Modulation of Inflammatory Pathways and Adipogenesis by the Action of Gentisic Acid in RAW 264.7 and 3T3-L1 Cell Lines.

Gentisic acid (GA), a benzoic acid derivative present in various food ingredients, has been shown to have diverse pharmaceutical activities such as anti-carcinogenic, antioxidant, and hepatoprotective effects. In this study, we used a co-culture system to investigate the mechanisms of the anti-inflammatory and anti-adipogenic effects of GA on macrophages and adipocytes, respectively, as well as its effect on obesity-related chronic inflammation. We found that GA effectively suppressed lipopolysaccharide-stimulated inflammatory responses by controlling the production of nitric oxide and pro-inflammatory cytokines and modulating inflammation-related protein pathways. GA treatment also inhibited lipid accumulation in adipocytes by modulating the expression of major adipogenic transcription factors and their upstream protein pathways. Furthermore, in the macrophage-adipocyte co-culture system, GA decreased the production of obesity-related cytokines. These results indicate that GA possesses effective anti-inflammatory and anti-adipogenic activities and may be used in developing treatments for the management of obesity-related chronic inflammatory diseases.

Anti-inflammatory effect of lovastatin is mediated via the modulation of NF-κB and inhibition of HDAC1 and the PI3K/Akt/mTOR pathway in RAW264.7 macrophages.

Lovastatin is a 3-hydroxy-3-methylglutaryl-CoA reductase inhibitor that is clinically used for the prevention of cardiovascular diseases. Although it has been reported that lovastatin has anti-inflammatory properties in several studies, how lovastatin regulates the inflammation is still unclear. To evaluate the effect of lovastatin on nitric oxide production (NO) in RAW264.7 macrophages, NO production assay was performed. Also, cell viability was measured to confirm cytotoxicity. Level of tumor necrosis factor-α (TNF-α) transcription was measured by reverse transcription polymerase chain reaction (RT-PCR) from total RNA in RAW264.7 cells. Western blot analysis and immunofluorescence staining were used to investigate the regulation of lovastatin on the expression, phosphorylation, and nuclear translocation of cellular proteins. The results of the present study revealed that lovastatin reduced nitric oxide production via the reduction of inducible nitric oxide synthase (iNOS) expression in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophage cells. The mRNA level of TNF-α was reduced in presence of lovastatin. In addition, lovastatin downregulated histone deacetylase 1 (HDAC1), resulting in the accumulation of acetylated histone H3 and heat shock protein 70. Furthermore, the expression of phosphoinositide 3-kinase catalytic subunits α and ß was reduced under lovastatin treatment, and the phosphorylation of Akt and mammalian target of rapamycin was consequently inhibited. Lovastatin also inhibited the phosphorylation of inhibitor of nuclear factor (NF)-κBα and the translocation of NF-κB into the nucleus. Therefore, the present study demonstrates that lovastatin inhibits the expression of pro-inflammatory mediators, including iNOS and TNF-α, through the suppression of HDAC1 expression, PI3K/Akt phosphorylation and NF-κB translocation in LPS-stimulated RAW264.7 macrophage cells.

The Anti-Adipogenic Activity of a New Cultivar, Pleurotus eryngii var. ferulae 'Beesan No. 2', through Down-Regulation of PPAR γ and C/EBP α in 3T3-L1 Cells.

Adipogenesis is one of the cellular processes and a highly controlled program. Nowadays, inhibition of adipogenesis has received attention as an effective way to regulate obesity. In the current study, we investigated the inhibition effect of a chloroform extract of Pleurotus eryngii var. ferulae 'Beesan No. 2' (CEBT) on adipogenesis in 3T3-L1 murine preadipocytes. Pleurotus eryngii var. ferulae is one of many varieties of King oyster mushroom and has been reported to have various biological activities, including antitumor and anti-inflammation effects. Biological activities of 'Beesan No. 2', a new cultivar of Pleurotus eryngii var. ferulae, have not yet been reported. In this study, we found that CEBT suppressed adipogenesis in 3T3-L1 cells through inhibition of key adipogenic transcription factors, such as peroxisome proliferator-activated receptor γ and CCAAT/enhancer binding protein α. Additionally, CEBT reduced the expression of the IRS/PI3K/Akt signaling pathway and its downstream factors, including mammalian target of rapamycin and p70S6 kinase, which stimulate adipogenesis. Furthermore, ß-catenin, a suppressor of adipogenesis, was increased in CEBT-treated cells. These results indicate that Pleurotus eryngii var. ferulae 'Beesan No. 2' effectively inhibited adipogenesis, so this mushroom has potential as an anti-obesity food and drug.

