Near-ultraviolet photolysis of beta-phenylpyruvic acid generates free radicals and results in DNA damage.

Ultraviolet A (UVA) light (315-400 nm) is ubiquitously found in our environment and constitutes about 95% of the total solar UV; all UVC and most UVB being absorbed by the stratospheric ozone layer. Compared with UVB and C, UVA does not show any direct effect on biological systems. Indirect effects of UVA, however, have been recognised overwhelmingly and this includes photosensitization of biological and non-biological compounds and production of free radicals many of which include oxygen and are hence known as reactive oxygen species or ROS. Several types of free radicals have been identified although their impacts on various macro- and micro-biomolecules are yet to be fully elucidated. beta-Phenylpyruvic acid is ubiquitously found in eukaryotic cells as a metabolite of phenylalanine, which is subsequently converted to phenyllactate and/or to 2-hydroxyphenylacetate and mandelate. In patients suffering from phenylketonuria the hydroxylation of phenylalanine to tyrosine is defective due to lack of phenylalanine hydroxylase. These result in accumulation and excretion of this compound in the urine. Here we present evidence that photolysis of beta-phenylpyruvic acid by a skin tanning lamp, emitting 99% UVA (315-400 nm) and 1% UVB (290-315 nm) generates carboxyl radicals (CO(2)(*)) and also possibly causes direct electron transfer (or type 1) reactions. Electron paramagnetic resonance was used to detect the free radicals. To determine the biological effects of this photolytic reaction, T7 was exposed to these photolytic reactive agents and found to lead to high levels of phage inactivation. Damage to DNA and/or components such as tail fibre proteins may be involved in T7 inactivation. In addition, our unpublished data suggest that certain phenylketonuria cell lines are more sensitive to PPA+NUV, lending importance to photolytic studies of this agent.

Anticandidal activity of ethanolic root extract of Juglans regia (L.): Effect on growth, cell morphology, and key virulence factors.

The emergence of drug-resistant strains has encouraged several studies on natural products with antifungal activity and low toxicity. In this study, the antifungal effect of methanolic root extract of Juglans regia (JRE) was investigated against 9 strains of Candida (one reference and 8 clinical strains) through MIC90 and spot assays. To gain insight into the mechanism of antifungal action, we carried out confocal scanning laser microscopy (CLSM), transmission electron microscopy (TEM), and then examined the effect of JRE on hydrolytic enzyme secretion. Additionally, JRE was subjected to various phytochemical tests, chemically characterized by gas chromatography-mass spectrometry analysis (GC-MS) and its toxicity was tested against H9c2 rat cardiac myoblasts. The phytochemical tests showed the presence of phenols, alkaloids, steroids, saponins, and tannins in JRE. In the GC-MS analysis, a total of 40 compounds were identified. JRE was found to be effective in liquid media with MICs ranging from 300 to 700µg/mL. Spot assay results revealed that Candida cells show increased sensitivity to JRE. CSLM experiments showed that cells exposed to JRE (MIC) exhibited cell membrane disruption. TEM micrograph of treated cells showed extensive breakage in the cell wall and cell membrane. Average inhibition of proteinase and phospholipase secretion (of five C. albicans strains) at MIC/2 values of JRE was 45.17%, and 34.29%, respectively. Cellular toxicity of JRE against H9c2 rat cardiac myoblasts was less than 10% at the highest MIC value. These findings encourage further development of JRE.

Clinical investigation of hypoglycemic effect of seeds of Azadirachta-inidca in type-2 (NIDDM) diabetes mellitus.

The present study was designed to investigate clinically the hypoglycemic effect of seeds of Azadirachta indica in Type-2 diabetes mellitus. After assaying fasting plasma and urinary glucose, 10 patients of type-2 diabetes mellitus with no previous medication, 10 patients of type-2 diabetes mellitus taking oral hypoglycemic agents with history of inadequate control and six control subjects were given low (0.5 g tid) and high (2 g tid) doses of powdered part, aqueous extract and alcoholic extract of Azadirachta indica for 14 days. On 15th day blood and urine samples for glucose were taken. Based on results obtained it was found that Azadirachta indica has significant hypoglycemic activity in high dose and can be successfully combined with oral hypoglycemic agents in type-2 diabetic patients whose diabetes is not controlled by these agents.

