Phenolic Contents and Antioxidant Properties from Aerial Parts of Achyranthes coynei Sant.

Aim of the study was to evaluate antioxidant activity and total phenolic content of Achyranthes coynei; an endemic plant used in treatment of several diseases in the same lines that of Achyranthes aspera by traditional practitioners of Belgaum region. Efficiency of extraction methods was studied for aerial parts (leaves, stem, and inflorescence) extracted in methanol using continuous shaking, microwave assisted and ultra sonic extraction technique, by exposing it for different time period. Total phenolic content was measured by Folin-Ciocalteu method and antioxidant activity using 2,2'-diphenyl-1-picryl hydrazyl radical scavenging assay and ferric reducing antioxidant power assay. Extracts of A. coynei revealed highest yield of total phenolic content in continuous shaking method compared to other methods. Significantly higher amount of phenolic content (467.07±23.35 tannic acid equivalent and 360.83±18.04 caffic acid equivalent mg/100 g FW) was estimated at 360 min of continuous shaking extraction. In 2,2'-diphenyl-1-picryl hydrazyl radical scavenging assay and ferric reducing antioxidant power assay, inflorescence and leaf showed highest potential activity, respectively. Stem extracts showed lower yield of total phenolic content and antioxidant activity. Results also showed 2,2'-diphenyl-1-picryl hydrazyl radical scavenging assay had significant correlation with total phenolic content. This is first report of total phenolic content and antioxidant studies in A. coynei.

A morphological study of variations in the branching pattern and termination of the radial artery.

INTRODUCTION: Coronary artery bypass grafting is an established means of treating advanced coronary artery disease. In recent years, there has been an increased interest in the radial artery as an entry route during coronary angiography. Accurate knowledge of the branching pattern of this artery and its relation to surrounding structures is of great importance in the care of surgical patients. METHODS: This study was conducted on 75 formalin-fixed upper limbs in order to note the variations in the branching pattern and termination of the radial artery. RESULTS: The radial artery divided into three branches in 2.7% of cases and into two branches in 52.0% of cases. The radial recurrent artery originated from the brachial artery instead of the radial artery in 12.0% of cases. The radial recurrent artery, palmar carpal artery, first dorsal metacarpal artery and superficial palmar artery were absent in 1.3%, 26.7%, 9.3% and 5.3% of cases, respectively. 6.7% of cases had a high origin of the superficial palmar artery. CONCLUSION: The rich photographic documentation of the variation of branching pattern and termination of radial artery is not only of academic interest but also useful to surgeons and radiologists working in the same area.

A preliminary survey of the median artery in human cadavers of South Indian origin.

BACKGROUND: The median artery is a transitory vessel that represents the arterial axis of the forearm during early embryonic life. It normally regresses in the second embryonic month. Its persistence in the human adult has been recorded in 2 different patterns: as a large, long vessel (palmar type) which reaches the hand; or as a small and short vessel (antebrachial type) which ends before reaching the wrist joint. The palmar type is of major clinical significance. OBJECTIVE: This study was undertaken to investigate the incidence and course of the palmar type of the median artery in South Indian cadavers. MATERIAL AND METHODS: 25 upper limbs of South Indian cadavers were taken to study the median artery. RESULTS: The occurrence of median artery was 8%; of which 4% was on the right side and the other 4% was on the left side. On both sides, the artery originated from the ulnar artery. On the right side, the artery was involved in the formation of superficial palmar arch, whereas the artery on the left side did not join the arch; it terminated as 1st and 2nd common palmar digital arteries. CONCLUSION: Persistent median artery is closely related to the anterior interosseous nerve, it is possible that the artery may compress the anterior interosseous nerve and cause the anterior interosseous nerve syndrome (Fig. 2, Ref. 17).

Identification of viable and non-viable Mycobacterium tuberculosis in mouse organs by directed RT-PCR for antigen 85B mRNA.

