Purification and characterization of a lectin from the beetle, Allomyrina dichotoma

A lectin was purified from the hemolymph of Allomyrina dichotoma larvae by affinity chromatography on acid-treated Sepharose 4B. The purified lectin showed two protein bands on polyacrylamide gel electrophoresis. These two lectin bands (allo A-I and -II) were separated by DEAE-Cellulofine column chromatography. By gel filtration on Sephadex G-100, the molecular weights of allo A-I and -II were estimated to be 65,000 and 66,500, respectively. On the other hand, by SDS-polyacrylamide gel electrophoresis after cross-linking of subunits with glutaraldehyde, they are estimated to be 38,000 and 39,000, respectively. On SDS-polyacrylamide gel electrophoresis, it was proved that both allo A-I and -II lectin consisted of two subunits, respectively. The molecular weights were 17,500 and 20,000 for allo A-I, and 19,000 and 20,000 for allo A-II. The isoelectric points of allo A-I and -II were estimated to be 6.4 and 5.9, respectively. On double immunodiffusion, allo A-I and -II gave single precipitin lines, which fused completely with each other, against the antibody to crude allo A. The hemagglutinating activity of allo A-I and -II was inhibited only by beta-linked D-galactose such as lactose and lactulose.     

Carbohydrate binding specificity of a beetle (Allomyrina dichotoma) lectin

Carbohydrate binding specificity of a lectin, allo A, isolated from a beetle (Allomyrina dichotoma), was investigated by means of lectin affinity chromatography. Sialylated complex-type and hybrid-type oligosaccharides/glycopeptides, and sialyllactose were retained by the column, whereas desialylated ones were retarded but not retained by the column. The association constants of allo A for biantennary oligosaccharides from human serum transferrin, determined by frontal analysis, were 8.0 X 10(5) M-1, 4.5 X 10(5) M-1, and 2.5 X 10(5) M-1 for disialo-, monosialo-, and asialo-oligosaccharides, respectively. Removal of the beta-galactose residues markedly reduced the association constant to 3.5 X 10(3) M-1. Furthermore, allo A was found to have no affinity for mucin-type glycopeptides carrying the sialylated Gal beta 1----3 GalNAc sugar sequence (Ka: 3.5 X 10(3) M-1). The results of this study indicated that allo A strongly binds to the trisaccharide structure, NeuAc alpha 2-3(6)Gal-beta 1-4GlcNAc, and that its binding potency is affected by the inner core structures of oligosaccharides and glycopeptides, because the presence of a bisecting N-acetyl-glucosamine residue and an alpha-fucose residue linked to the innermost N-acetylglucosamine residue reduced the association constants for oligosaccharides and glycopeptides.     

Affinity chromatography of human serum proteins using immobilized lectin from Allomyrina dichotoma

The beta-D-galactoside-specific lectin from Allomyrina dichotoma reacts with serum proteins which contain the corresponding carbohydrate moieties. By affinity chromatography of human serum using the insolubilized lectin coupled to Sepharose, it is possible to fractionate human serum proteins in 2 groups: those which react with the lectin (alpha 1-acid glycoprotein, haptoglobin, etc.) and those which do not (albumin, Gc-globulin, etc.). IgG is the only serum protein that can be found in both groups.     

Exosome isolation from hemolymph of Korean rhinoceros beetle,Allomyrina dichotoma (Coleoptera: Scarabaeidae)

Exosomes are 30–150 nm vesicles that are secreted from a range of cells. Recently, exosomes have been the subject of considerable research because there is mounting awareness of their diverse functions, including a role in cell–cell communication and presenting pathogens for immune responses. Exosomes contain diverse nucleic acid and protein cargos, derived not only from the organism but also from pathogens, making them suitable for use in disease diagnosis. The Korean rhinoceros beetle, Allomyrina dichotoma (Coleoptera: Scarabaeidae), is commercially reared in Korea for the pet trade and is used in traditional medicine for liver‐related diseases. However, several insect diseases caused by bacteria, fungi and viruses have been reported in A. dichotoma mass‐rearing facilities. Identifying these diseases with accuracy and in a timely manner is of paramount importance. Such diagnosis can be accomplished by identifying the nucleic acid or amino acid fragments from these disease‐causing pathogens in the exosome of A. dichotoma. We isolated exosomes from the hemolymph of A. dichotoma and used them to analyze exosome RNA and proteins. We confirmed the isolation of exosomes through RNA profiling, protein analysis and Western blotting. Our research established a solid foundation for using insect exosome protein and RNA analyses for the accurate diagnosis of insect diseases. To our knowledge, this is the first report of exosome isolation from insect hemolymph.     

