Relationship between rates of synthesis and intracellular distribution of ribosomal proteins during oogenesis in the mouse

Ribosomal proteins are synthesized continuously in nonequimolar amounts during oogenesis in the mouse (M. J. LaMarca and P. M. Wassarman, 1979, Develop. Biol. 73, 103), even though ribosomal proteins are found in equimolar amounts in ribosomes. In this report, the distribution of newly synthesized ribosomal proteins between the cytoplasm and germinal vesicle (nucleus) of fully grown mouse oocytes has been examined. As compared to total newly synthesized protein, ribosomal proteins were found to be highly concentrated in the oocyte's germinal vesicle. Furthermore, an inverse relationship was found between rates of synthesis of individual ribosomal proteins and percentages of newly synthesized ribosomal proteins associated with germinal vesicles. As a result of this relationship, the amounts of newly synthesized ribosomal proteins associated with germinal vesicles approximated an equimolar situation. Even in the presence of actinomycin D, oocytes continued to synthesize ribosomal proteins which were found associated with germinal vesicles in amounts similar to those observed in the absence of the drug. These results suggest that, although synthesis of ribosomal proteins by mouse oocytes is not coordinately regulated, a post-translational mechanism exists for adjusting the stoichiometry of these proteins within the oocyte's germinal vesicle; this mechanism apparently is not dependent upon concomitant ribosomal-RNA synthesis.     

Reciprocal and nonreciprocal recombination at the glucocerebrosidase gene region: implications for complexity in Gaucher disease

Gaucher disease results from an autosomal recessive deficiency of the lysosomal enzyme glucocerebrosidase. The glucocerebrosidase gene is located in a gene-rich region of 1q21 that contains six genes and two pseudogenes within 75 kb. The presence of contiguous, highly homologous pseudogenes for both glucocerebrosidase and metaxin at the locus increases the likelihood of DNA rearrangements in this region. These recombinations can complicate genotyping in patients with Gaucher disease and contribute to the difficulty in interpreting genotype-phenotype correlations in this disorder. In the present study, DNA samples from 240 patients with Gaucher disease were examined using several complementary approaches to identify and characterize recombinant alleles, including direct sequencing, long-template polymerase chain reaction, polymorphic microsatellite repeats, and Southern blots. Among the 480 alleles studied, 59 recombinant alleles were identified, including 34 gene conversions, 18 fusions, and 7 downstream duplications. Twenty-two percent of the patients evaluated had at least one recombinant allele. Twenty-six recombinant alleles were found among 310 alleles from patients with type 1 disease, 18 among 74 alleles from patients with type 2 disease, and 15 among 96 alleles from patients with type 3 disease. Several patients carried two recombinations or mutations on the same allele. Generally, alleles resulting from nonreciprocal recombination (gene conversion) could be distinguished from those arising by reciprocal recombination (crossover and exchange), and the length of the converted sequence was determined. Homozygosity for a recombinant allele was associated with early lethality. Ten different sites of crossover and a shared pentamer motif sequence (CACCA) that could be a hotspot for recombination were identified. These findings contribute to a better understanding of genotype-phenotype relationships in Gaucher disease and may provide insights into the mechanisms of DNA rearrangement in other disorders.     

Altered differentiation and paracrine stimulation of mammary epithelial cell proliferation by conditionally activated Smoothened

The Hedgehog (Hh) signaling network is critical for patterning and organogenesis in mammals, and has been implicated in a variety of cancers. Smoothened (Smo), the gene encoding the principal signal transducer, is overexpressed frequently in breast cancer, and constitutive activation in MMTV-SmoM2 transgenic mice caused alterations in mammary gland morphology, increased proliferation, and changes in stem/progenitor cell number. Both in transgenic mice and in clinical specimens, proliferative cells did not usually express detectable Smo, suggesting the hypothesis that Smo functioned in a non-cell autonomous manner to stimulate proliferation. Here, we employed a genetically tagged mouse model carrying a Cre-recombinase-dependent conditional allele of constitutively active Smo (SmoM2) to test this hypothesis. MMTV-Cre- or adenoviral-Cre-mediated SmoM2 expression in the luminal epithelium, but not in the myoepithelium, was required for the hyper-proliferative phenotypes. High levels of proliferation were observed in cells adjacent or in close-proximity to Smo expressing cells demonstrating that SmoM2 expressing cells were stimulating proliferation via a paracrine or juxtacrine mechanism. In contrast, Smo expression altered luminal cell differentiation in a cell-autonomous manner. SmoM2 expressing cells, purified by fluorescence activated cell sorting (FACS) via the genetic fluorescent tag, expressed high levels of Ptch2, Gli1, Gli2, Jag2 and Dll-1, and lower levels of Notch4 and Hes6, in comparison to wildtype cells. These studies provide insight into the mechanism of Smo activation in the mammary gland and its possible roles in breast tumorigenesis. In addition, these results also have potential implications for the interpretation of proliferative phenotypes commonly observed in other organs as a consequence of hedgehog signaling activation.     

