Quantitative analysis of the temporal pattern of somite formation in the mouse and rat. A simple and accurate method for age determination

The relation between the logarithm of the volume of the embryo and the number of somites was studied in the mouse and rat in the period of about 0-43 somites, a period characterized by a very rapid volume increase. A linear relation was found, and the variability of the individual observations around the straight line was rather small. Using a previously defined age estimate, i.e. the 'developmental age' based on the embryonic volume, we also found a linear relation between this age estimate and the number of somites. With the help of this straight line, the developmental age can be estimated from the number of somites with an accuracy of less than 0.1 day. Therefore, the developmental age can be considered a better age estimate than those based on the usual methods. The time needed to form a new somite was found to be constant in both species during the period studied. The length of the period differed, and was 1.68 h in the mouse and 2.24 h in the rat. However, the volume of the embryo at the time when a specific somite was formed (for instance the 25th) appeared to be the same in both species. Comparison of the volume of embryos, embedded in paraffin and Epon and sectioned, enabled us to estimate the shrinkage in Epon embryos. The volume after processing was approximately 62% of the original volume.     

Diminished Reovirus Capsid Stability Alters Disease Pathogenesis and Littermate Transmission

Reovirus is a nonenveloped mammalian virus that provides a useful model system for studies of viral infections in the young. Following internalization into host cells, the outermost capsid of reovirus virions is removed by endosomal cathepsin proteases. Determinants of capsid disassembly kinetics reside in the viral σ3 protein. However, the contribution of capsid stability to reovirus-induced disease is unknown. In this study, we found that mice inoculated intramuscularly with a serotype 3 reovirus containing σ3-Y354H, a mutation that reduces viral capsid stability, succumbed at a higher rate than those infected with wild-type virus. At early times after inoculation, σ3-Y354H virus reached higher titers than wild-type virus at several sites within the host. Animals inoculated perorally with a serotype 1 reassortant reovirus containing σ3-Y354H developed exaggerated myocarditis accompanied by elaboration of pro-inflammatory cytokines. Surprisingly, unchallenged littermates of mice infected with σ3-Y354H virus displayed higher titers in the intestine, heart, and brain than littermates of mice inoculated with wild-type virus. Together, these findings suggest that diminished capsid stability enhances reovirus replication, dissemination, lethality, and host-to-host spread, establishing a new virulence determinant for nonenveloped viruses.     

HPLC photofingerprinting of conformational peculiarities and transitions in oligonucleotide duplexes.

Two self-complementary sequence-isomeric decadeoxyribonucleotides were exposed to UV light under conditions in which they assume duplex structures. After that they were analyzed in the denatured state by reversed-phase high-performance liquid chromatography (HPLC). Characterization of the separated photoproducts allowed localization of cyclobutane pyrimidine dimers in the sequences of the modified oligonucleotides. For [d(GGAAATTTCC)]2, which is known to contain in its central part a stretch of rigid B'-conformation with decreased mobility of constituent bases, lower yields of thymine dimers, as compared with that for ordinary B-form [d(CCTTTAAAGG)]2, were found. On the contrary, mixed thymine-cytosine heterodimers generated in the former oligonucleotide demonstrate the increase in photoreactivity of these residues at the B'-B junction. This is probably due to the peculiar conformation adopted by this decanucleotide. Stimulation of B'-B transition, by increasing the temperature before melting, reduced an inhibition of thymine photodimer formation. During the melting of both oligonucleotides yields of all identified photoinduced cyclobutadipyrimidines were reduced. Possible influences of some metal cations on the stability of the B'-form were also studied by this photoprobing technique. The present study demonstrates the feasibility of HPLC photofingerprinting as a new approach for structural analysis of nucleic acids.     

Environmental regulation of surface conductance for evaporation from vegetation

We examine conductances for evaporation from both vegetation and soil in response to environmental variables. Data from a vertically-structured pristine forest of Nothofagus are presented as an example of the effects of biodiversity on the scaling of conductances between tiers of plant organisation. Available data sets of maximum leaf stomatal conductances (g _lmax ) and bulk vegetation surface conductances (G _smax ) are compared. Overall, the ratio G _smax /g _lmax is consistently close to 3 for seven major vegetation types of diverse structure. An analytical model accounts for this close relationship, and in particular how G _smax is conservative against changes in leaf area index because of the compensating decrease in plant canopy transpiration and increase in soil evaporation as leaf area index diminishes. The model is also successfully tested by comparison with canopy conductances of emergent trees measured in the Nothofagus forest. The constraint of vegetation surface conductance and evaporation via environmental regulation by irradiance, air saturation deficit and root zone water supply are discussed.     

Isomeric lipid signatures reveal compartmentalized fatty acid metabolism in cancer

The cellular energy and biomass demands of cancer drive a complex dynamic between uptake of extracellular FAs and their de novo synthesis. Given that oxidation of de novo synthesized FAs for energy would result in net-energy loss, there is an implication that FAs from these two sources must have distinct metabolic fates; however, hitherto, all FAs have been considered part of a common pool. To probe potential metabolic partitioning of cellular FAs, cancer cells were supplemented with stable isotope-labeled FAs. Structural analysis of the resulting glycerophospholipids revealed that labeled FAs from uptake were largely incorporated to canonical (sn-) positions on the glycerol backbone. Surprisingly, labeled FA uptake also disrupted canonical isomer patterns of the unlabeled lipidome and induced repartitioning of n-3 and n-6 PUFAs into glycerophospholipid classes. These structural changes support the existence of differences in the metabolic fates of FAs derived from uptake or de novo sources and demonstrate unique signaling and remodeling behaviors usually hidden from conventional lipidomics.     

