New production process of the antifungal chaetoglobosin A using cornstalks

Abstract Chaetoglobosin A is an antibacterial compound produced by Chaetomium globosum, with potential application as a biopesticide and cancer treatment drug. The aim of this study was to evaluate the feasibility of utilizing cornstalks to produce chaetoglobosin A by C. globosum W7 in solid-batch fermentation and to determine an optimal method for purification of the products. The output of chaetoglobosin A from the cornstalks was 0.34 mg/g, and its content in the crude extract was 4.80%. Purification conditions were optimized to increase the content of chaetoglobosin A in the crude extract, including the extract solvent, temperature, and pH value. The optimum process conditions were found to be acetone as the extractant, under room temperature, and at a pH value of 13. Under these conditions, a production process of the antifungal chaetoglobosin A was established, and the content reached 19.17%. Through further verification, cornstalks could replace crops for the production of chaetoglobosin A using this new production process. Moreover, the purified products showed great inhibition against Rhizoctonia solani, with chaetoglobosin A confirmed as the main effective constituent (IC50 = 3.88 µg/mL). Collectively, these results demonstrate the feasibility of using cornstalks to synthesize chaetoglobosin A and that the production process established in this study was effective.

New production process of the antifungal chaetoglobosin A using cornstalks.

Chaetoglobosin A is an antibacterial compound produced by Chaetomium globosum, with potential application as a biopesticide and cancer treatment drug. The aim of this study was to evaluate the feasibility of utilizing cornstalks to produce chaetoglobosin A by C. globosum W7 in solid-batch fermentation and to determine an optimal method for purification of the products. The output of chaetoglobosin A from the cornstalks was 0.34mg/g, and its content in the crude extract was 4.80%. Purification conditions were optimized to increase the content of chaetoglobosin A in the crude extract, including the extract solvent, temperature, and pH value. The optimum process conditions were found to be acetone as the extractant, under room temperature, and at a pH value of 13. Under these conditions, a production process of the antifungal chaetoglobosin A was established, and the content reached 19.17%. Through further verification, cornstalks could replace crops for the production of chaetoglobosin A using this new production process. Moreover, the purified products showed great inhibition against Rhizoctonia solani, with chaetoglobosin A confirmed as the main effective constituent (IC50=3.88µg/mL). Collectively, these results demonstrate the feasibility of using cornstalks to synthesize chaetoglobosin A and that the production process established in this study was effective.

Spatial Distribution of Selected Chemical Cell Wall Components in the Embryogenic Callus of Brachypodium distachyon.

Brachypodium distachyon L. Beauv. (Brachypodium) is a species that has become an excellent model system for gaining a better understanding of various areas of grass biology and improving plant breeding. Although there are some studies of an in vitro Brachypodium culture including somatic embryogenesis, detailed knowledge of the composition of the main cell wall components in the embryogenic callus in this species is missing. Therefore, using the immunocytochemical approach, we targeted 17 different antigens of which five were against the arabinogalactan proteins (AGP), three were against extensins, six recognised pectic epitopes and two recognised hemicelluloses. These studies were complemented by histological and scanning electron microscopy (SEM) analyses. We revealed that the characteristic cell wall components of Brachypodium embryogenic calli are AGP epitopes that are recognised by the JIM16 and LM2 antibodies, an extensin epitope that is recognised by the JIM11 antibody and a pectic epitopes that is recognised by the LM6 antibody. Furthermore, we demonstrated that AGPs and pectins are the components of the extracellular matrix network in Brachypodium embryogenic culture. Additionally, SEM analysis demonstrated the presence of an extracellular matrix on the surface of the calli cells. In conclusion, the chemical compositions of the cell walls and ECMSN of Brachypodium callus show spatial differences that correlate with the embryogenic character of the cells. Thus, the distribution of pectins, AGPs and hemicelluloses can be used as molecular markers of embryogenic cells. The presented data extends the knowledge about the chemical composition of the embryogenic callus cells of Brachypodium.

Callus formation is associated with hyperproliferation and incomplete differentiation of keratinocytes, and increased expression of adhesion molecules.

BACKGROUND: A callus is a local thickening of skin, characterized by accelerated keratinization and a reduced rate of desquamation. However, the mechanism of callus formation is not fully understood. OBJECTIVES: To evaluate the expression patterns, in callused skin, of genes that are implicated in keratinization and adhesion/desquamation. METHODS: Samples of skin from the dorsum of the foot (DF), centre of the plantar arch (CP) and anterior aspect of the heel (AH) were obtained from fresh cadavers, and protein and gene expression were determined by immunohistochemistry and reverse transcription-polymerase chain reaction, respectively. RESULTS: The stratum corneum in the DF showed a splitting phenotype by conventional haematoxylin and eosin staining, while the stratum corneum was normal in the AH. Cells of the stratum corneum in the AH were nonsquamous. Expression of cornification-related molecules including involucrin, filaggrin, caspase 14 and calcium-sensing receptor was higher in the AH. Similarly, expression of adhesive proteins such as corneodesmosin, desmoglein 1 and desmocollin 1 was increased in the AH. However, protease-activated receptor 2 expression was reduced in the stratum granulosum in the AH. The number of proliferating cells in the stratum basale was significantly increased in the AH, compared with the DF and CP. CONCLUSIONS: Our data suggest that calluses form as a result of hyperproliferation and incomplete differentiation of epidermal keratinocytes, and increased expression of adhesion molecules.

