Two-day Culture Research Articles

Influence of Xanthomonas campestris pv. campestris on the growth of etiolated and photosynthetic seedlings of Brassica oleracea

Relevance. In recent years, physiologists are very interested in studying the characteristics of the growth and development of seedlings under conditions of etiolation. Etiolated seedlings, as a model object, are interesting in studying the responses of a plant organism to the effects of various biotic environmental factors. In this regard, a comprehensive study of the characteristics of the influence of Xanthomonas campestris pv. campestris (Хсс) on growth processes in the early stages of development of white cabbage plants, depending on the racial composition of the pathogen, is very important.Material and method. The study was carried out on the basis of the immunity and plant protection laboratory of the Federal State Budgetary Scientific Institution of the Federal Scientific Vegetable Center (FSVC). The material of research were seeds of two varieties of white cabbage with different resistance to vascular bacteriosis and four races of Xanthomonas campestris pv. campestris (Хсс1, Хсс3, Хсс4 и Хсс6). Sterile seeds of each variety were laid out in Petri cups. Infection with different races of Xcc was carried out by introducing into each Petri cup 1 ml of a bacterial suspension of a two-day culture at a concentration of 106 cells/ml. Half of the Petri cups were placed in a thermostat without illumination, the other was exposed to light under a 16- hour regime of illumination with diffused light. The magnitude and orientation of biological effects of studied factors on biometric parameters of seedlings were evaluated by the relative deviation of the difference between parameters of the experimental and control variants.Results. A different degree of influence of the resistance of the variety and the aggressiveness of the pathogen race on the growth of individual parts of the seedling has been established, and the response of etiolated and photosynthetic seedlings to infection is often the opposite. In the susceptible cultivar Slava 1305, acceleration of root and hypocotyl growth in the dark (Хсс3, Хсс4 and Хсс1) and inhibition of these processes in the light (Хсс1, Хсс4 and Хсс6) were noted. Relatively stable SB-3 F1 hybrid has more pronounced raspecificity of the effect of Xcc on root growth and stimulation of hypocotyl growth in all variants, which was 2-8 times lower in etiolated seedlings when infected with Xcc6 and Xcc1 races than in light ones, and when infected by the races Xcc1 and Xcc4, the effect was comparable.

Bi-layer blood vessel mimicking microfluidic platform for antitumor drug screening based on co-culturing 3D tumor spheroids and endothelial layers.

Two-dimensional (2D) cell culture is not ideal for traditional drug screening, because 2D culture does not accurately mimic the physiological microenvironment of tumor cells. Thus, a drug-screening system which more closely mimics the microenvironment of in vivo tumors is necessary. Here, we present a biomimicking bilayer microfluidic device that can facilitate antitumor drug screening. The microfluidic device consists of two polydimethylsiloxane (PDMS) pieces with channels which are separated by a semipermeable membrane to allow water, oxygen, and nutrition supply, while preventing cell migration. The channels embedded on the two PDMS pieces overlap each other over a long distance to ensure a larger exchange area to mimic the blood vessel-tumor model. High concentrations of endothelial cells (EC) are first seeded onto the membrane through the apical channel, and after a two-day culture, a confluent EC monolayer forms. Tumor spheroid-laden Matrigel is then seeded into the basal channel. After the Matrigel is cured, the device is ready for drug testing. Paclitaxel is used as the model drug for testing. Confocal microscopy and ImageJ are used to assess the efficacy of different concentrations of paclitaxel, and optical coherence tomography (OCT) is employed to determine the tumor volumetric change after the drug treatment. The results indicate that the proposed bilayer microfluidic device in combination with confocal and OCT optical characterization provide an efficient platform for antitumor drug testing.

Impact of Sequential Culture Results on Diagnosis and De-Escalation of the Antibiotic Regimen in Joint and Bone Infections.

