Hexokinase in developing rabbit erythroid cells

The activity and isozyme distribution of hexokinase were studied in bone marrow cells from normal and anemic rabbits seperated by density centrifugation or by unit-gravity sedimentation. The specific activity of the enzyme was found to be about 150-fold higher in the basophilic erythroblasts as compared with the mature circulating erythrocytes. Mos of the falls in hexokinase activity take place whent the cell completes its final division and matures from the polychromatic stage to the orthochromatic stage. Concomitant with this strong decrease in enzyme activity, qualitative as well as quantitative changes in the hexokinase isozymic pattern become apparent. While in the basophilic and polychromatic erythroblasts the only hexokinase isozyme present is hexokinase type I, the orthochromatic cells also contain hexokinase Ib. This last isozymic form, which increases further at the reticulocyte stage, is also present in the circulating reticulocytes but not in mature red blood cells.     

兔胚胎时期特异性基因相关新片段的克隆

阶段特异性基因的表达是早期胚胎发育过程中的重要事件,对植入前胚胎基因表达模式的研究是进一步研究植入前胚胎发育调控机制的前提。本实验利用mRNA差异显示技术来研究兔(Oryctolagus cuniculus domestica)植入前各期胚胎的基因表达差异。在获得的42个阳性阶段特异性表达的基因中,有5个在NCBI和EMBL数据库中没有同源序列,登录EMBL,申请了登录号。这些新基因片段都是桑葚期特异表达的基因,而且在以后的囊胚期也有表达。兔由母源型调控向合子型调控的过渡是在8～16细胞期开始的,在桑葚期开始表达的这些基因片段应该是兔胚胎时期特异性基因。这些基因的克隆将为进一步研究兔的植入前胚胎发育模式奠定基础。     

Haemoglobin producing cells in freshwater snails

Summary One of the cell types occurring in the connective tissue of gastropods and bivalves is thepore cell. Cells of this type have a characteristic morphology in that their plasmamembrane has many invaginations bridged by cytoplasmic tongues. In the well developed granular endoplasmic reticulum and in these invaginations fine granular material, sometimes arranged in crystalline patterns, is present. InLymnaea stagnalis this material has been identified as haemocyanin on a morphological basis. Histochemical and ultrastructural results on pore cells of 2 snail species(Biomphalaria glabrata, Planorbarius corneus) having haemoglobin in their blood indicate that the pore cells in these species synthesize haemoglobin. Possible other functions of pore cells are briefly discussed.     

Autophagy of mitochondria in rat bone marrow erythroid cells Relation to nuclear extrusion

Summary Late erythroblasts and reticulocytes from bone marrow of male Wistar rats were studied by electron-microscopic stereology. Late erythroblasts with morphological signs of nuclear extrusion (EN+erythroblasts) and late erythroblasts without these signs (EN-erythroblasts) were analysed separately. The volumes of mitochondria, autophagosomes, autophagocytosed mitochondria, autophagocytosed cytoplasm and degraded material inside autophagosomes were calculated per unit volume of cytoplasm.The results demonstrate that (1) the volume density of mitochondria in the cytoplasm decreases by 34% during maturation from (EN-)- to (EN+)-erythroblasts (p< 0.001) and by 60% during differentiation from (EN+)-erythroblasts to reticulocytes (p<0.001), (2) a fivefold increase in the volume density of autophagosomes in the cytoplasm is noted during maturation from (EN-)- to (EN+)-erythroblasts (p<0.01), whereas the value of this parameter remains essentially unchanged during the subsequent differentiation to reticulocytes, (3) no mitochondria are found inside autophagosomes of (EN-)-erythroblasts, whereas mitochondria occupy 26% and 35%, respectively, of the autophagosomal volume in (EN+)-erythroblasts and in reticulocytes.Our results show that autophagocytosis of mitochondria starts at the moment of nuclear extrusion and continues in the bone marrow reticulocytes.     

