Antibacterial and antibiotic potentiating capabilities of extracts isolated from Burkillanthus malaccensis, Diospyros hasseltii and Cleisthanthus bracteosus against human pathogenic bacteria.

Antibiotics which once a boon in medicine and saved millions of lives are now facing an ever-growing menace of antibacterial resistance, which desperately needs new antibacterial drugs which are innovative in chemistry and mode of action. For many years, the world has turned to natural plants with antibacterial properties to combat antibiotic resistance. On that basis, we aimed to identify plants with antibacterial and antibiotic potentiating properties. Seventeen different extracts of 3 plants namely Burkillanthus malaccensis, Diospyros hasseltii and Cleisthanthus bracteosus were tested against multi-drug resistant Acinetobacter baumannii, Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae, Methicillinresistant Staphylococcus aureus (MRSA) and methicillin-susceptible Staphylococcus aureus (MSSA). Antibacterial activity of hexane, methanol and chloroform extracts of bark, seed, fruit, flesh and leaves from these plants were tested using, disk diffusion assay, minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) assays. Antibiotic potentiating capabilities were tested using time-kill assay. B. malaccensis fruit chloroform extract showed the biggest zone of inhibition against MRSA (13.00±0.0 mm) but C. bracteosus bark methanol extract showed the biggest inhibition zone against MSSA (15.33±0.6 mm). Interestingly, bark methanol extract of C. bracteosus was active against MRSA (8.7±0.6 mm), MSSA (7.7±0.6 mm) (Gram-positive) and A. baumannii (7.7±0.6 mm) (Gram-negative). Overall, the leaf methanol and bark methanol extract of C. bracteosus warrants further investigation such as compound isolation and mechanism of action for validating its therapeutic use as antibiotic potentiator importantly against MRSA and A. baumannii.

Antibacterial and antibiotic potentiating capabilities of extracts isolated from Burkillanthus malaccensis, Diospyros hasseltii and Cleisthanthus bracteosus against human pathogenic bacteria

@#Antibiotics which once a boon in medicine and saved millions of lives are now facing an ever-growing menace of antibacterial resistance, which desperately needs new antibacterial drugs which are innovative in chemistry and mode of action. For many years, the world has turned to natural plants with antibacterial properties to combat antibiotic resistance. On that basis, we aimed to identify plants with antibacterial and antibiotic potentiating properties. Seventeen different extracts of 3 plants namely Burkillanthus malaccensis, Diospyros hasseltii and Cleisthanthus bracteosus were tested against multi-drug resistant Acinetobacter baumannii, Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae, Methicillinresistant Staphylococcus aureus (MRSA) and methicillin-susceptible Staphylococcus aureus (MSSA). Antibacterial activity of hexane, methanol and chloroform extracts of bark, seed, fruit, flesh and leaves from these plants were tested using, disk diffusion assay, minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) assays. Antibiotic potentiating capabilities were tested using time-kill assay. B. malaccensis fruit chloroform extract showed the biggest zone of inhibition against MRSA (13.00±0.0 mm) but C. bracteosus bark methanol extract showed the biggest inhibition zone against MSSA (15.33±0.6 mm). Interestingly, bark methanol extract of C. bracteosus was active against MRSA (8.7±0.6 mm), MSSA (7.7±0.6 mm) (Gram-positive) and A. baumannii (7.7±0.6 mm) (Gram-negative). Overall, the leaf methanol and bark methanol extract of C. bracteosus warrants further investigation such as compound isolation and mechanism of action for validating its therapeutic use as antibiotic potentiator importantly against MRSA and A. baumannii.

Evaluation of bactericidal and virucidal activity of novel disinfectant Aaride AGT-1 compared to other commercially available disinfectants against hospital-acquired infections (HAIs).

