Effects of epidermal growth factor (EGF), transforming growth factor-alpha (TGFalpha), and 2,3,7,8-tetrachlorodibenzo-p-dioxin on fusion of embryonic palates in serum-free organ culture using wild-type, EGF knockout, and TGFalpha knockout mouse strains

BACKGROUND: 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is teratogenic in mice, producing cleft palate (CP). TCDD exposure disrupts expression of epidermal growth factor (EGF) receptor, EGF, and transforming growth factor-alpha (TGFalpha) in the palate and affects proliferation and differentiation of medial epithelial cells. EGF knockout embryos are less susceptible to the induction of CP by TCDD. This study used palate organ culture to examine the hypothesis that EGF enables a response to TCDD. METHODS: The midfacial tissues from wild-type (WT), EGF knockout, C57BL/6J, and TGFalpha knockout embryos were placed in organ culture on gestational day (GD) 12. Palatal explants were cultured for 4 days in serum-free Bigger's (BGJ) medium with 0.1% dimethyl sulfoxide (DMSO) or 1 x 10(-8) M TCDD with or without 2 ng of EGF/ml, 1 or 2 ng of TGFalpha/ml. Effects on palatal fusion were evaluated on day 4 of culture. EGF levels in explants and medium were determined using Luminex technology. RESULTS: In serum-free, control medium, palates from all of the strains fused. EGF knockout palates cultured with TCDD (no EGF) fused, but those cultured with TCDD + 2 ng of EGF/ml failed to fuse (p < 0.05 vs. control or TCDD without EGF). TGFalpha knockout palates failed to fuse when cultured with TCDD + 2 ng of TGFalpha/ml. EGF levels increased in tissue and accumulated in the medium after 24 hr of culture. CONCLUSIONS: This study demonstrated that providing EGF to the palates of EGF knockout mice restored the response to TCDD. These studies support the hypothesis that the mechanism for induction of CP by TCDD is mediated via the EGFR pathway.     

The crystal structure of <Emphasis Type="Italic">Escherichia coli</Emphasis> TdcF,a member of the highly conserved YjgF/YER057c/UK114 family

Background  

The YjgF/YER057c/UK114 family of proteins is widespread in nature, but has as yet no clearly defined biological role. Members of the family exist as homotrimers and are characterised by intersubunit clefts that are delineated by well-conserved residues; these sites are likely to be of functional significance, yet catalytic activity has never been detected for any member of this family. The gene encoding the TdcF protein of E. coli, a YjgF/YER057c/UK114 family member, resides in an operon that strongly suggests a role in the metabolism of 2-ketobutyrate for this protein.     

Molecular characterization of alpha-lactalbumin folding variants that induce apoptosis in tumor cells

This study characterized a protein complex in human milk that induces apoptosis in tumor cells but spares healthy cells. The active fraction was purified from casein by anion exchange chromatography. Unlike other casein components the active fraction was retained by the ion exchanger and eluted after a high salt gradient. The active fraction showed N-terminal amino acid sequence identity with human milk alpha-lactalbumin and mass spectrometry ruled out post-translational modifications. Size exclusion chromatography resolved monomers and oligomers of alpha-lactalbumin that were characterized using UV absorbance, fluorescence, and circular dichroism spectroscopy. The high molecular weight oligomers were kinetically stable against dissociation into monomers and were found to have an essentially retained secondary structure but a less well organized tertiary structure. Comparison with native monomeric and molten globule alpha-lactalbumin showed that the active fraction contains oligomers of alpha-lactalbumin that have undergone a conformational switch toward a molten globule-like state. Oligomerization appears to conserve alpha-lactalbumin in a state with molten globule-like properties at physiological conditions. The results suggest differences in biological properties between folding variants of alpha-lactalbumin.     

Effect of abrupt weaning at housing on leukocyte distribution,functional activity of neutrophils,and acute phase protein response of beef calves

Background  

Sixteen, spring-born, single suckled, castrated male calves of Limousin × Holstein-Friesian and Simmental × Holstein-Friesian dams respectively, were used to investigate the effect of weaning on total leukocyte and differential counts, neutrophil functional activity, lymphocyte immunophenotypes, and acute phase protein response. Calves grazed with their dams until the end of the grazing season when they were housed in a slatted floor shed. On the day of housing, calves were assigned to a treatment, (i) abruptly weaned (W: n = 8) or (ii) non-weaned (controls) (C: n = 8). Weaned calves were housed in pens without their dams, whereas non-weaned (control) calves were housed with their dams. Blood was collected on day -7, 0 (housing), 2, 7, and 14 to determine total leukocyte and differential counts and concentration of fibrinogen and haptoglobin. Lymphocyte immunophenotypes were characterised using selected surface antigens (CD4⁺, CD8⁺, WC1⁺ (γδ T cells), MHC Class II⁺ lymphocytes), and the functional activities of neutrophils (surface expression of L-selectin (CD62L), phagocytic and oxidative burst activity) were investigated using flow cytometry.     