Size dependent predatory pressure in the Japanese horned beetle,Allomyrina dichotoma L. (Coleoptera; Scarabaeidae)

We surveyed male survival and reproductive performances associated with dimorphism in the Japanese horned beetleAllomyrina dichotoma L. in a secondary forest in 1996. Morphological comparisons between living marked and prey individuals indicated that the larger horned males suffered higher predatory pressure than the smaller ones. The dominant predators of the beetles were suspected to be 2 crow species. The small-horned males showed lower recapture rates than the large-horned ones. This suggested that the former was more sensitive to disturbance, and/or dispersed more than the latter. Fighting behavior was rarely seen because of the low population density of the beetles in the study area. These results suggested that the large-horned males suffer not only the injury risk of intrasexual competition but also more predatory risk than the small-horned ones.     

Mate securing tactics and the cost of fighting in the Japanese horned beetle,Allomyrina dichotoma L. (Scarabaeidae)

Males of the horned beetleAllomyrina dichotoma L. show a bimodal frequency distribution with respect to horn size. The 2 morphs distinguished by this criteria showed different mate-securing tactics. Major males fought for possession of areas on oak trees that exuded sap. Fights escalated through a series of stereotyped encounters before entering the potentially damaging phase of close-quarter combat when the largest males in particular risked serious damage. Minor males, on the other hand, were never observed to fight with conspecific males, but retreated after making contact with them. Minor males arrived at sap sites earlier in the diurnal cycle than major males and so avoided them temporally as well as behaviourly. Minors appeared to be relatively as successful at gaining copulations as majors, but did so earlier in the diurnal cycle. Since females showed a slight tendency to remate on the same night, minors may lose fertilization opportunities if last male sperm precedence is high. Actual sperm precedence values are not know so the reproductive payoffs for the 2 morphs could not be assessed.     

A novel, sensitive, and specific assay for abasic sites, the most commonly produced DNA lesion.

Free radicals produce a wide spectrum of damages; among these are DNA base damages and abasic (AP) sites. Although several methods have been used to detect and quantify AP sites, they either are relatively laborious or require the use of radioactivity. A novel reagent for detecting abasic sites in DNA was prepared by reacting O-(carboxymethyl)hydroxylamine with biotin hydrazide in the presence of carbodiimide. This reagent, called Aldehyde Reactive Probe (ARP), specifically tagged AP sites in DNA with biotin residues. The number of biotin-tagged AP sites was then determined colorimetrically by an ELISA-like assay using avidin/biotin complex conjugated to horseradish peroxidase as the indicator enzyme. With heat/acid-depurinated calf thymus or bacteriophage f1 DNA, ARP detected femtomoles of AP sites in DNA. Using this assay, DNA damages generated in calf thymus, phi X174 RF, and f1 single-stranded DNA, X-irradiated in phosphate buffer, were easily detectable at 10 rad (0.1 Gy). Furthermore, ARP sites were detectable in DNA isolated from heat-inactivated X-irradiated (10 Gy) and methyl methanesulfonate (MMS)-treated (5 microM) Escherichia coli cells. The rate of production of ARP sites was proportional to the X-ray dose as well as to the concentration of MMS. Thus, the sensitivity and simplicity of the ARP assay should provide a potentially powerful method for the quantitation of AP sites or other DNA lesions containing an aldehyde group.     

The first report on the characterization of a virus isolated from Allomyrina dichotoma in the Republic of Korea

This report reveals the structure of a virus extracted from the Korean horn beetle Allomyrina dichotoma. The purified virus particle was 100% identical to Allomyrina virus lef‐8 sequence registered as KM_233709.1. The structure of this virus was confirmed to be closely related to that of the Nudiviridae family, and it was rod shaped and enveloped, and observed to be of approximately the mean length of a single viral nucleocapsid of 200–210 nm and mean diameter of 100–110 nm. These results provide an insight into the structural characteristics of the Nudiviridae family that can be used for nudiviral identification.     