Plasma membrane sialoglycoproteins of human corneal epithelium in culture.

A number of studies, using either rat or rabbit models, have focused on delineating the role of cell surface glycoconjugates in corneal epithelial cell migration and wound healing. We have recently identified the cell surface sialoglycoproteins of rabbit corneal epithelium and have shown that the levels of at least three membrane glycoproteins are markedly altered during cell migration. Because species-related differences may be present, it is important to select an appropriate animal model for studies designed to contribute to the understanding of the mechanism of human corneal wound healing. The purpose of this study was to identify the cell surface sialoglycoproteins of human corneal epithelium. Plasma membrane sialoglycoproteins of primary cultures of human corneal epithelium were labeled with NaB3H4 after oxidation by mild NaIO4 treatment. The labeled glycoproteins were analyzed by SDS-polyacrylamide gel electrophoresis and fluorography. Four different preparations of human corneal epithelial cell cultures were analyzed, and results were compared with those of rabbit corneal epithelium. Of the 11 radiolabeled sialoglycoproteins we identified recently in rabbit corneal epithelium, eight were also present in human corneal epithelium. These similarities in the electrophoretic patterns of the plasma membrane glycoproteins of the two species suggest that rabbit is most likely an appropriate animal model for studies designed to contribute to the understanding of the structure and function of cell surface glycoproteins of human corneal epithelium.

Isolation and analysis of a mutant of Escherichia coli hyper-resistant to near-ultraviolet light plus 8-methoxypsoralen.

A mutant of Escherichia coli K12 was isolated which shows enhanced resistance towards near-ultraviolet (NUV) light plus 8-methoxypsoralen (MPS) compared with its wild-type parent strain. The PUVA (NUV + MPS)-resistant strain remains as sensitive for far-ultraviolet (FUV) light as its parent strain. A recA- derivative of this mutant strain was as sensitive to PUVA as its reca- parental strain. A polyacrylamide gel electrophoresis study of total cell lysates from the mutant bacteria showed that a protein of approximately 55 kd was synthesised in higher concentrations compared with its synthesis in the wild-type parent strain. Furthermore, synthesis of this protein was reduced in the recA- derivative of the mutant strain suggesting that the recA gene product might be acting as a regulator of the synthesis of the 55-kd protein. It is suggested that in E. coli damage to DNA by PUVA can be repaired by a specific RecA LexA-inducible repair system and the repair efficiency is enhanced if the 55-kd protein is present in concentrations higher than that synthesised by the wild-type parent E. coli.

Inducible DNA polymerase I synthesis in a UV hyper-resistant mutant of Escherichia coli.

A mutant of Escherichia coli which is more resistant to shortwave UV light than its wild-type parent strain and which can synthesise DNA polymerase I constitutively has been further analysed. It carries two mutational alleles which are located about 1.5 min apart and cotransducible by P1 with the argH locus. The two mutational alleles have been segregated and their analysis shows that one of them is responsible for UV hyper-resistance whereas the other mutation confers UV sensitivity. Recombinant plasmids carrying various sections of the polA regulatory region, linked to a galK gene, were introduced into the mutant strains. Analysis of galactokinase shows that the enzyme activity in the UV hyper-resistant mutant is increased. The results suggest that the synthesis of DNA polymerase I in E. coli is inducible.

DNA damage by 8-methoxypsoralen plus near ultraviolet light (PUVA) and its repair in Escherichia coli: genetic analysis.

