Heme, which exists widely in living organisms, is a porphyrin compound with a variety of physiological functions. Bacillus amyloliquefaciens is an important industrial strain with the characteristics of easy cultivation and strong ability for expression and secretion of proteins. In order to screen the optimal starting strain for heme synthesis, the laboratory preserved strains were screened with and without addition of 5-aminolevulinic acid (ALA). There was no significant difference in the heme production of strains BA, BAΔ6 and BAΔ6ΔsigF. However, upon addition of ALA, the heme titer and specific heme production of strain BAΔ6ΔsigF were the highest, reaching 200.77 μmol/L and 615.70 μmol/(L·g DCW), respectively. Subsequently, the hemX gene (encoding the cytochrome assembly protein HemX) of strain BAΔ6ΔsigF was knocked out to explore its role in heme synthesis. It was found that the fermentation broth of the knockout strain turned red, while the growth was not significantly affected. The highest ALA concentration in flask fermentation reached 82.13 mg/L at 12 h, which was slightly higher than that of the control 75.11 mg/L. When ALA was not added, the heme titer and specific heme production were 1.99 times and 1.45 times that of the control, respectively. After adding ALA, the heme titer and specific heme production were 2.08 times and 1.72 times higher than that of the control, respectively. Real-time quantitative fluorescent PCR showed that the expressions of hemA, hemL, hemB, hemC, hemD, and hemQ genes at transcription level were up-regulated. We demonstrated that deletion of hemX gene can improve the production of heme, which may facilitate future development of heme-producing strain.
Gene Deletion ; Bacillus amyloliquefaciens/metabolism* ; Aminolevulinic Acid/metabolism* ; Heme/metabolism* ; Fermentation

5-aminolevulinic acid (5-ALA) plays an important role in the fields of medicine and agriculture. 5-ALA can be produced by engineered Escherichia coli and Corynebacterium glutamicum. We systematically engineered the C4 metabolic pathway of C. glutamicum to further improve its ability to produce 5-ALA. Firstly, the hemA gene encoding 5-ALA synthase (ALAS) from Rhodobacter capsulatus and Rhodopseudomonas palustris were heterologously expressed in C. glutamicum, respectively. The RphemA gene of R. palustris which showed relatively high enzyme activity was selected. Screening of the optimal ribosome binding site sequence RBS5 significantly increased the activity of RphemA. The ALAS activity of the recombinant strain reached (221.87±3.10) U/mg and 5-ALA production increased by 14.3%. Subsequently, knocking out genes encoding α-ketoglutarate dehydrogenase inhibitor protein (odhI) and succinate dehydrogenase (sdhA) increased the flux of succinyl CoA towards the production of 5-ALA. Moreover, inhibiting the expression of hemB by means of sRNA reduced the degradation of 5-ALA, while overexpressing the cysteine/O-acetylserine transporter eamA increased the output efficiency of intracellular 5-ALA. Shake flask fermentation using the engineered strain C. glutamicum 13032/∆odhI/∆sdhA-sRNAhemB- RBS5RphemA-eamA resulted in a yield of 11.90 g/L, which was 57% higher than that of the original strain. Fed-batch fermentation using the engineered strain in a 5 L fermenter produced 25.05 g/L of 5-ALA within 48 h, which is the highest reported-to-date yield of 5-ALA from glucose.
Aminolevulinic Acid/metabolism* ; Corynebacterium glutamicum/metabolism* ; Fermentation ; Metabolic Engineering ; Rhodobacter capsulatus/enzymology* ; Rhodopseudomonas/enzymology*

