Detection of age-related changes in the distributions of keratan sulfates and chondroitin sulfates in developing chick limbs: an immunocytochemical study

A panel of four separate monoclonal antibodies, all known to specifically recognize epitopes on keratan sulfate glycosaminoglycans, were employed in an immunocytochemical study of developing chick hind limbs. In addition, two monoclonal antibodies specific for epitopes on chondroitin/dermatan sulfate glycosaminoglycans were employed on equivalent sections to determine the degree of colocalization of keratan and chondroitin/dermatan sulfates. The spatial distributions of keratan sulfate and chondroitin/dermatan sulfate differed to some extent. In younger embryos, high extracellular concentrations of keratan sulfate occurred in joints and articular cartilages, with diminishing amounts being present in epiphyseal and diaphyseal regions. The high concentration of keratan sulfate in joints and articular cartilage corresponded to equally high concentration of chondroitin-6 sulfate. With advancing age, the above mentioned distribution was modified, most notably by increased amounts of keratan sulfate within diaphyseal regions. Finally, the use of four different anti-keratan sulfate monoclonal antibodies made it possible to compare keratan sulfate epitope expression. Differences in keratan sulfate epitopes were noted in some regions of bones, mostly in diaphyseal regions of younger bones and epiphyseal regions of older bones. This pattern of keratan sulfate expression suggests that different types of keratan sulfate may be present and their expression may be developmentally regulated.     

Enhancement of acetaldehyde-protein adduct formation by L-ascorbate

The effect of L-ascorbate on the binding of [14C]acetaldehyde to bovine serum albumin was examined. In the absence of ascorbate, acetaldehyde reacted with albumin to form both unstable (Schiff bases) and stable adducts. Ascorbate (5 mM) caused a time-dependent increase in the formation of total acetaldehyde-albumin adducts, which were comprised mainly of stable adducts. Significant enhancement of adduct formation by ascorbate was observed at acetaldehyde concentrations as low as 5 microM. An ascorbate concentration as low as 0.5 mM was still effective in stimulating stable adduct formation. The electron acceptor, 2,6 dichlorophenolindophenol, prevented the ascorbate-induced increase in albumin-adduct formation. Ascorbate also caused enhanced acetaldehyde adduct formation with other purified proteins, including cytochrome c and histones, as well as the polyamino acid, poly-L-lysine. These results indicate that ascorbate, acting as a reducing agent, can convert unstable acetaldehyde adducts to stable adducts, and can thereby increase and stabilize the binding of acetaldehyde to proteins.     

Azole-resistant Aspergillus fumigatus is highly prevalent in the environment of Vietnam,with marked variability by land use type

Azole-resistant environmental Aspergillus fumigatus presents a threat to public health but the extent of this threat in Southeast Asia is poorly described. We conducted environmental surveillance in the Mekong Delta region of Vietnam, collecting air and ground samples across key land-use types, and determined antifungal susceptibilities of Aspergillus section Fumigati (ASF) isolates and azole concentrations in soils. Of 119 ASF isolates, 55% were resistant (or non-wild type) to itraconazole, 65% to posaconazole and 50% to voriconazole. Azole resistance was more frequent in A. fumigatus sensu stricto isolates (95%) than other ASF species (32%). Resistant isolates and agricultural azole residues were overrepresented in samples from cultivated land. cyp51A gene sequence analysis showed 38/56 resistant A. fumigatus sensu stricto isolates carried known resistance mutations, with TR₃₄/L98H most frequent (34/38).     

Age-related changes in the proteoglycans of human skin. Specific cleavage of decorin to yield a major catabolic fragment in adult skin