Mutants of Escherichia coli, hyper-resistant and sensitive to 8-methoxypsoralen plus near ultraviolet light (PUVA) have been isolated and studied. Results show that a mutation, located at 57.2 min on the linkage map of E. coli, is responsible for the hyper-resistant phenotype. It is also responsible for the synthesis of a 55-kdal protein in high concentrations. In a wild-type cell the synthesis of this enzyme is inducible by mitomycin C. There are indications that the mutation may have occurred in a regulatory gene, puvR, and as a result the operon, including a putative puvA gene (the structural gene for the synthesis of the 55-kdal protein), is expressed constitutively. A model for the control of the PUV operon is proposed.

A mutant of Escherichia coli hyper-resistant to a number of DNA damaging agents: location of the mutational site.

A mutant of Escherichia coli, isolated as hyper-resistant to UVC, is found to be hyper-resistant to UVA, H2O2, low concentrations of nalidixic acid, novobiocin, UVA plus H2O2 and UVA plus 8-methoxypsoralen. A mutational site (uvh) conferring the hyper-resistance phenotype to UVC, and presumably to other DNA damaging agents, has been mapped at the 89.9 min region on the chromosome. Complementation analysis with an F-prime uvh+/uvh- diploid strain showed that the uvh+ allele is dominant over uvh- in trans. Studies with a variety of plasmids, carrying various LexA regions, introduced into the UV hyper-resistant strain show that mutation at the uvh locus may be responsible for derepression of the SOS inducible repair system. Based on the results, it is suggested that uvh is a part of the SOS inducible system. A plausible explanation for the hyper-resistance phenotypes for various DNA damaging agents and a model for the genetic control of a second set of putative SOS regulons are presented.

Synergistic action of near-UV and phenylalanine, tyrosine or tryptophan on the inactivation of phage T7: role of superoxide radicals and hydrogen peroxide.

Near ultraviolet (NUV) light can cause a variety of damage to biological systems. The effects of NUV are significantly enhanced in the presence of sensitizers. One of the most important targets of such synergistic effects is DNA. Cellular DNA exposed to NUV plus sensitizers is damaged in a variety of ways, DNA strand breaks and interstrand cross-links being the most common effects. In this study, phenylalanine, tyrosine and tryptophan are shown to act as sensitizers for NUV action of phage T7; superoxide anions are produced. The reactive species probably interacts with phage DNA causing damage responsible for phage inactivation. Superoxide dismutase reverses the synergistic activities of phenylalanine and tyrosine on NUV-induced phage inactivation, but catalase is additionally required to reverse the effect of tryptophan. Therefore, it is probable that NUV photolysis of tryptophan causes the production of superoxide ions and hydrogen peroxide, both of which contribute to phage inactivation. The ubiquitous nature of NUV in our environment and the presence of amino acids in skin cells suggests that an important mechanism for the induction of skin cancer in humans by solar exposure is amino acid photolysis by NUV.

Near-ultraviolet photolysis of L-mandelate, formation of reactive oxygen species, inactivation of phage T7 and implications on human health.

Compared with ultraviolet B and C, UVA is considered to have little direct effects on biological systems. However, damaging effects of UVA on biological systems are often synergistically enhanced in the presence of sensitizers. Production of reactive oxygen species (ROS) has been implicated in the process. Several ROS have been identified but their involvement in inducing cellular damage is yet to be fully evaluated. Although membranes and proteins are affected, DNA is an important target and a variety of types of damage have been reported. Here, we present evidence that L-mandelate can act as a near UV (NUV) sensitizer, when activated by a lamp emitting 99% UVA and 1% UVB. Although evidence is available that H(2)O(2) and a small amount of *OH are produced, an alternative effect of the sensitization reaction may involve direct electron transfer. Studies have shown that NUV photolysis of mandelate can inactivate phage T7. Employment of tetrazolium blue test to detect superoxide anion may not be sufficient evidence as this agent may be reduced by alternative routes.

Generation of reactive oxygen species from the photolysis of histidine by near-ultraviolet light: effects on T7 as a model biological system.

