The high mutation rate at the D614G hotspot-furin cleavage site region increases the priming efficiency of the Spike protein by furin protease: analysis of Indonesian SARS-CoV-2 G614 variants obtained during the early COVID-19 pandemic.

D614G mutation plays a significant role in the transmissibility of SARS-CoV-2. Identification of other mutations related to D614G mutation within the Spike protein is pivotal as they might contribute to the pathogenicity of SARS-CoV-2. This study aims to analyze the mutation rate of furin cleavage site (FCS) region of Indonesian origin SARS-CoV-2 and to predict the effect of mutation against Spike priming efficiency by furin. A total of 375 sequences of Indonesian isolates obtained during the early pandemic were used for mutation analysis. Mutation analysis includes mutation pattern, variability, frequency of mutation, amino acid conservation, and mutation rate. The effect of mutation against Spike priming efficiency by furin protease from eight sequences with mutation in the FCS region was analyzed by protein-protein docking. We showed that mutations related to the G614 variant were increasing through time, in contrast to the D614 variant. The FCS region at the position 675-692 contained the most variable (66.67%) as well as the highest mutation frequency (85.92%) and has been observed to be the hotspot mutations linked to the D614G mutation. The D614G hotspot-FCS region (residue 600-700) had the highest amino acid change per site (20.8%) as well as the highest mutation rate as 1.34 × 10-2 substitution per site per year (95% CI 1.79 × 10-3-2.74 × 10-2), compared with other Spike protein regions. Mutations in the FCS region were the most common mutation found after the D614G mutation. These mutations were predicted to increase the Spike priming efficiency by furin. Thus, this study elucidates the importance of D614G mutation to other mutations located in the FCS region and their significance to Spike priming efficiency by furin. Supplementary Information: The online version contains supplementary material available at 10.1007/s13337-023-00827-w.

Draft Genome Sequence of the Marine-Derived, Anticancer Compound-Producing Bacterium Streptomyces sp. Strain GMY01.

The marine Streptomyces sp. strain GMY01 was isolated from Indonesian marine sediment. Genome mining analysis revealed that GMY01 has 28 biosynthetic gene clusters, dominated by genes encoding nonribosomal peptide synthetase and polyketide synthase.

Growth in chikungunya virus-related research in ASEAN and South Asian countries from 1967 to 2022 following disease emergence: a bibliometric and graphical analysis.

BACKGROUND: ASEAN (Association of Southeast Asian Nations) is composed of ten Southeast Asian countries bound by socio-cultural ties that promote regional peace and stability. South Asia, located in the southern subregion of Asia, includes nine countries sharing similarities in geographical and ethno-cultural factors. Chikungunya is one of the most significant problems in Southeast and South Asian countries. Much of the current chikungunya epidemic in Southeast Asia is caused by the emergence of a virus strain that originated in Africa and spread to Southeast Asia. Meanwhile, in South Asia, three confirmed lineages are in circulation. Given the positive correlation between research activity and the improvement of the clinical framework of biomedical research, this article aimed to examine the growth of chikungunya virus-related research in ASEAN and South Asian countries. METHODS: The Scopus database was used for this bibliometric analysis. The retrieved publications were subjected to a number of analyses, including those for the most prolific countries, journals, authors, institutions, and articles. Co-occurrence mapping of terms and keywords was used to determine the current state, emerging topics, and future prospects of chikungunya virus-related research. Bibliometrix and VOSviewer were used to analyze the data and visualize the collaboration network mapping. RESULTS: The Scopus search engine identified 1280 chikungunya-related documents published by ASEAN and South Asian countries between 1967 and 2022. According to our findings, India was the most productive country in South Asia, and Thailand was the most productive country in Southeast Asia. In the early stages of the study, researchers investigated the vectors and outbreaks of the chikungunya virus. In recent years, the development of antivirus agents has emerged as a prominent topic. CONCLUSIONS: Our study is the first to present the growth of chikungunya virus-related research in ASEAN and South Asian countries from 1967 to 2022. In this study, the evaluation of the comprehensive profile of research on chikungunya can serve as a guide for future studies. In addition, a bibliometric analysis may serve as a resource for healthcare policymakers.