Role of tissue-type plasminogen activator in salicylic acid-induced sloughing of human corn tissue.

BACKGROUND: Plasminogen activators (PAs) and their regulatory counterparts, PA inhibitors (PAIs), play a role in normal differentiation processes and various pathophysiologic conditions of the epidermis. Normal desquamation of corneocytes from the skin3s surface may, in part, be regulated by the balanced activities of tissue-type PA (tPA) and PAI-2. Salicylic acid (SA) is commonly used to remove the hyperkeratotic tissue of corns, calluses, and verrucae, and it may disrupt intercellular adhesion structures; however, its exact mechanism of keratolytic action is poorly defined. We sought to determine the effects of SA by comparing the levels of PA and PAI messenger RNA (mRNA) in normal skin, untreated corns, and SA-treated corns. METHODS: Untreated and SA-treated human corn tissue samples were obtained from patients electing surgery to repair bony defects that underlay their lesions. Histopathologic examination of corns was performed by staining the tissue sections with hematoxylin and eosin and by light microscopy. Polymerase chain reaction was used to compare mRNA expression of PAs and PAIs in normal skin, untreated corns, and SA-treated corns. RESULTS: We demonstrated lower tPA and higher PAI-2 mRNA levels in corn tissue compared with normal skin. In corn tissue treated with SA, the expression of tPA mRNA increased and of PAI-2 mRNA decreased to the levels found in normal skin. CONCLUSION: An altered balance in tPA and PAI-2 levels contributes to the induction of hyperkeratotic corn tissue and suggests that the keratolytic action of SA is associated with its ability to stimulate proteinase-meditated desquamation processes.

Accumulation of sunscreens and other compounds in keratinous substrates.

Several cosmetic ingredients, especially sunscreens, should be substantive, which means they are to be adsorbed to specific binding sites within the upper skin layers, particularly keratinized structures of the stratum corneum, and thus show resistance to washing off. We investigated the affinity of 10 non-ionic compounds, among these UV-absorbing chemicals, antioxidants, antimicrobial compounds and a repellent to animal keratin and human callus. In each case a linear relationship between the drug amount, which has accumulated in the respective keratin, and the remaining free concentration of the applied solution could be established. Moreover, drug affinities to keratinous substrates are in direct proportion to the octanol/vehicle partition coefficients, pointing to the fact, that drug enrichment in keratinic substrates is clearly governed by lipophilicity, while specific adsorption, i.e. genuine substantivity, does not seem to occur. After application of a saturated solution non-ionic compounds with a pronounced keratin/vehicle partition coefficient will build up the highest concentration within the stratum corneum. If these compounds show, at the same time, a high solubility in the vehicle, they will penetrate the skin most easily. The used callous tissue seems to be a suitable substrate to simulate and quantify solute uptake into human skin.

Biochemical analysis of callus tissue in osteogenesis imperfecta type IV. Evidence for transient overmodification in collagen types I and III.

We analyzed tissue and cells from a stationary and a rapidly growing hyperplastic callus from a patient with osteogenesis imperfecta (OI) type IV and compared the results with those of compact bone and skin fibroblasts of an age-matched control. Collagen and protein contents per cell were low in the callus tissues and collagen I and III were overmodified as evidenced by an elevated level of hydroxylysine. The degree of lysyl hydroxylation was highest in those regions that appeared most immature by histological examination. Lysyl hydroxylation approached normal levels in collagen from the stationary callus and from the center of the growing callus. Overmodification of collagen was not seen in compact bone or cell cultures (neither skin fibroblasts nor callus cells) from the patient. Elevation of hydroxylysine in collagen from OI patients is generally attributed to mutations that delay triple helix formation. Our observations suggest that the varying degree of collagen modifications may occur in consequence of regulatory mechanisms during bone development and tissue repair. These mechanisms may be defective in some patients with OI as seen in this case with hyperplastic callus formation.

Study of hydration of stratum corneum in leprosy.

The study of hydration power of stratum corneum of lesions show highly significant poor water-uptake at low temperatures (p less than 0.001), a defect not recorded after removal of water soluble fractions. Secondly, the lesions show significant poor water-diffusive power (p less than 0.001). The findings suggest a qualitative alteration in the stratum corneum of leprosy patients, probably in its water-soluble protein fraction.