A sensitive, specific assay for tissue collagenase using telopeptide-free [3H]acetylated collagen

Collagenase is assayed by incubation with soluble, telopeptide-free collagen extracted from rat skin and labeled with [2-3H]acetic anhydride. Collagen is cleaved by collagenase and the resulting fragments are digested with trypsin and chymotrypsin. Undigested collagen is recovered by precipitation with trichloroacetic acid, collected on glass-fiber filters, and quantitated by liquid scintillation spectrometry. This procedure combines features of the Cawston and Barrett (T.E. Cawston and A.J. Barrett, 1979, Anal. Biochem. 99, 340-345) and the Ryh?nen et al. (L. Ryh?nen et al., 1982, Collagen Rel. Res. 2, 117-130) methods. The first method provides a simple way to prepare large quantities of uniform substrate, while the second increases the specificity of the assay by removal of the labeled telopeptides. The assay is reproducible and linear with time and enzyme concentration. It is approximately 10X more sensitive than the Cawston and Barrett method and can readily detect 1-8 mU collagenase (1 unit equals 1 microgram collagen cleaved/min at 30 degrees C). The substrate is resistant to elastase, trypsin, and chymotrypsin and is completely degraded by bacterial collagenase. Collagenase is the only tissue metalloprotease found, to date, that cleaves the substrate.     

Thymic shared antigen-1. A novel thymocyte marker discriminating immature from mature thymocyte subsets.

In a previous study, we raised a mAb (MTS 35) reacting with a plasma membrane Ag expressed on both cortical thymocytes and a subset of thymic medullary epithelial cells. In view of the shared expression of this molecule, we have defined it as thymic shared Ag-1 (TSA-1). Considering its selective reactivity with cortical, but not medullary thymocytes, the relevance of TSA-1 as a marker of immature T cells was investigated in detail in this study, using multicolor flow cytometric analysis. TSA-1 was found on all immature thymocyte subsets (CD3-4-8-, CD3-4+8-, CD3-4-8+, CD3-4+8+, CD3low4+8+). Conversely, CD3high4+8- and CD3high4-8+ thymocytes, early thymic migrants and peripheral T cells were TSA-1-. More refined gating and analysis of the transitional CD3intermediate/high4+8+ thymocytes, proposed candidates for negative selection, demonstrated that approximately one half were TSA-1-. In fact, there was a directly inverse relationship between TSA-1 and CD3 expression on thymocytes. In the periphery, TSA-1 was detected on B lymphocytes. TSA-1 is PI-linked and has a molecular mass of 17 kDa nonreduced, or 12 to 13 kDa reduced. Through cross-correlation analysis, this molecule was distinct from H-2K, PNA-R, CD5, CD11a/18, Thy-1, HSA, Ly6A/E, Ly6C, ThB, CD25, CD44. Hence TSA-1 appears to be a unique marker which exquisitely separates mature from immature thymocytes.     

The effect of a seasonal time constraint on development time, body size, condition, and morph determination in the horned beetle Allomyrina dichotoma L. (Coleoptera: Scarabaeidae)

Abstract.  1. In horned beetles selection favours males that adjust their investment in horn development in relation to cues that predict adult body size. Here it is shown that in the Japanese horned beetle, Allomyrina dichotoma . There is a significant discontinuity in the horn length body size allometry. This can be described as a linear relationship that is shifted towards an increased horn length to body length ratio in males with horns longer than 16 mm.
2. Larval nutrition explains morph determination in A. dichotoma . However, unlike other species, variation in larval nutrition was the result of a seasonal time constraint that limits the time available for feeding prior to the onset of winter diapause.
3. Even when eggs were reared with an ad libitum food supply, minor morphs were still observed. Individuals that were oviposited later in the season had less time to feed, shorter development times, eclosed as smaller individuals and, in the case of males, were more likely to be hornless. Major morphs, minor morphs, and females all reduced their body size in response to seasonal time constraints in the same way. However, males that were laid later in the season had faster development times than females laid at the same time, but showed no reduction in their size relative to females, suggesting seasonal time constraints increase growth rates in males but not in females.
4. No evidence was found that seasonal time constraints resulted in a reduction of size-corrected fat reserves at eclosion, or that minor morphs gained any developmental advantage by reducing investment in horn length.     