Irreversible denaturation mapping of a pyrimidine-rich domain of a complex satellite DNA

The highly complex G + C-rich satellite DNA of the Bermuda land crab Gecarcinus lateralis has been studied by denaturation mapping. Following digestion of the satellite with EndoR.Eco RI, the major 2.07-kilobase pair (kbp) basic repeating unit and a minor 4.14-kbp fragment were exposed to 254 nm light in the presence of silver ions, conditions which resulted in essentially irreversible denaturation of regions rich in adjacent pyrimidines by the formation of pyrimidine dimers. The positions and sizes of the denatured regions were determined in electron micrographs of partially denatured 2.07-kbp and 4.14-kbp fragments spread in the presence of formamide. After 15 min exposure to UV, 90% of the 2.07-kbp fragments had a denaturation bubble averaging 0.17 kbp centered around one-third (0.64 kbp) the total length; 20% exhibited another in the region from 1.8 kbp to 2.07 kbp. Similarly, about 90% of the 4.14-kbp fragments had denatured regions centered at 0.64 kbp and 2.75 kbp and 20% of the fragments had denaturation bubbles in regions centered at 1.92 kbp and 3.9 kbp. The positions of the denaturation bubbles in the 4.14-kbp fragments support restriction enzyme mapping evidence that it is a dimer of the 2.07-kbp fragment arranged head to tail. Sequencing data show that the predominant sequence of a 0.29-kbp region centered around 0.64 kbp in the basic repeat unit is 49% A + T and that 42% of the bases are adjacent TTs and CTs capable of dimerization under the conditions used.     

Quantitative and qualitative analysis of RNA synthesis in stage 6 and stage 4 oocytes of Xenopus laevis

RNA synthesis has been studied in oocytes taken from Xenopus laevis females which have not recently ovulated. Such females contain a population of large (stage 6) oocytes which exhibit white equatorial bands and which are considered to represent the terminal stage of oocyte development. Rates of RNA synthesis in these “banded” oocytes were measured by analyzing the kinetics of incorporation of ³H-guanosine into acid-precipitable, alkaline-labile material, and changes in precursor pool (GTP) specific activity during incubations. In additional experiments, rates of RNA synthesis were measured after ³H-GTP was injected directly into stage 6 oocytes. For comparison, rates of RNA synthesis were measured in lampbrush chromosome stage oocytes (stage 4; 0.5–0.6 mm diameter). The results show that, under the in vitro conditions employed, stage 6 oocytes are not metabolically dormant, but synthesize total RNA at a rate at least as great as the stage 4 oocytes.Qualitative studies on newly synthesized RNA in the two oocyte classes have been performed using sucrose density gradient centrifugation and acrylamide gel electrophoresis. Both stage 4 and stage 6 oocytes exhibited similar patterns, and the bulk of the RNA synthesized and accumulated during 12-hr pulses appears to be ribosomal. These observations are discussed in terms of existing concepts concerning synthetic activity in stage 6 oocytes.     

Brain grafts and Parkinson's disease

In animal models, grafts derived from several different tissues, principally fetal substantia nigra and adrenal medulla from young adults, have been found to be effective in alleviating some of the manifestations of lesions of the substantia nigra. It has been suggested that these grafts function by diffusely secreting dopamine, by exerting trophic effects on the host brain, or by producing a new innervation of the host corpus striatum. Evidence for each of these modes of action is briefly reviewed. Several brain tissue transplantation techniques have been described. Each of these techniques has significant limitations in animal models. The significance of these limitations for human application is described, and possibilities for improving the efficacy of brain tissue transplantation in animal models and for human application are discussed.     

Magnesium Sulfate Treatment Reverses Seizure Susceptibility and Decreases Neuroinflammation in a Rat Model of Severe Preeclampsia

Eclampsia, defined as unexplained seizure in a woman with preeclampsia, is a life-threatening complication of pregnancy with unclear etiology. Magnesium sulfate (MgSO₄) is the leading eclamptic seizure prophylactic, yet its mechanism of action remains unclear. Here, we hypothesized severe preeclampsia is a state of increased seizure susceptibility due to blood-brain barrier (BBB) disruption and neuroinflammation that lowers seizure threshold. Further, MgSO₄ decreases seizure susceptibility by protecting the BBB and preventing neuroinflammation. To model severe preeclampsia, placental ischemia (reduced uteroplacental perfusion pressure; RUPP) was combined with a high cholesterol diet (HC) to cause maternal endothelial dysfunction. RUPP+HC rats developed symptoms associated with severe preeclampsia, including hypertension, oxidative stress, endothelial dysfunction and fetal and placental growth restriction. Seizure threshold was determined by quantifying the amount of pentylenetetrazole (PTZ; mg/kg) required to elicit seizure in RUPP+HC±MgSO₄ and compared to normal pregnant controls (n = 6/group; gestational day 20). RUPP+HC rats were more sensitive to PTZ with seizure threshold being ∼65% lower vs. control (12.4±1.7 vs. 36.7±3.9 mg/kg PTZ; p<0.05) that was reversed by MgSO₄ (45.7±8.7 mg/kg PTZ; p<0.05 vs. RUPP+HC). BBB permeability to sodium fluorescein, measured in-vivo (n = 5–7/group), was increased in RUPP+HC vs. control rats, with more tracer passing into the brain (15.9±1.0 vs. 12.2±0.3 counts/gram ×1000; p<0.05) and was unaffected by MgSO₄ (15.6±1.0 counts/gram ×1000; p<0.05 vs. controls). In addition, RUPP+HC rats were in a state of neuroinflammation, indicated by 35±2% of microglia being active compared to 9±2% in normal pregnancy (p<0.01; n = 3–8/group). MgSO₄ treatment reversed neuroinflammation, reducing microglial activation to 6±2% (p<0.01 vs. RUPP+HC). Overall, RUPP+HC rats were in a state of augmented seizure susceptibility potentially due to increased BBB permeability and neuroinflammation. MgSO₄ treatment reversed this, increasing seizure threshold and decreasing neuroinflammation, without affecting BBB permeability. Thus, reducing neuroinflammation may be one mechanism by which MgSO₄ prevents eclampsia during severe preeclampsia.     

