Localization of the gene for the vitamin B12 binding protein,transcobalamin II,near the centromere on mouse chromosome 11, linked with the hemoglobin alpha-chain locus

Somatic cell hybrids, recombinant inbred (RI) mouse strains, and backcross breeding experiments were used to locate the gene of transcobalamin II (Tcn-2), the vitamin B12 binding protein in mouse serum. TCN-2 was found to be a useful genetic marker in the somatic cell hybrids. Selected hybrid clones were derived from fusions between GR mouse cells and the Chinese hamster cell line E36. Analysis of mouse specific chromosomal enzyme markers in relationship to TCN-2 secretion, in the hybrid clones, provided provisional evidence for assignment of the Tcn-2 locus to chromosome 11. The strain distribution pattern of the TCN-2 variants S and F in the RI series CXS, constructed from the cross of BALB/cHeA (TCN-2S) with STS/A (TCN-2F), implied a close linkage with the hemoglobin alpha-chain locus (Hba) on chromosome 11. Backcross breeding using inbred strains confirmed these findings and located the Tcn-2 gene closest to the centromere, linked with waved 2 (wa-2) and Hba with recombination frequencies of 6.9 and 19.2% each. The linkage group Tcn-2/wa-2/Hba was established.This work was supported by Swiss National Science Foundation Grants 3.023-0.81 and 3.728-0.80, Fritz Hoffmann-La Roche Stiftung 173, the Prof. Dr. Max Cloëtta Foundation, and the Kantonale Zürcher Liga für krebsbekämpfung, Switzerland. M. Prochazka was supported by the Postgraduate Training Program for Experimental Medicine and Biology of the University of Zürich, Switzerland.     

Burning Forests,Rising Power: Towards a Constitutionality Process in Mount Carmel Biosphere Reserve

Powerful states and elites frequently manage protected areas with little or no concern for historic land uses, people, or governance practices, justified by ideologies that portray these areas as “pure nature” to be protected from humans. New international participatory platforms, such as the UNESCO Man and Biosphere Program, coupled with strategic active agency, have provided an opportunity for challenging the fortress model of conservation in Israel. We examine the change in Israel’s government ecological policies following its failure in managing the Carmel forests, whereby its bargaining power with the local Druze-Arab minority was significantly reduced, opening a window of opportunity for the Druze to take advantage of new UNESCO rules on local participation to create management institutions for the local forest commons.     

Day‐flying moths are smaller: evidence for ecological costs of being large

Research on evolutionary forces determining optimal body sizes has primarily relied on experimental evaluation of respective selective pressures. Accounting for among‐species variation through application of phylogenetic comparative methods is a complementary although little used approach. It enables the direct association of body size values with particular environments. Using phylogenetically explicit comparative analyses, we show that small body size is associated with diurnal (rather than nocturnal) activity of adults among temperate species of the moth family Geometridae. The association of an exclusively adult trait with species‐specific body size suggests that optimal body sizes are at least partly determined by the costs being a large adult, as opposed to the more frequently considered costs of attaining large size. It appears likely that size‐selective predation by insectivorous birds is the primary factor responsible for selection against large body size in day‐flying moths.     

Hydrilla control with split treatments of fluridone in Lake Harris, Florida

After several unsuccessful management efforts, a split treatment of fluridone was applied to the 6700 ha Lake Harris in March and June 1987, at a rate of 3.4 kg ha^–1 (680 and 340 kg fluridone, respectively) to two 3 m deep, hydrilla-infested bays. Fluridone concentrations in the water were sampled following the June treatment. Average fluridone concentrations were 2.1 µg l^–1 prior to this second application, and a maximum concentration of 30.2 µg l^–1 was detected in the treated area on the day following application. Fluridone residues dissipated out of the plot quickly due to dilution but concentrations declined lake-wide more slowly, following a logarithmic model, with an estimated fluridone half-life of 97 days. Control of hydrilla in Lake Harris resulted from the long exposure (over 25 weeks due to the split application) to fluridone concentrations of 2 µg l^–1, well below the maximum labelled rate of 150 µg l^–1.     