Near-ultraviolet (NUV) light (280-400 nm) has a variety of effects on biological systems; these effects are increased, often synergistically, in the presence of sensitizers. A variety of both man-made and naturally occurring sensitizers have been identified, but their precise roles and relative contributions to cellular damage are not yet fully established. DNA seems to be a major target and a variety of types of damage have been observed. In this report we present evidence that histidine can also act as a sensitizer of NUV. Upon NUV photolysis a variety of reactive oxygen species, including superoxide anions, hydroxyl radicals and hydrogen peroxide, are produced as determined by the effects of various scavengers. pH influences the reaction, alkaline media being most effective, as has previously been reported for the photolysis of H2O2, tyrosine, phenylalanine and tryptophan. Exposure of phage T7 to a combination of histidine and NUV leads to synergistic inactivation and scavengers of O2.-, .OH and H2O2 reduce this effect. These results point to a possible involvement of sunlight-induced histidine photolysis in cellular damage. The fact that photolysis is maximal at high pH indicates that biological effects are likely to be highly localized, e.g., at enzyme active sites.

Isolation and analysis of UV and radio-resistant bacteria from Chernobyl.

The accident at the Chernobyl nuclear power station in 1986 led to the dispersal of large amounts of a variety of radioactive materials, most importantly uranium, plutonium, 137Cs, 131I and 90Sr, over very large distances estimated to reach as far as Sweden, Norway, Turkey and possibly the USA. As a consequence, the soil on which the radioactive materials fell was contaminated and the degree of contamination varied with distance from the station, the direction and strength of the wind and the amount of atmospheric scavenging by rainfall at that time. Some of the radioactive materials have left a significant impact on mankind in the form of chromosomal aberrations including trisomy, various forms of cancers and death, whilst others are still in the ground where they will remain for a prolonged period to continue to exert their effects. Likewise, microbes living in the soil and exposed to radioactive materials may have been affected in a number of ways; some perished, and others survived due to the acquisition of advantageous mutation. Six years after the accident, soil samples contaminated with different levels of radioactivity were obtained from five regions within a 30 km radius of the nuclear power plant. From these soil samples spore-forming bacilli were isolated, quantified, identified and tested for resistance to X-rays, UVC and 4-nitroquinoline 1-oxide (4NQO). As a control, spore-forming bacilli were obtained from 'Zeleny mys' (an area 50 km south-east of the power station and emitting basal levels of radioactivity). A mutant of Escherichia coli hyper-resistant to a variety of DNA-damaging agents and its parent strain were also included in the study. Analysis of results reveals that a proportion of isolates of the same species from near the power station and the E. coli mutant SA236 were more resistant to X-rays, UVC and 4NQO compared with isolates from the control site and the E. coli parent strain, KL14, respectively.

Control of skin infections by a combined action of ultraviolet A (from sun or UVA lamp) and hydrogen peroxide (HUVA therapy), with special emphasis on leprosy.

Despite its abundance and certain therapeutic value, the importance of sunlight in the treatment of infectious skin diseases has not been fully exploited. One reason is that a sufficient amount of the damaging components of sunlight (UVC and most UVB) cannot reach us and the band of UV that can reach (UVA) is a poor inactivator of living cells. UVA, however, can be deleterious to cells in the presence of sensitizers and a number of biological and chemical sensitizers have been identified which can inactivate microbes in the presence of UVA. Of several known agents, I have selected hydrogen peroxide (H(2)O(2)) as a UVA sensitizer and propose that a combined action of H(2)O(2)and UVA (HUVA therapy) can be utilized in controlling skin infections of various types. Of particular interest is infection by Mycobacterium leprae, which is known to affect many millions of humans globally. H(2)O(2)being relatively cheap (and UVA from the sun being free) the cost of application, particularly in third-world countries where leprosy is more common, would be low and therefore the treatment can be employed on a wide scale. A further reason for proposing the use of H(2)O(2)is that, out of several agents we have tested, this was found to be the most potent; it is also easily able to reach target sites, very cheap, relatively safe and there is no known microbial resistance to HUVA.

Treatment of post-burns bacterial infections by bacteriophages, specifically ubiquitous Pseudomonas spp. notoriously resistant to antibiotics.