Health comparison between guinea pigs raised in uncontrolled and controlled environments.

Background and Aim: Guinea pigs (GPs) (Cavia porcellus) are not only kept as pets but also widely used in biological and biomedical research. At present, GPs are also used as a species for animal-assisted therapy (AAT). Consequently, assessing their health status is vital to determining their quality of life, usability for research, and prevention of spread of potential zoonotic diseases to patients using them for AAT. GPs are mainly sourced from animal markets supplied by traditional farms, where environmental factors and sanitation are not properly controlled. This study aimed to compare health status between GPs raised in uncontrolled (conventional farm) and controlled (animal facility) environments. Materials and Methods: Sample animals were obtained from a local animal market and transported to an animal facility. After 1 week of acclimatization, the health status of the animals, including general health condition, body weight, body temperature, complete blood count, liver function (alanine aminotransferase and bilirubin), renal function (blood urea nitrogen and creatinine), and presence of ectoparasites and endoparasites, was assessed. Then, the animals were maintained in the animal facility following the standard procedure for laboratory animals. After 2 months, the animals' health status was re-examined, assessing the same parameters. Results: Based on the evaluated parameters, GPs raised in an uncontrolled environment were found to have poorer health status than those raised in a controlled environment. There were significant differences in almost all parameters between GPs raised in controlled and uncontrolled environments. We found that the populations of two ectoparasites, Gyropus ovalis and Gliricola porcelli, and one endoparasite, Eimeria caviae, decreased significantly following the movement of the animals from an uncontrolled to a controlled environment. Conclusion: GPs raised in an uncontrolled environment have poor health status. However, a controlled environment with better care management can improve the health status of GPs.

Evolutionary dynamics of SARS-CoV-2 circulating in Yogyakarta and Central Java, Indonesia: sequence analysis covering furin cleavage site (FCS) region of the spike protein / Dinámica evolutiva del SARS-CoV-2 que circula en Yogyakarta y Java Central, Indonesia: análisis de secuencia que cubre la región del sitio de escisión de furina (FCS) de la proteína espiga

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a new virus responsible for the COVID-19 pandemic. The emergence of the new SARS-CoV-2 has been attributed to the possibility of evolutionary dynamics in the furin cleavage site (FCS) region. This study aimed to analyze the sequence of the FCS region in the spike protein of SARS-CoV-2 isolates that circulated in the Special Region of Yogyakarta and Central Java provinces in Indonesia. The RNA solution extracted from nasopharyngeal swab samples of confirmed COVID-19 patients were used and subjected to cDNA synthesis, PCR amplification, sequencing, and analysis of the FCS region. The sequence data from GISAID were also retrieved for further genome analysis. This study included 52 FCS region sequences. Several mutations were identified in the FCS region, i.e., D614G, Q675H, Q677H, S680P, and silent mutation in 235.57 C > T. The most important mutation in the FCS region is D614G. This finding indicated the G614 variant was circulating from May 2020 in those two provinces. Eventually, the G614 variant totally replaced the D614 variant from September 2020. All Indonesian SARS-CoV-2 isolates during this study and those deposited in GISAID showed the formation of five clade clusters from the FCS region, in which the D614 variant is in one specific cluster, and the G614 variant is dispersed into four clusters. The data indicated there is evolutionary advantage of the D614G mutation in the FCS region of the spike protein of SARS-CoV-2 circulating in the Special Region of Yogyakarta and Central Java provinces in Indonesia.(AU)