Tick-borne thogoto virus infection in mice is inhibited by the orthomyxovirus resistance gene product Mx1.

We show that tick-transmitted Thogoto virus is sensitive to interferon-induced nuclear Mx1 protein, which is known for its specific antiviral action against orthomyxoviruses. Influenza virus-susceptible BALB/c mice (lacking a functional Mx1 gene) developed severe disease symptoms and died within days after intracerebral or intraperitoneal infection with a lethal challenge dose of Thogoto virus. In contrast, Mx1-positive congenic, influenza virus-resistant BALB.A2G-Mx1 mice remained healthy and survived. Likewise, A2G, congenic B6.A2G-Mx1 and CBA.T9-Mx1 mice (derived from influenza virus-resistant wild mice) as well as Mx1-transgenic 979 mice proved to be resistant. Peritoneal macrophages and interferon-treated embryo cells from resistant mice exhibited the same resistance phenotype in vitro. Moreover, stable lines of transfected mouse 3T3 cells that constitutively express Mx1 protein showed increased resistance to Thogoto virus infection. We conclude that an Mx1-sensitive step has been conserved during evolution of orthomyxoviruses and suggest that the Mx1 gene in rodents may serve to combat infections by influenza virus-like arboviruses.     

Dominant-negative mutants of human MxA protein: domains in the carboxy-terminal moiety are important for oligomerization and antiviral activity.

Human MxA protein is an interferon-induced 76-kDa GTPase that exhibits antiviral activity against several RNA viruses. Wild-type MxA accumulates in the cytoplasm of cells. TMxA, a modified form of wild-type MxA carrying a foreign nuclear localization signal, accumulates in the cell nucleus. Here we show that MxA protein is translocated into the nucleus together with TMxA when both proteins are expressed simultaneously in the same cell, demonstrating that MxA molecules form tight complexes in living cells. To define domains important for MxA-MxA interaction and antiviral function in vivo, we expressed mutant forms of MxA together with wild-type MxA or TMxA in appropriate cells and analyzed subcellular localization and interfering effects. An MxA deletion mutant, MxA(359-572), formed heterooligomers with TMxA and was translocated to the nucleus, indicating that the region between amino acid positions 359 and 572 contains an interaction domain which is critical for oligomerization of MxA proteins. Mutant T103A with threonine at position 103 replaced by alanine had lost both GTPase and antiviral activities. T103A exhibited a dominant-interfering effect on the antiviral activity of wild-type MxA rendering MxA-expressing cells susceptible to infection with influenza A virus, Thogoto virus, and vesicular stomatitis virus. To determine which sequences are critical for the dominant-negative effect of T103A, we expressed truncated forms of T103A together with wild-type protein. A C-terminal deletion mutant lacking the last 90 amino acids had lost interfering capacity, indicating that an intact C terminus was required. Surprisingly, a truncated version of MxA representing only the C-terminal half of the molecule exerted also a dominant-negative effect on wild-type function, demonstrating that sequences in the C-terminal moiety of MxA are necessary and sufficient for interference. However, all MxA mutants formed hetero-oligomers with TMxA and were translocated to the nucleus, indicating that physical interaction alone is not sufficient for disturbing wild-type function. We propose that dominant-negative mutants directly influence wild-type activity within hetero-oligomers or else compete with wild-type MxA for a cellular or viral target.     

Moderate exercise leads to decreased expression of β1 and β2 integrins on leucocytes

