Characteristics of Biogas Production and Synergistic Effect of Primary Sludge and Food Waste Co-Digestion.

Co-digestion implementation in wastewater treatment plants enhances biogas yield, so this research investigated the optimal ratio of biodegradable waste and sewage sludge. The increase in biogas production was investigated through batch tests using basic BMP equipment, while synergistic effects were evaluated by chemical oxygen demand (COD) balance. Analyses were performed in four volume basis ratios (3/1, 1/1, 1/3, 1/0) of primary sludge and food waste with added low food waste: 3.375%, 4.675%, and 5.35%, respectively. The best proportion was found to be 1/3 with the maximum biogas production (618.7 mL/g VS added) and the organic removal of 52.8% COD elimination. The highest enhancement rate was observed among co-digs 3/1 and 1/1 (105.72 mL/g VS). A positive correlation between biogas yield and COD removal is noticed while microbial flux required an optimal pH, value of 8 significantly decreased daily production rate. COD reductions further supported the synergistic impact; specifically, an additional 7.1%, 12.8%, and 17% of COD were converted into biogas during the co-digestions 1, 2, and 3, respectively. Three mathematical models were applied to estimate the kinetic parameters and check the accuracy of the experiment. The first-order model with a hydrolysis rate of 0.23-0.27 indicated rapidly biodegradable co-/substrates, modified Gompertz confirmed immediate commencement of co-digs through zero lag phase, while the Cone model had the best fit of over 99% for all trials. Finally, the study points out that the COD method based on linear dependence can be used for developing relatively accurate model for biogas potential estimation in anaerobic digestors. Supplementary Information: The online version contains supplementary material available at 10.1007/s12155-023-10620-8.

Whole genome sequence analysis of the TALLYHO/Jng mouse.

BACKGROUND: The TALLYHO/Jng (TH) mouse is a polygenic model for obesity and type 2 diabetes first described in the literature in 2001. The origin of the TH strain is an outbred colony of the Theiler Original strain and mice derived from this source were selectively bred for male hyperglycemia establishing an inbred strain at The Jackson Laboratory. TH mice manifest many of the disease phenotypes observed in human obesity and type 2 diabetes. RESULTS: We sequenced the whole genome of TH mice maintained at Marshall University to a depth of approximately 64.8X coverage using data from three next generation sequencing runs. Genome-wide, we found approximately 4.31 million homozygous single nucleotide polymorphisms (SNPs) and 1.10 million homozygous small insertions and deletions (indels) of which 98,899 SNPs and 163,720 indels were unique to the TH strain compared to 28 previously sequenced inbred mouse strains. In order to identify potentially clinically-relevant genes, we intersected our list of SNP and indel variants with human orthologous genes in which variants were associated in GWAS studies with obesity, diabetes, and metabolic syndrome, and with genes previously shown to confer a monogenic obesity phenotype in humans, and found several candidate variants that could be functionally tested using TH mice. Further, we filtered our list of variants to those occurring in an obesity quantitative trait locus, tabw2, identified in TH mice and found a missense polymorphism in the Cidec gene and characterized this variant's effect on protein function. CONCLUSIONS: We generated a complete catalog of variants in TH mice using the data from whole genome sequencing. Our findings will facilitate the identification of causal variants that underlie metabolic diseases in TH mice and will enable identification of candidate susceptibility genes for complex human obesity and type 2 diabetes.

Calculating the costs of waste collection: A methodological proposal.

Waste collection and transport can generate up to 70% of the total costs of the system. Separated collection of recyclables implies additional costs for which the sale of recycled waste often does not compensate, but there is increased pressure to reach the long-term recycling objectives set by law. The proper estimation and monitoring of waste collection costs are essential to define the most cost-effective waste collection system. The aim of this study is to propose and implement a management tool to determine waste collection costs for different waste collection schemes. Based on input data, such as waste quantity and composition, the number of waste bins, the location of collection points, the type of collection vehicle, crew, collection route, etc., the developed tool can calculate the time and costs of waste collection (per vehicle, collection point or tonne of collected waste). This tool uses Excel spreadsheets and it was tested on a district in the central area of the city of Kragujevac to calculate the costs of waste collection for two scenarios: Collecting all waste as mixed waste, and collecting separately recyclables and residual waste. The developed tool can be useful for municipal solid waste management companies, since it allows benchmarking and variance analysis.

Using multi-criteria decision making for selection of the optimal strategy for municipal solid waste management.

