Multiple Myeloma-Like Spinal MRI Findings in Skeletal Fluorosis: An Unusual Presentation of Fluoride Toxicity in Human.

Endemic fluorosis is a worldwide environmental problem due to excessive fluoride, commonly due to increased drinking water fluoride levels but sometimes due to other sources such as food with high fluoride content. In India, 21 of the 35 states are known to have health problems associated with fluoride toxicity. The present report is a case of a 50-year-old female who was seen with progressive spinal complications and a MRI of the spine suggestive of multiple myeloma. The MRI of the lumbosacral spine showed a diffuse and heterogeneous marrow signal of the lower dorsal and lumbosacral vertebrae. The MRI was also suggestive of coarse trabeculation and appeared predominantly hypointense on the T1W image and had mixed signal intensity on the T2W image. These findings were suggestive of neoplastic bone marrow infiltration and the presence of a proliferative disorder, with multiple myeloma being the most likely. During the patient workup, it was found that other family members were also having similar complications and, after investigation of these family members, it was found that they are suffering from systemic fluorosis. The patient was then evaluated for skeletal fluorosis, and this condition was found to be present. Multiple myeloma was ruled out by the finding of a negative serum protein electrophoresis. The spinal complications appeared to be mainly due to the compression of the spinal cord and nerve roots by protruding osteophytes, thickening of the posterior longitudinal ligament, and thickening of the ligamentum flavum resulting in a compressive myeloradiculopathy and compressive myelopathy. The finding of multiple myeloma-like findings on the spinal MRI in association with skeletal fluorosis was considered to be a very rare event. This case report underlines the need to consider the presence of spinal skeletal fluorosis when evaluating spinal complications with unusual pseudo-multiple myeloma-like changes on the spinal MRI.

Structural and Functional Changes in the Tight Junctions of Asymptomatic and Serology-negative First-degree Relatives of Patients With Celiac Disease.

BACKGROUND: Ten to 15% of first-degree relatives (FDRs) of celiac disease (CeD) patients develop CeD. Although intestinal barrier functions (intestinal permeability) are abnormal in the subset of serology-negative FDRs, what leads to the abnormal barrier function is not known. GOALS: To study the ultrastructure and functions of tight junctions in serology-negative FDRs of CeD patients. STUDY: The intestinal permeability was measured in 97 asymptomatic and anti-tissue transglutaminase antibody (anti-tTG Ab)-negative FDRs (using the lactulose mannitol ratio) and in 75 controls. The ultrastructure of tight junctions using transmission electron microscopy, and the expression of key tight junction proteins (claudin-2, claudin-3, occludin, JAM-A, and ZO-1) and zonulin using real-time PCR and immunohistochemistry were assessed in anti-tTG Ab-negative, HLA-DQ2/-DQ8-positive FDRs having normal villi and in disease controls. In addition, the serum zonulin level was measured in 172 anti-tTG Ab-negative FDRs and 198 controls. RESULTS: The intestinal permeability was significantly increased in FDRs than in controls. Ultrastructural abnormalities such as dilatation of the tight junction (P=0.004) and loss of the pentalaminar structure (P=0.001) were more common in FDRs than in disease controls. There was significant underexpression of tight junction proteins ZO-1 (P=0.040) and occludin (P=0.041) in FDRs. There was no significant difference in the serum zonulin level between FDRs and controls (P=0.154). CONCLUSIONS: Even asymptomatic, anti-tTG-Ab-negative FDRs with a normal villous histology have both ultrastructural and functional abnormalities in tight junctions. These findings are indirect evidence of the presence of tight junction abnormalities before the onset of the disease and may have therapeutic implications.

Surface deterioration and elemental composition of retrieved orthodontic miniscrews.

