Effect of different proteins on reabsorption of yellow fluorescent protein in the kidney of the brown frog <Emphasis Type="Italic">Rana temporaria</Emphasis>

Protein reabsorption in the proximal tubules (PT) of the frog kidney was studied by immunohistochemistry, fluorescent and confocal microscopy. The yellow fluorescent protein (YFP) was introduced in combination with other proteins. Reabsorption of YFP co-injected with lysozyme or the green fluorescent protein (GFP) was indistinguishable from that of YFP injection alone. Preliminary lysozyme injection did not change YFP absorption in contrast to YFP uptake reduced after GFP pretreatment. Lysozyme loading for 4 days led to a significant reduction in YFP absorption. The results show that receptor-mediated endocytosis in the frog kidney depends on the molecular nature of absorbable ligands, conditions of their competitive absorption and lysosomal accumulation in PT epithelial cells.     

Receptor-Mediated Endocytosis of Lysozyme in Renal Proximal Tubules of the Frog Rana Temporaria

The mechanism of protein reabsorption in the kidney of lower vertebrates remains insufficiently investigated in spite of raising interest to the amphibian and fish kidneys as a useful model for physiological and pathophysiological examinations. In the present study, we examined the renal tubular uptake and the internalization rote of lysozyme after its intravenous injection in the wintering frog Rana temporaria using immunohisto- and immunocytochemistry and specific markers for some endocytic compartments. The distinct expression of megalin and cubilin in the proximal tubule cells of lysozyme-injected frogs was revealed whereas kidney tissue of control animals showed no positive immunoreactivity. Lysozyme was detected in the apical endocytic compartment of the tubular cells and colocalized with clathrin 10 min after injection. After 20 min, lysozyme was located in the subapical compartment negative to clathrin (endo-somes), and intracellular trafficking of lysozyme was coincided with the distribution of megalin and cubilin. However, internalized protein was retained in the endosomes and did not reach lysosomes within 30 min after treatment that may indicate the inhibition of intra-cellular trafficking in hibernating frogs. For the first time, we provided the evidence that lysozyme is filtered through the glomeruli and absorbed by receptor-mediated clathrin-dependent endocytosis in the frog proximal tubule cells. Thus, the protein uptake in the amphibian mesonephros is mediated by megalin and cubilin that confirms a critical role of endocytic receptors in the renal reabsorption of proteins in amphibians as in mammals.Key words: Endocytic receptor, frog, kidney, lysozyme, protein uptake, proximal tubule     

Tubular GFP uptake pattern in the rat and frog kidneys

The renal tubular uptake of green fluorescent protein (GFP) after its bolus intravenous injection was studied in both frogs and rats. GFP fluorescence in the proximal tubule (PT) was revealed by fluorescent and confocal microscopy. Granular GFP fluorescence was observed nearby in the apical membrane of PT cells featuring distribution over the cytoplasm. GFP was internalized into endosomes and lysosomes as determined by immunocytochemistry in frogs. The tubular uptake and accumulation of GFP were dose- and time-dependent in both rats and frogs. Intralymphatic sac injection of arginine vasotocin (AVT) decreased the uptake of GFP in hydrated frogs. A high negative correlation between the AVT dose and the uptake of GFP was revealed. The effect of AVT was inhibited by a V(1)-receptor antagonist. A noted decrease in the average number of fluorescent PT profiles per kidney section and their irregular distribution after AVT injections suggest that not all of the glomeruli or preglomerular vessels are equally responsive to AVT. GFP may serve as a good marker for tubular uptake and intracellular traffic in the amphibian kidney for use in in vivo studies.     

Renal filtration and reabsorption of GFP in <Emphasis Type="Italic">Rana temporaria</Emphasis>: Effect of arginine-vasotocin

The renal tubular uptake of green fluorescent protein (GFP) in frog Rana temporaria was studied by laser confocal microscopy. The specific green fluorescence was revealed in cells of proximal tubules 30 min after intravenous GFP injection. The GFP fluorescence was located predominantly in the apical part of the cytoplasm in the form of intensively fluorescing vesicles. The GFP injections increased dose-dependently the GFP tubular uptake. This was confirmed by the quantitative assessment of intensity of the specific fluorescence, its relative vesicular density, and by correlation analysis. Preliminary administration of arginine-vasotocin into the dorsal lymphatic sack decreased significantly the GFP absorption. The effect of arginine-vasotocin was inhibited by pretreatment with an antagonist of vasopressin V_i-receptors. The results of this work together with literature data allow believing that a decrease in the GFP absorption in the frog kidney under effect of arginine-vasotocin is due to a fall of the AVT-dependent glomerular filtration rate and to a decrease in the input of protein into the lumen of tubules. The action of arginine-vasotocin seems to be mediated via the V_i-like receptors of preglomerular blood vessels.     

