Matrix-Assisted Refolding,Purification and Activity Assessment Using a ‘Form Invariant’ Assay for Matrix Metalloproteinase 2 (MMP2)

Matrix metalloproteinases expression is used as biomarker for various cancers and associated malignancies. Since these proteinases can cleave many intracellular proteins, overexpression tends to be toxic; hence, a challenge to purify them. To overcome these limitations, we designed a protocol where full length pro-MMP2 enzyme was overexpressed in E. coli as inclusion bodies and purified using 6xHis affinity chromatography under denaturing conditions. In one step, the enzyme was purified and refolded directly on the affinity matrix under redox conditions to obtain a bioactive protein. The pro-MMP2 protein was characterized by mass spectrometry, CD spectroscopy, zymography and activity analysis using a simple in-house developed ‘form invariant’ assay, which reports the total MMP2 activity independent of its various forms. The methodology yielded higher yields of bioactive protein compared to other strategies reported till date, and we anticipate that using the protocol, other toxic proteins can also be overexpressed and purified from E. coli and subsequently refolded into active form using a one step renaturation protocol.     

Characterization of Upstream Sequences of the <Emphasis Type=Italic>LOJ</Emphasis> Gene Leads to Identification of a Novel Enhancer Element Conferring Lateral Organ Junction-Specific Expression in <Emphasis Type=Italic>Arabidopsis thaliana</Emphasis>

Isolation and characterization of promoters are important in understanding gene regulation and genetic engineering of crop plants. Earlier, a pentatricopeptide repeat protein (PPR) encoding gene (At2g39230), designated as Lateral Organ Junction (LOJ) gene, was identified through T-DNA promoter trapping in Arabidopsis thaliana. The upstream sequence of the LOJ gene conferred on the reporter gene a novel LOJ-specific expression. The present study was aimed at identifying and characterizing the cis-regulatory motifs responsible for tissue-specific expression in the −673 and +90 bases upstream of the LOJ gene recognized as LOJ promoter. In silico analysis of the LOJ promoter revealed the presence of a few relevant regulatory motifs and a unique feature like AT-rich inverted repeat. Deletion analysis of the LOJ promoter confirmed the presence of an enhancer-like element in the distal region (−673/−214), which stimulates a minimal promoter-like sequence in the −424/−214 region in a position and orientation autonomous manner. The −136/+90 region of the LOJ promoter was efficient in driving reporter gene expression in tissues like developing anthers and seeds of Arabidopsis. A positive regulation for the seed- and anther-specific expression module was contemplated within the 5′ untranslated region of the LOJ gene. However, this function was repressed in the native context by the lateral organ junction-specific expression. The present study has led to the identification of a novel lateral organ junction-specific element and an enhancer sequence in Arabidopsis with potential applications in plant genetic engineering.     

Purification and characterization of a mucin-binding mycelial lectin from <Emphasis Type=Italic>Aspergillus nidulans</Emphasis> with potent mitogenic activity

Mucin-specific lectin from mycelium of Aspergillus nidulans was purified using anion exchange and gel filtration chromatographic techniques with an overall recovery of 32% and 21.97-fold purification. The purified lectin migrated as a single band in SDS–PAGE with an apparent molecular mass of 34 kDa. Carbohydrate analysis revealed that it is a glycoprotein with total sugar content of 2.54%. Optimal agglutination was observed when serially diluted lectin was incubated with human type O erythrocyte suspension at pH 7.0–8.0 and temperature 20–30°C. Lectin was found to be completely stable within pH 5.0–8.0 and temperature at or below 40°C. Demetallization by extensive dialysis against EDTA did not alter its haemagglutination activity. Lectin activity was reduced to half after 24 h incubation with urea and thiourea, with no such effect of guanidine HCl. The lectin showed potent mitogenic response towards mouse splenocytes, attaining a maximum at 200 μg/ml as compared to untreated control cells. Mitogenic lectins are invaluable tools to assess the functioning of immune cells. None of the microfungal lectin has yet been investigated for mitogenic activity. This is the first report on mitogenic activity of lectin from Aspergillus sp.     

Oral toxicity of symbiotic bacteria Photorhabdus spp. against immature stages of insects

The oral toxicity of 5 Photorhabdus spp. strains collected in different regions of Korea was determined in the larvae of Plodia interpunctella, Galleria mellonella, Lucilia caesar, and Culex pipiens pallens. When diet or water containing culture media containing 1 of the 5 different strains was ingested by immature insects, the first instar larvae of both G. mellonella and L. caesar and young larvae of C. pipiens pallens died within 3–5 days after treatment. However, mortality of P. interpunctella neonate larvae was slightly slower and reached 94.4%–100% within 7 days after treatment. The mortality rate of a control group given a diet containing water, the medium without cultured bacteria, or Escherichia coli culture medium was not affected. The mortality rates were 100%, 45.3%, 2.8%, and 0% for Galleria, Lucilia, Plodia, and Culex, respectively, in another control group given a culture medium of Photorhabdus luminescens ssp. laumondii (TT01). In addition, culture media containing Photorhabdus strains significantly inhibited molting of third instar Plodia larvae by as much as 88% 7 days after treatment, whereas molting inhibition was reduced by 0%, 4%, and 20% following treatments with water, E. coli, or TT01 culture media, respectively. Our results suggest that the oral administration of Photorhabdus bacterial medium was highly effective for controlling various immature insects.     

