Production and characterization of a trehalolipid biosurfactant produced by the novel marine bacterium Rhodococcus sp., strain PML026.

AIMS: The aim of this study was to evaluate biosurfactant production by a novel marine Rhodococcus sp., strain PML026 and characterize the chemical nature and properties of the biosurfactant. METHODS AND RESULTS: A novel marine bacterium (Rhodococcus species; strain PML026) was shown to produce biosurfactant in the presence of hydrophobic substrate (sunflower oil). Biosurfactant production (identified as a trehalolipid) was monitored in whole-batch cultures (oil layer and aqueous phase), aqueous phase (no oil layer) and filtered (0·2 µm) aqueous phase (no oil or cells; extracellular) and was shown to be closely associated with growth/biomass production. Extracellular trehalolipid levels increased postonset of stationary growth phase. Purified trehalolipid was able to reduce the surface tension of water to 29 mN m(-1) at Critical Micellar Concentration (CMC) of c. 250 mg l(-1) and produced emulsions that were stable to a wide range of conditions (pH 2-10, temperatures of 20-100°C and NaCl concentrations of 5-25% w/v). Separate chemical analyses of the intact trehalolipid and its constituents demonstrated the compound was in fact a mixture of homologues (>1180 MW) consisting of a trehalose moiety esterified to a series of straight chain and hydroxylated fatty acids. CONCLUSIONS: The trehalolipid biosurfactant produced by the novel marine strain Rhodococcus sp. PML026 was characterized and exhibited high surfactant activity under a wide range of conditions. SIGNIFICANCE AND IMPACT OF STUDY: Strain PML026 of Rhodococcus sp. is a potential candidate for bioremediation or biosurfactant production for various applications.

Three-dimensional structure of a lipoyl domain from the dihydrolipoyl acetyltransferase component of the pyruvate dehydrogenase multienzyme complex of Escherichia coli.

The structure of a lipoyl domain from the pyruvate dehydrogenase multienzyme complex of Escherichia coli has been determined by means of nuclear magnetic resonance spectroscopy. A total of 549 nuclear Overhauser effect distance restraints, 52 phi torsion angle restraints and 16 slowly exchanging amide protons were employed as input for the structure calculations. These were performed using a combined distance geometry-simulated annealing strategy. The domain is a hybrid between the N and C-terminal halves of the first and third lipoyl domains, respectively, of the dihydrolipoyl acetyltransferase component of the E. coli multienzyme complex, representing residues 1 to 33 and 238 to 289 (wild-type numbering). The lipoyl-lysine residue was also replaced by glutamine. Nonetheless, its structure, two four-stranded beta-sheets forming a flattened beta-barrel, closely resembles that of the lipoyl domain from the pyruvate dehydrogenase multienzyme complex from Bacillus stearothermophilus determined previously. As before, the lipoylation site is physically exposed in a tight turn in one of the beta-sheets, and the N and C-terminal residues are close together at the other end of the molecule in adjacent strands of the other beta-sheet. Another prominently conserved feature of the structure is the 2-fold axis of quasi-symmetry relating the N and C-terminal halves of the domain. Consistent with the high level of sequence similarity between lipoyl domains of 2-oxo acid dehydrogenase multienzyme complexes from many different sources, these results confirm that all lipoyl domains are likely to have closely related structures.

The treatment of rosacea with topical ivermectin.

The treatment of rosacea is challenging because several pathophysiologic processes may be involved, including neurovascular dysregulation and alterations in innate immune status. Demodex mites may play a role in the latter mechanism. Topical ivermectin is a new therapeutic modality which demonstrates antiparasitic and anti-inflammatory properties. This article reviews published evidence related to the efficacy and safety of topical ivermectin. PubMed was utilized to search for key words "topical ivermectin", "ivermectin cream" and "rosacea". Three clinical trials were found that studied topical ivermectin as a treatment option for rosacea. Ivermectin was effective, safe and well tolerated.

Altered lipid accumulation in Nannochloropsis salina CCAP849/3 following EMS and UV induced mutagenesis.