Hand hygiene is the topmost crucial procedure to prevent hospital-acquired infections. Choosing an effective hand disinfectant is necessary in enforcing good hand hygiene practice especially in hospital settings. The aim of the study was to investigate the efficacy of Aaride AGT-1 as a hand disinfectant for the inhibition of pathogenic microorganisms' transmission among both patients and personnel in the health care system compared to other commercially available disinfectants. In the present study, a new hand disinfectant Aaride AGT-1 was tested against several bacterial and viral pathogens to evaluate its antimicrobial activity profile. The results revealed that Aaride AGT-1 displayed the highest antibacterial activity against five pathogenic bacteria including MRSA when compared to other commercially available hand sanitizers. Aaride AGT-1 showed the lowest percentage needed to inhibit the growth of bacterial pathogens. In addition, results obtained from time killing assay revealed that Aaride AGT-1 demonstrated the best killing kinetics, by eradicating the bacterial cells rapidly within 0.5 min with 6 log reduction (>99.99% killing). Also, Aaride AGT1 was able to reduce 100% plaque formed by three viruses namely HSV-1, HSV-2 and EV-71. In conclusion, Aaride AGT-1 is capable of killing wide-spectrum of pathogens including bacteria and viruses compared to other common disinfectants used in hospital settings. Aaride AGT-1's ability to kill both bacteria and viruses contributes as valuable addition to the hand disinfection portfolio.

Evaluation of bactericidal and virucidal activity of novel disinfectant Aaride AGT-1 compared to other commercially available disinfectants against hospital-acquired infections (HAIs)

@# Hand hygiene is the topmost crucial procedure to prevent hospital-acquired infections. Choosing an effective hand disinfectant is necessary in enforcing good hand hygiene practice especially in hospital settings. The aim of the study was to investigate the efficacy of Aaride AGT-1 as a hand disinfectant for the inhibition of pathogenic microorganisms’ transmission among both patients and personnel in the health care system compared to other commercially available disinfectants. In the present study, a new hand disinfectant Aaride AGT-1 was tested against several bacterial and viral pathogens to evaluate its antimicrobial activity profile. The results revealed that Aaride AGT-1 displayed the highest antibacterial activity against five pathogenic bacteria including MRSA when compared to other commercially available hand sanitizers. Aaride AGT-1 showed the lowest percentage needed to inhibit the growth of bacterial pathogens. In addition, results obtained from time killing assay revealed that Aaride AGT-1 demonstrated the best killing kinetics, by eradicating the bacterial cells rapidly within 0.5 min with 6 log reduction (>99.99% killing). Also, Aaride AGT1 was able to reduce 100% plaque formed by three viruses namely HSV-1, HSV-2 and EV-71. In conclusion, Aaride AGT-1 is capable of killing wide-spectrum of pathogens including bacteria and viruses compared to other common disinfectants used in hospital settings. Aaride AGT-1’s ability to kill both bacteria and viruses contributes as valuable addition to the hand disinfection portfolio.

Application of rapid microbiological methods for the risk assessment of controlled biopharmaceutical environments.

AIMS: To assess the different operational states within a biopharmaceutical grade clean room, using a rapid microbiological method. The method was a novel system, based on spectrometry, designed for sampling, discriminating, and enumerating airborne particles. Central to the study was the aim to determine the microbiological levels as a clean room went from standard use through maintenance and shutdown, disinfection, and then back to standard use. The objective was to evaluate whether a rapid method could replace conventional environmental monitoring using growth-based media. METHODS AND RESULTS: The instrument evacuated was a BioVigilant IMD-A(®) System, which is a real-time and continuous monitoring technology based on optical spectroscopy that can differentiate between biological particles and inert ones (biological particles expressed as bio-counts based on the detection of microbial metabolites). The results indicated that certain activities lead to a high generation of biological particles and in showing an increase over the baseline, would be regarded as presenting a microbiological risk to the cleanroom. These activities include removing HEPA filter grilles, turning off an air handing unit, and tasks which requires an active personnel presence, such as cleaning and disinfection. CONCLUSIONS: The optical instrument can be used to process sufficient information, so that clean rooms can be returned to use following a period of unexpected downtime or following maintenance without the need to wait for the results from growth-based methods. As such, this type of rapid microbiological method is worth exploring further for clean room air monitoring. SIGNIFICANCE AND IMPACT OF THE STUDY: Few studies have been undertaken which examine air-monitoring devices that can both enumerate and discriminate particulates, in a volume of air as 'inert' or 'biological'. This study extends this limited field. Furthermore, the data collected in relation to cleanrooms is of interest in helping microbiologists understand that risks posed by different activities in relation to clean air-handling systems and personnel particle shedding.

