Integrated approach for obtaining bioactive peptides from whey proteins hydrolysed using a new proteolytic lactic acid bacteria.

In this study, we investigated the ability of Enterococcus faecalis 2/28, isolated from artisan cheese, to release biopeptides from whey proteins. We used an in silico approach for predicting the bioactivities of peptides generated by E. faecalis. The results of the in vitro study showed that the whey protein hydrolysates (WPHs) obtained had angiotensin-I-converting enzyme (ACE) and dipeptidyl peptidase IV (DPP-IV) inhibitory activities, with inhibition of ACE being stronger than that of DPP-IV. To identify peptides that may be potential inhibitors of ACE, WPH with the highest ACE inhibitory activity was analysed using Sephadex G-75 gel filtration chromatography, Superdex peptide 10/300 GL size exclusion chromatography, and liquid chromatography-electrospray ionisation tandem mass spectrometry (LC-ESI-MS/MS). Among the identified peptides were ACE-inhibitory peptides (LDAQSAPLR, LKGYGGVSLPEW, and LKALPMH), antimicrobial peptides (AASDISLLDAQSAPLR, IIAEKTKIPAVF, IDALNENK, and VLVLDTDYK), DPP-IV-inhibitory peptides (LKALPMH, LKPTPEGDLEIL, LKGYGGVSLPE, LKPTPEGDLE, ILDKVGINY, and VLVLDTDYK), proliferation stimulating peptide (IDALNENK), and cytotoxic peptide (LIVTQTMK).

Identification and partial characterization of proteolytic activity of Enterococcus faecalis relevant to their application in dairy industry.

Bacteria of the genus Enterococcus are lactic acid bacteria (LAB), which occur ubiquitous in many traditional fermented foods, especially artisanal cheeses, playing positive role in the development of cheese flavor. Moreover, several enterococci are successfully used as a pharmaceutical probiotic and some of them are able to produce bacteriocin and bioactive peptides, thanks to which the possibilities of application of enterococci in dairy technology and biotechnology are increased. The aims of the study were to investigate the proteolytic potential and identify the key enzymes of proteolytic system of Enterococcus faecalis isolated from artisan Polish cheeses. An extracellular - secreted (E) and a cell envelope proteinase (CEP) were isolated and enzyme activity depending on bacterial growth phase was evaluated. CEP showed a higher protease activity than E and this fraction has been purified 70-fold by a method including precipitation, diafiltration and gel filtration chromatography. The molecular mass of the enzyme has been estimated to be ~25 kDa by SDS-PAGE. Maximum enzyme activity of the proteinase has been observed at pH 6,9 and 37 ºC. The enzyme was able to hydrolyze: casein, bovine serum albumin, α-lactalbumin, ß-lactoglobulin, but not Leu-pNa. The results of zymography, SDS- PAGE and LC-MS-MS/MS data allowed us to identify the key enzymes of proteolytic system of E. faecalis as coccolysin and glutamylendopeptidase. To asses microbiological safety of the tested strain, the evaluation of the presence of virulence factors and antibiotic susceptibility was also conducted.

Pichia cactophila and Kluyveromyces lactis are Highly Efficient Microbial Cell Factories of Natural Amino Acid-Derived Aroma Compounds.

The pivotal role of non-conventional yeast (NCY) species in formation of valuable aroma compounds in various food commodities is widely acknowledged. This fact inspires endeavors aiming at exploitation of food-derived NCYs as biocatalysts in natural aromas production. In this study, we isolated, characterized and evaluated aroma-producing capacity of two NCY representatives-Pichia cactophila 7.20 and Klyuveromyces lactis 6.10 strains. The strains were isolated from food-related habitats-goat-milk regional cheese and Swiss-type ripening cheese, respectively. Aroma profiles generated by the two strains cultured in a general rich medium were analyzed through solvent extraction and GC-MS analysis of the compounds retained in the culture media. Finally, the strains were tested in bioconversion cultures with branched chain- or aromatic amino acids as the sole nitrogen source, to assess capability of the strains towards formation of amino acid-derived aromas. The results showed extraordinary capacity of both strains for production of 2-phenylethanol (at more than 3 g/L) and isoamyl alcohol (approx. 1.5 g/L). A distinctive trait of 2-phenylethyl acetate synthesis at high concentrations (0.64 g/L) was revealed for P. cactophila 7.20 strain. Highly valued disulfide dimethyl as well as methionol acetate were identified amongst the aroma compounds synthesized by the strains.

