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PubMed2003

Quantitative analyses of principal and secondary compound parieto-occipital feedback pathways in cat.

The purpose of our study was to quantify the magnitude of principal and secondary pathways emanating from the middle suprasylvian (MS) region of visuoparietal cortex and terminating in area 18 of primary visual cortex. These pathways transmit feedback signals from visuoparietal cortex to primary visual cortex. (1) WGA-HRP was injected into area 18 to identify inputs from visual structures. In terms of numbers of neurons, feedback projections to area 18 from MS sulcal cortex (areas PMLS, AMLS and PLLS) comprise 26% of inputs from all visual structures. Of these neurons, between 21% and 34.9% are located in upper layers 2-4 and the dominant numbers are located in deep layers 5 and 6. Areas 17 (11.8%) and 19 (11.2%) provide more modest cortical inputs, and another eight areas provide a combined total of 4.3% of inputs. The sum of neurons in all subcompartments of the lateral geniculate nucleus (LGN) accounts for another 34.8% of the input to area 18, whereas inputs from the lateral division of the lateral-posterior nucleus (LPl) account for the final 11.9%. (2) Injection of tritiated-((3)H)-amino acids into MS sulcal cortex revealed substantial direct projections from MS cortex that terminated in all layers of area 18, but with a markedly lower density in layer 4. Projections from MS cortex to both areas 17 and 19 are of similar density and characteristics, whereas those to other cortical targets have very low densities. Quantification also revealed minor-to-modest axon projections to all components of LGN and a massive projection throughout the LP-Pul complex. (3) Superposition of the labeled terminal and cell fields identified secondary, compound feedback pathways from MS cortex to area 18. The largest secondary pathway is massive and it includes the LPl nucleus. Much more modest secondary pathways include areas 17 and 19, and LGN. The relative magnitudes of the secondary pathways suggest that the one through LPl exerts a major influence on area 18, whereas the others exert more modest or minor influences. MS cortex in the contralateral hemisphere also innervates area 18 directly. These data are important for interpreting the impact of deactivating feedback projections from visuoparietal cortex on occipital cortex.

Animals↗
PubMed2003

Interferon-gamma pharmacokinetics and pharmacodynamics in patients with colorectal cancer.

PURPOSE: The study objectives were to define subcutaneous (s.c.) interferon gamma (IFN-gamma) disposition in patients with gastrointestinal malignancies receiving 5-fluorouracil (5-FU) and leucovorin (LV) and to examine the relationship between IFN-gamma exposures and Fas upregulation in vivo and in vitro. METHODS: Patients received IFN-gamma (10, 25, 50, 75, and 100 microg/m(2)) with LV and 5-FU, and serial samples were collected after the first dose. IFN-gamma concentrations were measured by ELISA. A linear one-compartment model with a lag was fitted to the IFN-gamma plasma concentration-time data. To examine the relationship between IFN-gamma systemic exposure and biological activity in vivo, cell surface Fas upregulation was assessed in peripheral blood mononuclear cell (PBMC) subcompartments. RESULTS: The median (range) apparent IFN-gamma clearance was 46 l/m(2) per hour (2.6-92 l/m(2) per hour). With increasing IFN-gamma dosages, the area under the concentration-time curve (AUC(0--> infinity )) and C(max) increased; however, significant interpatient variability was observed. IFN-gamma AUC(0--> infinity ) and time above 33.3 pg/ml significantly correlated with Fas upregulation in several PBMC compartments, but dosage was significantly correlated with this pharmacodynamic marker only in CD4(+) and CD56(+) cells. In vitro studies in HT29 cells demonstrated that clinically relevant IFN-gamma concentrations (1 to 10 U/ml for 6.5 h) with 5-FU/LV upregulated Fas expression 3.5-fold, similar to that in PBMC in vivo. CONCLUSIONS: We characterized IFN-gamma disposition and developed a limited sampling model for use in future pharmacokinetic studies. Our results showed that IFN-gamma upregulates Fas in PBMC in vivo and in HT29 cells in vitro at tolerable, clinically relevant exposures and that monitoring IFN-gamma pharmacokinetics/pharmacodynamics may be warranted in IFN-gamma clinical use.

Adult↗
PubMed2005

Characterization of nuclear compartments identified by ectopic markers in mammalian cells with distinctly different karyotype.

