Mechanistic studies of the translational elongation cycle in mammalian mitochondria

Polyclonal antibodies have been prepared against both components of the bovine liver mitochondrial translational elongation factor Tu and Ts complex (EF-Tu x Ts(mt)). The antibodies against EF-Tu(mt) cross-react somewhat with Escherichia coli EF-Tu and wheat germ EF-1alpha. The antibodies against EF-Ts(mt) cross-react little, if at all, with E. coli EF-Ts or with EF-Ts from Euglena gracilis chloroplasts. These polyclonal antibodies have been used to investigate the relative amounts of EF-Tu(mt) and EF-Ts(mt) in bovine liver mitochondria and in cultured cells. The results of this analysis suggest that there is a 1:1 ratio of EF-Tu(mt) to EF-Ts(mt) in mammalian mitochondria. Intermediate complexes formed during the elongation cycle of protein synthesis in bovine liver mitochondria have also been investigated. The EF-Tu x Ts(mt) complex is quite resistant to dissociation by guanine nucleotides. This complex will, however, dissociate in the presence of GTP and Phe-tRNA resulting in the formation of a ternary complex comparable to that observed in prokaryotes. Kinetic data suggest that the use of the ternary complex in chain elongation increases the rate of Phe-tRNA binding to ribosomes, suggesting that it is a true intermediate in the elongation cycle. Sucrose gradient analysis indicates that the binding of EF-Tu(mt) to ribosomes can be detected in the presence of Phe-tRNA and a non-hydrolyzable analog of GTP. These results suggest that, in contrast to previous thinking, the basic features of the elongation cycle in mammalian mitochondria are quite similar to those in prokaryotes.     

Hypothalamic monoaminergic systems in ontogenesis: development and functional significance

This paper summarizes recent data on the development of hypothalamic monoaminergic (MArgic) systems which play a key role in neuroendocrine regulation in adult mammals. Hypothalamic catecholaminergic (CArgic) and 5-hydroxytryptamine (=serotonin, 5-HT) systems undergo synchronous development that begins from the origin of dopaminergic neurons in the hypothalamus and 5-HT neurons in the raphe nucleus long before birth. Moreover, some hypothalamic neurons of fetuses and young rats partly express the 5-HT phenotype patterns: the 5-HT specific uptake and synthesis from 5-hydroxytryptophan. Differentiation of MArgic neurons is manifested in expression of specific enzymes and MA synthesis as well as in the onset of the MA specific uptake and potassium-stimulated release. In the course of differentiation, MArgic neurons send their axons to target neurons, hypophysial portal circulation, the third ventricle and adenohypophysis, followed by the establishment of specialized contacts: synapses, axo-vascular, axo-ventricular and axo-glandular. The hypothalamic CAs and 5-HT firstly appeared in embryogenesis control phenotype expression of target neurons: gene expression, specific synthesis, uptake and release, axonal growing, etc. In turn, the development of the hypothalamic MArgic systems is under control of MAs (autoregulation) and hormones of the peripheral endocrine glands (androgens). Conversely, there is a minor role, if any, of the maternal and placental neurohumoral factors in differentiation of MArgic neurons.     

Inheritance of the mammalian Golgi apparatus during the cell cycle

The creation and propagation of the intricate Golgi architecture during the cell cycle poses a fascinating problem for biologists. Similar to the inheritance process for nuclear DNA, the inheritance of the Golgi apparatus consists of biogenesis (replication) and partitioning (mitosis/meiosis) phases, in which Golgi components must double in unit mass, then be appropriately divided between nascent daughter cells during cytokinesis. In this article we focus discussion on the recent advances in the area of Golgi inheritance, first outlining our current understanding of the behaviour of the Golgi apparatus during cell division, then concluding with a more conceptual discussion of the Golgi biogenesis problem. Throughout, we attempt to integrate ultrastructural and biochemical findings with more recent information obtained using live cell microscopy and morphological techniques.     

