Protein Synthesis in Mycobacterium tuberculosis H37Rv and the Effect of Streptomycin in Streptomycin-Susceptible and -Resistant Strains

An efficient in vitro amino acid-incorporating system from Mycobacterium tuberculosis H37Rv was standardized. Ribonucleic acid (RNA) isolated from phage-infected M. smegmatis cells served as natural messenger RNA and directed the incorporation of 14C-amino acids into protein. The effects of various antitubercular drugs and “known inhibitors” of protein synthesis on amino acid incorporation were studied. Antibiotics like chloramphenicol and tetracycline inhibited mycobacterial protein synthesis, though they failed to prevent the growth of the organism. This failure was shown to be due to the impermeability of mycobacteria to these drugs by use of “membrane-active” agents along with the antibiotics in growth inhibition studies. Several independent streptomycin-resistant mutants of M. tuberculosis H37Rv were isolated. Streptomycin inhibited the incorporation of 14C-amino acids into proteins by whole cells of a streptomycin-susceptible strain by more than 90%, whereas very little or no inhibition was observed in either high-level or low-level streptomycin-resistant strains.

Degradation of substituted benzoic acids by a Micrococcus species

Abstract a Micrococcus sp. isolated by isophthalate enrichment, utilized 8 of the 13 substituted benzoic acids tested as the sole source of carbon and energy. The organism degraded benzoic acid and anthranilic acid through the intermediate formation of catechol. While salicylate is metabolized through genetisic acid, p-hydroxybenzoic acid is degraded through protocatechuic acid. The organism grew well on isophthalate but failed to utilize phthalate and terphthalate. Catechol disoxygenase, gentisate dioxygenase and protocatechuate dioxygenase activities were shown in the cell-free extracts. Catechol and protocatechuate are further metabolized through an ortho-cleavage pathway.

Molecular basis of nitrate reductase induction in Candida utilis

Temporal separaton of transcription and translation during nitrate reductase induction oin Candida utilis was achieved by the use of actinomycin D and cycloheximide. The yeast failed to synthesize nitrate reductase when nitrate was not provided during transcription. Nitrate thus appeared to induce during transcription the capacity to synthesize nitrate reductase. Presence of nitrate, on the other hand, was not obligatory during translation except for its essential role in maintaining the stability of nitrate reductase after its formation as well as its mRNA.

Nature of induction of tryptophan pyrrolase in cold exposure

The activity of hepatic tryptophan pyrrolase in rats exposed to cold increased rapidly and reached a maximum of three-fold at 8 h. On continued exposure up to 48 h stress, the activity partly decreased but remained at a level higher than the initial. Withdrawal from the cold stress reversed the change. Adrenalectomy or treatment with inhibitors of protein synthesis abolished the increase in the enzyme activity during cold stress indicating a possible involvement of corticosteroids and de novo protein synthesis. Treatment with drugs known to block autonomic nervous system failed to inhibit the cold-mediated increase in enzyme activity. The results suggest that the increase in enzyme activity obtained on cold exposure is mediated by corticosteroids and not by either indoleaklylamines or autonomic nervous system. The changes in the enzyme obtained under cold stress with respect to the overshoot phenomenon, relationship to the degree of stress and reversibility on withdrawal from the stress indicate the "adaptate" nature of the response.

Primary structure of a Thomsen-Friedenreich-antigen-specific lectin, jacalin [Artocarpus integrifolia (jack fruit) agglutinin]. Evidence for the presence of an internal repeat

