Incorporation of axonally transported glycoproteins into axolemma during nerve regeneration

The insertion of axonally transported fucosyl glycoproteins into the axolemma of regenerating nerve sprouts was examined in rat sciatic motor axons at intervals after nerve crush. [(3)H]Fucose was injected into the lumbar ventral horns and the nerves were removed at intervals between 1 and 14 d after labeling. To follow the fate of the “pulse- labeled” glycoproteins, we examined the nerves by correlative radiometric and EM radioautographic approaches. The results showed, first, that rapidly transported [(3)H]fucosyl glycoproteins were inserted into the axolemma of regenerating sprouts as well as parent axons. At 1 d after delivery, in addition to the substantial mobile fraction of radioactivity still undergoing bidirectional transport within the axon, a fraction of label was already associated with the axolemma. Insertion of labeled glycoproteins into the sprout axolemma appeared to occur all along the length of the regenerating sprouts, not just in sprout terminals. Once inserted, labeled glycoproteins did not undergo extensive redistribution, nor did they appear in sprout regions that formed (as a result of continued outgrowth) after their insertion. The amount of radioactivity in the regenerating nerves decreased with time, in part as a result of removal of transported label by retrograde transport. By 7-14 d after labeling, radioautography showed that almost all the remaining radioactivity was associated with axolemma. The regenerating sprouts retained increased amounts of labeled glycoproteins; 7 or 14 d after labeling, the regenerating sprouts had over twice as much of radioactivity as comparable lengths of control nerves or parent axons. One role of fast axonal transport in nerve regeneration is the contribution to the regenerating sprout of glycoproteins inserted into the axolemma; these membrane elements are added both during longitudinal outgrowth and during lateral growth and maturation of the sprout.     

Somatic mosaicism in a patient with bilateral retinoblastoma.

We describe two cell lines with different deletions of the retinoblastoma gene in a patient with bilateral retinoblastoma. This patient has transmitted the mutation less frequent in his lymphocytes to two affected children. We cloned, mapped, and sequenced the junction fragments of the two deletions and found that they share one breakpoint but extend into opposite directions. An insertion of 4 bp of unknown origin is present between the breakpoints in one of the deletions. The second deletion shows a more complex rearrangement, including an inversion at the 5' end. Short regions of homology were found at the breakpoints and flanking the inversion. These results support the notion that bilateral retinoblastoma may not only be due to a germ-line mutation but also to a postzygotic mutation leading to somatic mosaicism.     

Relationship between 5'-nucleotidase, adenosine deaminase, AMP deaminase, ATP-(Mg2+)-ase activities and dTMP kinase activity in rat liver mitochondria.

The activities of dTMP kinase (ATP-deoxythymidine monophosphate phosphotransferase, EC 2.7.4.9), 5'-nucleotidase (5'-ribonucleoside phosphohydrolase, EC 3.1.3.5), adenosine deaminase (adenosine aminohydrolase, EC 3.5.4.4), AMP deaminase (AMP aminohydrolase, EC 3.5.3.6) and ATP-(Mg2+)-ase (ATP phosphohydrolase, EC 3.6.1.3) were assayed in mitochondria of normal and regenerating rat liver. In regenerating mitochondria, the dTMP kinase activity increased 20 times, 5'-nucleotidase (5'Nase) activity for dTMP diminished by 65% and its activity for other nucleoside monophosphates did not change; adenosine deaminase activity for adenosine (AR) increased by 40%, but for deoxyadenosine (AdR) decreased by 70%. AMP deaminase and ATP-(Mg2+)-ase activities behaved similarly in mitochondria from regenerating liver, decreasing by 70 and 64% respectively. The changes of the amount of dTMP in mitochondria depend on enzyme activities which regulate the AdR concentration.     

