Cinchonicine-derived alkaloids from the bark of the Peruvian Ladenbergia oblongifolia.

The isolation of cinchonicine-derived alkaloids epicinchonicinol (1), cinchonidicinol (2) and a mixture of dihydrocinchonicinol and dihydrocinchonidicinol (3) from the dried bark of Ladenbergia oblongifolia, is reported along with (1)H and (13)C-NMR data.

Drug targeting Mycobacterium tuberculosis cell wall synthesis: genetics of dTDP-rhamnose synthetic enzymes and development of a microtiter plate-based screen for inhibitors of conversion of dTDP-glucose to dTDP-rhamnose.

An L-rhamnosyl residue plays an essential structural role in the cell wall of Mycobacterium tuberculosis. Therefore, the four enzymes (RmlA to RmlD) that form dTDP-rhamnose from dTTP and glucose-1-phosphate are important targets for the development of new tuberculosis therapeutics. M. tuberculosis genes encoding RmlA, RmlC, and RmlD have been identified and expressed in Escherichia coli. It is shown here that genes for only one isotype each of RmlA to RmlD are present in the M. tuberculosis genome. The gene for RmlB is Rv3464. Rv3264c was shown to encode ManB, not a second isotype of RmlA. Using recombinant RmlB, -C, and -D enzymes, a microtiter plate assay was developed to screen for inhibitors of the formation of dTDP-rhamnose. The three enzymes were incubated with dTDP-glucose and NADPH to form dTDP-rhamnose and NADP(+) with a concomitant decrease in optical density at 340 nm (OD(340)). Inhibitor candidates were monitored for their ability to lower the rate of OD(340) change. To test the robustness and practicality of the assay, a chemical library of 8,000 compounds was screened. Eleven inhibitors active at 10 microM were identified; four of these showed activities against whole M. tuberculosis cells, with MICs from 128 to 16 microg/ml. A rhodanine structural motif was present in three of the enzyme inhibitors, and two of these showed activity against whole M. tuberculosis cells. The enzyme assay was used to screen 60 Peruvian plant extracts known to inhibit the growth of M. tuberculosis in culture; two extracts were active inhibitors in the enzyme assay at concentrations of less than 2 microg/ml.

A longitudinal study of C3, C3d and factor Ba in burn patients in Hong Kong Chinese.

A longitudinal study of serum C3, C3d and fragment Ba was carried out in 53 burn patients of Chinese origin whose total burn surface area ranged from I to 45%. Complement C3 was found to be activated on or before day 7 post-burn. The sharp increase in C3d suggested an acute inflammatory response. In addition factor Ba was increased in these patients, suggesting that the alternative pathway was also activated following thermal injuries. The fluctuations of C3, C3d and Factor Ba observed about 1 year after injury suggested chronic inflammation associated with long term of outcome of healing in the burn sites.

Stimulatory effect of 660 nm low level laser energy on hypertrophic scar-derived fibroblasts: possible mechanisms for increase in cell counts.

BACKGROUND AND OBJECTIVE: Varying effects of red light wavelengths on in vitro cells have been reported. Low level lasers (LLL) are employed to assist wound healing especially for indolent ulcers. On healing, burn wounds may become hypertrophic, resulting in excessive wound contraction, poor cosmesis, and functional impairment. This study enquired whether 660 nm LLL affected hypertrophic scar-derived fibroblasts. STUDY DESIGN/MATERIALS AND METHODS: The experiments investigated the effect of a 660 nm, 17 mW laser diode at dosages of 2.4 J/cm2 and 4 J/cm2 on cell counts of two human fibroblast cell lines, derived from hypertrophic scar tissue (HSF) and normal dermal (NDF) tissue explants, respectively. The protocol avoided transfer of postirradiated cells. Estimation of fibroblasts utilized the methylene blue bioassay. RESULTS/CONCLUSION: The post-660 nm-irradiated HSFs exhibited very significantly higher cell counts than controls P < 0.01 on days 1-4 (Mann-Whitney U-test), and P < 0.01 on days 1-3 for similarly irradiated NDFs.

Analysis of charge distribution of lambda- and kappa-IgA in IgA nephropathy by focused antigen capture immunoassay.