Uptake and intracellular distribution of iron from transferrin and chelators in erythroid cells

Summary Iron chelators of different physicochemical properties were studied for their ability to donate iron in vitro to uninduced K562 cells, human bone marrow cells and purified human erythroblasts. To a large extent uptake was found to be related to lipophilicity and those chelators able to deliver iron to the cells in significant amounts were also able to deliver iron to ferritin and haem. Some differences in the distribution of iron delivered was observed but no chelator showed exclusive delivery to or rejection of a particular cellular iron compartment. Several chelators could probably substitute for transferrin and be used to probe metabolic events subsequent to iron removal from transferrin. Two chelators which were excellent iron donors were also found to cause considerable inhibition of iron incorporation into haem from transferrin. The implications of this for in vivo toxicity are briefly discussed.     

Keratin and vimentin expression in early organogenesis of the rabbit embryo

Summary The expression of vimentin and keratins is analysed in the early postimplantation embryo of the rabbit at 11 days post conceptionem (d.p.c.) using a panel of monoclonal antibodies specific for single intermediate filament polypeptides (keratins 7, 8, 18, 19 and vimentin) and a pan-epithelial monoclonal keratin antibody. Electrophoretic separation of cytoskeletal preparations obtained from embryonic tissues, in combination with immunoblotting of the resulting polypeptide bands, demonstrates the presence of the rabbit equivalents of human keratins 8, 18, and vimentin in 11-day-old rabbit embryonic tissues. Immunohistochemical staining shows that several embryonic epithelia such as notochord, surface ectoderm, primitive intestinal tube, and mesonephric duct, express keratins, while others (neural tube, dermomyotome) express vimentin, and a third group (coelomic epithelia) can express both. Similarly, of the mesenchymal tissues sclerotomal mesenchyme expresses vimentin, while somatopleuric mesenchyme (abdominal wall) expresses keratins, and splanchnopleuric mesenchyme (dorsal mesentery) expresses both keratins and vimentin. While these results are in accordance with most results of keratin and vimentin expression in embryos of other species, they stand against the common concept of keratin and vimentin specificity in adult vertebrate tissues. Furthermore, keratin and vimentin are not expressed in accordance with germ layer origin of tissues in the mammalian embryo; rather the expression of these proteins seems to be related to cellular function during embryonic development.Supported by the Deutsche Forschungsgemeinschaft and by the Netherlands Cancer Foundation     

The interstitial cells in the colon of the rabbit

Summary The interstitial cells associated with the myenteric plexus of the rabbit colon were studied by scanning and transmission electron microscopy. It was demonstrated that the interstitial cells were stellate or fusiform in shape and located over the ganglia, over nerve bundles and between muscle cells. They were characterized by many slender processes, and resemble fibroblasts. No basal lamina was observed between the interstitial cells and muscle cells. It was concluded that structural features of the interstitial cells are distinctly different from those of neurons, Schwann cells, or of smooth muscle cells, while they show clear similarities to those of fibroblasts. By scanning electron microscopy the shapes and the relations of these cells could be demonstrated in great detail.     

Changes in proteins synthesized by rabbit endometrial epithelial cells following primary culture

Summary Morphological and biochemical changes occurring in rabbit endometrial epithelial cells when placed in culture were investigated. Cells were examined by scanning- and transmission electron microscopy and freeze-fracture. Morphologically, cultured cells are shorter and broader than the columnar epithelial cells in vivo, but retain their polarity as indicated by the presence of apical microvilli and a well-developed junctional belt. To study changes in biochemical function, proteins synthesized by cells in primary culture were analyzed by two-dimensional gel electrophoresis. Proteins were labeled during a 24-h incubation with ³⁵S-methionine and gels examined by fluorography. The pattern of proteins changed after cells had been in culture for 48 h. On day 3 new proteins were synthesized and several protein species labeled during days 1 or 2 of culture, including uteroglobin, no longer appeared. On days 3–8 of culture the protein patterns were similar. Addition of progesterone, estradiol, prolactin, or combinations of these hormones to the culture medium for 24–144 h failed to elicit consistent changes in the pattern of labeled proteins established after 3 days of culture. Minor differences in protein patterns among unrelated cultures appear to have been derived from the original cells of the culture. These results indicate that after 48 h in primary culture, cells grown in vitro resemble endometrial epithelial cells morphologically, but no longer reflect functionally the character of epithelial cells in the uterus.     