A glutamine/asparagine-rich fragment of Gln3, but not the full-length protein,aggregates in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

The amino acid sequence of protein Gln3 in yeast Saccharomyces cerevisiae has a region enriched with Gln (Q) and Asn (N) residues. In this study, we analyzed the effects of overexpression of Gln3 and its Q/N-rich fragment fused with yellow fluorescent protein (YFP). Being overexpressed, full-length Gln3-YFP does not form aggregates, inhibits vegetative growth, and demonstrates nuclear localization, while the Q/N-rich fragment (Gln3QN) fused with YFP forms aggregates that do not colocalize with the nucleus and do not affect growth of the cells. Although detergent-resistant aggregates of Gln3QN are formed in the absence of yeast prions, the aggregation of Gln3QN significantly increases in the presence of [PIN⁺] prion, while in the presence of two prions, [PSI⁺] and [PIN⁺], the percentage of cells with Gln3QN aggregates is significantly lower than in the strain bearing only [PIN⁺]. Data on colocalization demonstrate that this effect is mediated by interaction between Gln3QN aggregates and [PSI⁺] and [PIN⁺] prions.     

Sex-related differences in the handling of fluorescent ovalbumin by the proximal tubule of the rat kidney

Sex-dependent protein handling in the rat renal tubular system was studied both qualitatively and quantitatively using the method of direct fluorescent protein tracing. The protein tracer, fluorescent ovalbumin, was synthesized by conjugating hen ovalbumin with fluorescein isothiocyanate (FITC), and the fluorescence characteristics of fluoresceinthiocarbamyl (FTC)-ovalbumin conjugates with different degrees of labelling were studied. Heavily labelled tracer was intravenously injected into male and female rats, and both kidneys were perfused; the right kidney was then homogenized and used for quantitative fluorometric measurements, while the left kidney was perfusion fixed and prepared for fluorescence microscopy. The tubular reabsorption of fluorescent ovalbumin was studied 4 min and 10 min after the injection of different doses (1.4, 7.0 and 14.0 mg/kg body weight) of the tracer, and the tubular catabolism was investigated in animals killed 60 and 120 min after the injection. Fluorescence microscopy demonstrated that, in both sexes and regardless of the dose administered and the time after injection, specifically fluorescent protein or its degradation products was only present in the epithelial cells of the proximal tubule. With regard to sex-dependent differences in protein handling, fluorometry indicated that at 4 min (7.0 mg) and at 10 min (all doses) after injection, female animals had reabsorbed more fluorescent protein than males. With regard to the catabolic phase, both the fluorescence microscopy and the fluorometric results showed that the female rats had degraded the fluorescent tracer at a significantly higher rate than males. The results are discussed in connection with sex-dependent proteinuria in rats.     

Sex-related differences in the handling of fluorescent ovalbumin by the proximal tubule of the rat kidney

Summary Sex-dependent protein handling in the rat renal tubular system was studied both qualitatively and quantitatively using the method of direct fluorescent protein tracing. The protein tracer, fluorescent ovalbumin, was synthesized by conjugating hen ovalbumin with fluorescein isothiocyanate (FITC), and the fluorescence characteristics of fluores-ceinthiocarbamyl (FTC)-ovalbumin conjugates with different degrees of labelling were studied. Heavily labelled tracer was intravenously injected into male and female rats, and both kidneys were perfused; the right kidney was then homogenized and used for quantitative fluorometric measurements, while the left kidney was perfusion fixed and prepared for fluorescence mciroscopy. The tubular reabsorption of fluorescent ovalbumin was studied 4 min and 10 min after the injection of different doses (1.4, 7.0 and 14.0 mg/kg body weight) of the tracer, and the tubular catabolism was investigated in animals killed 60 and 120 min after the injection. Fluorescence microscopy demonstrated that, in both sexes and regardless of the dose administered and the time after injection, specifically fluorescent protein or its degradation products was only present in the epithelial cells of the proximal tubule. With regard to sex-dependent differences in protein handling, fluorometry indicated that at 4 min (7.0 mg) and at 10 min (all doses) after injection, female animals had reabsorbed more fluorescent protein than males. With regard to the catabolic phase, both the fluorescence microscopy and the fluorometric results showed that the female rats had degraded the fluorescent tracer at a significantly higher rate than males. The results are discussed in connection with sex-dependent proteinuria in rats.In honour of Prof. P. van DuijnSupported by the Deutsche Forschungsgemeinschaft (SFB 105)     

