Endovascular Aneurysm Repair for Infrarenal Abdominal Aortic Aneurysm: New Horizon in Endovascular Surgery in Bangladesh.

Abdominal aortic aneurysm remains mostly asymptomatic. It is usually detected incidentally with imaging studies. Here we present a 55 years old hypertensive, non smoker, non diabetic, male patient who was diagnosed as a case of infrarenal abdominal aortic aneurysm. He was treated by endovascular means using endograft without laparatomy. Endografts were deployed through bilateral femoral artery cut down technique under general anesthesia. Completion angiogram following this endovascular aneurysm repair (EVAR) technique revealed good technical success with no endoleak. This hybrid procedure was performed in a cathlab having surgical instruments in hand. Three years after the procedure, patient is doing well.

Vermicomposting of Tea Factory Coal Ash: metal accumulation and metallothionein response in Eisenia fetida (Savigny) and Lampito mauritii (Kinberg).

Earthworms can accumulate heavy metals in their intestines to a great extent. Impact of feed materials and duration of metal exposure on natural activity of earthworms are rather unclear; this investigation therefore addresses the impact of metal rich Tea Factory Coal Ash (TFCA) on reproduction, composting and metal accumulation ability of Eisenia fetida and Lampito mauritii. Earthworm count and cocoon production increased significantly during vermicomposting. pH of the vermicomposted mixtures shifted toward neutrality, total organic C decreased substantially and total N enhanced significantly compared to composting. High heavy metal (Mn, Zn, Cu, As) accumulation was recorded in the intestine of both the earthworm species. Moreover, gradual increase in the metal-inducible metallothionein concentration indicated the causal mechanism of metal accumulation in these species. TFCA+cow dung (CD) (1:1) were most favorable feed mixture for E. fetida and TFCA+CD (1:2) were good for L. mauritii in regard to metal accumulation and compost quality.

Inactivation of indispensable bacterial proteins by early proteins of bacteriophages: implication in antibacterial drug discovery.

Bacteriophages utilize host bacterial cellular machineries for their own reproduction and completion of life cycles. The early proteins that phage synthesize immediately after the entry of their genomes into bacterial cells participate in inhibiting host macromolecular biosynthesis, initiating phage-specific replication and synthesizing late proteins. Inhibition of synthesis of host macromolecules that eventually leads to cell death is generally performed by the physical and/or chemical modification of indispensable host proteins by early proteins. Interestingly, most modified bacterial proteins were shown to take part actively in phage-specific transcription and replication. Research on phages in last nine decades has demonstrated such lethal early proteins that interact with or chemically modify indispensable host proteins. Among the host proteins inhibited by lethal phage proteins, several are not inhibited by any chemical inhibitor available today. Under the context of widespread dissemination of antibiotic-resistant strains of pathogenic bacteria in recent years, the information of lethal phage proteins and cognate host proteins could be extremely invaluable as they may lead to the identification of novel antibacterial compounds. In this review, we summarize the current knowledge about some early phage proteins, their cognate host proteins and their mechanism of action and also describe how the above interacting proteins had been exploited in antibacterial drug discovery.

Antimicrobial activity of saponins from Acacia auriculiformis.

Acaciaside A and B, two acylated bisglycoside saponins originally isolated from the funicles of Acacia auriculiformis, are known to have antihelminthic activity. Their antifungal and antibacterial activities were investigated. Complete inhibition of conidial germination of Aspergillus ochraceous and Curvularia lunata was recorded at 300 microg/ml or less whereas to inhibit the growth of Bacillus megaterium, Salmonella typhimurium and Pseudomonas aeruginosa 700 microg/ml or higher concentrations of the mixture was required. Two catabolic enzymes, phosphofructokinase and isocitrate dehydrogenase, responded differentially in fungi and bacteria against sublethal concentrations of the compound when assayed from their cell free extracts. An increased specific activity of the enzymes in bacteria and a decrease activity in fungi indicate a possible different mechanism of inhibition of saponins on the organisms tested.