Evaluation of Antioxidant, Anti-cholinesterase, and Anti-inflammatory Effects of Culinary Mushroom Pleurotus pulmonarius.

Culinary mushroom Pleurotus pulmonarius has been popular in Asian countries. In this study, the anti-oxidant, cholinesterase, and inflammation inhibitory activities of methanol extract (ME) of fruiting bodies of P. pulmonarius were evaluted. The 1,1-diphenyl-2-picryl-hydrazy free radical scavenging activity of ME at 2.0 mg/mL was comparable to that of butylated hydroxytoluene, the standard reference. The ME exhibited significantly higher hydroxyl radical scavenging activity than butylated hydroxytoluene. ME showed slightly lower but moderate inhibitory activity against acetylcholinesterase (AChE) and butyrylcholinesterase than galantamine, a standard AChE inhibitor. It also exhibited protective effect against cytotoxicity to PC-12 cells induced by glutamate (10~100 µg/mL), inhibitory effect on nitric oxide (NO) production and inducible nitric oxide synthase protein expression in lipopolysaccharide-stimulated RAW 264.7 macrophages, and carrageenan-induced paw edema in a rat model. High-performance liquid chromatography analysis revealed the ME of P. pulmonarius contained at least 10 phenolic compounds and some of them were identified by the comparison with known standard phenolics. Taken together, our results demonstrate that fruiting bodies of P. pulmonarius possess antioxidant, anti-cholinesterase, and inflammation inhibitory activities.

Toluhydroquinone from Aspergillus sp. suppress inflammatory mediators via nuclear factor-κB and mitogen-activated protein kinases pathways in lipopolysaccharide-induced RAW264.7 cells.

OBJECTIVES: The purpose of this study is to investigate anti-inflammatory effects of toluhydroquinone in lipopolysaccharide (LPS)-stimulated RAW264.7 cells. METHODS: Toluhydroquinone was purified from a fungal strain, Aspergillus sp. We investigated that levels of nitric oxide (NO) using Griess reagent, production of prostaglandin E2 (PGE2 ) and pro-inflammatory cytokines using ELISA assay. We conducted Western blot analysis to investigate regulatory effects of toluhydroquinone on expression of inducible nitric oxide synthase (iNOS), cyclooxyganse-2 (COX-2), nuclear factor-κB (NF-κB), Akt and mitogen-activated protein kinases (MAPKs) in LPS-stimulated RAW264.7 cells. The translocation of NF-κB was detected by immunofluorescence staining. KEY FINDINGS: Toluhydroquinone inhibited production of NO and PGE2 via suppressing protein expression of iNOS and COX-2, respectively. Secretion and expression of inflammatory cytokines were down-regulated by toluhydroquinone as well. Toluhydroquinone reduced phosphorylation of Akt, NF-κB and MAPKs. Moreover, toluhydroquinone inhibited translocation of NF-κB from the cytosol into the nucleus. CONCLUSIONS: We revealed that inhibitory effects of toluhydroquinone on expression of inflammatory mediators are induced through inactivation of Akt, NF-κB and MAPKs. Thus, our results suggest that toluhydroquinone may be used for a potential anti-inflammatory reagent.

Veratric acid inhibits iNOS expression through the regulation of PI3K activation and histone acetylation in LPS-stimulated RAW264.7 cells.