Nucleolar silver-stained granules in maturing erythroid and granulocytic cells

Summary Human and rabbit erythroid and granulocytic precursors in bone marrow have been investigated to provide information concerning the number of nucleolar silverstained granules (SSGs), which represent active interphasic nucleolar organizer regions (NORs). The differentiation and maturation of precursor cells of both investigated cell lines are characterized by a gradual decrease in number of nucleolar SSGs. In advanced maturation stages of erythroblasts or granulocytes, which are known to lose the capacity to divide, the number of nucleolar SSGs is smaller than the reported average or maximal values of NORs determined for human or rabbit cells. Since committed stem cells from both cell lines contain several times the number of nucleolar SSGs than the last dividing maturation and differentiation stages, the number of active parts of interphasic NORs in committed stem cells seems to be increased and might represent a stock for the later stages. In addition, the number of nucleolar SSGs appear to be a very convenient marker of nucleolar biosynthetic activity in individual differentiating and maturing blood cells. The differences between erythroid and granulocytic stem cells with respect to the number of nucleolar SSGs disappear during the course of further differentiation and maturation.     

The follicular oocyte and its granulosa cells in domestic pig

Summary Pig oocytes and their surrounding granulosa cells obtained from mature Graafian follicles at a stipulated time near to ovulation were studied in some details electronmicroscopically. Particular emphasis is given to the corona radiata cell processes and to the heterogeneous population of mitochondria in the oocyte.The corona radiata cell processes contain various components such as filaments, mitochondria, multivesicular bodies and lipid droplets in their matrix. The contact relationship of the corona radiata cell processes to the oocytes is maintained by desmosomes. Usually, the two parallel surface membranes forming the desmosome are separated by a space of about 200 Å. Occasionally, the two membranes approximate each other to form a junction having a gap of about 70 Å. Apparently the membranes become fused in some regions.Of particular interest is the distribution and structural characteristics of the single-membrane-bounded structures, and their relationship to the cytomembranes and the mitochondria. On the basis of the present and earlier (Norberg, 1972) observations, the question arises whether the formation and development of mitochondria of pig oocytes depend, at least partly, on a metamorphosis of single-membrane-bounded structures derived from less complex membraneous elements. Final conclusions concerning this problem demand integrated morphological and biochemical investigation regarding the biosynthesis of mitochondria.This work was supported by the Agricultural Research Council of Norway.     

Calbindin-immunopositive cells are cholinergic interneurons in the myenteric plexus of rabbit ileum

The 28-kDa calcium-binding protein (calbindin) is a widely studied neuronal marker in the enteric nervous system of numerous species. Calbindin has previously been detected in myenteric neurons of rabbit ileum in which 3% of all myenteric neurons are calbindin-immunopositive. We have studied the detailed morphology and chemical coding of calbindin-immunopositive neurons in this segment of the gut. We have found calbindin immunoreactivity in both strongly and weakly stained neurons. Of these, the strongly immunoreactive neurons belong to the Dogiel type I category. These neurons project only to other ganglia and primary strands of the plexus and their processes never run to the muscle or mucosal layers. The neurons within this group are 29.5±6.6 m in length and 14.7±3.8 m in width. The second smaller group of immunoreactive cells (27%) label faintly and have different morphological properties. They are characterized by their round medium-sized cell bodies (long axis: 24.4±5.2 m; short axis: 15.5±2.9 m) and do not exhibit immunoreactivity either in their dendrites or in their axonal processes. Double-label studies show that all calbindin-immunopositive neurons lack immunoreactivity for nitric oxide synthase, vasoactive intestinal peptide and substance P but all are immunoreactive for the synthesizing enzyme of acetylcholine, choline acetyltransferase. Thus, populations of neurons containing calbindin are cholinergic interneurons in the myenteric plexus of rabbit ileum.This study was supported by grant OTKA T 34160     

