Structural analysis of turkey tendon collagen upon removal of the inorganic phase

Calcified leg flexor tendons in which the inorganic phase content had been lowered by progressive demineralization were studied by small angle X-ray diffraction and thermogravimetry. The X-ray diffraction results agree very well with the data previously obtained on calcified turkey tendon indicating that the method used to decalcify tendons provides good correspondence with the process of calcification. Up to five thermal processes can be detected in the thermogravimetric scans: (1) water release; (2) collagen decomposition; (3 and 4) combustion of the residual organic components; (5) carbonate removal from the apatitic phase. The temperature of collagen decomposition decreases at lower inorganic phase content in agreement with the higher thermal stability of calcified collagen fibrils compared with uncalcified ones. The decrease of collagen thermal stability upon decalification is paralleled by a decrease of the structural order of the collagen fibrils as indicated by small angle X-ray diffraction data. Decalcification down to about 40% wt of inorganic phase does not significantly alter the inorganic blocks that are regularly arranged inside the gap zone of the collagen. Further removal of inorganic phase down to about 15% wt provokes a variation of the intensity distribution of the small angle meridional reflections that can be ascribed to a reduction of the mean height of the inorganic blocks. At inorganic phase contents below 15% wt the gap region is more free to contract upon air drying as a result of the reduction of the mean length of the inorganic blocks.     

Transduction of human embryonic stem cells by ecotropic retroviral vectors

The steadily increasing availability of human embryonic stem (hES) cell lines has created strong interest in applying available tools for gene transfer in murine cells to human systems. Here we present a method for the transduction of hES cells with ecotropic retroviral vectors. hES cells were transiently transfected with a construct carrying the murine retrovirus receptor mCAT1. Subsequently, the cells were exposed to replication-deficient Moloney murine leukemia virus (MoMuLV) derivatives or pseudotyped lentiviral vectors. With oncoretroviral vectors, this procedure yields overall transduction efficiencies of up to 20% and permits selection of permanently transduced clones with high frequency. Selected clones maintained expression of pluripotency-associated markers and exhibited multi-germ layer differentiation both in vitro and in vivo. HES cell-derived somatic cells including neural progeny maintained high levels of transgene expression. Lentiviral vectors pseudotyped with the MoMuLV envelope could be introduced in the same manner with efficiencies of up to 33%. Transgene expression of lentivirally transduced hES cells remained permanent after differentiation even without selection pressure. Bypassing the regulatory issues associated with the use of amphotropic retroviral systems and exploiting the large pool of existing murine vectors, this method provides a safe and versatile tool for gene transfer and lineage analysis in hES cells and their progeny.     

Novel role for Netrins in regulating epithelial behavior during lung branching morphogenesis

The development of many organs, including the lung, depends upon a process known as branching morphogenesis, in which a simple epithelial bud gives rise to a complex tree-like system of tubes specialized for the transport of gas or fluids. Previous studies on lung development have highlighted a role for fibroblast growth factors (FGFs), made by the mesodermal cells, in promoting the proliferation, budding, and chemotaxis of the epithelial endoderm. Here, by using a three-dimensional culture system, we provide evidence for a novel role for Netrins, best known as axonal guidance molecules, in modulating the morphogenetic response of lung endoderm to exogenous FGFs. This effect involves inhibition of localized changes in cell shape and phosphorylation of the intracellular mitogen-activated protein kinase(s) (ERK1/2, for extracellular signal-regulated kinase-1 and -2), elicited by exogenous FGFs. The temporal and spatial expression of netrin 1, netrin 4, and Unc5b genes and the localization of Netrin-4 protein in vivo suggest a model in which Netrins in the basal lamina locally modulate and fine-tune the outgrowth and shape of emergent epithelial buds.     

Isotopic tracking of foraging and long-distance migration in northeastern Pacific pinnipeds

We investigated the impact of foraging location (nearshore vs offshore) and foraging latitude (high vs middle) on the carbon (δ¹³C) and nitrogen (δ¹⁵N) isotope compositions of bone collagen of northern fur seals (Callorhinus ursinus), harbor seals (Phoca vitulina), California sea lions (Zalophus californianus), and northern elephant seals (Mirounga angustirostris). Nearshore-foraging harbor seals from California had δ¹³C values 2.0‰ higher than female northern elephant seals foraging offshore at similar latitudes. Likewise, nearshore-foraging harbor seals from Alaska had values 1.7‰ higher than male northern fur seals, which forage offshore at high latitudes. Middle-latitude pinnipeds foraging in either the nearshore or offshore were ¹³C enriched by ∼1.0‰ over similar populations from high latitudes. Male northern elephant seals migrate between middle and high latitudes, but they had δ¹³C values similar to high-latitude, nearshore foragers. Female northern fur seal δ¹³C values were intermediate between those of high- and middle-latitude offshore foragers, reflecting their migration between high- and middle-latitude waters. The δ¹³C values of California sea lions were intermediate between nearshore- and offshore-foraging pinnipeds at middle latitudes, yet there was no observational support for the suggestion that they use offshore food webs. We suggest that their “intermediate” values reflect migration between highly productive and less-productive, nearshore ecosystems on the Pacific coasts of California and Mexico. The relative uniformity among all of these pinnipeds in δ¹⁵N values, which are strongly sensitive to trophic level, reveals that the carbon isotope patterns result from differences in the δ¹³C of organic carbon at the base of the food web, rather than differences in trophic structure, among these regions. Finally, the magnitude and direction of the observed nearshore-offshore and high-to middle-latitude differences in δ¹³C values suggest that these gradients may chiefly reflect differences in rates and magnitudes of phytoplankton production as well as the δ¹³C value of inorganic carbon available for photosynthesis, rather than the input of ¹³C-enriched macroalgal carbon to nearshore food webs. Received: 8 September 1998 / Accepted: 24 February 1999     