The functional organization of chromatin in cell nuclei is a fundamental question in modern cell biology. Individual chromosomes occupy distinct chromosome territories in interphase nuclei. Nuclear bodies localize outside the territories and colocalize with ectopically expressed proteins in a nuclear subcompartment, the interchromosomal domain compartment. In order to investigate the structure of this compartment in mammalian cells with distinctly different karyotypes, we analyzed human HeLa cells (3n+ = 71 chromosomes) and cells of two closely related muntjac species, the Chinese muntjac (2n = 46 chromosomes) and the Indian muntjac (2n = 6/7 chromosomes). The distribution of ectopically expressed intermediate filament proteins (vimentin and cytokeratins) engineered to contain a nuclear localization sequence (NLS) and a nuclear particle forming protein (murine Mx1) fused to a yellow fluorescent protein (YFP) was compared. The proteins were predominantly localized in regions with poor DAPI staining independent of the cells' karyotype. In contrast to NLS-vimentin, the NLS-modified cytokeratins were also found close to the nuclear periphery. In Indian muntjac cells, NLS-vimentin colocalized with Mx1-YFP as well as the NLS-cytokeratins. Since the distribution of the ectopically expressed protein markers is similar in cells with distinctly different chromosome numbers, the property of the delineated, limited compartment might indeed depend on chromatin organization.

Animals↗
PubMed1999

Functional compartmentalization of the nucleus in the budding yeast Saccharomyces cerevisiae.

By combining cryofixation and cryosubstitution in a structural and functional analysis of the nucleus of Saccharomyces cerevisiae, we identified morphological subcompartments in the nucleolus. These were similar to those of nucleoli of higher eukaryotes, such as the fibrillar centre (FC), the dense fibrillar component (DFC) and the granular component (GC). In situ hybridization and immunocytochemistry revealed RNA polymerase I and proteins involved in early steps of ribosomal maturation along the DFC, while the ribosomal genes were detected at the FCs. Our results also suggest that ribosomal transcripts are distributed along a nucleolar network that might include both DFC and GC. We also show that pre-ribosomal subunits may be exported along tracks to the cytoplasm. Export takes place through all the pores of the nuclear envelope, not just those in contact with the nucleolus. Moreover, comparison of the nucleolar organization in S. cerevisiae and in Schizosaccharomyces pombe demonstrated than the distribution of the 5S genes with respect to the 35S transcription unit does not modify the organization of the nucleolus. We also report, for the first time, the ultrastructural localization of RNA polymerase II in yeast. The distribution of RNA polymerase II and morphological details that could be observed in the extra-nucleolar region of cryofixed cells provided cytological evidence of a peripheral region extending along the nuclear envelope that could correspond to heterochromatin in higher eukaryotes.

Cell Compartmentation↗
PubMed2005

Distribution of different phosphorylated forms of RNA polymerase II in relation to Cajal and PML bodies in human cells: an ultrastructural study.

The mammalian nucleus is a highly organised organelle that contains many subcompartments with roles in DNA replication and repair, gene expression and RNA processing. Cajal and promyelocytic leukaemia (PML) bodies are discrete nuclear structures with specific molecular signatures. RNA polymerase II and many transcription factors have been identified within these compartments by immunofluorescence microscopy, suggesting a role in polymerase II assembly or transcriptional activity. Here, we have examined the presence of different phosphorylated forms of polymerase II and newly made RNA in Cajal and PML bodies using high-resolution imaging of ultrathin cryosections (approximately 120 nm thick) with fluorescence and electron microscopes. We show that Cajal bodies contain polymerase II phosphorylated on Ser5, and not the Ser2-phosphorylated (active) form or newly made RNA. The presence of polymerase II in the absence of transcriptional activity suggests that Cajal bodies have roles in polymerase assembly or transport, but not in gene transcription. PML bodies contain no detectable polymerase II or nascent RNA in HeLa cells, at the resolution achieved by electron microscopy, but are often surrounded by these markers at distances>25 nm. These results support the view that although PML bodies are present in transcriptionally active areas of the nucleus, they are not generally sites of polymerase II assembly, transport or activity.

Antibodies↗
PubMed1998

Ribosomal gene transcription is organized in foci within nucleolar components.

