Human gingiva-derived mesenchymal stem cells are superior to bone marrow-derived mesenchymal stem cells for cell therapy in regenerative medicine

Mesenchymal stem cells (MSCs) are capable of self-renewal and differentiation into multiple cell lineages. Presently, bone marrow is considered as a prime source of MSCs; however, there are some drawbacks and limitations in use of these MSCs for cell therapy. In this study, we demonstrate that human gingival tissue-derived MSCs have several advantages over bone marrow-derived MSCs. Gingival MSCs are easy to isolate, homogenous and proliferate faster than bone marrow MSCs without any growth factor. Importantly, gingival MSCs display stable morphology and do not loose MSC characteristic at higher passages. In addition, gingival MSCs maintain normal karyotype and telomerase activity in long-term cultures, and are not tumorigenic. Thus, we reveal that human gingiva is a better source of MSCs than bone marrow, and large number of functionally competent clinical grade MSCs can be generated in short duration for cell therapy in regenerative medicine and tissue engineering.     

Dielectric studies of wheat in powder form at microwave frequencies

Dielectric constant and loss factor of Raj-4120 variety of Indian wheat were determined in powder form (grain size 125 to 150 micron) at room temperature. Microwaves at three different frequencies were employed in C-band, X-band and Ku-band respectively for investigating frequency dependence of dielectric parameters of the sample. Bulk dielectric values of the sample were determined by employing the dielectric mixture relations, such as, half power mixture equation, Landau and Lifshitz, Looyenga equation etc.     

Comparative genomic analysis of a neurotoxigenic Clostridium species using partial genome sequence: Phylogenetic analysis of a few conserved proteins involved in cellular processes and metabolism

Clostridial organisms produce neurotoxins, which are generally regarded as the most potent toxic substances of biological origin and potential biological warfare agents. Clostridium tetani produces tetanus neurotoxin and is responsible for the fatal tetanus disease. In spite of the extensive immunization regimen, the disease is an important cause of death especially among neonates. Strains of C. tetani have not been genetically characterized except the complete genome sequencing of strain E88. The present study reports the genetic makeup and phylogenetic affiliations of an environmental strain of this bacterium with respect to C. tetani E88 and other clostridia. A shot gun library was constructed from the genomic DNA of C. tetani drde, isolated from decaying fish sample. Unique clones were sequenced and sequences compared with its closest relative C. tetani E88.A total of 275 clones were obtained and 32,457 bases of non-redundant sequence were generated. A total of 150 base changes were observed over the entire length of sequence obtained, including, additions, deletions and base substitutions. Of the total 120 ORFs detected, 48 exhibited closest similarity to E88 proteins of which three are hypothetical proteins. Eight of the ORFs exhibited similarity with hypothetical proteins from other organisms and 10 aligned with other proteins from unrelated organisms. There is an overall conservation of protein sequences among the two strains of C. tetani and. Selected ORFs involved in cellular processes and metabolism were subjected to phylogenetic analysis.     

Digital karyotyping: an update of its applications in cancer

DNA copy number alterations, including entire chromosomal changes and small interstitial DNA amplifications and deletions, characterize the development of cancer. These changes usually affect the expression of target genes and subsequently the function of the target proteins. Since the completion of the human genome project, the capacity to comprehensively analyze the human cancer genome has expanded significantly. Techniques such as digital karyotyping have been developed to allow for the detection of DNA copy number alterations in cancer at the whole-genome scale. When compared with conventional methods such as spectral karyotyping, representational difference analysis, comparative genomic hybridization (CGH), or the more recent array CGH; digital karyotyping provides an evaluation of copy number of genetic material at higher resolution. Digital karyotyping has therefore promised to enhance our understanding of the cancer genome. This article provides an overview of digital karyotyping including the principle of the technology and its applications in identifying potential oncogenes and tumor suppressor genes.     

Cytologic features of granulosa cell tumors in fluids and fine needle aspiration specimens

