscopy. GFP-ATP7B colocalized with late endosome markers, but not with endoplasmic reticulum, Golgi, or lysosome markers in both the steady and copper-loaded states. ATP7B mainly localized at the perinuclear regions in both states. These results suggest that the main localization of ATP7B is in the late endosomes in both the steady and copper-loaded states. ATP7B seems to translocate copper from the cytosol to the late endosomal lumen, thus participating in biliary copper excretion via lysosomes.     

Internalization of nanopolymeric tracers does not alter characteristics of placental cells

In the cell therapy scenario, efficient tracing of transplanted cells is essential for investigating cell migration and interactions with host tissues. This is fundamental to provide mechanistic insights which altogether allow for the understanding of the translational potential of placental cell therapy in the clinical setting. Mesenchymal stem/stromal cells (MSC) from human placenta are increasingly being investigated for their potential in treating patients with a variety of diseases. In this study, we investigated the feasibility of using poly (methyl methacrylate) nanoparticles (PMMA‐NPs) to trace placental MSC, namely those from the amniotic membrane (hAMSC) and early chorionic villi (hCV‐MSC). We report that PMMP‐NPs are efficiently internalized and retained in both populations, and do not alter cell morphofunctional parameters. We observed that PMMP‐NP incorporation does not alter in vitro immune modulatory capability of placental MSC, a characteristic central to their reparative/therapeutic effects in vitro. We also show that in vitro, PMMP‐NP uptake is not affected by hypoxia. Interestingly, after in vivo brain ischaemia and reperfusion injury achieved by transient middle cerebral artery occlusion (tMCAo) in mice, iv hAMSC treatment resulted in significant improvement in cognitive function compared to PBS‐treated tMCAo mice. Our study provides evidence that tracing placental MSC with PMMP‐NPs does not alter their in vitro and in vivo functions. These observations are grounds for the use of PMMP‐NPs as tools to investigate the therapeutic mechanisms of hAMSC and hCV‐MSC in preclinical models of inflammatory‐driven diseases.     

Synchronization of 9L rat brain tumor cells by centrifugal elutriation

Asynchronous 9L cells were separated into relatively homogeneously-sized populations using centrifugal elutriation with both a conventional collection method and a long collection method. A substantial increase in the homogeneity of the volume distributions and in the degree of synchrony of the separated fractions was obtained using the long collection method. Autoradiographic data indicated that fractions containing greater than or equal to 97% G1 cells, greater than or equal to 80% S cells, and 70-75% G2 cells could be routinely recovered with this procedure. Recovery in these fractions varied from 5 to 8% of the total number of cells elutriated. The colony forming efficiency (CFE) of cells from fractions representing each phase of the cell cycle was a constant 60-70%, which was comparable to the 60-80% usually found for asynchronous 9L cells. The percentage of cells in the G1, S, and G2 phases in the elutriated fractions was more accurately determined from the volume distribution than from computer fits of the DNA histogram obtained from flow cytometry. In genereal, the degree of synchrony was related to the coefficient of variation (CV) of the volume distributions of the elutriated fractions. The CV was about 14% for all elutriated fractions. When the greater than or equal to 97% G1 population was allowed to progress to S and G2, the CVs were about 17 and 20.2%, respectively. Thus, the best nonperturbing method for obtaining synchronous 9L cells in the S or G2 phases was direct elutriation with the long collection method.     

