Evidence of two forms of poly(A) polymerase in germinated wheat embryos and their regulation by a novel protein kinase

Two forms of poly(A) polymerase (PAPI and PAPII) from germinated wheat embryos have been resolved on DEAE-cellulose ion-exchange chromatography by a linear gradient of 0-500 mM (NH(4))(2)SO(4). Further purification shows that both forms are monomeric in nature with an identical molecular weight, approximately 65 kDa. The phosphoprotein nature of PAPI and PAPII has been established by in vivo labelling with (32)P-orthophosphate. Acid hydrolysis of both (32)P-labelled purified PAPI and PAPII has revealed that phosphorylations generally take place in serine and threonine residues. PAPI and PAPII have also been characterised with respect to V(max) and K(m) for poly(A). The V(max) and K(m) values of PAPI are 28.57 and 11.37 microg, respectively, whereas 34.48 and 7.04 microg of PAPII. In vitro dephosphorylation of the purified enzyme by alkaline phosphatase leads to a significant loss of the enzyme activity, which is regained upon phosphorylation by a 65 kDa protein kinase (PK) purified from wheat embryos. The extent of phosphorylation by protein kinase shows that PK has similar affinity towards both PAPI and PAPII, whereas the phosphate incorporation in PAPII is twofold higher than PAPI suggesting their distinct chemical nature.     

The ars operon of Escherichia coli confers arsenical and antimonial resistance.

The chromosomally encoded arsenical resistance (ars) operon subcloned into a multicopy plasmid was found to confer a moderate level of resistance to arsenite and antimonite in Escherichia coli. When the operon was deleted from the chromosome, the cells exhibited hypersensitivity to arsenite, antimonite, and arsenate. Expression of the ars genes was inducible by arsenite. By Southern hybridization, the operon was found in all strains of E. coli examined but not in Salmonella typhimurium, Pseudomonas aeruginosa, or Bacillus subtilis.     

Structure and Evolution of the Archaeal Lipid Synthesis Enzyme sn-Glycerol-1-phosphate Dehydrogenase

One of the most critical events in the origins of cellular life was the development of lipid membranes. Archaea use isoprenoid chains linked via ether bonds to sn-glycerol 1-phosphate (G1P), whereas bacteria and eukaryotes use fatty acids attached via ester bonds to enantiomeric sn-glycerol 3-phosphate. NAD(P)H-dependent G1P dehydrogenase (G1PDH) forms G1P and has been proposed to have played a crucial role in the speciation of the Archaea. We present here, to our knowledge, the first structures of archaeal G1PDH from the hyperthermophilic methanogen Methanocaldococcus jannaschii with bound substrate dihydroxyacetone phosphate, product G1P, NADPH, and Zn²⁺ cofactor. We also biochemically characterized the enzyme with respect to pH optimum, cation specificity, and kinetic parameters for dihydroxyacetone phosphate and NAD(P)H. The structures provide key evidence for the reaction mechanism in the stereospecific addition for the NAD(P)H-based pro-R hydrogen transfer and the coordination of the Zn²⁺ cofactor during catalysis. Structure-based phylogenetic analyses also provide insight into the origins of G1PDH.     

The lectin nature of α-galactosidases from Vicia faba seeds

α-Galactosidase from Vicia faba seeds has been resolved into three molecular forms, I, II¹ and II², respectively. Enzyme I is a tetramer (M_r 160 000) consisting of identical sub-units (M_r 44000 ± 2000). All three forms display lectin activity with glucose/mannose specificity. Enzyme I has been further studied with respect to its lectin specificity and various factors affecting this property. The results indicate that the catalytic and the lectin sites reside in the same protein molecule. The results presented are unique in that the enzyme activity is specific for galactose and its lectin activity is specific for glucose/mannose.     

