Preservation of key biomolecules in the fossil record: current knowledge and future challenges.
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We have developed a model based on the analyses of modern and Pleistocene eggshells and mammalian bones which can be used to understand the preservation of amino acids and other important biomolecules such as DNA in fossil specimens. The model is based on the following series of diagenetic reactions and processes involving amino acids: the hydrolysis of proteins and the subsequent loss of hydrolysis products from the fossil matrix with increasing geologic age; the racemization of amino acids which produces totally racemized amino acids in 10(5)-10(6) years in most environments on the Earth; the introduction of contaminants into the fossil that lowers the enantiomeric (D:L) ratios produced via racemization; and the condensation reactions between amino acids, as well as other compounds with primary amino groups, and sugars which yield humic acid-like polymers. This model was used to evaluate whether useful amino acid and DNA sequence information is preserved in a variety of human, amber-entombed insect and dinosaur specimens. Most skeletal remains of evolutionary interest with respect to the origin of modern humans are unlikely to preserve useful biomolecular information although those from high latitude sites may be an exception. Amber-entombed insects contain well-preserved unracemized amino acids, apparently because of the anhydrous nature of the amber matrix, and thus may contain DNA fragments which have retained meaningful genetic information. Dinosaur specimens contain mainly exogenous amino acids, although traces of endogenous amino acids may be present in some cases. Future ancient biomolecule research which takes advantage of new methologies involving, for example, humic acid cleaving reagents and microchip-based DNA-protein detection and sequencing, along with investigations of very slow biomolecule diagenetic reactions such as the racemization of isoleucine at the beta-carbon, will lead to further enhancements of our understanding of biomolecule preservation in the fossil record. (+info)
Salmonella enteritidis phage types 1 and 4: pheno- and genotypic epidemiology of recent outbreaks in Finland.
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In the 1990s, Salmonella enterica subsp. enterica serovar Enteritidis has caused 15 outbreaks in Finland; 12 of them were caused by phage type 1 (PT1) and PT4. Thus far, there has been no clear evidence as to the source of these Salmonella Enteritidis PT1 and PT4 strains, so it was necessary to try to characterize them further. Salmonella Enteritidis PT1 (n = 57) and PT4 (n = 43) isolates from different sources were analyzed by genomic pulsed-field gel electrophoresis (PFGE), plasmid profiling, and antimicrobial resistance testing to investigate the distribution of their subtypes in Finland. It was also hoped that this investigation would help in identifying the sources of the infections, especially the sources of the outbreaks caused by PT1 and PT4 in the 1990s. The results showed that both PFGE and plasmid profiling, but not antimicrobial susceptibility testing, were capable of differentiating isolates of Salmonella Enteritidis PT1 and PT4. By genotypic methods, it was possible to divide both PT1 and PT4 isolates into 12 subtypes. It could also be shown that all PT1 outbreak isolates were identical and, at least with this collection of isolates, that the outbreaks did not originate from the Baltic countries or from Russia, where this phage type predominates. It was also established that the outbreaks caused by PT4 all had different origins. Valuable information for future investigations was gained on the distribution of molecular subtypes of strains that originated from the tourist resorts that are popular among Finns and of strains that were isolated from livestock. (+info)
Lysyl oxidase coupled with catalase in egg shell membrane.
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The activity of lysyl oxidase was found in egg shell membrane (ESM) of hens. The activity was determined by measuring the enzymatic conversion of n-butylamine and Nalpha-acetyl-L-lysine to n-butyraldehyde and Nalpha-acetyl-L-allysine, respectively. ESM lysyl oxidase was significantly inhibited by beta-aminopropionitrile, chelating agents, and deoxygenation, consistent with the known properties of lysyl oxidase. Nevertheless, ESM lysyl oxidase was insoluble in urea solution, suggesting that it complexes with ESM. These findings support previous reports indicating the presence of lysine-derived cross-links in ESM and the necessity of lysyl oxidase located in the isthmus of the hen oviduct for the biosynthesis of ESM. Lysyl oxidase secreted around the egg white from the isthmus may initiate the cross-linking reaction of ESM protein, and remain as the constituent of ESM. Moreover, the H(2)O(2) released by lysyl oxidase in ESM was completely decomposed by coexisting catalase activity. ESM lysyl oxidase activity was greatly elevated in the presence of H(2)O(2), probably due to the O(2) produced by catalase. These findings indicate that lysyl oxidase is coupled with catalase in ESM. This coupling enzyme system was considered to be involved in the biosynthesis of ESM and to protect the embryo against H(2)O(2). (+info)
Histology and ultrastructure of the chorioallantoic membrane of the mallard duck (Anas platyrhynchos).