Post-burn microbial infections are a major problem in recovering from the trauma of third-degree burns, and the survival of patients can depend upon the severity of the burn and the infections encountered. Within 24 hours, patients can start suffering from opportunistic bacterial attacks, which can vary from simple infection, such as those easily treatable by antibiotics, to more complicated types, which may have natural or acquired resistance to drugs. Infection by multiple drug-resistant bacteria can create additional complexity to the problem. As an alternative to treating bacterial infections by antibiotics, bacteriophages have been in use in certain parts of the world, such as at Tbilisi in Georgia and in Poland, and this approach has now been more widely recognized. Results have shown that phage therapy has an 80% success rate against Enterococcus infections and up to 90% against Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli and Klebsiella pneumoniae. Here it is proposed that bacteriophages can effectively be used for the treatment of post-burn infections, particularly the ubiquitous opportunistic pathogens, Pseudomonas spp., known to be notoriously resistant to a variety of antibiotics. This kind of treatment may be of particular importance in Third World countries where the incidence of burns and infections, due to lack of stringent safety regulations and proper hygiene respectively, may be more common and where cocktails of antibiotics may be less affordable. Phages that can possibly be employed in the treatment and their advantages compared to the use of antibiotics are also highlighted.

Treatment of post-burns bacterial infections by Fenton reagent, particularly the ubiquitous multiple drug resistant Pseudomonas spp.

Post-burn microbial infections are a major problem in burns, and in cases of third degree burns, the survival of patients can depend not only upon the severity but also upon the extent and the type of infections. If proper measures are not employed, patients may suffer from opportunistic bacterial attacks, which can vary from simple infection, such as those easily treatable by antibiotics, to more complicated types, which may have natural or acquired resistance to drugs. Infection by multiple drug resistant (MDR) bacteria can create further complexity to the treatment. It is proposed that a combination of diluted hydrogen peroxide (H(2)O(2)) and ferrous sulphate (FeSO(4)), which generates hydroxyl radicals (*OH) via Fenton reaction, can effectively be used for the treatment of post-burns bacterial infections. It should be particularly useful for the ubiquitous opportunistic pathogen, Pseudomonas aeruginosa, known to be notoriously resistant to various antibiotics. This reactive oxygen species (ROS)-induced inactivation of the bacterial skin infections may be of particular importance in Third World countries where the incidence of burns and post-burns infections by MDR bacteria (due to the indiscriminate use of antibiotics, lack of stringent safety regulations and proper hygiene) may be more prevalent and where cocktails of antibiotics may be less affordable. Also, since the putative lack of development of bacterial resistance to *OH is not known, it provides an added advantage to the treatment. Finally, although this work addresses the control of bacterial infections in burns cases, it is envisaged that this ROS-induced chemotherapy may also be useful in combating other kinds of skin infections particularly those resisting antibiotic treatment.

Hydrogen peroxide: a potent cytotoxic agent effective in causing cellular damage and used in the possible treatment for certain tumours.

H2O2, a highly reactive agent, can react under certain conditions with a variety of cellular components. These reactions include the lipid peroxidation of membrane and hydroxylation of proteins and DNA. The reactions can take place in the presence of oxygen and are fairly rapid, the H2O2 being converted to water and oxygen. Experiments were carried out in vitro to assess the ability of this agent to destroy cancer cells without generating dangerous by-products. The direct administration of aqueous H2O2 into solid tumours has the potential to cause tumour cell death. The efficacy of the use of H2O2 for treating 'solid' cancers will necessitate its delivery to the tumour site, for example by direct special multiple injection of H2O2 into a detectable tumour mass. We anticipate that, if suggested mode of delivery can be obtained, H2O2 can act as an anti-cancer drug with two distinct advantages over conventional chemotherapeutic agents: to produce minimal short- and long-term side-effects and is relatively cheap and cost effective.

Circadian variation--increased morning incidence of acute myocardial infarction in patients with coronary artery disease.