Dynamics of absorption of yellow fluorescent protein in intestine and reabsorption in kidney in rat postnatal ontogenesis

In experiments of the 5, 12 and 25-day old rat pups and adult rats in has been shown that after administration of yellow fluorescent protein (YFP) into stomach, its partial absorption in the non-degraded state in the small intestine takes place, with subsequent transport to kidney with blood flow and accumulation in cells of the proximal nephron segment. With age of rats, intensity of the intestinal YFP absorption decrease; the YFP accumulation in the kidney is more active in rats of the younger age groups than in adult animals. No accumulation of YFP in liver was revealed. The obtained data indicate an intensive absorption of YFP in the non-hydrolyzed form in the rat pup small intestine in early postnatal ontogenesis and an important role of kidney in protein metabolism and in proteolysis of exogenous proteins.     

Renal tubular sites of increased phosphate transport and NaPi-2 expression in the juvenile rat

To determine the tubular sites and mechanisms involved in enhanced renal phosphate (P(i)) reabsorption seen in the juvenile animal, renal micropuncture experiments were performed in acutely thyroparathyroidectomized adult (>14 wk old) and juvenile (4 wk old) male Wistar rats fed either a normal P(i) diet (NPD, 0.6% P(i)) or low P(i) diet (0.07% P(i)) for 2 days, in the presence and absence of parathyroid hormone (PTH). P(i) reabsorption was greater in proximal convoluted (PCT) and straight tubules (PST) of the juvenile compared with adult rats fed NPD, whether or not PTH was present. These findings were consistent with a greater P(i) uptake in brush-border membrane (BBM) vesicles from both superficial (SC) and outer juxtamedullary (JMC) cortices of juvenile animals. Western blot analysis revealed a 2- and 1.8-fold higher amount of NaPi-2 protein in the SC and JMC, respectively, in juvenile rats. Immunofluorescence microscopy also indicated that NaPi-2 protein expression was present in the proximal tubule (PT) BBM to a greater extent in juvenile rats. Dietary P(i) restriction in juvenile rats resulted in a significant increase in P(i) reabsorption in the PCT and PST segments. NaPi-2 expression in the PT BBM was also increased, as was the expression of intracellular NaPi-2 protein. These studies indicate that P(i) reabsorption in both the PCT and PST segments of the renal tubule contributes to the attenuated response to PTH in the normal juvenile animal. In addition, dietary P(i) restriction in the juvenile rat upregulates BBM NaPi-2 expression, which is associated with a further increase in proximal tubular P(i) reabsorption.     

Evaluation of the physiological activity of venom from the Eurasian water shrew <Emphasis Type="Italic">Neomys fodiens</Emphasis>

Background

Animal toxins can have medical and therapeutic applications. Principally, toxins produced by insects, arachnids, snakes and frogs have been characterized. Venomous mammals are rare, and their venoms have not been comprehensively investigated. Among shrews, only the venom of Blarina brevicauda has been analysed so far, and blarina toxin has been proven to be its main toxic component. It is assumed that Neomys fodiens employs its venom to hunt larger prey. However, the toxic profile, properties and mode of action of its venom are largely unknown. Therefore, we analysed the cardio-, myo- and neurotropic properties of N. fodiens venom and saliva of non-venomous Sorex araneus (control tests) in vitro in physiological bioassays carried out on two model organisms: beetles and frogs. For the first time, we fractionated N. fodiens venom and S. araneus saliva by performing chromatographic separation. Next, the properties of selected compounds were analysed in cardiotropic bioassays in the Tenebrio molitor heart.