Jacalin [Artocarpus integrifolia (jack fruit) agglutinin] is made up of two types of chains, heavy and light, with M(r) values of 16,200 +/- 1200 and 2090 +/- 300 respectively (on the basis of gel-permeation chromatography under denaturing conditions). Its complete amino acid sequence was determined by manual degradation using a 4-dimethylaminoazobenzene 4'-isothiocyanate double-coupling method. Peptide fragments for sequence analysis were obtained by chemical cleavages of the heavy chain with CNBr, hydroxylamine hydrochloride and iodosobenzoic acid and enzymic cleavage with Staphylococcus aureus proteinase. The peptides were purified by a combination gel-permeation and reverse-phase chromatography. The light chains, being only 20 residues long, could be sequenced without fragmentation. Amino acid analyses and carboxypeptidase-Y-digestion C-terminal analyses of the subunits provided supportive evidence for their sequence. Computer-assisted alignment of the jacalin heavy-chain sequence failed to show sequence similarity to that of any lectin for which the complete sequence is known. Analyses of the sequence showed the presence of an internal repeat spanning residues 7-64 and 76-130. The internal repeat was found to be statistically significant.

The role of His-134, -147, and -150 residues in subunit assembly, cofactor binding, and catalysis of sheep liver cytosolic serine hydroxymethyltransferase

In an attempt to unravel the role of conserved histidine residues in the structure-function of sheep liver cytosolic serine hydroxymethyltransferase (SHMT), three site-specific mutants (H134N, H147N, and H150N) were constructed and expressed, H134N and H147N SHMTs had K-m values for L-serine, L-allo-threonine and beta-phenylserine similar to that of wild type enzyme, although the k(cat) values were markedly decreased, H134N SHMT was obtained in a dimeric form with only 6% of bound pyridoxal 5'-phosphate (PLP) compared with the wild type enzyme, Increasing concentrations of PLP (up to 500 mu M) enhanced the enzyme activity without changing its oligomeric structure, indicating that His-134 may be involved in dimer-dimer interactions, H147N SHMT was obtained in a tetrameric form but with very little PLP (3%) bound to it, suggesting that this residue was probably involved in cofactor binding, Unlike the wild type enzyme, the cofactor could be easily removed by dialysis from H147N SHMT, and the apoenzyme thus formed was present predominantly in the dimeric form, indicating that PLP binding is at the dimer-dimer interface, H150N SHMT was obtained in a tetrameric form with bound PLP, However, the mutant had very little enzyme activity (<2%). The k(cat)/K-m values for L-serine, L-allo-threonine and beta-phenylserine were 80-, 56-, and SS-fold less compared with wild type enzyme, Unlike the wild type enzyme, it failed to form the characteristic quinonoid intermediate and was unable to carry out the exchange of 2-S proton from glycine in the presence of H-4-folate. However, it could form an external aldimine with serine and glycine, The wild type and the mutant enzyme had similar K-d values for serine and glycine, These results suggest that His-150 may be the base that abstracts the alpha-proton of the substrate, leading to formation of the quinonoid intermediate in the reaction catalyzed by SHMT.

Interaction of surfactants with DNA. Role of hydrophobicity and surface charge on intercalation and DNA melting

A probe, 9-(anthrylmethyl)trimethylammonium chloride, 1, was prepared. 1 binds to calf-thymus DNA or Escherichia coli genomic DNA with high affinity, as evidenced from the absorption titration. Strong hypochromism, spectral broadening and red-shifts in the absorption spectra were observed. Half-reciprocal plot constructed from this experiment gave binding constant of 5±0.5×104 M−1 in base molarity. We employed this anthryl probe-DNA complex for studying the effects of addition of various surfactant to DNA. Surfactants of different charge types and chain lengths were used in this study and the effects of surfactant addition to such probe-DNA complex were compared with that of small organic cations or salts. Addition of either salts or cationic surfactants led to structural changes in DNA and under these conditions, the probe from the DNA-bound complex appeared to get released. However, the cationic surfactants could induce such release of the probe from the probe-DNA complex at a much lower concentration than that of the small organic cations or salts. In contrast the anionic surfactants failed to promote any destabilization of such probe-DNA complexes. The effects of additives on the probe-DNA complexes were also examined by using a different technique (fluorescence spectroscopy) using a different probe ethidium bromide. The association complexes formed between the cationic surfactants and the plasmid DNA pTZ19R, were further examined under agarose gel electrophoresis and could not be visualized by ethidium bromide staining presumably due to cationic surfactant-induced condensation of DNA. Most of the DNA from such association complexes can be recovered by extraction of surfactants with phenol-chloroform. Inclusion of surfactants and other additives into the DNA generally enhanced the DNA melting temperatures by a few °C and at high [surfactant], the corresponding melting profiles got broadened.