An epidemiological study of the risk factors of occupational diseases in coal handling plant of a thermal power station.

Anthropometry and morbidity profile of fifty randomly selected workers of coal handling plant at a thermal power station of West Bengal having at least five years experience were studied. The mean value of their age, duration of present job and Body Mass Index (BMI) were 34.8 years, 7.86 years and 19.24 respectively. As they were exposed to coal dust (Av. concentration 300-350 mg/M3 of air), warmth (Ambient temperature 40 +/- 5 degrees C), humidity (Relative humidity 80 +/- 5%) and noise [Av. 80 dB(A)], respiratory system was most commonly (72%) effected followed by greying of hair (5.6%), Cloth dusters used by some could not render any discernable protective effect. Addiction was also prevent (62%). Use of proper protective equipments (PPE) supplemented by adequate preplacement and periodic medical examination followed by successful rehabilitation were suggested.

A study of energetics of cooperative interaction using a mutant lambda-repressor.

A lambda-repressor mutant, S228N, which is defective in tetramer formation in the free state but retains full cooperativity, was studied in detail. Isolated single operator-bound S228N repressor shows association properties similar to those of the wild-type repressor. Fluorescence anisotropy studies with dansyl chloride-labeled repressor show a dimer-monomer dissociation constant of around 10(-5) M. The structure of the mutant repressor was studied by circular dichroism, acrylamide quenching and sulfhydryl reactivity at protein concentrations of < or =10(-6) M, where it is predominantly monomeric. The results suggest no significant perturbations in the structure of the S228N mutant repressor from that of the wild-type repressor. Urea denaturation studies also indicate no significant change in the stability of the repressor. The results were used to calculate energetics of loop formation in the cooperative binding process.

Amino acid changes in the repressor of bacteriophage lambda due to temperature-sensitive mutations in its cI gene and the structure of a highly temperature-sensitive mutant repressor.

The mutant cIts genes from seven different lambdacIts phages carrying tsU50, tsU9, tsU46, ts1, tsU51, tsI-22 and ts2 mutations were cloned in plasmid. The positions of these mutations and the resulting changes of amino acids in the repressor were determined by DNA sequencing. The first four mutations mapping in the N-terminal domain show the following changes: I21S, G53S, A62T and V73A, respectively. Of the three remaining mutations mapping in the C-terminal domain, cItsI-22 and cIts2 show N207T and K224E substitutions respectively, while the mutant cItsU51 gene carries F141I and P153L substitutions. Among these ts repressors, CIts2 having the charge-reversal change K224E was overexpressed from tac promoter in a plasmid and purified, and its structure and function were studied. Operator-binding studies suggest that the ts2 repressor is somewhat defective in monomer-dimer equilibrium and/or cooperativity even at permissive temperatures and loses its operator-binding ability very rapidly above 25 degrees C. Comparative studies of fluorescence and CD spectra, sulfhydryl group reactivity and elution behaviour in size-exclusion HPLC of both wild-type and ts2-mutant repressors at permissive and non-permissive temperatures suggest that the C-terminal domain of the ts2 repressor carrying a K224E substitution has a structure that does not favor tetramer formation at non-permissive temperatures.

Identification of an early positive regulatory gene of mycobacteriophage L1.

Among 14 temperature-sensitive, growth-defective mutants of mycobacteriophage L1 showing a lysis-defective phenotype at 42 degrees C, six are, in addition, defective in phage DNA synthesis at 42 degrees C. In the present study, we show that one of the latter six mutants, L1G27ts901, is also defective in the synthesis of both an L1-specific exonuclease (a representative delayed early protein), and of RNA in both the delayed early and late periods but not in the immediate early period. The results of a temperature-shift experiment suggest that the synthesis of L1 exonuclease is regulated by G27 at the level of transcription. Furthermore, the temperature-sensitive defect in delayed early and late RNA synthesis could be largely overcome when the L1G27ts901-infected culture was shifted from 32 to 42 degrees C at 10 min but not at zero time post-infection. These results suggest that the primary effect of the G27ts901 mutation is to make the phage defective in transcription of delayed early genes at 42 degrees C, and the defect in late RNA synthesis by this mutant is a secondary effect which is caused by its inability to express regulatory gene products. We conclude that G27 is involved in the positive regulation of expression of the delayed early genes of L1 at the transcriptional level.