The effect of unilateral destruction of the head of the caudate nucleus with kainic acid on the structure of the wakefulness-sleep cycle and the EEG in rats

The wakefulness-sleep cycle characteristics changes in Wistar line rats after unilateral lesions of the neuronal elements of the caudate nucleus head with kainic acid (at sparing the conducting ways going through this structure) were studied. It was shown that in 4 days after acid injection the share of the active wakefulness and light slow-wave sleep is significantly increased, whereas the percentage of the passive wakefulness, deep slow-wave stage, and paradoxal sleep (up to 80%) is reduced. The wakefulness-sleep cycle in rats is completely normalized on the 7th day after injection. The obtained data allow to consider the caudate nucleus as a modulator for wakefulness-sleep cycle, that is the structure involved in the motive activity regulation.     

Expression of heterogeneous nuclear ribonucleoprotein A2/B1 changes with critical stages of mammalian lung development

Recent reports have demostrated a link between expression of members of the family of heterogeneous nuclear ribonucleoproteins (hnRNPs) and cancer. Overexpression of hnRNP A2/B1 correlated with the eventual development of lung cancer in three different clinical cohorts. We have studied the expression of hnRNP A2/B1 messenger RNA (mRNA) and protein during mammalian development. The expression of hnRNP A2/B1 mRNA and protein are parallel but change dynamically during critical periods in mouse pulmonary development. hnRNP A2/B1 is first detected in the lung in the early pseudoglandular period, peaks at the beginning of the canalicular period, and remains high during the saccular (alveolar) period. In mouse and rat, hnRNP A2/B1 expression is first evident in the earliest lung buds. As lung development progresses, the cuboidal epithelial cells of the distal primitive alveoli show high levels of the ribonucleoprotein, which is almost undetectable in the proximal conducting airways. The expression of hnRNP A2/ B1 is restricted in mature lung. Similar dynamic pattern of expression through lung development was also found in rat and human lung. Upregulated expression of hnRNP A2/B1 at critical periods of lung development was comparable to the level of expression found in lung cancers and preneoplastic lesions and is consistent with hnRNP A2/B1 overexpression playing an oncodevelopmental role.     

A molecular marker for centriole maturation in the mammalian cell cycle

The centriole pair in animals shows duplication and structural maturation at specific cell cycle points. In G1, a cell has two centrioles. One of the centrioles is mature and was generated at least two cell cycles ago. The other centriole was produced in the previous cell cycle and is immature. Both centrioles then nucleate one procentriole each which subsequently elongate to full-length centrioles, usually in S or G2 phase. However, the point in the cell cycle at which maturation of the immature centriole occurs is open to question. Furthermore, the molecular events underlying this process are entirely unknown. Here, using monoclonal and polyclonal antibody approaches, we describe for the first time a molecular marker which localizes exclusively to one centriole of the centriolar pair and provides biochemical evidence that the two centrioles are different. Moreover, this 96-kD protein, which we name Cenexin (derived from the Latin, senex for "old man," and Cenexin for centriole) defines very precisely the mature centriole of a pair and is acquired by the immature centriole at the G2/M transition in prophase. Thus the acquisition of Cenexin marks the functional maturation of the centriole and may indicate a change in centriolar potential such as its ability to act as a basal body for axoneme development or as a congregating site for microtubule-organizing material.     

An alternate pathway for T cell development supported by the bone marrow microenvironment: recapitulation of thymic maturation

In the principal pathway of alpha/beta T cell maturation, T cell precursors from the bone marrow migrate to the thymus and proceed through several well-characterized developmental stages into mature CD4+ and CD8+ T cells. This study demonstrates an alternative pathway in which the bone marrow microenvironment also supports the differentiation of T cell precursors into CD4+ and CD8+ T cells. The marrow pathway recapitulates developmental stages of thymic maturation including a CD4+CD8+ intermediary cell and positive and negative selection, and is strongly inhibited by the presence of mature T cells. The contribution of the marrow pathway in vivo requires further study in mice with normal and deficient thymic or immune function.     

The mammalian cell cycle in normal and abnormal growth

Cell division, a complex array of intracellular events, occurs in a highly ordered and carefully coordinated manner. This regulation is achieved by the sequential activation and deactivation of the members of a family of serine-threonine-specific protein kinases that consist of regulatory and enzymatic subunits, the cyclins and cyclin-dependent kinases. These enzymes, in turn, regulate the activity of other proteins involved in the mitogenic pathway. Mutations in the components of the regulatory pathways can lead to aberrant growth, including malignancies.     