According to existing guidelines, orthopedic specimens collected in joint and bone infections (JBI) in our institution are cultured on several media sets and incubated for two, seven, and 14 days. The optimal timing for de-escalation of the first-line antibiotic combination according to the culture results needs to be defined. Single-center, retrospective analysis of all adult patients with a first documented episode of JBI between May 2012 and April 2013. Ninety patients were included, 51 males (57%), median age 58 y (range 18-87 y), with prosthesis infection in 62 cases (69%). Rapidly growing pathogens (Staphylococcus aureus [n = 36] and Enterobacteriaceae [n = 12]) usually were diagnosed within two days, whereas coagulase-negative staphylococci (n = 25) and Propionibacterium acnes (n = 13) generally were identified after seven days (p < 10-5). Positive culture results at day 2 fit with definitive microbiological diagnosis in 95% of cases, and prolonged incubation led to the identification of additional micro-organisms in only four of 76 patients (5%) with day-2-positive cultures. Conversely, for those with negative two-day culture (n = 14), the seven-day culture allowed identification of less virulent pathogens in eight cases (57%). Our results suggest that, in JBI, de-escalation of the empirical antibiotic regimen can be based on micro-organisms identified on the two-day culture set. The impact of such a strategy on clinical outcomes, antibiotic consumption, and costs needs to be assessed in larger studies.

Oxygenated thawing and rewarming alleviate rewarming injury of cryopreserved pancreatic islets

Background/AimsPancreatic islet transplantation is an effective treatment for Type 1 diabetic patients to eliminate insulin injections; however, a shortage of donor organs hinders the widespread use. Although long-term islet storage, such as cryopreservation, is considered one of the key solutions, transplantation of cryopreserved islets is still not practical due to the extensive loss during the cryopreservation-rewarming process. We have previously reported that culturing islets in a hyperoxic environment is an effective treatment to prevent islet death from the hypoxic injury during culture. In this study, we explored the effectiveness of thawing and rewarming cryopreserved islets in a hyperoxic environment. MethodsFollowing cryopreservation of isolated human islets, the thawing solution and culture media were prepared with or without pre-equilibration to 50% oxygen. Thawing/rewarming and the pursuant two-day culture were performed with or without oxygenation. Short-term recovery rate, defined as the volume change during cryopreservation and thawing/rewarming, was assessed. Ischemia-associated and inflammation-associated gene expressions were examined using qPCR after the initial rewarming period. Long-term recovery rate, defined as the volume change during the two-day culture after the thawing/rewarming, was also examined. Islet metabolism and function were assessed by basal oxygen consumption rate and glucose stimulated insulin secretion after long-term recovery. ResultsOxygenated thawing/rewarming did not alter the short-term recovery rate. Inflammation-associated gene expressions were elevated by the conventional thawing/rewarming method and suppressed by the oxygenated thawing/rewarming, whereas ischemia-associated gene expressions did not change between the thawing/rewarming methods. Long-term recovery rate experiments revealed that only the combination therapy of oxygenated thawing/rewarming and oxygenated culture alleviated islet volume loss. These islets showed higher metabolism and better function among the conditions examined. ConclusionOxygenated thawing/rewarming alleviated islet volume loss, with the help of oxygenated culture.

Comparison of the effect of honey and miconazole against Candida albicans in vitro

Background:One of the most common causes of vaginitis is candidiasis. The aim of this study is to compare the effect of honey and miconazole against Candida albicans, in vitro.Materials and Methods:The different W/V concentrations of honey were prepared at 20, 40, 60, 80, and 95% and different dilutions of miconazole were prepared in 0.05, 5, and 50 μg/ml. A microdilution of 100/000 cells per ml of a two-day old culture of Candida albicans was prepared in normal saline, after culturing the strain of PTCC 5027 in RPMI 1640 medium. Ten microliters of this dilution was added to 1 ml of the RPMI 1640 medium containing different concentrations of honey and to 1 ml of the RPMI 1640 medium containing different dilutions of miconazole. The cultures were incubated at 35°C for 12, 24, and 48 hours.Results:The growth rate of Candida albicans was determined in the cultures. The results indicated that the honey prevented the growth of C. albicans greatly only at an 80% concentration, whereas, miconazole inhibited it completely.Conclusions:As Candida albicans is a normal vaginal flora, the inhibitory effect of honey without the fungicide effect is a very good trend in the treatment of vaginal candidiasis.