Schwannomin isoform-1 interacts with syntenin via PDZ domains.

The neurofibromatosis type 2 gene (NF2) is involved in the pathogenesis of benign tumors of the human nervous system. The NF2 protein, called schwannomin or merlin, is inactivated in virtually all schwannomas and meningiomas. The molecular mechanisms by which schwannomin functions as a tumor suppressor is unknown but believed to involve plasma membrane-cytoskeletal interactions. Two major alternatively spliced isoforms of schwannomin differing in their C termini have been reported. Using the yeast two-hybrid system, we have identified syntenin as a binding partner for schwannomin isoform-1 (sch-1). Syntenin is an adapter protein that couples transmembrane proteoglycans to cytoskeletal components and is involved in intracellular vesicle transport. The C terminus 25 amino acids of sch-1 and the two PDZ domains of syntenin mediate their binding, and mutations introduced within the VAFFEEL region of sch-1 defined a sequence crucial for syntenin recognition. We have showed that the two proteins interacted in vitro and in vivo and localized underneath the plasma membrane. Fibroblast cells expressing heterologous antisense syntenin display alterations in the subcellular distribution of sch-1. Together, these results provide the first functional clue to the existence of schwannomin isoforms and could unravel novel pathways for the transport and subcellular localization of schwannomin in vivo.

Specific loss of protein kinase activities in senescent erythrocytes.

Rabbit erythrocytes of progressively increasing age were isolated using an avidin-biotin affinity technique and the activity of protein kinases and other enzymes was analyzed in cytosols and membranes from the isolated cells. The activities of cytosolic protein kinase C (PKC), cAMP-dependent kinase (PKA), and casein kinase type I and II (CKI and II) were all found to undergo an age-dependent decrease of twofold to fourfold over the 8-week lifespan of the cells. Membrane-associated tyrosine kinase showed little or no decrease, but membrane-associated CKI showed a dramatic eightfold decrease over the 8-week period. By contrast, various cytosolic enzymes, including lactate dehydrogenase, phosphoglycerate kinase, pyruvate kinase, and acid phosphatase, showed no change in activity over the same time period. Density-separated human erythrocytes showed qualitatively similar decreases in cytosolic protein kinase activities in the densest fractions, which contain the oldest cells. Our results show that aging erythrocytes undergo progressive loss of protein kinases that may adversely affect various cellular processes. The age-dependent loss of kinase activity reported here is one of the most striking manifestations of erythrocyte senescence yet to be reported.

Mutations in the NTP-binding motif of minute virus of mice (MVM) NS-1 protein uncouple ATPase and DNA helicase functions.

The NS-1 protein of minute virus of mice (MVM) is required for viral DNA replication and transcriptional regulation. To define the domain structure of NS-1, we have generated point mutations in its putative NTP-binding/ATPase domain. We show that all mutants were unable to support replication of MVM DNA in a transient DNA replication assay. Furthermore, all mutants, except for the K405S substitution, were able to transactivate the P38 promoter in transient transfection experiments. NS-1 proteins bearing COOH-terminal deletions of 29 and 33 amino acid residues were also transcriptionally inert. Biochemical analysis of recombinant NS-1 expressed in insect cells shows that mutations in the putative NTP-binding/ATPase domain severely reduced helicase activity in vitro. However, affinity labeling experiments indicate that none of these mutations, except for K469T, impaired NTP-binding activity. Finally, all point mutants retained significant levels of ATPase activity, except for the E444Q mutant (1%). These findings suggest that the replication and transcription activities of NS-1 reside in separate functional domains. In addition, NS-1 proteins with mutations in the putative nucleotide binding fold have lost helicase activity, whereas most retain nucleotide binding and ATPase functions, suggesting that the mutations have uncoupled the ATPase and helicase activities.

The role of primer recognition proteins in DNA replication: association with nuclear matrix in HeLa cells.