Mycobacterium tuberculosis (MTB) the causative organism of tuberculosis can remain dormant as a non-culturable organism, reactivate and cause disease in man and animals. There is a need for proof of viability of such organisms in order to understand the process of reactivation. PCR for bacterial DNA cannot distinguish between viable and non-viable bacilli. We have tested a previously described two tube directed reverse transcriptase polymerase chain reaction (RT-PCR) for the detection of mRNA of antigen 85B (Ag85B) of MTB that can distinguish between viable and non-viable organisms. Using a set of external and internal primers for Ag85B, a cDNA amplified product (216 bp) was seen among simulated samples containing only viable cfus at a sensitivity of >10 and <100 cfu/ml. Eucaryotic DNA rich normal mouse lung homogenate did not interfere among these samples. The method amplified the 216 bp product also among cfu positive tissues of naturally infected mice. Finally, in a mouse model of dormancy, direct RT-PCR detected a signal among multiple tissues that were negative for cfus and hence non-culturable. Ag85B is abundantly secreted by MTB and hyper-expressed under stress conditions. Thus the method to identify its mRNA message may be useful to detect viable but dormant bacteria.

Interaction of the Rb tumor suppressor protein with the c-fos promoter in c-fos transfected cells overexpressing c-fos and Rb.

The Rb tumor suppressor protein is overexpressed in HeLa cell lines permanently transfected with a constitutively expressed c-fos gene. CP17-14 cell overexpression of Rb may be due to a balancing response to overexpression of the stimulatory effects of c-fos overexpression on transcription. The cis-acting retinoblastoma control element (RCE, -97 to -86 bp) in the human c-fos promoter is thought to allow regulation of c-fos by Rb. Gel-shift assays were performed with a 168 bp fragment encoding the c-fos RCE. Competition assays with increasing mass of unlabeled probe or dose-dependence assays using increasing mass of nuclear proteins, demonstrated sequence-specific complex formation. Indistinguishable complexes were formed between the c-fos RCE fragment in transfected cells, but at higher levels (> 50%), compared to proteins from parental cells. Supershift analysis utilizing epitope-specific Rb-monoclonal antibodies indicated the presence of Rb protein bound to the RCE-containing DNA fragment. In contrast, polyclonal anti-Rb antibodies enhanced the amounts of nuclear protein-DNA complexes detected but did not result in a supershift. These results suggested the presence of Rb and/or Rb-like peptides involved in complex formation and the presence of multiple variants of RCE-binding complexes in response to c-fos over-expression.

Overexpression of c-fos induces expression of the retinoblastoma tumor suppressor gene Rb in transfected cells.

Hypophosphorylated Rb, the product of the tumor suppressor gene associated with hereditary retinoblastoma, is thought to act as a suppressor of cell growth and proliferation during G1 phase. We investigated whether Rb expression was dependent on the expression level of the immediate early cell growth gene, c-fos, which is transiently expressed as cells re-enter G1 phase from quiescence. To explore the functional relationship between c-fos and Rb, a eukaryotic expression plasmid was constructed containing the c-fos gene under control of the SV40 promoter complex. This plasmid was co-transfected with plasmids encoding pRSV cat and G418 resistance, into HeLa S3 cells, and clonal populations of transfected (RSfos) cells selected. High levels of c-fos expression in transfected cells were confirmed by both western and northern blot. Rb protein content per cell was determined by flow cytometry using an Rb-specific primary antibody and a FITC-conjugated secondary antibody. Higher expression of Rb (2-6 fold/cell) in approximately 20% of RSfos transfected cells was observed in comparison to parental HeLa cells. Rb content per cell increased approximately 2-fold during the cell cycle in both parental HeLa cells and RSfos cell clones which overexpressed Rb. Rb accumulation occurred in a manner consistent with normal mass accumulation during the cell cycle. Overexpression of Rb in RSfos cells was also confirmed by western blot analysis. Because one possible function of RB may be to act as a brake on cell growth, it is possible that overexpression of RB acts as an inhibitory counter activity to overexpression of the growth promoting activity of c-fos. This possible balancing activity of Rb was further suggested when Rb protein expression levels were measured in different clonal lines of RSfos transfected cells overexpressing increasing levels of c-fos. Overexpression of Rb was proportional to the level of overexpression of c-fos in each clonal cell line. Such evidence suggests that Rb was proportional to the level of overexpression of c-fos in each clonal cell line. Such evidence suggests that Rb expression may be regulated in a manner which balances the transcription stimulatory effects of c-fos overexpression and its effects on the transcription of other genes during the cell cycle.

c-fos expression is required during all phases of the cell cycle during exponential cell proliferation.

