This study examines the hypoglycemic and hypolipidemic effects of rhizome extract from Aristolochia bracteolata Lam (Doman Duste) on rats with alloxan-induced diabetes. Aqueous extracts of the rhizome were given to groups of rats with and without diabetes, and blood glucose and lipid profiles were then measured. The results showed that both the diabetes untreated and treated groups saw a mean decrease in body weight from 143.33 ± 5.77 to 136.67 ± 4.73 g and 167.67 ± 1.53 to 159.67 ± 0.58 g, respectively, following the induction of the alloxan. After the diabetic rats were treated, their body weight climbed from 159.67 ± 0.58 to 169.67 ± 0.58 g, and all of them regained their previous body weight. Following alloxan induction, the diabetic untreated rat group’s serum glucose levels were higher (7.91 ± 0.07 mmol) than those of the normal untreated (6.13 ± 0.49 mmol/L), normal treated (6.70 ± 0.50 mmol/L), and diabetic treated (6.30 ± 0.17 mmol/L) groups. The diabetic untreated rats had lower HDL-cholesterol (2.51 ± 0.45 mmol/L) than the other groups, but greater triacylglyceride (3.19 ± 0.79 mmol/L), VLDL-cholesterol (1.77 ± 0.56 mmol/L), and LDL-cholesterol (1.44 ± 0.36 mmol/L). Out of the four groups, the diabetic untreated rat group’s atherogenic index (AI) seems to be the highest (1.27 ± 1.75). This study emphasizes A. bracteolata potential as a natural treatment for lipid regulation and diabetic management.
Using Drosophila melanogaster as an experimental organism, this study examined the bioactivity and possible harmful consequences of Bryophyllum pinnatum leaves powder. In order to assess phenotypic differences among five filial generations (F1–F5), the leaves powder was added to culture media at different concentrations (1.0 g/g, 0.5 g/g, and 0.25 g/g). The leaves powder caused developmental abnormalities, such as apterous wings, small stature, and narrow abdomens as seen by phenotypic traits. Significant impacts of leaf powder concentration were shown by statistical studies, such as one-way ANOVA, on both the total number of flies (p = 0.004) and the total number of mutant flies (p = 0.048). These parameters, however, were not significantly impacted by the filial generation itself. The findings imply that B. pinnatum metabolites affect D. melanogaster phenotypic plasticity, possibly as a result of secondary plant chemicals that function as growth inhibitors or modulators of allelochemical plasticity. These results support the wider implications of B. pinnatum in pharmacological and toxicological applications by highlighting its capacity to modify insect physiology. The underlying molecular mechanisms and their possible use in the creation of drugs and biopesticides should be investigated in future studies.
Cysteine protease inhibitors (CPIs) have a vital role in the strict regulation and control of cysteine protease activity. In this work, a cysteine protease inhibitor (CPI) from watermelon (Citrullus lanatus) seeds was isolated, purified, and partially characterised. Ammonium sulfate precipitation was used to extract CPI, then size exclusion chromatography and ion exchange were used to further purify it. Papain was used to assess the inhibitory action, and identifying the ideal temperature stability and method of inhibition were part of the biochemical characterisation. The protein content of the fractionated samples ranged from 3.6 to 6.5 mg/L, whereas the crude sample of C. lanatus seed had 9.2 mg/L. Cysteine protease inhibitor (CPI) was isolated from C. lanatus seeds, and the highest inhibitory activity occurred at 60–100% saturation of (NH4)2SO4. The CPI inhibitor exhibits a competitive mode of inhibition on papain, with the same Vmax = 1.06 μmol/min, Km = 2.5 μM, and Ki = 4.00 μM. Its activity is retained up to 65°C, but it drastically decreases beyond 70°C. The work advances knowledge of the biochemical characteristics of CPIs obtained from plants and highlights their applicability in the agricultural and pharmaceutical sectors. Keywords: Ammonium sulfate, Cysteine protease inhibitor (CPI), Isolation, Purification Watermelon (Citrullus lanatus)