Primer recognition proteins (PRP) enable DNA polymerase alpha to utilize efficiently DNA substrates with low primer to template ratios. We have previously identified the protein-tyrosine kinase substrate annexin II, and the glycolytic enzyme 3-phosphoglycerate kinase as components of PRP. As a step towards elucidation of the role of PRP in the process of DNA replication, we have investigated the subcellular distribution and specific association of these proteins with the nuclear matrix in HeLa cells. Nuclear extracts prepared from HeLa cells in S phase contain the enzymatic activity of 3-phosphoglycerate kinase (PGK) and phospholipase A2 inhibitory activity of annexin II. Monomer annexin II is approximately equally distributed between the nuclear and cytoplasmic fractions, while a majority of PGK is in the cytoplasm. Immunoblot analyses reveal the presence of these two proteins in nuclei, specifically associated with the nuclear matrix. This is further confirmed by observation of the presence of annexin II and PGK in isolated nuclear matrices by immunoelectron microscopy. The phospholipase A2 inhibitory activity of annexin II colocalizes with the nuclear matrix-bound annexin II. A related protein, annexin I, is not detectable in the nuclear extracts and nuclear matrix. A slower-migrating (perhaps modified) form of annexin II is found to be associated with the nuclear matrix. Attempts to dissociate PGK and annexin II from the nuclear matrix with octyl-beta-glucoside, high salt or metal ion chelators were unsuccessful, suggesting that the interaction is very strong.

Expression of minute virus of mice major nonstructural protein in insect cells: purification and identification of ATPase and helicase activities.

The gene encoding the major nonstructural (NS-1) protein of minute virus of mice (MVM) has been expressed in insect cells using a baculovirus expression system. This 83-kDa polypeptide was found to be localized in the soluble (cytosolic) fraction in insect cells, in contrast with the nuclear localization of NS-1 expressed in MVM-infected mouse LA-9 cells. The protein was purified by immunoaffinity chromatography using a monoclonal antibody (MAb) prepared to an NS-1 fusion peptide [(Yeung et al., Virology 185, 35-45 (1991)]. Recombinant NS-1 was eluted using either low pH or a synthetic peptide corresponding to the epitope of the MAb. The peptide-eluted material is greater than 95% pure and biologically active in that it has ATPase activity and ATP-dependent helicase activity as determined by a strand displacement assay.

The protein-tyrosine kinase substrate, calpactin I heavy chain (p36), is part of the primer recognition protein complex that interacts with DNA polymerase alpha.

Primer recognition proteins (PRP) stimulate the activity of DNA polymerase alpha on DNA substrates with long single-stranded template containing few primers. Purified PRP from HeLa cells and human placenta are composed of two subunits of 36,000 (PRP 1) and 41,000 (PRP 2) daltons. By amino acid sequence homology, we have identified PRP 2 as the glycolytic enzyme 3-phosphoglycerate kinase. Here we present data that establishes PRP 1 to be the protein-tyrosine kinase substrate, calpactin I heavy chain. Amino acid sequence analysis of six tryptic peptides of PRP 1 followed by homology search in a protein sequence data base revealed 100% identity of all six peptides with the deduced amino acid sequence of human calpactin I heavy chain. The activities of PRP and calpactin I coelute on gel filtration columns, and a high correlation of PRP and calpactin I activities was seen at different stages of purification. A rabbit polyclonal anti-chicken calpactin I antibody was shown to cross-react with PRP 1 polypeptide at various stages of PRP purification, and the homogeneous preparation of PRP exhibits 3-phosphoglycerate kinase (PRP 2) and calpactin I (PRP 1) activities. PRP activity is neutralized by a mouse monoclonal anti-calpactin II antibody although having no effect on the polymerase alpha activity itself. Calpactin II has a 50% amino acid sequence homology with calpactin I. However, PRP 1 is not calpactin II as shown by lack of cross-reaction to a monoclonal anti-calpactin II antibody on Western blots. Calpactin I and 3-phosphoglycerate kinase, purified independently, cannot be efficiently reconstituted into the PRP complex, indicating that their association in the PRP complex involves specific protein-protein interactions that remain to be elucidated. The biochemical and immunological data presented here revealing the identity of PRP 1 as calpactin I provide evidence for one physiological role of calpactin I in the cell.

