Glucosylation of esculetin by plant cell suspension cultures

Cell suspension cultures of Lithospermum erythrorhizon, Gardenia jasminoides and Nicotiana tabacum were capable of glucosylating esculetin to esculin (7-hydroxycoumarin-6-O--D-glucoside). Especially, a culture strain of Lithospermum erythrorhizon was superior in the esculetin glucosylating capability; 40 to 50% of esculetin administered to the culture medium at early stationary growth stage was converted into esculin within 24 h. The rate of glucosylation was also dependent on the growth stage and the medium composition especially growth hormones and sugar.     

A study of lipid- and protein- bound sialic acids for the diagnosis of bladder cancer and their relationships with the severity of malignancy

Background:

The gold standard for detection of bladder cancer is cystoscopy, which is an invasive and complicated procedure. Our study was conducted to find a tumor marker with high specificity, sensitivity, and accuracy for the diagnosis of bladder cancer.

Methods:

Serum samples were collected from 58 bladder cancer patients and 60 healthy control subjects. Levels of lipid-bound sialic acid (LBSA), and protein-bound sialic acid (PBSA) were measured spectrophotometrically by Aminoff’s method.

Results:

Mean levels of both markers were found to be significantly higher in the patients than the healthy controls. Positive correlations were observed between serum levels of lipid- (r=0.283, p<0.05) and protein- bound (r=0.56, p<0.05) sialic acids and the grade of malignancy. To differentiate patients with bladder tumors from healthy controls, cut-offpoints were determined for each of the two parameters based on Receiver Operating Characteristic (ROC) curve analysis (LBSA=21.25 mg/dL, PBSA=6.15 mg/dL). The data showed good sensitivities (LBSA=89%, PBSA=79%), specificities (LBSA=70%, PBSA=70%) and accuracies (LBSA=83%, PBSA=81%) for both markers.

Conclusion:

Measuring serum LBSA and PBSA by this simple, reproducible, noninvasive, and inexpensive method can accurately discriminate cancer patients from healthy individuals. Key Words: Urinary Bladder Neoplasms, N-Acetylneuraminic Acid, Tumor Markers     

Effect of Homocysteine Thiolactone on Structure and Aggregation Propensity of Bovine Pancreatic Insulin

Homocysteine thiolactone (HCTL) is a cyclic thioester of homocysteine, showing high reactivity toward lysine residues of proteins. In the present study the structural properties and aggregation propensity of bovine pancreatic insulin were studied in the presences of increasing concentration of HCTL (0–500 μM), using different spectroscopic techniques. As shown in this study, HCTL induces gross structural alterations and subsequently aggregation of insulin in a dose dependent manner. Also induction of insulin aggregation by HCTL occurs in a sequential process, where native protein with alpha-helical abundant structure gradually transforms into partially folded conformations with the significant amount of beta-sheet. Since C-terminal B-chain of insulin plays a critical role in stability of this protein, the structural alteration/aggregation induced by HCTL can be consequence of homocysteinylation of the only Lysine residue (Lys29) on its B-chain. This study may have important implications regarding the effect of HCTL on structure of insulin particularly in the pathological states linked to hyperhomocysteinemia.     

Interferon Gamma Release Assay in response to PE35/PPE68 proteins: a promising diagnostic method for diagnosis of latent tuberculosis

Tuberculosis control relies on the identification and preventive treatment of people who are latently infected with Mycobacterium tuberculosis (Mtb). PE/PPE proteins have been reported to elicit CD4 and/or CD8 responses either in the form of whole recombinant proteins or as individual peptides. Very few of the PE and PPE proteins have been previously tested for responses in patients with TB and healthy donors. This is the first study to evaluate the Interferon Gamma Release Assay (IGRA) after stimulation with PE35 and PPE68. The antigenspecific levels of IFN-γ following stimulation with QuantiFERON-TB gold in-tube (QFT-G-IT) antigens, and PE35 and PPE68 recombinant proteins were evaluated in 79 children and 102 adults, respectively. Using QFT-G-IT kit, latent tuberculosis infection (LTBI) was detected in 26 children (33%) and 41 adults (40%); IGRA following stimulation with PE35 and PPE68 recombinant proteins, was positive, respectively, in 36 (46%) and 32 (40.5%) children, respectively. In addition, 53 adults (52%) had positive results following stimulation with these two proteins. The sensitivity and specificity ofIGRAfollowing stimulation with recombinant PE35 in children were76%and 80%, and following stimulation with recombinant PPE68 in this group, it was 73% and 75%, respectively. Meanwhile, there is no gold standard test for LTBI. Our designed tests using PE35 and PPE68 PE/PPE proteins, two PE/PPE proteins not present in BCG vaccins, which elicit CD4 and/or CD8 responses, might be helpful for rapid diagnosis of TB and improve the detection of LTBI. However, further validation studies to determine the advantage of IGRAs using these proteins, alone or combined, are highly recommended.     