In the present study, we investigated regulatory effects of veratric acid on the production of nitric oxide (NO) in lipopolysaccharide (LPS)-stimulated RAW264.7 cells. NO production was significantly decreased by veratric acid in the LPS-stimulated RAW264.7 cells in a dose-dependent manner. The reduction in nitric oxide production was induced by the downregulation of inducible NO synthase (iNOS) expression. Veratric acid suppressed the LPS-induced effects on the regulatory and catalytic subunits of phosphoinositide 3-kinase (PI3K), comprised of p85, p110α, p110ß and Akt. The acetylation of p300 and the phosphorylation of activating transcription factor 2 (ATF-2) induced by LPS were downregulated following treatment with veratric acid; similar effects were observed following treatment with LY294002, a specific inhibitor of PI3K/Akt. The LPS-induced expression of histone deacetylase (HDAC)3 decreased to basal levels following treatment with veratric acid, and its expression was also downregulated by LY294002. In the measurement of histone acetylation levels, the LPS-stimulated acetylation of histone H4 was significantly attenuated by veratric acid, and was also reduced following the inhibition of PI3K/Akt with LY294002. From our data, it can be concluded that veratric acid exerts a regulatory effect on LPS-induced iNOS expression. Our results suggest that veratric acid impedes the PI3K/Akt-mediated histone acetyl-transferase (HAT) activation and HDAC expression induced by LPS, thereby abrogating iNOS expression.

Induction of the endoplasmic reticulum stress and autophagy in human lung carcinoma A549 cells by anacardic acid.

Anacardic acid (AA, 2-hydroxy-6-pentadecylbenzoic acid), a constituent of the cashew-nut shell, has a variety of beneficial effects on the treatment of cancer and tumors. However, the fact that AA induces ER stress and autophagy in cancer cell is not known. We investigated the effect of AA on ER-stress and autophagy-induced cell death in cancer cells. Because of our interest in lung cancer, we used the non-small cell lung adenocarcinoma A549 cells treated with 3.0 µg/ml of AA for this research. In this research we found that AA induces intracellular Ca(2+) mobilization and ER stress. AA induced the ER stress-inducing factors, especially IRE1α, and the hallmarks of UPR, Grp78/Bip and GADD153/CHOP. AA inhibited the expression of p-PERK and its downstream substrate, p-elF2α. We also demonstrated that AA induces autophagy. Up-regulation of autophagy-related genes and the appearance of autophagosome in transfected cells with green fluorescent protein (GFP)-LC3 and GFP-Beclin1 plasmids showed the induction of autophagy in AA-treated A549 cells. The morphological analysis of intracellular organelles by TEM also showed the evidence that AA induces ER stress and autophagy. For the first time, our research showed that AA induces ER stress and autophagy in cancer cells.

Anacardic acid induces mitochondrial-mediated apoptosis in the A549 human lung adenocarcinoma cells.

Anacardic acid (AA) is a constituent of the cashew nut shell and is known as an inhibitor of nuclear factor-κB (NF-κB). We investigated the cytotoxicity of AA on cancer cells and more experiments to reveal the cell death mechanism focused on A549 lung adenocarcinoma cells for our interest in lung cancer. To examine the molecular mechanism of cell death in AA treated A549 cells, we performed experiments such as transmission electron microscopy (TEM), western blot analysis, fluorescence-activated cell sorting (FACS), genomic DNA extraction and staining with 4',6-diamidino-2-phenylindole (DAPI). For the first time we revealed that AA induces caspase-independent apoptosis with no inhibition of cytotoxicity by pan-caspase inhibitor, Z-VAD-fmk, in A549 cells. Our results showed the possibility of mitochondrial-mediated apoptosis through the activation of apoptosis-inducing factor (AIF) and an intrinsic pathway executioner such as cytochrome c. This study will be helpful in revealing the cell death mechanisms and in developing potential drugs for lung cancer using AA.

Immunomodulating and Antitumor Activities of Panellus serotinus Polysaccharides.

This study was initiated in order to investigate the anticancer and immunomodulating activities of crude polysaccharides extracted in methanol, neutral saline, and hot water (hereinafter referred to as Fr. MeOH, Fr. NaCl, and Fr. HW, respectively) from the fruiting bodies of Panellus serotinus. Content of ß-glucan and protein in Fr. MeOH, Fr. NaCl, and Fr. HW extracts of P. serotinus ranged from 22.92~28.52 g/100 g and 3.24~3.68 g/100 g, respectively. In vitro cytotoxicity tests, none of the various fractions of crude polysaccharides were cytotoxic against sarcoma 180, HT-29, NIH3T3, and RAW 264.7 cell lines at the tested concentration. Intraperitoneal injection with crude polysaccharides resulted in a life prolongation effect of 23.53~44.71% in mice previously inoculated with sarcoma 180. Treatment with Fr. HW resulted in an increase in the numbers of spleen cells by 1.3 fold at the concentration of 50 µg/mL compared with control. Treatment with Fr. NaCl resulted in improvement of the immuno-potentiating activity of B lymphocytes by increasing the alkaline phosphatase activity by 1.4 fold, compared with control, at the concentration of 200 µg/mL. Among the three fractions, maximum nitric oxide (13.48 µM) was recorded at 500 µg/mL in Fr. HW. Production of tumor necrosis factor alpha, interleukin-1ß, and interleukin-6 was significantly higher, compared to the positive control, concanavalin A, at the tested concentration. Therefore, treatment with crude polysaccharides extracted from the fruiting body of P. serotinus could result in improvement of antitumor activity.