Structural insights into thermostable direct hemolysin of Vibrio parahaemolyticus using single-particle cryo-EM

Thermostable direct hemolysin (TDH) is a ~19 kDa, hemolytic pore-forming toxin from the gram-negative marine bacterium Vibrio parahaemolyticus, one of the causative agents of seafood-borne acute gastroenteritis and septicemia. Previous studies have established that TDH exists as a tetrameric assembly in physiological state; however, there is limited knowledge regarding the molecular arrangement of its disordered N-terminal region (NTR)—the absence of which has been shown to compromise TDH's hemolytic and cytotoxic abilities. In our current study, we have employed single-particle cryo-electron microscopy to resolve the solution-state structures of wild-type TDH and a TDH construct with deletion of the NTR (NTD), in order to investigate structural aspects of NTR on the overall tetrameric architecture. We observed that both TDH and NTD electron density maps, resolved at global resolutions of 4.5 and 4.2 Å, respectively, showed good correlation in their respective oligomeric architecture. Additionally, we were able to locate extra densities near the pore opening of TDH which might correspond to the disordered NTR. Surprisingly, under cryogenic conditions, we were also able to observe novel supramolecular assemblies of TDH tetramers, which we were able to resolve to 4.3 Å. We further investigated the tetrameric and inter-tetrameric interaction interfaces to elaborate upon the key residues involved in both TDH tetramers and TDH super assemblies. Our current structural study will aid in understanding the mechanistic aspects of this pore-forming toxin and the role of its disordered NTR in membrane interaction.     

Morphological and molecular divergence of Indian hill trout,Barilius bendelisis (Hamilton, 1822) stocks from different Rivers in Indo‐Burma biodiversity hotspot: Does river altitude and Dam play a role?

The present study explains the intraspecific variation in Indian Hill trout (Barilius bendelisis) on the basis of image based truss network system and D‐loop region of mtDNA. A total of 210 samples were collected from three different rivers (Teesta, Kameng and Myntudu River) of North East India in Indo‐Burma Biodiversity Hotspot. By using the software applications (tpsDig version 2.1 and PAST), a total of 25 morphometric measurements were generated from 10 landmarks. The Analysis of Variance (ANOVA), Factor Analysis (FA) and Discriminate Function Analysis (DFA) showed, out of the total variations, factor 1 explained 46.74% while factor 2 and factor 3 explained 27.14% and 11.92%, respectively. Using these variables 83.33% of the cross‐validated specimens were classified into distinct groups. Analysis of Molecular Variance (AMOVA) and pairwise Fst value for D‐loop region of mtDNA also showed high to medium level of genetic variation among the stocks and within the stocks. We conclude that the observed discrete stocks might be the result of changing environmental conditions in different rivers of the hotspot as the rivers are present at different altitudinal labels. It is also believed that the variation might be due to the construction of barrages across the river which hinder the mixing among the stocks.     

The effect of osteoporosis on residual ridge resorption and masticatory performance in denture wearers

doi: 10.1111/j.1741‐2358.2011.00610.x The effect of osteoporosis on residual ridge resorption and masticatory performance in denture wearers Aim: To compare masticatory performance, masticatory efficiency and residual ridge resorption (RRR) in osteoporotic and non‐osteoporotic edentulous subjects after rehabilitation with complete dentures. Method: Thirty subjects fulfilling the inclusion criteria were enrolled from the patients visiting the Department of Prosthodontics for complete denture fabrication. Two groups consisting of control subjects (group I; N = 15) and osteoporotic subjects (group II; N = 15) were formed. Complete dentures satisfying certain criteria were fabricated for both groups. Masticatory performance and efficiency were measured 6 months after denture insertion. Areal measurements were taken on lateral cephalograms before and 6 months after denture fabrication. The data were then computed to analyse differences between groups I and II using SPSS statistical software version 15.0. Results: Six months after denture fabrication, the masticatory performance and efficiency were significantly higher (p < 0.001) for group I, with a significant decrease in maxillary and mandibular sagittal area seen in both groups. The rate of bone loss was more in group II compared with group I. Conclusion: Greater masticatory function was demonstrated by the non‐osteoporotic group, and the rate of RRR was more in the osteoporotic group compared with the normal group. In this pilot study, osteoporosis leads to greater RRR, decreased masticatory performance and efficiency in edentulous subjects 6 months after denture insertion. Screening for osteoporosis is suggested as a routine procedure for all edentulous subjects undergoing rehabilitation. Recall check‐ups for osteoporotic patients should be more frequent, and these patients may require more frequent denture remakes.     

Amelioration of cognitive impairment and neurodegeneration by catechin hydrate in rat model of streptozotocin-induced experimental dementia of Alzheimer’s type

Alzheimer’s disease (AD) is a progressive neurodegenerative disorder resulting in cognitive decline and enhancement of oxidative loads in the brain. Flavonoids have been considered to exert human health benefits by anti-oxidant and anti-inflammatory properties. The present study is aimed to elucidate the neuroprotective effect of catechin hydrate (CH), a natural flavanoid with potential antioxidant and anti-inflammatory properties, on intracerebroventricular streptozotocin (ICV-STZ) induced neuronal loss and memory impairment. To test this hypothesis, male Wistar rats were pretreated with CH (10 and 20 mg/kg bwt) orally once daily for 21 days and then bilaterally injected with ICV-STZ (3 mg/kg bwt), while sham group rats receive the same volume of vehicle. After 2 weeks of ICV-STZ infusion, rats were tested for cognitive performance using Morris water maze (MWM) test and then sacrifice for biochemical and histopathological assays. CH was found to be successful in upregulating the antioxidant status and prevented the memory loss. The expression of choline acetyl transferase (ChAT) was decreased in ICV-STZ group and CH pretreatment increases the expression of ChAT. Moreover, inflammatory mediators like TNF-α, IL-1β levels and expression of iNOS were significantly attenuated by CH pretreatment. The study suggests that CH is effective in preventing memory loss, ameliorating the oxidative stress and might be beneficial for the treatment of sporadic dementia of Alzheimer’s type (SDAT).