Transformation of distinct mycobacterial species by shuttle vectors derived from theMycobacterium fortuitum pAL5000 plasmid

Mycobacterium tuberculosis H37Ra,M. smegmatisATCC 607,M. smegmatis MC²155,M. aurum A +,M. aurum A11, and one representative strain ofM. flavescens were transformed by electroporation with plasmid pMY10 and cosmid pDC100. Plasmid pMY 10 contained the origin of replication of pAL5000, the origin of replication of pBR322, a kanamycin resistance gene, and the origin of transfer of the Inc plasmid RK2; the cosmid pDC100 contained the pHC79 SS cosmid, the origin of replication of pAL5000, and a kanamycin resistance gene. The efficiency of transformation varied with the recipient cells used and was in decreasing order: 7×10⁵ forM. smegmatis MC²155, 6×10³ forM. tuberculosis H37Ra, 10³ forM. aurum, 50 forM. smegmatis ATCC 607, and 5 forM. flavescens. A rapid protocol for plasmid extraction from mycobacteria was developed.The satisfactory transformation of the nonvirulentM. tuberculosis strain H37Ra was of interest for future studies on cloning of virulence genes, while the satisfactory transformation ofM. aurum was of interest for future studies on the genetics of drug resistance because these bacteria are sensitive to drugs specifically used in the treatment of tuberculosis and leprosy. However, neither vector was stably maintained inM. smegmatis, indicating that further investigations are still necessary to resolve this difficulty.     

Evolution of several cytoskeletal proteins of astrocytes in primary culture: Effect of prenatal alcohol exposure

In the present work we have analyzed, using immunoblotting and immunofluorescence techniques, the evolution of several cytoskeletal proteins during the development of astrocytes in primary culture. The effect of prenatal exposure to alcohol on these proteins was also evaluated. Microtubular protein -tubulin decreased approximately 47% from 4 to 7 days after which its content remained practically constant. Immunofluorescence studies showed also that the content of -tubulin was greater at day 4 of culture. This increase in fluorescence was coincident with the presence of globular particles which were found in interphase astrocytes and stained with both anti - and anti--tubulin. These structures appeared only in proliferating cells. Glial fibrillary acidic protein (GFAP) and vimentin were analyzed as intermediate filament (IF) proteins. GFAP, in cytoskeletal preparations, increased regularly for 14 days followed by a decrease to day 21. In contrast, vimentin showed a progressive increase throughout the entire culture period. Fluorescence studies revealed some differences between the IF distribution patterns of GFAP and vimentin.In astrocytes obtained from rats prenatally exposed to ethanol, decreases in the amounts of all the cytoskeletal proteins studied were found during the entire culture period. In these cells a striking disorganization of cytoskeleton was also observed. The alcohol-induced decrease of GFAP in cultured astrocytes was also found when this protein was studied in preparations from whole brain developed in vivo.Special issue dedicated to Dr. Santiago Grisolia     

Palmar flexion creases in schizophrenia

Palmar flexion creases have been studied in schizophrenics with a family history of schizophrenia or other psychiatric disorders and without such a background, and compared to a control population. Palmar flexion creases have been analyzed according to the method suggested byBali & Chaube (1971). When compared to controls, differences in the DRBC and TRBC frequencies are significant in the subgroup with no family history, supporting the existence of biological heterogeneity in schizophrenia, and of congenital factors when there is no known genetic background.     

Simultaneous purification and characterization of aspartate aminotransferase isoenzymes from chicken liver

Cytosolic and mitochondrial isoenzymes of aspartate aminotransferase (EC 2.6.1.1) were purified to homogeneity from chicken liver, without previous fractionation of the subcellular components. The procedure includes initial heat treatment and ammonium sulfate fractionation. The two isoenzymes can then be separated by a DEAE-Sepharose chromatography using a linear gradient of L-aspartate (reaction substrate). The separated fractions can be further purified by a parallel step with HA-Ultrogel prior to octyl-Sepharose (c-AAT) and CM-Sepharose (m-AAT) chromatographies. Michaelis constants, pI values, inhibition by adipate and subforms generation with time were studied for both isoenzymes.     

Chemotaxis of Rhizobium meliloti to the plant flavone luteolin requires functional nodulation genes.