The finding that eluted mesangial IgA and serum IgA from patients with IgA nephropathy (IgAN) had a restricted anionic charge contrasting with normal serum IgA prompted us to examine the charge of kappa- and lambda-subclasses of IgA. Previous studies are not totally satisfactory because either total IgA without further dividing into subclass or only kappa- and lambda-IgA1 was examined. A new approach is described to study the electrostatic property of total IgA and its light chain subclasses. The new focused antigen capture immunoassay (FACIA) allows us to separate the immunoglobulins by isoelectric point, then to capture by the heavy chain class and finally to visualize according to the light chain class. This method works well with whole blood or serum without the need of prior purification by affinity chromatography. The serum total IgA and lambda-IgA levels in patients with IgAN were significantly higher than that of healthy controls. Elevated lambda-IgA levels in patients with IgAN resulted in reduced kappa/lambda ratio of total IgA. Similar to our previous findings in IgA1 with O-linked oligosaccharide side chain, lambda-IgA from patients with IgAN or health controls is highly anionic whereas kappa-IgA is relatively cationic. The higher anionic/cationic ratio observed in total IgA from patients compared with controls was due to the higher concentration of lambda-IgA from the former group. Raised anionic lambda-IgA in IgAN may be contributory to the immunopathogenesis through its selective mesangial binding.

Study of free iron and pyridinoline in hypertrophic scars and normal skin.

In order to investigate the possible involvement of free radicals in the formation of hypertrophic scar, normal skin sections were subjected in vitro to a free radical generating system and the pyridinoline levels determined by enzyme-linked immunoassay. Pyridinoline concentration increased from 5.123 pmol/mg wet weight to 8.760 pmol/mg wet weight after treatment with a free radical generating system. Free iron, a catalyst for the generation of hydroxyl radicals from hydrogen peroxide, was found to be significantly higher in hypertrophic scar tissue (n = 6, mean 50.70 ng/mg wet weight) compared to normal skin (n = 12, mean 7.89 ng/mg wet weight, P = 0.0093, unpaired t-test). It is hypothesised that the occurrence of increased pyridinoline cross-links in hypertrophic scar tissue may result from free radical generation related to inflammatory processes. Further work is necessary to test this hypothesis, but these results suggest that it is tenable.

DNA polymorphism at the ABO locus in the Chinese population of Hong Kong.

DNA polymorphism at the ABO locus was investigated using denaturing gradient gel electrophoresis of polymerase-chain-reaction-amplified DNA products from 315 healthy individuals of the Chinese population of Hong Kong. Five different alleles were identified: ABO*O1, O2, A1, A2 and B1. The ABO*A2 is a new allele not previously reported in the literature. The allele frequencies found were 0.3698 for O1, 0.2508 for O2, 0.1952 for A1, 0.0159 for A2, and 0.1683 for B1. The Chinese population being investigated was in Hardy-Weinberg equilibrium. The genotyping method identifies many more alleles in the ABO locus and thus makes it a more useful genetic marker in linkage analysis, paternity testing and individualization in forensic work than when the locus is genotyped using conventional DNA-based methods or phenotyped using serological methods.

Duplex PCR system for simultaneous detection of Neisseria gonorrhoeae and Chlamydia trachomatis in clinical specimens.

AIMS: To evaluate the use of a duplex polymerase chain reaction (PCR) assay for the simultaneous detection of Neisseria gonorrhoeae and Chlamydia trachomatis in clinical samples. METHODS: Genital swab specimens were obtained from both China (203 swabs) and Hong Kong (202 swabs). N gonorrhoeae and C trachomatis were detected in each specimen with a number of tests including enzyme immunoassays (IDEIA) and PCR assays using both single and double primer pairs. The primer pair for N gonorrhoeae was derived from the cppB gene on its cryptic plasmid and the PCR product was 390 base pairs long. For C trachomatis, the PCR product was 473 base pairs long, resulting from amplification of a sequence in the common 7.4 kilobase plasmid present in all serovars. For N gonorrhoeae, PCR results were also compared with those obtained by culture and Gram's smear of the discharges. RESULTS: For the 203 specimens collected in China, similar numbers of positive results (177) were obtained by both Gonozyme and duplex PCR for the detection of N gonorrhoeae. No discrepant results were found among the cultured specimens when Gonozyme and duplex PCR were compared. C trachomatis was detected in 47 specimens by duplex PCR, but was detected in only 28 by IDEIA. Of the 202 Hong Kong specimens, 46 were positive for N gonorrhoeae, detected by both Gonozyme and duplex PCR; 34 were positive for C trachomatis, 25 of which were detected by IDEIA and the remainder by duplex PCR. CONCLUSIONS: The duplex PCR assay is a satisfactory diagnostic tool for the simultaneous detection of N gonorrhoeae and C trachomatis in clinical swab samples. Further evaluation is suggested.

Diagnosis of urogenital gonorrhoea: evaluation of an enzyme immunoassay and use of urine as a non-invasive specimen.