Suramin affects DNA synthesis in HeLa cells by inhibition of DNA polymerases.

Suramin, a polysulfonated naphthylurea widely used in the treatment of trypanosomiasis and onchocerciasis, is currently being investigated as an antitumor agent for the treatment of advanced cancer. Suramin exerts a wide variety of biological effects. We have shown that suramin inhibits cell proliferation and DNA synthesis in cultured HeLa cells. The replication in vitro of SV40 DNA is completely abolished by 40 microM suramin. The inhibition of DNA replication is due to inhibition of DNA polymerases alpha and delta, the replicative enzymes in eukaryotic cells. DNA polymerase alpha is sensitive to lower concentrations of suramin [concentration to achieve 50% inhibition (IC50) of 8 microM] than is DNA polymerase delta (IC50 36 microM), whereas DNA polymerase beta is relatively insensitive to the drug (IC50 of 90 microM). Suramin inhibits other replicative DNA polymerases such as Escherichia coli polymerase I (Klenow fragment) and Thermus aquaticus polymerase. Suramin is noncompetitive with both substrate deoxyribonucleotides and template-primers with respect to DNA polymerase inhibition. Much lower concentrations (8-30 microM) of the drug are required for 50% inhibition of DNA polymerases than for 50% inhibition of other enzymes such as protein kinase C and reverse transcriptase. These results show an important biological effect of this drug and indicate the need for more studies before its clinical use as an antitumor agent.

Purification and characterization of primer recognition proteins from HeLa cells.

We have purified to homogeneity the primer recognition proteins (PRP) from human HeLa cells. PRP is associated with DNA polymerase alpha complex in HeLa cells. Purified PRP is free of DNA polymerases alpha, beta, and delta, deoxyribonuclease, DNA primase, ATPase, topoisomerase, and DNA ligase activities. The protein structure of the PRP was defined by sodium dodecyl sulfate gel electrophoresis, which revealed two polypeptides of 36,000 Da (PRP 1) and 41,000 Da (PRP 2). The two polypeptides are associated in a complex in the native state. The Stokes radius of the PRP complex by gel filtration is 40.5 A and the sedimentation coefficient in glycerol gradients is 5.7 S. Purified PRP, which exhibits no DNA polymerase activity, completely restores the activity of DNA polymerase alpha on templates with low primer to template ratios such as heat-denaturated DNA, poly(dA)-oligo(dT), and singly primed M13 single-stranded DNA. Experiments using various amounts of PRP, DNA polymerase alpha, and DNA indicate that a concentration dependence exists between these components in the DNA replication process. Amino acid composition analysis indicates that the PRP is rich in hydrophobic amino acids.

Functional identity of a primer recognition protein as phosphoglycerate kinase.

Primer recognition proteins (PRP) are cofactors of DNA polymerase alpha and may have a role in lagging strand DNA replication. Purified PRP from HeLa cells and human placenta are composed of two subunits of 36,000 (PRP 1) and 41,000 (PRP 2) daltons. Upon tryptic digestion, amino acid sequencing of tryptic peptides, and homology search against a protein sequence data base, we have identified PRP 2 to be the glycolytic enzyme, phosphoglycerate kinase (PGK). The activities of PRP and PGK increase coordinately in the PRP purification procedure. PRP activity is inhibited by the PGK substrate 3-phosphoglycerate and the competitive inhibitor of substrate binding, DL-alpha-glycerol 3-phosphate. 5'-p-Fluorosulfonylbenzoyl adenosine, which inactivates PGK by binding to the nucleotide binding site, also inhibits PRP. For PRP activity, the two substrate binding sites of PGK are necessary in addition to the as yet unidentified PRP 1 polypeptide.

Clinical findings in an Arab boy with ring (14)(mos 46,XY,r(14)/45,XY,-14).

A five-month male Arab child with clinical features of ring(14) is reported. He had recurrent seizures and chest infections, microcephaly, elongated face, short palpebral fissures, broad nasal bridge, long philtrum, fish-like mouth with thin lips, micrognathia, low-set ears and retinal pigmentation with yellow-white spots on the maculae. In addition brachydactyly of fingers and toes, hypoplastic scrotum and mental deterioration were present.