The effect of eubiotic feed additives on the performance of growing pigs and the activity of intestinal microflora.

The aim of this study was to compare the effect of probiotic bacteria, prebiotics, phytobiotics and their combinations on performance and microbial activity in the digestive tract of growing pigs. The experiment was conducted over 28 d on 48 male pigs of about 12 kg body weight (BW), which were allocated to following treatments.: (1) Control Group (Con) without additive, (2) Group I, addition of a prebiotic (inulin), (3) Group Ph, a phytobiotic (herbal water extracts), (4) Group P, a probiotic composed of four strains of lactic acid bacteria, (5) Group PhP, phytobiotic and probiotic bacteria and (6) Group PhPI, a phytobiotic, probiotic bacteria and a prebiotic. Animal performance was recorded and at d 28 six pigs from each group were euthanised to collect digesta samples. In all groups except for Group I, diarrhoea incidents were observed. Groups Ph and P had significantly higher daily gains and final BW, and Group Ph utilised feed better than other groups. The pH of ileal digesta was significantly lower in Group PhPI. In the caecal digesta of Groups I, P and PhP, the pH level was lower than in the other groups but dry matter contents was significantly higher in Groups Con and I. The short-chain fatty acids and particular acid content differed significantly only in the colonic digesta. The yeast and mould numbers in caecal digesta was highest in Group Con. No treatment effects were observed for the number of lactic acid bacteria, coli group bacteria or Clostridium. However, the observed significantly higher number of total bacteria suggests that a multi-component eubiotic treatment changes the bacterial composition and distribution more effectively. Our findings indicated that all used additives changed the intestinal microflora, but the multi-component eubiotics were not beneficial as feed additives offered separately. Moreover, supplementation of phytobiotics and probiotic bacteria also improved the animal performance significantly.

Group II intron-mediated deletion of lactate dehydrogenase gene in an isolated 1,3-propanediol producer Hafnia alvei AD27.

Our previous studies showed that glycerol fermentation by Hafnia alvei AD27 strain was accompanied by formation of high quantities of lactate. The ultimate aim of this work was the elimination of excessive lactate production in the 1,3-propanediol producer cultures. Group II intron-mediated deletion of ldh (lactate dehydrogenase) gene in an environmental isolate of H. alvei AD27 strain was conducted. The effect of the Δldh genotype in H. alvei AD27 strain varied depending on the culture medium applied. Under lower initial glycerol concentration (20 gL-1), lactate and 1,3-propanediol production was fully abolished, and the main carbon flux was directed to ethanol synthesis. On the other hand, at higher initial glycerol concentrations (40 gL-1), 1,3-propanediol and lactate production was recovered in the recombinant strain. The final titers of 1,3-propanediol and ethanol were similar for the recombinant and the WT strains, while the Δldh genotype displayed significantly decreased lactate titer. The by-products profile was altered upon ldh gene deletion, while glycerol utilization and biomass accumulation remained unaltered. As indicated by flow-cytometry analyses, the internal pH was not different for the WT and the recombinant Δldh strains over the culture duration, however, the WT strain was characterized by higher redox potential.

L-Phenylalanine catabolism and 2-phenylethanol synthesis in Yarrowia lipolytica--mapping molecular identities through whole-proteome quantitative mass spectrometry analysis.

A world-wide effort is now being pursued towards the development of flavors and fragrances (F&F) production independently from traditional sources, as well as autonomously from depleting fossil fuel supplies. Biotechnological production of F&F by microbes has emerged as a vivid solution to the current market limitations. Amongst a wide variety of fragrant chemicals, 2-PE is of significant interest to both scientific and industrial community. Although the general overview of the 2-PE synthesis pathway is commonly known, involvement of particular molecular identities in this pathway has not been elucidated in Yarrowia lipolytica to date. The aim of this study was mapping molecular identities involved in 2-PE synthesis in Y. lipolytica. To acquire a comprehensive landscape of the proteins that are directly and indirectly involved in L-Phe degradation and 2-PE synthesis, we took advantage of comprehensibility and sensitivity of high-throughput LC-MS/MS-quantitative analysis. Amongst a number of proteins involved in amino acid turnover and the central carbon metabolism, enzymes involved in L-Phe conversion to 2-PE have been identified. Results on yeast-to-hyphae transition in relation to the character of the provided nitrogen source have been presented.

