Open Access Original Research Article

Analyses of Homing Endonucleases and Mechanism of Action of CRISPR-Cas9 HNH Endonucleases

Peramachi Palanivelu

International Journal of Biochemistry Research & Review, Page 1-25
DOI: 10.9734/ijbcrr/2020/v29i630192

Aim: To analyze different HNH endonucleases from various sources including the HNH endonuclease regions of CRISPR-Cas9 proteins for their conserved motifs, metal-binding sites and catalytic amino acids and propose a plausible mechanism of action for HNH endonucleases, using CRISPR-Cas9 as the model enzyme.

Study Design: Multiple sequence analysis (MSA) of homing endonucleases including the CRISPR-Cas9 using Clustal Omega was studied. Other biochemical, Site-directed mutagenesis (SDM) and X-ray crystallographic data were also analyzed.

Place and Duration of Study: School of Biotechnology, Madurai Kamaraj University, Madurai, India, between 2007 and 2013.

Methodology: Bioinformatics, Biochemical, SDM and X-ray crystallographic data of the HNH endonucleases from different organisms including CRISPR-Cas9 enzymes were analyzed. The advanced version of Clustal Omega was used for protein sequence analysis of different HNH endonucleases from various sources. The conserved motifs identified by the bioinformatics analysis were analyzed further with the data already available from biochemical and SDM and X-ray crystallographic analyses of this group of enzymes and to confirm the possible amino acids involved in the active sites and catalysis.

Results: Different types of homing endonucleases from various sources including the HNH endonuclease regions of CRISPR-Cas9 enzymes exhibit different catalytic regions and metal-binding sites. However, the catalytic amino acid, i.e., the proton acceptor histidine (His), is completely conserved in all homing endonucleases analyzed. From these data, a plausible mechanism of action for HNH endonucleases, using CRISPR-Cas9 from Streptococcus pyogenes, as the model enzyme is proposed. Furthermore, multiple sequence alignment (MSA) of various homing endonucleases from different organisms showed many highly conserved motifs also among them. However, some of the HNH endonucleases showed consensus only around the active site regions. Possible catalytic amino acids identified among them belong to either -DH---N or -HH--N types. There are at least two types of metal-binding sites and bind Mg2+ or Zn2+ or both. The CRISPR-Cas9 enzyme from S. pyogenes belongs to the -DH- based HNH endonucleases and possesses –DxD- type metal-binding site where it possibly binds to a Mg2+ ion. The other HNH enzymes possess one or two invariant Zn binding CxxC/ CxxxC motifs.

Conclusions: The CRISPR-Cas9 enzymes are found to be -DH- type where the first D is likely to involve in metal-binding and the second invariant H acts as the proton acceptor and the N in –HNH- Cas9 confers specificity by interacting with the nucleotide near the catalytic region. In this communication, a metal-bound water molecule is shown as the nucleophile initiating catalysis. Homing endonucleases may be used as novel DNA binding and cleaving reagents for a variety of genome editing applications and Zinc finger nucleases have already found applications in genome editing.

Open Access Original Research Article

Study of Staining Capability of Alcoholic and Aqueous Hibiscus sabdariffa Extract in Demonstration of Selected Fungal Moulds in Some Selected Tissue Section of Wistar Rat

S. Y. Ma’aruf, M. O. Mohammed, O. G. Avwioro, A. T. Muhammad, R. I. Tsamiya, U. Abubakar, I. Mohammed, A. Umar, S. M. Sani, H. Kabir, B. A. Bello, S. D. Abubakar, F. A. Dogondaji, N. Okorie, S. Garba

International Journal of Biochemistry Research & Review, Page 26-33
DOI: 10.9734/ijbcrr/2020/v29i630193

Introduction: Hibiscus sabdariifa is a natural dye, generally called roselle. Most of Synthetic dye  have been established to cause toxicity. The aim of this research was to determine the staining effect of different concentration of Hibiscus sabdariffa extracts at various pH, duration on fungal staining.

Methods: Standard Gomori’s metenaine silver stains as control and Hibiscus sabdariffa extracts (alcoholic and aqueous) were used to stain a fungal (Aspergillus spp) positive lung tissue, using various concentration (5% and 10%) of the extract, with change of pH achieved by treating the extracts with ammonium hydroxide and glacial acetic acid at various duration (30 seconds and 1 hour respectively), each was used for staining Aspergillus spp in tissue section as a primary stain replacing (4% Chromic acid, sodium metabisulphite, Hexamine working silver solution and Sodium thiosulphate).

