Mechanisms governing maintenance of Cdk1/cyclin B1 kinase activity in cells infected with human cytomegalovirus

Previous work has demonstrated dysregulation of key cell cycle components in human cytomegalovirus (HCMV)-infected human fibroblasts, resulting in cell cycle arrest (F. M. Jault, J.-M. Jault, F. Ruchti, E. A. Fortunato, C. L. Clark, J. Corbeil, D. D. Richman, and D. H. Spector, J. Virol. 69:6697-6704, 1995). The activation of the mitotic kinase Cdk1/cyclin B, which was detected as early as 8 h postinfection (p.i.) and maintained throughout the time course, was particularly interesting. To understand the mechanisms underlying the induction of this kinase activity, we have examined the pathways that regulate the activation of Cdk1/cyclin B1 complexes. The accumulation of the cyclin B1 subunit in HCMV-infected cells is the result of increased synthesis and reduced degradation of the protein. In addition, the catalytic subunit, Cdk1, accumulates in its active form in virus-infected cells. The decreased level of the Tyr15-phosphorylated form of Cdk1 in virus-infected fibroblasts is due in part to the down-regulation of the expression and activity of the Cdk1 inhibitory kinases Myt1 and Wee1. Increased degradation of Wee1 via the proteasome also accounts for its absence at 24 h p.i. At late times, we observed accumulation of the Cdc25 phosphatases that remove the inhibitory phosphates from Cdk1. Interestingly, biochemical fractionation studies revealed that the active form of Cdk1, a fraction of total cyclin B1, and the Cdc25 phosphatases reside predominantly in the cytoplasm of infected cells. Collectively, these data suggest that the maintenance of Cdk1/cyclin B1 activity observed in HCMV-infected cells can be explained by three mechanisms: the accumulation of cyclin B1, the inactivation of negative regulatory pathways for Cdk1, and the accumulation of positive factors that promote Cdk1 activity.     

Rh-RhAG/ankyrin-R,a new interaction site between the membrane bilayer and the red cell skeleton,is impaired by Rh(null)-associated mutation

Several studies suggest that the Rh complex represents a major interaction site between the membrane lipid bilayer and the red cell skeleton, but little is known about the molecular basis of this interaction. We report here that ankyrin-R is capable of interacting directly with the C-terminal cytoplasmic domain of Rh and RhAG polypeptides. We first show that the primary defect of ankyrin-R in normoblastosis (nb/nb) spherocytosis mutant mice is associated with a sharp reduction of RhAG and Rh polypeptides. Secondly, our flow cytometric analysis of the Triton X-100 extractability of recombinant fusion proteins expressed in erythroleukemic cell lines suggests that the C-terminal cytoplasmic domains of Rh and RhAG are sufficient to mediate interaction with the erythroid membrane skeleton. Using the yeast two-hybrid system, we demonstrate a direct interaction between the cytoplasmic tails of Rh and RhAG and the second repeat domain (D2) of ankyrin-R. This finding is supported by the demonstration that the substitution of Asp-399 in the cytoplasmic tail of RhAG, a mutation associated with the deficiency of the Rh complex in one Rhnull patient, totally impaired interaction with domain D2 of ankyrin-R. These results identify the Rh/RhAG-ankyrin complex as a new interaction site between the red cell membrane and the spectrin-based skeleton, the disruption of which might result in the stomato-spherocytosis typical of Rhnull red cells.     

Human leukocyte formin: a novel protein expressed in lymphoid malignancies and associated with Akt

The very large family of Formin proteins is involved in processes such as morphogenesis, embryonic differentiation, cell polarity, and cytokinesis. A novel human gene from the Formin family, denominated human leukocyte formin gene, was cloned. The cDNA of the gene was determined to be 3959bp long with an open reading frame of 3302bp and computational analysis located this gene on chromosome 17, suggesting that it is composed of 27 exons. Northern blot analysis revealed a restricted expression of mRNA in the thymus, spleen, and peripheral blood leukocytes in normal human tissues. Western blot analysis demonstrated that the protein encoded by this gene is overexpressed in lymphoid malignancies; cancer cell lines and peripheral blood leukocyte from chronic lymphocytic leukemia (CLL) patients. Furthermore, the human leukocyte formin protein was observed to associate with Akt, a critical survival regulator in many different cell types.     

Lack of species-specificity in mammalian sperm chemotaxis

Attraction of spermatozoa by way of chemotaxis to substances secreted from the egg or its surrounding cells has been demonstrated in marine species, amphibians, and mammals. This process is species- or family-specific in marine invertebrates: a chemoattractant for one marine species is usually not recognized by another species or by a member of another family. It is not known whether this selectivity is also the rule in other phyla. Furthermore, it is not at all obvious that such selectivity would be advantageous to species with internal fertilization. Here, using a directionality-based assay for chemotaxis, we studied in vitro the chemotactic response of human and rabbit spermatozoa to human, rabbit, and bovine egg-related factors. We found that spermatozoa from each of the two sources responded similarly well to egg-related factors obtained from any of the three species examined. These results indicate lack of chemotaxis-related, species specificity between these species, suggesting that their sperm chemoattractants are common or very similar. The findings further suggest that mammals do not rely on species specificity of sperm chemotaxis for avoidance of interspecies fertilization.     

sGC and PDE5 are elevated in lambs with increased pulmonary blood flow and pulmonary hypertension