Microalgae have potential as a chemical feed stock in a range of industrial applications. Nannochloropsis salina was subject to EMS mutagenesis and the highest lipid containing cells selected using fluorescence-activated cell sorting. Assessment of growth, lipid content and fatty acid composition identified mutant strains displaying a range of altered traits including changes in the PUFA content and a total FAME increase of up to 156% that of the wild type strain. Combined with a reduction in growth this demonstrated a productivity increase of up to 76%. Following UV mutagenesis, lipid accumulation of the mutant cultures was elevated to more than 3 fold that of the wild type strain, however reduced growth rates resulted in a reduction in overall productivity. Changes observed are indicative of alterations to the regulation of the omega 6 Kennedy pathway. The importance of these variations in physiology for industrial applications such as biofuel production is discussed.

Evidence for two protein-lipoylation activities in Escherichia coli.

The lipoate acyltransferase subunits of the 2-oxo acid dehydrogenase complexes are post-translationally modified with one or more covalently-bound lipoyl cofactors. Two distinct lipoate-protein ligase activities, LPL-A and LPL-B, have been detected in E. coli by their ability to modify purified lipoyl apo-domains of the bacterial pyruvate dehydrogenase complex. Both enzymes require ATP and Mg2+, use L-lipoate, 8-methyllipoate, lipoyl adenylate and octanoyl adenylate as substrates, and both activate lipoyl-deficient pyruvate dehydrogenase complexes. In contrast, only LPL-B uses D-lipoate and octanoate and there are differences in the metal-ion and phosphate requirements. It is suggested that LPL-B may be responsible for the octanoylation of lipoyl domains observed previously under lipoate-deficient conditions.

Further constituents from the bark of Holarrhena pubescens.

Three compounds, pubadysone [11 alpha-hydroxy-18,20-oxido-3-oxo-pregna-1,4,17(20)-triene] (1), puboestrene [3-acetoxy-17-oxo-1,3,5(10)-estratriene] (2) and pubamide [3,18-dioxo-11 alpha-hydroxycona-1,4-diene] (3), have been isolated from the bark of Holarrhena pubescens. Their structures have been established through spectroscopic studies.

HIV-1 associated neuropathies in males; impotence and penile electrodiagnosis.

Value of the bulbocavernosus reflex latency versus nerve conduction velocity of the dorsal nerve of penis and penile brachial index was evaluated in HIV-1 infected asymptomatic and symptomatic men with and without an objective evidence of neuropathy. These studies revealed striking results in neuropathic group. Both asymptomatic and symptomatic patients with neuropathy exhibited a significant decrease (P < 0.0005) in the values of the nerve conduction velocity of the dorsal nerve of penis as well as penile brachial index in comparison with the controls of the same age group. However both types of non neuropathic patients showed a non significant difference in the above mentioned parameters with their respective controls. The latency of bulbocavernosus reflex showed no significant difference between the groups and was within the normal limits. A non significant association in the values of the study parameters among asymptomatic and symptomatic patients with and without neuropathy was also observed. These findings suggest an exclusively apparent sexual pathway for the penile dorsal nerve conduction and penile brachial index in both HIV-1 infected asymptomatic and AIDS positive men affected by neuropathic conditions, irrespective of their disease state. We thus conclude that a primary defective neuropathic mechanism may play an etiological role in the pathogenesis of erectile impotence in these patients.

A comparative study of the effects of ethanol, acetylcholine and adrenaline on various parameters of intestinal contractions of rabbit.

A comparative study was carried out to observe the effects of ethanol, acetylcholine and adrenaline on various parameters of the contraction cycle of rabbit intestine. Acetylcholine and adrenaline were used in concentrations of 10(-3), either alone or in combination with absolute ethanol. The results showed that ethanol and acetylcholine had no effect on the rate of intestinal contractions while adrenaline increased it significantly. However, all these drugs decreased the active tension of the contracting intestine, the decrease being minimum for ethanol, moderate for acetylcholine and maximum for adrenaline. On the contrary, a small transient decrease in resting tension was produced by ethanol and this occurred at a very fast rate while adrenaline produced a marked decrease in this parameter, its rate being 6-10 times slower. Ethanol and acetylcholine were also found to increase the resting tension, it being 2 times greater and occurring at a faster rate with acetylcholine. It is suggested that these drugs produce their effect by altering resting membrane potential, Ca++ and other ion fluxes.