Characterization of keratin polypeptides of normal and psoriatic horny cells.

Keratin was extracted from normal human horny cells of the leg, calluses of the sole, and psoriatic scales. After dissociation in sodium dodecyl sulfate the polypeptides were separated by Laemmli's gel electrophoresis method and their molecular weights and relative amounts determined. Normal horny cells contained 3 polypeptide chains of Mr 67K, 59K, and 57K, while those of callus contained 9 polypeptides of Mr 67K, 66K, 63K, 62K, 58K, 54K, 52K, 48K, and 45K. In both cases all keratin polypeptides participated in filament reassembly in vitro and were recovered from the filaments. In psoriatic scale keratin, 7 prominent polypeptides were detected having Mr 67K, 59K, 57K, 50K, 48K, 42K, and 40K. The 67K polypeptide could not be recovered from reassembled filaments. Ultrastructural studies revealed that these filaments are imperfect and readily aggregate into thick fibrils. These observations indicate that there are significant differences in composition of keratin of normal horny cells, calluses, and psoriatic scales.

A simplified method for studying fibrous proteins in psoriatic scales obtained by tape stripping.

Polyacrylamide gel electrophoretic investigations were carried out on solubilized proteins from psoriatic and normal stratum corneum obtained by adhesive tape stripping. The proteins in the scales adhering to the tape were solubilized by incubating the tape in 1% sodium dodecyl sulphate (SDS) solution. The electrophoretic behaviour of these solubilized proteins on SDS-polyacrylamide gel was compared with the alpha-fibrous proteins (keratin) of callus. The proteins isolated from callus of normal human heel showed six main bands which were similar to those of the keratin isolated by the 8 M urea-mercaptoethanol method. The lesional skin of forty-five psoriatic patients consistently showed nine main bands on polyacrylamide gels, but only two main bands were observed in the non-lesional, non-heel skin. Six of these nine bands had mobilities and relative intensities almost identical with those of alpha-keratin extracted by the mercaptoethanol method, but the other three bands had greater mobilities on the gels. These results suggest that this technique may have considerable potential for studying changes in alpha-keratin in patients with psoriasis and other disorders of keratinization.

Structural changes of human epidermal alpha-keratin in disorders of keratinization.

The chemistry and structure of the epidermal alpha-keratin extracted from the skin of patients with a variety of disorders of keratinization have been investigated using biochemical, biophysical, and electron microscopic techniques developed for the characterization of normal mammalian epidermal keratin. Generally, the alpha-keratin polypeptides of the diseased epidermis differed from those of uninvolved epidermis or of normal volunteers in having varying numbers of polypeptide components of lower molecular weights, numerous free amino acids, higher contents of alpha-helix, and only limited facility for polymerization in vitro into native-type epidermal keratin filaments. As the alpha-helix-enriched fragments, which represent up to two-thirds of the polypeptide chains, isolated after limited tryptic digestion of the keratin filaments of normal, uninvolved, and involved epidermis, were physicochemically identical, it seems that the end-terminal non-alpha-helical regions of the polypeptides of diseased epidermis are abnormal. These differences may be a result of degradation or of altered protein synthesis.

Composition and function of "hornmark" in cutaneous horns.

In an attempt to elucidate the mechanism underlying the formation of cutaneous horns, 43 cases of cutaneous horns were examined histologically and histochemically. Three of 43 cases were also investigated by using direct immunofluorescence technique and electron microscopy. The structure identical to "Hornmark" was found in all of 43 cases of cutaneous horns except two which consisted only of horny masses. The substance found in the intercellular space of "Hornmark" was homogenous, eosinophilic and mostly diastase-resistant PAS positive, and showed strongly positive immunofluorescence for immunoglobulins, complement, and fibrinogen. Electron microscopically, it was moderately electron dense, fine granular and in part fine fibrillar, and was similar to the contents of capillary lumen. From these findings, it was suggested that the substance found in "Hornmark" consisted mainly of the components of plasma protein, and that coagulated plasma protein might play an important role on the increased cohesiveness of the horny cells, i.d. on the formation of cutaneous horns.

Cholestrol and cholesteryl ester content in normal and pathologic scale.

Using a sensitive new assay, we have measured the cholesteryl ester and cholesterol contents of stratum corneum from callus, normal skin, psoriatic lesions (plantar and nonplantar), and lamellar ichthyotic lesions (plantar and nonplantar). Cholesteryl ester content of normal stratum corneum was significantly higher than that of callus, suggesting that callus was not a suitable control tissue for further biochemical studies involving sterol content of stratum corneum. Both psoriatic and lamellar ichthyotic scale have increased levels of free cholesterol and decreased levels of esterified cholesterol when compared to appropriate controls.