Assembly of physalis mottle virus capsid protein in Escherichia coli and the role of amino and carboxy termini in the formation of the icosahedral particles

The coat protein gene of physalis mottle tymovirus (PhMV) was over expressed in Escherichia coli using pET-3d vector. The recombinant protein was found to self assemble into capsids in vivo. The purified recombinant capsids had an apparent s value of 56.5 S and a diameter of 29(±2) nm. In order to establish the role of amino and carboxy-terminal regions in capsid assembly, two amino-terminal deletions clones lacking the first 11 and 26 amino acid residues and two carboxy-terminal deletions lacking the last five and ten amino acid residues were constructed and overexpressed. The proteins lacking N-terminal 11 (PhCPN1) and 26 (PhCPN2) amino acid residues self assembled into T = 3 capsids in vivo, as evident from electron microscopy, ultracentrifugation and agarose gel electrophoresis. The recombinant, PhCPN1 and PhCPN2 capsids were as stable as the empty capsids formed in vivo and encapsidated a small amount of mRNA. The monoclonal antibody PA3B2, which recognizes the epitope within region 22 to 36, failed to react with PhCPN2 capsids while it recognized the recombinant and PhCPN1 capsids. Disassembly of the capsids upon treatment with urea showed that PhCPN2 capsids were most stable. These results demonstrate that the N-terminal 26 amino acid residues are not essential for T = 3 capsid assembly in PhMV. In contrast, both the proteins lacking the C-terminal five and ten amino acid residues were present only in the insoluble fraction and could not assemble into capsids, suggesting that these residues are crucial for folding and assembly of the particles.

Plant regeneration from protoplasts of Capsicum annuum L cv. California Wonder

Plant regeneration from mesophyll protoplasts of pepper, Capsicum annuum L. cv. California Wonder has been demonstrated via shoot organogenesis, Protoplasts isolated from fully expanded leaves of 3-week-old axenic shoots when cultured in TM medium supplemented with 1 mgl(-1) NAA, 1 mgl(-1) 2, 4-D, 0.5 mgl(-1) BAP (CM 1) resulted in divisions with a frequency ranging from 20-25%. Antioxidant ascorbic acid and polyvinylpyrrolidone (PVP) in the medium and incubation in the dark helped overcome browning of protoplasts. Microcalli and macrocalli were formed in TM medium containing 2 mgl(-1) NAA and 0.5 mgl(-1) BAP (CM II) and MS gelled medium containing 2 mgl(-1) NAA and 0.5 mgl(-1) BAP (CM III), respectively, Regeneration of plantlets was possible via caulogenesis, Microshoots, 2-5 per callus appeared on MS gelled medium enriched with 0.5 mgl(-1) IAA, 2 mgl(-1) GA and 10 mgl(-1) BAP (CM IVc). Rooting of microshoots was obtained on half strength gelled medium containing 1 mgl(-1) NAA and 0.5 mgl(-1) BAP, Protoplasts isolated from cotyledons failed to divide and degenerated eventually.

Role of the Central Metal Ion and Ligand Charge in the DNA Binding and Modification by Metallosalen Complexes

