Differential immunolocalization between lingual antimicrobial peptide and lactoferrin in mammary gland of dairy cows

Lingual antimicrobial peptide (LAP), one of the β-defensins in bovines, and lactoferrin (LF) are synthesized in mammary epithelium and have bactericidal and bacteriostatic functions. However, it is not known whether they have similar expression patterns. Therefore, the present study was undertaken to compare (1) immunolocalization of LAP and LF in the mammary gland and (2) changes in concentration of these two components in milk after lipopolysaccharide (LPS) challenge. Bovine mammary tissues without LPS challenge were collected and their sections were immunostained with antibodies to LAP or LF. Milk from our previous study was collected every hour up to 12h and twice daily from d 1 to 7 after LPS challenge (the day of infusion was considered as d 0). These milk samples were measured for LAP but not LF in our previous report. Therefore, concentration of LF was measured by enzyme immunoassay in the present study. Epithelial cells of some alveoli showed immunopositive reaction for LF, but negative for LAP. Conversely, some alveoli were LAP positive in their epithelial cells but LF negative. Many alveoli had immunoreactions for neither LAP nor LF. The concentration of LAP in milk was elevated significantly at 3h after LPS infusion compared with pre-infusion values and remained at a high level until 12h. However, LF concentration in milk remained low at d 0 and increased at d 2. These results suggest that LAP and LF were mostly differentially localized in the alveolar epithelium in mammary glands. The different spatial expressions between them may be associated with their different temporal expression mechanisms.     

A simulation study for a linear measurement error model when error variances vary between measurements

When predicting scores in the Draize eye irritation test based on measurements of in vitro alternative tests, we are often faced with estimating parameters in a linear measurement error model with heterogeneous error variances. This article proposes a new statistical method for parameter estimation to address this issue. The proposed method is an extension of an earlier proposal that applied a linear measurement error model with homogeneous error variances, to cases with heterogeneous error variances. A simulation study to examine the performance of the proposed method was conducted in a framework that was adaptable to the data, which was obtained in a validation study of alternative methods to animal experiments conducted in Japan. The proposed method reduced the biases of estimates in comparison with an ordinary regression analysis method and three other methods under the assumption of homogeneous error variances. Although the proposed method did not fit the real data well, the resulting prediction formula was far better than those obtained by other methods.     

Effect of EDTA alone and in combination with polygodial on the growth of Saccharomyces cerevisiae

The antifungal activity of ethylenediaminetetraacetic acid (EDTA) against Saccharomyces cerevisiae was significantly affected by various conditions such as inoculum size, pH, and metal ions (Mg(2+), Ca(2+)). EDTA was found to be effective against this yeast at the inoculum size of 10(5) colony forming units (CFU)/mL with the minimum inhibitory concentration of 400 mug/mL and the minimum fungicidal concentration of 6400 mug/mL, but it was not effective at 10(7) CFU/mL up to 6400 mug/mL. The fungicidal activity of EDTA against S. cerevisiae was significantly enhanced in combination with polygodial. Isobolograms, fractional inhibitory concentration, and fractional fungicidal concentration indices were used for evaluating the interaction between combined compounds. This synergistic effect is likely due to polygodial's destructive action on the cellular membrane, which facilitates the transmembrane transport of foreign compounds (EDTA) into yeast cells. Once inside the cells, EDTA forms chelation with divalent metals such as Mg(2+) and Ca(2+), which are required by various essential enzymes.     

Evaluation of free radical scavenging activities of antioxidants with an H(2)O(2)/NaOH/DMSO system by electron spin resonance.

An H(2)O(2)/NaOH/DMSO system has been developed for the formation of three free radicals, and the application of the system was examined with the antioxidants ascorbic acid and tocopherol. Superoxide anion, hydroxyl radical,and methyl radical are simultaneously generated in this system. The scavenging activity of ascorbic acid and tocopherol for these radicals was estimated by 5, 5'-dimethyl-1-pyrroline-N-oxide spin trapping electron spin resonance. Both water-soluble and oil-soluble antioxidants could be evaluated by using this system. Ascorbic acid specifically inhibited the superoxide anion and hydroxyl radical, whereas tocopherol suppressed the methyl radical.     

Structural and Retrogradation Properties of Rice Endosperm Starch Affect Enzyme Digestibility of Steamed Milled-Rice Grains Used in Sake Production

Structural and physicochemical characteristics of endosperm starch from milled rice grains of seven Japanese cultivars used in sake production were examined. Amylose content was 15.2–20.2%, number-average degree of polymerization (DP_n) of amylose was 900–1,400, and the ratio of short-to-long chain amylopectin was 2.7–3.5, respectively. The degree of retrogradation of purified starch stored for seven days at 4°C after gelatinization was 20–31%. The degree of retrogradation correlated negatively with the ratio of short-to-long chain amylopectin. The effect of holding time after steaming on enzyme digestibility and starch retrogradation of steamed rice grains was investigated. The longer the holding time after steaming, the greater the extent of retrogradation, and the less the degree of enzymatic digestibility. The decreased rate of enzyme digestibility correlated with amylopectin chain length distribution. Samples with short-chain amylopectin exhibited a slow decrease in enzyme digestibility. It was determined that the structure and retrogradation properties of endosperm starch in Japanese rice cultivars affect the decreasing rate of enzyme digestibility of the steamed, milled rice grains.     