To investigate the effect of 5-aminolevulinic acid photodynamic therapy (ALA-PDT) on () biofilm. biofilms were constructed on a cell slide and treated with ALA-PDT.According to different light doses,the biofilms were divided into six groups:ALA-PDT group [ALA-PDT1 (50 J/cm),ALA-PDT2 group (100 J/cm),ALA-PDT3 group (200 J/cm)],ALA-only group (ALA group),light-only group (LED),and a negative control group (ALA-PDT-group).The biofilm structure and the ratio of the dead bacteria/live bacteria were observed using a laser confocal microscope (CLSM).Biofilm viability was measured using the XTT assay. CLSM showed that the biofilm structures of ALA group and LED group were not significantly different from that of ALA-PDT-group,whereas the biofilm structure was more seriously damaged in ALA-PDT1 group,ALA-PDT2 group,and ALA-PDT3 group than in the ALA-PDT-group.The ratios of the dead/live bacteria in ALA-PDT-group,ALA group,LED group,ALA-PDT1 group,ALA-PDT2 group,and ALA-PDT3 group were 0.350±0.033, 0.305±0.046, 0.330±0.032, 1.525±0.439, 2.293±0.148 and 3.092±0.189,respectively.ALA group(=0.003, =1.000)and LED group(=-0.025, =1.000)did not significantly differ from the ALA-PDT-group.However,the ratio of dead/live bacteria in ALA-PDT-group was significantly lower than those in ALA-PDT1 group (=-0.162, <0.001),ALA-PDT2 group (=-0.254, <0.001),and ALA-PDT3 group (=-0.352, <0.001).The values of the XTT assay were were 0.462±0.028,0.465±0.044,0.437±0.047,0.301±0.040,0.207±0.001,and 0.110±0.007,respectively,in ALA-PDT-group,ALA group,LED group,ALA-PDT1 group,ALA-PDT2 group,and ALA-PDT3 group.Although the values of XTT assay in ALA(=-0.044, =1.000)and LED groups (=-0.020, =1.000)did not significantly differ from that in ALA-PDT-group,it was significantly higher in ALA-PDT-group than in ALA-PDT1 group (=1.175, <0.001),ALA-PDT2 group (=1.942, <0.001),and ALA-PDT3 group (=-0.352, =2.742, <0.001). ALA-PDT has an inhibitory effect on biofilm.ALA-PDT destroys biofilm structure and inhibits biofilm viability.
Aminolevulinic Acid ; Biofilms ; Photochemotherapy ; Photosensitizing Agents ; Propionibacterium acnes

STUDY DESIGN: A retrospective study. PURPOSE: We report our experience with 5-aminolevulinic acid (5-ALA)–assisted resection of spinal cord ependymomas in adults. OVERVIEW OF LITERATURE: Ependymoma is the most frequent primary spinal cord tumor in adults. Surgery is the treatment of choice in most cases. However, while complete resection is achieved in approximately 80% of cases, clinical improvement is achieved in 15% only. Five-ALA fluorescence–guided surgery seems to be useful for this tumor type. METHODS: We studied 14 patients undergoing 5-ALA fluorescence-guided surgery for spinal cord ependymomas in our service. The modified McCormick classification was used to determine clinical status and the degree of resection was assessed with magnetic resonance imaging. RESULTS: Of the 14 patients, the tumor showed an intense emission of fluorescence in 12 and the fluorescence was weak and nonuniform in two. Complete resection was achieved in 11 cases. According to the McCormick classification, 10 patients improved, two remained the same, and two deteriorated. CONCLUSIONS: Our results confirm that 5-ALA fluorescence-guided resection is useful in spinal cord ependymoma resection. Although the rate of complete resections is similar to that in published series without 5-ALA, clinical results are better when using 5-ALA with a lower percentage of clinical deterioration.
Adult ; Aminolevulinic Acid ; Classification ; Ependymoma ; Fluorescence ; Humans ; Magnetic Resonance Imaging ; Retrospective Studies ; Spinal Cord Neoplasms ; Spinal Cord

With recent developments in photosensitizers and light delivery systems, topical 5-aminolevulinic acid-mediated photodynamic therapy (ALA-PDT) has become the fourth alternative therapeutic approach in the management of oral leucoplakia (OLK) due to its minimally invasive nature, efficacy, and low risk of systemic side effects and disfigurement. This report presents step-by-step guidelines for applying topical ALA-PDT in the management of OLK based on both the clinical experience of the authors and a systematic review of the current literature. Studies using protocols with standardized parameters and randomized clinical trials at multiple centres with adequate sample sizes and both interim and long-term follow-ups are needed before universally applicable guidelines can be produced in this field.
Aminolevulinic Acid ; administration & dosage ; therapeutic use ; Humans ; Leukoplakia, Oral ; therapy ; Photochemotherapy ; methods ; Photosensitizing Agents ; administration & dosage ; therapeutic use ; Practice Guidelines as Topic

OBJECTIVETo evaluate the photodynamic therapy (PDT) against multi-antibiotic-resistant Staphylococcus aureus (S. aureus) and Staphylococcus epidermidis (S.epidermidis) obtained from patients with chronic rhinosinusitis (CRS).

METHODSForty-five CRS patients who had been given medical treatment but still needed endoscopic surgery were included in this study. The mucus from middle meatus was collected from these patients during surgery, followed by separation of S. aureus and S. epidermidis and drug sensitive test. The strains which could form biofilm were selected. Light emitting diode (LED) array with a major wavelength of (633±10) nm was used as light source and 5-Aminolevulinic acid (ALA) was used as photosensitizer in this PDT experiment. The safe range of LED dose and ALA concentration which were not toxic to bacteria by themselves were confirmed, and then did PDT experiment on S. aureus and S. epidermidis. The data of bacterial colony forming unit were transformed to lgCFU before statistical analysis.The Graph Pad Prism 5 software was used to analyzed the data.