Cranberries (Oxycoccus quadripetalus) inhibit lipid metabolism and modulate leptin and adiponectin secretion in 3T3-L1 adipocytes.

It has previously been shown that lyophilized cranberries (LCB) decreased lipid accumulation in 3T3-L1 cells and inhibited preadipocyte differentiation by down-regulation of the expression of key transcription factors (PPARγ, C/EBPα, SREBP1) of the adipogenesis pathway. To elucidate the molecular basis of anti-lipogenic activity of LCB, the expression of several genes involved in lipid metabolism, such as adipocyte fatty acid-binding protein (aP2), lipoprotein lipase (LPL), fatty acid synthase (FAS), hormone sensitive lipase (HSL) and perilipin 1 (PLIN1), was examined in the present study. Additionally, the effects of LCB on adiponectin and leptin expression and protein secretion were also investigated. LCB reduced lipid accumulation during preadipocyte differentiation by down-regulation of the mRNA level of aP2, FAS, LPL, HSL and PLIN1. Moreover, LCB decreased leptin gene expression and increased adiponectin gene expression and protein secretion in a dose-dependent manner. Therefore cranberries could be considered as bioactive factors, which are effective in the inhibition of adipose tissue mass production.

Antioxidant capacity of broccoli sprouts subjected to gastrointestinal digestion.

BACKGROUND: Broccoli is a common vegetable recognized as a rich source of antioxidants. To date, research on the antioxidant properties of broccoli, predominantly conducted on extracts, has not considered the lesions of composition and this activity after gastrointestinal digestion. Here the stability of antioxidants during gastrointestinal digestion was evaluated in conjunction with the protective effects of broccoli sprouts (BS) against oxidative stress in human colon cells. RESULTS: The obtained data suggest that, among the biocompounds identified in BS, glucosinolates were mainly degraded under gastrointestinal digestion, while phenolics, particularly hydroxycinnamic acid derivatives, were the most resistant constituents. The antioxidant capacity of BS extract subjected to gastrointestinal digestion was similar to or higher than that determined for non-digested BS. Gastrointestinal digested BS extract exhibited reactive oxygen species (ROS)-inhibitory capacity in NCM460 human colon cells, with 1 mg mL(-1) showing an ROS clearance of 76.59%. A 57.33% reduction in oxidative DNA damage in NCM460 cells due to treatment with digested BS extract was observed. CONCLUSION: The results lend support to the possible application of BS as a rich source of antioxidants to improve the defensive system against oxidative stress in the human colon mucosa.

Cloning, expression, and purification of insect (Sitophilus oryzae) alpha-amylase, able to digest granular starch, in Yarrowia lipolytica host.

Raw-starch-digesting enzymes (RSDE) are of major importance for industrial applications, as their usage greatly simplifies the starch processing pipeline. To date, only microbial RSDE have gained considerable attention, since only microbial production of enzymes meets industrial demands. In this study, α-amylase from rice weevil (Sitophilus oryzae), the major rice pest, was cloned and expressed in Yarrowia lipolytica Po1g strain. The enzyme was secreted into the culture medium, and the peak activity (81 AU/L) was reached after only 29 h of culturing in 5-L bioreactors. Through simple purification procedure of ammonium sulfate precipitation and affinity chromatography, it was possible to purify the enzyme to apparent homogeneity (25-fold purification factor, at 5 % yield). The optimal conditions for the α-amylase activity were pH 5.0 and a temperature of 40 °C. The α-amylase studied here did not show any obligate requirement for Ca(2+) ions. The recombinant α-amylase appeared to efficiently digest granular starch from pea, amaranth, waxy corn, and waxy rice.

Impurities of crude glycerol and their effect on metabolite production.

Glycerol is a valuable raw material for the production of industrially useful metabolites. Among many promising applications for the use of glycerol is its bioconversion to high value-added compounds, such as 1,3-propanediol (1,3-PD), succinate, ethanol, propionate, and hydrogen, through microbial fermentation. Another method of waste material utilization is the application of crude glycerol in blends with other wastes (e.g., tomato waste hydrolysate). However, crude glycerol, a by-product of biodiesel production, has many impurities which can limit the yield of metabolites. In this mini-review we summarize the effects of crude glycerol impurities on various microbial fermentations and give an overview of the metabolites that can be synthesized by a number of prokaryotic and eukaryotic microorganisms when cultivated on glycerol.