Evolutionary dynamics of SARS-CoV-2 circulating in Yogyakarta and Central Java, Indonesia: sequence analysis covering furin cleavage site (FCS) region of the spike protein.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a new virus responsible for the COVID-19 pandemic. The emergence of the new SARS-CoV-2 has been attributed to the possibility of evolutionary dynamics in the furin cleavage site (FCS) region. This study aimed to analyze the sequence of the FCS region in the spike protein of SARS-CoV-2 isolates that circulated in the Special Region of Yogyakarta and Central Java provinces in Indonesia. The RNA solution extracted from nasopharyngeal swab samples of confirmed COVID-19 patients were used and subjected to cDNA synthesis, PCR amplification, sequencing, and analysis of the FCS region. The sequence data from GISAID were also retrieved for further genome analysis. This study included 52 FCS region sequences. Several mutations were identified in the FCS region, i.e., D614G, Q675H, Q677H, S680P, and silent mutation in 235.57 C > T. The most important mutation in the FCS region is D614G. This finding indicated the G614 variant was circulating from May 2020 in those two provinces. Eventually, the G614 variant totally replaced the D614 variant from September 2020. All Indonesian SARS-CoV-2 isolates during this study and those deposited in GISAID showed the formation of five clade clusters from the FCS region, in which the D614 variant is in one specific cluster, and the G614 variant is dispersed into four clusters. The data indicated there is evolutionary advantage of the D614G mutation in the FCS region of the spike protein of SARS-CoV-2 circulating in the Special Region of Yogyakarta and Central Java provinces in Indonesia.

Immunogenicity Response of Mus musculus var. Balb/C after Immunization using Flagellin Salmonella typhi Serovar Semarang.

<b>Background and Objective:</b> The flagellin of <i>Salmonella typhi</i> is potentially developed as an identifying antigen in a rapid diagnostic test instrument that may be more accurate than conventional serological tests. Therefore, this study aims to analyze the immunogenicity of flagellin <i>S. typhi</i> as the basis for developing a typhoid fever diagnostic. <b>Materials and Methods:</b> Flagellin was isolated from the bacterial culture of <i>S. typhi</i> serovar Semarang and used as the primary antigen for vaccine assembly. Native flagellin antigen was immunized in Balb/C mice with injection doses of 2, 3, 4, 5 and 6 g/100 L in each group (K0-K5), respectively, via intraperitoneal cavity. Blood serum was collected to ELISA based-measurement for IL-6 and TNF-a titers. Then, specific immunoglobulin (Ig) of anti-flagellin was detected using in-house ELISA and western blotting. <b>Results:</b> The findings in this study showed that immunization at the dose of 4-5 g/100 L significantly decreased the IL-6 titer, i.e., 8.33±0.87 pg mL¹, compared to control. The antibody titer test analysis showed the highest Ig-G anti-flagellin was found in K4 mice after immunization using a dose of 5 g/100 L with an average absorbance of Ig-G reaching 1.19 ±0.32. <b>Conclusion:</b> The results indicated that the flagellin protein of <i>S. typhi</i> serovar Semarang induces adaptive immune responses and produces specific antibodies against flagellin. The immunogenic properties of the flagellin protein of <i>S. typhi</i> serovar Semarang potentially developed as a specific diagnostic marker. Further research may also focus on a beneficial feature of flagellin as a vaccine candidate.

Detection of SARS-CoV-2 spike protein D614G mutation by qPCR-HRM analysis.

