A critical element of the light‐induced quaternary structural changes in YtvA‐LOV

YtvA, a photosensory LOV (light‐oxygen‐voltage) protein from Bacillus subtilis, exists as a dimer that previously appeared to undergo surprisingly small structural changes after light illumination compared with other light‐sensing proteins. However, we now report that light induces significant structural perturbations in a series of YtvA‐LOV domain derivatives in which the Jα helix has been truncated or replaced. Results from native gel analysis showed significant mobility changes in these derivatives after light illumination; YtvA‐LOV without the Jα helix dimerized in the dark state but existed as a monomer in the light state. The absence of the Jα helix also affected the dark regeneration kinetics and the stability of the flavin mononucleotide (FMN) binding to its binding site. Our results demonstrate an alternative way of photo‐induced signal propagation that leads to a bigger functional response through dimer/monomer conversions of the YtvA‐LOV than the local disruption of Jα helix in the As‐LOV domain.     

Production of biocommodities and bioelectricity by cell‐free synthetic enzymatic pathway biotransformations: Challenges and opportunities

Cell‐free synthetic (enzymatic) pathway biotransformation (SyPaB) is the assembly of a number of purified enzymes (usually more than 10) and coenzymes for the production of desired products through complicated biochemical reaction networks that a single enzyme cannot do. Cell‐free SyPaB, as compared to microbial fermentation, has several distinctive advantages, such as high product yield, great engineering flexibility, high product titer, and fast reaction rate. Biocommodities (e.g., ethanol, hydrogen, and butanol) are low‐value products where costs of feedstock carbohydrates often account for ～30–70% of the prices of the products. Therefore, yield of biocommodities is the most important cost factor, and the lowest yields of profitable biofuels are estimated to be ca. 70% of the theoretical yields of sugar‐to‐biofuels based on sugar prices of ca. US$ 0.18 per kg. The opinion that SyPaB is too costly for producing low‐value biocommodities are mainly attributed to the lack of stable standardized building blocks (e.g., enzymes or their complexes), costly labile coenzymes, and replenishment of enzymes and coenzymes. In this perspective, I propose design principles for SyPaB, present several SyPaB examples for generating hydrogen, alcohols, and electricity, and analyze the advantages and limitations of SyPaB. The economical analyses clearly suggest that developments in stable enzymes or their complexes as standardized parts, efficient coenzyme recycling, and use of low‐cost and more stable biomimetic coenzyme analogs, would result in much lower production costs than do microbial fermentations because the stabilized enzymes have more than 3 orders of magnitude higher weight‐based total turn‐over numbers than microbial biocatalysts, although extra costs for enzyme purification and stabilization are spent. Biotechnol. Bioeng. 2010. 105: 663–677. © 2009 Wiley Periodicals, Inc.     

High‐efficiency delivery of CRISPR‐Cas9 by engineered probiotics enables precise microbiome editing

Antibiotic resistance threatens our ability to treat infectious diseases, spurring interest in alternative antimicrobial technologies. The use of bacterial conjugation to deliver CRISPR‐cas systems programmed to precisely eliminate antibiotic‐resistant bacteria represents a promising approach but requires high in situ DNA transfer rates. We have optimized the transfer efficiency of conjugative plasmid TP114 using accelerated laboratory evolution. We hence generated a potent conjugative delivery vehicle for CRISPR‐cas9 that can eliminate > 99.9% of targeted antibiotic‐resistant Escherichia coli in the mouse gut microbiota using a single dose. We then applied this system to a Citrobacter rodentium infection model, achieving full clearance within four consecutive days of treatment.     

Photosynthetic activity including the thermal‐ and chilling‐light sensitivities of a temperate Japanese brown alga Sargassum macrocarpum