Role of the C-terminal tail region in the self-assembly of lambda-repressor.

Acrylamide quenching of the tryptophan fluorescence of the lambda-repressor at different protein concentrations indicates that one of the three tryptophan residues, W129, W142, and W230, undergoes a change in environment upon self-assembly, from dimer to associated species. Quenching data suggest that this tryptophan residue is inaccessible to low concentrations of acrylamide and is blue-shifted in the associated form. In the dimer, this tryptophan residue is highly accessible to acrylamide and is red-shifted. NBS oxidation, at protein concentrations which favor the associated form, showed that this tryptophan is also significantly protected from NBS oxidation. HPLC peptide mapping of NBS-oxidized lambda-repressor, amino acid analysis, and sequencing indicate that the protected, blue-shifted tryptophan is tryptophan 230. A mutant repressor (F235C) was specifically labeled at Cys 235 with an environment-sensitive probe, acrylodan. The acrylodan fluorescence of the labeled F235C lambda-repressor undergoes a significant blue-shift, accompanied by fluorescence enhancement, upon protein association. Along with other genetic evidence, these results suggest involvement of the C-terminal tail region in the self-assembly of the lambda-repressor.

Isolation, characterization, and mapping of temperature-sensitive mutations in the genes essential for lysogenic and lytic growth of the mycobacteriophage L1.

Forty temperature-sensitive mutations affecting lytic growth and eight affecting both establishment and maintenance of lysogeny of the temperate mycobacteriophage L1 have been isolated. All of the latter mutations form one complementation group and map within a very short region around the 15% coordinate of the L1 genome; these affect a single gene, cl, coding for the L1 repressor. The former 40 mutations form 28 complementation groups, identifying 28 different genes, G1-G28, essential for the lytic growth of L1. These genes have been mapped using the Gts mutations. Of the 28 Gts mutants, 14 are defective in host lysis at 42 degrees but not at 32 degrees while the other 14 can lyse the host at both temperatures. Among the former 14 Gts mutants, 6 are also defective in L1 DNA synthesis at 42 degrees, and they map in two different clusters, 4 around 65% and 2 around 84% of the L1 genome.

A fluorescence anisotropy study of tetramer-dimer equilibrium of lambda repressor and its implication for function.

Tetramer-dimer equilibrium of lambda repressor has been studied by fluorescence anisotropy techniques. We have chosen 1-dimethylamino naphthalene-5-sulfonyl chloride (dansyl chloride)-labeled repressor to study the dissociation-association equilibrium, because of relatively long life-time of the probe (> 10 ns). Polarization of the dansyl-labeled repressor decreases with decreasing protein concentrations in the range of 20 to 0.2 microM. The decrease of anisotropy was shown to be due to reversible dissociation of the protein. Size exclusion high-performance liquid chromatography studies and polyacrylamide gel electrophoresis under native conditions (Ferguson plot) confirmed that at around 20 microM concentrations the repressor exists in predominantly tetrameric form, whereas in lower concentrations it exists in predominantly dimer form. A dissociation constant of 2.3 +/- 0.9 microM was estimated in 0.1 M potassium phosphate, pH 8.0, at 25 degrees C. A stoichiometric amount of isolated single operator shifted the tetramer-dimer equilibrium toward the dimer. Increased ionic strength had only a modest effect on the dissociation constant. The thermodynamic constants for the dissociation reaction calculated from the Van't Hoff plot was +26.6 kcal/mol for delta H and +64.7 e.u. for delta S. The rotational correlation times derived from isothermal Perrin plot indicated elongated dimers and tetramers.

Multiphasic denaturation of the lambda repressor by urea and its implications for the repressor structure.