Biomimetic Calcium Carbonate Concentric Microgrooves with Tunable Widths for Promoting MC3T3‐E1 Cell Functions

Biomimetic, self-assembled calcium carbonate (CaCO(3) ) concentric microgrooves with groove widths of 5.0 and 10 μm were fabricated through simply controlling incubation temperature. Mouse pre-osteoblastic MC3T3-E1 cells were cultured on flat and microgrooved substrates of CaCO(3) and their adhesion, spreading, proliferation, alkaline phosphatase activity, and calcium content were remarkably enhanced by the microgrooves, in particular, the narrower ones. Furthermore, focal adhesions and actin filaments of MC3T3-E1 cells could be aligned on both 5.0-μm and 10-μm-wide CaCO(3) grooves. Compared with the original round nuclei on the flat substrates and expanded round nuclei on the narrower microgrooves, the MC3T3-E1 cell nuclei on 10-μm-wide CaCO(3) grooves demonstrated preferred entrapment in the grooves and significant alignment with a smaller area after two-day culture.

Bioactive Collagen Membrane as a Carrier for Sustained Release of PDGF

The objective of this study was to evaluate the ability of a collagen membrane (CM) as a carrier to successfully deliver platelet-derived growth factor (PDGF) and to observe the subsequent effects of the factor on preosteoblasts in vitro. MC3T3-E1 mouse preosteoblasts were cultured with a commercially available CM containing PDGF. After a two-day cell culture, cell viability was investigated by the MTT assay and cell proliferation was assessed by the crystal violet proliferation assay. Expression levels of the following osteoblastic differentiation marker genes were measured by real-time PCR: runt-related transcription factor 2 (RUNX2), osteopontin (OPN), bone sialoprotein (BSP), and osteocalcin (OCN). A cell proliferation assay was conducted, and osteoblastogenesis was determined by alkaline phosphatase (ALP) activity. A sustained release of PDGF from a CM was observed for ~3 weeks. Gene expression of all RUNX2, OPN, BSP, and OCN in CM with PDGF was significantly upregulated compared to those in CM without PDGF (all p < 0.05). Interestingly, CM without PDGF also significantly increased gene expression of RUNX2 and OPN in MC3T3-E1 cells compared to the cell control (both p < 0.05). Furthermore, it was observed that the PDGF released from CM significantly promoted ALP activity and cell proliferation with little cytotoxicity. These results suggest that a CM can be utilized for sustained delivery of PDGF. Also, released PDGF can promote MC3T3-E1 cell activities. This strategy may lead to an improvement in the current clinical treatment of bone defects in periodontal and implant therapy.

Stimulation of polygalacturonase production in an immobilized system by Aspergillus sp.: effect of pectin and glucose

The synthesis of polygalacturonases (PG) is known to be influenced by Aspergillus growth conditions, namely, environmental factors and pectin content in the cultivation medium containing a mixed carbon source. Optimal conditions were attained at a temperature of 30 °C and an initial pH of 4.5. PG activity (3.29 and 2.48 U/mL) was determined after a two-day culture of Aspergillus sp. HC1 and Aspergillus sp. CC1, respectively, in a basic medium containing 2% citrus pectin as the sole carbon source. The addition of glucose (2% w/v) to the basic medium led to a 2-fold increase in PG production. However, enzyme synthesis was repressed when a higher concentration of glucose was used in the medium containing the mixed carbon source. Spores from the two fungi were immobilized in a 3% Ca–alginate system and the mechanical strength of the gel beads allowed the use of this process system 6-fold longer (288 h) than the free culture. In the Aspergillus sp. CC1 immobilized system, PG production increased nearly 10-fold in the medium with 2% glucose added (5.95 U/mL) in comparison to the medium without sugar (0.55 U/mL). The results demonstrate that a different response in activity was produced by free and entrapped spore systems. PG production remained approximately constant throughout the six 48 h cycles in the medium containing citrus pectin (2% w/v) as the sole carbon source.

