Role of proteases in fermentation of the fused protein staphylococcal protein A-human insulin-like growth factor I in Staphylococcus aureus

Summary Fermentation of the fused protein staphylococcal protein A-human insulin-like growth factor I (SpA-IGF-I) was investigated. The fused protein was expressed in Staphylococcus aureus and secreted to the culture medium. The production of SpA-IGF-I was growth related and started after a short lag phase. Examination of the product quality by SDS-PAGE after IgG affinity purification showed that the product was partially degraded. Exoproteases appeared later than SpA-IGF-I and were responsible for some, but probably not all, of the degradation of the product. It was possible to influence the exoprotease activity and thereby the product degradation by varying the cultivation conditions.     

Symptomatology and histopathology of fibrous roots of rough lemon (citrus limon) infected with Fusarium solani

Fibrous-root decay and epidermal and cortical sloughing were common on blight trees in two citrus groves with severe symptoms in October 1975. These symptoms were present to varying extent on blight trees in other groves, and some fibrous-root decline also was present on apparently healthy trees. Fusarium solani was present in declining fibrous roots of diseased and healthy trees and occurrence appeared to be limited to the fibrous roots. Fusarium infection resulted in early cortical degeneration, leaving epidermal sleeves on intact, infected steles. Generally, in the xylem and cortex with advanced symptoms, only chlamydospores were present. Sites of early infection conained abundant hyphae and immature chlamydospores. Hyphae penetrated the cortex intracellularly and intercellularly, and all cell types of the xylem appeared to be infected predominantly through pits. Fusarium was found on the surface of the epidermis and in broken epidermal cells of healthy-appearing and diseased roots.This paper reports the results of research only. Mention of a pesticide in this paper does not constitute a recommendation for use by the U.S. Department of Agriculture, nor does it imply registration under FIFRA as amended. Mention of a trademark or proprietary product does not constitute a guarantee or warranty of the product by the U.S. Department of Agriculture and does not imply its approval to the exclusion of other products that may also be suitable.     

Behavior of cultured fusion products fromZygnema andSpirogyra protoplasts

Summary Protoplasts fromZygnema andSpirogyra were fused intergenerically by polyethylene glycol treatment. Nuclei from the two algae did not co-exist in the intergeneric fusion products. Nuclei from one alga degenerated when a lesser number of its protoplasts was involved. When a product involved oneZygnema and oneSpirogyra protoplast, theSpirogyra nucleus was preferentially lost, although nuclei from both tended to be lost. Following degeneration of one algal nucleus, chloroplasts from that alga degenerated gradually, whereas chloroplasts from the other alga remained intact. During degeneration, theSpirogyra chloroplast appeared not grow in length but to swell and accumulated starch grains abundantly, whileZygnema chloroplasts became rounded losing their lobes. A bright green color was maintained in morphologically degenerating chloroplasts. Stable growth took place in the fusion products. Except for the possession of degenerating chloroplasts of the other alga, the elongating products appeared to be the same as elongating, unfused protoplasts or intraspecific fusion products of either alga.     

Metabolism of 2,5-dichlorobenzoic acid inPseudomonas stutzeri

4-Chroropyrocatechol is formed as a results of the oxidation of 2,5-dichlorobenzoate byPseudomonas stutzeri. 3-Chloro-cis,cis-muconic acid is the product of the oxidation of 4-chloropyrocatechol. Pyrocatechol 1,2-dioxygenase, gentisate 1,2-dioxygenase, but not pyrocatechol 2,3-dioxygenase or protocatechuate 3,4-dioxygenase activities were found in cell-free extracts. Theortho cleavage activity for catechols appeared to involve induction of isoenzymes with different stereospecificity towards chlorocatechols. A catablic pathway for the degradation of 2,5-dichlorobenzoate by a newly isolated strain ofP. stutzeri was proposed.     