This study looked at the impact of Anisopus mannii methanolic leaf extract on Wistar rats’ organ weights and alloxan-induced diabetes. For this investigation, a total of 20 male Wistar (albino) rats weighing between 80 and 150 g were used. Four groups of rats were randomly assigned. 1: Normal control; 2: Negative control; 3: Positive control (treated alloxan induced-diabetic with 5 mg/kg metformin); 4: alloxan induced-diabetic treated with 200 mg/kg of methanolic A. mannii leaf extract. The findings of the study show there was a significant difference in the blood glucose levels between the diabetic rats treated with metformin and the diabetic rats treated with A. mannii methanolic leaves extract on days 7 and no significant difference in day 14. The fasting blood glucose level in the diabetic rats treated with A. mannii on day 7 reduced from 167.00±19.76 mg/dL to 88.08±11.78 mg/dL on day 14. the liver weights (3.69±0.21 g) of the diabetic rats treated with the extract were significantly lower than those of the diabetic rats treated with metformin (4.86±0.16 g), and it was also within the weight for normal control which were non-diabetic (3.93±0.29 g). The kidney weights of the diabetic rats treated with the extract (1.13±0.18 g) were significantly higher than those of the diabetic rats treated with metformin (0.96±0.03 g). This work unequivocally shows that, in rats given alloxan to produce diabetes, A. mannii extract was able to substantially lower fasting blood glucose levels and shield the kidneys and liver from harm in just two weeks..
In this work, physicochemical characteristics of the oils extracted from Cucumeropsis manni (Egusi) and Citrullus lanatus (Watermelon) seeds were determined, and the output for both vegetable oils was compared. Unshelled seeds of both C. mannii and C. lanatus were purchased from modern market, in Lafia local government area of Nasarawa state, Nigeria. Following dehulling, oil was extracted from both seed samples using the cold extraction technique. The physicochemical properties such as density, viscosity, iodine value (IV), acid value (AV), peroxide value (PV), and saponification value (SV) of the seed oil samples were then examined. The result revealed that the density for both C. mannii and C. lanatus seed oil were 0.98 ± 0.16 and 0.98 ± 0.16 g/ml, and their viscosity are 72.5 ± 0.03 and 57.5 ± 0.11 Cp respectively. Both C. mannii and C. lanatus seed oil IV was 84.92 ± 0.24 and 80.91 ± 0.12 g/I2/100 g, AV was 2.92 ± 0.26 and 2.44 ± 0.04 mgKOH/g, and PV was 3.77 ± 0.01 and 4.28 ± 0.05 meq peroxide/g respectively. The SV for C. mannii is 243.85 ± 0.31 is higher than that of C. lanatus which is 187.12 ± 0.19 mgKOH/g. In conclusion, the iodine, acid, and peroxide values for both samples show that they are safe to consume. The C. mannii SV demonstrates that it is superior for soap manufacturing. Finally, the PV demonstrates that both samples are suitable for biodiesel production, which can help reduce the impact of climate change on our society as a whole.
This study set out to assess the nutritional (amino acids and proximate) composition of burukutu, a locally prepared alcoholic drink from 4 selected areas of Jos metropolis in Plateau State, Nigeria. Burukutu samples were collected from Kugiya, Tundunwada, and Hwolshe and Angwan Rukuba in Jos, Plateau State. Samples were subjected to proximate analysis to investigate the following: ash, moisture, content, crude fat, crude fibre, crude protein and total carbohydrate. Amino acids profile of each sample was also examined. The chemical composition of the burukutu samples exhibited varying levels of: Ash (0.05-0.60%), Crude fibre (1.10-2.50%), Crude fat (0.50-0.70%), Crude protein (1.34-1.99%), Moisture (4.50-5.00%), and Total carbohydrate (89.36-92.50%). Leucine (5.86-7.21 g/100g protein), alanine (4.55-5.61 g/100g protein), proline (4.26-5.89 g/100g protein) and aspartic acid (6.02-7.79 g/100g protein) alongside glutamic acid (2.01-18.24 g/100g protein) were the amino acids that appear in some substantial concentration of all the amino acids that were present in all the 4 burukutu samples. In conclusion, all the burukutu samples are low in fibre, fat and protein, but they are rich source of carbohydrate. Also, from this study it could be said that the burukutu is a rich source of the following amino acids: glutamic acid, leucine, alanine, proline, and aspartic acid..