Transient transcription of the c-fos gene is induced by serum stimulation of quiescent cells during the earliest part of Gl phase, reaching maximum levels of mRNA within 30 min. To determine whether expression of c-fos is required, or has any regulatory role in continuous exponential cell proliferation following re-entry into the cell cycle, a chimeric plasmid was constructed containing the human c-fos gene such that transcription was under control of the SV40 promoter complex. The plasmid was co-transfected into HeLa S3 cells (RSfos cells) along with plasmids encoding pRSVcat and G418 resistance followed by clonal propagation. The regulatory role of c-fos in an exponentially growing transfected RSfos cell clone (CP17-14) and parental HeLa cells was assessed through studies of c-fos overexpression or suppression of c-fos translation by treatment with a c-fos antisense 16-mer oligonucleotide. Transfected cells grew normally despite excess expression of c-fos. In contrast, antisense oligonucleotide treatment efficiently suppressed proliferation in normal exponentially growing cells by more than 70% for approximately 60 hr. Beyond this time oligonucleotides were ineffective likely due to degradation/depletion. In contrast, transfected cells grew normally, indicating overexpression of c-fos was sufficient to neutralize the effects of c-fos antisense oligonucleotides. Flow cytometric analysis of cell cycle phase distribution and determination of proliferation rate in oligonucleotide treated HeLa cells revealed a virtually complete inhibition of cell proliferation without a block in any specific cell cycle phase. In addition, no effect of oligonucleotide treatment on cell cycle phase distribution was observed in CP17-14 cells. Overexpression of c-fos rendered these cells resistant as the antisense c-fos oligonucleotides were unable to impose proliferative inhibition. These results demonstrate that c-fos expression is required during all phases of the continuous cell cycle in exponentially growing cells suggesting an important maintenance role for c-fos in addition to its role during re-entry into the cell cycle from quiescence.

Characterization of monoclonal antibodies to the outer membrane protein (OmpD) of Salmonella typhimurium.

A panel of monoclonal antibodies, seven against the trimeric and seven against the monomeric forms to outer membrane protein D (OmpD) of Salmonella typhimurium were produced. The specificities of these monoclonal antibodies for the porin proteins of S. typhimurium and their cross-reactions with Salmonella porins OmpC and OmpF were determined by Western immunoblotting and enzyme-linked immunosorbent assay. We observed that OmpD shared more epitopes and had greater structural similarity with OmpC than with OmpF.

Growth of HeLa S3 cells cotransfected with plasmids containing a c-fos gene under the control of the SV40 promoter complex, pRSVcat, and G418 resistance.

HeLa S3 cells, which have been fractionated into sequential and synchronous cell cycle phase-specific fractions, express c-fos at twice the basal levels in the earliest part of G1 phase. To determine whether this peak in c-fos synthesis has regulatory significance, a DNA construct was prepared which contained the human c-fos gene under the transcriptional control of the SV40 promoter complex. The pc-fos(human)-1 gene (9 kilobases) was inserted into the eukaryotic expression vector pSG5 (4.076 kilobases) at the EcoRI site. Electroporation with an exponentially decaying pulse was employed to cotransfect this construct into HeLa S3 cells along with the plasmids pRSVcat and the neomycin-resistance plasmid pF beta fos3' neo. The level of transient expression of each plasmid was determined. Transfection efficiency was determined as percentage fluorescent cells by measurement of immunofluorescence with a chloramphenicol acetyltransferase (CAT) antibody. Efficiency of transfection ranged up to approximately 5% of the cells. Transfected cells were selected on the basis of resistance to Geneticin (G418) at 400 micrograms/mL. CAT fluorescence and Geneticin resistance were employed to select permanently transformed cell lines. Compared with exponentially growing cells, successfully transfected cell lines expressed more than twice the level of c-fos mRNA as determined by dot-blot analysis and 16 times more of the 62-kilodalton c-fos protein as determined by Western blot analysis. As all cells in the population were not stable c-fos transfectants, this value is likely to be an underestimate of the overexpression level. In addition, expression was under the control of a strong serum induction insensitive promoter, unlike the native c-fos promoter.(ABSTRACT TRUNCATED AT 250 WORDS)

Catabolic pools in Escherichia coli.

Methods are described for measuring soluble pool magnitudes in steady-state or exponentially growing cultures, and for distinguishing between anabolic, catabolic and total metabolic pools within cells. These methods were applied to the measurement of pool magnitudes for several amino acids and other precursors in Escherichia coli THU. Our results support the independence of the magnitudes of total metabolic pools and growth rate in steady-state cultures. Our results also show that the total metabolic pool size is much larger than previously published estimates, which failed to include the contribution of catabolic pools. The average value of the total soluble material in exponential-phase cells is estimated to be 8 to 9% of the cell dry mass; pool values could be almost twice as large during midcycle because of the known increase in the magnitudes of protein and RNA precursor pool during the cell cycle.