Utilizing aortopulmonary vascular graft placement, we established a lamb model of pulmonary hypertension that mimics congenital heart disease with increased pulmonary blood flow. We previously demonstrated that endothelial nitric oxide synthase (eNOS) is increased in lambs at age 4 wk. However, these lambs display a selective impairment of endothelium-dependent pulmonary vasodilation that is suggestive of a derangement downstream of NO release. Thus our objective was to characterize potential alterations in the expression and activity of soluble guanylate cyclase (sGC) and phosphodiesterase type 5 (PDE5) induced by increased pulmonary blood flow and pulmonary hypertension. Late-gestational fetal lambs (n = 10) underwent in utero placement of an aortopulmonary vascular graft (shunt). Western blotting analysis on lung tissue from 4-wk-old shunted lambs and age-matched controls showed that protein for both subunits of sGC was increased in shunted lamb lungs compared with age-matched controls. Similarly, cGMP levels were increased in shunted lamb lungs compared with age-matched controls. However, PDE5 expression and activity were also increased in shunted lambs. Thus although cGMP generation was increased, concomitant upregulation of PDE5 expression and activity may have (at least partially) limited and accounted for the impairment of endothelium-dependent pulmonary vasodilation in shunted lambs.     

Effects of oral aluminum on essential trace elements metabolism during pregnancy

The present study was conducted to assess in rats the effects of oral aluminum (Al) exposure on calcium (Ca), magnesium (Mg), manganese (Mn), copper (Cu), zinc (Zn), and iron (Fe) accumulation and urinary excretion. Three groups of plug-positive Sprague-Dawley (SD) rats were given by gavage 0, 200, and 400 mg/kg/d of Al(OH)₃ on gestational days 1–20. Three groups of nonpregnant female SD rats of the same age received Al(OH)₃ by gavage at the same doses for 20 consecutive days. At the end of the treatment period, 24-h urine samples were collected for analysis of Al and essential elements. Subsequently, all animals were sacrificed and samples of liver, bone, spleen, kidneys, and brain were removed for metal analyses. With some exceptions, the urinary amounts of Al, Mn, and Cu excreted by pregnant animals as well as the urinary levels of Al excreted by nonpregnant rats were higher in the Al-treated groups than in the respective control groups. Although higher Al levels were found in the liver of pregnant rats, the concentrations of Al in the brain of these animals were lower than those found in the same tissues of nonpregnant rats. With regard to the essential elements, tissue accumulation was most affected in pregnant than in nonpregnant animals. In pregnant rats, the hepatic and renal concentrations of Ca, Mg, Mn, Cu, Zn, and Fe, as well as the levels of Ca in bone, and the concentrations of Cu in brain were significantly higher in the Al-exposed groups than in the control group. According to the current results, oral Al exposure during pregnancy can produce significant changes in the tissue distribution of a number of essential elements.     

Autoradiographic localization of angiotensin II receptors in developing rat cerebellum and brainstem

The role of Angiotensin II (Ang II) as a growth promoting or modulating factor has recently become a field of intensive research. A central issue in developmental neurobiology is the understanding of mechanisms governing the formation of spatially ordered connections. In this study, we show the localization of Ang II receptor subtypes by autoradiography in 2-week-old rat hindbrains confronting these data with membrane binding assays. Competition studies done on membrane preparations evidence no major changes on the relative affinities for both receptor subtypes between 2-week-old and adult rat tissues. By autoradiography, we found that all the areas (1-10) of the 2-week-old cerebellum showed both receptor subtypes present in complementary adjacent layers. Areas expressing a high level of AT2 receptors follow: inferior colicullus (IC), dorso tegmental nucleus, central (DTgC), subcoeruleus, alpha, sensory root of the trigeminal nerve, principal sensory root trigeminal nucleus (Pr5, Pr5VL) supragenual nucleus, genu facial nerve, facial nucleus, cerebellar peduncles, vestibular and lateral nuclei. Spinal trigeminal, (oral) and Raphe nuclei express AT1 receptor subtype. The high level of Ang II AT2 receptors present in the cerebellar peduncles might have a meaning on the establishment of the olivo-cerebellar connection. The high expression of Ang II AT2 receptors on 2-week-old rat hindbrains, a critical age on development, as well as its disappearance in the adult, strongly suggests a probable role of these receptors in cell migration and neuronal synaptogenesis.     

Formulations of single or multiple H. pylori antigens with DC Chol adjuvant induce protection by the systemic route in mice. Optimal prophylactic combinations are different from therapeutic ones

The ability to induce a protective response against Helicobacter pylori infection has been investigated by systemic immunization of mice with urease formulated with the cationic lipid DC Chol. This compound acts both as a formulating agent and as an adjuvant and induces a balanced Th1/Th2 response shown to be more effective for protection in our previous studies. Urease-DC Chol induced a significant protection in prophylaxis but not in therapeutic immunization. The protection level was between 1.5 and 2 log reduction of bacterial density measured by quantitative culture compared to unimmunized-infected mice. In parallel, the protective efficacy of other H. pylori antigens formulated in a similar way and administered with DC Chol was tested. These antigens were tested alone or in combination in prophylactic and therapeutic regimens. Some combinations of antigens induced a better prophylactic or therapeutic activity than urease alone (0.5-1.5 log further reduction in prophylaxis and therapy respectively, P<0.05). The combinations that induced the best protection were different in prophylaxis and therapy. In conclusion, DC Chol provides a convenient and efficient method to formulate different antigens even when they are present in non-compatible buffers initially. Moreover, the results obtained in protection against H. pylori with such formulations should lead the way to future clinical trials.