 Intravascular adhesion of leucocytes plays a role in the pathogenesis of acute and chronic vascular disease. Regular aerobic exercise seems to protect against vascular disease. Since leucocyte adhesion is mediated by integrins, we tested the hypothesis that surface expression of the integrin adhesive receptors LFA-1 (cd11a/cd18), MAC-1 (cd11b/cd18), gp 150/95 (cd11c/cd18), and VLA-4 (cd29/cd49) is decreased by moderate endurance exercise. Surface expression of integrins was measured by FACS analysis in 19 healthy subjects (16 males, 3 females, 36.6 ± 8.7 years, 177.1 ± 7.5 cm, 70.3 ± 8.1 kg) before and after submaximal exercise (3 h run) using monoclonal antibodies against cd11a, cd11b, cd11c, cd18, cd29 and cd49. In addition, we compared resting integrin expression in this group with a group of sedentary subjects (19 males, 6 females, 29.3 ± 5.3 years). White blood cell count increased from 5300 ml^–1 to 9740 ml^–1 during exercise (P<0.001). Nevertheless, the expression (indicated by the mean log fluorescence) of cd11a (94 ± 24 vs. 78 ± 14) and cd18 (128 ± 31 vs. 102 ± 21) on lymphocytes and of cd11a (104 ± 25 vs. 85 ± 16), cd11c (497 ± 171 vs. 408 ± 126) cd29 (109 ± 16 vs. 89 ± 16), cd49 (69± 8 vs. 54 ± 11) on monocytes was decreased after exercise (all P<0.05). In contrast, integrin expression on granulocytes was not altered by exercise. Comparison of exercising and sedentary subjects showed a significantly decreased expression of integrins in exercising subjects. Our results demonstrate that moderate exercise leads to decreased expression of integrin receptors on leucocytes. This decreased expression of adhesion molecules may result in decreased adhesion and infiltration of leucocytes into the vessel wall. This phenomenon may play a role in the beneficial effect of moderate exercise in prevention of acute and chronic vascular disease. Accepted: 18 March 1997     

The impact of autophagy on the development of senescence in primary tubular epithelial cells

Autophagy and senescence are 2 distinct pathways that are importantly involved in acute kidney injury and renal repair. Recent data indicate that the 2 processes might be interrelated. To investigate the potential link between autophagy and senescence in the kidney we isolated primary tubular epithelial cells (PTEC) from wild-type mice and monitored the occurrence of cellular senescence during autophagy activation and inhibition. We found that the process of cell isolation and transfer into culture was associated with a strong basal autophagic activation in PTEC. Specific inhibition of autophagy by silencing autophagy-related 5 (Atg5) counteracted the occurrence of senescence hallmarks under baseline conditions. Reduced senescent features were also observed in Atg5 silenced PTEC after γ-irradiation and during H-Ras induced oncogenic senescence, but the response was less uniform in these stress models. Senescence inhibition was paralleled by better preservation of a mature epithelial phenotype in PTEC. Interestingly, treatment with rapamycin, which acts as an activator of autophagy, also counteracted the occurrence of senescence features in PTEC. While we interpret the anti-senescent effect of rapamycin as an autophagy-independent effect of mTOR-inhibition, the more specific approach of Atg5 silencing indicates that overactivated autophagy can have pro-senescent effects in PTEC. These results highlight the complex interaction between cell culture dependent stress mechanisms, autophagy and senescence.     

Effect of hydroxychloroquine and characterization of autophagy in a mouse model of endometriosis