RESULTSThirteen S. aureus and 16 S. epidermidis were included in this experiment(from 45 patients), all of them were multi-antibiotic-resistant bacteria, and four of S. aureus and five of S. epidermidis could form biofilm in each group. In planktonic S. aureus experiment, the mean lgCFU was 8.32±0.31 in control group whereas the experiment group was 6.47±0.67 (t=9.01, P<0.01), and in planktonic S. epidermidis experiment the final data was 8.34±0.20 (control group) and 6.97±0.59 (experiment group) (t=8.84, P<0.01). In biofilm S. aureus experiment, the mean lgCFU was 8.68±0.05 (control group), 6.90±0.96(experiment group) (t=3.68, P<0.05); and in biofilm S. epidermidis experiment the data was 8.67±0.05 (control group), 7.29±0.61 (experiment group, t=5.07, P<0.01).

CONCLUSIONOur results demonstrated that ALA-mediated PDT on multi-antibiotic-resistant S. aureus and S. epidermidis from CRS patients was effective in vitro. Additional work defining if the PDT treatment would damage the nasal mucosa and further checking the effectiveness of PDT in vivo is still needed.

Aminolevulinic Acid ; therapeutic use ; Anti-Bacterial Agents ; Biofilms ; Drug Resistance, Multiple, Bacterial ; Humans ; Light ; Photochemotherapy ; Photosensitizing Agents ; therapeutic use ; Rhinitis ; drug therapy ; microbiology ; Sinusitis ; drug therapy ; microbiology ; Staphylococcal Infections ; complications ; drug therapy ; Staphylococcus

OBJECTIVETo investigate the lead exposure, its effects, and the relationships between biomarkers of susceptibility in the workers with low-level occupational lead exposure, and to explore its sensitivity and practical value to evaluate the health hazard.

METHODSThe concentrations of lead fume and lead dust in workplaces of a lead acid storage battery enterprise in Jiangsu Province, China, were measured by occupational health monitoring method. The blood samples of 233 workers with occupational lead exposure and 76 non-occupational lead exposure were collected to measure the blood lead (Pb-B) level using graphite furnace atomic absorption spectrometry (GFAAS), the zinc Protoporphyrin (ZPP) level with blood fluorescence assay, and the delta-aminolevulinic acid dehydratase (ALAD) concentration by a spectrophotometer, and to determine the gene polymorphism of ALAD with TaqMan real-time polymerase chain reaction. At the same time, their urine samples were collected to measure urine lead (Pb-U) concentration with GFAAS and delta-aminolevulinic acid (ALA-U) concentration with a spectrophotometer. The correlations between the above indices were analyzed by multiple linear regression method.

RESULTSThe concentration of lead fume in 18 testing sites and the concentration of lead dust in 30 testing sites were 0.002-0.019 mg/m3 and 0.004-0.013 mg/m3, respectively. Pb-B level was positively correlated with Pb-U concentration (r=0.62, P<0.01) and ZPP level (r=0.47, P<0.01) and was negatively correlated with ALAD concentration (r=-0.77, P<0.01) in 233 workers with occupational lead exposure. Among 233 workers, 218 (93.6%) had ≤70 µg/L Pb-U, and 15 (6.9%) had ≥400≥g/L Pb-B. Pb-B level was not correlated with ZPP level as Pb-B level was <190 µg/L (r=0.18, P=0.068 ), while Pb-B level was positively correlated with ZPP level as Pb-B level was ≥190 µg/L (r=0.36, P<0.01). Pb-U concentration was positively correlated with ALA-U concentration (r=0.49, P<0.01) and ZPP level (r=0.47, P<0.01). ZPP level was negatively correlated with ALAD concentration (r=-0. 19, P<0.01), and was positively correlated with ALA-U concentration (r=0.27, P<0.01). ALAD concentration was not correlated with ALA-U concentration (r =-0. 11, P>0.05). And in 233 workers with occupational lead exposure, there were no significant differences in Pb-B level, ZPP level, and ALAD activity between the workers with ALAD1-2 genotype and the workers with ALAD1-1 genotype (P>0.05). In 76 workers with non-occupational lead exposure, there was no significant difference in Pb-B level between the workers with ALAD1-2 genotype and the workers with ALAD1-1 genotype (P >0.05). The workers with ALAD1-2 genotype had a significantly lower ALAD activity, and a significantly higher ZPP level compared with those ALAD1-1 genotype (P<0.01).

CONCLUSIONIn the workers with low-level occupational lead exposure, ZPP level is positively correlated with Pb-B level when Pb-B level was ≥190 µ/L. ALAD could be used as an effect biomarker of low Pb-B level. ALAD gene polymorphism shows different effects on the Pb-B level and the toxic effects between the workers with occupational lead exposure and the workers with non-occupational lead exposure.