Luteolin is a phenolic compound from plants that acts as a potent and specific inducer of nodABC gene expression in Rhizobium meliloti. We have found that R. meliloti RCR2011 exhibits positive chemotaxis towards luteolin. A maximum chemotactic response was observed at 10(-8) M. Two closely related flavonoids, naringenin and apigenin, were not chemoattractants. The presence of naringenin but not apigenin abolished chemotaxis of R. meliloti towards luteolin. A large deletion in the nif-nod region of the symbiotic megaplasmid eliminated all chemotactic response to luteolin but did not affect general chemotaxis, as indicated by swarm size on semisoft agar plates and chemotaxis towards proline in capillary tubes. Transposon Tn5 mutations in nodD, nodA, or nodC selectively abolished the chemotactic response of R. meliloti to luteolin. Agrobacterium tumefaciens GMI9050, a derivative of the C58 wild type lacking a Ti plasmid, responded chemotactically to 10(-8) M luteolin. The introduction of a 290-kilobase nif-nod-containing sequence of DNA from R. meliloti into A. tumefaciens GMI9050 enabled the recipient to respond to luteolin at concentrations peaking at 10(-6) M as well as at concentrations peaking at 10(-8) M. The response of A. tumefaciens GMI9050 to luteolin was also abolished by the presence of naringenin.     

Analysis of the aromatic 1H NMR spectrum of chicken plasminogen kringle 4

The intact kringle 4 domain of chicken plasminogen has been characterized by 1H NMR spectroscopy at 300 and 620 MHz in both the presence and absence of epsilon-aminocaproic acid, an antifibrinolytic drug. The study focuses on the aromatic resonances. Comparisons with spectra from human, porcine and bovine kringle 4 homologs indicates a strict conservancy of conformation, reflecting the underlying primary sequence homology, and leads to an unambiguous assignment of all the aromatic resonances, including those of Phe15 and His40 which are unique to the chicken domain. Conclusive evidence is found that the Tyr9 ring fluctuates between two states, one in which it flips fast and other in which it is severely hindered. Similarly, the Tyr64 side chain finds itself in a structurally constrained locus. The Trp62, Tyr64, and Trp72 aromatic resonances are most sensitive to ligand presence, supporting a previously reported model of the kringle 4 lysine-binding site. His40, Phe41, and Tyr74 are also perturbed by ligand indicating proximity to the site. In contrast, the Phe15 aromatic spectrum indicates a rather mobile phenyl ring which is insensitive to ligand presence, thus confirming the lesser importance of the corresponding segment within the first kringle loop in determining kringle structure and/or function.     

Amino acid sequence of the N-terminus and selected tryptic peptides of the active subunit of human plasma carboxypeptidase N: comparison with other carboxypeptidases

Human plasma carboxypeptidase N was purified to homogeneity and its active and inactive subunits were separated. By introducing a novel technique, both forms of the active subunit (Mr = 55,000 and Mr = 48,000) were isolated. N-terminal sequencing of the active subunit of human carboxypeptidase N revealed significant homology with the N-terminal sequence of bovine carboxypeptidase H (43% identity) and to a lesser extent with carboxypeptidase A (29% identity) or carboxypeptidase B (18% identity). The active subunit of carboxypeptidase N was hydrolyzed with trypsin and 4 of the tryptic peptides were isolated by HPLC and sequenced. The sequences of the four peptides were homologous (39-64% identity) with regions of carboxypeptidase H corresponding to the middle (residues 148-175) and C-terminal portion (residues 321-408). These regions had essentially no homology with carboxypeptidase A or B. These data indicate that carboxypeptidase H and the active subunit of carboxypeptidase N may have diverged from a common ancestral gene.     

Yeast cAMP-dependent protein kinase can be associated to the plasma membrane

Using an anti-yeast regulatory subunit antibody and the synthetic peptide Kemptide as specific substrate we show in this work that purified preparations of yeast plasma membrane have an associated form of the regulatory subunit and cAMP-dependent protein kinase activity. Treatment of the plasma membrane "in vitro" with 1 microM cAMP releases cAMP-independent protein kinase activity while regulatory subunit remains on the membrane as revealed by immunoblotting. Incubation of the plasma membrane with [gamma-32P]ATP results in the phosphorylation of the regulatory subunit.