We investigated how only three morphologically distinguished nucleolar components can integrate the many necessary tasks in ribosome biogenesis. For the mapping of ribosomal (r)DNA transcription loci, we combined non-autoradiographic in situ transcription assays with the immunological analysis of the ultrastructural distribution of transcription-associated proteins, i.e., polymerase I, the human polymerase I-specific upstream binding factor, and topoisomerase I. Furthermore, we visualized the nascent transcripts simultaneously with the rDNA in the nucleoli. All tested transcription proteins were found in both the fibrillar center and the dense fibrillar component (DF) of nucleoli in human cells. In the DF the nascent transcripts, detected by bromouridine incorporation, were found colocalized with the transcription proteins only within circumscribed regions. We did not observe colocalization of rDNA with nascent transcripts within the fibrillar centers, which corroborates the view that transcription proteins in this component are rather inactive. Our results suggests that only a minor portion of the DF is involved in transcriptional activity. Transcription appears to be confined to small foci, which exist close to or associated with the DF. Our results are in favor of the view that the DF has different functions which are localized in subcompartments of the DF.

Bromodeoxyuridine↗
PubMedDate not supplied

Golgi-disturbing agents.

Pharmacological agents have proven useful for gaining fundamental insights into the biology of the Golgi apparatus. This review summarizes pertinent and recent work on the effects on this organelle of monensin, brefeldin A, bafilomycin, ilimaquinone, okadaic acid, retinoic acid, and nocodazole. The molecular targets of monensin, brefeldin A, ilimaquinone, and retinoic acid remain to be elucidated whereas those for bafilomycin (vacuolar H+-ATPase), okadaic acid (serine/threonine phosphatases types 1, 2a, and 2b), and nocodazole (microtubules) are reasonably well understood. The molecular target of brefeldin has not been defined, but has been suggested to involve guanine nucleotide exchange proteins acting on ADP-ribosylation factor 1. Whether a defined molecular target can be found for monensin must be questioned since its main action consists in exchanging protons for Na+ which leads to osmotic swelling of post-Golgi endosomal structures and Golgi subcompartments by virtue of its membrane-associated effect as a cationophore. Brefeldin A was one of the most thoroughly investigated Golgi-disturbing agents and proved instrumental in unraveling retrograde flow mechanisms in the secretory pathways. Okadaic acid attracted interest for its properties mimicking mitotic fragmentation of the Golgi apparatus. Nocodazole was instrumental in establishing the cytoskeletal anchoring of the Golgi apparatus close to the microtubular organizing center.

Animals↗
PubMed1999

Two-photon Na+ imaging in spines and fine dendrites of central neurons.

Dendritic spines are assumed to be the smallest units of neuronal integration. Because of their miniature size, however, many of their functional properties are still unclear. New insights in spine physiology have been provided by two-photon laser-scanning microscopy which allows fluorescence imaging with high spatial resolution and minimal photodamage. For example, two-photon imaging has been employed successfully for the measurement of activity-induced calcium transients in individual spines. Here, we describe the first application of two-photon imaging to measure Na+ transients in spines and dendrites of CA1 pyramidal neurons in hippocampal slices. Whole-cell patch-clamped neurons were loaded with the Na(+)-indicator dye SBFI (sodium-binding benzofuran-isophthalate). In situ calibration of SBFI fluorescence with ionophores enabled the determination of the actual magnitude of the [Na+]i changes. We found that back-propagating action potentials (APs) evoked Na+ transients throughout the proximal part of the dendritic tree and adjacent spines. The action-potential-induced [Na+]i transients reached values of 4 mM for a train of 20 APs and monotonically decayed with a time constant of several seconds. These results represent the first demonstration of activity-induced Na+ accumulation in spines. Our results demonstrate that two-photon Na+ imaging represents a powerful tool for extending our knowledge on Na+ signaling in fine cellular subcompartments.

Action Potentials↗
PubMed2000

The development of the avian vertebral column.

Segmentation of the paraxial mesoderm leads to somite formation. The underlying molecular mechanisms involve the oscillation of "clock-genes" like c-hairy-1 and lunatic fringe indicative of an implication of the Notch signaling pathway. The cranio-caudal polarity of each segment is already established in the cranial part of the segmental plate and accompanied by the expression of genes like Delta1, Mesp1, Mesp2, Ulicx-1, and EphA4 which are restricted to one half of the prospective somite. Dorsoventral compartmentalization of somites leads to the development of the dermomyotome and the sclerotome, the latter forming as a consequence of an epithelio-to-mesenchymal transition of the ventral part of the somite. The sclerotome cells express Pax-1 and Pax-9, which are induced by notochordal signals mediated by sonic hedgehog (Shh) and noggin. The craniocaudal somite compartmentalization that becomes visible in the sclerotomes is the prerequisite for the segmental pattern of the peripheral nervous system and the formation of the vertebrae and ribs, whose boundaries are shifted half a segment compared to the sclerotome boundaries. Sclerotome development is characterized by the formation of three subcompartments giving rise to different parts of the axial skeleton and ribs. The lateral sclerotome gives rise to the laminae and pedicles of the neural arches and to the ribs. Its development depends on signals from the notochord and the myotome. The ventral sclerotome giving rise to the vertebral bodies and intervertebral discs is made up of Pax-1 expressing cells that have invaded the perinotochordal space. The dorsal sclerotome is formed by cells that migrate from the dorso-medial angle of the sclerotome into the space between the roof plate of the neural tube and the dermis. These cells express the genes Msx1 and Msx2, which are induced by BMP-4 secreted from the roof plate, and they later form the dorsal part of the neural arch and the spinous process. The formation of the ventral and dorsal sclerotome requires directed migration of sclerotome cells. The regionalization of the paraxial mesoderm occurs by a combination of functionally Hox genes, the Hox code, and determines the segment identity. The development of the vertebral column is a consequence of a segment-specific balance between proliferation, apoptosis and differentiation of cells.