Multiple coordination modes of hemilabile 3-dimethylaminopropyl chalcogenolates in platinum(II) complexes: Synthesis, spectroscopy and structures

The reactions of N,N-dimethylaminopropyl chalcogenolates with platinum(II) compounds have been carried out and complexes of the types [PtCl(ECH₂CH₂CH₂NMe₂)]₂ (1) (E = S (1a) and Se (1b)), [Pt(ECH₂CH₂CH₂NMe₂)₂]_n (2) (E = S (2a) and Se (2b)), [(PtCl₂)₂{(Me₂NCH₂CH₂CH₂E)₂}]_n (3), [PtX(SeCH₂CH₂CH₂NMe₂)]₂ (4) (X = SePh (4a) and OAc (4b)) and [PtCl(ECH₂CH₂CH₂NMe₂)(PR₃)]_n (5) (E = S, Se, Te) have been isolated. These complexes have been characterized by elemental analysis, IR, UV-Vis, NMR (¹H, ¹³C, ³¹P, ⁷⁷Se, ¹⁹⁵Pt) spectroscopy and FAB mass spectral data. The structures of [PtCl(SeCH₂CH₂CH₂NMe₂)]₂ (1b) and [PtCl(SCH₂CH₂CH₂NMe₂)(PPr₃)]₂ (5a) have been established by single crystal X-ray diffraction data. Both the molecules have dimeric structures. In 1b, two platinum atoms are held together by symmetrically bridging Se atoms of the chelating selenolate groups. In 5a, two thiolates form a four-membered Pt₂S₂ bridge with dangling NMe₂ groups.     

The development of tone in the smooth muscle of guinea-pig isolated tracheal preparations may be influenced by prostanoids released from the adjacent airway cartilage

Guinea-pig tracheal strip preparations containing cartilage, placed under an applied load , develop tone spontaneously. The finding that spontaneous tone is reduced by indomethacin suggests that one or more prostanoids are involved in the development of spontaneous tone in this species. In this study we examined the effects of removing the cartilage component of the preparations on changes in tone induced by indomethacin and isoproterenol. In contrast to preparations containing cartilage, tissues devoid of cartilage, did not develop tone after the application of an initial 1 g resting load. Indomethacin (1 μM) reduced resting tone by 0.62 ± 0.14 g in cartilage-containing tissues but, in contrast, reduced tone by only 0.03 ± 0.01 g in tissues devoid of cartilage. Furthermore, relaxation responses (0.38 ± 0.05 g) to isoproterenol (1 μM) could be produced in cartilage-containing preparations but not in cartilage-free preparations. Radioimmunoassays indicated that the release of PGE₂, PGF_2α and 6-keto PGF_1α, the end-product of PGI₂ breakdown, was diminished in preparations lacking cartilage. Thus, in guinea-pig airway preparations cartilage is apparently a source of sufficient prostanoids to induce spontaneous tone     

Fundamental differences in promoter CpG island DNA hypermethylation between human cancer and genetically engineered mouse models of cancer

Genetic and epigenetic alterations are essential for the initiation and progression of human cancer. We previously reported that primary human medulloblastomas showed extensive cancer-specific CpG island DNA hypermethylation in critical developmental pathways. To determine whether genetically engineered mouse models (GEMMs) of medulloblastoma have comparable epigenetic changes, we assessed genome-wide DNA methylation in three mouse models of medulloblastoma. In contrast to human samples, very few loci with cancer-specific DNA hypermethylation were detected, and in almost all cases the degree of methylation was relatively modest compared with the dense hypermethylation in the human cancers. To determine if this finding was common to other GEMMs, we examined a Burkitt lymphoma and breast cancer model and did not detect promoter CpG island DNA hypermethylation, suggesting that human cancers and at least some GEMMs are fundamentally different with respect to this epigenetic modification. These findings provide an opportunity to both better understand the mechanism of aberrant DNA methylation in human cancer and construct better GEMMs to serve as preclinical platforms for therapy development.     