Islet culture and counter-culture

The publication of the Edmonton protocol caused a lot of excitement by reporting an almost overnight improvement in the rate of success of clinical islet of Langerhans transplantation [1]. Conventional wisdom holds that steroid-free immunosuppression and an increased transplanted islet mass, thanks to the use of multiple donors for each recipient, were the key determinants for the success of the Edmonton protocol. However, the protocol incorporated several additional strategies in the effort to improve the results of the method. One of these was the transplantation of freshly isolated islets within 2 h of the end of the isolation procedure [1,2]. This approach was novel, but based on intuition rather than on hard data. Indeed, in the previous years, islet preparations were commonly cultured prior to transplantation [3]. In the wake of the Edmonton report, many islet transplant centers attempted to reproduce its results by implementing the same protocol, including immediate transplantation of fresh islets, as was the case in the first ever multicenter trial of islet transplantation, funded by the Immune Tolerance Network [2]. There are, however, obvious logistical advantages in culturing islets prior to transplantation: it provides flexibility in the organization and planning of the procedure, which involves interventional radiology access and anesthesiology standby, and it allows the pooling of islet preparations sequentially isolated from different donor pancreata, to increase the islet graft functional mass. Indeed, it could be shown that transplantation of cultured islets could lead to seemingly similar success rates as those described by the Edmonton group [4,5]. The question thus arises whether culturing islets for a period of time could have a beneficial or detrimental impact on islet viability and function, and on what mechanistic basis. This question is addressed by Dr. Ihm et al. in a recent issue of Transplant International [6] [Correction added after publication 31 March 2009: in the preceding sentence, this issue was corrected to a recent issue]. This is an important question and it is noteworthy that this is one of very few publications that have addressed this topic studying human islets. Results obtained in animal models may not necessarily be extrapolated to the human, because of the species-specific impact of culture on islet function [7]. The authors show that short-term culture has a beneficial effect on islet vitality and viability. Two-day culture increased islet cell ATP contents and decreased the activity of protein kinases involved in pro-apoptotic signalling. These positive findings are balanced by the increased expression of certain pro-inflammatory genes induced by short-term culture. What matters to the islet transplant community, however, is the overall impact of short-term culture on in vivo islet function. It is well known and a matter of concern that culturing islets leads to a loss of function and mass Correspondence Thierry Berney MD, MSc, Division of Visceral/ Transplantation Surgery, Geneva University Hospitals, 24 rue MIcheli-du-Crest, CH-1211 Geneva 14. Tel.: +41 22 37 23 404; fax: +41 22 37 27 707; e-mail: thierry. berney@hcuge.ch

Screening of secreted proteases of Paenibacillus larvae by using substrate-SDS-polyacrylamide gel electrophoresis

SummaryThis study is focused on a primary proteolytic screening of the secreted products of P. larvae cultures by means of substrate SDS-PAGE and using specific inhibitors for the determination of protease classes. Two- and five-day cultures (cultivation medium: MYPGP broth) were used for primary screening. Proteolytic activity was detected only in gels containing gelatin in the area of 87, 74 and 42 kDa in two-day culture and in the area of 87,74,42 and 40 kDa in five-day culture. By using various protease inhibitors, it was considered that the 87 and 74 kDa stable proteases (most active at pH 7) presented in two-day and five-day culture can be metalloproteases, by virtue of their sensitivity to EDTA, 1, 10-phenantroline and EGTA (metal chelators) in standard concentrations, and by the lack of sensitivity to inhibitors of the other classes of protease. The 40 kDa protease in two-day culture was inhibited also by standard concentration of metal chelators, and was most active at pH 6. The 40 and 42 kDa proteases in five-day culture (most active also at pH 6) were inhibited by 1, 10- phenantroline in 2 mM concentration; the EDTA partially inhibited them at 8 mM concentration. All proteases were partially inhibited by 5 mM DTT in incubation buffers. On the other hand, the 2-mercaptoethanol in sample buffer did not alter the proteolytic activity. The inhibited 87 and 74 kDa proteases (by means of EDTA) can be reactivated by Ca2+ and partial by Fe2+ ions. The 40 kDa proteases in twoday culture can be reactivated by Zn2+ ions. No proteolytic activity was detected on the casein substrate gels.