OBJECTIVES: Monitoring the spread of the G614 in specific locations is critical as this variant is highly transmissible and can trigger the emergence of other mutations. Therefore, a rapid and accurate method that can reliably detect the D614G mutation will be beneficial. This study aims to analyze the potential use of the two-step Reverse Transcriptase quantitative polymerase chain reaction - high resolution melting analysis (RT-qPCR-HRM) to detect a specific mutation in the SARS-CoV-2 genome. METHODS: Six SARS-CoV-2 RNA samples were synthesized into cDNA and analyzed with the qPCR-HRM method in order to detect the D614G mutation in Spike protein of SARS-CoV-2. The primers are designed to target the specific Spike region containing the D614G mutation. The qPCR-HRM analysis was conducted simultaneously, and the identification of the SARS-CoV-2 variant was confirmed by conventional PCR and Sanger sequencing methods. RESULTS: The results showed that the melting temperature (Tm) of the D614 variant was 79.39 ± 0.03 °C, which was slightly lower than the Tm of the G614 variant (79.62 ± 0.015 °C). The results of the HRM analysis, visualized by the normalized melting curve and the difference curve were able to discriminate the D614 and G614 variant samples. All samples were identified as G614 variants by qPCR-HRM assay, which was subsequently confirmed by Sanger sequencing. CONCLUSIONS: This study demonstrated a sensitive method that can identify the D614G mutation by a simple two-step RT-qPCR-HRM assay procedure analysis, which can be useful for active surveillance of the transmission of a specific mutation.

Detection of HBB:c.92+5G>C and HBB:c.108delC mutations in ß-thalassemia carriers using high-resolution melting analysis.

The HBB:c.92+5G>C [known as IVSI-5 (GC)] and HBB:c.108delC [known as Cd 35 (del C)] are the ß-globin gene mutations that commonly found in Indonesia. Detection of the mutation in the ß-thalassemia carriers can be useful to prevent an increase in the number of ß-thalassemia patients. High-Resolution Melting Analysis (HRMA) is the method that can detect the mutation rapidly. The aim of this study was to detect HBB:c.92+5G>C and HBB:c.108delC mutations of the ß-thalassemia carriers using HRMA. DNA was isolated from blood archive of ten hematologically proved ß-thalassemia carriers. Detection of mutations was carried out by HRMA and then confirmed by sequencing. HRMA was performed by using two pairs of specific primers. One pair of primer targeted the region of HBB:c.92+5G>C and the other targeted the region of HBB:c.108delC. The results of detection of mutation using HRMA then were confirmed by sequencing. A specific primer pair covering the region of HBB:c.92+5G>C to HBB:c.108delC were used for sequencing. The results of HRMA showed that the HBB:c.92+5G>C and HBB:c.108delC mutations found in 50% and 30% samples, respectively. The HRMA results can be confirmed by sequencing in all samples. It can be concluded that HRMA can be used to detect HBB:c.92+5G>C heterozygote and HBB:c.108delC heterozygote mutations.

The neuroprotective effect of Ocimum sanctum Linn. ethanolic extract on human embryonic kidney-293 cells as in vitro model of neurodegenerative disease.

AIM: This study aimed to analyze the neuroprotective effect of Ocimum sanctum Linn. ethanolic extract (OSE) on human embryonic kidney-293 (HEK-293) cells as the in vitro model of neurodegenerative diseases. MATERIALS AND METHODS: In this research, HEK-293 cells divided into five groups consisting of normal and healthy cells (NT), cells treated with Camptothecin 500 µM as the negative control, cells treated with trimethyltin 10 µM (TMT), cells treated with OSE 75 µg/ml, and cells pre-treated with OSE 75 µg/ml then induced by TMT 10 µM (OSE+TMT). MTT assay and phase contrast microscopy were applied to observe the cell viability quantitatively and morphological after Ocimum sanctum Linn extract treatment. Finally, the reverse transcription polymerase chain reaction was employed to study the expression of choline acetyltransferase (ChAT). RESULTS: The MTT assay and phase contrast microscopy showed that OSE pre-treatment significantly increased the viability of TMT-induced apoptotic cells and maintained cell viability of the normal HEK-293 cells. Expression of ChAT markedly reduced on TMT treatment group, but OSE administration stabilized ChAT expression in TMT-induced HEK-293 cells. CONCLUSION: This present study proved that OSE administration has neuroprotective effect by increased HEK-293 cells viability and maintain ChAT expression.