Effect of triturated dilutions of ethanol on the peristaltic contractions of isolated rabbit intestine.

Effect of triturated dilutions of ethanol from to 10(-3) to 10(-24) were studied on the peristaltic contractions of isolated rabbit intestinal pieces. Triturated dilutions of ethanol were applied in two doses. It was generally observed that the addition of first dose was followed by a small increase of 1.1 gms. in the resting tension which occurred at a slower rate in about 36.5 sec. The second doses however, increased the resting tension by 24 gms, in about 21 sec. In addition, the active tensions showed large variations on the application of first doses of triturated ethanol, while the second doses always decreased this parameter below the first dose levels. The rate of contractions was however, unaltered by both the first and second doses of ethanol, although an increase in the contraction times was found to be balanced by a simultaneous decrease either in the peak duration or relaxation times. The results are discussed in terms of electrophysiological, active state and free ionic Ca++ release phenomena.

Estimation of nor-adrenaline content of human penile tissue in diabeticmen with/without neuropathy.

Penile tissue consisting of corps cavernosum (cc) and tunica albuginea (TA) was obtained from 35 impotent patients undergoing surgery for implantation of penile prostheses and was examined for nor adrenaline content. 10 patients were classified as a non diabetic non neuropathic group, on the basis of their clinical history and differential diagnostic symptoms which included Peyronie's disease, vascular disease, hypertension and psychogenic impotence. The nor adrenaline content was found to be significantly lower in tunica albuginea than the corpus cavernosum (P<0.02) in this group. The nor adrenaline content of corpus cavernosum from insulin dependent (IDDM) and non insulin dependent (NIDDM) diabetic neuropathic patients was also found to be significantly lower (P <0.02) than that of non diabetic non neuropathic patients. The nor adrenaline content of tunica albuginea however, was similar in both groups. A non significant association in the content of nor adrenaline in corpus cavernosum and tunica albuginea among IDDM and NIDDM diabetic neuropathics was also observed. These results provide evidence that an underlying neuropathic factor itself causes vascular as well as metabolic changes in the adrenergic nerves of the penis in diabetics due to neuropathy in addition to the effect of the disease and thus may contribute to the development of impotence in these patients irrespective of their type of diabetes.

Panoramic radiomorphometric indices as reliable parameters in predicting osteoporosis.

BACKGROUND: The purpose was to evaluate the significance of panoramic radiomorphometric indices (mandibular cortical index [MCI], mental index [MI] and panoramic mandibular index [PMI]) as useful tools for identifying osteoporosis. METHODS: One hundred healthy women aged ≥ 30 years were included. Digital panoramic images and bone mineral density (BMD) of the lumbar spines were recorded. Radiomorphometric indices (MCI, MI and PMI) were measured and categorized. RESULTS: Interobserver agreements were kappa = 0.922 for the MCI and alpha = 0.902 and 0.702 for the PMI and MI, respectively. The indices MI, PMI and BMD showed a statistically significant positive correlation with the t score (r = 0.47, 0.36 and 0.96, respectively). The MI showed a statistically significant positive correlation with the PMI (r = 0.72). Sensitivity, specificity and diagnostic accuracy of the MI at a cutoff point of 4.5 mm were 76.9%, 54.1% and 63%, respectively, whereas for the MCI were 66.7%, 75.4% and 72%, respectively. CONCLUSIONS: Panoramic indices (MI, PMI and MCI) were positively correlated with the t score and BMD of the lumbar spines. The MCI was found to be the most reproducible index.

Neurologic disorders are prevalent in HIV-positive outpatients in the Asia-Pacific region.