Dramatic changes occur in skin as a function of age, including changes in morphology, physiology, and mechanical properties. Changes in extracellular matrix molecules also occur, and these changes likely contribute to the overall age-related changes in the physical properties of skin. The major proteoglycans detected in extracts of human skin are decorin and versican. In addition, adult human skin contains a truncated form of decorin, whereas fetal skin contains virtually undetectable levels of this truncated decorin. Analysis of this molecule, herein referred to as decorunt, indicates that it is a catabolic fragment of decorin rather than a splice variant. With antibody probes to the core protein, decorunt is found to lack the carboxyl-terminal portion of decorin. Further analysis by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry shows that the carboxyl terminus of decorunt is at Phe(170) of decorin. This result indicates that decorunt represents the amino-terminal 43% of the mature decorin molecule. Such a structure is inconsistent with alternative splicing of decorin and suggests that decorunt is a catabolic fragment of decorin. A neoepitope antiserum, anti-VRKVTF, was generated against the carboxyl terminus of decorunt. This antiserum does not recognize intact decorin in any skin proteoglycan sample tested on immunoblots but recognizes every sample of decorunt tested. The results with anti-VRKVTF confirm the identification of the carboxyl terminus of decorunt. Analysis of collagen binding by surface plasmon resonance indicates that the affinity of decorunt for type I collagen is 100-fold less than that of decorin. This observation correlates with the structural analysis of decorunt, in that it lacks regions of decorin previously shown to be important for interaction with type I collagen. The detection of a catabolic fragment of decorin suggests the existence of a specific catabolic pathway for this proteoglycan. Because of the capacity of decorin to influence collagen fibrillogenesis, catabolism of decorin may have important functional implications with respect to the dermal collagen network.     

Spatial and temporal changes in the microbial community in an anaerobic swine waste treatment lagoon

Microorganisms are central to both the beneficial (organic degradation, nutrient removal, biogas production) and detrimental (odor production, pathogen contamination) effects of swine waste storage systems. In this study, both quantitative (real-time polymerase chain reaction) and qualitative (denaturing gradient gel electrophoresis, cloning, sequence analysis) molecular analyses were used to track spatial and temporal changes in the microbial community of swine slurry from a 0.4 ha anaerobic lagoon. The lagoon, located in a region of western Kentucky which has a humid, subtropical environment, was sampled on a monthly basis (n = 10) over a period of one year at four different depths (top, 51 cm from the top, 152 cm from the top, and bottom >198 cm). The concentration and diversity of Bacteroides sp. was seasonal (up to 90% decrease between March and June). Hespellia sp. and other clostridial species, on the other hand, were endemic in the slurry (concentrations up to 1.0 × 10⁷ cells mL^?1 slurry) regardless of time of the year or lagoon depth. Results suggest that there were seasonal effects on the microbial community in the swine lagoon, while the effect of depth was not as pronounced. Seasonal changes in the microbial community in stored wastes may be (directly or indirectly) correlated with changes in malodor emissions from lagoons.     

Production of human CD59-transgenic pigs by embryonic germ cell nuclear transfer

This study was performed to produce transgenic pigs expressing the human complement regulatory protein CD59 (hCD59) using the nuclear transfer (NT) of embryonic germ (EG) cells, which are undifferentiated stem cells derived from primordial germ cells. Because EG cells can be cultured indefinitely in an undifferentiated state, they may provide an inexhaustible source of nuclear donor cells for NT to produce transgenic pigs. A total of 1980 NT embryos derived from hCD59-transgenic EG cells were transferred to ten recipients, resulting in the birth of fifteen piglets from three pregnancies. Among these offspring, ten were alive without overt health problems. Based on PCR analysis, all fifteen piglets were confirmed as hCD59 transgenic. The expression of the hCD59 transgene in the ten living piglets was verified by RT-PCR. Western analysis showed the expression of the hCD59 protein in four of the ten RT-PCR-positive piglets. These results demonstrate that hCD59-transgenic pigs could effectively be produced by EG cell NT and that such transgenic pigs may be used as organ donors in pig-to-human xenotransplantation.     

Synthesis, antifungal and antimicrobial activity of alkylphospholipids

The antifungal, antibacterial and haemolytic activity of a series of alkylphosphocholines (e.g., miltefosine) and alkylglycerophosphocholines (e.g., edelfosine) has been investigated. These compound classes exhibit significant antifungal and moderate antibacterial activities. Several new alkylphosphocholine derivatives with amide or ester bonds in the alkyl chain have been synthesised. These compounds show much lower haemolytic activity than miltefosine. Alkylphosphocholines and alkylglycerophosphocholines show significant promise as novel orally available antifungal and antibacterial therapeutics.