INTRODUCTION: This study provides insight into surface and elemental analyses of orthodontic retrieved miniscrew implants (MSIs). The sole purpose was to investigate the behavior of MSIs while they are in contact with bone and soft tissues, fluids, and food in the oral cavity. The information thus gathered may help to understand the underlying process of success or failure of MSIs and can be helpful in improving their material composition and design. METHODS: The study was carried out on 28 titanium-alloy MSIs (all from the same manufacturer) split into 3 groups: 18 MSIs were retrieved after successful orthodontic treatment, 5 were failed MSIs, and 5 were as-received MSIs serving as the controls. All MSIs were subjected to energy dispersive x-ray microanalysis to investigate the changes in surface elemental composition and to scanning electron microscopy to analyze their surface topography. Data thus obtained were subjected to suitable statistical analyses. RESULTS: Scanning electron microscope analysis showed surface manufacturing imperfections of the as-received MSIs in the form of stripes. Their elemental composition was confirmed to the specifications of the American Society for Testing of Materials for surgical implants. Retrieved MSIs exhibited generalized surface dullness; variable corrosion; craters in the head, neck, body, and tip regions; and blunting on tips and threads. Energy dispersive x-ray analyses showed deposition of additional elements: calcium had greater significance in its proportion in the body region by 0.056 weight percent; iron was seen in greater proportion in the failed retrieved MSIs compared with the successful miniscrews; cerium was seen in greater proportions in the head region by 0.128 weight percent and in the neck region by 0.147 weight percent than in the body and tip regions of retrieved MSIs. CONCLUSIONS: Retrieved MSIs showed considerable surface and structural alterations such as dullness, corrosion, and blunting of threads and tips. Their surfaces showed interactions and adsorption of several elements, such as calcium, at the body region. A high content of iron was found on the failed MSIs, and cerium was seen in the head and neck regions of retrieved MSIs.

Comparative evaluation of effects of bleaching on color stability and marginal adaptation of discolored direct and indirect composite laminate veneers under in vivo conditions.

STATEMENT OF PROBLEM: Change in color and loss of marginal adaptation of tooth colored restorative materials is not acceptable. Bleaching is commonly used for treating discolored teeth. However, the literature is scanty regarding its effect on color and marginal adaptation of direct and indirect composite laminate veneers (CLVs) under in vivo conditions. PURPOSE: Purpose of the study was to determine the effect of bleaching on color change and marginal adaptation of direct and indirect CLVs over a period of time when exposed to the oral environment. MATERIALS AND METHODS: For this purpose, a total of 14 subjects irrespective of age and sex indicated for CLV restorations on maxillary anterior teeth were selected following the inclusion and exclusion criteria. For each subject, indirect CLVs were fabricated and looted in the first quadrant (Group 1) and direct CLV's (Group 2), were given in the second quadrant. Color change was assessed clinically using intra-oral digital spectrophotometer and marginal adaptation was assessed on epoxy resin replica of the tooth-restoration interface under scanning electron microscope. After 6 months, the subjects underwent a home bleaching regimen for 14 days using 10% carbamide peroxide. The assessment of color change and marginal adaptation was done at 6 months after veneering (0-180 days), immediately after the bleaching regimen (0-194 days) and 3 months after the bleaching regimen (0-284 days). RESULTS: The difference in median color change (ΔE) between the groups was tested using Wilcoxon rank sum test while the median color change with time within the groups was tested using Wilcoxon signed rank test. The difference in the rates of marginal adaptation was tested between the groups using Chi-square/Fisher's exact test. Bleaching led to statistically significant color change at cervical (CE), middle and incisal (IE) regions when direct and indirect composites were compared (P < 0.05). During intra-group comparison, direct CLV's showed significant color change at CE and IE regions when ΔE was compared at 180 days and 284 days (CE 10 vs. CE 30, P = 0.008, IE 10 vs. IE 30, P = 0.003). No significant differences were found when within group comparison was made for indirect laminates. Intergroup comparison between the groups showed significant difference in marginal adaptation at CE margin at all.time points (at baseline, P = 0.005; at 180 days, P = 0.007; 194 days, P = 0.025; at 284 days, P = 0.067). CONCLUSION: After bleaching, indirect CLVs performed better in terms of color stability whereas direct CLVs performed better in terms of marginal adaptation. CLINICAL SIGNIFICANCE: Indirect composites should be preferred to direct composites as veneering materials as they have better color stability. Special attention should be given to their marginal adaptation especially in the CE region. Bleaching should be avoided in patients with composite restorations in the mouth.

clpC operon regulates cell architecture and sporulation in Bacillus anthracis.