Quick and high-efficiency electroelution of nucleic acid fragments.

We describe a quick, simple, and inexpensive technique for the electroelution of nucleic acid fragments that provides a high yield of DNA by using common laboratory components. The quantity of buffer used for the recovery of nucleic acid fragments is relatively small, the quality of the DNA recovered is relatively high, and more than one electroelution can be carried out at the same time.

The phosphate pool in Escherichia coli THU.

The phosphate pool of Escherichia coli was determined as a fraction of the total cell phosphate. This relative pool size was found to be essentially independent of cell age.

Response of lymphoid cells to thymic hormonal factors isolated from mouse thymus.

Thymic hormonal factors were isolated from mouse thymus by two methods. (1) Thymic cytosols in phosphate buffer saline were filtered through Sephadex G100 with 0.1 M NH4HCO3 (pH 8.0) as buffer and the protein peaks were collected. (2) Protein having thymosin activity (F5) was isolated from thymic cytosols after heat inactivation, salt fractionation and desalting on Sephadex G25. Molecular weights of all the proteins were determined on SDS-PAGE. Biological activity of thymic proteins was studied by in vitro and in vivo assays, using synthetic thymosin alpha 1 as the standard. Thymocytes treated with different thymic proteins showed maximum stimulation at 16 h of incubation period. Preincubation of the thymocytes with the thymic proteins and subsequent incubation with Con-A decreased the stimulation index. Incubation of spleen lymphocytes with thymic proteins increased the percentage of Tdt+ cells. The antitumor effects of thymic proteins carried out on animals having leukemia, showed statistically significant results. Clinically however, the antitumor effects of the thymic proteins alone and in combination chemotherapy were negligible at 1 mg/kg body weight dose level.

Activation of amino acid transport during steady-state growth of Escherichia coli.

Accumulation of amino acids in exponentially increasing cultures of Escherichia coli was linear, supporting the interpretation that the biphasic response observed when cultures grew without these acids reflects a transient perturbation in accumulation. Rates of accumulation of glutamine, histidine and glycine were compared in steady-state and non-steady-state cultures. Their uptake rates were markedly enhanced in steady-state cultures at low exogenous concentrations, 10 microM or less. The results support the activation of amino acid transport systems by low concentrations of the particular amino acid present during growth. This activation was decreased when exogenous concentrations of the amino acid were markedly increased or when cells were washed free of the amino acid. Upon readdition of the amino acid after washing, recovery of enhanced transport required several generations, supporting a process of recovery other than enzymatic induction. The observation of amino acid enhancement of transport for eight other amino acids examined in steady-state culture suggests that this enhancement is a common process.

Variation in precursor pool size during the division cycle of Escherichia coli: further evidence for linear cell growth.

The magnitudes of several pools of radioactively labeled precursors for RNA and protein synthesis were determined as a function of cell age during the division cycle of Escherichia coli 15 THU. Uracil, histidine, and methionine pools increased from low initial values for cells at birth to maxima during midcycle and then subsided again. These pools were small or nonexistent at the beginning and the end of the cycle, and their average values during the cycle were less than 4% of the total cellular radioactivity. The results are consistent with a linear pattern of growth for cells during the division cycle and provide strong evidence against exponential or bilinear growth of E. coli cells.

Acceleration of neoplasms of reticular tissue with syngeneic thymic graft under renal capsule and gamma radiation.

Weanling ICRC female mice were given syngeneic neonatal thymus graft under renal capsule and two weeks later whole-body gamma-radiation (group ITR). The findings on ITR were compared with three controls--untreated (C), thymus grafted (IT) and irradiated (IR). In ITR group 15/15 developed generalized neoplasms of reticular tissue at 5 to 8 months and single mammary tumor at 7 months. In IR group 12/13, 21/25 in IT and 5/23 in C had neoplastic changes at 7-month onwards. Microscopically these neoplasms had diffuse or focal proliferation of modified reticulum cells, histiocytes, lymphocytes and giant cells. Localized thymic tumors showed either the same picture or prominence of small lymphocytes. Mammary tumors developed in 12/25 IT, and 8/25 in C and nil in IR.