This study aims at isolating and molecularly characterizing Aspergillus flavus from maize (Zea mays L.) varieties (Samaz 15,20, 37, 52, Oba super 6, 9, 39, 98, and DK 777) obtained across Abuja, Nigeria. Maize varieties were obtained across the district areas of Abuja namely; Kuje, Kwali, Gwagalada, AMAC, Abaji, Bwari and UniAbuja Experimental field. The potato dextrose agar as a medium was used in growing the fungi from the maize varieties seeds for 7 days at 25oC and then sub-cultured to obtain an isolate into potato dextrose broth. The mycelial mass produced was processed to extract its DNA using genomic DNA extraction of Zymo, D6005 for fungi and bacteria. Molecular Identification of the Internal transcribed spacer (ITS) region for each A. flavus isolate was carried out with ITS1 and ITS4 primers. anf18S rRNA gene region was also amplified with ITS5 and ITS4 primers. 2.0% agarose gels were electrophoresed for 30 minutes in 0.5× buffer, Dye and 60 mM EDTA at 80 V. A gel documentation system was used in determining PCR product sizes by evaluating with internal Mol. Wt. Standards of Thermo Scientific GeneRuler 100 bp DNA Ladder. Fifteen (15) of the twenty (20) fungal isolates were found to be positive with PCR amplification of ITS regions and amplicon sizes of 600 base pairs (bp) for A. flavus were obtained. All the twenty isolates have the presence of 18S rRNA 300-400bp. Thirteen of the fungal growth from the fourteen maize varieties obtained across Abuja were identified as A. flavus.
This study aims to examine aflD (Nor-1) and aflP (omt) genes that could be present in species of Aspergillus flavus isolated from varieties of maize seeds obtained across Abuja, Nigeria, utilizing the PCR method and Sanger sequencing method. The DNA extraction of16 isolates (14 were procured maize varieties and 2 known aflatoxin infected varieties of yellow and white maize from the laboratory to serve as positive control) was carried out with Zymo, D6005 DNA extraction kit. All 16 A. flavus isolates were tested for amplification of Aflatoxin genes with oligonucleotide primers (FwOmt-1:5’, RvOmt-1:5’, FwNor-1: 5′, and Rv Nor-1: 5′). The PCR analysis was carried out with a DreamTaq Green PCR Master Mix (2x). The sanger sequencing was carried out using ExoSAP-IT™ Express PCR Product Cleanup Sequencing Kit. The findings of this study showed that all A. flavus isolated from the various maize varieties had the presence of NOR (afl D) gene except for the A. flavus isolated from Maize-Samaz 15, Maize-Oba super 39 and Maize-Oba super 6 (UNIAbuja). In addition, Maize-DK 777, Maize-Samaz 52 (Abaji) and Maize-Samaz 37 varieties had the OMT (aflP) gene. This study has demonstrated that an Aspergillus flavus attack may either be accompanied with aflatoxin production or not, with the Maize-Oba super 39, Maize-Samaz 15 and Maize-Oba super 6 (UNIAbuja) although infected with A. flavus they had no aflD and aflP gene present in them. In addition, this study revealed that the maize varieties across Abuja were prone to A. flavus contamination which have tendency to produce aflatoxin.