A Dysfunctional Sense of Smell: The Irreversibility of Olfactory Evolution in Free-Living Pigs

Artificial selection began to override natural selection in domesticated wild boar and other species about 10,000 years ago. The intentional selection of a desired phenotypic trait is a complex process, and comes along with unexpected or even unwanted changes in other traits, because of epistatic gene effects, and ontogenetic constraints. The loss of brain mass in domestic ungulates is related to selection for reduced reaction to external stimuli. Evolutionary losses in body structures and genes were once considered mostly irreversible, in keeping with Dollo’s law. Here we studied the biochemical and the histological functioning of the free-living pigs (FLPs) olfactory system, to see if and to what extent does FLPs regain a full sense of smell, as compared to the domestic pigs and wild boar Sus scrofa. In our samples both wild boars and FLPs have significantly larger brain per unit mass than domestic pigs, and FLPs’ brains are not significantly smaller than wild boar’s. Similarly, both wild boars and FLPs have significantly higher cell density than domestic pigs in the olfactory mucosa. Yet, at the functional level, olfactory marker protein and neuropeptide Y, both of which are important to the correct functioning of the sense of smell, are fully expressed only in wild boar. These results suggest that FLPs reacquired structural, but not the biochemical capability in their olfactory system.     

Cadmium uptake in Elodea canadensis leaves and its interference with extra‐ and intra‐cellular pH

This study investigated cadmium (Cd) uptake in Elodea canadensis shoots under different photosynthetic conditions, and its effects on internal (cytosolic) and external pH. The plants were grown under photosynthetic (light) or non‐photosynthetic (dark or in the presence of a photosynthetic inhibitor) conditions in the presence or absence of CdCl₂ (0.5 μm ) in a medium with a starting pH of 5.0. The pH‐sensitive dye BCECF‐AM was used to monitor cytosolic pH changes in the leaves. Cadmium uptake in protoplasts and leaves was detected with a Cd‐specific fluorescent dye, Leadmium Green AM, and with atomic absorption spectrophotometry. During cultivation for 3 days without Cd, shoots of E. canadensis increased the pH of the surrounding water, irrespective of the photosynthetic conditions. This medium alkalisation was higher in the presence of CdCl₂. Moreover, the presence of Cd also increased the cation exchange capacity of the shoots. The total Cd uptake by E. canadensis shoots was independent of photosynthetic conditions. Protoplasts from plants exposed to 0.5 μm CdCl₂ for 3 days did not exhibit significant change in cytosolic [Cd²⁺] or pH. However, exposure to CdCl₂ for 7 days resulted in increased cytosolic [Cd²⁺] as well as pH. The results suggest that E. canadensis subjected to a low CdCl₂ concentration initially sequesters Cd into the apoplasm, but under prolonged exposure, Cd is transported into the cytosol and subsequently alters cytosolic pH. In contrast, addition of 10–50 μm CdCl₂ directly to protoplasts resulted in immediate uptake of Cd into the cytosol.     

Long-Term Human Impact and Vegetation Changes in a Boreal Forest Reserve: Implications for the Use of Protected Areas as Ecological References

Northern boreal forest reserves that display no signs of modern forest exploitation are often regarded as pristine and are frequently used as ecological reference areas for conservation and restoration. However, the long-term effects of human utilization of such forests are rarely investigated. Therefore, using both paleoecological and archaeological methods, we analyzed temporal and spatial gradients of long-term human impact in a large old-growth forest reserve in the far north of Sweden, comparing vegetational changes during the last millennium at three sites with different land use histories. Large parts of the forest displayed no visible signs of past human land use, and in an area with no recognized history of human land use the vegetation composition appears to have been relatively stable throughout the studied period. However, at two locations effects of previous land use could be distinguished extending at least four centuries back in time. Long-term, but low-intensity, human land use, including cultivation, reindeer herding and tree cutting, has clearly generated an open forest structure with altered species composition in the field layer at settlement sites and in the surrounding forest. Our analysis shows that past human land use created a persistent legacy that is still visible in the present forest ecosystem. This study highlights the necessity for ecologists to incorporate a historical approach to discern underlying factors that have caused vegetational changes, including past human activity. It also indicates that the intensity and spatial distribution of human land use within the landscape matrices of any forests should be assessed before using them as ecological references. The nomenclature of vascular plants follows Krok and Almquist (Svensk flora. Fanerogamer och ormbunksväxter, 2001).     