The fine structure of smooth muscle cells and their relationship to connective tissue in the rabbit portal vein

Summary The smooth muscle of rabbit portal vein was studied by electron microscopy with particular emphasis on the mechanical linkage between the muscle cells and on the distribution of connective tissue.The media of this vein is composed of inner circular and outer longitudinal muscle layers which are orientated almost perpendicularly to each other. The muscle of the inner circular layer shows very irregular contours with much branching and anastomosing of the cytoplasmic processes, which often make membrane contacts with neighbouring cells to form an extensive network of cytoplasmic processes. The muscle cells of the outer longitudinal layer are arranged in densely packed bundles and are spindle-shaped, with no branching processes. Opposing dense areas from neighbouring cells, with variable gap distances (30–100 nm) and close membrane contacts (intermediate junctions) with a gap of 11 nm were observed in both circular and longitudinal muscle layers.In the terminal regions of muscle cells in both circular and longitudinal layers a specialized anchoring structure was present which was closely related to extracellular elastic tissue. Muscle cells in the longitudinal layer showed the most elaborate structure, the tapering end of the muscle cell showing a honeycomb-like structure penetrated by columns of connective tissue compounds. The functional implications of these structures are discussed.     

Immunocytochemical studies of subtypes of pulmonary endocrine cells in diethylnitrosamine-treated rabbits

New Zealand White rabbits were injected subcutaneously with 20 mg/kg body weight of diethylnitrosamine (DEN), twice per week, starting when they were 1 week old. The animals were sacrificed 6 to 12 months after the first injection and lung tissues were processed for light microscopy. Using serotonin (5HT) and neuron specific enolase (NSE) as markers for the endocrine cells, tissue sections were stained immunocytochemically by the avidin-biotin complex method. Numerous neuroepithelial bodies (NEBs) positive for 5HT, but negative for NSE, were seen in the alveolar duct regions of DEN-treated rabbits. On the other hand, an increased number of solitary endocrine cells immunoreactive for NSE was found in bronchial or bronchiolar epithelia. The results indicate that DEN induced increases in two distinct types of endocrine cells: the component cells of NEBs are positive for 5HT and solitary cells are positive for NSE.     

Enhancement of juvenile hormone biosynthesis in locusts following electrostimulation of cerebral neurosecretory cells

Electrostimulation of the medial neurosecretory cells of day-1 adult female Locusta migratoria resulted in a significant enhancement of juvenile hormone biosynthesis by the corpora allata within 2–3 days of the operation, as determined by a radiochemical assay for juvenile hormone biosynthesis. This elevation in the rate of juvenile hormone biosynthesis was also reflected in basal oöcyte length, with the oöcytes of stimulated animals significantly larger than the sham-operated animals. Radio-frequency cautery of the cerebral axonal tracts of the medial neurosecretory cells prevented this enhancement in juvenile hormone biosynthesis and in basal oöcyte growth in both stimulated and sham-operated animals.Stimulation of the lateral neurosecretory cells resulted in a slight elevation in rates of juvenile hormone biosynthesis 2 days after the operation. However, after cautery of the medial cell tracts, a significant elevation in juvenile hormone biosynthesis was observed 1 and 2 days after stimulation. Basal oöcyte length in stimulated animals differed significantly from sham-operated animals only on day 6. Cautery of the medial cell tracts again attenuated oöcyte growth. Our results suggest that the medial neurosecretory cells are the source of an allatotropin that can be released by electrostimulation. This substance appears to operate directly on the corpus allatum, causing a change in the juvenile hormone biosynthetic machinery.     