Synchronization of 9L rat brain tumor cells by centrifugal elutriation

Asynchronous 9L cells were separated into relatively homogeneously-sized populations using centrifugal elutriation with both a conventional collection method and a long collection method. A substantial increase in the homogeneity of the volume distributions and in the degree of synchrony of the separated fractions was obtained using the long collection method. Autoradiographic data indicated that fractions containing ≥97% G₁ cells, ≥80% S cells, and 70–75% G₂ cells could be routinely recovered with this procedure. Recovery in these fractions varied from 5 to 8% of the total number of cells elutriated. The colony forming efficiency (CFE) of cells from fractions representing each phase of the cell cycle was a constant 60–70%, which was comparable to the 60–80% usually found for asynchronous 9L cells. The percentage of cells in the G₁, S, and G₂ phases in the elutriated fractions was more accurately determined from the volume distribution than from computer fits of the DNA histogram obtained from flow cytometry. In general, the degree of synchrony was related to the coefficient of variation (CV) of the volume distributions of the elutriated fractions. The CV was about 14% for all elutriated fractions. When the ≥97% G₁ population was allowed to progress to S and G₂, the CVs were about 17 and 20.2%, respectively. Thus, the best nonperturbing method for obtaining synchronous 9L cells in the S or G₂ phases was direct elutriation with the long collection method.     

Cryopreservation does not alter the allogenicity and development of vasculopathy in post-transplant rat aortas

BACKGROUND: Cryopreservation is a valuable technique for storing heart valve and vascular allografts. However, the biological ramifications of cryopreservation are still unclear; therefore, using animal experiments we assessed how 'cryopreservation' influences graft allogenicity and cell viability. METHODS: Thoracic aortas of Lewis rats were prepared as fresh (F) or cryopreserved (CP) grafts, and implanted into the infrarenal aorta of Lewis or Brown Norway rats (BNs). The grafts and spleens were harvested at post-operative day 7 and 28 (POD7, POD28) for analyses. RESULTS: First, the systemic immune response to transplantation was estimated by mixed lymphocyte reaction analyses using spleen cells from na?ve or recipient BNs. The alloreactivity of the recipients increased to 1.5 times that of the na?ve BNs at POD7 and POD28, when stimulated by mitomycin C-treated Lewis spleen cells. Second, local immune response was estimated by TNFalpha, IFNgamma, and iNOS mRNA expression in the grafts by quantitative PCR, which revealed 20- to 40-fold increases at POD28 after allotransplantation. Third, endothelial cell viability was estimated by endothelial NOS mRNA expression level: it was similar and highest in F and CP grafts before transplantation then significantly decreased after both syngeneic and allogeneic transplantation. Finally, intimal hyperplasia, expressed by I/M ratio, developed over time after allotransplantation, reaching 2.5 times the thickness of F grafts before transplantation. The results of these experiments revealed no difference between F and CP grafts before and after transplantation. CONCLUSION: Cryopreservation did not modify the allogenicity of vascular allografts and had minimal adverse impacts on graft cell viability.     

Short-term carnitine deficiency does not alter aerobic rat heart function but depresses reperfusion recovery after ischemia.

Clinical and experimental studies have shown that long-term carnitine deficiency is often associated with cardiomyopathy and ischemic failure. The present study was designed to determine whether cardiac dysfunction is seen in an experimental model of short-terrm carnitine deficiency. Carnitine deficiency was induced in Sprague-Dawley rats by supplementing the drinking water with sodium pivalate for a period of 2 weeks. This resulted in a 25% depletion of total myocardial carnitine content. When isolated working hearts from these animals were paced and subjected to increments in left atrial filling pressure, there were no differences in mechanical function compared with control hearts. Following no-flow ischemia, however, recovery of cardiac output and relaxation parameters was depressed in hearts from pivalate-treated animals. Under these conditions, L-carnitine prevented the depressions of function from occurring. Our results show that short-term carnitine deficiency is not associated with cardiac dysfunction under normoxic conditions. However, hearts from pivalate-treated animals are more susceptible to ischemic injury and thus may prove to be useful for the study of metabolic and functional aspects of carnitine deficiency.     