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The histology and fine structure of the chorioallantoic membrane of the mallard duck (Anas platyrhynchos), and the density of vessels per millimeter of membrane were assessed between days 12 and 24 of incubation. Light and transmission electron microscopy of the chorioallantoic membrane of the mallard duck after various days of incubation was carried out. Blood vessels within the mesoderm were counted per millimeter of membrane by light microscopy (40x). The chorioallantoic membrane had three distinct layers from day 12 to 24 of incubation, the chorionic epithelium, the mesoderm, and the allantoic epithelium. After day 12, chorionic epithelium consisted of two layers of flattened, elongated epithelial cells interfaced by numerous desmosomes, and separated from the underlying mesoderm by a basement membrane. At this stage, the allantoic epithelium consisted of a single layer of flattened, overlapping cells. Blood capillaries were observed in the mesoderm close to the chorionic epithelium on days 12 and 13; by day 14, these capillaries were located within the chorionic epithelium, forming a capillary sinus. Between days 14 and 16, the chorion underwent cellular and cytological differentiation into three cell types: capillary covering cells, villus cavity cells, and less differentiated basal cells. The mesoderm was composed of a loose matrix of mesenchymal cells and collagen fibrils through which coursed blood and lymphatic vessels. The vascular density in the mesoderm increased rapidly from 4.2+/-0.6 vessels per mm (n = 12) on day 12 to a maximum of 9.4+/-0.3 vessels per mm (n = 15) by day 16. From day 16, the allantoic epithelium had two to three layers of elongated and overlapping cells. The luminal layer of allantoic epithelial cells had microvillus projections and varying numbers of membrane-bound dense vesicles at all stages from day 12 onward. The histologic and ultrastructural features of mallard duck chorioallantoic membrane from day 12 to 24 of incubation were very similar to those described in the chorioallantoic membrane of the chicken (Gallus gallus) from day 8 to 20 of incubation. Much of the information available concerning the CAM of the chicken also may apply to the CAM of the mallard, with timing adjusted to match the developmental time-frame recorded here. (+info)
Osteogenic protein-1, a bone morphogenetic protein, induces angiogenesis in the chick chorioallantoic membrane and synergizes with basic fibroblast growth factor and transforming growth factor-beta1.
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Capillary invasion is a vital regulatory signal during bone morphogenesis that is influenced by angiogenic molecules such as fibroblast growth factor (FGF) and some members of the transforming growth factor-beta (TGF-beta) superfamily, including TGF-betas themselves. Bone morphogenetic proteins (BMPs), which are members of the TGF-beta superfamily, have previously not been shown to possess direct angiogenic properties. Osteogenic protein-1 (OP-1; BMP-7) is a potent regulator of cartilage and bone differentiation in vivo. The osteogenic and angiogenic properties of OP-1 at both ortho- and heterotopic sites in adult chacma baboons (Papio ursinus) are enhanced synergistically by the simultaneous application of relatively low doses of TGF-beta1. The single application of relatively high doses of TGF-beta1 (20 ng), and bFGF (500 ng) or relatively low (100 ng) and high (1,000 ng) doses of OP-1 in the chick chorioallantoic membrane (CAM) assay elicited a prominent and (for OP-1) dose-dependent angiogenic response. The binary application of a relatively low dose of OP-1 (100 ng) with a relatively low dose of bFGF (100 ng) or with a relatively low (5 ng) or high (20 ng) dose of TGF-beta1 resulted in a synergistic enhancement of the angiogenic response. The angiogenic effect of the relatively low doses of the combined morphogens was distinctly more pronounced than that of the single application of the relatively high doses of the respective factors. The present findings suggest that these morphogens may be deployed in binary combination in order to accentuate experimental angiogenesis. The cooperative interaction of the different morphogens in the CAM assay may provide important biological clues towards the control of clinical angiogenesis. (+info)
Drosophila dec-1 eggshell proteins are differentially distributed via a multistep extracellular processing and localization pathway.