Urea denaturation of the lambda repressor has been studied by fluorescence and circular dichroic spectroscopies. Three phases of denaturation could be detected which we have assigned to part of the C-terminal domain, N-terminal domain and subunit dissociation coupled with further denaturation of the rest of the C-terminal domain at increasing urea concentrations. Acrylamide quenching suggests that at least one of the three tryptophan residues of the lambda repressor is in a different environment and its emission maximum is considerably blue-shifted. The transition in low urea concentration (midpoint approximately 2 M) affects the environment of this tryptophan residue, which is located in the C-terminal domain. Removal of the hinge and the N-terminal domain shifts this transition towards even lower urea concentrations, indicating the presence of interaction between hinge on N-terminal and C-terminal domains in the intact repressor.

An operator-induced conformational change in the C-terminal domain of the lambda repressor.

4,4'-bis(1-anilino-8-naphthalenesulfonic acid (Bis-ANS), an environment-sensitive fluorescent probe for hydrophobic region of proteins, binds specifically to the C-terminal domain of lambda repressor. The binding is characterized by positive cooperativity, the magnitude of which is dependent on protein concentration in the concentration range where dimeric repressor aggregates to a tetramer. In this range, positive cooperativity becomes more pronounced at higher protein concentrations. This suggests a preferential binding of Bis-ANS to the dimeric form of the repressor. Binding of single operator OR1 to the N-terminal domain of the repressor causes enhancement of fluorescence of the C-terminal domain bound Bis-ANS. The binding of single operator OR1 also leads to quenching of fluorescence of tryptophan residues, all of which are located in the hinge or the C-terminal domain. Thus two different fluorescent probes indicate an operator-induced conformational change which affects the C-terminal domain. The significance of this conformational change with respect to the function of lambda repressor has been discussed.

Bacteriophage lambda P gene shows host killing which is not dependent on lambda DNA replication.

Bacteriophage lambda, having a mutation replacing glycine by glutamic acid at the 48th codon of cro, kills the host under N- conditions; we call this the hk mutation. In lambda N-N-cl-hk phage-infected bacteria, the late gene R is expressed to a significant level, phage DNA synthesis occurs with better efficiency, and the Cro activity is around 20% less, all compared to those in lambda N-N-cl-hk(+)-infected bacteria. Segments of lambda DNA from the left of pR to the right of tR2, carrying cro, cII, O, P, and the genes of the nin5 region from the above hk and hk+ phages, were cloned in pBR322. Studies with these plasmids and their derivatives having one or more of the lambda genes deleted indicate that the hk mutation is lethal only when a functional P gene is also present. When expression of P from pR is elevated, due to the deletion of tR1, host killing also occurs without the hk mutation. We conclude that the higher levels of P protein, produced either (1) when cro has the hk mutation or (2) when tR1 is deleted, are lethal to the host. We also show that due to the hk mutation, the Cro protein becomes partially defective in its negative regulation at pR, resulting in the expression of P to a lethal level even in the absence of N protein-mediated antitermination. This P protein-induced host killing depends neither on lambda DNA replication nor on any other gene functions of the phage.

Isolation and preliminary characterization of Escherichia coli mutants resistant to lethal action of the bacteriophage lambda P gene.

Both spontaneous and NTG-induced mutants of Escherichia coli 594 insensitive to the lethal action of lambda P gene were isolated and called rpl (resistant to P lethality). These mutants were of two types, showing different phenotypes. On type I rpl mutants, lambda cl- and lambda v1v3 did not plate, while lambda vir, lambda cl- c17, lambda imm434, and lambda imm21 did; plasmid pMR45 carrying the lambda P gene could not complement lambda imm21P- phage in type I mutants. On the other hand, the type II rpl mutants support the growth of all the above phages including lambda cl-. Neither type of rpl mutation affects growth of the bacteria.

Enzymes of carbohydrate metabolism in fast-growing Rhizobium grown on hexoses or succinate.

Enzymatic evidence supports that succinate mediates repression of hexose-catabolising enzymes in fast-growing Rhizobium sp. (Cicer arietinum). Enzymes of the Embden-Myerhof-Parnas, Entner-Doudoroff and pentose phosphate pathways were found present in hexose-grown cells but not in succinate-grown cells. These however could be induced by the presence of hexoses.