Halide localization in the teleost chloride cell and its identification by selected area electron diffraction

Further evidence defining an active role for the chloride cell in teleost osmoregulation is presented in this study. Gill filaments from salt water adaptedFundulus heteroclitus were fixed in a solution of silver acetate-osmium tetroxide in an attempt to localize chloride at the level of light and electron microscopes. Characteristically, a reaction product in the form of dense granules appeared concentrated and localized near the margin of the chloride cell apical cavity. Selected area electron diffraction patterns obtained from the localized accumulations of reaction product match diffraction patterns from control (known) silver chloride preparations. It should be emphasized that since the reaction product is not concentrated in other regions of the branchial epithelium, these observations strongly support an electrolyte regulating function for the chloride cell.Supported by a Training Grant (2 G-707) to K. R. Porter from the United States Public Health Service.     

Light and electron microscope localization of beta-glucuronidase activity in the stomach and digestive gland of the marine gastropod Littorina littorea

Summary An azo dye technique was used to investigate localization of the acid hydrolase,-glucuronidase, at light and electron microscope level in the stomach and digestive gland of the marine periwinkleLittorina littorea. Activity for-glucuronidase was located principally within digestive cells of the digestive gland and also associated with the microvillous border and epithelial cells lining the stomach. At the light microscope level all digestive tubules showed activity which appeared essentially restricted to the large heterolysosomes of the digestive cells. However not all digestive cells showed activity. In the electron microscope, reaction product was apparent in all types of macrovesicle in the digestive cells although not all stained positively. Heterophagosomes typically showed reaction product around their periphery or associated with the electron opaque contents. Activity was commonly seen around the apical edge of heterolysosomes where merging of heterophagosomes into heterolysosomes was apparent. Reaction product was commonly located within small electron lucent vesicles which lined the internal membrane of the heterolysosomes but sometimes also associated with flocculent, electron opaque contents. In the stomach dense clusters of reaction product were visible in lysosomes in the basal region of the epithelial cells and in the large granular inclusions of the secretory cells.     

Inhibiting Effect of Browned Processed Foods on Trypsin

Inhibition of proteases was examined in browned foods including soy pastes, soy sauces, fish sauce, sauces, teas, coffees, and others in association with melanoidin, a Maillard product, which had been found to be a potent inhibitor of trypsin. Most of the foods were effective in restraining trypsin activity based on hydrolysis of N-α-benzoyl-DL-arginine-p-nitroanilide, while being ineffective on chymotrypsin except for black tea and cola. Especially, teas were noted for their strong inhibitory effect on trypsin. The color intensity of the foods in terms of optical density at 400 nm appeared not to correlate with the extent of inhibition except for soy sauces.     

Purification of Staphylococcal Alpha Toxin by Electrofocusing

Concentrated, crude alpha toxin from Staphylococcus aureus was purified by two electrofocusing steps. Wide-range followed by narrow-range electrofocusing yielded a product possessing high specific activity, which appeared to be pure by the criteria of ultracentrifugation and immunoelectrophoresis. The apparent isoelectric point of the toxin was 8.65.     

Flavour profile of idli batter prepared from defined microbial starter cultures

Idli is a traditional cereal/legume-based naturally fermented steamed product with a soft and spongy texture which is highly popular and widely consumed as a snack food item in India. The predominant fermentation microflora comprises lactic acid bacteria and yeasts and causes an improvement in the nutritional, textural and flavour characteristics of the final product. The flavour profile of idli batter prepared with initial levels of 2 × 10⁴ c.f.u. g⁻¹ of Candida versatilis CFR 505 and 2 × 10¹ c.f.u. g⁻¹ of Pediococcus pentosaceus CFR 2123 in 500 g idli batter, packed in polyester polylaminate pouches and stored at 30 ± 2 °C was periodically analysed by GC-MS. The desirable flavour compounds such as ketones, diols and acids were found to be present upto 8 days of storage, whereas undesirable flavours like sulphurous and oxazolidone compounds, ethanone and thiazole appeared in the batter subsequent to 6 days of storage. The sensory attributes of idlis (final product) prepared from the stored batter related well to the determined flavour profile. The present study appeared to indicate that the flavour profile of traditional fermented foods can be a reliable qualitative and quantitative parameter for objective assessment. This revised version was published online in July 2006 with corrections to the Cover Date.     