Colorectal cancer (CRC), the third most common cancer worldwide, also has the highest rate of cancer-related morbidity and mortality. WNT signaling is initiated by binding of WNT to various receptors, including frizzleds (FZDs), and plays a critical role in CRC and other tumor development by regulating proliferation, differentiation, migration, apoptosis, and polarity. Among the members of the FZD family, FZD6 is broadly expressed in various tissues, and its overexpression has been reported in several cancers, suggesting an important role in cancer development. In this study, we investigated the expression of FZD6 in patients with CRC and found it to be increased in tumors, as compared to paired adjacent non-tumor tissues. Additionally, we found that FZD6 expression was negatively regulated by miR199a5p in CRC cells. These results suggest that overexpression of FZD6, mediated by reduced expression of miR-199a-5p, may play an important role in the development of CRC. [BMB Reports 2015; 48(6): 360-366]
Telomerase plays a pivotal role in the pathology of aging and cancer by maintaining genome integrity, controlling cell proliferation, and regulating tissue homeostasis. Telomerase is essentially composed of an RNA component, Telomerase RNA or TERC, which serves as a template for telomeric DNA synthesis, and a catalytic subunit, telomerase reverse transcriptase (TERT). The canonical function of TERT is the synthesis of telomeric DNA repeats, and the maintenance of telomere length. However, accumulating evidence indicates that TERT may also have some fundamental functions that are independent of its enzymatic activity. Among these telomere-independent activities of hTERT, the role of hTERT in gene transcription has been investigated in detail. Transcriptional regulation is a fundamental process in biological systems. Several studies have shown a direct involvement of hTERT in gene transcription. This mini-review will focus on the role of hTERT in gene transcription regulation, and discuss its possible mechanisms.
Seed storage proteins (SSPs) are important for seed germination and early seedling growth. We studied the accumulation and degradation profiles of four major Arabidopsis 12S SSPs using a 2-DE scheme combined with mass spectrometric methods. On the 2-DE map of 23 dpa (days post anthesis) siliques, 48 protein spots were identified as putative full-length or partial alpha, beta subunits. Only 9 of them were found in 12 dpa siliques with none in younger than 8 dpa siliques, indicating that the accumulation of 12S SSPs started after the completion of cell elongation processes both in siliques and in developing seeds. The length and strength of transcription activity for each gene determined the final contents of respective SSP. At the beginning of imbibition, 68 SSP spots were identified while only 2 spots were found at the end of the 4 d germination period, with alpha subunits degraded more rapidly than the beta subunits. The CRC alpha subunit was found to degrade from its C-terminus with conserved sequence motifs. Our data provide an important basis for understanding the nutritional value of developing plant seeds and may serve as a useful platform for other species.
Cold acclimation improves freezing tolerance in plants. In higher plants, many advances have been made toward identifying the signaling and regulatory pathways that direct the low-temperature stress response; however, similar insights have not yet been gained for simple nonvascular plants, such as bryophytes. To elucidate the pathways that regulate cold acclimation in bryophytes, we used two PCR-based differential screening techniques, cDNA amplified fragment length polymorphism (cDNA-AFLP) and suppression subtractive hybridization (SSH), to isolate 510 ESTs that are differentially expressed during cold acclimation in Physcomitrella patens. We used realtime RT-PCR to further analyze expression of 29 of these transcripts during cold acclimation. Our results show that cold acclimation in the bryophyte Physcomitrella patens is not only largely similar to higher plants but also displays distinct differences, suggests significant alteration during the evolution of land plants.
Three Angelica sinensis polysaccharide fractions (APFs), named APF1, APF2 and APF3, were isolated and purified from Radix A. sinensis and their antioxidant activities were evaluated in isolated mouse peritoneal macrophages by pretreatment with APFs before exposure to 0.2 mM tertbutylhydroperoxide (t-BHP). The results showed that pretreatment of the macrophages with APFs as low as 10 microg/ml could significantly enhance t-BHP-decreased cell survival, intracellular glutathione (GSH) content and superoxide dismutase (SOD) activity, and also inhibited t-BHP-increased lactate dehydrogenase (LDH) leakage and malondialdehyde (MDA) formation (p < 0.05), and APF3 was the most active fraction, followed by APF2 and APF1 in decreasing order. Furthermore, we found for the first time that the bound-protein in APF3 was associated closely with the protective effects and the polysaccharide inhibited the excess NO release from t-BHP-activated macrophages to protect host cells.