Preparation and dynamic viscoelasticity characterization of alkali-solubilized collagen from shark skin

Alkali-solubilized collagens, prepared by alkali-acid extraction and alkali direct extraction (abbreviated AASC and ALSC, respectively), were characterized by dynamic viscoelastic measurement of collagen solution (10 mg/mL). The optimum preparative conditions in terms of yield and polypeptide size are as follows: for the alkali-acid extraction, a pretreatment with 0.5 or 1 M NaOH containing 15% Na(2)SO(4) within 5 days at 20 degrees C followed by the subsequent acid extraction, and for the alkaline direct extraction, a treatment with 0.5 M NaOH containing 10% NaCl at 4 degrees C for 20-30 days. A major portion of the polypeptide sizes of AASC and ALSC is composed of alpha chains (alpha1 and alpha2). Dynamic viscoelasticity of collagen solution was measured as a function of temperature. AASC showed a greater contribution of elastic behavior rather than viscous behavior. On the contrary, ALSC exhibits a stronger viscous behavior than elastic behavior.     

Modes of antifungal action of (2E)-alkenals against Saccharomyces cerevisiae

A series of aliphatic (2E)-alkenals from C(5) to C(14) were tested for their antifungal activity against Saccharomyces cerevisiae ATCC 7754. (2E)-Undecenal (C(11)) was found to be the most effective with the minimum fungicidal concentration (MFC) of 6.25 microgram/mL, followed by (2E)-decenal (C(10)) with an MFC of 12.5 microgram/mL. The time-kill curve study showed that (2E)-undecenal was fungicidal against S. cerevisiae at any growth stage, and this activity was not influenced by pH values. The (2E)-alkenals inhibited glucose-induced acidification by inhibiting the plasma membrane H(+)-ATPase. The primary antifungal action of medium-chain (C(9)-C(12)) (2E)-alkenals against S. cerevisiae comes from their ability to function as nonionic surface-active agents (surfactants), disrupting the native membrane-associated function nonspecifically. Hence, the antifungal activity of (2E)-alkenals is mediated by biophysical processes, and the maximum activity can be obtained when the balance between the hydrophilic and hydrophobic portions becomes the most appropriate.     

Bioaugmentation and biostimulation effects on PAH dissipation and soil ecotoxicity under controlled conditions

The degradation of spiked anthracene (ANT), pyrene (PYR) and benzo[a]pyrene (B[a]P) in soil (3000 mg ∑ 3 PAHs kg⁻¹ dry soil) was studied in aerobically incubated microcosms for 120 d. The applied treatments aimed at enhancing PAH removal from the heavily contaminated soils are: (i) bioaugmentation by adding aged PAH-contaminated soil (ACS) containing activated indigenous degraders; and (ii) combined bioaugmentation/biostimulation by incorporating sewage sludge compost (SSC) and decaying rice straw (DRS). The adopted treatments produced higher PAH dissipation rates than those observed in unamended PAH-spiked soils, especially for ANT and PYR in the presence of DRS or ACS (>96%). However, B[a]P was the most recalcitrant hydrocarbon to biodegradation. Extracellular enzyme investigation revealed the existence of ligninolytic activities in all soil treatments, including control but no relationship could be found with PAH dissipation. The ecotoxicological assessment indicated that regardless of applied treatment, PAH-spiked soils were chronically lethal to ostracod Heterocypris incongruens and confirmed the sensitivity of the microcrustacean to the concomitant presence of these three hydrocarbons. Lettuce root elongation inhibition was correlated with PAH level but the presence of SSC conferred a strong phytotoxic capacity to PAH-spiked soils. DRS amendment may constitute a cost-effective alternative for hydrocarbon bioremediation as it has impacted positively on soil microbial activity and enhanced PAH removal with no apparent changes in soil physico-chemical properties.     

Molecular design of antifungal agents

In a rational approach to the design of antifungal agents against Saccharomyces cerevisiae, a series of alkyl gallates (3,4,5-trihydroxybenzoates) were synthesized and assayed. Nonyl gallate (1) was found to be the most effective with a minimum fungicidal concentration (MFC) of 12.5 microg/mL (42 microM), followed by octyl gallate (2) with an MFC of 25 microg/mL (89 microM). These MFCs are little influenced by pH values. A time-kill curve study indicates that nonyl gallate exhibits fungicidal activity against S. cerevisiae at any growing stage. The antifungal activity of nonyl gallate is due primarily to its ability to act as a nonionic surface-active agent (surfactant). The length of the alkyl group is not a major contributor but plays a role in eliciting the activity to a large extent. As far as alkyl gallates are concerned, their antimicrobial spectra and potency depend largely on the hydrophobic portion of the molecules.