The clpC operon is known to regulate several processes such as genetic competence, protein degradation and stress survival in bacteria. Here, we describe the role of clpC operon in Bacillus anthracis. We generated knockout strains of the clpC operon genes to investigate the impact of CtsR, McsA, McsB and ClpC deletion on essential processes of B. anthracis. We observed that growth, cell division, sporulation and germination were severely affected in mcsB and clpC deleted strains, while none of deletions affected toxin secretion. Growth defect in these strains was pronounced at elevated temperature. The growth pattern gets restored on complementation of mcsB and clpC in respective mutants. Electron microscopic examination revealed that mcsB and clpC deletion also causes defect in septum formation leading to cell elongation. These vegetative cell deformities were accompanied by inability of mutant strains to generate morphologically intact spores. Higher levels of polyhydroxybutyrate granules accumulation were also observed in these deletion strains, indicating a defect in sporulation process. Our results demonstrate, for the first time, the vital role played by McsB and ClpC in physiology of B. anthracis and open up further interest on this operon, which might be of importance to success of B. anthracis as pathogen.

Comparison of marginal fidelity and surface roughness of porcelain veneers fabricated by refractory die and pressing techniques.

PURPOSE: The aim of the present study was to compare the marginal fidelity and surface roughness of porcelain veneers fabricated by the refractory die and pressing techniques under in vivo conditions. MATERIALS AND METHODS: A total of 72 veneers were prepared for anterior teeth in 12 participants. Veneers on anterior teeth in the first and second quadrants were fabricated using refractory die (group I) and pressing techniques (group II), respectively. Surface roughness was evaluated using a profilometer in three areas (cervical, mesio-incisal, disto-incisal) for each veneer. Marginal adaptation of all the veneers (N = 36/group) was evaluated at each margin (cervical, incisal, mesial, and distal) at 7 days and at 3 months after cementation under a scanning electron microscope (SEM) at 200× magnification. RESULTS: The mean surface roughness of veneers in cervical, mesio-incisal, and disto-incisal areas was 0.41 ± 0.25, 0.33 ± 0.14, and 0.32 ± 0.14 µm, respectively, for group I; and 0.31 ± 0.11, 0.36 ± 0.18, and 0.29 ± 0.11 µm, respectively, for group II. Intra- and intergroup comparisons showed no statistically significant values for all areas (p > 0.05). In 144 margins evaluated for each group, a visible gap was present in 15 (10.4%) and 18 (12.5%) recordings at 7 days for groups I and II, respectively. They increased to 19 (13.1%) and 20 (13.8%) after 3 months. These gaps were further broken down into percent distribution of total recordings at the cervical, incisal, mesial, and distal margins. Intragroup comparison was made using the Cochrane test. The chi-square test and Fisher's exact test were used for intergroup comparison of margins, revealing no statistical difference (p > 0.05) CONCLUSION: Within the limitations of the study, the surface roughness and marginal fidelity of porcelain veneers fabricated by refractory die technique and pressing technique were comparable.

Factor VIII can be synthesized in hemophilia A mice liver by bone marrow progenitor cell-derived hepatocytes and sinusoidal endothelial cells.

Hemophilia A (HA) is caused by mutation in factor VIII (FVIII) gene in humans; it leads to inadequate synthesis of active protein. Liver is the primary site of FVIII synthesis; however, the specific cell types responsible for its synthesis remain controversial. We propose that the severity of the bleeding disorder could be ameliorated by partial replacement of mutated liver cells by healthy cells in HA mice. The aim of this investigation was to study the cellular origin of FVIII by examining bone marrow cell therapy for treatment of HA in mice. Recipient liver was perturbed with either acetaminophen or monocrotaline to facilitate the engraftment and differentiation of lineage-depleted (Lin(-)) enhanced green fluorescent protein-expressing bone marrow cells. Immunohistochemical analysis of liver tissue was conducted to identify the donor-derived cells that expressed FVIII. This identification was confirmed by transmission electron microscopy and quantitative gene expression analysis. The phenotypic correction in HA mice was determined by tail-clip challenge and FVIII level in plasma by Chromogenix and activated partial thromboplastin time assays. Immunohistochemical analysis showed that von Willebrand factor and cytokeratin-18-expressing endothelial cells and hepatocytes, respectively, were obtained from BM-derived cells. Both cell types expressed FVIII light chain mRNA and protein, which was further confirmed by transmission electron microscopy. The transplanted HA mice showed FVIII activity in plasma (P<0.01) and survived tail-clip challenge (P<0.001). Thus, we conclude that BM-derived hepatocytes and endothelial cells can synthesize FVIII in liver and correct bleeding phenotype in HA mice.

Ultrastructural variations in platelets and platelet mitochondria: a novel feature in amyotrophic lateral sclerosis.