Modulation of FGF-2 binding to chondrocytes from the developing growth plate by perlecan

FGF-2 is a regulator of chondrocyte proliferation in the developing growth plate and has been shown to bind to perlecan, a heparan sulfate proteoglycan. We evaluated the effect of perlecan isolated from the growth plate on the binding of FGF-2 to its low and high affinity receptors on resting and proliferating chondrocytes. Chondrocytes were isolated by pronase/collagenase digestion of 1 mm thick slices from the resting and proliferating zones of fetal bovine ribs and were plated in serum-free DMEM. Chondrocytes maintained their zone-specific level of DNA and matrix synthesis over a two-day culture period. The collagen, aggrecan, and perlecan components of the matrix produced were associated with the cell layer and were secreted into the medium. Most of the perlecan made by the chondrocytes was secreted into the medium. Western blots showed medium perlecan to contain two high molecular weight core proteins and overlay assays showed only the large core protein bound FGF-2. Cell layer perlecan contained only the smaller core protein. Immunoprecipitation assays of media showed that the medium perlecan bound (125)I-FGF-2, that the bound FGF-2 was eluted from perlecan by 2 M NaCl at pH 7.4, and that this binding was eliminated by prior digestion with heparatinase. This indicates that the perlecan secreted into the medium is a low affinity receptor for FGF-2. (125)I-FGF-2 also bound to the chondrocytes in cell culture. Competition studies showed exogenous FGF-2 reduced (125)I-FGF-2 binding to high affinity receptor but not the low affinity receptor in the cell layer. Exogenous perlecan, however, reduced (125)I-FGF-2 binding to both the low and the high affinity receptors in the cell layer by approximately 60%. The results suggest that perlecan made by growth plate chondrocytes is a low affinity receptor for FGF-2 and acts to sequester FGF-2 away from the high affinity receptor.

Cytokine and chemokine expression profiles of maturing dendritic cells using multiprotein platform arrays.

Understanding the whole process of dendritic cell (DC) activation might help in the development of more efficient immunotherapeutic strategies for tumor patients. Part of this process is cytokine secretion, which has important effects on innate and adaptive immune response. Here, we cultured circulating monocytes for five days with interleukin-4 and GM-CSF followed by two-day culture with or without CD40 ligand and LPS to create a mature DC (mDC) and an immature DC (iDC) phenotype, respectively, characterized by differential expression of co-stimulatory molecules (CD80, CD83). We then compared the cytokine expression profile of the mDC and iDC using two protein platform arrays. Twelve supernatants from mDC paired with 12 from iDC were compared. The mDC protein expression profile showed significant increases in 16 out of 34 factors tested, including TNFalpha, IL-10, IL-12, IFNgamma, MIP1alpha, MIP1beta, IL-8, MDC, RANTES, and IL-6, which play a crucial role in the regulation of the innate immune response as well as the recruitment and activation of adaptive immune effectors. Interestingly, some of the cytokines expressed during maturation were also found in the gene expression profile identified in tumor metastases following IL-2 therapy using cDNA arrays. This finding suggests a possible role for resident DC maturation as a mediator of systemic IL-2 effects. Most important, the array of cytokines secreted during DC maturation may be considered an important component during adoptive transfer. Further characterization of the kinetics and persistence of their secretion should be undertaken in the future.