Ethylene performs an important function in plant growth and development. 1-aminocyclopropane-1-carboxylate (ACC) synthase (ACS), the key enzyme involved in ethylene biosynthesis, has been the focus of most ethylene studies. Here, a cotton ACS gene referred to as Gossypium hirsutum ACS1 (GhACS1), was isolated. The full-length cDNA of GhACS1 encodes for a 476-amino acid protein which harbors seven conserved regions, 11 invariant amino acid residues, and the PLP binding active site, all of which characterize ACC synthases. Alignment analysis showed that GhACS1 shared a high degree of identity with other known ACC synthases from different species. Two introns were detected in the genomic DNA sequence, and the results of Southern blot analysis suggested that there might be a multi-gene family encoding for ACC synthase in cotton. From the phylogenetic tree constructed with 24 different kinds of ACC synthases, we determined that GhACS1 falls into group II, and was closely associated with the wound-inducible ACS of citrus. The analysis of the 5' flanking region of GhACS1 revealed a group of putative cis-acting elements. The results of expression analysis showed that GhACS1 displayed its transient expression nature after wounding, abscisic acid (ABA), and $CuCl_2$ treatments. These results indicate that GhACS1, which was transiently expressed in response to certain stimuli, may be involved in the production of ethylene for the transmission of stress signals.
There were many different families of zinc finger proteins that contained multiple cysteine and/or histidine residues and used zinc to stabilize their folds. The classical C2H2 zinc finger proteins were the founding members of this superfamily and were among the most abundant proteins in eukaryotic genomes. C2H2 proteins typically contained several C2H2 fingers that made tandem contacts along the DNA. Here we reported a novel C2H2 type zinc finger gene, ZNF438, which encoded 828 amino acids that formed five zinc finger domains. Bioinformatics analysis revealed that the ZNF438 was mapped to human chromosome 10p11.2 and shared 62% identity with rat and mouse homologues. RT-PCR analysis indicated that it was ubiquitously expressed in 18 human adult tissues. With immunofluorescence assay, it was shown that the exogenous Flag-tagged ZNF438 was located in nucleus of COS-7 cells. To further explore the function of ZNF438, we examined the transcriptional activity of ZNF438 protein by transfecting recombinant pM-ZNF438 into mammalian cells. The subsequent analysis based on the duel luciferase assay system showed that ZNF438 was a transcriptional repressor.
Breast cancer is the most common malignancy among women, and mutations in the BRCA1 gene produce increased susceptibility to these malignancies in certain families. In this study, the forward 1-13 exons of breast cancer associated gene BRCA1 were cloned from breast cancer cell line ZR-75-30 by RT-PCR method. Sequence analysis showed that nine BRCA1 splice forms were isolated and characterized, compared with wild-type BRCA1 gene, five splice forms of which were novel. These splice isoforms were produced from the molecular mechanism of 5' and 3' alternative splicing. All these splice forms deleting exon 11b and the locations of alternative splicing were focused on two parts:one was exons 2 and 3, and the other was exons 9 and 10. These splice forms accorded with GT-AG rule. Most these BRCA1 splice variants still kept the original reading frame. Western blot analysis indicated that some BRCA1 splice variants were expressed in ZR-75-30 cell line at the protein level. In addition, we confirmed the presence of these new transcripts of BRCA1 gene in MDA-MB-435S, K562, Hela, HLA, HIC, H9, Jurkat and human fetus samples by RT-PCR analysis. These results suggested that breast cancer associated gene BRCA1 may have unexpectedly a large number of splice variants. We hypothesized that alternative splicing of BRCA1 possibly plays a major role in the tumorigenesis of breast and/or ovarian cancer. Thus, the identification of cancer-specific splice forms will provide a novel source for the discovery of diagnostic or prognostic biomarkers and tumor antigens suitable as targets for therapeutic intervention.
A new full-length cDNA encoding hyoscyamine 6beta-hydroxylase (designated as aah6h, GenBank Accession No. EF187826), which catalyzes the last committed step in the scopolamine biosynthetic pathway, was isolated from young roots of Anisodus acutangulus by rapid amplification of cDNA ends (RACE) for the first time. The full-length cDNA of aah6h was 1380 bp and contained a 1035 bp open reading frame (ORF) encoding a deduced protein of 344 amino acid residues. The deduced protein had an isoelectric point (pI) of 5.09 and a calculated molecular mass of about 38.7 kDa. Sequence analyses showed that AaH6H had high homology with other H6Hs isolated from some scopolamine-producing plants such as Hyoscyamus niger, Datura metel and Atropa belladonna etc. Bioinformatics analyses results indicated AaH6H belongs to 2-oxoglutarate-dependent dioxygenase superfamily. Phylogenetic tree analysis showed that AaH6H had closest relationship with H6H from A. tanguticus. Southern hybridization analysis of the genomic DNA revealed that aah6h belonged to a multi-copy gene family. Tissue expression pattern analysis firstly founded that aah6h expressed in all the tested tissues including roots, stems and leaves and indicated that aah6h was a constitutive-expression gene, which was the first reported tissue-independent h6h gene compared to other known h6h genes.