Anti-angiogenic effect of Artocarpus heterophyllus seed methanolic extract in ex ovo chicken chorioallantoic membrane

Objective: To examine the anti-angiogenic potential of Artocarpus heterophyllus (A. heterophyllus) seed extract in chicken chorioallantoic membrane (CAM). Methods: This study used chicken CAM ex ovo culture to examine the potential anti-angiogenic activity of A. heterophyllus seed methanolic extract. Basic fibroblast growth factor was used to induce the ectopic formation of blood vessels on CAM treated with extract. Blood vessel number was assessed by macroscopic and microscopic observation, and compared and analyzed for all treatments and controls. Results: Macroscopic observation revealed that a dose of 35 μg/mL of methanolic extract of A. heterophyllus seeds could inhibit basic fibroblast growth factor-induced angiogenesis by 61% in chicken CAM ex ovo culture. This concurred with micro-scopic observations on the histological structure of blood vessels, which indicated that extract treatment repressed the formation of new blood vessels. Conclusions: This is the first study to report the anti-angiogenic effect of methanolic extract derived from A. heterophyllus seeds and its potential as a candidate for future anticancer therapy.

Analysis of viral protein-2 encoding gene of avian encephalomyelitis virus from field specimens in Central Java region, Indonesia.

AIM: Avian encephalomyelitis (AE) is a viral disease which can infect various types of poultry, especially chicken. In Indonesia, the incidence of AE infection in chicken has been reported since 2009, the AE incidence tends to increase from year to year. The objective of this study was to analyze viral protein 2 (VP-2) encoding gene of AE virus (AEV) from various species of birds in field specimen by reverse transcription polymerase chain reaction (RT-PCR) amplification using specific nucleotides primer for confirmation of AE diagnosis. MATERIALS AND METHODS: A total of 13 AEV samples are isolated from various species of poultry which are serologically diagnosed infected by AEV from some areas in central Java, Indonesia. Research stage consists of virus samples collection from field specimens, extraction of AEV RNA, amplification of VP-2 protein encoding gene by RT-PCR, separation of RT-PCR product by agarose gel electrophoresis, DNA sequencing and data analysis. RESULTS: Amplification products of the VP-2 encoding gene of AEV by RT-PCR methods of various types of poultry from field specimens showed a positive results on sample code 499/4/12 which generated DNA fragment in the size of 619 bp. Sensitivity test of RT-PCR amplification showed that the minimum concentration of RNA template is 127.75 ng/µl. The multiple alignments of DNA sequencing product indicated that positive sample with code 499/4/12 has 92% nucleotide homology compared with AEV with accession number AV1775/07 and 85% nucleotide homology with accession number ZCHP2/0912695 from Genbank database. Analysis of VP-2 gene sequence showed that it found 46 nucleotides difference between isolate 499/4/12 compared with accession number AV1775/07 and 93 nucleotides different with accession number ZCHP2/0912695. CONCLUSIONS: Analyses of the VP-2 encoding gene of AEV with RT-PCR method from 13 samples from field specimen generated the DNA fragment in the size of 619 bp from one sample with sample code 499/4/12. The sensitivity rate of RT-PCR is to amplify the VP-2 gene of AEV until 127.75 ng/µl of RNA template. Compared to Genbank databases, isolate 499/4/12 has 85% and 92% nucleotide homology.

DC-SIGN (CD209) Carbohydrate Recognition Domain Is Not Polymorphic in Dengue Virus-Infected Indonesian Patients.