The iron-sulfur cluster-free hydrogenase (Hmd) is a metalloenzyme with a novel iron binding motif

The iron-sulfur cluster-free hydrogenase (Hmd) from methanogenic archaea harbors an iron-containing cofactor of yet unknown structure. X-ray absorption spectroscopy of the active, as isolated enzyme from Methanothermobacter marburgensis (mHmd) and of the active, reconstituted enzyme from Methanocaldococcus jannaschii (jHmd) revealed the presence of mononuclear iron with two CO, one sulfur and one or two N/O in coordination distance. In jHmd, the single sulfur ligand is most probably provided by Cys176, as deduced from a comparison of the activity and of the x-ray absorption and M?ssbauer spectra of the enzyme mutated in any of the three conserved cysteines. In the isolated Hmd cofactor, two CO, one sulfur, and two nitrogen/oxygen atoms coordinate the iron, the sulfur ligand being most probably provided by mercaptoethanol, which is absolutely required for the extraction of the iron-containing cofactor from the holoenzyme and for the stabilization of the extracted cofactor. In active mHmd holoenzyme, the number of iron ligands increased by one when one of the Hmd inhibitors (CO or KCN) were present, indicating that in active Hmd, the iron contains an open coordination site, which is proposed to be the site of H2 interaction.     

Unprecedented intraspecific diversity of the MHC class I region of a teleost medaka, Oryzias latipes

The major histocompatibility complex (MHC) is present at a single chromosomal locus of all jawed vertebrate analyzed so far, from sharks to mammals, except for teleosts whose orthologs of the mammalian MHC-encoded genes are dispersed at several chromosomal loci. Even in teleosts, several class IA genes and those genes directly involved in class I antigen presentation preserve their linkage, defining the teleost MHC class I region. We determined the complete nucleotide sequence of the MHC class I region of the inbred HNI strain of medaka, Oryzias latipes (northern Japan population-derived), from four overlapping bacterial artificial chromosome (BAC) clones spanning 540,982 bp, and compared it with the published sequence of the corresponding region of the inbred Hd-rR strain of medaka (425,935 bp, southern Japan population-derived) as the first extensive study of intraspecies polymorphisms of the ectotherm MHC regions. A segment of about 100 kb in the middle of the compared sequences encompassing two class Ia genes and two immunoproteasome subunit genes, PSMB8 and PSMB10, was so divergent between these two inbred strains that a reliable sequence alignment could not be made. The rest of the compared region (about 320 kb) showed a fair correspondence, and an approximately 96% nucleotide identity was observed upon gap-free segmental alignment. These results indicate that the medaka MHC class I region contains an ∼100-kb polymorphic core, which is most probably evolving adaptively by accumulation of point mutations and extensive genetic rearrangements such as insertions, deletions and duplications. The nucleotide sequence data of HNI MHC class I region reported in this paper have been submitted to the DDBJ/EMBL/GenBank and were assigned the accession number AB183488.     

The production of cloned fish in the medaka (Oryzias latipes)

The measurement of cellular DNA content by DNA microfluorometry revealed that medaka embryos that were fertilized with normal sperm and exposed to heat shock (41 degrees C for 3 min) or hydrostatic pressure (700 kg/cm2 for 10 min) at 85-95 min after insemination were tetraploid. Embryos fertilized with normal sperm and exposed to heat shock (41 degrees C for 2 min at 2-3 min after insemination) were triploid. These results suggest that heat shock or hydrostatic pressure at 85-95 min after insemination arrests the first cleavage, while heat shock at 2-3 min after insemination arrests the second meiotic division. Medaka clones have been produced by the following method: Eggs from orange-red or variegated variety were activated by UV-irradiated, genetically impotent sperm of wild-type fish (UV sperm). The haploid eggs obtained were diploidized by preventing the first cleavage with heat shock or hydrostatic pressure to produce homozygous females. Each of the two homozygous females was mated with vasectomized male in isotonic balanced salt solution to collect unfertilized eggs. The collected eggs were activated with UV sperm and converted from haploid to diploid by arrest of the second meiotic division with heat shock. Hatched fry of each homozygous diploid (all females) were fed with a methyltestosterone-containing diet (40 micrograms/gm diet) to produce sex-reversed males, which were mated with brood females, and thus two cloned lines were obtained.