Multi-criteria decision making (MCDM) is a relatively new tool for decision makers who deal with numerous and often contradictory factors during their decision making process. This paper presents a procedure to choose the optimal municipal solid waste (MSW) management system for the area of the city of Kragujevac (Republic of Serbia) based on the MCDM method. Two methods of multiple attribute decision making, i.e. SAW (simple additive weighting method) and TOPSIS (technique for order preference by similarity to ideal solution), respectively, were used to compare the proposed waste management strategies (WMS). Each of the created strategies was simulated using the software package IWM2. Total values for eight chosen parameters were calculated for all the strategies. Contribution of each of the six waste treatment options was valorized. The SAW analysis was used to obtain the sum characteristics for all the waste management treatment strategies and they were ranked accordingly. The TOPSIS method was used to calculate the relative closeness factors to the ideal solution for all the alternatives. Then, the proposed strategies were ranked in form of tables and diagrams obtained based on both MCDM methods. As shown in this paper, the results were in good agreement, which additionally confirmed and facilitated the choice of the optimal MSW management strategy.

Fast methodology to design the optimal collection point locations and number of waste bins: A case study.

This paper concerns the development of a methodology aimed at determining the optimal number of waste bins as well optimizing the location of collection points. The methodology was based on a geographic information system, which handled different sets of information, such as street directions, spatial location of objects and number of inhabitants, location of waste bins, and radius of their coverage. The study was conducted in a district in the central area of the city of Kragujevac. Due to a lack of information about the existing situation, all necessary data was collected by fieldwork and by using GPS equipment. By using the developed methodology, the results indicated a reduction of 24% in the number of collection points and 33.5% in the number of waste bins, without reducing the quality of the provided services. It has led to cost and time savings for waste collection and environmental benefits. All users of the services were covered within a 75-m radius, and the usage of bins is more efficient. According to the reduction in the number of waste bins, a total amount of €26,000 may be achieved. In addition, the time for waste collection was reduced, resulting in a €1700 saving per year in fuel costs, as well as 4.5 tons of emitted CO2 into the atmosphere.

Optimization of thiamethoxam adsorption parameters using multi-walled carbon nanotubes by means of fractional factorial design.

The aim of this work was to evaluate significant factors affecting the thiamethoxam adsorption efficiency using oxidized multi-walled carbon nanotubes (MWCNTs) as adsorbents. Five factors (initial solution concentration of thiamethoxam in water, temperature, solution pH, MWCNTs weight and contact time) were investigated using 2V(5-1) fractional factorial design. The obtained linear model was statistically tested using analysis of variance (ANOVA) and the analysis of residuals was used to investigate the model validity. It was observed that the factors and their second-order interactions affecting the thiamethoxam removal can be divided into three groups: very important, moderately important and insignificant ones. The initial solution concentration was found to be the most influencing parameter on thiamethoxam adsorption from water. Optimization of the factors levels was carried out by minimizing those parameters which are usually critical in real life: the temperature (energy), contact time (money) and weight of MWCNTs (potential health hazard), in order to maximize the adsorbed amount of the pollutant. The results of maximal adsorbed thiamethoxam amount in both real and optimized experiments indicate that among minimized parameters the adsorption time is one that makes the largest difference. The results of this study indicate that fractional factorial design is very useful tool for screening the higher number of parameters and reducing the number of adsorption experiments.

Identification of the PS1 Thr147Ile Variant in a Family with Very Early Onset Dementia and Expressive Aphasia.

BACKGROUND: Early onset dementias have variable clinical presentations and are often difficult to diagnose. We established a family pedigree that demonstrated consistent recurrence of very early onset dementia in successive generations. OBJECTIVE AND METHOD: In order to refine the diagnosis in this family, we sequenced the exomes of two affected family members and relied on discrete filtering to identify disease genes and the corresponding causal variants. RESULTS: Among the 720 nonsynonymous single nucleotide polymorphisms (SNPs) shared by two affected members, we found a C to T transition that gives rise to a Thr147Ile missense substitution in the presenilin 1 (PS1) protein. The presence of this same mutation in a French early-onset Alzheimer's disease family, other affected members of the family, and the predicted high pathogenicity of the substitution strongly suggest that it is the causal variant. In addition to exceptionally young age of onset, we also observed significant limb spasticity and early loss of speech, concurrent with progression of dementia in affected family members. These findings extend the clinical presentation associated with the Thr147Ile variant. Lastly, one member with the Thr147Ile variant was treated with the PKC epsilon activator, bryostatin, in a compassionate use trial after successful FDA review. Initial improvements with this treatment were unexpectedly clear, including return of some speech, increased attentional focus, ability to swallow, and some apparent decrease in limb spasticity. CONCLUSIONS: Our findings confirm the role of the PS1 Thr147Ile substitution in Alzheimer's disease and expand the clinical phenotype to include expressive aphasia and very early onset of dementia.