Dengue virus (DENV) infection is a significant burden in Indonesia and other tropical countries. DENV infection has a wide spectrum of clinical manifestations, i.e. asymptomatic, dengue fever, dengue hemorrhagic fever and dengue shock syndrome. The variety of clinical manifestations may be due to the diversity of genetic constitution of the host. The C-type lectin DC-SIGN (CD209) has been identified as the major dengue receptor on human dendritic cells. There are at least five polymorphisms in exon 5 and 6 of the DC-SIGN encoded gene which have been identified and recorded in dbSNP. The aim of this work is to measure the frequency of these polymorphisms among asymptomatic and hospitalized DENV-infected patients. We enrolled 23 hospitalized and 73 asymptomatic DENV-infected patients. Among the subjects, we performed PCR amplification and DNA direct seqencing for 23 hospitalized DENV-infected patients and 24 asymptomatic DENV-infected patients. The result showed that there were no polymorphic nucleotides in the CD209 encoded gene among the patients.

An atypical NF-kappa B-regulated pathway mediates phorbol ester-dependent heme oxygenase-1 gene activation in monocytes.

Heme oxygenase (HO)-1 catalyzes the rate-limiting step of heme degradation and plays an important anti-inflammatory role via its enzymatic products carbon monoxide and biliverdin. In this study it is reported that the HO-1 gene is transcriptionally induced by the phorbol ester PMA in cell cultures of monocytic cells with a regulatory pattern that is different from that of LPS-dependent HO-1 induction in these cells. Activation of HO-1 by PMA was mediated via a newly identified kappaB element of the proximal rat HO-1 gene promoter region (-284 to -275). This HO-kappaB element was a nuclear target for the NF-kappaB subunit p65/RelA as determined by nuclear binding assays and transfection experiments with luciferase reporter gene constructs in RAW264.7 monocytes. Moreover, PMA-dependent induction of endogenous HO-1 gene expression and promoter activity was abrogated in embryonic fibroblasts from p65(-/-) mice. PMA-dependent HO-1 gene activation was reduced by an overexpressed dominant negative mutant of IkappaBalpha, but not by dominant negative IkappaB kinase-2, suggesting that the classical NF-kappaB pathway was not involved in this regulation. The antioxidant N-acetylcysteine and inhibitors of p38 MAPK or serine/threonine kinase CK2 blocked PMA-dependent HO-1 gene activation. Finally, it is demonstrated by luciferase assays with a Gal4-CHOP fusion protein that the activation of p38 MAPK by PMA was independent of CK2. Taken together, induction of HO-1 gene expression by PMA is regulated via an IkappaB kinase-independent, atypical NF-kappaB pathway that is mediated via the activation of p38 MAPK and CK2.

Inhibition of phorbol ester-dependent peroxiredoxin I gene activation by lipopolysaccharide via phosphorylation of RelA/p65 at serine 276 in monocytes.

Peroxiredoxin I (Prx I) is an antioxidant enzyme with thioredoxin-dependent peroxidase activity which is involved in various cellular processes such as regulation of cell proliferation. Here, it is shown that the proinflammatory mediator lipopolysaccharide (LPS) inhibits the induction of Prx I expression and promoter activity by the phorbol ester 12-O-tetradecanoylphorbol- 13-acetate (TPA) in RAW264.7 monocytes, but not that of cyclooxygenase-2. LPS-dependent repression of Prx I induction by TPA was mediated via a newly identified kappaB site in the Prx I promoter, but the "classical" NF-kappaB cascade was not involved in this regulatory pathway, because IkappaB did not affect LPS-mediated Prx I repression. By contrast, phosphorylation of p65 at serine 276, which enhances the transcriptional activity of NF-kappaB, was up-regulated by TPA and was reduced by simultaneous exposure to LPS. Functional studies with Gal4-p65 constructs revealed that serine 276 is crucial to confer LPS-dependent repression of TPA-mediated induction of p65 transactivation. Finally, repression of TPA-dependent Prx I induction by LPS was mediated via Bruton's tyrosine kinase as indicated by studies with the pharmacological inhibitor LFM-A13. In summary, LPS-dependent inhibition of Prx I gene activation by TPA in monocytes is regulated via a pathway that involves phosphorylation of the NF-kappaB subunit p65 at serine 276.