Results

The venom of N. fodiens caused a high decrease in the conduction velocity of the frog sciatic nerve, as well as a significant decrease in the force of frog calf muscle contraction. We also recorded a significant decrease in the frog heart contractile activity. Most of the selected compounds from N. fodiens venom displayed a positive chronotropic effect on the beetle heart. However, one fraction caused a strong decrease in the T. molitor heart contractile activity coupled with a reversible cardiac arrest. We did not observe any responses of the insect heart and frog organs to the saliva of S. araneus. Preliminary mass spectrometry analysis revealed that calmodulin-like protein, thymosin β-10, hyaluronidase, lysozyme C and phospholipase A2 are present in the venom of N. fodiens, whereas thymosin β4, lysozyme C and β-defensin are present in S. araneus saliva.

Conclusion

Our results showed that N. fodiens venom has stronger paralytic properties and lower cardioinhibitory activity. Therefore, it is highly probable that N. fodiens might use its venom as a prey immobilizing agent. We also confirmed that S. araneus is not a venomous mammal because its saliva did not exhibit any toxic effects.

     

Cloning and expression analysis of c-type and g-type lysozymes in yellow catfish (<Emphasis Type="Italic">Pelteobagrus fulvidraco</Emphasis>)

Lysozymes have important roles in innate immune system. Here, a c-type and a g-type lysozyme were identified from yellow catfish (Pelteobagrus fulvidraco). The deduced amino acid sequences of both lysozymes were conserved in catalytic sites and structural features as compared to their counterparts from other species. It was interesting that the g-type lysozyme possessed a signal peptide. The c-type and g-type lysozymes had the highest identity 89.4 and 76.2 % with that from channel catfish respectively. Phylogenetic analysis showed that the two lysozymes had a closely relationship with that from channel catfish and Astyanax mexicanus. Lysozymes from one order could form more than one clade in the phylogenetic tree, which indicated the gene duplications in evolution. Expression analysis with real time quantitative PCR revealed that the two lysozyme genes were constitutively expressed in all the tested tissues. The highest expression of c-type lysozyme was observed in liver, followed by spleen, head kidney, and trunk kidney, while the g-type lysozyme had highest expression in intestine, followed by spleen, head kidney, and trunk kidney. The mRNA levels of both genes were all up-regulated after challenging with Aeromonas hydrophila. However, there were differences in tissues and time points when the mRNA levels reached its peak between the two lysozymes. It indicated the diversity in regulation mechanisms and detailed functions among lysozymes. Taking together, these results will benefit the understanding of yellow catfish lysozymes.     

Impairment of <Emphasis Type="Italic">Wnt11</Emphasis> function leads to kidney tubular abnormalities and secondary glomerular cystogenesis

Background

Wnt11 is a member of the Wnt family of secreted signals controlling the early steps in ureteric bud (UB) branching. Due to the reported lethality of Wnt11 knockout embryos in utero, its role in later mammalian kidney organogenesis remains open. The presence of Wnt11 in the emerging tubular system suggests that it may have certain roles later in the development of the epithelial ductal system.

Results

The Wnt11 knockout allele was backcrossed with the C57Bl6 strain for several generations to address possible differences in penetrance of the kidney phenotypes. Strikingly, around one third of the null mice with this inbred background survived to the postnatal stages. Many of them also reached adulthood, but urine and plasma analyses pointed out to compromised kidney function. Consistent with these data the tubules of the C57Bl6 Wnt11 ^?/? mice appeared to be enlarged, and the optical projection tomography indicated changes in tubular convolution. Moreover, the C57Bl6 Wnt11 ^?/? mice developed secondary glomerular cysts not observed in the controls. The failure of Wnt11 signaling reduced the expression of several genes implicated in kidney development, such as Wnt9b, Six2, Foxd1 and Hox10. Also Dvl2, an important PCP pathway component, was downregulated by more than 90 % due to Wnt11 deficiency in both the E16.5 and NB kidneys. Since all these genes take part in the control of UB, nephron and stromal progenitor cell differentiation, their disrupted expression may contribute to the observed anomalies in the kidney tubular system caused by Wnt11 deficiency.

Conclusions

The Wnt11 signal has roles at the later stages of kidney development, namely in coordinating the development of the tubular system. The C57Bl6 Wnt11 ^?/? mouse generated here provides a model for studying the mechanisms behind tubular anomalies and glomerular cyst formation.