Platelets are characterized as a systemic tool to elucidate mitochondria-allied perturbance in neurological diseases. The authors studied ultrastructural changes in platelets and platelet mitochondria using a case-control approach in amyotrophic lateral sclerosis (ALS). Subjects were sporadic ALS cases (n = 22) and age- and sex-matched controls (n = 16). Phlebotomy was performed, platelet concentrates (PCs) were prepared, and mitochondria were extracted. PCs and mitochondria were processed for ultrastructure study using transmission electron microscopy. Image analysis was done using Image-J. Transmission electron microscopy demonstrated both qualitative and quantitative variations in ALS platelets and platelet mitochondria. Heterogeneous distribution of granules, formation of vacuoles, blebs, pseudopodia, loose demarcation of cell membrane with a significant increase in area (20.3%), perimeter (17.82%), integrated density (21.44%), electron-lucent granules (41.79%), and vacuoles (36.58%) were observed in ALS platelets. Conversely, control platelets exhibited an increase of circularity (11.7%) and electron-dense granules (36.89%). In parallel, nonuniformity of matrix, faint cristae, greater lysosomal bodies, and lesser intramitochondrial granules were seen in ALS platelet mitochondria. Significantly greater area (26.88%), perimeter (15%), circularity (3.76%), and integrated density (25.18%) were observed in control platelet mitochondria. Ultastructural divergence in platelets of ALS patients underlines a potential dependence of platelets on modest mitochondrial functioning. These observations also support the view that systemic involvement might be a novel feature in ALS pathophysiology.

Mitochondrial perturbance and execution of apoptosis in platelet mitochondria of patients with amyotrophic lateral sclerosis.

Role of platelets have been evinced as a systemic tool in a variety of neurological disorders. Oxidative phosphorylation contributes approximately 80% of total adenosine-tri-phosphate (ATP) production in resting platelets suggesting potential dependence of platelets on modest mitochondrial functioning. Since mitochondria play a pivotal role in regulating metabolic and apoptotic pathways in various neurodegenerative disorders including amyotrophic lateral sclerosis (ALS), we assessed mitochondrial membrane potential (MMP) associated alterations and apoptotic status of platelet mitochondria in ALS patients using case-control approach. Confocal microscopy reflected heterogeneous distribution of JC-1 aggregates and monomers indicating altered MMP in ALS platelets. Our flow cytometry results confirmed greater percentage of mitochondrial depolarization in ALS platelets. Greater exposure of phosphatidyl serine (PS) residue vindicated by annexin V binding and lesser accumulation of mitotracker red in mitochondrial matrix demonstrated initiation of apoptosis in ALS platelets. Our findings corroborate mitochondrial abnormalities such as perturbance of MMP, mitochondrial depolarization, and apoptosis in ALS platelet mitochondria. In conclusion, our study further evinces the involvement of mitochondrial dysfunction in the pathogenesis of ALS and suggests implication of cell death in peripheral tissues apart from motor neurons in ALS.

Sturge-Weber syndrome with congenital glaucoma and cytochrome P450 (CYP1B1) gene mutations.

UNLABELLED: Sturge-Weber syndrome (SWS) is a progressive condition of mesodermal phakomatosis. This preliminary study is the first report of CYP1B1 mutation analysis in SWS with congenital glaucoma. PURPOSE: Mutations in CYP1B1 gene are the major cause of congenital glaucoma. CYP1B1 is involved in metabolism of melatonin, retinol, and other endogenous/exogenous substrates. Mutations in CYP1B1 adversely affect signal transduction pathways and thus impair development/differentiation of anterior segment structures. This results in impaired aqueous outflow. CYP1B1 has higher expression in fetal eyes and plays major role in morphogenesis of iris, ciliary body, and anterior chamber angle. Hence, we decided to evaluate SWS cases with buphthalmos for 6 most prevalent CYP1B1 mutations by polymerase chain reaction-restriction-fragment length polymorphism followed by sequencing. Trabecular meshwork was studied for morphological alterations by scanning electron microscopy. RESULTS: All patients had normal 46, XY karyotype. Polymerase chain reaction-restriction-fragment length polymorphism showed CYP1B1 mutations in 2 of 5 SWS cases. Scanning electron microscopy findings were suggestive of trabecular dysgenesis. DISCUSSION: No CYP1B1 mutation has been reported in any SWS case till date because syndromic cases were not analyzed for mutations in earlier studies. Earlier studies have reported that onset of glaucoma in SWS shows a bimodal pattern. The results from this pilot study show that SWS cases with gyral calcification, buphthalmos, and early onset glaucoma should be analyzed for CYP1B1 mutations. The effect of vascular malformation-induced venous engorgement and raised intraocular pressure may only be additive and may result in a much more severe phenotype. CONCLUSION: SWS with buphthalmos and gyral calcification should undergo CYP1B1 mutation analysis to identify an underlying genetic pathology for glaucoma. This will aid in determining the prognosis and management and will also help to provide comprehensive counseling in such cases.