Expression of bacteriocin divercin AS7 in Escherichia coli and its functional analysis.

Bacteriocins are small peptides with antimicrobial activity, that are produced by bacteria. Four classes of bacteriocins produced by lactic acid bacteria have been defined. Class IIa bacteriocins are promising candidates for industrial applications due to their high biological activity and their physicochemical properties. Divercin AS7 is a class IIa bacteriocin produced by Carnobacterium divergens AS7. It shows antibacterial activity against pathogens and food spoilage flora including Listeria spp. Little is known about the impact of class IIa bacteriocins upon eukaryotic cells. The safe use of bacteriocins as food biopreservatives requires the absence of cytotoxicity to human cells. To analyze the impact of divercin AS7 on human enterocytes, we expressed the recombinant divercin AS7 in the Escherichia coli BL21DE3pLys strain and conducted in vitro studies to evaluate the safety of recombinant divercin AS7. No cytotoxic effect on differentiated monolayer Caco-2 cells and no apoptotic appearance were observed when recombinant divercin AS7 was used at a concentration of 2 µg ml-1. In our study, divercin AS7 also did not interfere with differentiated Caco-2 cells monolayer integrity. The obtained results suggest that divercin AS7 is a promising peptide for the food industry.

Cranberries (Oxycoccus quadripetalus) inhibit adipogenesis and lipogenesis in 3T3-L1 cells.

Cranberries (Oxycoccus quadripetalus) are a valuable source of bioactive substances with high antioxidant potential and well documented beneficial health properties. In the present study, the activity of cranberries, in terms of the inhibiting effects of adipogenesis, was investigated using the 3T3-L1 cell line. The obtained results showed that cranberries reduced proliferation and viability of 3T3-L1 preadipocytes in a dose-dependent manner. Treatment with cranberries decreased the number of adipocytes and reduced lipid accumulation in maturing 3T3-L1 preadipocytes, demonstrating an inhibitory effect on lipogenesis. Moreover, it was found that cranberries directly induced lipolysis in adipocytes and down-regulated the expression of major transcription factors of the adipogenesis pathway, such as PPARγ, C/EBPα and SREBP1. These findings indicate that cranberries are capable of suppressing adipogenesis and therefore they seem to be natural bioactive factors effective in adipose tissue mass modulation.

A novel multigene expression construct for modification of glycerol metabolism in Yarrowia lipolytica.

BACKGROUND: High supply of raw, residual glycerol from biodiesel production plants promote the search for novel biotechnological methods of its utilization. In this study we attempted modification of glycerol catabolism in a nonconventional yeast species Yarrowia lipolytica through genetic engineering approach. RESULTS: To address this, we developed a novel genetic construct which allows transferring three heterologous genes, encoding glycerol dehydratase, its reactivator and a wide-spectrum alcohol oxidoreductase under the control of glycerol-induced promoter. The three genes, tandemly arrayed in an expression cassette with a marker gene ura3, regulatory and targeting sequences (G3P dh promoter and XPR-like terminator, 28S rDNA as a target locus), were transferred into Yarrowia lipolytica cells. The obtained recombinant strain NCYC3825 was characterized at the molecular level and with respect to its biotechnological potential. Our experiments indicated that the novel recombinant strain stably borne one copy of the expression cassette and efficiently expressed heterologous alcohol oxidoreductase, while glycerol dehydratase and its reactivator were expressed at lower level. Comparative shake flask cultivations in glucose- and glycerol-based media demonstrated higher biomass production by the recombinant strain when glycerol was the main carbon source. During bioreactor (5 L) fed-batch cultivation in glycerol-based medium, the recombinant strain was characterized by relatively high biomass and lipids accumulation (up to 42 gDCW L(-1), and a peak value of 38%LIPIDS of DCW, respectively), and production of high titers of citric acid (59 g L(-1)) and 2-phenylethanol (up to 1 g L(-1) in shake flask cultivation), which are industrially attractive bioproducts. CONCLUSIONS: Due to heterogeneous nature of the observed alterations, we postulate that the main driving force of the modified phenotype was faster growth in glycerol-based media, triggered by modifications in the red-ox balance brought by the wide spectrum oxidoreductase. Our results demonstrate the potential multidirectional use of a novel Yarrowia lipolytica strain as a microbial cell factory.