The effects of irradiance, temperature, thermal‐ and chilling‐light sensitivities on the photosynthesis of a temperate alga, Sargassum macrocarpum (Fucales) were determined by a pulse amplitude modulation (PAM)‐chlorophyll fluorometer and dissolved oxygen sensors. Oxygenic photosynthesis–irradiance curves at 8, 20, and 28°C revealed that the maximum net photosynthetic rates (NP _max) and saturation irradiance were highest at 28°C, and lowest at 8°C. Gross photosynthesis and dark respiration determined over a range of temperatures (8–36°C) at 300 μmol photons m^?2 s^?1 revealed that the maximum gross photosynthetic rate (GP_max) occurred at 27.8°C, which is consistent with the highest seawater temperature in the southern distributional limit of this species in Japan. Additionally, the maximum quantum yields of photosystem II (F _v/F _m) during the 72‐h temperature exposures were stable at 8–28°C, but suddenly dropped to zero at higher temperatures, indicative of PSII deactivation. Continuous exposure (12 h) to irradiance of 200 (low) and 1000 (high) μmol photons m^?2 s^?1 at 8, 20, and 28°C revealed greater declines in their effective quantum yields (Φ _PSII) under high irradiance. While Φ _PSII under low irradiance were very similar with the initial F _v/F _m under 20 and 28°C, values rapidly decreased with exposure duration at 8°C. At this temperature, F _v/F _m did not recover to initial values even after 12 h of dark acclimation. Final F _v/F _m of alga at 28°C under high irradiance treatment also did not recover, suggesting its sensitivity to photoinhibition at both low and high temperatures. These photosynthetic characteristics reflect both the adaptation of the species to the general environmental conditions, and its ability to acclimate to seasonal changes in seawater temperature within their geographical range of distribution.     

Inactivation of thioredoxin f1 leads to decreased light activation of ADP‐glucose pyrophosphorylase and altered diurnal starch turnover in leaves of Arabidopsis plants

Chloroplast thioredoxin f (Trx f) is an important regulator of primary metabolic enzymes. However, genetic evidence for its physiological importance is largely lacking. To test the functional significance of Trx f in vivo, Arabidopsis mutants with insertions in the trx f1 gene were studied, showing a drastic decrease in Trx f leaf content. Knockout of Trx f1 led to strong attenuation in reductive light activation of ADP‐glucose pyrophosphorylase (AGPase), the key enzyme of starch synthesis, in leaves during the day and in isolated chloroplasts, while sucrose‐dependent redox activation of AGPase in darkened leaves was not affected. The decrease in light‐activation of AGPase in leaves was accompanied by a decrease in starch accumulation, an increase in sucrose levels and a decrease in starch‐to‐sucrose ratio. Analysis of metabolite levels at the end of day shows that inhibition of starch synthesis was unlikely due to shortage of substrates or changes in allosteric effectors. Metabolite profiling by gas chromatography–mass spectrometry pinpoints only a small number of metabolites affected, including sugars, organic acids and ethanolamine. Interestingly, metabolite data indicate carbon shortage in trx f1 mutant leaves at the end of night. Overall, results provide in planta evidence for the role played by Trx f in the light activation of AGPase and photosynthetic carbon partitioning in plants.     

Binding of B‐cell maturation antigen to B‐cell activating factor induces survival of multiple myeloma cells by activating Akt and JNK signaling pathways

B‐cell maturation antigen (BCMA) is expressed on normal and malignant plasma cells and represents a potential target for therapeutic intervention. In this study, we characterized the mechanism underlying the protein kinase B (Akt) and c‐Jun N‐terminal kinase (JNK) pathways and BCMA interactions in regulating multiple myeloma (MM) cell survival. It was found that the expression levels of B cell‐activating factor (BAFF) and BCMA were increased in MM cells as compared with those in normal controls. The proliferation of U266 cells was induced by recombinant human BAFF (rhBAFF) and could also be decreased by BCMA siRNA. The expression of Bcl‐2 protein was up‐regulated, and Bax protein was down‐regulated after rhBAFF treatment, which could be reversed by BCMA siRNA. Similarly, the protein p‐JNK and p‐Akt were activated by rhBAFF and could be changed by BCMA siRNA. In addition, the BCMA mRNA and protein expression levels were decreased after treatment with Akt and JNK pathway inhibitors. These results suggest that Akt and JNK pathways are involved in the regulation of BCMA. A novel BAFF/BCMA signalling pathway in MM may be a new therapeutic target for MM. Copyright © 2016 John Wiley & Sons, Ltd.     