The mitogen-activated protein kinase (MAPK) cascade is one of the major and evolutionally conserved signaling pathways and plays pivotal role in the regulation of stress and developmental signals in plants. Here, a novel gene, termed Gossypium hirsutum MAPK (GhMAPK), was isolated from cotton. The full-length cDNA of GhMAPK encodes for a 372 amino acid protein that contains all 11 of the MAPK conserved subdomains and the phosphorylation-activation motif, TEY. Amino acid sequence alignment revealed that GhMAPK shared high identity with group-C MAPK in plants and showed 83 approximately 89% similarities with MAPKs from Arabidopsis, apricot, pea, petunia, and tobacco. Southern blot analysis indicated that the GhMAPK belonged to a multygene family in cotton. Two introns were found within the region of genomic sequence. Northern blot analysis revealed that the transcripts of GhMAPK accumulated markedly when the cotton seedlings were subjected to various abiotic stimuli such as wounding, cold (4 degrees C), or salinity stress; Furthermore, GhMAPK was upregulated by the exogenous signaling molecules, such as salicylic acid (SA) and hydrogen peroxide (H(2)O(2)), as well as pathogen attacks. These results indicate that the GhMAPK, which has a high degree of identity with group-C plant MAPKs, may also play an important role in response to environmental stresses.
To investigate whether phospholipase D (PLD, EC 3.1.4.4) plays a role in adaptive response of post-harvest fruit to environment, a PLD gene was firstly cloned from grape berry (Vitis Vinifera L. cv. Chardonnay) using RT-PCR and 3'- and 5'-RACE. The deduced amino acid sequence (809 residues) showed 84.7% identity with that of PLD from Ricinus communis. The secondary structures of this protein showed the characteristic C2 domain and two active sites of a phospholipid-metabolizing enzyme. The PLD activity and its expression in response to heat acclimation were then assayed. The results indicated PLD was significantly activated at enzyme activity, as well as accumulation of PLD mRNA and synthesis of new PLD protein during the early of heat acclimation, primary suggesting that the grape berry PLD may be involved in the heat response in post-harvest grape berry. This work offers an important basis for further investigating the mechanism of post-harvest fruit adaptation to environmental stresses.
Tuberculosis, caused by Mycobacterium tuberculosis, continues to be one of the leading infectious diseases to humans. It is urgent to discover novel drug targets for the development of antitubercular agents. The 2-C-methyl-D-erythritol-4-phosphate (MEP) pathway for isoprenoid biosynthesis has been considered as an attractive target for the discovery of novel antibiotics for its essentiality in bacteria and absence in mammals. MEP cytidyltransferase (IspD), the third-step enzyme of the pathway, catalyzes MEP and CTP to form 4-diphosphocytidyl-2-C-methylerythritol (CDP-ME) and PPi. In the work, ispD gene from M. tuberculosis H37Rv (MtIspD) was cloned and expressed. With N-terminal fusion of a histidine-tagged sequence, MtIspD could be purified to homogeneity by one-step nickel affinity chromatography. MtIspD exists as a homodimer with an apparent molecular mass of 52 kDa. Enzyme property analysis revealed that MtIspD has high specificity for pyrimidine bases and narrow divalent cation requirements, with maximal activity found in the presence of CTP and Mg(2+). The turnover number of MtIspD is 3.4 s(-1). The Km for MEP and CTP are 43 and 92 muM, respectively. Furthermore, MtIspD shows thermal instable above 50 degrees C. Circular dichroism spectra revealed that the alteration of tertiary conformation is closely related with sharp loss of enzyme activity at higher temperature. This study is expected to help better understand the features of IspD and provide useful information for the development of novel antibiotics to treat M. tuberculosis.