OBJECTIVE: To describe the cytologic features of granulosa cell tumors in fluids and fine needle aspiration specimens, with histologic confirmation. STUDY DESIGN: Histologically confirmed granulosa cell tumors, 6 adult type and 1 juvenile type, were identified. All patients had local recurrences or metastases. Eleven specimens from 7 patients, including cytologic samples, cell blocks and histology, were reviewed. Inhibin immunostaining was performed on cell blocks to aid identification of this group of tumors in the cytologic and histologic samples. RESULTS: The patients were 22-72 years old. Sites included ovary and peritoneum; there were pelvic recurrences and metastatic lesions in the spleen, liver, perirectum and cervical lymph node. Cytologic features of adult granulosa cell tumors included 3-dimensional clusters, resettes loose monolayers and individual cells. Other features were Call-Exner bodies, vacuolated cytoplasm, exuberant capillaries associated with papillarylike fronds, a second population of elongated theca cells, and prominent or rare nuclear grooves. In juvenile granulosa cell tumor the features observed were monolayers, loosely cohesive sheets, single cells, occasional larger pleomorphic cells with nuclear clefting and nuclear protrusions, vacuolated cytoplasm, finely granular chromatin and frequent mitoses. The overall cytologic and histologic correlation was good. Inhibin was focally positive in one peritoneal fluid, correlating with the focal pattern of staining seen on histology. CONCLUSION: A definitive cytologic diagnosis of granulosa cell tumor can be made based on the above criteria. Aggressive tumors are discohesive and show pleomorphism and nuclear protrusions. Inhibin stain may be helpful in identifying granulosa cell tumors in cell block specimens.     

Defects in human immunodeficiency virus budding and endosomal sorting induced by TSG101 overexpression

Retrovirus budding is greatly stimulated by the presence of Gag sequences known as late or L domains. The L domain of human immunodeficiency virus type 1 (HIV-1) maps to a highly conserved Pro-Thr-Ala-Pro (PTAP) sequence in the p6 domain of Gag. We and others recently observed that the p6 PTAP motif interacts with the cellular endosomal sorting protein TSG101. Consistent with a role for TSG101 in virus release, we demonstrated that overexpressing the N-terminal, Gag-binding domain of TSG101 (TSG-5') suppresses HIV-1 budding by blocking L domain function. To elucidate the role of TSG101 in HIV-1 budding, we evaluated the significance of the binding between Gag and TSG-5' on the inhibition of HIV-1 release. We observed that a mutation in TSG-5' that disrupts the Gag/TSG101 interaction suppresses the ability of TSG-5' to inhibit HIV-1 release. We also determined the effect of overexpressing a panel of truncated TSG101 derivatives and full-length TSG101 (TSG-F) on virus budding. Overexpressing TSG-F inhibits HIV-1 budding; however, the effect of TSG-F on virus release does not require Gag binding. Furthermore, overexpression of the C-terminal portion of TSG101 (TSG-3') potently inhibits budding of not only HIV-1 but also murine leukemia virus. Confocal microscopy data indicate that TSG-F and TSG-3' overexpression induces an aberrant endosome phenotype; this defect is dependent upon the C-terminal, Vps-28-binding domain of TSG101. We propose that TSG-5' suppresses HIV-1 release by binding PTAP and blocking HIV-1 L domain function, whereas overexpressing TSG-F or TSG-3' globally inhibits virus release by disrupting the cellular endosomal sorting machinery. These results highlight the importance of TSG101 and the endosomal sorting pathway in virus budding and suggest that inhibitors can be developed that, like TSG-5', target HIV-1 without disrupting endosomal sorting.     

Sucrose mobilization in relation to essential oil biogenesis during palmarosa (<Emphasis Type="Italic">Cymbopogon martinii</Emphasis> Roxb. Wats. var.<Emphasis Type="Italic">motia</Emphasis>) inflorescence development

Palmarosa inflorescence with partially opened spikelets is biogenetically active to incorporate [U-¹⁴C]sucrose into essential oil. The percent distribution of¹⁴C-radioactivity incorporated into geranyl acetate was relatively higher as compared to that in geraniol, the major essential oil constituent of palmarosa. At the partially opened spikelet stage, more of the geraniol synthesized was acetylated to form geranyl acetate, suggesting that majority of the newly synthesized geraniol undergoes acetylation, thus producing more geranyl acetate.In vitro development of palmarosa inflorescence, fed with [U-¹⁴C]sucrose, resulted in a substantial reduction in percent label from geranyl acetate with a corresponding increase in free geraniol, thereby suggesting the role of an esterase in the production of geraniol from geranyl acetate. At time course measurement of¹⁴CO₂ incorporation into geraniol and geranyl acetate substantiated this observation. Soluble acid invertase was the major enzyme involved in the sucrose breakdown throughout the inflorescence development. The activities of cell wall bound acid invertase, alkaline invertase and sucrose synthase were relatively lower as compared to the soluble acid invertase. Sucrose to reducing sugars ratio decreased till fully opened spikelets stage, concomitant with increased acid invertase activity and higher metabolic activity. The phenomenon of essential oil biosynthesis has been discussed in relation to changes in these physiological parameters.     