Intersectin‐s interaction with DENND2B facilitates recycling of epidermal growth factor receptor

Epidermal growth factor (EGF) activates the EGF receptor (EGFR) and stimulates its internalization and trafficking to lysosomes for degradation. However, a percentage of EGFR undergoes ligand‐independent endocytosis and is rapidly recycled back to the plasma membrane. Importantly, alterations in EGFR recycling are a common hallmark of cancer, and yet, our understanding of the machineries controlling the fate of endocytosed EGFR is incomplete. Intersectin‐s is a multi‐domain adaptor protein that is required for internalization of EGFR. Here, we discover that intersectin‐s binds DENND2B, a guanine nucleotide exchange factor for the exocytic GTPase Rab13, and this interaction promotes recycling of ligand‐free EGFR to the cell surface. Intriguingly, upon EGF treatment, DENND2B is phosphorylated by protein kinase D and dissociates from intersectin‐s, allowing for receptor targeting to degradation. Our study thus reveals a novel mechanism controlling the fate of internalized EGFR with important implications for cancer.     

The evolutionary characteristics and structural biology of Gallus toll‐like receptor 21

Toll‐like receptors (TLRs) are an important part of the innate immune system, acting as a first line of defense against many invading pathogens. The ligand known to bind Gallus toll‐like receptor 21 (gTLR21) is the unmethylated cytosine phosphate guanine dideoxy nucleotide motif; however, the evolutionary characteristics and structural biology of gTLR21 are poorly elaborated. Our results suggest that gTLR21 is phylogenetically and evolutionarily related to the TLR11 family and is perhaps a close ortholog of the Mus TLR13. Structural biology of homology modeling of the gTLR21 ectodomain structure suggests that it has no Z‐loop like that seen in Mus TLR9. The cytosolic toll‐IL‐1 receptor region of gTLR21 contains a central 4‐stranded parallel β‐sheet (βA‐βD) surrounded by 5 α‐helices (αA‐αE) on both sides, a highly conserved structure also seen in other TLRs. Molecular docking analysis reveals that the gTLR21 ectodomain has the potential to distinguish between different ligands. Homodimer analysis results also suggest that Phe842 and Pro844 of the BB loop and Cys876 of the αC helix in gTLR21 are conserved in other cytosolic toll‐IL‐1 receptor domains of other TLRs and may contribute to the docking of homodimers. Our study on the evolutionary characteristics and structural biology of gTLR21 reveals that the molecule may have a broader role to play in innate immune system; however, further experimental validation is required to confirm our findings.     

Conformation control of the histidine kinase BceS of Bacillus subtilis by its cognate ABC‐transporter facilitates need‐based activation of antibiotic resistance

Bacteria closely control gene expression to ensure optimal physiological responses to their environment. Such careful gene expression can minimize the fitness cost associated with antibiotic resistance. We previously described a novel regulatory logic in Bacillus subtilis enabling the cell to directly monitor its need for detoxification. This cost‐effective strategy is achieved via a two‐component regulatory system (BceRS) working in a sensory complex with an ABC‐transporter (BceAB), together acting as a flux‐sensor where signaling is proportional to transport activity. How this is realized at the molecular level has remained unknown. Using experimentation and computation we here show that the histidine kinase is activated by piston‐like displacements in the membrane, which are converted to helical rotations in the catalytic core via an intervening HAMP‐like domain. Intriguingly, the transporter was not only required for kinase activation, but also to actively maintain the kinase in its inactive state in the absence of antibiotics. Such coupling of kinase activity to that of the transporter ensures the complete control required for transport flux‐dependent signaling. Moreover, we show that the transporter likely conserves energy by signaling with sub‐maximal sensitivity. These results provide the first mechanistic insights into transport flux‐dependent signaling, a unique strategy for energy‐efficient decision making.     

Role of N‐ and L‐type calcium channels in depolarization‐induced activation of tyrosine hydroxylase and release of norepinephrine by sympathetic cell bodies and nerve terminals

Multiple types of voltage‐activated calcium (Ca²⁺) channels are present in all nerve cells examined so far; however, the underlying functional consequences of their presence is often unclear. We have examined the contribution of Ca²⁺ influx through N‐ and L‐ type voltage‐activated Ca²⁺ channels in sympathetic neurons to the depolarization‐induced activation of tyrosine hydroxylase (TH), the rate‐limiting enzyme in norepinephrine (NE) synthesis, and the depolarization‐induced release of NE. Superior cervical ganglia (SCG) were decentralized 4 days prior to their use to eliminate the possibility of indirect effects of depolarization via preganglionic nerve terminals. The presence of both ω‐conotoxin GVIA (1 μM), a specific blocker of N‐type channels, and nimodipine (1 μM), a specific blocker of L‐type Ca²⁺ channels, was necessary to inhibit completely the stimulation of TH activity by 55 mM K⁺, indicating that Ca²⁺ influx through both types of channels contributes to enzyme activation. In contrast, K⁺ stimulation of TH activity in nerve fibers and terminals in the iris could be inhibited completely by ω‐conotoxin GVIA alone and was unaffected by nimodipine as previously shown. K⁺ stimulation of NE release from both ganglia and irises was also blocked completely when ω‐conotoxin GVIA was included in the medium, while nimodipine had no significant effect in either tissue. These results indicate that particular cellular processes in specific areas of a neuron are differentially dependent on Ca²⁺ influx through N‐ and L‐type Ca²⁺ channels. © 1999 John Wiley & Sons, Inc. J Neurobiol 40: 137–148, 1999     