     

High-level Recombinant Expression and Refolding of Lysozyme from the Marine Shrimp <Emphasis Type="Italic">Marsupenaeus japonicus</Emphasis>

Shrimp lysozyme is as an antibacterial enzyme that participates in the innate defense against the invasion of bacterial pathogens. In this study, the lysozyme gene from hemocytes of the shrimp Marsupenaeus japonicus was isolated and characterized. The M. japonicus lysozyme (MjLys) encodes a polypeptide of 158 amino acids (aa) that includes an 18 aa signal peptide. The gene fragment encoding the mature MjLys protein was subcloned into the expression vector pET-32a(+) and transformed into E. coli BL21(DE3)pLysS, and the protein was strongly expressed in insoluble inclusion bodies. Following extraction using urea, the denatured recombinant protein was refolded by on-column Ni²⁺ affinity chromatography or dialysis with a gradient of decreasing urea concentration. Approximately 50% of the recombinant MjLys was successfully refolded into monomeric protein using urea gradient dialysis, while 30% was salvaged using on-column refolding. Purified MjLys exhibited significant antibacterial activity against Gram-positive bacteria Micrococcus lysodeikticus and Staphylococcus aureus. This efficient over-expression and refolding method can provide the large quantities of biologically active protein required for further biochemical and structural studies and potential biotechnological applications.     

Characteristics and Gel Properties of Gelatin from Skin of Asian Bullfrog (<Emphasis Type="Italic">Rana tigerina</Emphasis>)

Characteristics and gel properties of gelatin from frog skin as influenced by extraction temperatures (45–75 °C) were investigated. Yield of gelatin increased as the extraction temperature increased (P < 0.05). All gelatins contained α- and β-chains as the predominant components and showed a high imino acid content (215 residues/1000 residues). Fourier transform infrared (FTIR) spectra indicated that all gelatin samples had major peaks in amide regions. Gelatin extracted at 55 °C exhibited the highest gel strength (P < 0.05), which was similar to that of commercial bovine gelatin (P > 0.05). Gelling and melting temperatures of frog skin gelatin were 23.47–24.87 and 33.22–34.66 °C, respectively. Gels became more yellowish with increasing extraction temperatures (P < 0.05). All gelatin gels were sponge or coral-like in structure but varied in patterns as visualized by scanning electron microscopy (SEM). Gelatin from frog skin could be used as a replacement for land animal counterpart.     

Ubiquitous distribution of fluorescent protein in muscles of four species and two subspecies of eel (genus <Emphasis Type="Italic">Anguilla</Emphasis>)

In this study, the localization of fluorescent protein (FP) was characterized in the muscles of four species and two subspecies of eels Anguilla anguilla, A. australis, A. bicolor bicolor (b.), A. bicolor pacifica (p.) and A. mossambica in addition to the previously reported A. japonica. The open reading frame of each eel FP was 417 bp encoding 139 amino acid residues. The deduced amino acid sequences among the four species and two subspecies exhibited 91.4–100% identity, and belonged to the fatty-acid-binding protein (FABP) family. The gene structure of eel FPs in A. japonica, A. anguilla, A. australis, A. bicolor b., A. bicolor p. and A. mossambica have four exons and three introns, and were common to that of FABP family. The apo eel FPs expressed by Escherichia coli with recombinant eel FP genes were analysed for the fluorescent properties in the presence of bilirubin. The excitation and emission spectra of holo eel FPs had the maximum wavelengths of 490–496 and 527–530 nm, respectively. The holo eel FPs indicated that the fluorescent intensities were stronger in A. japonica and A. bicolor than in A. mossambica, A. australis and A. anguilla. The comparison of amino acid sequences revealed two common substitutions in A. mossambica, A. australis and A. anguilla with weak fluorescent intensity.     

Variability of quantitative morphological traits of mosquito larvae in some species of the <Emphasis Type="Italic">Culex pipiens</Emphasis> complex (Diptera,Culicidae)

The variability of 13 larval morphological characteristics in the urban mosquito, Culex pipiens f. molestus and Culex torrentium, was investigated. These species, belonging to the Culex pipiens complex, are characterized by rapid ecological and evolutionary processes following the human-induced environmental changes. A comparison of 4 geographical larval populations of C. pipiens f. molestus and Culex torrentium revealed different rates of divergence of different morphological traits. In the older form in terms of evolutionary development, C. torrentium, the two local populations differed in the structures of the trophic morphological complex characterized by a higher functional trophic load, while the structures with a lower functional load did not differ between the populations. On the contrary, the populations of the younger form, C. pipiens f. molestus, which had recently colonized new urban sites, differed in the structures with a low functional trophic load (some structures of siphon and abdominal segment IX), whereas structures of the trophic morphological complex were similar in different populations. The possible mechanisms of these morphological transformations and their role in the evolution of mosquito larvae are discussed.     