Results: All extracts after treatment were acidic, the fungi were  best demonstrated with Aqueous hibiscus stains (5% untreated, 10% untreated and 5% glacial acetic acid treated) at longer duration, staining the capsule, light to dark-brown in a light-green background when compared with the alcoholic hibiscus stain. Few of the alcoholic hibiscus stains show metachromatic property. The Hibiscus stains stained better at longer duration and the change of pH was indirectly proportional to the staining ability of the hibiscus stains.

Conclusion: The results obtained indicate that hibiscus plant extract have the potential for use in the morphological identification of fungi in formalin-fixed paraffin-embedded tissue section.

Open Access Original Research Article

Inhibitory Potential and Antidiabetic Activity of Leaf Extracts of Justicia carnea

Onuabuchi Nnenna Ani, Stanley Chidi Udedi, Kelechi Kingsley Asogwa, Michael Okey Enemali, Chigozie Michael Onwelumadu, Kenneth Segun Ikedife

International Journal of Biochemistry Research & Review, Page 34-45
DOI: 10.9734/ijbcrr/2020/v29i630194

The aim of this study was to evaluate the in-vitro inhibitory potential of various leaf extracts of Justicia carnea on alpha-amylase and alpha-glucosidase activities and the anti-diabetic activity of the ethanol leaf extract using albino wistar rats. The analyses were carried out using standard biochemical methods. The oral acute toxicity test (LD50) in rats of the ethanol leaf extract was determined using Lorke’s method. Diabetes was induced in the rats by a single intraperitoneal dose of 120 mg/kg. b.w of alloxan. Six (6) experimental groups of rats (n=6) were used for the study. Three groups of diabetic rats received oral daily doses of 200 mg/kg, 400 mg/kg and 600 mg/kg ethanol leaf extract of Justicia carnea respectively while gilbenclamide (5 mg/ml); a standard diabetic drug was administered to a specific group. Treatment lasted for 14 days. From the results of the in-vitro inhibitory activity, the different extracts of Justicia carnea demonstrated dose dependent strong inhibitory activity against α-amylase but moderate inhibitory activity against α-glucosidase. The ethanol leaf extract was the most potent inhibitor of both α-amylase and α-glucosidase in comparison with other extracts and acarbose; the standard. The ethanol leaf extract exhibited maximum of 82.18% inhibition with IC50 of 2.99 ± 1.14 mg/ml for α-amylase and 41.66% with IC50 of 9.66 mg/ml ± 0.32 mg/ml for α-glucosidase. From the analysis of the Lineweaver-Burk plot, the ethanol leaf extract exhibited mixed noncompetitive inhibition of α-amylase and competitive inhibition of α-glucosidase activities. The acute toxicity study showed that the extract had an LD50 > 5000 mg/kg. From the result of the anti-diabetic study, the fasting blood glucose levels significantly (p<0.05) decreased in the animals treated with the ethanol leaf extract of Justicia carnea when compared to the untreated rats. It can be concluded from the results that the leaf extracts of Justicia carnea can be used in the management of diabetes.

Open Access Original Research Article

Impact of Illegal Crude Oil Refining in Jike-ama River of Bille Kingdom, Rivers State, Nigeria

Nwachoko, Ndidi, Davies, Bekinbo, Tetam, Jack Gbenenee

International Journal of Biochemistry Research & Review, Page 46-51
DOI: 10.9734/ijbcrr/2020/v29i630195