Reduced content of homogalacturonan does not alter the ion-mediated increase in xylem hydraulic conductivity in tobacco

Xylem hydraulic conductivity (K(s)) in stems of tobacco (Nicotiana tabacum) wild-type SR1 was compared to that of PG7 and PG16, two transgenic lines with increased levels of expression of the gene encoding the Aspergillus niger endopolygalacturonase (AnPGII). Activity of AnPGII removes in planta blocks of homogalacturonan (HG) with deesterified carboxyls, thus increasing the degree of neutrality of pectins. The effect of K+ was tested in increasing stem K(s) using model plants with more neutral polysaccharides in primary walls and, hence, in intervessel pit membranes. K(s) measured with deionized water was compared to that with KCl solutions at increasing concentrations (DeltaK(s), %). Plants transformed for HG degree of neutrality showed a dwarfed phenotype, but DeltaK(s) did not differ among the three experimental groups. The ion-mediated hydraulic effect saturated at a KCl concentration of 25 mm in SR1 plants. All the three tobacco lines showed DeltaK(s) of around +12.5% and +17.0% when perfused with 10 and 25 mm KCl, respectively. Because modification of HG content did not influence ion-mediated hydraulic enhancement, we suggest that pectin components other than HG, like rhamnogalacturonan-I and/or rhamnogalacturonan-II, might play important roles in the hydrogel behavior of pit membranes.     

Modification of cell cholesterol content of rat tumour cells

Summary Among the different constituents of the cell membrane, lipids have been poorly studied with respect to their role in the immunogenicity of tumour cells and their influence on the expression on tumour-associated antigens. Since liposome-associated antigens are more potent immunogens when the lipid matrix is in a rigid state, we have modified the lipid composition of rat hepatoma cells by incorporation of cholesteryl hemisuccinate (CH) into the lipid matrix, and studied its effect on the tumorigenicity and immunogenicity of these tumour cells in syngeneic animals. A slight and significant decrease of tumorigenicity of CH-enriched D23 cells was observed when 2×10³ cells were injected SC, whereas with a higher tumour cell challenge there was no difference in the tumorigenicity of untreated or treated cells. The immunogenicity of CH-treated cells was tested by IP immunization with 10⁷ or 10⁶ cells followed 1 week later by an SC challenge with 2×10⁴ viable D23 cells. No statistical difference was observed between the immunogenicity of CH-enriched cells and that of control cells on either tumour incidence or tumour growth rate. In addition, similar experiments performed with the spontaneous mammary carcinoma SP4 showed that CH-enriched SP4 cells were of lower immunogenicity and unable to induce a significant memory immunity. This lack of effect of the CH treatment on the immunogenicity was not related to the absence of incorporation of CH, since the CH treatment increased the cell lipid rigidity as determined by the increase of fluorescence anisotropy of the diphenyl hexatriene probe. These results obtained in two weak immunogenic tumour models underlined the need for further studies before such a lipid modification of cancer cells is applied in human immunotherapy trials.Attaché de Recherche au CNRS, Fellow of the Royal Society (European Science Exchange Programm) from 1. 4. 1981 to 30. 9. 1981     

Caffeine does not inhibit substance P-evoked intracellular Ca2+ mobilization in rat salivary acinar cells

We used theCa²⁺-sensitive fluorescent dyefura 2, together with measurements of intracellularD-myo-inositol1,4,5-trisphosphate [Ins(1,4,5)P₃],to assess the inhibitory effects of caffeine on signal transduction viaG protein-coupled receptor pathways in isolated rat mandibular salivaryacinar cells. ACh, norepinephrine (NE), and substance P (SP) all evokedsubstantial increases in the intracellular freeCa²⁺ concentration([Ca²⁺]_i).Responses to ACh and NE were markedly inhibited by prior application of20 mM caffeine. The inhibitory effect of caffeine was not reproduced byphosphodiesterase inhibition with IBMX or addition of cell-permeantdibutyryl cAMP. In contrast to the ACh and NE responses, the[Ca²⁺]_iresponse to SP was unaffected by caffeine. Despite this, SP and AChappeared to mobilize Ca²⁺ from acommon intracellular pool. Measurements of agonist-induced changes inIns(1,4,5)P₃levels confirmed that caffeine inhibited the stimulus-response couplingpathway at a point beforeIns(1,4,5)P₃ generation. Caffeine did not, however, inhibit[Ca²⁺]_iresponses evoked by direct activation of G proteins with 40 mMF. These data show thatcaffeine inhibits G protein-coupled signal transduction in these cellsat some element that is common to the muscarinic and -adrenergicsignaling pathways but is not shared by the SP signaling pathway. Wesuggest that this element might be a specific structural motif on the Gprotein-coupled muscarinic and -adrenergic receptors.     