Endogenous aryl hydrocarbon receptor promotes basal and inducible expression of tumor necrosis factor target genes in MCF-7 cancer cells.

The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor that upon activation by the toxicant 2,3,7,8 tetrachlorodibenzo-p-dioxin (TCDD) stimulates gene expression and toxicity. AHR is also important for normal mouse physiology and may play a role in cancer progression in the absence of environmental toxicants. The objective of this report was to identify AHR-dependent genes (ADGs) whose expression is regulated by AHR in the absence of toxicants. RNA-Seq analysis revealed that AHR regulated the expression of over 600 genes at an FDR<10% in MCF-7 breast cancer cells upon knockdown with short interfering RNA. Pathway analysis revealed that a significant number of ADGs were components of TCDD and tumor necrosis factor (TNF) pathways. We also demonstrated that siRNA knockdown of AHR modulated TNF induction of MNSOD and cytotoxicity in MCF-7 cells. Collectively, the major new findings of this report are: (1) endogenous AHR promotes the expression of xenobiotic metabolizing enzymes even in the absence of toxicants and drugs, (2) AHR by modulating the basal expression of a large fraction of TNF target genes may prime them for TNF stimulation and (3) AHR is required for TNF induction of MNSOD and the cellular response to cytotoxicity in MCF-7 cells. This latter result provides a potentially new role for AHR in MCF-7 cancer progression as a mediator of TNF and antioxidant responses.

RGS16, a novel p53 and pRb cross-talk candidate inhibits migration and invasion of pancreatic cancer cells.

Data collected since the discovery of p53 and pRb/RB1 suggests these tumor suppressors cooperate to inhibit tumor progression. Patients who have mutations in both p53 and RB1 genes have increased tumor reoccurrence and decreased survival compared to patients with only one tumor suppressor gene inactivated. It remains unclear how p53 and pRb cooperate toward inhibiting tumorigenesis. Using RNA expression profiling we identified 179 p53 and pRb cross-talk candidates in normal lung fibroblasts (WI38) cells exogenously coexpressing p53 and pRb. Regulator of G protein signaling 16 (RGS16) was among the p53 and pRb cross-talk candidates and has been implicated in inhibiting activation of several oncogenic pathways associated with proliferation, migration, and invasion of cancer cells. RGS16 has been found to be downregulated in pancreatic cancer patients with metastases compared to patients without metastasis. Expression of RGS16 mRNA was decreased in the pancreatic cancer cell lines tested compared to control. Expression of RGS16 inhibited migration of the BxPC-3 and AsPC-1 but not PANC-1 cells and inhibited invasion of BxPC-3 and AsPC-1 cells with no impact on cell viability. We have identified for the first time p53 and pRb cross-talk candidates and a role for RGS16 to inhibit pancreatic cancer migration and invasion.

Inhibition of nuclear factor kappa B activation in early-stage chronic lymphocytic leukemia by omega-3 fatty acids.

Targeting the nuclear factor kappa B (NFκB) pathway is proposed as therapy for chronic lymphocytic leukemia (CLL). We hypothesized that an omega-3 fatty acids (n-3) supplement would suppress NFκB activation in lymphocytes of Rai Stage 0-1 CLL patients. The initial dose of 2.4 g n-3/day was gradually increased to 7.2 g n-3/day. After n-3 consumption: 1) plasma n-3 increased; 2) NFκB activation was suppressed in lymphocytes; 3) in vitro sensitivity of lymphocytes to doxorubicin was increased; and 4) expression of 32 genes in lymphocytes was significantly decreased.

High throughput DNA sequencing to detect differences in the subgingival plaque microbiome in elderly subjects with and without dementia.