In endometriosis, the increased survival potential of shed endometrial cells (which normally undergo anoikis) is suggested to promote lesion development. One mechanism that may alter anoikis is autophagy. Using an autophagic flux inhibitor hydroxychloroquine (HCQ), we identified that it reduces the in vitro survival capacity of human endometriotic and endometrial T-HESC cells. We also identified that HCQ could decrease lesion numbers and disrupt lesion histopathology, as well as increase the levels of peritoneal macrophages and the IP-10 (10 kDa interferon-γ-induced protein) chemokine in a mouse model of endometriosis. We noted that RNA levels of a subset of autophagic markers were reduced in lesions relative to uterine horns from endometriosis-induced (untreated) mice. In addition, the RNA levels of autophagic markers were decreased in uterine horns of endometriosis-induced mice compared with those from controls. However, we noted that protein expression of LC3B (microtubule-associated protein 1 light-chain 3β; an autophagic marker) was increased in uterine horns of endometriosis-induced mice compared with uterine horns of controls. By immunohistochemical staining of a human endometriosis-focused tissue microarray, we observed LC3B expression predominantly in epithelial relative to stromal cells in both eutopic and ectopic endometria. Via transmission electron microscopy, cells from eutopic endometria of endometriosis-induced mice contained more lipid droplets (rather than autophagosomes) compared with uterine horns from controls. Collectively, our findings indicate that the autophagic pathway is dysregulated in both ectopic and eutopic endometrium in a murine model of endometriosis and that HCQ has potential as a therapeutic agent for women afflicted with endometriosis.Endometriosis is a chronic, painful, and debilitating disease in which endometrium-like glandular and stromal cells grow outside the uterine cavity.^{1, 2} It is an inflammatory and estrogen-dependent disease that affects 6–10% of women during their reproductive years and up to 50% of women receiving fertility treatments.³ Sampson''s hypothesis (the most accepted theory) states that shed endometrial tissue during menses reaches the peritoneal cavity by exiting the uterus through the fallopian tubes by retrograde menstruation.^{4, 5, 6} These shed endometrial cells survive, implant, and grow at ectopic locations, developing into endometriotic lesions.^{5, 7}Epithelial cells normally undergo anoikis, a mechanism of programmed cell death, upon detachment from the extracellular matrix.⁸ One mechanism that we propose could potentially alter the anoikis response in endometrial cells is autophagy. This cellular pathway needs to be carefully regulated to maintain cellular homeostasis.^{9, 10} Under conditions of stress, changes in autophagic flux can lead to altered cellular survival.^{9, 10} Autophagy is a complex process that begins with the formation of double-membrane vesicles, termed autophagosomes, which engulf cytoplasmic components. For a comprehensive review of the autophagic pathway, refer to Feng et al.¹⁰ Briefly, autophagosomes fuse with lysosomes to degrade and recycle their cargo comprised of oxidized proteins, lipids, and damaged organelles. Presently, there is limited evidence that autophagy contributes to the development and progression of endometriosis. In a surgical induction model of murine endometriosis, increased expression of ATG9A, an autophagic mediator that is involved in vesicle formation,¹¹ was detected in the eutopic endometria from endometriosis-induced mice.¹² In human endometriomas (ovarian endometriosis), there was a reduction of LC3-II (the conjugated form of LC3) protein compared with control eutopic endometrial tissue.¹³ In contrast, an independent study reported that the protein expression of LC3-II was elevated, while p62 (which binds ubiquitinated cargo for degradation) was decreased in ovarian endometriomas compared with eutopic endometria of disease-free participants.¹⁴Herein, our main aim was to provide further evidence for a role of autophagy in endometriosis development. Specifically, we sought to determine the therapeutic effects of a lysosomotropic agent and known autophagic flux inhibitor, hydroxychloroquine (HCQ),^{15, 16, 17} on human endometriotic cells and in an established mouse model of endometriosis. The results presented herein are of high clinical translational value, as we identify a potential new non-hormonal treatment for this still incurable and common disease.     

Protein kinase C translocation in intact vascular smooth muscle strips.

Using intact muscle strips from the bovine carotid artery, the time course of translocation of protein kinase C (PKC) from the cytosol to the membrane fraction was measured in response to various agonists that induce contractile responses. PKC activity was assessed by Ca2+/phospholipid-dependent phosphorylation of histone. Exposure of the muscle strips to phorbol ester (12-deoxyphorbol 13-isobutyrate) induced a rapid and sustained translocation of PKC from the cytosol to the membrane fraction, and a slowly developing but sustained contractile response. Histamine induced a comparable initial translocation of PKC to the membrane which then decreased somewhat to a stable plateau significantly above basal values. Histamine also led to a rapid and sustained increase in tension. Angiotensin I, which caused a rapid but transient contraction, induced a rapid initial translocation of PKC to the membrane. The membrane-associated PKC then declined to a stable plateau significantly lower than that seen after a histamine-induced response, and only slightly above the basal value. Endothelin, which induced a sustained contraction, caused a sustained translocation of PKC from the cytosol to the membrane. In contrast, although exposure to 35 mM-KCl induced a rapid and sustained contraction, it caused only a transient translocation of PKC; the membrane-associated PKC returned to its basal value within 20 min. These results demonstrate that PKC in intact smooth muscle can be rapidly translocated to the membrane and remains membrane-bound during sustained phorbol ester- or agonist-induced contractions, but that such a sustained translocation of PKC does not occur during prolonged stimulation with KCl.