Heme oxygenase-1 gene activation by the NAD(P)H oxidase inhibitor 4-(2-aminoethyl) benzenesulfonyl fluoride via a protein kinase B, p38-dependent signaling pathway in monocytes.

Heme oxygenase (HO)-1 is the inducible isoform of the rate-limiting enzyme of heme degradation and modulates the inflammatory immune response. Because HO-1 is up-regulated by NAD(P)H oxidase activators such as lipopolysaccharide and 12-O-tetradecanoylphorbol-13-acetate in monocytic cells, we investigated the gene regulation of HO-1 by the chemical NAD(P)H oxidase inhibitor 4-(2-aminoethyl) benzenesulfonyl fluoride (AEBSF). Unexpectedly, AEBSF induced endogenous gene expression and promoter activity of HO-1 in cell cultures of human and mouse monocytes. Inhibition of the phosphatidylinositol 3-kinase/protein kinase B (PKB) pathway by pharmacological inhibitors and cotransfection of an expression vector for a dominant negative mutant of PKB reduced the AEBSF-dependent induction of HO-1 gene transcription. Accordingly, overexpressed constitutively active PKB markedly up-regulated HO-1 promoter activity. AEBSF activated the mitogen-activated protein kinases (MAPK) JNK and p38. Inhibition of p38alpha and p38beta, but not that of JNK or p38gamma and p38delta, prevented the induction of HO-1 gene expression by AEBSF. p38 was stimulated by AEBSF in a PKB-dependent manner as demonstrated by a luciferase assay with a Gal4-CHOP fusion protein. Finally, AEBSF- and PKB-dependent induction of HO-1 promoter activity was reduced by simultaneous mutation of an E-box motif (-47/-42) and a cAMP response element/AP-1 element (-664/-657) of the proximal HO-1 gene promoter. Overexpression of the basic helix-loop-helix transcription factor USF2 and coactivator p300 enhanced the AEBSF-dependent response of the HO-1 promoter. The data suggest that the transcriptional induction of HO-1 gene expression by AEBSF is mediated via activation of a PKB, p38 MAPK signaling pathway.

The antioxidant defense protein heme oxygenase 1 is a novel target for statins in endothelial cells.

Cholesterol-independent, pleiotropic actions of hydroxymethylglutaryl coenzyme A (HMG-CoA) reductase inhibitors (statins) exert anti-inflammatory and antioxidant action by as yet unidentified mechanisms. This study explores the role of heme oxygenase 1 (HO-1) as a target and mediator of statins. In cultured endothelial cells derived from human umbilical vein, simvastatin and lovastatin increased HO-1 mRNA levels in a concentration- and time-dependent fashion. HO-1 induction by statins remained unaffected by mevalonate and N-nitro-l-arginine methyl ester, precluding the involvement of isoprenoid- and NO-dependent pathways. HO-1 mRNA induction was abrogated in the presence of actinomycin D and cycloheximide. In cells transfected with a reporter gene construct containing the proximal 4 kB of the HO-1 gene promoter 5'-flanking region, significant upregulation of promoter activity was detected, indicating that regulatory elements binding to this region were involved in transcriptional HO-1 induction by statins. Increased transcriptional expression of HO-1 was associated with elevated HO-1 protein levels and reduction of free radical formation. Our results show that the antioxidant defense protein HO-1 is a target site of statins in endothelial cells. Statins lead to HO-1 promoter activation, transcript and protein accumulation. This novel pathway may contribute to and explain the pleiotropic antioxidant, anti-inflammatory, and antiatherogenic actions of statins.

Role of NF-kappaB and p38 MAP kinase signaling pathways in the lipopolysaccharide-dependent activation of heme oxygenase-1 gene expression.