Potential of fecal microbiota for early-stage detection of colorectal cancer

Several bacterial species have been implicated in the development of colorectal carcinoma (CRC), but CRC-associated changes of fecal microbiota and their potential for cancer screening remain to be explored. Here, we used metagenomic sequencing of fecal samples to identify taxonomic markers that distinguished CRC patients from tumor-free controls in a study population of 156 participants. Accuracy of metagenomic CRC detection was similar to the standard fecal occult blood test (FOBT) and when both approaches were combined, sensitivity improved > 45% relative to the FOBT, while maintaining its specificity. Accuracy of metagenomic CRC detection did not differ significantly between early- and late-stage cancer and could be validated in independent patient and control populations (N = 335) from different countries. CRC-associated changes in the fecal microbiome at least partially reflected microbial community composition at the tumor itself, indicating that observed gene pool differences may reveal tumor-related host–microbe interactions. Indeed, we deduced a metabolic shift from fiber degradation in controls to utilization of host carbohydrates and amino acids in CRC patients, accompanied by an increase of lipopolysaccharide metabolism.     

Source-sink relations in maize mutants with starch-deficient endosperms

Partitioning and translocation of photosynthates were compared between a nonmutant genotype (Oh 43) of corn (Zea mays L.) and two starch-deficient endosperm mutants, shruken-2 (sh2) and brittle-1 (bt1), with similar genetic backgrounds. Steady-state levels of ¹⁴CO₂ were supplied to source leaf blades for 2-hour periods, followed by separation and identification of ¹⁴C-assimilates in the leaf, kernel, and along the translocation path. An average of 14.1% of the total ¹⁴C assimilated was translocated to normal kernels, versus 0.9% in sh2 kernels and 2.6% in btl kernels. Over 98% of the kernel ¹⁴C was in free sugars, and further analysis of nonmutant kernels showed 46% of this label in glucose and fructose. Source leaves of mutant plants exported significantly less total photosynthate (24.0% and 36.3% in sh2 and bt1 compared to 48.0% in the normal plants) and accumulated greater portions of label in the insoluble (starch) fraction. Mutant plants also showed lower percentages of photosynthate in the leaf blade and sheath below the exposed blade area. The starch-deficient endosperm mutants influence the partitioning and translocation of photosynthates and provide a valuable tool for the study of source-sink relations.     

Plant Ontogeny as a Conditionality Factor in the Protective Effect of Ants on a Neotropical Tree

The outcome of any interspecific interaction is often determined by the ecological context in which the interacting species are embedded. Plant ontogeny may represent an important source of variation in the outcome of ant–plant mutualisms, as the level of investment in ant rewards, in alternative (non‐biotic) defenses, or both, may be modulated by the plant's developmental stage. In addition, the abundance and identities of the ants involved in the interaction may change during ontogeny of the host‐plant. Here, we evaluated if plant ontogeny affects the interaction between ants and a savanna tree species (Caryocar brasiliense) that produces extrafloral nectar. We found fewer ants per branch and fewer species of ants per tree in juvenile than in reproductive trees of medium and large size. In addition, large‐sized reproductive trees were more likely to host more aggressive ants than were medium‐sized reproductive or juvenile trees. Such differences strongly affected the outcome of the interaction between ants and their host‐plants, as the magnitude of the effect of ants on herbivory was much stronger for large trees than for juvenile ones. The fact that we did not find significant ontogenetic variation in the concentration of leaf tannins suggests that the observed differences in herbivory did not result from a differential investment in chemical defenses among different‐sized plants. Overall, the results of our study indicate that the developmental stage of the host plant is an important factor of conditionality in the interaction between C. brasiliense and arboreal foraging ants.     

C-Photosynthate Partitioning and Translocation in Soybeans during Reproductive Development

Partitioning and translocation of ¹⁴C-photosynthates were examined during flowering and seed maturation in soybean (Glycine max [L.]Merr.) plants to quantify allocation to sugars, amino acids, organic acids, and starch and to study transport of C and N from leaves to reproductive sinks. The trifoliolate leaf at the eighth node was exposed to steady state levels of ¹⁴CO₂ for 2 hours, followed by immediate extraction and identification of radioactive assimilates in the fed leaf blade, tissues of the transport path (e.g. petiole and stem), and fruits if they were present. About one-third of the total ¹⁴C recovered from the leaf blades was in starch until late pod-filling, after which the proportion dropped to 16%. Sugars comprised 70% to 86% of the recovered ¹⁴C from soluble assimilates of the source leaf, with highest proportions occurring during late flowering and early pod-filling. Amino acids accounted for 8% to 17% of the ¹⁴C recovered from the soluble fraction, and were most evident during early flowering and mid to late pod-filling. The ¹⁴C-organic acids comprised from 3% to 14% of the soluble ¹⁴C-assimilates in leaves. Petioles consistently contained a higher percentage of recovered radioactivity in sugars (87-97%) and a lower percentage in amino acids (3-12%) than did leaf blades. ¹⁴C-Amino acids in petioles attained their highest levels during mid and late pod-filling, while ¹⁴C-organic acids comprised 2% or less of the recovered radioactivity after pod initiation. The distribution of ¹⁴C-assimilates in the internode below the source leaf was similar to that found in petioles. A comparison of the above data to calculated C and N requirements for seed development suggests that ¹⁴C-amino acids derived from current photosynthesis and translocated from source leaves supply at least 12% to 48% of the seed N depending on the stage of pod-filling.