Occurrence of novel Cu2+-dependent sialic acid-specific lectin,on the outer surface of mature caprine spermatozoa

Effects of several bivalent metal ions on the autoagglutination event in mature caprine epididymal sperm cells have been investigated using a chemically defined medium. This study demonstrates for the first time that Copper (Cu²⁺) ion (300 μM) has high specificity for autoagglutination of mature cauda-epididymal sperm. Head-to-head interaction of the male gametes is responsible for this event. Studies on the effect of various sugars reveal that the autoagglutinated cells can be dissociated specifically with neutralized sialic acid (50 mM), which also inhibits the sperm cell autoagglutination phenomenon. Blood serum protein fetuin, that contains terminal sialic acid residue, showed high efficacy for inhibiting this autoagglutination event at 4 μM concentration. However, asialofetuin is not capable of inhibiting this Cu²⁺-dependent cellular event. Mature sperm cells bound with caprine erythrocytes at their head region in presence of Cu²⁺ ion. The purified sperm membrane fraction isolated by aqueous two phase polymer method showed high efficacy to agglutinate erythrocytes. These sperm-erythrocyte interactions as well as sperm membrane induced haemagglutination were strongly blocked by neutralized sialic acid (50 mM). The results confirm the occurrence of unique Cu²⁺ dependent, sialic acid-specific lectin on the outer surface of a mammalian cell using caprine sperm as the model. The observed Cu²⁺-mediated cellular autoagglutination is caused by the interaction of the cell surface lectin with the lectin receptor on the surface of the neighboring homologous cell.     

Bile acid amides derived from chiral amino alcohols: novel antimicrobials and antifungals

Cholic and deoxycholic acid amides 10-17 have been synthesised from (1R,2R)-1-phenyl-2-amino-1,3-propanediol 2, (1S,2S)-1-phenyl-2-amino-1,3-propanediol 4, (1R,2R)-1-para-nitrophenyl-2-amino-1,3-propanediol 3, (1S,2S)-1-para-nitrophenyl-2-amino-1,3-propanediol 5. Amide 12 derived from N-succinimidyl ester 9 of deoxycholic acid and (1R,2R)-1-phenyl-2-amino-1,3-propanediol 2, found to be active against Cryptococcus neoformans and the amide 17 obtained from N-succinimidyl ester 9 of deoxycholic acid and (1S,2S)-1-para-nitrophenyl-2-amino-1,3-propanediol 5, is found to be potent against various gram-positive bacteria.     

Fractal dimension in aspiration cytology smears of breast and cervical lesions

OBJECTIVE: To standardize the automated measurement of fractal dimension on cytologic smears and compare the fractal dimension of benign and malignant breast cells and cervical lesions on cytologic material to evaluate its role in the discrimination of benign from malignant cells. STUDY DESIGN: We randomly selected fine needle aspiration cytology smears of 42 cases of infiltrating duct carcinoma and 38 cases of fibroadenoma of the breast. Similarly, 16 cervical carcinoma and 20 normal cervical smears were selected for study. Ten cells were selected randomly from each case. Box counting of fractal dimension of malignant and benign cells was achieved with an image cytometer (Leica, Cambridge, England) using Quantimet 600 software (Leica). Then a well-spaced grid with multiple small boxes of a particular pixel length was superimposed on the cell. The dimension of the box was selected as 4, 8 and 16 pixels. With the help of a logical "AND" operation, we counted the number of boxes touching the peripheral margin of the cell nuclei. For each cell, the log-log graph of 1 per box size was plotted against the number of boxes touching the peripheral rim of the cell. The slope of each graph was identified using the least-squares method of regression analysis. RESULTS: The mean fractal dimension of malignant cells was 0.8536 +/- 0.1120 as compared to 0.8403 +/- 0.1115 in benign cell groups. The Mann-Whitney U test showed a significant difference in fractal dimension in these 2 groups (P = .05). The mean fractal dimension of malignant cells from the cervix was 0.8656 +/- 0.1499 as compared to 0.8315 +/- 0.1312 in benign cells. The Mann-Whitney U test showed a significant difference in fractal dimension in these 2 groups (P < .02). CONCLUSION: Fractal dimension may be a helpful adjunctive technique to discriminate between benign and malignant cells.