Animals↗
PubMed1998

Structure and dynamics of human interphase chromosome territories in vivo.

A new approach is presented which allows the in vivo visualization of individual chromosome territories in the nuclei of living human cells. The fluorescent thymidine analog Cy3-AP3-dUTP was microinjected into the nuclei of cultured human cells, such as human diploid fibroblasts, HeLa cells and neuroblastoma cells. The fluorescent analog was incorporated during S-phase into the replicating genomic DNA. Labelled cells were further cultivated for several cell cycles in normal medium. This well-known scheme yielded sister chromatid labelling. Random segregation of labelled and unlabelled chromatids into daughter nuclei resulted in nuclei exhibiting individual in vivo detectable chromatid territories. The territories were composed of subcompartments with diameters ranging between approximately 400 and 800 nm which we refer to as subchromosomal foci. Time-resolved in vivo studies demonstrated changes of positioning and shape of territories and subchromosomal foci. The hypothesis that subchromosomal foci persist as functionally distinct entities was supported by double labelling of chromatin with CldU and IdU, respectively, at early and late S-phase and subsequent cultivation of corresponding cells for 5-10 cell cycles before fixation and immunocytochemical detection. This scheme yielded segregated chromatid territories with distinctly separated subchromosomal foci composed of either early- or late-replicating chromatin. The size range of subchromosomal foci was similar after shorter (2 h) and longer (16 h) labelling periods and was observed in nuclei of both living and fixed cells, suggesting their structural identity. A possible functional relevance of chromosome territory compartmentalization into subchromosomal foci is discussed in the context of present models of interphase chromosome and nuclear architecture.

Bromodeoxyuridine↗
PubMed2002

Cellular autophagic capacity changes during azaserine-induced tumour progression in the rat pancreas. Up-regulation in all premalignant stages and down-regulation with loss of cycloheximide sensitivity of segregation along with malignant transformation.

The knowledge of alterations in regulation of autophagy during tumorigenesis may also help our understanding of its normal control. We established an experimental system and reported recently that autophagic capacity, measured as the cell's capability of increasing segregation (formation of autophagosomes) and subsequent degradation of cytoplasmic quanta were highly increased in premalignant nodule cells 6 months after initiation by azaserine in the rat pancreas in vivo. In the present study, we followed changes of these autophagic functions throughout the tumour progression. We carried out electron-microscopic morphometrical analysis of the expansion of autophagic vacuole compartment and subcompartments induced by vinblastine (an in vivo segregation enhancer), as well as their regression upon segregation-inhibitor cycloheximide post-treatment. Premalignant tumour samples were taken at month 5, month 8 (nodules), month 10 and month 15 (adenomas) after initiation. In all these stages, a highly increased and varying autophagic capacity was found compared with the host tissue. The basal (non-stimulated) autophagic compartment was measurable only at month 5 and month 15, and its regression upon cycloheximide was consistent with increased basal autophagic activity. Compared with the host tissue, autophagic capacity profoundly decreased in the differentiated and anaplastic adenocarcinomas at month 20, when, surprisingly, cycloheximide was unable to inhibit segregation. Our conclusion is that down-regulation of the cycloheximide sensitive segregation and a partly compensatory up-regulation of an alternative pathway of segregation might occur along with malignant transformation.

Adenocarcinoma↗
PubMed2003

Ultrastructure of pigment-dispersing hormone-immunoreactive neurons in a three-dimensional model of the accessory medulla of the cockroach Leucophaea maderae.