Urethral swabs and urine samples were collected from 42 male patients attending a social hygiene clinic in Hong Kong and used for gonococcal culture and detection of antigens by enzyme immunoassay. Of the patients, 21 were suffering from gonorrhoea as indicated by positive gonococcal culture from both urethral swabs and urine. Twenty of the positive cases were detected by both swab and urine specimens using Gonozyme, an enzyme immunoassay kit. One culture-positive case failed detection by both swab and urine specimens. Gonozyme showed sensitivity and specificity of 95.2% and 100% respectively. With male patients, the use of urine as an alternative specimen to urethral swabs appears to have potential value for both culture and Gonozyme testing. Genital swabs and urine samples were subsequently collected from 108 patients (89 male, 19 female) attending the sexually transmitted disease monitoring centre in Guangzhou, China. Gonozyme tests were performed on both types of specimen, while only swabs were cultivated. When compared with swab culture, Gonozyme had sensitivity and specificity of 100% and 89.2% for males, and 100% and 90.9% for females, respectively. When urine was compared with swabs in the Gonozyme test, urine had sensitivity and specificity of 83.6% and 89.2% for males, and 62.5% and 81.8% for females, respectively. Hence, Gonozyme merits further investigation as an acceptable rapid diagnostic method for detection of Neisseria gonorrhoeae, especially when using urine as an alternative non-invasive specimen from male patients.

Focused antigen capture immunoassay.

A new approach is described to immunoblotting in which antigens after isoelectric focusing are capture on nitrocellulose membranes coated with a specific antibody. The method has been applied to the analysis of human IgA kappa and lambda from whole blood and serum without prior purification. The visualized bands were quantified by laser densitometry in four pI ranges. It is concluded that, under the conditions used, close to 100% of serum IgA is captured. The method has a coefficient of variation of 4.5-12.8%, mean 8.0%.

Circulating immune complexes of xanthine oxidase in normal subjects.

An antigen-specific enzyme-linked immunosorbent assay (ELISA) was developed to detect immune complexes of xanthine oxidase (XODIC), and applied to assay serum XODIC in normal subjects. It was found that XODIC of both IgM and IgG isotypes could be detected in all the 85 sera tested. XODIC levels as a percentage of total xanthine oxidase antibody (XODAb) in the free and bound forms were mostly less than 20%, and there was also significant correlation in XODIC and XODAb levels for both IgM and IgG isotypes. The implications of these findings are discussed.

Basic, quantitative and experimental research phases of future ethnobotany with reference to the medicinal plants of South America.

Ethnobotany of the future will encompass what we perceive as three interrelated research phases. Basic ethnobotany includes the compilation and organization of information about biota obtained from indigenous and other peoples, such as obtaining data about useful plants and animals, understanding how peoples manage their environments and learning about their lexicons and classifications. This is what we try to do in the best possible way, directly in the field from original sources. These results can then be organized in many ways once species determinations are completed. They may also be organized using other types of information, the most obvious being chemical, medical and linguistic. Quantitative ethnobotany develops methods to allow quantitative description and to evaluate and analyse primary data sets. Original field research must be sufficiently structured and consistent, for example in relation to forest habitat and composition or to oral exchanges between informant and listener, so that statistical techniques may be used to test proposed hypotheses rigorously. This aspect of ethnobotany is in its infancy, yet it can be broadly utilized to comprehend more meaningfully and usefully ethnobotanically valued plants, particularly in the exceedingly diversified environments of tropical regions where because of community isolation practitioners are still most knowledgeable. Experimental ethnobotany involves the use of biota in search of products for industrial, medical and other purposes. Plant ethnomedicinal findings may set the stage for targeting materials which can be meaningfully analysed for chemical activity using appropriate biodirected assays. This approach in search of new pharmaceuticals is woefully underutilized today to the detriment of human health and a number of new strategies should be considered for future advancements in drug discovery. These aspects of ethnobotany will be evaluated largely in relation to current and future research in South America.

Charge distribution of IgA-lambda in IgA nephropathy.

The finding that eluted mesangial IgA and serum IgA from patients with IgA nephropathy had a restricted anionic charge contrasting with normal serum IgA prompted us to study the charge of the kappa and lambda subclasses of IgA. Serum IgA from 11 patients with IgA nephropathy and 11 controls was purified by affinity chromatography by binding to jacalin. The charges of IgA were studied by a novel method. The spectrotype of total IgA was first studied by isoelectric focusing and immunoblotting. IgA lambda and IgA kappa was further analyzed by reacting with specific monoclonal antibodies. The amount of IgA with different pIs was analyzed by computerized densitometry. The anionic:cationic (A:C) ratio of IgA using pI 5.6 as the dividing point was greater in patients (at clinical quiescence and during exacerbation) than in controls (1.67 +/- 0.31 versus 1.36 +/- 0.27, p < 0.025). IgA lambda in both groups was anionic (A:C ratio 2.22 +/- 0.77 versus 2.36 +/- 0.36) and IgA kappa was cationic (A:C ratio 1.15 +/- 0.36 versus 1.04 +/- 0.39) but no difference in the A:C ratio was demonstrated. The increased A:C ratio in total IgA in patients was due to raised serum IgA lambda (kappa/lambda ratio 1.11 +/- 0.14 in patients and 1.51 +/- 0.16 in controls, p < 0.01). We had previously shown a predominant mesangial deposition of IgA lambda in IgA nephropathy. Animal experiments have revealed the preferential mesangial deposition of immune complexes is related to their anionic charges. Our data of raised anionic IgA lambda in IgA nephropathy may be important in determining its selective mesangial binding that could contribute to the immunopathogenesis.