Beneficial and harmful roles of bacteria from the Clostridium genus.

Bacteria of the Clostridium genus are often described only as a biological threat and a foe of mankind. However, many of them have positive properties and thanks to them they may be used in many industry branches (e.g., in solvents and alcohol production, in medicine, and also in esthetic cosmetology). During the last 10 years interest in application of C. botulinum and C. tetani in medicine significantly increased. Currently, the structure and biochemical properties of neurotoxins produced by these bacterial species, as well as possibilities of application of such toxins as botulinum as a therapeutic factor in humans, are being intensely researched. The main aim of this article is to demonstrate that bacteria from Clostridium spp. are not only pathogens and the enemy of humanity but they also have many important beneficial properties which make them usable among many chemical, medical, and cosmetic applications.

Development of a PCR-based assay for rapid detection of class IIa bacteriocin genes.

AIMS: We have developed a PCR-based assay using custom designed panel of primers which allows rapid detection of class IIa bacteriocin-coding genes. To demonstrate the applicability of the developed assay, the method was applied on 40 metagenomic DNA preparations isolated from native microbiota of Polish artisanal cheeses produced in the Tatra Mountains. METHODS AND RESULTS: The developed assay was designed on the basis of a large scale alignment of class IIa bacteriocin-coding genes. A panel of seven primer pairs with confirmed ability to detect class IIa bacteriocin-coding sequences was obtained. The following study has revealed a superb bacteriocinogenic potential of all forty analysed cheese samples. CONCLUSIONS: The majority of obtained sequences were lactic acid bacteria (LAB) related, although some sequences showed significant similarity to bacteriocin-coding sequences present in non-LAB bacteriocin producers. The results suggest that several potentially new bacteriocin-coding sequences were found. SIGNIFICANCE AND IMPACT OF THE STUDY: The developed assay can be extremely helpful in establishing whether isolates from the environment of interest have a potential of synthesizing antilisterial class IIa bacteriocins. Application of the approach may represent a useful tool contributing to ecological studies looking for valuable probiotic, bacteriocinogenic microbiota developing in foods.

Evaluation of quantitative PCR measurement of bacterial colonization of epithelial cells.

Microbial colonization is an important step in establishing pathogenic or probiotic relations to host cells and in biofilm formation on industrial or medical devices. The aim of this work was to verify the applicability of quantitative PCR (Real-Time PCR) to measure bacterial colonization of epithelial cells. Salmonella enterica and Caco-2 intestinal epithelial cell line was used as a model. To verify sensitivity of the assay a competition of the pathogen cells to probiotic microorganism was tested. The qPCR method was compared to plate count and radiolabel approach, which are well established techniques in this area of research. The three methods returned similar results. The best quantification accuracy had radiolabel method, followed by qPCR. The plate count results showed coefficient of variation two-times higher than this of qPCR. The quantitative PCR proved to be a reliable method for enumeration of microbes in colonization assay. It has several advantages that make it very useful in case of analyzing mixed populations, where several different species or even strains can be monitored at the same time.

[The role of natural dietary compounds in colorectal cancer chemoprevention]. / Rola naturalnych skladników diety w chemioprewencji nowotworów jelita grubego.

This review discusses the preventive and therapeutic potential of natural dietary compounds against colorectal cancer. The chemopreventive properties of many natural food matrices and purified bioactive compounds have been evaluated. Prominent among the dietary constituents that are the focus of interest in colorectal cancer chemoprevention are dietary fiber, probiotics and prebiotics, methionine and folate, vitamins D and E, calcium and selenium, anthocyanins, procyanidins, phytoestrogens, isothiocyanates, epigallocatechin gallate, curcumin, and resveratrol. Laboratory studies provide strong evidence for the antitumor potential of these dietary agents. The mechanisms of their chemopreventive action are associated with, for example, the modulation of gene expression involved in the regulation of cell proliferation, differentiation, and apoptosis and the suppression of metastasis and angiogenesis. The anti-carcinogenic properties of these food compounds are also related to inhibition of many inflammatory agents, including the expression of cyclooxygenase-2. In vitro and animal studies showed that most of them can protect against various carcinogens mediating colon cancer and suggest that they can also sensitize tumors to chemotherapy and radiation. Although experimental studies have clearly demonstrated their anticancer activity, not many clinical trials have provided satisfying results, not only because of the lack of efficiency of the chemopreventive agents, but also due to the lack of precise biomarkers monitoring their effects on colon cancer. Despite the lack of strong evidence for the anticancer potential of natural food compounds, clinicians have high hopes for using these factors in colon cancer chemoprevention and decreasing the incidence of this common malignancy in the future.