BACKGROUND: To investigate the potential association between oral health and cognitive function, a pilot study was conducted to evaluate high throughput DNA sequencing of the V3 region of the 16S ribosomal RNA gene for determining the relative abundance of bacterial taxa in subgingival plaque from older adults with or without dementia. METHODS: Subgingival plaque samples were obtained from ten individuals at least 70 years old who participated in a study to assess oral health and cognitive function. DNA was isolated from the samples and a gene segment from the V3 portion of the 16S bacterial ribosomal RNA gene was amplified and sequenced using an Illumina HiSeq1000 DNA sequencer. Bacterial populations found in the subgingival plaque were identified and assessed with respect to the cognitive status and oral health of the participants who provided the samples. RESULTS: More than two million high quality DNA sequences were obtained from each sample. Individuals differed greatly in the mix of phylotypes, but different sites from different subgingival depths in the same subject were usually similar. No consistent differences were observed in this small sample between subjects separated by levels of oral health, sex, or age; however a consistently higher level of Fusobacteriaceae and a generally lower level of Prevotellaceae was seen in subjects without dementia, although the difference did not reach statistical significance, possibly because of the small sample size. CONCLUSIONS: The results from this pilot study provide suggestive evidence that alterations in the subgingival microbiome are associated with changes in cognitive function, and provide support for an expanded analysis of the role of the oral microbiome in dementia.

Bone marrow osteoblast damage by chemotherapeutic agents.

Hematopoietic reconstitution, following bone marrow or stem cell transplantation, requires a microenvironment niche capable of supporting both immature progenitors and stem cells with the capacity to differentiate and expand. Osteoblasts comprise one important component of this niche. We determined that treatment of human primary osteoblasts (HOB) with melphalan or VP-16 resulted in increased phospho-Smad2, consistent with increased TGF-ß1 activity. This increase was coincident with reduced HOB capacity to support immature B lineage cell chemotaxis and adherence. The supportive deficit was not limited to committed progenitor cells, as human embryonic stem cells (hESC) or human CD34+ bone marrow cells co-cultured with HOB pre-exposed to melphalan, VP-16 or rTGF-ß1 had profiles distinct from the same populations co-cultured with untreated HOB. Functional support deficits were downstream of changes in HOB gene expression profiles following chemotherapy exposure. Melphalan and VP-16 induced damage of HOB suggests vulnerability of this critical niche to therapeutic agents frequently utilized in pre-transplant regimens and suggests that dose escalated chemotherapy may contribute to post-transplantation hematopoietic deficits by damaging structural components of this supportive niche.

Identification of the early VIP-regulated transcriptome and its associated, interactome in resting and activated murine CD4 T cells.

More than 40 years after the discovery of vasoactive intestinal peptide (VIP), its transcriptome in the immune system has still not been completely elucidated. In an attempt to understand the biological role of this neuropeptide in immunity, we chose CD4 T cells as a cellular system. Agilent Mouse Whole Genome microarrays were hybridized with fluorescently labeled total RNA isolated from resting CD4 T cells cultured +/-10(-7)M VIP for 5h or PMA/ionomycin activated CD4 T cells cultured +/-10(-7)M VIP for 5h. These VIP-regulated transcriptomes were analyzed by Significance Analysis of Microarrays (SAM) and Ingenuity Pathway Analysis (IPA) software to identify relevant signaling pathways modulated by VIP in the absence and presence of T cell activation. In resting CD4 T cells, VIP-modulated 368 genes, ranging from 3.49 to -4.78-fold. In the PMA/ionomycin activated CD4 T cells, 326 gene expression levels were changed by VIP, ranging from 2.94 to -1.66-fold. IPA analysis revealed that VIP exposure alters cellular function through EGFR signaling in resting CD4 T cells, and modulates immediate early genes, Fos and CREM/ICER, in activated CD4 T cells. These gene expression changes are suggested to explain at a molecular level how VIP can regulate T cell homing to the gut and induce regulatory T cell generation.

Chmp 1A is a mediator of the anti-proliferative effects of all-trans retinoic acid in human pancreatic cancer cells.