Variation in carbon isotope composition among years in the riparian tree Populus fremontii

Water availability is an important factor limiting the productivity of desert plants but little is known about the impact of water-limiting conditions on the physiology of plants in mesic environments. Riparian ecosystems of the western US receive significantly more water than the surrounding desert environments but experience dramatic interannual fluctuations in water availability because both stream flow and precipitation are highly variable over time. This variability results in different growing conditions each year which may influence the physiology of riparian species such as Fremont cottonwood (Populus fremontii), the dominant, native canopy tree species in lowland southwestern US river systems. We wished to determine if the physiology of this species varies among years, what climatic parameters are related to any observed physiological variation and if individuals within a P. fremontii population differ in their physiological response to variation through time. We collected tree ring cores from a central New Mexico cottonwood population and analyzed carbon isotope composition (δ¹³C) in each year from 1981 to 1995. We used δ¹³C analysis in this study because it allowed us to obtain multi-year estimates of physiological activity. During these years, mean stream flow at our study site ranged over two orders of magnitude from 0.82 to 80.94 m³ s⁻¹, precipitation ranged fourfold from 49 to 215 ccmm and mean temperature ranged from 20.5 to 22.6°C during the growing season. δ¹³C varied from a low of −26.7‰ in 1984 to a high of −24.7‰ in 1981. Low δ¹³C values were associated with years in which stream flow and/or precipitation were high and temperature was low. The opposite was true of years with high δ¹³C values. We observed a strong linear relationship between δ¹³C and stream flow during years when stream flow was <25 m³ s⁻¹ but no significant relationship between these variables when stream flow was >25 m³ s⁻¹. Additionally, there was a linear relationship between δ¹³C and precipitation during years when stream flow was <25 m³ s⁻¹ but not in years when stream flow was >25 m³ s⁻¹. These data suggest that above a threshold of total stream flow, increased flow does not influence physiology. Below this threshold, precipitation can be an important water source. The ten individuals within our study population varied significantly in mean δ¹³C values but responded to interannual variation in a similar manner (i.e., all individuals had low δ¹³C values when water was abundant). These results suggest that precipitation as well as stream flow are important factors influencing the physiology of this riparian tree. Received: 25 November 1998 / Accepted: 9 February 1999     

A novel sulfoglycosphingolipid of mouse small intestine, IV3-sulfogangliotetraosylceramide, demonstrated by negative ion fast atom bombardment mass spectrometry

In a comparative study of acidic glycosphingolipids in the small intestine of several species it was found that the mouse contained a complex sulfoglycolipid as a major component (Breimer, M.E., Hansson, G.C., Karlsson, K.-A., and Leffler, H. (1983) J. Biochem. (Tokyo) 93, 1473-1485). Fast atom bombardment negative ion mass spectrometry proved the presence and location of the sulfate group and also showed the saccharide sequence and ceramide composition. Combined with NMR spectroscopy of the intact structure and degradative studies the structure was shown to be -O3SO----3Galp beta 1----3GalNAcp beta 1----4Galp beta 1----4Glcp beta 1----1Cer. The sulfoglycolipid was enriched in epithelial cells of mouse small intestine where it constituted at least 90% of the acidic glycolipids and 4-8 mol% of the total glycosphingolipids.     

Molecular phylogenetic evidence that the phylum Haplosporidia has an alveolate ancestry

The phylogenetic position of the phylum Haplosporidia among other protists was investigated with the complete 16S-like rRNA gene sequences from two species in the phylum: Haplosporidium nelsoni, a parasite of oysters, and Minchinia teredinis, a parasite of shipworms. Because the lack of obvious morphological homologies with other protists hampered decisions regarding taxonomic composition for sequence alignment and phylogenetic analysis, the complete sequences for these two haplosporidians were directed as search queries to the blast/ncbi.nlm.nih.gov electronic mail server. The results of this heuristic similarity search provided a basis for constructing a preliminary higher-taxonomic-level analysis comparing the haplosporidians with species from the slime molds, fungi, algae, amoebae, ciliates, dinoflagellates, and apicomplexans. Maximum parsimony yielded equivocal results, whereas transversionally weighted parsimony suggested an affinity with the alveolates (i.e., the ciliates, dinoflagellates, and apicomplexans). Multiple alignment of the two haplosporidian sequences against 17 taxa in a secondary analysis focusing on the alveolates and subsequent parsimony analysis placed the phylum Haplosporidia as a monophyletic group within the Alveolata and as a taxon of equal rank with the other three alveolate phyla. The precise placement within the Alveolata was sensitive to weighting.