The influence of the rye genome on expression of heat shock proteins in triticale

Summary The heat shock protein profiles from Secale cereale L. cv Imperial, Triticum aestivum L. cv Chinese Spring, S. cereale x T. aestivum amphiploid, and the seven disomic S. cereale addition lines to T. aestivum were used to compare the wheat, rye, and triticale Heat Shock Protein profiles and to study the influence of the rye genome on heat shock protein expression in triticale. Three-day-old seedlings were heat shocked for 2 h at 40 °C in the presence of ³⁵S-methionine, and polypeptides from root tissues were subjected to one- or two-dimensional gel electrophoresis. The wheat and rye heat shock protein profiles each consisted of > 150 heat shock proteins, of which 94 were sufficiently reproducible to construct a standard map. There were 11 unique rye heat shock proteins compared to 22 unique wheat heat shock proteins. The triticale heat shock protein profile resembled the rye parent more than the wheat parent. There were 22 heat shock proteins expressed uniquely by wheat that were not expressed in triticale. Rye chromosomes 1 and 3 exhibited a substantial repressive influence on the expression of 95% of the unique wheat heat shock proteins in triticale, while rye chromosome 4 appeared to have the least repressive influence on expression of the unique wheat heat shock proteins in triticale.Mention of a trade name or proprietary product does not constitute a guarantee, warranty, or recommendation of the product by the United States Department of Agriculture or the University of Missouri and does not imply its approval to the exclusion of other products that may be suitable     

Interrelationship of kernel water activity,soil temperature,maturity, and phytoalexin production in preharvest aflatoxin contamination of drought-stressed peanuts

Samples of Florunner peanuts were collected throughout a period of late-season drought stress with mean geocarposphere temperatures of 29 and 25 °C, and determinations of maturity, kernel water activity (a_w), percent moisture, capacity for phytoalexin production, and aflatoxin contamination were made. Results showed an association between the loss of the capacity of kernels to produce phytoalexins and the appearance of aflatoxin contamination. Kernel a_w appeared to be the most important factor controlling the capacity of kernels to produce phytoalexins. Mature peanuts possessed additional resistance to contamination that could not be attributed solely to phytoalexin production. Kernel moisture loss was accelerated in the 29 °C treatment compared to the 25 °C treatment, and data indicated that the higher soil temperature also favored growth and aflatoxin production by Aspergillus flavus in peanuts susceptible to contamination.Mention of a trademark or proprietary product does not constitute a guarantee or warranty of the product by the U.S. Department of Agriculture and does not imply its approval to the exclusion of other products that may also be suitable.     

Different shake flask closures alter gas phase composition and ajmalicine production in Catharanthus roseus cell suspensions

The type of closure chosen for plant cell cultures can significantly alter the headspace gas composition of a culture, leading to major differences in the production of secondary metabolites. In cell suspension cultures of Catharanthus roseus, ethylene accumulated in cultures with limited gas exchange and appeared to inhibit the production of ajmalicine. The variability in product yields between replicates can also be attributed to gas composition differences.     

Colpidium-Produced RNA as a Growth Stimulant for Tetrahymena*

SYNOPSIS. During logarithmic growth, Colpidium campylum produced a complex, heat-stable material which elevated the reproductive ate of Tetrahymena subsequently inoculated into the culture. This material stimulated only early stages in the growth of Tetrahymena—effect accompanied by an increased number and size of food vacuoles. The Colpidium product was found to be RNA, precipitable from the medium by acetone. Lipids and proteins or peptides were also present in the complex; these appeared to protect the RNA from the action of chemical and physical agents.     