Anticancer and Immunopotentiating Activities of Crude Polysaccharides from Pleurotus nebrodensis on Mouse Sarcoma 180.

Pleurotus nebrodensis is an edible and commercially available mushroom in Korea. This study was conducted in order to evaluate the anticancer and immunopotentiating activities of crude polysaccharides, extracted in methanol, neutral saline, and hot water (hereafter referred to as Fr. MeOH, Fr. NaCl, and Fr. HW, respectively) from the fruiting bodies of P. nebrodensis. ß-Glucan and protein contents in Fr. MeOH, Fr. NaCl, and Fr. HW extracts of P. nebrodensis ranged from 23.79~36.63 g/100 g and 4.45~6.12 g/100 g, respectively. Crude polysaccharides were not cytotoxic against sarcoma 180, HT-29, NIH3T3, and RAW 264.7 cell lines at a range of 10~2,000 µg/mL. Intraperitoneal injection with crude polysaccharides resulted in a life prolongation effect of 11.76~27.06% in mice previously inoculated with sarcoma 180. Treatment with Fr. NaCl resulted in an increase in the numbers of spleen cells by 1.49 fold at the concentration of 50 µg/mL, compared with control. Fr. HW improved the immuno-potentiating activity of B lymphocytes through an increase in alkaline phosphatase activity by 1.65 fold, compared with control at 200 µg/mL. Maximum production of nitric oxide (14.3 µM) was recorded in the Fr. NaCl fraction at 200 µg/mL. Production of tumor necrosis factor-α (TNF-α), interleukin-1ß (IL-1ß), and interleukin-6 (IL-6) was significantly higher, compared to control, and IL-6 production was highest, in contrast to TNF-α, IL-1ß, and positive control, concanavalin at the tested concentration of the various fractions. Results of the current study suggest that polysaccharides extracted from P. nebrodensis have a strong anticancer effect and may be useful as an ingredient of biopharmaceutical products for treatment of cancer.

The regulatory effect of veratric acid on NO production in LPS-stimulated RAW264.7 macrophage cells.

Veratric acid, a simple benzoic acid derived from plants and fruits, has been reported to have anti-oxidant, anti-inflammation, and blood pressure-lowering effects. This study was designed to evaluate the inhibitory effects of veratric acid on nitric oxide (NO) production in LPS-stimulated RAW264.7 cells. It was found that veratric acid inhibited NO production and inducible nitric oxide synthase (iNOS) expression in LPS-stimulated RAW264.7 cells. The inhibitory effects of veratric acid on the generation of interleukin-6 (IL-6) and interferon-γ (IFN-γ) was determined. Furthermore, veratric acid facilitated the inactivation of glycogen synthase kinase-3ß (GSK-3ß), STAT-1, and STAT-3 in dose-dependent manner. Notably, NF-κB and members of the mitogen activated protein kinase (MAPK) family including p38, ERK, and JNK were dephosphorylated by veratric acid. These findings suggest that the treatment of veratric acid might be effective in neutralizing the over-expression of NO in inflammatory disorders.

Comparative genomics of the mating-type loci of the mushroom Flammulina velutipes reveals widespread synteny and recent inversions.