Repulsive axon guidance molecule Sema3A inhibits branching morphogenesis of fetal mouse lung

Semaphorin III/collapsin-1 (Sema3A) guides a specific subset of neuronal growth cones as a repulsive molecule. In this study, we have investigated a possible role of non-neuronal Sema3A in lung morphogenesis. Expression of mRNAs of Sema3A and neuropilin-1 (NP-1), a Sema3A receptor, was detected in fetal and adult lungs. Sema3A-immunoreactive cells were found in airway and alveolar epithelial cells of the fetal and adult lungs. Immunoreactivity for NP-1 was seen in fetal and adult alveolar epithelial cells as well as endothelial cells. Immunoreactivity of collapsin response mediator protein CRMP (CRMP-2), an intracellular protein mediating Sema3A signaling, was localized in alveolar epithelial cells, nerve tissue and airway neuroendocrine cells. The expression of CRMP-2 increased during the fetal, neonate and adult periods, and this pattern paralleled that of NP-1. In a two-day culture of lung explants from fetal mouse lung (E11.5), with exogenous Sema3A at a dose comparable to that which induces growth cone collapse of dorsal root ganglia neurons, the number of terminal buds was reduced in a dose-dependent manner when compared with control or untreated lung explants. This decrease was not accompanied with any alteration of the bromodeoxyuridine-positive DNA-synthesizing fraction. A soluble NP-1 lacking the transmembrane and intracellular region, neutralized the inhibitory effect of Sema3A. The fetal lung explants from neuropilin-1 homozygous null mice grew normally in vitro regardless of Sema3A treatment. These results provide evidence that Sema3A inhibits branching morphogenesis in lung bud organ cultures via NP-1 as a receptor or a component of a possible multimeric Sema3A receptor complex.

Extracellular products as mediators of the formation and detachment of Pseudomonas fluorescens biofilms

Pseudomonas fluorescens B52 produces substantial biofilms at the air/liquid/solid interface of glass coverslips clamped vertically and partly submerged in liquid medium at 21°C. Biofilm formation was maximal ca. 20–50 h after inoculation of the liquid medium and, as indicated by environmental scanning electron microscopy (ESEM), contained large numbers of bacterial cells that were embedded within an extensive exopolymeric matrix. Incubation beyond 50 h led to reductions in biofilm which ESEM related primarily to losses of exopolymer. Both biofilm formation and the subsequent decline in exopolymer deposition was more rapid, and occurred to greater extents, when supernatants from two-day old cultures of B52 were used as the initial growth media. The addition of N-acyl-hexanoyl homoserine lactone to fresh growth medium had a similar effect upon biofilm formation as using spent culture medium. Homoserine lactones could not be demonstrated in spent culture supernatants by an Agrobacterium tumefaciens bioassay. An exopolysaccharide lyase was detected in spent culture media taken from dense biofilm cultures whose action was specifically directed towards biofilm exopolysaccharide. Results suggest that (i) cell-cell signals such as homoserine lactones are associated with the formation of P. fluorescens biofilms, (ii) the enzymic degradation of exopolymers has a specific role in the detachment of cells under starvation conditions, and (iii) whilst short chain (C 6) exogenous homoserines can trigger such responses in P. fluorescens, its own signal substance is likely to possess a longer (>C 8) fatty acyl chain.

Extracellular products as mediators of the formation and detachment of Pseudomonas fluorescens biofilms.

Pseudomonas fluorescens B52 produces substantial biofilms at the air/liquid/solid interface of glass coverslips clamped vertically and partly submerged in liquid medium at 21 degrees C. Biofilm formation was maximal ca. 20-50 h after inoculation of the liquid medium and as indicated by environmental scanning electron microscopy (ESEM), contained large numbers of bacterial cells that were embedded within an extensive exopolymeric matrix. Incubation beyond 50 h led to reductions in biofilm which ESEM related primarily to losses of exopolymer. Both biofilm formation and the subsequent decline in exopolymer deposition was more rapid, and occurred to greater extents, when supernatants from two-day old cultures of B52 were used as the initial growth media. The addition of N-acyl-hexanoyl homoserine lactone to fresh growth medium had a similar effect upon biofilm formation as using spent culture medium. Homoserine lactones could not be demonstrated in spent culture supernatants by an Agrobacterium tumefaciens bioassay. An exopolysaccharide lyase was detected in spend culture media taken from dense biofilm cultures whose action was specifically directed towards biofilm exopolysaccharide. Results suggest that (i) cell-cell signals such as homoserine lactones are associated with the formation of P. fluorescens biofilms, (ii) the enzymic degradation of exopolymers has a specific role in the detachment of cells under starvation conditions, and (iii) whilst short chain (C6) exogenous homoserines can trigger such response in P. fluorescens, its own signal substance is likely to possess a longer (> C8) fatty acyl chain.

