Fine needle aspiration cytologic diagnosis of erythema nodosum leprosum: a case report

BACKGROUND: Erythema nodosum leprosum (ENL), the type 2 lepra reaction occurring in lepromatous or borderline lepromatous leprosy, presents clinically with acute manifestations that compel the patient to seek medical attention. Recognition and timely management of these patients is critical in order to avoid permanent disability. Fine needle aspiration cytology (FNAC) is a simple, effective tool that aids in correct diagnosis and management of ENL. CASE: A 30-year-old woman presented with history of fever, reddening of the face, and multiple raised, reddish, painful swellings of the bilateral forearms and legs for 7 days. One year previously, she was diagnosed and treated for lepromatous leprosy with type 2 reaction. After a thorough clinical examination a diagnosis of ENL was made. FNA smears from the forearm swellings showed pus-like material with intact and degenerated polymorphonuclear leukocytes and many foamy macrophages with strong granular acid-fast bacillus (AFB) positivity. A cytologic diagnosis of ENL was given, which was confirmed on histopathologic examination of skin biopsy. CONCLUSION: Cytologic features such as a large number of intact and degenerated neutrophils with foamy macrophages and strong granular AFB positivity, in an appropriate clinical background, allows a confident diagnosis of ENL.     

Interdomain contacts control folding of transcription factor RfaH

Escherichia coli RfaH activates gene expression by tethering the elongating RNA polymerase to the ribosome. This bridging action requires a complete refolding of the RfaH C-terminal domain (CTD) from an α-helical hairpin, which binds to the N-terminal domain (NTD) in the free protein, to a β-barrel, which interacts with the ribosomal protein S10 following RfaH recruitment to its target operons. The CTD forms a β-barrel when expressed alone or proteolytically separated from the NTD, indicating that the α-helical state is trapped by the NTD, perhaps co-translationally. Alternatively, the interdomain contacts may be sufficient to drive the formation of the α-helical form. Here, we use functional and NMR analyses to show that the denatured RfaH refolds into the native state and that RfaH in which the order of the domains is reversed is fully functional in vitro and in vivo. Our results indicate that all information necessary to determine its fold is encoded within RfaH itself, whereas accessory factors or sequential folding of NTD and CTD during translation are dispensable. These findings suggest that universally conserved RfaH homologs may change folds to accommodate diverse interaction partners and that context-dependent protein refolding may be widespread in nature.     

Oral delivery of curcumin bound to chitosan nanoparticles cured Plasmodium yoelii infected mice

Curcumin has been shown to have anti malarial activity, but poor bioavailability and chemical instability has hindered its development as a drug. We have bound curcumin to chitosan nanoparticles to improve its bioavailability and chemical stability. We found that curcumin bound to chitosan nanoparticles did not degrade that rapidly in comparison to free curcumin when such particles were incubated in mouse plasma in vitro at room temperature. The uptake of bound curcumin from chitosan nanoparticles by mouse RBC was much better than from free curcumin. Oral delivery of curcumin bound chitosan nanoparticles to normal mice showed that they can cross the mucosal barrier intact and confocal microscopy detected the nanoparticles in the blood. Curcumin loaded chitosan nanoparticles when delivered orally improved the bioavailability of curcumin in the plasma and RBC. While mice infected with a lethal strain of Plasmodium yoelii (N-67) died between 8 and 9 days post infection, feeding of chitosan nanoparticles alone made them to survive for five more days. Feeding 1mg of native curcumin to infected mice per day for seven days resulted in survival of one third of mice but under the same condition when 1mg of curcumin bound to chitosan nanoparticles was fed all the mice survived. Like chloroquine, curcumin inhibited parasite lysate induced heme polymerization in vitro in a dose dependent manner and curcumin had a lower IC(50) value than chloroquine. We believe that binding of curcumin to chitosan nanoparticles increases its chemical stability and enhances its bioavailability when fed to mice. In vitro data suggest that it can inhibit hemozoin synthesis which is lethal for the parasite.     

Anti-hemolytic and Peroxyl Radical Scavenging Activity of Organoselenium Compounds: An In Vitro Study

Selenium-containing amino acids, selenocystine (CysSeSeCys), methylselenocysteine (MeSeCys), and selenomethionine (SeMet) have been examined for anti-hemolytic and peroxyl radical scavenging ability. Effect of these compounds on membrane lipid peroxidation, release of hemoglobin, and loss of intracellular K⁺ ion as a consequence of peroxyl radicals-induced oxidation of human red blood cells were used to evaluate their anti-hemolytic ability. The peroxyl radicals were generated from thermal degradation of 2,2′-azobis(2-methylpropionamidine) dihydrochloride. Significant delay (t _eff) was observed in oxidative damage in the presence of the selenium compounds. From the IC₅₀ values for the inhibition of hemolysis, lipid peroxidation, and K⁺ ion leakage, the relative anti-hemolytic ability of the compounds were found to be in the order of CysSeSeCys > MeSeCys > SeMet. The anti-hemolytic abilities of the compounds, when compared with sodium selenite (Na₂SeO₃) under identical experimental conditions, were found to be better than Na₂SeO₃. Relative rate constants estimated for the reaction of MeSeCys and SeMet with peroxyl radicals by competition kinetics using ABTS²⁻ as a reference confirmed that all the compounds are efficient peroxyl radical scavengers. Comparison of the GPx-like activity of these compounds, by NADPH–GSH reductase coupled assay, indicated that CysSeSeCys exhibits the highest activity. Based on these results, it is concluded that among the compounds examined, CysSeSeCys, possessing the ability to reduce peroxyl radicals and hydroperoxides showed efficient anti-hemolytic activity.     

Purification and properties of a novel broad substrate specific alcohol oxidase from Aspergillus terreus MTCC 6324

An alcohol oxidase was isolated from the microsome of n-hexadecane grown Aspergillus terreus and purified by ion exchange chromatography. The oxidase was found to act on short chain-, long chain-, secondary-, and aromatic-alcohol substrates with highest affinity for n-heptanol (K(M)=0.498 mM, K(cat)=2.7x10(2) s(-1)). The native protein molecular mass was 269+/-5 kDa and the subunit molecular masses were 85-, 63-, 43-, 27-, and 13-kDa. The isoelectric point of the proteins was within 8.3-8.5. High aggregating property of the protein was demonstrated by AFM, DLS and TEM analyses. Chemical analysis showed the presence of oleic acid and palmitic acid at a ratio of 2:1 in the purified protein. This lipoidic nature of the protein particles was correlated to the high aggregating property. In this flavoenzyme, flavin was non-covalently but avidly associated. Peptide mass fingerprinting studies showed the presence of two FAD binding domains in 63 kDa protein. Among these two FAD binding domain sequences only the YPVIDHEYDAVVVGAGGAGLR peptide shows 45-50% sequence homology with the reported N-terminal sequences of other known alcohol oxidases. Non-redundant database search of 63- and 43-kDa subunits peptide sequences showed no sequence similarity with the other alcohol oxidase protein reported till now.     

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.