Locomotor activity rhythms of the cockroach Leucophaea maderae are orchestrated by two bilaterally symmetric, mutually coupled, circadian pacemakers. They lie in the optic lobes of the brain and are confined to the accessory medulla (AMe), ventro-medially to the medulla. The AMe is innervated by approximately 12 pigment-dispersing hormone (PDH)-immunoreactive anterior medulla neurons (PDHMe), which are circadian pacemaker candidates in the fruitfly and the cockroach. We have developed a three-dimensional computer model of the AMe and associated structures as a framework for neuroanatomical studies. Our greatly improved understanding of this structure in space has allowed us further to subdivide the anterior PDHMe into three subgroups, i.e., large, medium-sized, and small anterior PDHMe. The synaptic connections of two of these subgroups have been examined within subcompartments of the AMe by light and electron microscopy. The large, intensely staining, anterior PDHMe contain medium-sized dense-core vesicles and form input and output synapses with profiles densely filled with clear vesicles primarily in the anterior and shell neuropil of the AMe. The medium-sized anterior PDHMe contain large dense-core vesicles and constitute input and output synapses either with profiles being densely filled with clear vesicles, or with profiles containing granular dense-core vesicles. The small, weakly staining anterior PDHMe belong to a morphological group different from the large and medium-sized PDHMe and cannot be further identified at the electron-microscopic level because of their weak PDH immunoreactivity.

Animals↗
PubMed2000

Poly-A-rich domains in adrenal cell nuclear matrix after acute and chronic ACTH stimulation: an in situ hybridization study.

The regulatory effects of adrenocorticotropin (ACTH) at the adrenal cell nucleus are not yet completely clarified. Recent studies showed that eukaryotic cell nucleus is composed of specific subcompartments organized by the nucleoskeleton-nuclear matrix. In this paper, the RNA polymerase II transcriptional domains were detected in the adrenal nucleus after acute and chronic ACTH stimulation by using in situ hybridization. Polydeoxythymidine (poly-T) probe, complementary to the polyadenylated (poly-A) chain of all the mRNAs and its precursors, was used. Hybridization was performed in intact adrenal cells and in nuclear matrices isolated from previously dispersed cells. The transcriptional domains, enriched in poly-A RNA, were evident in intact adrenal cells, the staining intensity being higher both in the acutely and in the chronically stimulated animals. In the nuclear matrices poly-A-rich areas were still visible, and were more intensely stained after ACTH stimulation.

Adrenal Glands↗
PubMed2002

Muscle fiber-type distribution pattern in the human cricopharyngeus muscle.

Our previous studies described that the human cricopharyngeus (CP) is composed of two neuromuscular compartments (NMCs), horizontal and oblique. The present study was designed to explore the differences in muscle fiber-type distribution between the NMCs within the human CP and to examine the oxidative capacity of the muscle fibers. Seven adult human CP muscles obtained from autopsies were stained for myofibrillar ATPase, reduced nicotinamide adenine dinucleotide tetrazolium reductase (NADH-TR), and succinic dehydrogenase (SDH) to analyze enzyme-histochemical fiber-type characteristics. Notable findings obtained from this study are as follows: (1) Different NMCs within the human CP contained different percentages of muscle fiber types. The horizontal CP (CPh) contained more slow-twitch fibers than the oblique CP (CPo). (2) Each of the NMCs was dividable histochemically into two layers or subcompartments: a slow fiber-type inner layer and a relatively fast fiber outer layer. (3) As a whole, type I fibers had higher levels of NADH-TR and SDH than type II fibers. However, in both type I and II muscle fiber types, different patterns of oxidative enzyme activity were seen. Histochemically defined fiber layers of the CP are not seen in other mammals, suggesting that CP function is more specialized in humans.

Aged↗
PubMed2002

Magnetoencephalography in presurgical epilepsy evaluation.

The introduction of whole-head magnetoencephalography (MEG) systems facilitating simultaneous recording from the entire brain surface has led to a major breakthrough of MEG in presurgical epilepsy evaluation. Localizations of the interictal spike zone with MEG showed excellent agreement with invasive electrical recordings, were useful to clarify the spatial relationship of the irritative spike zone to structural lesions, and could attribute epileptic activity to lobar subcompartments both in temporal lobe and extratemporal epilepsy. MEG was especially useful for the study of patients with non-lesional neocortical epilepsy and of patients with large lesions, where it provided unique information on the epileptogenic zone. It could reliably localize sensorimotor cortex prior to surgical procedures adjacent to central fissure. MEG language mapping yielded concordant results with the Wada test and cortical stimulation studies. MEG localizations of epileptic activity and essential brain regions were successfully integrated into frameless stereotaxy systems providing accurate functional information intraoperatively. Because MEG and EEG yield both complementary and confirmatory information, combined MEG-EEG recordings in conjunction with advanced source modeling techniques will further improve the noninvasive evaluation of epilepsy patients and constantly reduce the need for invasive procedures.