Presence of sourdough lactic acid bacteria in commercial total mixed ration silage as revealed by denaturing gradient gel electrophoresis analysis

Aims: To characterize the bacterial communities in commercial total mixed ration (TMR) silage, which is known to have a long bunk life after silo opening. Methods and Results: Samples were collected from four factories that produce TMR silage according to their own recipes. Three factories were sampled three times at 1‐month intervals during the summer to characterize the differences between factories; one factory was sampled 12 times, three samples each during the summer, autumn, winter and spring, to determine seasonal changes. Bacterial communities were determined by culture‐independent denaturing gradient gel electrophoresis. All silages contained lactic acid as the predominant acid, and the contents appeared stable regardless of factories and product seasons. Acetic acid and 1‐propanol contents were different between factories and indicated seasonal changes, with increases in warm seasons compared to cool seasons. Both differences and similarities existed among the bacterial communities from each factory and product season. Lactobacillus parabuchneri was found in the products from three of four factories. Various sourdough lactic acid bacteria (LAB) were identified in commercial TMR silage; Lactobacillus panis, Lactobacillus hammesii, Lactobacillus mindensis, Lactobacillus pontis, Lactobacillus frumenti and Lactobacillus farciminis were detected in many products. Moreover, changes owing to product season were distinctive, and Lact. pontis and Lact. frumenti became detectable in summer products. Conclusion: Sourdough LAB are involved in the ensiling of commercial TMR silage. Silage bacterial communities vary more by season than by factory. The LAB species Lact. parabuchneri was detected in the TMR silage but may not be essential to the product’s long bunk life after silo opening. Significance and Impact of the Study: Commercial TMR silage resembles sourdough with respect to bacterial communities and long shelf life. The roles of sourdough LAB in the ensiling process and aerobic stability are worth examining.     

The role of alcohol dehydrogenase in the fermentation of D-xylose byCandida shehatae ATCC 22984

Summary When the oxygen supply to an aerobic chemostat culture ofCandida shehatae growing on D-xylose was reduced to oxygen-limited and anoxic conditions, accumulation of ethanol occurred, the specific activity of alcohol dehydrogenase (ADH) increased up to four-fold and the number of ADH isozymes detected increased from one to three. ADH in crude extracts prepared from anoxically-grown cells had a lower affinity for but was more tolerant to ethanol than in extracts prepared from aerobically grown cells. ADH activity appeared to be sufficient for ethanol production byC. shehatae under anoxic conditions.Maintained in cooperation with the University of Wisconsin-Madison. The use of trade, firm or corporation names in this publication is for the information and convenience of the reader. Such use does not constitute an official endorsement or approval by the US Department of any product or service to the exclusion of others which may be suitable.     

Improved preservation of alkaline phosphatase in salivary glands of the cat

Synopsis The effects of tissue preparation on the localization of alkaline phosphatase were assessed at the light and electron microscopical levels. Glutaraldehyde-containing solutions were more inhibitory than formaldehyde alone but appeared to give betterin situ immobilization of the enzyme. The inhibitory effects of glutaraldehyde solutions were related especially to temperature and time and not necessarily to material absorbing at 235 nm. Distilled glutaraldehyde was the only form of glutaraldehyde to give consistently good results with least inhibition. Snap-freezing of pre-fixed tissue, after washing in a sucrose-containing solution, gave satisfactory results without undue ice-crystal formation. Immersion in dimethylsulphoxide before snap-freezing gave less good localization with greater diffusion of reaction product. Use of a Sorvall tissue-chopper on unfrozen tissue did not produce satisfactory sections. Free-floating sections of pre-fixed material appeared less inhibited than glass-mounted sections. The most satisfactory results were obtained after per-arterial perfusion with a 1% distilled glutaraldehyde-0.8% formaldehyde mixture, containing dextran, for up to 5 min followed by formaldehyde-dextran. The results were uniform; there was stronger staining of stromal surfaces of myoepithelial cells than previously, basal duct cells were also stained and occasional staining between acinar cells was now evident for the first time.     