Plastic properties of the venous bed of the brain following disengagement of the external jugular veins at different stages of ontogenesis

The capacity of the venous bed in the brain being great, even inconsiderable changes of the vein lumen can cause severe changes in the blood circulation without an enlargement of the cranial cavity volume. The progressing venous stagnation in the brain is a severe disease of not only the brain but also the whole organism. So, we attempted to study the character of compensatory redistribution of the pial venous system of the cerebrum in occlusion of both exterior jugular veins and to establish its developmental accomodative properties. The work has been performed on 45 dogs. The plasticity of the cerebral pial veins was found to drop with age, their reserving capacity-to grow and their carrying capacity-to decrease. Three successive phases are characteristic of the compensatory-adaptational changes: diffuse dilatation of the venous network, appearance of collaterals and their development against the background of reduction of the surrounding venous network.     

Adenomatous polyposis coli protein is expressed in alternate stages of the ameloblast life cycle

Mutations of the adenomatous polyposis coli gene protein (APC) are associated with familial polyposis and also sporadic colon adenomas, both preconditions to cancer formation. Some familial polyposis patients also develop Gardner's syndrome, a condition characterized by supernumerary teeth, mandibular osteomas, and other maladies. We investigated participation of APC in normal tooth development. Using a monoclonal antibody to study APC expression in the forming rat incisor, we found no APC staining in differentiating ameloblasts, then strong staining in secreting ameloblasts and stratum intermedium cells, followed by cells in the transition stage which did not stain. Intense APC staining resumes in maturation-stage ameloblasts and proximal papillary cells. APC staining disappears again in reduced ameloblasts at the conclusion of amelogenesis. APC staining was not seen in any other odontogenic cells. We report a unique system in which APC expression is upregulated and downregulated twice during the normal life cycle of ameloblasts. APC, therefore, is important in the normal maturation of both colonic epithelium and odontogenic epithelium. At this point, we cannot rule out any of the known functions of APC, which include: modulation of cell adhesion by binding to catenin, regulation of beta-catenin as a differentiative signaling molecule, and promotion of microtubule assembly. In this respect, the rat incisor enamel organ provides a unique tissue for studying the regulation and functions of APC.     

Cellular immune responses of jirds to extracts of life cycle stages and adult excretory secretory products during the early development of Brugia pahangi

The Brugia-jird model of lymphatic filariasis was used to examine the induction of cellular immune responses during the early premicrofilaremic phases of the infection. The intensity of the pulmonary granulomatous inflammatory response (PGRN) was determined by measuring granuloma areas around Sepharose beads coated with parasite extracts which were embolized in the lungs of jirds prior to necropsy. Necropsies were performed at 7, 14, 28, 56, and 150 days postinfection (DPI). These time points correspond to specific developmental changes in the life cycle. Lymphocyte blastogenesis assays were performed using cells from draining renal lymph nodes and splenocytes at 14 and 150 DPI. Soluble extracts of third stage larvae (L3), fourth stage larvae (L4), adult females, adult males, microfilariae (MF), and excretory secretory products (ES) of males and females were used in both measurements of cellular responsiveness. A marked granulomatous response to parasite extracts peaked at 7 DPI or 14 DPI followed by a gradual decrease to a hyporesponsive state at 120 DPI. The response of renal lymph node cells also was significantly elevated at 14 DPI and significantly decreased at > 150 DPI. The splenocyte responses were erratic and did not follow this pattern. Significant differences in PGRN responses to somatic extract preparations were not seen during the early stages of the infection (7, 14, 28 DPI), but those to MF and L3 were significantly less at 56 and 120 DPI. Although PGRN responses to ES followed a similar pattern, these were less than those to the somatic extract. The data indicated that a rapid, intense cell-mediated inflammatory response is induced early during a primary infection and that this response is rapidly downregulated. This downregulation begins prior to the maturation of adult parasites and microfilarial production. The early phase of the cellular response appears to be compartmentalized in that this response was consistently observed in the renal lymph nodes but not in the spleen. Soluble protein components of the parasites responsible for these responses are likely multiple and shared by all life cycle stages.