Effects of hyperthermia on DNA interstrand crosslinking after treatment with BCNU in 9L rat brain tumor cells

The effects of hyperthermia (42 degrees C) on 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU)-mediated DNA interstrand crosslink formation were investigated in 9L rat brain tumor cells using the technique of alkaline elution. When cells were treated with 60 microM BCNU for 1 hr at 37 degrees C and incubated for 6 hr in drug-free medium at 42 degrees C, there was a 50% increase in crosslinking; and when cells were treated at 42 degrees C and incubated at 37 degrees C, there was a 45% increase in crosslinking compared with the results for cells treated and incubated at 37 degrees C. When cells were treated and incubated at 42 degrees C, there was a 129% increase in DNA crosslinking. The same relative order of results was found for cell survival. These results suggest that hyperthermia can increase DNA interstrand crosslink formation and the consequent cell death through two independent mechanisms: an increase in the amount of initial alkylation because of the increased rate of hydrolysis of BCNU at higher temperatures, and the effect of heat on DNA structure that leads to an increase in the number of crosslinks formed.     

Changes in water content of two agricultural soils does not alter labile P and C pools

Aims

An incubation study was conducted to investigate how changes in soil water content affect labile phosphorus and carbon pools, mineralisation patterns and microbial community composition.

Methods

Two soils from different climatic histories were subjected to four long-term (15 weeks) soil water regimes (constant field capacity (m); 3 dry-rewet (DRW) cycles evenly spaced (intermittent, int); 3 DRW cycles with a shorter interval after a long dry period (false break, fb); constantly air-dry (d)) (incubation period 1). In the subsequent incubation period 2, a set of cores from each treatment were subjected to one DRW cycle (air-dry for 7 day; field capacity for 14 day) or maintained at field capacity.

Results

Long-term soil water regime altered soil respiration with the largest CO₂ pulse occurring in soil with the longest dry period. However, changing the distribution of the 3 DRW events within incubation period 1 (int/fb) did not alter cumulative CO₂. In addition, DRW during incubation period 2 did not affect cumulative CO₂ in either treatment (m, int, fb, d) (except for Hamilton int). Our results show that carbon and phosphorus availability and the size and community composition of the microbial biomass were largely unaffected by fluctuating soil water content.

Conclusions

Changes in soil water content altered respiration, phosphatase activity and microbial C:P ratio and indicate physiological and/or functional changes in the microbial community. However, it appeared that these would have little impact on plant P availability.     

Short-term hypergravity does not affect protein-ubiquitination and proliferation in rat L6 myoblastic cells.

We previously reported that spaceflight (STS-90) and tail-suspension stimulated muscle protein ubiquitination and accumulated the degradation fragments. However, in space experiments the side-effects of hypergravity on samples are inevitable during the launch of a space shuttle into space or the reentry. To examine whether hypergravity also caused protein-ubiquitination in skeletal muscle cells, we exposed rat myoblastic L6 cells to various hypergravity conditions. Immunoblot analysis showed that the centrifugation at 2, 3, 30 or 100 G for 10 min did not increase the amount of ubiquitinated proteins in L6 cells, whereas the centrifugation at 100 G for 1 or 2 hrs significantly induced the protein-ubiquitination. In contrast, heat shock protein 70 (HSP70), another stress-responsive protein, in L6 cells was accumulated only by centrifugation at 100 G for more than 10 min. Short-term (10 min) hypergravity including 3 or 100 G did not affect the proliferation and morphological changes in L6 cells. Our present results suggest that the ubiquitination of muscle proteins is less sensitive to hypergravity than the induction of HSP70, and that the effect of hypergravity on protein-ubiquitination and proliferation of skeletal muscle cells may be negligible, as far as its duration is short-term.