The evolution of proteinase substrates with special reference to dipeptidylpeptidase IV

Summary The design and development of specific substrates for proteolytic enzymes is reviewed. Particular attention is given to substrates containing the leaving groups 4-methoxy-2-naphthylamide (MNA) and 7-amino-4-trifluoromethylcoumarin (AFC). The MNA substrates are used for histochemical and cytochemical purposes, and they yield a coloured final reaction product when azo-coupled with a diazonium salt, an osmiophilic product for electron microscopy when coupled with hexazotized Pararosaniline, or a fluorescent final reaction product when coupled with 5-nitrosalicylaldehyde. AFC substrates are considerably more sensitive, and they yield the fluorescent product AFC after enzymatic cleavage of the substrate. AFC is not sufficiently water-insoluble to allow (intra)cellular localization, but AFC substrates are successfully used for incubations in microwells (Immu-Probe technique) and for the demonstration of banding patterns after gel electrophoresis (enzyme-directed overlay membrane technique). The methods are discussed with the example of the elucidation of the role of dipeptidylpeptidase IV in autoimmune diseases.As presented by R.E.S. for the Pearse Prize Lecture at the Annual Histochemistry Meeting of the Royal Microscopical Society on 8 January 1991 in London, UK. The oral presentation appeared in written form inProc. Roy. Microsc. Soc. 26, 135–43 (1991).     

Synthesis and Antifungal Activity of 2‐Hydroxy‐4,5‐methylenedioxyaryl Ketones as Analogues of Kakuol

In a study aiming to determine the structural elements essential to the antifungal activity of kakuol, we synthesized a series of 2‐hydroxy‐4,5‐methylenedioxyaryl ketones, and we assayed their in vitro antifungal activity. The most sensitive target organisms to the action of these class of compounds were Phytophthora infestans, Phytium ultimum, Cercospora beticola, Cladosporium cucumerinum, and Rhizoctonia solani. Most of the analogs showed a remarkable in vitro activity, and some of them appeared significantly more effective than the natural product. The biological activity was mainly affected by introducing structural modification on the carbonyl moiety of the natural‐product molecule. In particular, compound 5a , bearing a C?C bond conjugated to the C?O group, was found active with a MIC value of 10 μg ml^?1 against Cladosporium cucumerinum. The results suggest that 2‐hydroxy‐4,5‐methylenedioxyaryl ketones can be considered promising candidates in the development of new antifungal compounds.     

Distribution of Methanotrophs in the Phyllosphere

A polygalacturonase gene of Aspergillus awamori IFO 4033 was cloned by genomic Southern hybridization with a probe of a DNA fragment synthesized by PCR. This was done using primers constructed based on the N-terminal amino acid sequence of a polygalacturonase, protopectinase-AS, produced by the strain and the consensus internal amino acid sequence of fungal polygalacturonases. The cloned polygalacturonase gene, containing an ORF, encodes 362 amino acids, including a 52-bp intron. It contains the consensus nucleotide sequence of PacC binding sites, and its expression was appeared to be regulated by ambient pH. After the intron was excised, the cloned gene was inserted into an expression plasmid for yeast, pMA91, and introduced into Saccharomyces cerevisiae to be expressed. The expressed gene product was purified to a homogeneous preparation, and this confirmed that the polygalacturonase produced was the product of the cloned gene.     

Organelle DNA compositions and isoenzyme expression in an interspecific somatic hybrid of Daucus

Summary Cell suspension cultures of Daucus carota, D. capillifolius and a somatic hybrid of these lines were analyzed to determine their chloroplast and mitochondrial DNA compositions. The plastid DNAs (pDNA) from the somatic hybrid and D. carota were identical and were different from that of D. capillifolius when analyzed on agarose electrophoretic gels after digestion by the restriction endonuclease HpaII. The endonuclease restriction patterns of the mitochondrial DNAs (mtDNA) from each cell line were different. Although the restriction pattern of the mtDNA from the somatic hybrid contained fragments in common with one or both parents, unique fragments not found in the restriction pattern of either parent were also present.The amounts and feedback regulation of aspartokinase, homoserine dehydrogenase and dihydrodipicolinic acid synthase were quantified to define the effects of somatic hybridization upon the pathway leading to the biosynthesis of lysine, threonine, methionine and isoleucine. Regulation of each enzyme by end product inhibitors was not altered in the somatic hybrid, but levels of each enzyme appeared to be increased. However, isoenzyme analysis indicated two major forms of homoserine dehydrogenase were present in the hybrid, including one unique form not present in either parent.