A genetically encoded Förster resonance energy transfer biosensor for two-photon excitation microscopy

Pippi (phosphatidyl inositol phosphate indicator) is a biosensor based on the principle of FRET (Förster resonance energy transfer), which consists of a pair of fluorescent proteins, CFP (cyan fluorescent protein) and YFP (yellow fluorescent protein), the PH domain sandwiched between them, and K-Ras C-terminal sequence for plasma membrane localization. Due to marked cross-excitation of YFP with the conditions used to excite CFP, initial FRET images obtained by TPE (two-photon excitation) microscopy suffered from low signal-to-noise ratio, hampering the observation of lipids in three-dimensional structures. To solve this problem, YFP and CFP in the original Pippi-PI(3,4)P₂ was replaced by sREACh (super resonance energy accepting chromoprotein) and mTFP1 (monomeric teal fluorescent protein), respectively. The biosensor was also fused with an internal control protein, mKeima, where Keima/mTFP1 indicates the FRET efficiency, and indeed epidermal growth factor stimulation increased Keima/mTFP1 in HeLa cells. This biosensor successfully showed PI(3,4)P₂ accumulation to the lateral membrane in the MDCK cyst cultured in a three-dimensional environment. Furthermore, other FRET-based biosensors for PIP₃ distribution and for tyrosine kinase activity were developed based on this method, suggesting its broad application for visualizing signal transduction events with TPE microscopy.     

Calcium-dependent antibacterial activity of donkey’s milk against <Emphasis Type="Italic">Salmonella</Emphasis>

The aim of this study was to examine the antibacterial activity of raw donkey’s milk (DM) against Salmonella Enteritidis and Salmonella Typhimurium as well as to determine the dependence of this antibacterial activity on calcium, lysozyme and lactoferrin content. Antibacterial assays were conducted in DM artificially contaminated with these Salmonella serotypes and then incubated at 38 °C for 8 hours. Calcium concentration was found to have a strong influence on the antibacterial activity of the contaminated DM samples supplemented with CaCl₂ and EDTA. The antibacterial activity of DM against the tested Salmonella strains was observed to be strongly calcium dependent, with the addition of CaCl₂ to DM samples improving its antibacterial potential against both pathogens. Salmonella Enteritidis appeared to be less sensitive to the antimicrobial agents in DM than S. Typhimurium. One explanation is the calcium-dependant antibacterial activity of DM is attributable to the calcium-binding ability of its lysozyme. Lysozyme may be the main antibacterial agent, most probably via a nonenzymatic mode of action against tested Salmonella strains.     

Immunostimulatory characteristics of a novel adjuvant on the basis of cucumarioside A<Subscript>2</Subscript>-2 and monogalactosyldiacylgycerol

A novel antigen carrier has been formulated on the basis of a cucumarioside-A₂-2 triterpene glycoside (CD) complex with cholesterol and monogalactosyldiacylglycerol from Ahnfeltia tobuchiensis (MGDGAt) and Ulva fenestrate (MGDGUf). Morphological and immunostimulative characteristics of the carrier were studied. Electron microscopy experiments demonstrated the formation of homogeneous tubular structures in a mixture of CD, cholesterol, and MGDG in molar ratio of 1: 2: 3. In animals immunized by the carrier bearing pore forming protein monomer of pseudotuberculosis agent CD and MGDG synergically affected synthesis of specific antibodies, interleukin-2, and γ-interferon and delayed hypersensitivity reaction when compared to Freund’s complete adjuvant or to immunostimulatory complexes between Quillaja saponaria saponins and phosphatidylcholine from egg yolk. The immunostimulatory effect depends upon the composition of polyunsaturated fatty acids of MGDG. The new tubular adjuvant carrier is a competitive adjuvant, as it includes CD obtained from far-eastern sea cucumber commercial species Cucumaria japonica, and MGDG from seaweed.