Fuel ethanol production from granular corn starch using Saccharomyces cerevisiae in a long term repeated SSF process with full stillage recycling.

A major problem with fermentative ethanol production is the formation of large amounts of numerous organic pollutants. In an industrial distillery, stillage, fermenter and condenser cooling water are the main sources of wastewater. However, the selection of a proper technology makes it possible to almost completely avoid emissions of such kind of wastewater to the environment. This study examines the effect of stillage recirculation on fuel ethanol production. It is based on the use of Saccharomyces cerevisiae and a granular starch hydrolyzing enzyme in a simultaneous saccharification and fermentation process using a native starch obtained from corn flour. It was shown that the yield of the ethanol production was not influenced by the recycled stillage, a mean yield being 83.38% of the theoretical value. No significant trend for change in the ethanol concentration or in the residual starch was observed during any particular run, even after the 75% of fresh water was replaced with stillage. Thus, by applying this new clean technology it is possible to significantly reduce the rate of water consumption and in this way the production of by-products such as stillage.

In vitro effects of beetroot juice and chips on oxidative metabolism and apoptosis in neutrophils from obese individuals.

Oxidative stress and inflammation are involved in the development of obesity. Beetroot (Beta vulgaris var. rubra) is a food ingredient containing betalain pigments that show antioxidant activity. The in vitro effect of beetroot juice and chips on oxidative metabolism and apoptosis in neutrophils from obese individuals has been investigated. Fifteen obese women (aged 45 +/- 9 years, BMI >30 kg/m2) and nine healthy controls (women, aged 29 +/- 11 years, BMI = 22.2 +/- 1.6 kg/m2) were examined. The investigated products were used as concentrates and after transport and digestion in an artificial gastrointestinal tract. Neutrophil oxidant production, in response to phorbol 12-myristate 13-acetate, was characterized by luminol-dependent chemiluminescence and a flow cytometric dichlorofluorescin oxidation assay. Caspase-3 activity, a marker of apoptosis, was measured by cleavage of the fluorogenic substrate Ac-DEVD-AMC. Neutrophils from obese individuals had a significantly higher ROS production compared with the controls (p < 0.05). Beetroot products inhibited neutrophil oxidative metabolism in a concentration-dependent manner. Also observed were the pro-apoptotic effects of beetroot at a concentration range of 0.1-10% in 24 h culture of stimulated neutrophils. These natural products (in both the liquid and solid state) have antioxidant and antiinflammatory capacity, and could be an important adjunct in the treatment of obesity.

Regulatory elements in the juvenile hormone binding protein gene from Galleria mellonella--topography of binding sites for Usp and EcRDBD.

The juvenile hormone binding protein (JHBP) plays a key role in the protection and transport of the hormone to target tissues. In this report the sequence of the jhbp promoter comprising about 2000 bp is characterized. Using a minimized false positive algorithm, six putative regulatory elements, Hunchback, Heat shock factor binding element, Ultrabithorax, Broad-Complex Z3, Elf-1 and Chorion factor 1/ultraspiracle (CF1/Usp) were found in the distal promoter of the jhbp gene. Proteins from nuclear extract of Galleria mellonella fat body form four specific complexes with probe containing TATA box, five complexes with Inr probe and one protein complex with DPE probe. EMSA and footprinting analyses showed that one of the three CF1/Usp elements (starting at -1053) has an exceptionally high affinity to Usp protein. An unknown, high-affinity Usp/EcRDBD-binding element (TCAACA-AAC-TGTTCA), distinct from 20-hydroxyecdysone response elements, was identified in the jhbp gene promoter, based on a footprinting assay. Deletions of jhbp promoter in the regions containing the CF1/Usp elements enhance the transcriptional activity of luciferase reporter gene in the Trichoplusia ni High Five cell line. Obtained data suggest that jhbp promoter is TATA- and Inr-driven, CF1/Usp elements exhibit inhibitory effect on jhbp expression, and an interaction between Usp and DNA relies on recognition of the consensus sequence (GGGTCA) and on ionic interactions of several phosphate groups outside from this element.