Oil exploration and exploitation is a lucrative business and one of the major sources of revenue in Nigeria. Each year, hundreds of post-impact assessment (PIA) studies are conducted to assess the impact of the hazards generated by the oil industry on social environment and on human health. This hazardous impact is the associated with the activities of illegal refining of crude oil. Thus, this work examined the impact of illegal crude oil refining activities in Jike-Ama river of Jike-Ama community. Shellfish, fish, surface water and sediment samples were randomly collected in triplicates. Physiochemical parameters, heavy metal content, polycyclic aromatic hydrocarbons (PAHs) and total hydrocarbon content of the water in addition to heavy metal content and PAHs in fish, Shellfish and sediment were determined. The results of the physicochemical parameters showed that pH, total suspended solids, biological oxygen demand, turbidity and total hydrocarbon content of water were above FEPA permissible limits while chloride, nitrate and sulphate detected in water were within FEPA permissible limits. Dissolved oxygen was observed to be below FEPA permissible limit. The mean concentration of heavy metals and PAHs in sediment showed significant difference (p<0.05) when compared with values for Shellfish and fish and were above FEPA and WHO permissible limits. Shellfish had higher concentrations of heavy metals and PAHs than fish. PAHs values in shellfish showed significant difference (p<0.05). Heavy metals concentration in fish were above permissible limits except Cr, Zn and Pb. Concentration of PAHs in fish, shellfish, surface water and sediment were generally above FEPA and WHO permissible limits. This findings suggests high concentration of contaminants in Jike-Ama river.

Open Access Original Research Article

Serum Melatonin with Respect to Mental Health (Anxiety & Depression) Status of First Year M.B.B.S Students

Vinita H. Belsare, Arun Tadas, Sanjay Agrawal, Hrishikesh Belsare

International Journal of Biochemistry Research & Review, Page 52-60
DOI: 10.9734/ijbcrr/2020/v29i630198

Introduction: Melatonin is a pineal hormone secreted in circadian manner, with a peak during evening and night. Night-time light exposure is a powerful suppressor of nocturnal melatonin secretion. Dopamine and serotonin have complex relationship in melatonin synthesis and secretion. Reduced levels of melatonin have been associated with severe depression. Melatonin exhibits GABA-like effects may be responsible to reduce anxiety.

Objectives: To determine the Serum Melatonin levels with respect to bedtime and its correlation with the severity of anxiety and depression. Also, to find the difference in anxiety score, depression score and serum melatonin level with respect to gender.

Methods: A cross sectional study was done amongst first year medical students, and anxiety and depression score was calculated using Hamilton’s anxiety and depression scale. Estimation of Serum Melatonin was done on the fasting morning blood samples.

Results and Conclusion: The present study shows that there is no significant difference in melatonin secretion in the students with late bedtime. With the increasing severity of the anxiety the serum melatonin was found to be lower, but the difference was not statistically significant. Similar results were observed with depression. The results shows non-significant higher anxiety and depression scores in females as compared to males. The study results also shows the significant high levels of melatonin in males as compared to females. Low melatonin levels in females may be attributed to high anxiety and depression in females.

Open Access Original Research Article

Efficacy of Securidaca longipedunculata Fresen (Polygalaceae) against Two Standard Isolates of Neisseria gonorrhoeae

Wekesa Euginia Makinia, Michaiah Ojunga, Z. Ngalo Otieno- Ayayo

International Journal of Biochemistry Research & Review, Page 61-68
DOI: 10.9734/ijbcrr/2020/v29i630199

Aim: A study was carried out to determine the in vitro antibacterial activity of ethanol extract of root and bark of Securidaca longipedunculata against two standard isolates of Neisseria gonorrhoeae NO.0296 and WHO- K.

Materials and Methods: Plant materials were collected and dried at room temperature, followed by ethanol extraction using a rotary pump. Antibacterial activity was done on the isolates using agar disc-diffusion method with Dimethyl Sulfoxide (DMSO) as a negative control and ceftriaxone as a positive control. Zone of inhibition was measured in millimeters. Minimum bactericidal concentration of the extract was determined using different concentrations of the extract then plated on Neisseria gonorrhoeae (GC) medium and a standard protocol was used to determine the presence of phytochemical compounds.

Results and Discussion: The results obtained showed that both the root and bark extracts of S. longipedunculata have antibacterial activity against the two bacterial strains with a zone of inhibition of ≥ 10mm and a bactericidal activity at 0.01µg-ml of 10% crude extract. Comparison of susceptibility on tetracycline, penicillin and ciprofloxacin with the root and bark extract showed higher susceptibility to the extracts.  The extracts showed presence of saponins, steroids, glycosides, flavonoids, terpenes, alkanoids, phenolics and tanins. The demonstrated antibacterial activity of S. longipedunculata against N. gonorrhoeae provides a scientific basis for the traditional use in treating venereal diseases in western Kenya.

Conclusion: This investigation and further studies will pave the way for use of this plant in antibacterial drug development for alleviating human suffering. We recommend further studies to identify the specific compound(s) responsible for the antibacterial activity.