Truncated human cathepsin L, encoded by a novel splice variant, exhibits altered subcellular localization and cytotoxicity.

Cathepsin L (ctsl), a lysosomal cyteine protease over expressed and secreted by cancer cells, has been implicated in a number of physiological and pathological processes including tumor cell proliferation and metastasis. In the present study we demonstrate that an unknown mRNA of human origin (Gene Bank accession number AF 217997) is a splice variant of human cathepsin L mRNA (hCATL A IV) and encodes a truncated form of cathepsin L (Deltactsl) containing only 151 C-terminal amino acids. This isoform is cytotoxic to the mammalian cells. Transient transfection studies revealed that unlike ctsl, upon over expression in eukaryotic cells Deltactsl is not secreted in to the media. Immunogold electron microscopy exhibited its localization to nuclear, perinuclear and cytosolic region. In view of its cytotoxic property, targeted expression of Deltactsl in tumor cells may prove useful in the management of cancer.

Mutation spectrum of CYP1B1 in North Indian congenital glaucoma patients.

PURPOSE: Mutations in Cytochrome P450 (CYP1B1) are a predominant cause of congenital glaucoma. This study was planned with the aim to identify the mutation profile of CYP1B1 in North Indian primary congenital glaucoma (PCG) patients. METHODS: After ethical clearance, 50 congenital glaucoma patients and 50 ethnically matched controls were recruited in this study. Genomic DNA was isolated from the blood and trabecular meshwork, and CYP1B1 was screened for the six most prevalent mutations (termination at 223 [Ter@223], Gly61Glu, Pro193Leu, Glu229Lys, Arg368His, and Arg390Cys) by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). DNA sequencing was done to identify other mutations and for confirmation of RFLP positive samples. RESULTS: On PCR-RFLP, 21/50 cases (42%) were found positive for one or more of these mutations. However, on sequencing, we found that 23/50 (46%) harbored the CYPIB1 mutations. Ter@223 was found in 18%, p.R390H in 16%, and p.R368H in 8% of cases. Three novel mutations, p.L24R, p.F190L, and p.G329D, were identified by DNA sequencing. Leucine, phenylalanine, and glycine are conserved at the 24th, 190th, and 329th position in the CYP1B1 protein in different species, suggestive of important functions at these loci. Ter@223 was found to be the most prevalent mutation in our patients while p.R368H was most prevalent in southern India. The difference in frequency and mutation profile may be due to the heterogeneous Indian population. Pathogenic CYP1B1 mutations impair anterior chamber development and differentiation by blocking the aqueous outflow and raising intraocular pressure (IOP). CONCLUSIONS: Three novel mutations were identified in this study. Studies of pathogenic sequence variants in CYP1B1 in different populations may contribute to a better understanding of the disease pathogenesis. This may lead to the development of novel therapeutic approaches in the near future.

Properties of Bacillus anthracis spores prepared under various environmental conditions.

Bacillus anthracis makes highly stable, heat-resistant spores which remain viable for decades. Effect of various stress conditions on sporulation in B. anthracis was studied in nutrient-deprived and sporulation medium adjusted to various pH and temperatures. The results revealed that sporulation efficiency was dependent on conditions prevailing during sporulation. Sporulation occurred earlier in culture sporulating at alkaline pH or in PBS than control. Spores formed in PBS were highly sensitive towards spore denaturants whereas, those formed at 45 degrees C were highly resistant. The decimal reduction time (D-10 time) of the spores formed at 45 degrees C by wet heat, 2 M HCl, 2 M NaOH and 2 M H(2)O(2) was higher than the respective D-10 time for the spores formed in PBS. The dipicolinic acid (DPA) content and germination efficiency was highest in spores formed at 45 degrees C. Since DPA is related to spore sensitivity towards heat and chemicals, the increased DPA content of spores prepared at 45 degrees C may be responsible for increased resistance to wet heat and other denaturants. The size of spores formed at 45 degrees C was smallest amongst all. The study reveals that temperature, pH and nutrient availability during sporulation affect properties of B. anthracis spores.