BACKGROUND: Mating-type loci of mushroom fungi contain master regulatory genes that control recognition between compatible nuclei, maintenance of compatible nuclei as heterokaryons, and fruiting body development. Regions near mating-type loci in fungi often show adapted recombination, facilitating the generation of novel mating types and reducing the production of self-compatible mating types. Compared to other fungi, mushroom fungi have complex mating-type systems, showing both loci with redundant function (subloci) and subloci with many alleles. The genomic organization of mating-type loci has been solved in very few mushroom species, which complicates proper interpretation of mating-type evolution and use of those genes in breeding programs. METHODOLOGY/PRINCIPAL FINDINGS: We report a complete genetic structure of the mating-type loci from the tetrapolar, edible mushroom Flammulina velutipes mating type A3B3. Two matB3 subloci, matB3a that contains a unique pheromone and matB3b, were mapped 177 Kb apart on scaffold 1. The matA locus of F. velutipes contains three homeodomain genes distributed over 73 Kb distant matA3a and matA3b subloci. The conserved matA region in Agaricales approaches 350 Kb and contains conserved recombination hotspots showing major rearrangements in F. velutipes and Schizophyllum commune. Important evolutionary differences were indicated; separation of the matA subloci in F. velutipes was diverged from the Coprinopsis cinerea arrangement via two large inversions whereas separation in S. commune emerged through transposition of gene clusters. CONCLUSIONS/SIGNIFICANCE: In our study we determined that the Agaricales have very large scale synteny at matA (∼350 Kb) and that this synteny is maintained even when parts of this region are separated through chromosomal rearrangements. Four conserved recombination hotspots allow reshuffling of large fragments of this region. Next to this, it was revealed that large distance subloci can exist in matB as well. Finally, the genes that were linked to specific mating types will serve as molecular markers in breeding.

Antioxidant and antityrosinase activities of various extracts from the fruiting bodies of Lentinus lepideus.

Lentinus lepideus is an edible mushroom currently available in Korea. The acetone, methanol and hot water extracts were prepared and assayed for their antioxidant and antityrosinase inhibitory activities. The hot water extract showed the strongest ß-carotene-linoleic acid inhibition compared to the other extracts. At 8 mg/mL, the methanolic extract showed a high reducing power of 1.21. The acetone and methanol extracts were more effective in scavenging DPPH radicals than the hot water extract. The strongest chelating effect was obtained from the methanolic extract. Xanthine oxidase and tyrosinase inhibitory activities of the acetonic, methanol and hot water extracts increased with increasing concentration. Gallic acid, chlorogenic acid, vanillin, naringin, naringenin, formononetin, and biochanin-A were detected in the acetonitrile and hydrochloric acid (5:1) solvent extract. This study suggests that fruiting bodies of L. lepideus can potentially be used as a readily accessible source of natural antioxidants.

Assessment of Antioxidant and Phenolic Compound Concentrations as well as Xanthine Oxidase and Tyrosinase Inhibitory Properties of Different Extracts of Pleurotus citrinopileatus Fruiting Bodies.

Cellular damage caused by reactive oxygen species has been implicated in several diseases, thus establishing a significant role for antioxidants in maintaining human health. Acetone, methanol, and hot water extracts of Pleurotus citrinopileatus were evaluated for their antioxidant activities against ß-carotene-linoleic acid and 1,1-diphenyl-2-picrylhydrazyl (DPPH) radicals, reducing power, ferrous ion-chelating abilities, and xanthine oxidase inhibitory activities. In addition, the tyrosinase inhibitory effects and phenolic compound contents of the extracts were also analyzed. Methanol and acetone extracts of P. citrinopileatus showed stronger inhibition of ß-carotene-linoleic acid compared to the hot water extract. Methanol extract (8 mg/mL) showed a significantly high reducing power of 2.92 compared to the other extracts. The hot water extract was more effective than the acetone and methanole extracts for scavenging DPPH radicals. The strongest chelating effect (92.72%) was obtained with 1.0 mg/mL of acetone extract. High performance liquid chromatography analysis detected eight phenolic compounds, including gallic acid, protocatechuic acid, chlorogenic acid, ferulic acid, naringenin, hesperetin, formononetin, and biochanin-A, in an acetonitrile and hydrochloric acid (5 : 1) solvent extract. Xanthine oxidase and tyrosinase inhibitory activities of the acetone, methanol, and hot water extracts increased with increasing concentration. This study suggests that fruiting bodies of P. citrinopileatus can potentially be used as a readily accessible source of natural antioxidants.

Appraisal of Antihyperlipidemic Activities of Lentinus lepideus in Hypercholesterolemic Rats.