Hypertension in response to CD4(+) T cells from reduced uterine perfusion pregnant rats is associated with activation of the endothelin-1 system

We have shown that adoptive transfer of CD4(+) T cells from placental ischemia (reduction in uteroplacental perfusion, RUPP) rats causes hypertension and elevated inflammatory cytokines during pregnancy. In this study we tested the hypothesis that adoptive transfer of RUPP CD4(+) T cells was associated with endothelin-1 activation as a mechanism to increase blood pressure during pregnancy. CD4(+) T cells from RUPP or normal pregnant (NP) rats were adoptively transferred into NP rats on gestational day 13. Mean arterial pressure (MAP) was analyzed on gestational day 19, and tissues were collected for endothelin-1 analysis. MAP increased in placental ischemic RUPP rats versus NP rats (124.1 ± 3 vs. 96.2 ± 3 mmHg; P = 0.0001) and increased in NP recipients of RUPP CD4(+) T cells (117.8 ± 2 mmHg; P = 0.001 compared with NP). Adoptive transfer of RUPP CD4(+) T cells increased placental preproendothelin-1 mRNA 2.1-fold compared with NP CD4(+) T cell rats and 1.7-fold compared with NP. Endothelin-1 secretion from endothelial cells exposed to NP rat serum was 52.2 ± 1.9 pg·mg(-1)·ml(-1), 77.5 ± 4.3 pg·mg(-1)·ml(-1) with RUPP rat serum (P = 0.0003); 47.2 ± .16 pg·mg(-1)·ml(-1) with NP+NP CD4(+) T cell serum, and 62.2 ± 2.1 pg·mg(-1)·ml(-1) with NP+RUPP CD4(+) T cell serum (P = 0.002). To test the role of endothelin-1 in RUPP CD4(+) T cell-induced hypertension, pregnant rats were treated with an endothelin A (ET(A)) receptor antagonist (ABT-627, 5 mg/kg) via drinking water. MAP was 92 ± 2 mmHg in NP+ET(A) blockade and 108 ± 3 mmHg in RUPP+ET(A) blockade; 95 ± 5 mmHg in NP+NP CD4(+) T cells+ET(A) blockade and 102 ± 2 mmHg in NP+RUPP CD4(+) T cells+ET(A) blockade. These data indicate the importance of endothelin-1 activation to cause hypertension via chronic exposure to activated CD4(+) T cells in response to placental ischemia.     

Dietary salt enhances benzamil-sensitive component of myogenic constriction in mesenteric arteries

Recent work from our laboratory indicates that epithelial Na(+) channel (ENaC) function plays an important role in modulating myogenic vascular reactivity. Increases in dietary sodium are known to affect vascular reactivity. Although previous studies have demonstrated that dietary salt intake regulates ENaC expression and activity in epithelial tissue, the importance of dietary salt on ENaC expression in vascular smooth muscle cells (VSMCs) and its role in myogenic constriction is unknown. Therefore, the goal of the present study was to determine whether dietary salt modulates ENaC expression and function in myogenic vasoconstriction. To accomplish this goal, we examined ENaC expression in freshly dispersed VSMCs and pressure-induced vasoconstrictor responses in isolated mesenteric resistance arteries from normotensive Sprague-Dawley rats fed a normal-salt (NS; 0.4% NaCl) or high-salt (HS; 8% NaCl for 2 wk) diet. VSMCs from the mesenteric arteries of NS-fed animals express alpha-, beta-, and gamma-ENaC. The HS diet reduced whole cell alpha- and gamma-ENaC and induced a pronounced translocation of beta-ENaC from intracellular regions toward the VSMC membrane (approximately 336 nm). Associated with this change in expression was a change in the importance of ENaC in pressure-induced constriction. Pressure-induced constriction in NS-fed animals was insensitive to ENaC inhibition with 1 microM benzamil, suggesting that ENaC proteins do not contribute to myogenic constriction in mesenteric arteries under NS intake. In contrast, ENaC inhibition blocked pressure-induced constriction in HS-fed animals. These data suggest that dietary sodium regulates ENaC expression and the quantitative importance of the vascular ENaC signaling pathway contributing to myogenic constriction.