Differences in the cell wall architecture of melanin lacking and melanin producing Cryptococcus neoformans clinical isolates from India: an electron microscopic study

Cryptococcus neoformans is an important opportunistic fungal pathogen that causes life-threatening infection of the central nervous system. A major virulence factor for C. neoformans is the production of melanin in the cell wall. Using transmission electron microscopy, we studied the cell walls of three pairs of isolates obtained from patients with dual cryptococcal infections, where a melanotic and an albino strain were isolated from the CSF of each patient. Transmission Electron Microscopy revealed that the albino strains lacked a melanin layer whereas a melanin layer was associated with the cell wall of the melanotic strains, comprising approximately 75 percent of the cell wall area. The cell wall size of the melanin producing cells was approximately double the size the albino isolates' cell walls (p value <= 0.003). In this study TEM revealed that the differences in the ultrastructure of the melanin lacking and melanin producing isolates were associated to the cell wall and the melanin layer.

Cryptococcus neoformans é um importante fungo oportunista patogênico que causa infecção no sistema nervoso central, e que pode levar o paciente à morte. Um dos principais fatores de virulência do C. neoformans é a produção de melanina na parede celular. Utilizando microscopia eletrônica de transmissão, nós estudamos as paredes celulares de três pares de isolados obtidos de pacientes com dupla infecção pelo fungo, onde um isolado melanizado e um albino foram isolados do líquor de cada paciente. A microscopia eletrônica de transmissão revelou que as cepas albinas não apresentavam a camada de melanina enquanto que uma camada de melanina estava associada com a parede celular de cepas melanóticas, constituindo aproximadamente 75 por cento da área da parede celular. O tamanho da parede celular das células produtoras de melanina foi aproximadamente o dobro do tamanho da parede celular dos isolados albinos (p < 0,003). Neste estudo, a microscopia eletrônica de transmissão revelou que as diferenças na estrutura dos isolados albinos sem melanina e dos isolados produtores de melanina estava associada à parede celular e a camada de melanina.

Inhibition of protein aggregation: supramolecular assemblies of arginine hold the key.

BACKGROUND: Aggregation of unfolded proteins occurs mainly through the exposed hydrophobic surfaces. Any mechanism of inhibition of this aggregation should explain the prevention of these hydrophobic interactions. Though arginine is prevalently used as an aggregation suppressor, its mechanism of action is not clearly understood. We propose a mechanism based on the hydrophobic interactions of arginine. METHODOLOGY: We have analyzed arginine solution for its hydrotropic effect by pyrene solubility and the presence of hydrophobic environment by 1-anilino-8-naphthalene sulfonic acid fluorescence. Mass spectroscopic analyses show that arginine forms molecular clusters in the gas phase and the cluster composition is dependent on the solution conditions. Light scattering studies indicate that arginine exists as clusters in solution. In the presence of arginine, the reverse phase chromatographic elution profile of Alzheimer's amyloid beta 1-42 (Abeta(1-42)) peptide is modified. Changes in the hydrodynamic volume of Abeta(1-42) in the presence of arginine measured by size exclusion chromatography show that arginine binds to Abeta(1-42). Arginine increases the solubility of Abeta(1-42) peptide in aqueous medium. It decreases the aggregation of Abeta(1-42) as observed by atomic force microscopy. CONCLUSIONS: Based on our experimental results we propose that molecular clusters of arginine in aqueous solutions display a hydrophobic surface by the alignment of its three methylene groups. The hydrophobic surfaces present on the proteins interact with the hydrophobic surface presented by the arginine clusters. The masking of hydrophobic surface inhibits protein-protein aggregation. This mechanism is also responsible for the hydrotropic effect of arginine on various compounds. It is also explained why other amino acids fail to inhibit the protein aggregation.

Expression of mycobacterial cell division protein, FtsZ, and dormancy proteins, DevR and Acr, within lung granulomas throughout guinea pig infection.