Deletion of exon I of SMAD7 in mice results in altered B cell responses

The members of the TGF-beta superfamily, i.e., TGF-beta isoforms, activins, and bone morphogenetic proteins, regulate growth, differentiation, and apoptosis, both during embryonic development and during postnatal life. Smad7 is induced by the TGF-beta superfamily members and negatively modulates their signaling, thus acting in a negative, autocrine feedback manner. In addition, Smad7 is induced by other stimuli. Thus, it can fine-tune and integrate TGF-beta signaling with other signaling pathways. To investigate the functional role(s) of Smad7 in vivo, we generated mice deficient in exon I of Smad7, leading to a partial loss of Smad7 function. Mutant animals are viable, but significantly smaller on the outbred CD-1 mouse strain background. Mutant B cells showed an overactive TGF-beta signaling measured as increase of phosphorylated Smad2-positive B cells compared with B cells from wild-type mice. In agreement with this expected increase in TGF-beta signaling, several changes in B cell responses were observed. Mutant B cells exhibited increased Ig class switch recombination to IgA, significantly enhanced spontaneous apoptosis in B cells, and a markedly reduced proliferative response to LPS stimulation. Interestingly, LPS treatment reverted the apoptotic phenotype in the mutant cells. Taken together, the observed phenotype highlights a prominent role for Smad7 in development and in regulating the immune system's response to TGF-beta.     

SH2D2A Modulates T Cell Mediated Protection to a B Cell Derived Tumor in Transgenic Mice

Background

T cell specific adapter protein (TSAd), encoded by the SH2D2A gene, modulates signaling downstream of the T cell receptor (TCR). Young, unchallenged SH2D2A-deficient C57BL/6 mice exhibit a relatively normal immune phenotype. To address whether SH2D2A regulates physiologic immune responses, SH2D2A-deficient TCR-transgenic BALB/c mice were generated. The transgenic TCR recognizes a myeloma-derived idiotypic (Id) peptide in the context of the major histocompatibility complex (MHC) class II molecule I-E^d, and confers T cell mediated resistance to transplanted multiple myeloma development in vivo.

Principal Findings

The immune phenotype of SH2D2A-deficient C57BL/6 and BALB/c mice did not reveal major differences compared to the corresponding wild type mice. When challenged with myeloma cells, Id-specific TCR-transgenic BALB/c mice lacking SH2D2A displayed increased resistance towards tumor development. Tumor free TCR-transgenic SH2D2A-deficient mice had higher numbers of Id-specific single positive CD4+ thymocytes compared to TCR-transgenic wild-type mice.

Conclusion

Our results suggest a modulatory role for SH2D2A in T cell mediated immune surveillance of cancer. However, it remains to be established whether its effect is T-cell intrinsic. Further studies are required to determine whether targeting SH2D2A function in T cells may be a potential adjuvant in cancer immunotherapy.     

The impact of past climate change on genetic variation and population connectivity in the Icelandic arctic fox

Previous studies have suggested that the presence of sea ice is an important factor in facilitating migration and determining the degree of genetic isolation among contemporary arctic fox populations. Because the extent of sea ice is dependent upon global temperatures, periods of significant cooling would have had a major impact on fox population connectivity and genetic variation. We tested this hypothesis by extracting and sequencing mitochondrial control region sequences from 17 arctic foxes excavated from two late-ninth-century to twelfth-century AD archaeological sites in northeast Iceland, both of which predate the Little Ice Age (approx. sixteenth to nineteenth century). Despite the fact that five haplotypes have been observed in modern Icelandic foxes, a single haplotype was shared among all of the ancient individuals. Results from simulations within an approximate Bayesian computation framework suggest that the rapid increase in Icelandic arctic fox haplotype diversity can only be explained by sea-ice-mediated fox immigration facilitated by the Little Ice Age.     

Effects of dietary tin and aluminum on selenium utilization by adult males

The main purpose of these studies was to determine whether the amounts of tin and aluminum that can enter foods during processing and storage are sufficient to affect the utilization of selenium by human subjects. Two 40-day balance studies were conducted. The eight adult males who participated in the first study lost significantly more selenium in their feces when fed a test diet containing 50 mg tin daily than when fed the control diet containing 0.1 mg tin daily. During the first study subjects tended to excrete less selenium in the urine when fed the test diet rather than the control diet. In the second study, the dietary treatments (5 and 125 mg aluminum daily) had no effect on the excretion and apparent retention of selenium by eight adult males.