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In Drosophila the multilayered eggshell forms during late oogenesis between the oocyte and the overlaying follicle cells. Proper eggshell assembly requires wild-type dec-1 gene function. Alternatively spliced dec-1 transcripts encode three proproteins that are cleaved extracellularly in a stage-specific manner to at least five distinct derivatives. Using polyclonal antibodies raised against fusion proteins containing different regions of the dec-1 proteins, we have localized several dec-1 derivatives in the assembling and completed eggshell. Although all of the dec-1 derivatives are generated in the oocyte proximal vitelline membrane layer, they are differentially distributed in the mature egg. Some derivatives are gradually released from the vitelline membrane and become localized within distinct regions of the chorion, while others are taken up by the oocyte or become concentrated in the endochorionic spaces or cavities. The diverse distributions of the dec-1 derivatives suggest that each derivative plays a distinct role in eggshell assembly. These results also suggest that the vitelline membrane layer, by acting as a transient storage site, may control the availability of molecules active in eggshell assembly and by extension perhaps other follicle cell products important in early embryonic pattern formation. (+info)
Maternally derived yolk testosterone enhances the development of the hatching muscle in the red-winged blackbird Agelaius phoeniceus.
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Hatching asynchrony in avian species often leads to the formation of a size hierarchy that places last-hatched nestlings at a significant disadvantage. The hatching muscle (musculus complexus) is responsible for breaking the shell during hatching and for dorsal flexion of the neck during begging. An increase in its strength in last-hatched nestlings could mitigate the effects of hatching asynchrony by reducing the time required for hatching or enhancing the effectiveness of begging for parentally delivered food or both. We have previously found that yolk testosterone concentration increases with laying order in the red-winged blackbird Agelaius phoeniceus. In this study, we investigated the hypothesis that yolk testosterone has anabolic effects on the development of the complexus, thereby influencing competition among asynchronously hatched nestlings. We found that both yolk testosterone concentration and relative complexus mass (complexus mass/nestling body mass) increased with laying order and that these two variables were positively correlated in both newly hatched nestlings and in two-day-old broods. Moreover, direct injections of testosterone into egg yolks resulted in an increase in relative complexus mass, while injections of flutamide, a testosterone antagonist, resulted in a decrease in relative complexus mass. Neither yolk testosterone concentration nor relative complexus mass differed between male and female nestlings. (+info)
Interruption of cardiac output does not affect short-term growth and metabolic rate in day 3 and 4 chick embryos.
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The heart beat of vertebrate embryos has been assumed to begin when convective bulk transport by blood takes over from transport by simple diffusion. To test this hypothesis, we measured eye growth, cervical flexure and rates of oxygen consumption ( V(O2)) in day 3-4 chick embryos denied cardiac output by ligation of the outflow tract and compared them with those of embryos with an intact cardiovascular system. Eye diameter, used as the index for embryonic growth, increased at a rate of approximately 4.5-5 % h(-)(1) during the observation period. There was no significant difference (P>0.1) in the rate of increase in eye diameter between control (egg opened), sham-ligated (ligature present but not tied) and ligated embryos. Similarly, the normal progression of cervical flexure was not significantly altered by ligation (P>0.1). V(O2) (ml O(2 )g(-)(1 )h(-)(1)) at 38 degrees C, measured by closed respirometry, was not significantly different (P>0.1) on day 3 in sham-ligated (14.5+/-1.9 ml O(2 )g(-)(1 )h(-)(1)) and ligated 17.6+/-1.8 ml O(2 )g(-)(1 )h(-)(1)) embryos. Similarly, on day 4, V(O2) in sham-ligated and ligated embryos was statistically the same (sham-ligated 10. 5+/-2.9 ml O(2 )g(-)(1 )h(-)(1); ligated 9.7+/-2.9 ml O(2 )g(-)(1 )h(-)(1)). Expressed as a linear function of body mass (M), V(O2) in sham-ligated embryos was described by the equation V(O2)=-0.48M+24.06 (r(2)=0.36, N=18, P<0.01), while V(O2) in ligated embryos was described by the equation V(O2)=-0.53M+23.32 (r(2)=0.38, N=16, P<0.01). The regression line describing the relationship between body mass and V(O2) for pooled sham-ligated and ligated embryos (the two populations being statistically identical) was V(O2)=-0.47M+23.24. The slope of this regression line, which was significantly different from zero (r(2)=0.30, N=34, P<0.01), was similar to slopes calculated from previous studies over the same range of body mass.Collectively, these data indicate that growth and V(O2) are not dependent upon cardiac output and the convective blood flow it generates. Thus, early chick embryos join those of the zebrafish, clawed frog and axolotl in developing a heart beat and blood flow hours or days before required for convective oxygen and nutrient transport. We speculate that angiogenesis is the most likely role for the early development of a heart beat in vertebrate embryos. (+info)