The ability of Mycobacterium tuberculosis to persist in a dormant state is a hallmark of tuberculosis. An insight into the expression of mycobacterial proteins will contribute to our understanding of bacterial physiology in vivo. To this end, the expression of FtsZ, Acr and DevR was assessed in the lung granulomas of guinea pigs infected with M. tuberculosis. Antigen immunostaining was then compared with the detection of acid-fast bacilli (AFB) and mycobacterial DNA. Surprisingly, immunostaining for all three antigens was observed throughout the course of infection; maximum expression of all antigens was noted at 20 weeks of infection. The intensity of immunostaining correlated well with the presence of intact bacteria, suggesting that mycobacterial antigens in the extracellular fraction have a short half-life; in contrast to protein, extracellular bacterial DNA was found to be more stable. Immunostaining for bacterial division and dormancy markers could not clearly distinguish between replicating and non-replicating organisms during the course of infection. The detection of Acr and DevR from 4 weeks onwards indicates that the dormancy proteins are expressed from early on in infection. Both antigen staining and DNA detection from intact bacilli were useful for detecting intact mycobacteria in the absence of AFB.

Imaging and analysis of Bacillus anthracis spore germination.

External and internal changes occurring during the process of germination of Bacillus anthracis spores were observed through atomic force microscopy (AFM) and transmission electron microscopy (TEM), respectively. AFM studies showed that in response to L-alanine (4 mM), as a germinant, the spore germinates into a vegetative cell in 3 hours. The temporal size changes occurring during the germination were gradual but the major change in size was observed between the second and third hour. TEM of spores showed the presence of varied layers, which is in accordance with previous studies. However, the integrity of these layers was lost gradually during the process of germination. The inner spore membrane remains intact even until late stages of germination, whereas the coat, outer spore membrane, and the cortical layers are discarded at the second-hour stage. The results indicate that sequential changes during the germination of a B. anthracis spore are similar to other species of the Bacillus group.

Role of Mycobacterium tuberculosis Ser/Thr kinase PknF: implications in glucose transport and cell division.

Protein kinases have a diverse array of functions in bacterial physiology, with a distinct role in the regulation of development, stress responses, and pathogenicity. pknF, one of the 11 kinases of Mycobacterium tuberculosis, encodes an autophosphorylating, transmembrane serine/threonine protein kinase, which is absent in the fast-growing, nonpathogenic Mycobacterium smegmatis. Herein, we investigate the physiological role of PknF using an antisense strategy with M. tuberculosis and expressing PknF and its kinase mutant (K41M) in M. smegmatis. Expression of PknF in M. smegmatis led to reduction in the growth rate and shortening and swelling of cells with constrictions. Interestingly, an antisense strain of M. tuberculosis expressing a low level of PknF displayed fast growth and a deformed cell morphology compared to the wild-type strain. Electron microscopy showed that most of the cells of the antisense strain were of a smaller size with an aberrant septum. Furthermore, nutrient transport analysis of these strains was conducted using 3H-labeled and 14C-labeled substrates. A significant increase in the uptake of D-glucose but not of glycerol, leucine, or oleic acid was observed in the antisense strain compared to the wild-type strain. The results suggest that PknF plays a direct/indirect role in the regulation of glucose transport, cell growth, and septum formation in M. tuberculosis.

Identification, expression, localization and serological characterization of a tryptophan-rich antigen from the human malaria parasite Plasmodium vivax.

Plasmodium vivax is most common but non-cultivable human malaria parasite which is poorly characterized at the molecular level. Here, we describe the identification and characterization of a P. vivax Tryptophan-Rich Antigen (PvTRAg) which contains unusually high (8.28%) tryptophan residues and is expressed by all blood stages of the parasite. The pvtrag gene comprises a 978bp open reading frame interrupted by two introns. The first intron is located in the 5'-untranslated region while the second one is positioned 174bp downstream to the ATG codon. The encoded approximately 40kDa protein contains a transmembrane domain near the N-terminus followed by a tryptophan-rich domain with significantly high surface probability and antigenic index. It is localized in the parasite cytoplasm as well as in the cytoplasm of the parasitized erythrocyte. The purified E. coli expressed recombinant PvTRAg protein showed a very high seropositivity rate for the presence of antibodies amongst the P. vivax patients, indicating that the antigen generates significant humoral immune response during the natural course of P. vivax infection. Analysis of various field isolates revealed that the tryptophan-rich domain is highly conserved except for three-point mutations. The PvTRAg could be a potential vaccine candidate since similar tryptophan-rich antigens of P. yoelii have shown protection against malaria in murine model.