A sensitive assay for ABCA1-mediated cholesterol efflux using BODIPY-cholesterol

Studies have shown a negative association between cellular cholesterol efflux and coronary artery disease (CAD). Standard protocol for quantitating cholesterol efflux involves labeling cells with [(3)H]cholesterol and measuring release of the labeled sterol. Using [(3)H]cholesterol is not ideal for the development of a high-throughput assay to screen large numbers of serum as would be required in studying the link between efflux and CAD. We compared efflux using a fluorescent sterol (boron dipyrromethene difluoride linked to sterol carbon-24, BODIPY-cholesterol) with that of [(3)H]cholesterol in J774 macrophages. Fractional efflux of BODIPY-cholesterol was significantly higher than that of [(3)H]cholesterol when apo A-I, HDL(3), or 2% apoB-depleted human serum were used as acceptors. BODIPY-cholesterol efflux correlated significantly with [(3)H]cholesterol efflux (p < 0.0001) when apoB-depleted sera were used. The BODIPY-cholesterol efflux correlated significantly with preβ-1 (r(2) = 0.6) but not with total HDL-cholesterol. Reproducibility of the BODIPY-cholesterol efflux assay was excellent between weeks (r(2) = 0.98, inter-assay CV = 3.31%). These studies demonstrate that BODIPY-cholesterol provides an efficient measurement of efflux compared with [(3)H]cholesterol and is a sensitive probe for ABCA1-mediated efflux. The increased sensitivity of BODIPY-cholesterol assay coupled with the simplicity of measuring fluorescence results in a sensitive, high-throughput assay that can screen large numbers of sera, and thus establish the relationship between cholesterol efflux and atherosclerosis.     

A simple, sensitive, and specific assay for free choline in plasma.

This paper describes a sensitive and specific enzymatic-radioisotopic method for determining plasma choline. Assays may be performed without prior extraction of the tissue. Plasma is first heated to destroy enzymes that would otherwise produce free choline from that which is normally bound. The free choline in plasma is then converted to phosphorylcholine [³²P], in the presence of ATP-γ-³²P, in a reaction catalyzed by choline kinase. Phosphorylcholine [³²P], isolated by ion-exchange chromatography, is measured as an index of the concentration of free choline. The concentration of plasma choline in man and in several species of laboratory animals was determined, and found to range from 5.5 nmoles/ml in dogs to 15.4 nmoles/ml in guinea pigs. The concentration of free choline in plasma of adult rats raised on a choline-deficient diet was half that of littermate controls raised on a control diet supplemented with free choline.     

A simple, sensitive and specific radioreceptor assay for beta-adrenergic antagonist drugs.

A radioreceptor assay for β-adrenergic blocking drugs described here is based on the ability of the blood content of drugs to compete with the binding of ³H-dihydroalprenolol (³H-DHA) to β-adrenergic receptors in calf cerebellar membranes. Plasma protein greatly inhibits the binding of ³H-DHA to β-receptors by binding the ³H-DHA so it is unavailable to the β-receptors. As little as 0.01 ml of human plasma in a final volume of 1 ml reduces binding 25–45%. Assays conducted on plasma dialysates can be performed without such inhibition. The radioreceptor assay is simple to perform as 100 samples can be processed in a morning. It is sensitive, detecting low nanomolar concentrations of plasma propranolol, and it is specific. No drugs clinically employed other than β-blocking agents compete for β-receptor binding. The assay detects all pharmacologically active metabolites of β-blocking drugs as well as the parent drug.     