Heme oxygenase (HO)-1 is the inducible isoform of the rate-limiting enzyme of heme degradation, which is up-regulated by a host of stress stimuli. The bacterial cell membrane component lipopolysaccharide (LPS) is a prototypical activator of monocytic cells. Here, it is shown that LPS induced the endogenous HO-1 gene expression in RAW264.7 monocytic cells. To investigate the molecular mechanisms of HO-1 gene induction by LPS, we performed transfection experiments with reporter gene constructs containing sequences of the proximal rat HO-1 gene promoter. Deletion and mutation analysis indicated that a cyclic AMP response element/activator protein-1 site (-664/-657), but not an E-box motif (-47/-42), played a major role for LPS-dependent HO-1 gene induction. Up-regulation of HO-1 promoter activity by LPS was decreased by pharmacological nuclear factor-kappaB (NF-kappaB) inhibitors and by cotransfected expression vectors with dominant negative isoforms of NF-kappaB-inducing kinase, inhibitor of NF-kappaB (IkappaB) kinase beta, and IkappaBalpha. Moreover, the p38 mitogen-activated protein kinase (MAPK) inhibitor SB203580 and overexpressed dominant negative p38beta decreased, whereas dominant negative p38delta increased, LPS-dependent induction of HO-1 gene expression. The results suggest that the NF-kappaB and p38 MAPK signaling pathways mediate the LPS-dependent induction of HO-1 gene expression via DNA sequences of the proximal promoter region.

Biology of heme in health and disease.

Heme is an essential molecule with contradictory biological functions. In hemoproteins such as hemoglobin and cytochromes protein-bound heme is a prosthetic group serving physiological functions as a transporter for oxygen and electrons. On the other hand free heme can have deleterious effects by generating reactive oxygen species that cause oxidative stress. Consequently, heme homeostasis of the cell must be tightly controlled. The biosynthesis of heme is catalyzed by eight enzymes that are differentially regulated in liver and erythroid cells. Recent findings on proinflammatory functions of heme and its role in the pathogenesis of diseases, such as rhabdomyolysis or atherosclerosis are summarized. The regulation of gene expression by heme in yeast and mammalian cells and the underlying molecular mechanisms are presented. Finally, we discuss the functional significance of the heme-degrading enzyme heme oxygenase and heme-binding proteins for the regulation of heme homeostasis.

Phorbol ester-dependent activation of peroxiredoxin I gene expression via a protein kinase C, Ras, p38 mitogen-activated protein kinase signaling pathway.

The antioxidant protein peroxiredoxin (Prx) I is a thioredoxin peroxidase that is involved in the regulation of proliferation and differentiation of mammalian cells. Here, it is shown that Prx I gene expression was induced transcriptionally by the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) in cultured rat liver tissue macrophages and RAW264.7 monocytic cells. TPA-dependent induction of Prx I gene expression was mediated by two proximal activator protein-1 sites of the rat Prx I promoter region that were nuclear targets of c-Jun as determined by transfection studies with luciferase reporter gene constructs and electrophoretic mobility shift assays. The transcription factor Nrf2, however, was not involved in the regulation of Prx I promoter activity. Prx I gene induction by TPA was decreased by protein kinase C inhibitors and overexpressed dominant negative forms of Ras and MEKK1, but not Raf-1. The p38 MAPK inhibitor SB202190 and overexpression of dominant negative mutants of MAPK kinase 4 (MKK4), MKK6, and p38 inhibited the TPA-dependent induction of Prx I gene transcription. In contrast, inhibitors of the JNK, SP600125, and the NF-kappaB signaling pathway, caffeic acid phenethyl ester, respectively, as well as overexpressed dominant negative MKK7 and IkappaB, had no effect on the up-regulation of Prx I reporter gene activity by TPA. Cotransfection of wild-type p38alpha and p38beta, but not that of p38gamma and p38delta, increased Prx I promoter activity. The data indicate that a protein kinase C, Ras, MEKK1, p38 MAPK signaling pathway plays a major role for the transcriptional up-regulation of Prx I gene expression.