Aminolevulinic Acid ; blood ; Biomarkers ; blood ; China ; Electric Power Supplies ; Genotype ; Humans ; Lead ; blood ; Linear Models ; Occupational Exposure ; Polymorphism, Genetic ; Porphobilinogen Synthase ; blood ; genetics ; Protoporphyrins ; blood

OBJECTIVETo investigate the effect of 8-aminolevulinic acid (ALA) photodynamic therapy (PDT) on the cell proliferation, apoptosis and collagen secretion in keloid fibroblasts and to provide the theoretical base for ALA-PDT treatment of keloids.

METHODSFibroblasts from keloid patients were cultured to the third generation in vitro and incubated in 0, 1, 3, 6, 9 mmol/L of δ-aminolevulinic acid for 3 h in the darkness. Then they were exposed to 635 nm wavelength red light ( 30 J/cm2 ) and continued incubation 24 h after irradiation. CCK-8 assay was used to detect proliferation inhibition rate of fibroblasts. The content of hydroxyproline was measured by colorimetric method. The expression of p-Akt and programmed cell death 4 ( PDCD4) were detected by Western blot.

RESULTSThe inhibition rate of keloid fibroblasts were respectively 0, (8.30 ± 1.01)%, (29.48 ± 3.27)%, (52.01 ± 5.34)%, (79.99 ± 5.85)% with the presence of difference concentrations (0, 1, 3, 6, 9 mmol/L) of ALA. The content of hydroxyproline were respectively (9.540 0 ± 0.352 42), (6.242 5 ± 0.224 85 ), (5.107 5 ± 0.534 88), (3.490 0 ± 0.623 48), (2.945 0 ± 0.514 10) μg/mg. The relative expression of p-Akt were respectively 1, 0.75 ± 0.12, 0.52 ± 0.14, 0.41 ± 0.18, 0.32 ± 0.09. The relative expression of PDCD4 were respectively 1, 1.18 ± 0.19, 1.51 ± 0.22, 0.15 ± 0.30, 2.44 ± 0.22. The difference was statistically significant when compared the group of 1, 3, 6, 9 mmol/L with 0 mmol/L (P < 0.05).

CONCLUSIONSIn concentration within the range of 1-9 mmol/L, ALA could inhibit the proliferation of fibroblasts significantly, promote fibroblasts apoptosis and reduce the content of hydroxyproline in a dose-dependent manner, indicating that 8-aminolevulinic acid photodynamic therapy may be a potential treatment for keloid.

Aminolevulinic Acid ; pharmacology ; Apoptosis ; drug effects ; Cell Culture Techniques ; Cell Proliferation ; drug effects ; Collagen ; secretion ; Fibroblasts ; cytology ; drug effects ; secretion ; Humans ; Keloid ; drug therapy ; pathology ; Light ; Photochemotherapy ; methods ; Photosensitizing Agents ; pharmacology

Although 5-aminolevulinic acid (5-ALA)-mediated photodynamic therapy (PDT) has been demonstrated to be a novel and effective therapeutic modality for some human malignancies, its effect and mechanism on glioma are still controversial. Previous studies have reported that 5-ALA-PDT induced necrosis of C6 rat glioma cells in vitro. The aim of this study was to further investigate the effect and mechanism of 5-ALA-PDT on C6 gliomas implanted in rats in vivo. Twenty-four rats bearing similar size of subcutaneously implanted C6 rat glioma were randomly divided into 3 groups: receiving 5-ALA-PDT (group A), laser irradiation (group B), and mock procedures but without any treatment (group C), respectively. The growth, histology, microvessel density (MVD), and apoptosis of the grafts in each group were determined after the treatments. As compared with groups B and C, the volume of tumor grafts was significantly reduced (P<0.05), MVD was significantly decreased (P<0.001), and the cellular necrosis was obviously increased in group A. There was no significant difference in apoptosis among the three groups. The in vivo studies confirmed that 5-ALA-PDT may be an effective treatment for gliomas by inhibiting the tumor growth. The mechanism underlying may involve increasing the cellular necrosis but not inducing the cellular apoptosis, which may result from the destruction of the tumor microvessels.
Aminolevulinic Acid ; pharmacology ; therapeutic use ; Animals ; Brain Neoplasms ; blood supply ; drug therapy ; pathology ; Cell Line, Tumor ; Glioma ; blood supply ; drug therapy ; pathology ; Microvessels ; drug effects ; Photochemotherapy ; Photosensitizing Agents ; pharmacology ; therapeutic use ; Rats ; Rats, Wistar ; Xenograft Model Antitumor Assays

No abstract available.
Aminolevulinic Acid* ; Pemphigus, Benign Familial* ; Photochemotherapy*