BACKGROUND: We recently have shown that Charged multivesicular protein/Chromatin modifying protein1A (Chmp1A) functions as a tumor suppressor in human pancreatic tumor cells. Pancreatic cancer has the worst prognosis of all cancers with a dismal 5-year survival rate. Preclinical studies using ATRA for treating human pancreatic cancer suggest this compound might be useful for treatment of pancreatic cancer patients. However, the molecular mechanism by which ATRA inhibits growth of pancreatic cancer cells is not clear. The objective of our study was to investigate whether Chmp1A is involved in ATRA-mediated growth inhibition of human pancreatic tumor cells. RESULTS: We performed microarray studies using HEK 293T cells and discovered that Chmp1A positively regulated Cellular retinol-binding protein 1 (CRBP-1). CRBP-1 is a key regulator of All-trans retinoic acid (ATRA) through ATRA metabolism and nuclear localization. Since our microarray data indicates a potential involvement of Chmp1A in ATRA signaling, we tested this hypothesis by treating pancreatic tumor cells with ATRA in vitro. In the ATRA-responsive cell lines, ATRA significantly increased the protein expression of Chmp1A, CRBP-1, P53 and phospho-P53 at serine 15 and 37 position. We found that knockdown of Chmp1A via shRNA abolished the ATRA-mediated growth inhibition of PanC-1 cells. Also, Chmp1A silencing diminished the increase of Chmp1A, P53 and phospho-P53 protein expression induced by ATRA. In the ATRA non-responsive cells, ATRA did not have any effect on the protein level of Chmp1A and P53. Chmp1A over-expression, however, induced growth inhibition of ATRA non-responsive cells, which was accompanied by an increase of Chmp1A, P53 and phospho-P53. Interestingly, in ATRA responsive cells Chmp1A is localized to the nucleus, which became robust upon ATRA treatment. In the ATRA-non-responsive cells, Chmp1A was mainly translocated to the plasma membrane upon ATRA treatment. CONCLUSION: Collectively our data provides evidence that Chmp1A mediates the growth inhibitory activity of ATRA in human pancreatic cancer cells via regulation of CRBP-1. Our results also suggest that nuclear localization of Chmp1A is important in mediating ATRA signaling.

Proteomic and genomic analysis of PITX2 interacting and regulating networks.

Pituitary homeobox 2 (PITX2) is a homeodomain transcription factor that has a substantial role in cell proliferation and differentiation in various tissues. In this report, we have conducted a systematic study, using proteomic and genomic approaches, to characterize PITX2-interacting proteins and PITX2-regulating genes. We identified four novel PITX2-associated protein partners Y box binding factor-1, heterogeneous ribonucleoprotein K, nucleolin and heterogeneous nuclear ribonucleoprotein U in mass spectrometry analysis. We also found that overexpression of PITX2 upregulated 868 genes (2-25-fold) and downregulated 191 genes (2-15-fold) in DNA microarray analysis. These data provide an insightful perspective for further studying PITX2 function and mechanism of action.

Global analysis of gene expression changes during retinoic acid-induced growth arrest and differentiation of melanoma: comparison to differentially expressed genes in melanocytes vs melanoma.

BACKGROUND: The incidence of malignant melanoma has significantly increased over the last decade. Some of these malignancies are susceptible to the growth inhibitory and pro-differentiating effects of all-trans-retinoic acid (RA). The molecular changes responsible for the biological activity of RA in melanoma are not well understood. RESULTS: In an analysis of sequential global gene expression changes during a 4-48 h RA treatment of B16 mouse melanoma cells, we found that RA increased the expression of 757 genes and decreased the expression of 737 genes. We also compared the gene expression profile (no RA treatment) between non-malignant melan-a mouse melanocytes and B16 melanoma cells. Using the same statistical test, we found 1495 genes whose expression was significantly higher in melan-a than in B16 cells and 2054 genes whose expression was significantly lower in melan-a than in B16 cells. By intersecting these two gene sets, we discovered a common set of 233 genes whose RNA levels were significantly different between B16 and melan-a cells and whose expression was altered by RA treatment. Within this set, RA treatment altered the expression of 203 (87%) genes toward the melan-a expression level. In addition, hierarchical clustering showed that after 48 h of RA treatment expression of the 203 genes was more closely related to the melan-a gene set than any other RA treatment time point. Functional analysis of the 203 gene set indicated that RA decreased expression of mRNAs that encode proteins involved in cell division/cell cycle, DNA replication, recombination and repair, and transcription regulation. Conversely, it stimulated genes involved in cell-cell signaling, cell adhesion and cell differentiation/embryonic development. Pathway analysis of the 203 gene set revealed four major hubs of connectivity: CDC2, CHEK1, CDC45L and MCM6. CONCLUSION: Our analysis of common genes in the 48 h RA-treatment of B16 melanoma cells and untreated B16 vs. melan-a data set show that RA "normalized" the expression of genes involved in energy metabolism, DNA replication, DNA repair and differentiation. These results are compatible with the known growth inhibitory and pro-differentiating effects of RA. Pathway analysis suggests that CDC2, CHEK1, CDC45L and MCM6 are key players in mediating the biological activity of RA in B16 melanoma cells.

The morphology of the NiO-Al2O3 catalyst.