Green synthesis of enantiopure (S)‐1‐(benzofuran‐2‐yl)ethanol by whole‐cell biocatalyst

Optically active aromatic alcohols are valuable chiral building blocks of many natural products and chiral drugs. Lactobacillus paracasei BD87E6, which was isolated from a cereal‐based fermented beverage, was shown as a biocatalyst for the bioreduction of 1‐(benzofuran‐2‐yl) ethanone to (S)‐1‐(benzofuran‐2‐yl) ethanol with highly stereoselectivity. The bioreduction conditions were optimized using L. paracasei BD87E6 to obtain high enantiomeric excess (ee) and conversion. After optimization of the bioreduction conditions, it was shown that the bioreduction of 1‐(benzofuran‐2‐yl)ethanone was performed in mild reaction conditions. The asymmetric bioreduction of the 1‐(benzofuran‐2‐yl)ethanone had reached 92% yield with ee of higher than 99.9% at 6.73 g of substrate. Our study gave the first example for enantiopure production of (S)‐1‐(benzofuran‐2‐yl)ethanol by a biological green method. This process is also scalable and has potential in application. In this study, a basic and novel whole‐cell mediated biocatalytic method was performed for the enantiopure production of (S)‐1‐(benzofuran‐2‐yl)ethanol in the aqueous medium, which empowered the synthesis of a precious chiral intermediary process to be converted into a sophisticated molecule for drug production.     

LOV‐domain photoreceptor,encoded in a genomic island,attenuates the virulence of Pseudomonas syringae in light‐exposed Arabidopsis leaves

In Arabidopsis thaliana, light signals modulate the defences against bacteria. Here we show that light perceived by the LOV domain‐regulated two‐component system (Pst–Lov) of Pseudomonas syringae pv. tomato DC3000 (Pst DC3000) modulates virulence against A. thaliana. Bioinformatic analysis and the existence of an episomal circular intermediate indicate that the locus encoding Pst–Lov is present in an active genomic island acquired by horizontal transfer. Strains mutated at Pst–Lov showed enhanced growth on minimal medium and in leaves of A. thaliana exposed to light, but not in leaves incubated in darkness or buried in the soil. Pst–Lov repressed the expression of principal and alternative sigma factor genes and their downstream targets linked to bacterial growth, virulence and quorum sensing, in a strictly light‐dependent manner. We propose that the function of Pst–Lov is to distinguish between soil (dark) and leaf (light) environments, attenuating the damage caused to host tissues while releasing growth out of the host. Therefore, in addition to its direct actions via photosynthesis and plant sensory receptors, light may affect plants indirectly via the sensory receptors of bacterial pathogens.     

Insulin receptor kinase‐independent signaling via tyrosine phosphorylation of phosphatase PHLPP1