Rapid and sensitive detection of retrovirus entry by using a novel luciferase-based content-mixing assay

We describe a novel assay that permits measurement of entry of murine leukemia virus and pseudotypes with greater sensitivity and more rapidly than previously possible. To achieve this, we encapsulated a sensitive reporter enzyme, luciferase, directly into fully infectious, intact viral particles. The enzyme is specifically targeted to the viral lumen, as a C-terminal fusion on the viral envelope protein. Only when the incorporated luciferase is released from the viral lumen and gains access to its substrates is light emitted and readily detected. When cells are perfused with luciferin, quantitative measurements of entry can be made in real time on live cells. Uniquely, the amount of cell-bound virus can be determined in the same assay by addition of detergent to expose the luciferase. We demonstrate that virus carrying a mutation in the fusion peptide binds normally to cells but is unable to infect them and gives no entry signal. Using this assay, we show that inhibitors of endosomal acidification inhibit signal from vesicular stomatitis virus pseudotypes but not murine leukemia virus, consistent with a pH-independent mode of entry for the latter virus. Additionally, the fusion kinetics are rapid, with a half-life of 25 min after a delay of 10 to 15 min. The future use of this assay will permit a detailed examination of the entry mechanism of viruses and provide a convenient platform to discover novel entry inhibitors. The design also permits packaging of potential therapeutic protein cargoes into functional virus particles and their specific delivery to cellular targets.     

A direct, highly sensitive fluorometric assay for a microsomal cytochrome P450-mediated O-demethylation using a novel coumarin analog as substrate

A highly sensitive fluorometric assay for the determination of monooxygenase activity in liver microsomes is described. The assay is based on the use of 3-chloro-7-methoxy-4-methylcoumarin which is demethylated to 3-chloro-7-hydroxy-4-methylcoumarin. The rate of formation of 3-chloro-7-hydroxy-4-methylcoumarin was recorded as an increase of fluorescence (lambdaA = 380 nm, lambdaF = 480 nm) with time. When 3-chloro-7-methoxy-4-methylcoumarin was incubated in the presence of MgCl2 and NADPH with rat liver microsomes, a continuous increase of the fluorescence could be measured. The reaction proceeded linearly for about 10 min and at least up to a concentration of 0.1 mg/ml of microsomal protein. Besides 3-chloro-7-hydroxy-4-methylcoumarin a hydroxylated derivative of the substrate was formed as a second metabolite during the incubation. Using an excitation wavelength of 380 nm and a fluorescence/emission wavelength of 480 nm, the fluorescence of this substance (lambdaA = 338 nm, lambdaF = 422 nm) amounted only to about 1% of the fluorescence of the main product. The use of 3-chloro-7-methoxy-4-methylcoumarin as substrate enables the fluorometric determination of the O-dealkylation activity of a cytochrome P450-dependent monooxygenase system in rat liver which is inducible by phenobarbital but not by 3-methylcholanthrene.     

A sensitive and specific enzyme-based assay detecting HIV-1 virion fusion in primary T lymphocytes

As an early event in the viral life cycle, the entry of enveloped viruses into target cells has received considerable attention. Viral fusion to cellular targets has been studied principally with fusion assays in which cells engineered to express the viral envelope are cultured with the target cells. These assays yield valuable information but do not fully recapitulate all of the variables governing the fusion of actual virions to their cellular targets. The virion membrane and the plasma membrane, for example, differ strikingly in their lipid and protein compositions. Two virion-based fusion assays have been described. One is based on the redistribution of a self-quenching fluorophore, whereas the second depends on photosensitized activation of a hydrophobic probe by a fluorescent lipid loaded into the target membrane. These assays are complex and have not been adapted to study fusion in complex cell populations. We have developed a simple, rapid assay allowing the detection of HIV-1 virion fusion to biologically relevant target cells, including primary CD4(+) T lymphocytes. It is based on the incorporation of beta-lactamase-Vpr chimeric proteins (BlaM-Vpr) into HIV-1 virions and their subsequent delivery into the cytoplasm of target cells as a result of virion fusion. This transfer is then detected by enzymatic cleavage of the CCF2 dye, a fluorescent substrate of beta-lactamase (BlaM), loaded in the target cells. BlaM cleaves the beta-lactam ring in CCF2, changing its fluorescence emission spectrum from green (520 nm) to blue (447 nm) and thereby allowing fusion to be detected by fluorescence microscopy, flow cytometry, or UV photometry.