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.

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.

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.

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.

Serum lipids and lipoproteins in patients with documented coronary artery disease.

Levels of various lipoprotein subclasses can provide useful information on coronary artery disease (CAD) risk status, even when traditional risk factors are known. Elevated serum level of lipids, lipoproteins and lipoprotein (a) have been identified as risk factor for atherosclerosis resulting in coronary artery disease, cerebrovascular disease, peripheral vascular disease and venous thromboembolism. The objective of this study is to evaluate the levels of serum lipids, lipoprotein and lipoprotein (a) in-patients suffering from documented coronary artery disease and normal healthy subjects. A case control study was conducted to evaluate the levels of lipid, lipoprotein and lipoprotein (a) in patients from our local population suffering from documented coronary artery disease. The present study was earned out in Dr. HMI Institute of Pharmacology & Herbal Sciences in collaboration with National Institute of Cardiovascular Diseases, Karachi from January 2001 to June 2001. One hundred and fifty persons were included in this case control study. Out of these one hundred were patients suffering from documented (coronary angiography done 4 month before the study) coronary artery disease (mean age 46.38 +/- 1.38, 18 females and 82 males). 50 normal healthy subjects (mean age, 44.86 +/- 1.15, 2 females and 48 males) had no history of IHD ever before. Their serum lipid, lipoprotein and lipoprotein (a) were estimated and compared.

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.

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.

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.

Hypotensive effect of chemical constituents from Aloe barbadensis.

Hypotensive effects of aloeemodin, aloin A, elgonica dimer A and bisbenzopyran from Aloe barbadensis have been studied. Aloeemodin has emerged as a potent hypotensive agent in current pharmacological investigations and caused 26 %, 52 %, and 79 % falls in mean arterial blood pressure at the corresponding doses of 0.5, 1, and 3 mg/kg in rats. The paper also describes the absolute configuration of elgonica dimer A (1).

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.

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.

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.

Hypotensive, hypoglycaemic and toxicological studies on the flavonol C-glycoside shamimin from Bombax ceiba.

Shamimin, a C-flavonol glucoside from Bombax ceiba leaves showed significant potency as a hypotensive agent at the doses of 15 mg/kg, 3 mg/kg, 1 mg/kg and significant hypoglycaemic activity at 500 mg/kg in Sprague-Dawley rats. Further studies revealed that it did not cause any mortality in mice at the dose of 1 g/kg but in rats 500 mg/kg is a lethal dose. Aqueous and methanolic extracts of Bombax ceiba leaves and one of its fractions were also subjected to pharmacological and toxicological screening.

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.

Thymine metabolism and thymineless death in prokaryotes and eukaryotes.

For many years it has been known that thymine auxotrophic microorganisms undergo cell death in response to thymine starvation [thymineless death (TLD)]. This effect is unusual in that deprivation of many other nutritional requirements has a biostatic, but not lethal, effect. Studies of numerous microbes have indicated that thymine starvation has both direct and indirect effects. The direct effects involve both single- and double-strand DNA breaks. The former may be repaired effectively, but the latter lead to cell death. DNA damaged by thymine starvation is a substrate for DNA repair processes, in particular recombinational repair. Mutations in recBCD recombinational repair genes increase sensitivity to thymineless death, whereas mutations in RecF repair protein genes enhance the recovery process. This suggests that the RecF repair pathway may be critical to cell death, perhaps because it increases the occurrence of double-strand DNA breaks with unique DNA configurations at lesion sites. Indirect effects in bacteria include elimination of plasmids, loss of transforming ability, filamentation, changes in the pool sizes of various nucleotides and nucleosides and in their excretion, and phage induction. Yeast cells show effects similar to those of bacteria upon thymine starvation, although there are some unique features. The mode of action of certain anticancer drugs and antibiotics is based on the interruption of thymidylate metabolism and provides a major impetus for further studies on TLD. There are similarities between TLD of bacteria and death of eukaryotic cells. Also, bacteria have "survival" genes other than thy (thymidylate synthetase), and this raises the question of whether there is a relationship between the two. A model is presented for a molecular basis of TLD.

[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.

Partial-area method in bioequivalence assessment: naproxen.

Regulatory authorities require demonstration of bioequivalence through comparisons of different pharmacokinetic parameters, the area under the plasma concentration-time curve (AUC), the maximum plasma concentration (Cmax), and the time to reach peak concentration (Tmax). The applicability and validity of regulatory requirements have been widely criticized on statistical and clinical relevance grounds. For most noncomplicated absorption models, the AUC correlates well with the extent of absorption. However, in nonlinear models of absorption, in mechanisms involving recycling of drugs, and for drugs with long half-life, the use of total AUC (from zero to infinity) can give erroneous and clinically irrelevant results since the area is mostly determined by elimination phase or by recycling. The calculation of total AUC also involves prolonged sampling, adding to the cost and risks associated with bioequivalence studies. The use of Cmax or Tmax as a measure of rate of absorption, to correlate with clinical relevance, is widely criticized on logical, technical, and statistical grounds. For drugs used on a multiple-dose basis, Cmax and Tmax evaluations become redundant since the average plateau concentration is not affected by these parameters. To resolve the drawbacks in the traditional methodology of bioequivalence evaluation, the use of partial areas in lieu of total AUC, Tmax, and Cmax is suggested. This study investigates the logic and robustness of the partial-area method in establishing bioequivalence. We conclude that the 5h AUC is a more relevant parameter to establish naproxen bioequivalence than AUCinf. We recommend against using symmetrical confidence intervals and report excellent agreement among several methods of calculating confidence intervals, probability values, and nonparametric tests. We suggest that a single-point short-term AUC is a better indicator of the bioequivalence of generic products than the total AUC, Cmax, and Tmax as required currently by the regulatory authorities.