P53 and protein kinase C independent induction of growth arrest and apoptosis by bryostatin 1 in a highly metastatic mammary epithelial cell line: In vitro versus in vivo activity.

We have evaluated the effects of bryostatin 1 on growth of a highly malignant p53-null mouse mammary tumor line, 4T1, and the mechanism by which bryostatin 1 inhibits in vitro growth and in vivo development of tumor and metastases from the orthotopic site. Bryostatin 1 at 20-400 nM concentrations inhibits growth of 4T1 cells by approximately 60% in two-day cultures. Inhibition of growth is associated with an increase in the number of cells undergoing apoptosis with concomitant elevation in the steady state levels of bax protein and drop in bcl-2 levels. The cytotoxic effect of bryostatin 1 on 4T1 cells occurs independently of p53, since there was no evidence of p53-mediated transcriptional activity in 4T1 cells following treatment with bryostatin 1.4T1 cells respond in vivo to bryostatin 1 therapy (75 microg/kg body weight). Intraperitoneal administration of bryostatin 1 inhibits both primary and secondary tumor growth by approximately 50%. However, although bryostatin 1 has a remarkable capacity to slow tumor growth and progression, it is unable to completely eradicate tumor growth and progression due to in vivo development of tumor resistance to bryostatin 1. Levels and cellular distribution of PKCalpha and delta do not correlate with the growth inhibitory effects of bryostatin 1 on 4T1 cells; however, reduction in cytosolic PKCalpha and delta without associated increase in membrane compartment appear to correlate with bryostatin-resistance. Our results suggest that the therapeutic effects of bryostatin 1 in our system do not involve alterations in levels and distribution of PKC but rather a direct upregulation of bax/ bcl-2 ratios that is independent of p53.

Volatile fatty acids, metabolic by-products of periodontopathic bacteria, inhibit lymphocyte proliferation and cytokine production.

Short-chain fatty acids are a major by-product of anaerobic metabolism and can be detected in gingival fluid from periodontal pockets. Since most T cells are present subjacent to the pocket epithelium in conjunction with the plasma cells, it is important to know how these T cells are affected by short-chain fatty acids produced by subgingival plaque. The purpose of this study is to examine the effects of extracellular metabolites from periodontopathic bacteria on the proliferation and cytokine production of mouse splenic cells as a potential mechanism of imbalance among host-microbial interactions. A low-molecular-weight, heat-stable agent present in the two-day culture filtrate of Porphyromonas gingivalis, Prevotella loescheii, and Fusobacterium nucleatum significantly depressed Con A- and LPS- induced cell proliferation. To determine whether short-chain fatty acids present in the filtrate could account for the depression, we tested extracted volatile and non-volatile fatty acids for their effects on mitogenic activity. The volatile fatty acids extracted from immunosuppressive supernatants greatly inhibited T- and B- cell proliferation. Among these volatile fatty acids, butyric, propionic, valeric, and isovaleric acids impaired cell proliferation dose-dependently. From gas-liquid chromatographic analysis data, it is suggested that immuno-inhibitory activities in culture filtrates are mainly attributable to butyric and isovaleric acids in P. gingivalis, to propionic, butyric, and isovaleric acids in P. loescheii, and to butyric acid in F. nucleatum. Furthermore, these fatty acids significantly depressed interleukin 2 (IL-2), IL-4, IL-5, IL-6, and IL-10 production by Con A-stimulated splenic-T cells dose-dependently.(ABSTRACT TRUNCATED AT 250 WORDS)

Forskolin mimics TSH action on the expression of protein kinase C isozymes in pig thyroid cell cultures