Most insulin responses correlate well with insulin receptor (IR) Tyr kinase activation; however, critical exceptions to this concept have been presented. Specific IR mutants and stimulatory IR antibodies demonstrate a lack of correlation between IR kinase activity and specific insulin responses in numerous independent studies. IR conformation changes in response to insulin observed with various IR antibodies define an IR kinase‐independent signal that alters the C‐terminus. IR‐related receptors in lower eukaryotes that lack a Tyr kinase point to an alternative mechanism of IR signaling earlier in evolution. However, the implied IR kinase‐independent signaling mechanism remained obscure at the molecular level. Here we begin to define the molecular basis of an IR‐dependent but IR kinase‐independent insulin signal that is equally transmitted by a kinase‐inactive mutant IR. This insulin signal results in Tyr phosphorylation and catalytic activation of phosphatase PHLPP1 via a PI 3‐kinase‐independent, wortmannin‐insensitive signaling pathway. Dimerized SH2B1/PSM is a critical activator of the IR kinase and the resulting established insulin signal. In contrast it is an inhibitor of the IR kinase‐independent insulin signal and disruption of SH2B1/PSM dimer binding to IR potentiates this signal. Dephosphorylation of Akt2 by PHLPP1 provides an alternative, SH2B1/PSM‐regulated insulin‐signaling pathway from IR to Akt2 of opposite polarity and distinct from the established PI 3‐kinase‐dependent signaling pathway via IRS proteins. In combination, both pathways should allow the opposing regulation of Akt2 activity at two phosphorylation sites to specifically define the insulin signal in the background of interfering Akt‐regulating signals, such as those controlling cell proliferation and survival. J. Cell. Biochem. 107: 65–75, 2009. © 2009 Wiley‐Liss, Inc.     

Enhanced diastereoselective synthesis of t‐Butyl 6‐cyano‐(3R,5R)‐dihydroxyhexanoate by using aldo‐keto reductase and glucose dehydrogenase co‐producing engineered Escherichia coli

t‐Butyl 6‐cyano‐(3R,5R)‐dihydroxyhexanoate ((3R,5R)‐ 2 ) is a key chiral diol precursor of atorvastatin calcium (Lipitor®). We have constructed a Kluyveromyces lactis aldo‐keto reductase mutant KlAKR‐Y295W/W296L (KlAKRm) by rational design in previous research, which displayed high activity and excellent diastereoselectivity (de_p > 99.5%) toward t‐butyl 6‐cyano‐(5R)‐hydroxy‐3‐oxohexanoate ((5R)‐ 1 ). To realize in situ cofactor regeneration, a robust KlAKRm and Exiguobacterium sibiricum glucose dehydrogenase (EsGDH) co‐producer E. coli BL 21(DE3) pETDuet‐esgdh (MCS1)/pET‐28b (+)‐klakrm was constructed in this work. Under the optimized conditions, AKR and GDH activities of E. coli BL 21(DE3) pETDuet‐esgdh (MCS1)/pET‐28b (+)‐klakrm peaked at 249.9 U/g DCW (dry cellular weight) and 29100 U/g DCW, respectively. It completely converted (5R)‐ 1 at substrate loading size of up to 60.0 g/L (5R)‐ 1 in the absence of exogenous NADH, which was one‐fifth higher than that of the separately prepared KlAKRm and EsGDH under the same conditions. In this manner, a biocatalytic process for (3R,5R)‐ 2 with productivity of 243.2 kg/m³ d was developed. Compared with the combination of separate expressed KlAKRm with EsGDH, co‐expression of KlAKRm and EsGDH has the advantages of alleviating cell cultivation burden and elevating substrate load. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1235–1242, 2017     

Zingerone induced caspase‐dependent apoptosis in MCF‐7 cells and prevents 7,12‐dimethylbenz(a)anthracene‐induced mammary carcinogenesis in experimental rats

Breast cancer is a prevalent of tumoregenesis in women and reports for the maximum mortality and morbidity in the global. Ginger (Zingiber officinale) is the mainly widespread spice and herbal remedies used in the world. Since antique periods, ginger has been used in Greece, India and China for the curing of upset stomach, nausea, diarrhea, colds, and headaches. The current work was planned to explore the anticancer properties of zingerone (ZO) toward 7,12‐dimethylbenz(a)anthracene (DMBA)‐treated mammary carcinogenesis in Sprague‐Dawley (SD) rats and MCF‐7 mammary cancer cells. The mammary carcinogenesis was produced through a single dosage of DMBA (20 mg/kg bwt) mixed in soya oil (1 mL) administrated intragastrically with a gavage. We found improved concentrations of lipid peroxidation (LOOH and TBARS), carcinoembryonic antigen, lowered levels of enzymatic (CAT, GPx, and SOD), and nonenzymatic (vitamin E, GSH, and vitamin C) antioxidant in mammary tissues and plasma of DMBA‐induced cancer bearing animals. Moreover, augmented concentrations of phase I (Cyt‐b₅ and CYP₄₅₀) and reduced levels of phase II (GR and GST) detoxification microsomal proteins in mammary tissues were noticed. ZO administrations significantly reverted back to all these parameters in this way, showing efficient of anticancer effect. Furthermore, our in vitro study also supported the anticancer effect of the treatment of ZO were noticed loss of cell viability, improved reactive oxygen species formation, and reduced MMP. Furthermore, the status of apoptosis proteins such as Bcl‐2, Bax, and Bid expressions was determined by using Western blot analysis techniques. Overall, these results proposed the anticancer effect of ZO toward DMBA‐induced mammary cancer in SD animals and Michigan cancer foundation‐7 mammary cancer cells.     

