Functional insights by comparison of modeled structures of 18kDa small heat shock protein and its mutant in Mycobacterium leprae

In this work we are proposing Homology modeled structures of Mycobacterium leprae 18kDa heat shock protein and its mutant. The more closely related structure of the small heat shock protein (sHSP) belonging to the eukaryotic species from wheat sHSP16.9 and 16.3kDa ACR1 protein from Mycobacterium tuberculosis were used as template structures. Each model contains an N-terminal domain, alpha-crystalline domain and a C-terminal tail. The models showed that a single point mutation from serine to proline at 52nd position causes structural changes. The structural changes are observed in N-terminal region and alpha-crystalline domains. Serine in 52nd position is observed in β4 strand and Proline in 52nd position is observed in loop. The number of residues contributing α helix at N-terminal region varies in both models. In 18S more number of residues is present in α helix when compared to 18P. The loop regions between β3 and β4 strands of both models vary in number of residues present in it. Number of residues contributing β4 strand in both models vary. β6 strand is absent in both models. Major functional peptide region of alpha crystalline domains of both models varies. These differences observed in secondary structures support their distinct functional roles. It also emphasizes that a point mutation can cause structural variation

Functional characterization of a small heat shock protein from Mycobacterium leprae

<p>Abstract</p> <p>Background</p> <p>Small heat shock proteins are ubiquitous family of stress proteins, having a role in virulence and survival of the pathogen. <it>M. leprae</it>, the causative agent of leprosy is an uncultivable organism in defined media, hence the biology and function of proteins were examined by cloning <it>M. leprae </it>genes in heterologous hosts. The study on sHsp18 was carried out as the knowledge about the functions of this major immunodominant antigen of <it>M. leprae </it>is scanty.</p> <p>Results</p> <p>The gene encoding <it>Mycobacterium leprae </it>small heat shock protein (sHsp18) was amplified from biopsy material of leprosy patients, and cloned and expressed in <it>E. coli</it>. The localization and <it>in vitro </it>characterization of the protein are detailed in this report. Data show that major portion of the protein is localized in the outer membrane of <it>E. coli</it>. The purified sHsp18 functions as an efficient chaperone as shown by their ability to prevent thermal inactivation of restriction enzymes <it>Sma</it>I and <it>Nde</it>I. Physical interaction of the chaperone with target protein is also demonstrated. Size exclusion chromatography of purified protein shows that the protein can form multimeric complexes under <it>in vitro </it>conditions as is demonstrated for several small heat shock proteins.</p> <p>Conclusion</p> <p>The small heat shock protein sHsp18 of <it>M. leprae </it>is a chaperone and shows several properties associated with other small heat shock proteins. Membrane association and <it>in vitro </it>chaperone function of sHsp18 shows that the protein may play a role in the virulence and survival of <it>M. leprae </it>in infected host.</p

Multicenter Evaluation of Diagnostic Circulating Biomarkers to Detect Sight-Threatening Diabetic Retinopathy

Importance: It is a global challenge to provide regular retinal screening for all people with diabetes to detect sight-threatening diabetic retinopathy (STDR). Objective: To determine if circulating biomarkers could be used to prioritize people with type 2 diabetes for retinal screening to detect STDR. Design, Setting, and Participants: This cross-sectional study collected data from October 22, 2018, to December 31, 2021. All laboratory staff were masked to the clinical diagnosis, assigned a study cohort, and provided with the database containing the clinical data. This was a multicenter study conducted in parallel in 3 outpatient ophthalmology clinics in the UK and 2 centers in India. Adults 40 years and older were categorized into 4 groups: (1) no history of diabetes, (2) type 2 diabetes of at least 5 years' duration with no evidence of DR, (3) nonproliferative DR with diabetic macular edema (DME), or (4) proliferative DR. STDR comprised groups 3 and 4. Exposures: Thirteen previously verified biomarkers were measured using enzyme-linked immunosorbent assay. Main Outcomes and Measures: Severity of DR and presence of DME were diagnosed using fundus photographs and optical coherence tomography. Weighted logistic regression and receiver operating characteristic curve analysis (ROC) were performed to identify biomarkers that discriminate STDR from no DR beyond the standard clinical parameters of age, disease duration, ethnicity (in the UK) and hemoglobin A1c. Results: A total of 538 participants (mean [SD] age, 60.8 [9.8] years; 319 men [59.3%]) were recruited into the study. A total of 264 participants (49.1%) were from India (group 1, 54 [20.5%]; group 2, 53 [20.1%]; group 3, 52 [19.7%]; group 4, 105 [39.8%]), and 274 participants (50.9%) were from the UK (group 1, 50 [18.2%]; group 2, 70 [25.5%]; group 3, 55 [20.1%]; group 4, 99 [36.1%]). ROC analysis (no DR vs STDR) showed that in addition to age, disease duration, ethnicity (in the UK) and hemoglobin A1c, inclusion of cystatin C had near-acceptable discrimination power in both countries (area under the receiver operating characteristic curve [AUC], 0.779; 95% CI, 0.700-0.857 in 215 patients in the UK with complete data; AUC, 0.696; 95% CI, 0.602-0.791 in 208 patients in India with complete data). Conclusions and Relevance: Results of this cross-sectional study suggest that serum cystatin C had good discrimination power in the UK and India. Circulating cystatin-C levels may be considered as a test to identify those who require prioritization for retinal screening for STDR