The wild edible mushroom, Lentinus lepideus has recently been cultivated for commercial use in Korea. While the mushroom has been widely used for nutritional and medicinal purposes, the possible anti-hyperlipidemic action is unclear. The effects of dietary L. lepideus on plasma and feces biochemical and on the liver histological status were investigated in hypercholesterolemic rats. Six-wk-old female Sprague-Dawley albino rats were divided into three groups of 10 rats each. Biochemical and histological examinations were performed. A diet containing 5% L. lepideus fruiting bodies reduced plasma total cholesterol, triglyceride, low-density lipoprotein, total lipid, phospholipids, and the ratio of low-density to high-density lipoprotein. Body weight was reduced. The diet did not adversely affect plasma biochemical and enzyme profiles. L. lepideus reduced significantly plasma ß- and pre-ß-lipoprotein, while α-lipoprotein content was increased. A histological study of hepatic cells by conventional hematoxylin-eosin and oil red O staining revealed normal findings for mushroom-fed hypercholesterolemic rats. The present study suggests that a diet supplemented with L. lepideus can provide health benefits by acting on the atherogenic lipid profile in hypercholesterolemic rats.

Antioxidant Activities and Tyrosinase Inhibitory Effects of Different Extracts from Pleurotus ostreatus Fruiting Bodies.

We evaluated the antioxidant activity and tyrosinase inhibitory effects of Pleurotus ostreatus fruiting bodies extracted with acetone, methanol, and hot water. The antioxidant activities were tested against ß-carotene-linoleic acid, reducing power, 1,1-diphenyl-2-picrylhydrazyl free radical scavenging activity, and ferrous chelating ability. Furthermore, phenolic acid and flavonoid contents were also analyzed. The methanol extract showed the strongest ß-carotene-linoleic acid inhibition as compared to the other exracts. The acetone extract (8 mg/mL) showed a significantly high reducing power of 1.54 than the other extracts. The acetone extract was more effective than other extracts for scavenging on 1,1-diphenyl-2-picrylhydrazyl radicals. The strongest chelating effect (85.66%) was obtained from the acetone extract at 1.0 mg/mL. The antioxidant activities of the extracts from the P. ostreatus fruiting bodies increased with increasing concentration. A high performance liquid chromatography analysis detected seven phenolic compounds, including gallic acid, protocatechuic acid, chlorogenic acid, naringenin, hesperetin, formononetin, and biochanin-A in an acetonitrile and 0.1 N hydrochloric acid (5: 1) solvent extract. The total phenolic compound concentration was 188 µg/g. Tyrosinase inhibition of the acetone, methanol, and hot water P. ostreatus extracts increased with increasing concentration. The results revealed that the methanol extract had good tyrosinase inhibitory ability, whereas the acetone and hot water extracts showed moderate activity at the concentrations tested. The results suggested that P. ostreatus may have potential as a natural antioxidant.

Vegetative Growth and Phylogenetic Relationship of Commercially Cultivated Strains of Pleurotus eryngii based on ITS sequence and RAPD.

Pleurotus eryngii, known as king oyster mushroom has been widely used for nutritional and medicinal purposes. This study was initiated to screen the suitable conditions for mycelial growth and to determine the phylogenetic relationship of the selected strains. Optimal mycelial growth was observed at 30℃ and minimum mycelial growth observed at 10℃. This mushroom tolerates a broad pH range for mycelial growth, with most favorable growth observed at pH 6. Results also indicated that glucose peptone, yeast malt extract and mushroom complete media were favorable growth media, while Hennerberg and Hoppkins media were unfavorable. Dextrin was the best and xylose the least effective carbon sources. Results revealed that inorganic nitrogen sources were less effective than organic sources for the mycelial growth of P. eryngii. Investigation of genetic diversity is necessary to identify the strains. The ITS region of rDNA were amplified using PCR. The size of the ITS1 and ITS2 regions of rDNA from the different strains varied from 214 to 222 bp and 145 to 236 bp, respectively. The sequence of ITS2 was more variable than that of ITS1, and the 5.8S sequences were identical. A phylogenetic tree based on the ITS region sequences indicated that selected strains could be classified into six clusters. Fourteen IUM and ATCC-90212 strains were also analyzed by RAPD with 20 arbitrary primers. Fourteen of these primers were efficiently amplified the genomic DNA. The number of amplified bands varied with the primers and strains, with polymorphic fragments in the range from 0.2 to 2.3 kb.