Reclassification of "atypical" diagnoses in endoscopic retrograde cholangiopancreaticography-guided biliary brushings

OBJECTIVE: To evaluate the usefulness of reclassifying "atypical" diagnoses in reporting biliary cytology using strict morphologic criteria. STUDY DESIGN: Cytologic specimens from 139 patients (direct, alcohol-fixed smears or cytocentrifuge preparations) were evaluated. Diagnoses were benign (70), atypical (36) and malignant (33). Using strict criteria--major (nuclear contour, chromatin pattern) and minor (polarity, cell types, nuclear size, nuclear grooves, nucleoli, mitosis, nuclear/cytoplasmic [N/C] ratio)--atypical cases were reevaluated and reclassified. Follow-up (F/U) was available on all cases. RESULTS: Atypical cases, (36) were reclassified as malignant (26), atypical favor benign (2)/reactive (3) and atypical, not otherwise specified (NOS) (5). Cases reclassified as malignant showed irregular nuclear contours, chromatin irregularities and rare mitosis. Nuclear enlargement, nucleoli and cellularity varied widely in all groups. N/C ratio was increased in most reclassified malignant cases. All 26 malignant reclassifications correlated with F/U of malignancy. Benign and reactive cases (5) were negative for malignancy on F/U (4), and in 1 case a metastatic carcinoma involving the biliary tree was found. In the 5 atypical (NOS) cases, F/U showed malignancy (3) and pancreatitis (2). Cytocentrifuge preparations made in our laboratory were of superior quality when compared to other methods of cell preparation. CONCLUSION: Irregularities in nuclear membrane and abnormal chromatin pattern were the most consistently useful features correlating with malignancy. The sensitivity and specificity of biliary brush cytology can be enhanced by using strict cytomorphologic criteria and proper collection and fixation, all of which decrease atypical diagnoses.     

Purification and concentration of alkaline phosphatase by selective permeabilization of Escherichia coli using reverse micellar solutions

Recovery of alkaline phosphatase (AP) from the periplasm of Escherichia coli using reverse micellar solutions (RMSs) of sodium dioctyl sulfosuccinate (AOT) in aliphatic hydrocarbons has been attempted. A variety of surface-active agents, solvents, and reverse micellar conditions were screened, and an excellent recovery of the enzyme in a concentrated form, with a high purification factor, was obtained in a single-step process. The permeabilization process strongly depended on the water content of the RMS as well as on the amount of water coating the microbial cell surface. The product was almost free from nucleic acids. In addition, because of the low affinity of AOT and the organic solvent for the aqueous phase, contamination by the permeabilizing agents would also be negligible.     

Molecular pathology of dityrosine cross-links in proteins: Structural and functional analysis of four proteins

The dityrosine bond (DT) is an oxidative covalent cross-link between two tyrosines. DT cross-linking is increasingly identified as a marker of oxidative stress, aging and disease, and has been detected in diverse pathologies. While DT cross- linked proteins have been documented, the consequences of the DT link on the structure and function of the so modified proteins are yet to be understood. With this in view, we have studied the properties of intermolecular DT-dimers of four proteins of diverse functions, namely the enzyme ribonuclease A, the signal protein calmodulin, and the eye lens proteins alpha- and gamma B-crystallins. We find that DT is formed through radical reactions and type I photosensitization (including OH, O₂ ^– and OONO^–), but not by ¹O₂ and NO₂ ^– (which modify his, trp and met more readily). Tyr residues on the surface of the protein make DT bonds (intra- and intermolecular) most readily and preferentially. The conformation of each of these DT-dimers, monitored by spectroscopy, is seen not to be significantly altered in comparison to that of the parent monomer, but the structural stability of the DT cross-linked molecule is lower than that of the parent native monomer. The DT-dimer is denatured at a lower temperature, and at lower concentrations of urea or guanidinium chloride. The effect of DT-cross-linking on the biological activities of these proteins was next studied. The enzymatic activity of the DT-dimer of ribonuclease A is not lost but lowered. DT-dimerization of lens alpha-crystallin did not significantly affect the chaperone-like ability; it inhibits the self-aggregation and precipitation of target proteins just as well as the parent, unmodified alpha-crystallin does. DT-dimerization of gamma B-crystallin is however seen to lead to more ready aggregation and precipitation, a point of interest in cataract. In the case of calmodulin, we could generate both intermolecular and intramolecular DT cross-linking, and study both the DT-dimer and DT-monomer. The DT-dimer binds smooth muscle light chain kinase and also Ca²⁺, but less efficiently and over a broad concentration range than the native monomer. The intramolecular DT-monomer is weaker in all these respects, presumably since it is structurally more constrained. These results suggest that DT cross-linking of globular proteins weakens their structural stability and compromises (though does not abolish) their biological activity, both of which are pathologically relevant. The intramolecular DT cross-link would appear to lead to more severe structural and functional consequences.