No association found between C3 alleles and scar hypertrophy.

Serum C3 was examined for structural variants using high-voltage agarose gel electrophoresis at both pH 6.2 and 8.4, thermal stability and stability to attack by hydroxyl radicals, in 450 Chinese people in Hong Kong. The C3 allele frequencies for electrophoretic variants were found to be 0.9911 for C3 *S and 0.0089 for all other rare alleles. No variants were found in terms of stability at 54 degrees C in the absence of Ca2+ or at 62 degrees C in the presence of Ca2+, and in terms of stability to attack by hydroxyl radicals. Based on a recently proposed single gene hypothesis for scar hypertrophy, statistical analyses were carried out to compare the relevant frequency data of scar hypertrophy with C3 phenotype frequencies, both for the local Chinese population and a European Caucasian population. There is no evidence for any association between C3 alleles or phenotypes and the formation of hypertrophic scars.

Enhancement of wound healing by the alkaloid taspine defining mechanism of action.

Taspine (mol wt 369,000) is an alkaloid extracted from trees of Croton (family Euphorbiaceae) of the western Amazon region that has been used by natives and others as a vulnerary agent. Taspine was purified from tree sap to test its healing properties using different topical concentrations in the paired rat surgical incision model. Wound tensile strength and histology were evaluated. Samples treated with 250 micrograms, but not those treated with 50 micrograms or 10 micrograms, had significant higher values for MBS than paired controls (26%, P < 0.005, and 30%, P < 0.001, by Days 5 and 7, respectively). Taspine did not modify MBS at Day 12. Sample treated with 250 micrograms had significantly greater mononuclear cellular infiltration at Days 5 and 7 but not at Day 12. To better understand the effect of taspine as an enhancer of wound healing, we conducted in vitro studies in cell cultures. Taspine stimulated chemotaxis for fibroblasts. Taspine did not have an effect on specific assays for macrophage chemotaxis, neutrophil activation, fibroblast proliferation, or matrix assembly. Taken together, the data suggest that taspine promotes early phases of wound healing in a dose-dependent manner with no substantial modification thereafter. Its mechanism of action is probably related to its chemotactic properties on fibroblasts and is not mediated by changes in extracellular matrix.

Analysis of charge of kappa and lambda chain-containing serum immunoglobulin A by immunoblotting and densitometry.

A novel method for studying the charge distribution of different subclasses of serum immunoglobulin A (defined by the light chains) is described. Affinity-chromatography purified immunoglobulins were focused in polyacrylamide gel and were then transferred electrophoretically onto nitrocellulose membranes. The transferred immunoglobulins were detected by rabbit antiserum to human kappa (kappa) or lambda (lambda) chain, swine antirabbit IgG, rabbit anti-peroxidase antibodies and peroxidase, together with a substrate solution comprising H2O2 and diaminobenzidine. Finally, the developed membranes were made transparent with Triton-x 114, scanned at 485 nm with a densitometer to obtain quantitation of charge distribution.

Triterpenoid saponins from Gouania lupuloides.

The stems of Jamaican chawstick, Gouania lupuloides, have yielded two novel 16,17-seco-dammaranoid saponins, designated gouanoside A [4] and gouanoside B [5]. Structural assignments are based on spectroscopic data including 2D nmr experiments on the corresponding aglycones, gouanogenin A [1] and gouanogenin B [3].

C3d levels in normal individuals of Chinese origin.

The level of C3d in the urine and serum of normal healthy Chinese individuals was determined by enzyme-linked immunoassay. Urine levels were found to be in the range of 0.1-2.3 ng/mmol/l creatinine (n = 87, mean = 0.242 microgram /ml). Serum levels were in the range of 10.1-37.6 micrograms/ml (n = 30, mean = 22.6 micrograms/ml). C3d clearance was also determined in 10 selected normal healthy individuals with random urine levels in the upper, middle and lower range. Results suggest that there may be individual variability in the way in which C3d is cleared from blood in normal individuals and it is suggested that caution is necessary in interpreting single urine C3d estimations.