Brain↗
PubMed1982

Body fluid homeostasis in congestive heart failure and cirrhosis with ascites.

The urinary excretion of salt and water in man is regulated by a variety of renal and extrarenal mechanisms that respond to changes in dietary sodium intake as well as to alterations in the holding capacity of the vascular and interstitial compartments. Changes in extracellular fluid volume are detected by volume sensors located in the intrathoracic vascular bed, the kidney and other organs. These sensing mechanisms gauge the adequacy of intravascular volume relative to capacitance at various sites within the circulation. Congestive heart failure and cirrhosis with ascites are two disease states of man in which a hemodynamic disturbance within a given circulatory subcompartment is perceived by these sensing mechanisms and results in renal sodium retention. While the primary disturbance in both of these conditions originates outside the kidney, a variety of renal effector mechanisms respond to the perceived circulatory disturbance and result in enhanced tubule reabsorption of salt and water. These effector mechanisms involve physical adjustments in renal microvascular hemodynamics, tubule fluid composition and flow rate and transtubular ion gradients. These in turn are partially regulated by a variety of neural and humoral pathways including the renin-angiotensin-aldosterone axis, prostaglandins, and kinins.

Aldosterone↗
PubMed1986

Golgi-specific localization of transglycosylases engaged in glycoprotein biosynthesis in suspension-cultured cells of sycamore (Acer pseudoplatanus L.).

Golgi complex and endoplasmic reticulum (ER) were isolated from suspension-cultured cells of sycamore (Acer pseudoplatanus L.) by stepwise sucrose density gradient centrifugation using protoplasts as starting material. The purity of the two organelle fractions isolated was assessed by measuring marker enzyme activities. Localization of glycolipid and glycoprotein glycosyltransferase activities in the isolated Golgi and ER fractions was examined; three glycosyltransferases, i.e., galactosyltransferase, fucosyltransferase, and xylosyltransferase, proved to be almost exclusively confined to the Golgi, whereas the ER fractions contained glycolipid glycosyltransferase. The Golgi complex was further subfractionated on a discontinuous sucrose density gradient into two components, migrating at densities of 1.118 and 1.127 g/cm3. The two fractions differed in their compositional polypeptide bands discernible from Na-dodecylsulfate gel electrophoresis. Galactosyltransferase distributed nearly equally between the two protein peaks and xylosyltransferase activities using the endogenous acceptor also appeared to be localized in the two subcompartments. By contrast, fucosyltransferase, engaged in the terminal stage of glycosylation, banded in the lower density fractions. Golgi-specific alpha-mannosidase, which is presumably engaged in the sugar trimming of Asn-N-linked glycoprotein carbohydrate core, was enriched fourfold in specific activity in the fractions of the higher density. The overall experimental results indicate that the cotranslational glycosylation of Asn-N-linked glycoproteins, e.g., polyphenol oxidase (laccase), takes place in the ER, while subsequent post-translational processing of the oligosaccharide moiety proceeds successively in the two physically separable compartments of the Golgi complex.

Endoplasmic Reticulum↗
PubMed1994

Mitochondrial protein import: mechanisms, components and energetics.

The transport of nuclear-encoded proteins from the cytosol into mitochondria is mediated by targeting (signal) sequences present on precursor forms. Most precursors of the mitochondrial matrix possess amino-terminal signals which characteristically contain hydroxylated and basic amino acids and lack acidic residues. With a minority of precursor proteins, internal sequence motifs can direct proteins to the mitochondria (Pfanner, N., Hoeben, P., Tropschug, M. and Neupert, W. (1987) J. Biol. Chem. 262, 14851-14854). The presence of a mitochondrial targeting sequence alone, however, is not sufficient for specific targeting to the organelle and further to the various subcompartments. There is the need for components which recognise the targeting sequences and others which keep the precursor protein in a translocation-competent form. Beyond the recognition step, components are required which mediate translocation across the mitochondrial membranes. Mitochondria posses two translocation machineries, one in the outer membrane and one in the inner membrane. The matrix space harbors a number of factors which participate in the import of proteins, in their unfolding and folding. Energy is required at several steps of these processes.

Cytosol↗