OBJECTIVE: To examine whether a circadian variation exists in the onset of symptoms of Acute Myocardial Infarction (AMI) in patients with Coronary Artery Disease (CAD) and to review the characteristics of such variation. SETTING: Patients with AMI admitted to the coronary care units of two teaching hospitals in the city of Karachi, Dr. Ziauddin Medical University Hospital and National Institute of Cardiovascular Disease. METHOD: The study population included patients admitted to the coronary care unit. The diagnosis was based on the onset of AMI signs and symptoms and ECG changes. The inclusion criterion was an age less than 76 years. RESULTS: The incidence of Myocardial infarction assessed by the onset of clinical symptoms exhibited a marked circadian variation. Myocardial infarction occurred 2.8 times more frequently during morning hours (period of maximum incidence) as compared to evening (period of minimum incidence). CONCLUSION: This result extends previous observation of the circadian variation in the incidence of AMI onset. This study also helps in searching the potential triggers to physiological changes that may occur during morning hours. Design and timing of cardioprotective medication may play a crucial role in improving circadian variation and prevention of AMI.

Antibiotic susceptibility of Streptococcus pneumoniae in Karachi.

OBJECTIVE: To evaluate, record and analyse the comparative activity of a range of oral antibiotics against Streptococcus pneumoniae. SETTING: Specimen collected at a private laboratory and some received from different hospitals and clinics. MATERIALS: The specimen comprised of Sputum, Throat Swabs, C.S.F., Pus, Pleural fluid, Ear Swabs, Eye Swabs, Bronchial Wash, Prostatic discharge and Antral Secretions. METHODS: The isolates were tested for their antibiotic susceptibility using the E-test by the methods described in the study protocol. The groups of antibiotics tested were Amoxicillin--Clavulanate, Clarithromycin, Cefaclor, Cefuroxime, Ceftriaxone and Penicillin. RESULTS: A total of 116 strains of Streptococcus pneumoniae were isolated and susceptibility to Amoxicillin-Clavulanate was 100%, 99.1% strains were susceptible to Cefuroxime and 0.9% were intermediate. Susceptibility to Penicillin was 80.2% and 19.8% of the strains were in intermediate category, while susceptibility to Clarithromycin was 89.7%, 1.7% were in intermediate region and 8.6% of the strains were resistant to this antibiotic. The susceptibility pattern of Cefaclor was not calculated as there is no current NCCLS--97 Cefaclor breakpoints available. CONCLUSION: The percentage of drug resistant Streptococcus pneumoniae was negligible in Karachi during the study period, however it is important to monitor the susceptibility pattern to keep a check on an increase in the number of DRSP as reported at different centres in Turkey, Saudi Arabia, Singapore, Thailand, Hong Kong and Indonesia.

[Hyperrecombination of plasmids is characteristic for hyperresistant strains of Escherichia coli]. / Giperrekombinatsiia plazmid kharakterna dlia giperrezistentnykh shtammov Escherichia coli.

Intraplasmid recombination frequency was assessed in several Escherichia coli K12 strains by the kinetic bioluminescence method. Enhanced plasmid recombination was observed in two mutant E. coli K12 strains, which were hyperresistant to DNA-damaging agents, gamma-rays, photosensitizer 8-methoxypsoralen. Plasmid recombination frequency per one generation (P) was calculated. In mutant strains, this value was shown to exceed that in control isogenic strains with a standard resistance approximately by a factor of 15. Enhanced constitutive synthesis of specific proteins such as heat-shock proteins in Gamr444 and a 55-kDa protein in SA270 in hyperresistant mutant strains is assumed to promote activity of the recombinational RecF pathway system.

Effect of diet on transaminase and arginase activities.

The composition of the diet greatly influences the enzyme activities of the liver. The effect of low carbohydrate diet on AST, ALT and arginase activities in liver and serum of rats was determined in the present study. Liver ALT and serum AST were significantly increased in animals fed on low carbohydrate diet, the activities of other enzymes remained unchanged.