Intergeneric Conjugation in Streptomyces peucetius and Streptomyces sp. Strain C5: Chromosomal Integration and Expression of Recombinant Plasmids Carrying the chiC Gene

Intergeneric conjugal transfer of plasmid DNA from Escherichia coli to Streptomyces circumvents problems such as host-controlled restriction and instability of foreign DNA during the transformation of Streptomyces protoplasts. The anthracycline antibiotic-producing strains Streptomyces peucetius and Streptomyces sp. strain C5 were transformed using E. coli ET12567(pUZ8002) as a conjugal donor. When this donor species, carrying pSET152, was mated with Streptomyces strains, the resident plasmid was mobilized to the recipient and the transferred DNA was also integrated into the recipient chromosome. Analysis of the exconjugants showed stable integration of the plasmid at a single chromosomal site (attB) of the Streptomyces genome. The DNA sequence of the chromosomal integration site was determined and shown to be conserved. However, the core sequence, where the crossover presumably occurred in C5 and S. peucetius, is TTC. These results also showed that the φC31 integrative recombination is active and the phage attP site is functional in S. peucetius as well as in C5. The efficiency and specificity of φC31-mediated site-specific integration of the plasmid in the presence of a 3.7-kb homologous DNA sequence indicates that integrative recombination is preferred under these conditions. The integration of plasmid DNA did not affect antibiotic biosynthesis or biosynthesis of essential amino acids. Integration of a single copy of a mutant chiC into the wild-type S. peucetius chromosome led to the production of 30-fold more chitinase

Construction and genetic characterization of lambda specialized transducing particles carrying the rglB gene of Escherichia coli K-12

The rglB gene ofEscherichia coli codes for a restriction activity that cleaves the hydroxymethylated DNA of T2 and T4 phages. Earlier mapping data placed the gene at 98.39 min counterclockwise to the hsd operon. Genetic analysis of the in vivo gene fusions with λ fusion-transducing phages established the location of the rglB gene next to the hsdS gene of the hsdRMS cluster. The methodology used in this study could be extended to similar in vivo physical mapping of closely linked genes

<span style="font-size:11.0pt;font-family: "Times New Roman";mso-fareast-font-family:"Times New Roman";mso-bidi-font-family: Mangal;mso-ansi-language:EN-GB;mso-fareast-language:EN-US;mso-bidi-language: HI" lang="EN-GB">Rare codons caused poor expression of mammalian cell entry (Mce1A) gene cloned from <i>Mycobacterium leprae</i> in <i>Escherichia coli</i></span>

64-68<span style="font-size:11.0pt;font-family: " times="" new="" roman";mso-fareast-font-family:"times="" roman";mso-bidi-font-family:="" mangal;mso-ansi-language:en-gb;mso-fareast-language:en-gb;mso-bidi-language:="" hi"="" lang="EN-GB">Mammalian cell entry gene of mycobacterium helps its entry into epithelial and natural target cells. In order to carry out the functional assay of Mycobacterium leprae Mce1A, the whole gene was cloned in Escherichia coli. An overexpression vector carrying M. leprae mce1A gene, under IPTG inducible T5 promoter, was cloned in E. coli for expression of encoded protein with N-terminal 6xHis-tag. In the absence of IPTG, E. coli cells carrying the mce1A gene grew normally but induction of gene expression led to inhibition of cell growth. Western blot analysis using anti-His HRP conjugate showed full length Mce1A protein expressed in low amount. Deletion of N-terminal region having adjacent arginine rare codons resulted in overexpression of truncated protein as inclusion bodies without inhibiting cell division with size reduction of recombinant protein. However the full length protein poorly expressed without size reduction.</span

Assembly of mitochondria in yeast complementation of mitochondrial and cytosolic products in a temporal sequence in vitro

1: Sequential transfer of derepressing yeast spheroplasts from a medium containing chloramphenicol to one containing cycloheximide or vice versa, shows that the cytosolically and mitochondrially synthesized products are synthesized independent of each other and accumulate in the absence of their counterparts. 2: This has been demonstrated by immunoprecipitation using specific antisera for cytochrome oxidase and ATPase enzymes. 3: The independently accumulated products have been shown to complement each other for the expression of enzyme activity, upon mixing in vitro. 4: By varying the time of treatment with cycloheximide, thereby allowing the mitochondrial protein synthesis to proceed to different extent, a time sequence in the appearance of the mitochondrially synthesized products is demonstrated