Several metal complexes of three different functionalized salen derivatives have been synthesized. The salens differ in terms of the electrostatic character and the location of the charges. The interactions of such complexes with DNA were first investigated in detail by UV−vis absorption titrimetry. It appears that the DNA binding by most of these compounds is primarily due to a combination of electrostatic and other modes of interactions. The melting temperatures of DNA in the presence of various metal complexes were higher than that of the pure DNA. The presence of additional charge on the central metal ion core in the complex, however, alters the nature of binding. Bis-cationic salen complexes containing central Ni(II) or Mn(III) were found to induce DNA strand scission, especially in the presence of co-oxidant as revealed by plasmid DNA cleavage assay and also on the basis of the autoradiogram obtained from their respective high-resolution sequencing gels. Modest base selectivity was observed in the DNA cleavage reactions. Comparisons of the linearized and supercoiled forms of DNA in the metal complex-mediated cleavage reactions reveal that the supercoiled forms are more susceptible to DNA scission. Under suitable conditions, the DNA cleavage reactions can be induced either by preformed metal complexes or by in situ complexation of the ligand in the presence of the appropriate metal ion. Also revealed was the fact that the analogous complexes containing Cu(II) or Cr(III) did not effect any DNA strand scission under comparable conditions. Salens with pendant negative charges on either side of the precursor salicylaldehyde or ethylenediamine fragments did not bind with DNA. Similarly, metallosalen complexes with net anionic character also failed to induce any DNA modification activities.

Artifactual staining of proteins on polyacrylamide gels by nitrobluetetrazolium chloride and phenazine methosulfate

Different purified proteins were shown to give purple formazan bands corresponding to the protein stain following electrophoresis on polyacrylamide gels, in the presence of nitrobluetetrazolium (NBT) and phenazine methosulfate (PMS). Both PMS and NBT are needed for formazan production which has a favorable pH at 8.5. Sulfhydryl blockers in the incubation medium inhibited this color development to different extents. While proteins with free SH groups like bovine serum albumin, ovalbumin, and urease showed this pyridine nucleotide independent artifact, nonthiol proteins, viz., bovine pancreatic ribonuclease A, and riboflavin-binding protein from chicken egg white failed to do so. The nonenzymatic formazan formation observed with different proteins could also be shown in an in vitro assay system. It is clear that the “nothing dehydrogenase” phenomenon observed in several cases may be due to the thiol group-mediated artifactual staining of proteins.

Direct somatic embroygenesis from immature embryos of rosewood (Dalbergia latifolia Roxb.)

Direct regeneration of somatic embryos was obtained from immature zygotic embryos of Dalbergia latifolia. Immature embryos dissected from green pods 90 d after flowering gave the highest frequency of somatic embryo formation. Preculture on high 2,4-D medium for 4 weeks induced direct somatic embryogenesis, which was expressed during the second culture phase in the presence of low 2,4-D along with a high sucrose concentration. Embryos were separated and transferred to the maturation medium containing MS + 0.5-1.0 mg/L BAP, where embryos developed into plantlets. Somatic embryos failed to convert into complete plants without BAP treatment. This method of direct regeneration of somatic embryos without a callus phase has direct application for genetic manipulation studies.

Analysis of the fusA2 locus encoding EFG2 in Mycobacterium smegmatis

The translation elongation factor G (EFG) is encoded by the fusA gene.Several bacteria possess a second fusA-like locus,fusA2 which encodes EFG2. A comparison of EFG and EFG2 from various bacteria reveals that EFG2 preserves domain organization and maintains significant sequence homology with EFG, suggesting that EFG2 may function as an elongation factor. However, with the single exception of a recent study on Thermus thermophilus EFG2, this class of EFG-like factors has not been investigated. Here, we have characterized EFG2 (MSMEG_6535) from Mycobacterium smegmatis. Expression of EFG2 was detected in stationary phase cultures of M.smegmatis (Msm). Our in vitro studies show that while MsmEFG2 binds guanine nucleotides, it lacks the ribosome-dependent GTPase activity characteristic of EFGs. Furthermore,unlike MsmEFG (MSMEG_1400), MsmEFG2 failed to rescue an E. coli strain harboring a temperature-sensitive allele of EFG, for its growth at thenon-permissive temperature. Subsequent experiments showed that the fusA2 gene could be disrupted in M. smegmatis mc(2)155 with Kan(R)marker. The M. smegmatis fusA2::kan strain was viable and showed growth kinetics similar to that of the parent strain (wild-type for fusA2).However, in the growth competition assays, the disruption of fusA2 was found to confer a fitness disadvantage to M. smegmatis, raising the possibility that EFG2 is of some physiological relevance to mycobacteria.