BACKGROUND: A total of 8.3 million HIV-positive people live in the Asia-Pacific region. The burden of HIV-associated neurocognitive impairment and symptomatic sensory neuropathy in this region is unknown. METHODS: Between July 2005 and March 2006, we undertook a cross-sectional study at 10 sentinel sites within eight Asia-Pacific countries to determine the prevalence of moderate to severe HIV-related neurocognitive impairment and symptomatic sensory neuropathy. We clinically assessed and administered sensitive neuropsychological and peripheral neuropathy screening tools to 658 patients infected with HIV. Univariate and logistic regression analyses were applied to the data. RESULTS: The results showed that 76 patients (11.7%) (95% CI 9.3-14.2) were significantly neurocognitively impaired, 235 patients (36.4%) (95% CI 32.7-40.2) were depressed, and 126 patients (19.7%) (95% CI 16.6-22.8) had either definite or probable symptomatic sensory neuropathy; 63% of this last group had exposure to stavudine, didanosine, or zalcitabine. Several potential confounders including depression (OR 1.49, 95% CI 0.88-2.51, p = 0.11) and prior CNS AIDS illness (OR 1.28, 95% CI 0.50-2.89, p = 0.54) were not significantly associated with neurocognitive impairment. CONCLUSIONS: A total of 12% of patients had moderate to severe HIV-related neurocognitive impairment, 20% of patients had symptomatic sensory neuropathy, and 36% of patients had evidence of depression. This study provides a broad regional estimate of the burden of HIV-related neurologic disease and depression in the Asia-Pacific region.

Chemical constituents of Centella asiatica.

Studies on the chemical constituents of the aerial part of Centella asiatica have led to the isolation of three new compounds, named centellin (1), asiaticin (2), and centellicin (3). Their structures have been elucidated through spectral studies including 2D NMR experiments (HMQC, HMBC, (1)H-(1)H COSY, NOESY and J resolved).

Measurement of the residual urine index in insulin-dependent and non-insulin dependent diabetic men with and without neuropathy.

Value of the residual urine index was evaluated in 40 individuals both insulin-dependent (IDDM) and non-insulin dependent (NIDDM) diabetic male patients with and without an objective evidence of neuropathy and in 20 age matched non-diabetic men serving as controls using post void bladder ultrasonographic technique. These studies revealed striking results in the neuropathic group. Both IDDM and NIDDM diabetic patients with neuropathy exhibited a significant (P < 0.005) increase in residual-volume in comparison with the controls of the same age group and a direct correlation between residual urine retention and neurogenic bladder was found to be established thus suggesting a generalized massive hypotonia of the bladder in these patients. However, non of the two types of non-neuropathic diabetic patients showed significant difference in the above-mentioned parameters compared to that their respective controls. A non-significant association in the values of the study parameters between insulin dependent and non-insulin dependent diabetic men (with and without neuropathy) was also observed. These findings thus suggest a probable neuropathic involvement in the pathway of urinary tract in both IDDM and NIDDM diabetic men with neuropathy. The greater impairment of the values of residual urine index in these patients may be due to overall greater severity of neuropathy with sympathetic as well as parasympathetic damage irrespective of their type of diabetes.

Protease in normal human epidermal keratinocytes.

An in vitro normal human epidermal keratinocytes (NHEK) model was used to study and to characterize the protease stimulated by the mustards 2-chloroethyl ethyl sulphide (CEES), 2-chloro-N-(2-chloroethyl)-N-methylethanamine hydrochloride (nitrogen mustard, HN2), and Bis-2-chloroethyl sulfide (sulfur mustard, HD). The results obtained by using a chromozym (TRY) peptide substrate protease assay showed the optimum mustard concentration and time for protease stimulation to be about 200 microM CEES, 100 microM HN2 or HD, and 16 hours. The mustard-stimulated protease was membrane-bound, and was inhibited by adding a Ca2+ chelator EGTA (2 mM), BAPTA AM (50 microM) or a serine protease inhibitor diisopropyl fluoro-phosphate DFP (1 mM), or a protein synthesis inhibitor cycloheximide (10 microM) in the extracellular medium. These results suggest that one of the mechanisms of mustard toxicity is via the stimulation of a trypsin/chymotrypsin like serine protease, which is dependent on Ca2+ and new protein synthesis. Sodium dodecyl sulfate polyacrylamide gel electrophoresis revealed a mustard-stimulated approximately equal to 70-80 KDa protein band that was associated with protease activity which was inhibitable by EGTA, BAPTA, DFP or cycloheximide. This mustard-stimulated protein (protease) may serve as a diagnostic tool for mustard exposure as well as an assay for screening prospective antivesicant protease inhibitor drugs.