The morphology of the NiO-Al2O3 catalyst, prepared by coprecipitation, impregnation, and mechanical powder mixing with ca. 5, 10, and 20 wt% nickel treated at 400 degrees, 700 degrees, and 1100 degrees C, was studied. Scanning electron microscopy is a useful technique for determining the manner of catalyst preparation for particle size estimation of catalyst components, whereas their mutual interactions become visible in the obtained images.

T-box binding protein type two (TBX2) is an immediate early gene target in retinoic-acid-treated B16 murine melanoma cells.

Retinoic acid induces growth arrest and differentiation in B16 mouse melanoma cells. Using gene arrays, we identified several early response genes whose expression is altered by retinoic acid. One of the genes, tbx2, is a member of T-box nuclear binding proteins that are important morphogens in developing embryos. Increased TBX2 mRNA is seen within 2 h after addition of retinoic acid to B16 cells. The effect of retinoic acid on gene expression is direct since it does not require any new protein synthesis. We identified a degenerate retinoic acid response element (RARE) between -186 and -163 in the promoter region of the tbx2 gene. A synthetic oligonucleotide spanning this region was able to drive increased expression of a luciferase reporter gene in response to retinoic acid; however, this induction was lost when a point mutation was introduced into the RARE. This oligonucleotide also specifically bound RAR in nuclear extracts from B16 cells. TBX2 expression and its induction by retinoic acid was also observed in normal human and nonmalignant mouse melanocytes.

Retinoic acid-induced AP-1 transcriptional activity regulates B16 mouse melanoma growth inhibition and differentiation.

Retinoic acid (RA) inhibits growth and induces differentiation of B16 mouse melanoma cells. These effects are accompanied by a large increase in PKCalpha mRNA and protein levels and surprisingly an increase in activating protein-1 (AP-1) transcriptional activity. To further investigate the RA-induced AP-1 activity we established clones of B16 cells stably expressing an AP-1-luciferase reporter gene. Treatment of these clones with phorbol dibutyrate increased AP-1 activity which peaked at 2-4 h and returned to baseline level by 24 h. In contrast, RA treatment resulted in a slow increase in AP-1 activity that reached a maximum level at 48 h and was maintained for the duration of the treatment. We tested the importance of the RA-induced AP-1 activity by establishing clones which stably express a dominant negative fos gene (A-fos) and have greatly diminished AP-1 activity. Growth rates of untreated A-fos expressing cells were similar to wt B16 and clones not expressing A-fos. However, clones expressing the dominant-negative fos had a markedly decreased sensitivity to RA-induced inhibition of anchorage-dependent and -independent growth. Treatment of wt B16 cells for 48 h with RA increased melanin production by two to fourfold, but this effect was completely lost in the A-fos clones. The ability of RA to induce RARbeta and PKCalpha expression was retained in A-fos clones, suggesting that A-fos was not interfering with RAR transcription activation functions. We tested whether the RA-induced AP-1 activity might be mediated by the ERK1/2 MAPK pathway. Inhibition of ERK1/2 phosphorylation stimulated AP-1 activity, which was not additive to that induced by RA. This finding raises the possibility that this MAPK pathway may be a target of retinoid action. Our observations suggest that AP-1 transcriptional activity induced by RA likely plays an important role in the biological changes mediated by this retinoid in B16 melanoma cells.

Regulation of retinoic acid receptor alpha by protein kinase C in B16 mouse melanoma cells.

We have previously found that retinoic acid stimulates the expression of protein kinase C alpha (PKC) in B16 mouse melanoma cells. Because it has been reported that PKC can phosphorylate retinoic acid receptor (RAR) and alter its function, we determined whether changes in the level and/or activity of PKC could affect the expression or function of the RAR in B16 melanoma. Using in vivo phosphorylation and band shift techniques, we could not demonstrate that altering PKC activity and/or protein level changed the in vivo phosphorylation of RAR alpha. However activation of PKC resulted in increased RAR alpha protein. Increased receptor protein correlated with a phorbol dibutyrate-stimulated increase in receptor activation function-2 (AF-2)-dependent transcriptional activity. Use of enzyme inhibitors and dominant-negative PKCs indicated that enzyme activity was required for elevation in the RAR alpha. The PKC-mediated increase in RAR alpha was due to a 2.5-fold increase in the half-life of this protein. In contrast, the down-regulation of PKC diminished RAR alpha protein half-life and markedly inhibited AF-2-dependent transcriptional activity. The down-regulation of PKC also inhibited the binding of RAR to a retinoic acid response element and the retinoic acid induction of RAR beta expression. These findings suggest that PKC can influence retinoic acid signaling by altering the stability of RAR protein without directly phosphorylating this receptor.