Solution structure of the cysteine‐rich domain in Fn14, a member of the tumor necrosis factor receptor superfamily

Fn14 is the smallest member of the tumor necrosis factor (TNF) receptor superfamily, and specifically binds to its ligand, TWEAK (TNF‐like weak inducer of apoptosis), which is a member of the TNF superfamily. The receptor‐ligand recognition between Fn14 and TWEAK induces a variety of cellular processes for tissue remodeling and is also involved in the pathogenesis of some human diseases, such as cancer, chronic autoimmune diseases, and acute ischaemic stroke. The extracellular ligand‐binding region of Fn14 is composed of 53 amino acid residues and forms a single, cysteine‐rich domain (CRD). In this study, we determined the solution structure of the Fn14 CRD (Glu28‐Ala70) by heteronuclear NMR, with a ¹³C‐/¹⁵N‐labeled sample. The tertiary structure of the CRD comprises a β‐sheet with two strands, followed by a 3₁₀ helix and a C‐terminal α‐helix, and is stabilized by three disulfide bonds connecting Cys36‐Cys49, Cys52‐Cys67, and Cys55‐Cys64. Comparison of the disulfide bond connectivities and the tertiary structures with those of other CRDs revealed that the Fn14 CRD is similar to the fourth CRD of TNF receptor 1 (A1‐C2 module type), but not to the CRD of B‐cell maturation antigen and the second CRD of transmembrane activator and CAML (calcium modulator and cyclophilin ligand) interactor (A1‐D2 module type). This is the first structural report about the A1‐C2 type CRD that could bind to the known target.     

The chloroplast NADPH thioredoxin reductase C,NTRC, controls non‐photochemical quenching of light energy and photosynthetic electron transport in Arabidopsis

High irradiances may lead to photooxidative stress in plants, and non‐photochemical quenching (NPQ) contributes to protection against excess excitation. One of the NPQ mechanisms, qE, involves thermal dissipation of the light energy captured. Importantly, plants need to tune down qE under light‐limiting conditions for efficient utilization of the available quanta. Considering the possible redox control of responses to excess light implying enzymes, such as thioredoxins, we have studied the role of the NADPH thioredoxin reductase C (NTRC). Whereas Arabidopsis thaliana plants lacking NTRC tolerate high light intensities, these plants display drastically elevated qE, have larger trans‐thylakoid ΔpH and have 10‐fold higher zeaxanthin levels under low and medium light intensities, leading to extremely low linear electron transport rates. To test the impact of the high qE on plant growth, we generated an ntrc–psbs double‐knockout mutant, which is devoid of qE. This double mutant grows faster than the ntrc mutant and has a higher chlorophyll content. The photosystem II activity is partially restored in the ntrc–psbs mutant, and linear electron transport rates under low and medium light intensities are twice as high as compared with plants lacking ntrc alone. These data uncover a new role for NTRC in the control of photosynthetic yield.     

Resonance light scattering spectrometric study of poly diallyldimethylammonium chloride–antibody–antigen system

The interaction of antigen (Ag) and antibody (Ab) with poly diallyldimethylammonium chloride (PDDA) in aqueous solutions has been studied by optical absorption and resonance light‐scattering (RLS) spectroscopies. The formation of the three‐component‐complex is due to aggregates of Ab or Ag with PDDA by electrostatic interaction and aggregates of Ab with Ag by immunoreaction. The influences of some experimental factors, including incubation time, pH value, concentration of PDDA and concentration of Ab, on the aggregation process have also been studied. A linear relationship between the concentration of Ag and the RLS intensity was found. Under the optimal conditions, for a given concentration of Ab (4.6 µg/mL), the enhancement of RLS intensity is in proportion to the concentration of Ag in the range 0.03–0.83 µg/mL. The RLS could, in combination with immunoassay, be a rapid and sensitive detection method for Ag. Copyright © 2009 John Wiley & Sons, Ltd.