Isolation and characterization of lipoylated and unlipoylated domains of the E2p subunit of the pyruvate dehydrogenase complex of Escherichia coli.

The dihydrolipoamide acetyltransferase subunit (E2p) of the pyruvate dehydrogenase complex of Escherichia coli has three highly conserved and tandemly repeated lipoyl domains, each containing approx. 80 amino acid residues. These domains are covalently modified with lipoyl groups bound in amide linkage to the N6-amino groups of specific lysine residues, and the cofactors perform essential roles in the formation and transfer of acetyl groups by the dehydrogenase (E1p) and acetyltransferase (E2p) subunits. A subgene encoding a hybrid lipoyl domain was previously shown to generate two products when overexpressed, whereas a mutant subgene, in which the lipoyl-lysine codon is replaced by a glutamine codon, expresses only one product. A method has been devised for purifying the three types of independently folded domain from crude extracts of E. coli, based on their pH-(and heat-)stabilities. The domains were characterized by: amino acid and N-terminal sequence analysis, lipoic acid content, acetylation by E1p, tryptic peptide analysis and immunochemical activity. This has shown that the two forms of domain expressed from the parental subgene are lipoylated (L203) and unlipoylated (U203) derivatives of the hybrid lipoyl domain, whereas the mutant subgene produces a single unlipoylatable domain (204) containing the Lys-244----Gln substitution.

Wigner functions from the two-dimensional wavelet group.

Following a general procedure developed previously [Ann. Henri Poincaré 1, 685 (2000)], here we construct Wigner functions on a phase space related to the similitude group in two dimensions. Since the group space in this case is topologically homeomorphic to the phase space in question, the Wigner functions so constructed may also be considered as being functions on the group space itself. Previously the similitude group was used to construct wavelets for two-dimensional image analysis; we discuss here the connection between the wavelet transform and the Wigner function.

Assessment of a neuropathic factor in HIV-1 associated impotence: penile electrodiagnosis.

The value of bulbocavernosus reflex latency verses nerve conduction velocity of the dorsal nerve of penis and penile brachial index was evaluated in 50 impotent individuals infected with the human immunodeficiency virus, type 1 (both symptomatic and asymptomatic) and in 50 aged matched HIV-1 sero-negative impotent men serving as controls. All the subjects were neurologically asymptomatic and non demented. Both HIV-1 infected asymptomatic and symptomatic impotent patients exhibited a significant decrease (P < 0.0005) in the nerve conduction velocity of the dorsal nerve of penis as well as penile branchial index from the controls of the same age group. The latency of bulbocavernosus showed no significant difference between the groups and was within the normal limits. A non-significant association in the study parameters between HIV-1 infected asymptomatic and AIDS positive impotent men was also observed. These findings suggest that impotence and altered erectile electrodiagnostic responses are likely to be associated with an increased frequency to neuropathy in these patients irrespective of their disease state.

The study of cell-death proteins in the outer mitochondrial membrane by chemical cross-linking.

Chemical cross-linking was used to study the interactions of the anti-cell-death protein Bcl2 with other proteins in the outer mitochondrial membrane. Cross-linking of mitochondrial surface proteins produced a large Bcl2-containing complex (>200 kDa), and a Bcl2-derived peptide was shown to cross-link specifically with a mitochondrial protein identified by immunoblotting as Raf-1 kinase.