The costs of egg production and incubation in great tits (Parus major).

The costs of egg production and incubation may have a crucial effect on avian reproductive decisions, such as clutch size and the timing of reproduction. We carried out a brood-size enlargement experiment on the great tit (Parus major), in which the birds had to lay and incubate extra eggs (full costs), only incubate extra eggs (free eggs) or did not pay any extra cost (free chicks) in obtaining a larger brood. We used female fitness (half the recruits produced plus female survival) as a fitness measure because it is the female which pays the costs of egg production and incubation, and because clutch size is under female control. Female fitness decreased with increasing costs (fitness of free chicks females is higher than that of free eggs females which is higher than that of full costs females). These fitness differences were due to differences in female survival rather than in the number of recruits produced. This is the first time that the costs of egg production and incubation have been estimated using such a complete fitness measure, including, as our measure does, the local survival to the following year of both the female and her offspring. Our results emphasize that reproductive decisions cannot be understood without taking egg production and incubation costs into account.     

Role of kallikrein-kininogen system in insulin-stimulated glucose transport after muscle contractions.

Serum proteins [molecular weight (MW) > 10,000] are essential for increased insulin-stimulated glucose transport after in vitro muscle contractions. We investigated the role of the kallikrein-kininogen system, including bradykinin, which is derived from kallikrein (MW > 10,000)-catalyzed degradation of serum protein kininogen (MW > 10,000), on this contraction effect. In vitro electrical stimulation of rat epitrochlearis muscles was performed in 1) rat serum +/- kallikrein inhibitors; 2) human plasma (normal or kallikrein-deficient); 3) rat serum +/- bradykinin receptor-2 inhibitors; or 4) serum-free buffer +/- bradykinin. 3-O-methylglucose transport (3-MGT) was measured 3.5 h later. Serum +/- kallikrein inhibitors tended (P = 0.08) to diminish postcontraction insulin-stimulated 3-MGT. Contractions in normal plasma enhanced insulin-stimulated 3-MGT vs. controls, but contractions in kallikrein-deficient plasma did not. Supplementing rat serum with bradykinin receptor antagonist HOE-140 during contraction did not alter insulin-stimulated 3-MGT. Muscles stimulated to contract in serum-free buffer plus bradykinin did not have enhanced insulin-stimulated 3-MGT. Bradykinin was insufficient for postcontraction-enhanced insulin sensitivity. However, results with kallikrein inhibitors and kallikrein-deficient plasma suggest kallikrein plays a role in this improved insulin action.     

Polypyrimidine Tract Binding Protein Prevents Activity of an Intronic Regulatory Element That Promotes Usage of a Composite 3��-Terminal Exon

Alternative splicing of 3′-terminal exons plays a critical role in gene expression by producing mRNA with distinct 3′-untranslated regions that regulate their fate and their expression. The Xenopus α-tropomyosin pre-mRNA possesses a composite internal/3′-terminal exon (exon 9A9′) that is differentially processed depending on the embryonic tissue. Exon 9A9′ is repressed in non-muscle tissue by the polypyrimidine tract binding protein, whereas it is selected as a 3′-terminal or internal exon in myotomal cells and adult striated muscles, respectively. We report here the identification of an intronic regulatory element, designated the upstream terminal exon enhancer (UTE), that is required for the specific usage of exon 9A9′ as a 3′-terminal exon in the myotome. We demonstrate that polypyrimidine tract binding protein prevents the activity of UTE in non-muscle cells, whereas a subclass of serine/arginine rich (SR) proteins promotes the selection of exon 9A9′ in a UTE-dependent way. Morpholino-targeted blocking of UTE in the embryo strongly reduced the inclusion of exon 9A9′ as a 3′-terminal exon in the endogenous mRNA, demonstrating the function of UTE under physiological circumstances. This strategy allowed us to reveal a splicing pathway that generates a mRNA with no in frame stop codon and whose steady-state level is translation-dependent. This result suggests that a non-stop decay mechanism participates in the strict control of the 3′-end processing of the α-tropomyosin pre-mRNA.     

Production of N‐Acetyl‐Phosphinothricin: A Substance used for Inducing Male Sterility in Transgenic Plants

The non‐toxic compound N‐acetyl‐L‐phosphinothricin (N‐Ac‐L‐PPT) is used in a so‐called deacetylation system to induce male sterility in transgenic plants by tapetum specific deacetylation to the herbicide L‐phosphinothricin (L‐PPT). A procedure was developed to produce pure racemic and L‐isomeric N‐Ac‐PPT containing less than 30 ppm residual PPT. Experiments applied to wild type tobacco and PPT‐resistant tobacco showed that the maximal tolerated N‐Ac‐PPT concentration would be less than 45 mM of the L‐isomer. Otherwise unspecific deacetylation by several acylases, as well as by environmental conditions like higher temperatures or pHs beyond neutrality, increased the residual L‐PPT content to toxic concentrations. In contrast, N‐acetyl‐L‐phosphinothricyl‐alanyl‐alanine (N‐Ac‐L‐PPTT), a substance also occurring during the biosynthesis of phosphinothricyl‐alanyl‐alanine (PPTT) by some Streptomyces species, was tolerated up to 274 mM by wild type tobacco plants. However, the ArgE deacatylase from Escherichia coli originally used in the deacetylation system, as well as some other acylases, showed no activity towards N‐Ac‐L‐PPTT.