Mutational analysis of active-site residues in the Mycobacterium leprae RecA intein, a LAGLIDADG homing endonuclease: Asp(122) and Asp(193) are crucial to the double-stranded DNA cleavage activity whereas Asp(218) is not

Mycobacterium leprae recA harbors an in-frame insertion sequence that encodes an intein homing endonuclease (PI-MleI). Most inteins (intein endonucleases) possess two conserved LAGLIDADG (DOD) motifs at their ctive center. A common feature of LAGLIDADG-type homing endonucleases is that they recognize and cleave the same or very similar DNA sequences. However, PI-MleI is distinctive from other members of the family of LAGLIDADG-type HEases for its modular structure with functionally separable domains for DNA-binding and cleavage, each with distinct sequence preferences. Sequence alignment analyses of PI-MleI revealed three putative LAGLIDADG motifs; however, there is conflicting bioinformatics data in regard to their identity and specific location within the intein polypeptide. To resolve this conflict and to determine the active-site residues essential for DNA target site recognition and double-stranded DNA cleavage, we performed site-directed mutagenesis of presumptive catalytic residues in the LAGLIDADG motifs. Analysis of target DNA recognition and kinetic parameters of the wild-type PI-MleI and its variants disclosed that the two amino acid residues, Asp(122) (in Block C) and Asp(193) (in functional Block E), are crucial to the double-stranded DNA endonuclease activity, whereas Asp(218) (in pseudo-Block E) is not. However, despite the reduced catalytic activity, the PI-MleI variants, like the wild-type PI-MleI, generated a footprint of the same length around the insertion site. The D122T variant showed significantly reduced catalytic activity, and D122A and D193A mutations although failed to affect their DNA-binding affinities, but abolished the double-stranded DNA cleavage activity. On the other hand, D122C variant showed approximately twofold higher double-stranded DNA cleavage activity, compared with the wild-type PI-MleI. These results provide compelling evidence that Asp(122) and Asp(193) in DOD motif I and II, respectively, are bona fide active-site residues essential for DNA cleavage activity. The implications of these results are discussed in this report.

Cooperation of Notch and Ras/MAPK signaling pathways in human breast carcinogenesis

Background: Recent studies have implicated aberrant Notch signaling in breast cancers. Yet, relatively little is known about the pattern of expression of various components of the Notch pathway, or its mechanism of action. To better understand the role of the Notch pathway in breast cancer, we have undertaken a detailed expression analysis of various Notch receptors, their ligands, and downstream targets at different stages of breast cancer progression. Results: We report here that there is a general increase in the expression levels of Notch 1, 2, 4, Jagged1, Jagged2, and Delta-like 4 proteins in breast cancers, with simultaneous upregulation of multiple Notch receptors and ligands in a given cancer tissue. While Notch3 and Delta-like1 were undetectable in normal tissues, moderate to high expression was detected in several cancers. We detected the presence of active, cleaved Notch1, along with downstream targets of the Notch pathway, Hes1/Hes5, in similar to 75% of breast cancers, clearly indicating that in a large proportion of breast cancers Notch signaling is aberrantly activated. Furthermore, we detected cleaved Notch1 and Hes1/5 in early precursors of breast cancers - hyperplasia and ductal carcinoma in situ suggesting that aberrant Notch activation may be an early event in breast cancer progression. Mechanistically, while constitutively active Notch1 alone failed to transform immortalized breast cells, it synergized with the Ras/MAPK pathway to mediate transformation. This cooperation is reflected in vivo, as a subset of cleaved Notch positive tumors additionally expressed phopsho-Erk1/2 in the nuclei. Such cases exhibited high node positivity, suggesting that Notch-Ras cooperation may lead to poor prognosis. Conclusions: High level expression of Notch receptors and ligands, and its increased activation in several breast cancers and early precursors, places Notch signaling as a key player in breast cancer pathogenesis. Its cooperation with the Ras/MAPK pathway in transformation offers combined inhibition of the two pathways as a new modality for breast cancer treatment.