Structure and function of the repressor of bacteriophage lambda. III. Molecular cloning of the high-affinity mutant cI gene of lambda and studies of the properties of the clones.

The high-affinity mutant cI gene of lambda cIha (Nag et al. 1984) was cloned in the multicopy plasmid pBR322. In the resulting plasmid, pMD 102, a lacUV5 promoter was inserted giving the lacUV5-cIha fusion plasmid pMD 205. Bacteria carrying pMD 102 and pMD 205 contain 2.5 and 15 times, respectively, the level of repressor in a monolysogen of lambda cIha. Results of the study of certain properties of the bacteria carrying these plasmids suggest that the ha repressor also has a higher affinity for the virulent mutant operators as well as the prm promoter of lambda.

Structure and function of the repressor of bacteriophage lambda. II. Isolation and characterization of a lambda mutant which produces repressor having higher affinity for operators.

By mutagenizing a lambda cIts (lambda cI857) lysogen, a lambda mutant has been isolated with a wild-type phenotype. This mutant phage lysogenizes with low efficiency and produces a low burst. Though the initial rates of repressor synthesis in Escherichia coli after infection with wild-type and mutant lambda are the same, the maximum level of repressor that is synthesized in the latter case is only about 30% of that synthesized in the former. Virulent lambda plates on the lysogen of mutant lambda with slightly less efficiency producing very tiny plaques. Operator-binding studies made in vitro with purified mutant and wild-type repressors show that the binding curve of the former repressor is a rectangular hyperbola while that of the latter is sigmoid. The half-lives of the complexes of mutant and wild-type repressors with right operator are 133 and 27 min, respectively. All these results suggest that the mutant repressor possibly has a higher affinity for the operators. This mutant has been named lambda cIha (ha = high affinity).

A simple procedure for large-scale preparation of pure plasmid DNA free from chromosomal DNA from bacteria.

A very simple, inexpensive procedure for preparing pure plasmid DNA from bacteria is described. In this method, lysozyme-induced spheroplasts are made in presence of 833 micrograms/ml of ethidium bromide which are then lysed by a mixture of Brij 58 and sodium deoxycholate, and the lysate is centrifuged at 48,000 g for 25 min whereby about 99.9% of total chromosomal DNA is pelleted. From the supernatant containing plasmid DNA, the proteins are removed by phenol extraction and the major part of RNA by CaCl2 precipitation, and finally the small amount of residual RNA is removed by RNase treatment. The average yield of pBR322 DNA from 1 liter of amplified culture by this procedure is 2 to 2.5 mg and the preparation is highly pure, containing only about 0.005% of total yield as chromosomal DNA contaminant. Moreover, the substrate activity and the transforming ability of the plasmid DNA prepared by this method remain unaffected.

Studies on polylysogens containing lambda N-cI- prophages. II. Role of high multiplicities in lysogen formation by lambda N-cI- phage.

Results of the experiments presented in this paper show that lambda N-cI- phage can lysogenize a nonpermissive host Escherichia coli when it infects at very high multiplicities (around 100), and lambda N-cI-cII- and lambda cIII-N-cI- lysogenize poorly at similar high multiplicities. The latter two phages lysogenize with appreciable frequency when either lambda N-cI- or lambda int-cN-cI-cII- is used as helper. The phages, lambda N-cI-, lambda N-cI-cII-, and lambda cIII-N-cI- can lysogenize also at relatively low m.o.i. of 20 in presence of the above lambda int-c helper, and the lambda int-cN-cI-cII- phage alone forms converted lysogens at an m.o.i. as low as 12. All these results suggest that the establishment of prophage integration by lambda N-cI- is positively regulated, like lambda N+cI+ phage, by the cII/cIII-promoted expression of the int gene of lambda, and under the N- condition, high multiplicities are needed to provide optimum levels of cII and cIII products, especially the latter.