In porcine thyroid cell cultures, phospholipid-dependent protein kinases (PKCs) have the same characteristics as intact glands. The overall PKC activity, presence of PKC isozymes, chromatographic pattern and endogenous substrates specificity were not modified during the two-day culture period. Three PKC isozymes (cPKC epsilon, nPKC epsilon and aPKC zeta) were identified by immunoblot analysis in the two subcellular fractions: cytosol and particulate extract, both in intact glands and two-day-old cultures. In cells cultured for two days in the presence of TSH (0.1 mU/ml), the overall PKC activity was stimulated (ca. 200%) in the two compartments. This stimulation was parallel to the increase in protein expression of the three PKC isoforms (as demonstrated by immunoblot analysis) and was accompanied by a redistribution of cPKC alpha and nPKC epsilon toward the particulate fraction. In TSH-treated cells, hydroxyapatite chromatography of cytosolic PKC revealed an additional peak of PKC activity eluted at 195 mM potassium phosphate. Its elution molarity did not correspond to the molarity of any known PKC isozyme, and it did not cross-react with antibodies directed against cPKC isozymes--: alpha, beta, or gamma. When TSH was replaced by forskolin (10(-5) M), identical quantitative and qualitative modifications were obtained, suggesting that, in thyroid cells, the cyclic AMP-dependent regulatory cascade could be involved in the control of PKC isoforms expression by TSH.

Synergistic stimulation of macrophage proliferation by the monokines tumor necrosis factor-alpha and colony-stimulating factor 1

The effects of pure recombinant human tumor necrosis factor-alpha (TNF) on the CSF-1-stimulated proliferation of well-defined populations of murine macrophages are examined. Primary bone marrow-derived macrophages (BMM) from endotoxin-resistant C3H/HeJ mice were characterized for homogeneity in comparison with a cloned, growth factor-dependent macrophage cell line (S1) also derived from C3H/HeJ bone marrow cells. The mitogenic effects of each factor, alone and in combination, on the proliferation of both macrophage populations over a two-day culture period were studied. In contrast to CSF-1, TNF alone only slightly stimulated macrophage proliferation. However, the combination of CSF-1 and TNF stimulated proliferation of both primary BMM and S1 cells 1.5- to 2-fold greater than the sum of their predicted individual contributions. Such synergy was observed even at very high (plateau) levels of factors. TNF was found to transiently down-regulate CSF-1 receptor levels on both populations. Down-regulation was maximal at one hour; however, receptor numbers returned to initial, or greater, levels after 24 hours of incubation. Thus, TNF, an inducible monokine, greatly enhances the maximal mitogenic effects of CSF-1, an inducer of TNF production. These observations suggest an autocrine rule for TNF that involves synergy with (and perhaps obligatory cooperation with) CSF-1 in the regulation of macrophage proliferation.

Synergistic Stimulation of Macrophage Proliferation by the Monokines Tumor Necrosis Factor-Alpha and Colony-Stimulating Factor 1

The effects of pure recombinant human tumor necrosis factor-alpha (TNF) on the CSF-1-stimulated proliferation of well-defined populations of murine macrophages are examined. Primary bone marrow-derived macrophages (BMM) from endotoxin-resistant C3H/HeJ mice were characterized for homogeneity in comparison with a cloned, growth factor-dependent macrophage cell line (S1) also derived from C3H/HeJ bone marrow cells. The mitogenic effects of each factor, alone and in combination, on the proliferation of both macrophage populations over a two-day culture period were studied. In contrast to CSF-1, TNF alone only slightly stimulated macrophage proliferation. However, the combination of CSF-1 and TNF stimulated proliferation of both primary BMM and S1 cells 1.5- to 2-fold greater than the sum of their predicted individual contributions. Such synergy was observed even at very high (plateau) levels of factors. TNF was found to transiently down-regulate CSF-1 receptor levels on both populations. Down-regulation was maximal at one hour; however, receptor numbers returned to initial, or greater, levels after 24 hours of incubation. Thus, TNF, an inducible monokine, greatly enhances the maximal mitogenic effects of CSF-1, an inducer of TNF production. These observations suggest an autocrine rule for TNF that involves synergy with (and perhaps obligatory cooperation with) CSF-1 in the regulation of macrophage proliferation.