Origin of primordial germ cells in the prestreak chick embryo

The temporal and spatial pattern of segregation of the avian germline from the formation of the area pellucida to the beginning of primitive streak formation (stages VII–XIV, EG&K) was investigated using the culture of whole embryos and central and peripheral embryo fragments on vilelline membranes at stages VII–IX, immunohistological analysis of whole mount embryos and sections with monoclonal antibodies MC-480 against stage-specific embryonic antigen-1 (SSEA-1) and EMA-1, and with the culture of dispersed blastoderms at stages IX–XIV with and without an STO feeder layer. Whole embryos at intrauterine stages developed up to the formation of the primitive streak despite the absence of area pellucida expansion. Primordial germ cells (PGCs) appeared in the cultures of whole embryos and only in central fragments containing a partially formed area pellucida at stages VII–IX. When individual stage IX–XIV embryos were dispersed and cultured without a feeder layer, 25–45 PGCs/embryo were detected only with stage X–XIV, but not with stage IX blastoderms. However, the culture of dispersed cells from the area pellucida of stages IX–XIII on STO feeder layers yielded about 150 PGCs/embryo. The carbohydrate epitopes recognized by anti-SSEA-1 and EMA-1 first appeared at stage X on cells in association with polyingressing cells on the ventral surface of the epiblast and later on the dorsal surface of the hypoblast. The SSEA-1-positive hypoblast cells gave rise to chicken PGCs when cultured on a feeder layer of quail blastodermal cells. From these observations, we propose that the segregation and development of avian germline is a gradual, epigenetic process associated with the translocation of SSEA-1/EMA-1-positive cells from the ventral surface of the area pellucida at stage X to the dorsal side of the hypoblast at stages XI–XIV. © 1996 Wiley-Liss, Inc.     

Endocrine cells in atresic chick embryo intestine: histochemical and immunohistochemical study

Intestinal motility disorders are an important problem in the postoperative management of patients with intestinal atresia. Intestinal motility could be initiated by luminal factors that activate intrinsic and extrinsic primary afferent nerves involved in the peristaltic reflex. Endocrine cells act as a key point, because they transfer information regarding the intestinal contents and intraluminal pressure to nerve fibers lying in close proximity to the basolateral surface of the epithelium. In chick embryo, experimental intestinal atresia is associated with disorders in the development of the enteric nervous system, related to the severity of intestinal dilation. Our aim was to investigate the distribution pattern of endocrine cells in the developing endocrine system of chick embryo small intestine with experimentally-induced atresia on day 12 and on day 16. Changes in enteroendocrine population were examined in gut specimens (excised proximal and distal to the atresia) from experimental embryos 19 days old and in control sham-operated chick embryos at the same age. Sections from proximal and distal bowel and control bowel were stained with Grimelius silver stain, a valuable histochemical method for detecting the argyrophil and argentophilic cells, and with an immunohistochemical procedure for detecting serotonin and neurotensin immunoreactive cells. In chick embryo proximal bowel, intestinal dilation differed in the various embryos. We found significantly higher enteroendocrine cell counts in proximal bowel than in distal and control bowel. The differences depended on the precociousness of surgery and the severity of dilation. Considering the major contribution of enteroendocrine cells to the peristaltic reflex, our data may help to explain the pathogenesis of motility disorders related to intestinal atresia.     

Pyrimidine biosynthesis in normal and transformed cells

We have developed procedures for sensitive measurement of specific radioactivities of pyrimidine nucleosides excreted from cells in culture. The changes in the observed values reflect dilution of the added isotope through de novo biosynthesis of nonradioactive pyrimidine nucleosides or by shifting and equilibration of other nucleotide pools into the free uridine pool. It is thus possible to monitor uridine biosynthesis occurring in intact cells without destroying or disrupting the cell population. On comparing a series of normal and transformed lines, we have observed several growth-dependent patterns of change in specific activity and levels of uridine excretion and the temporal appearance of these changes. Hamster embyro fibroblasts slows pyrimidine biosynthesis at mid-growth while the hamster cell line V79 continues to dilute the pyrimidine pool at about 7% of the rate observed during exponential growth at confluence. Both cells exhibit Urd excretion beginning at one-half maximal growth. Passageable normal rat liver cells (IARC-20) also show a cessation of pyrimidine biosynthesis with a prior increase in uridine excretion. Two chemically transformed lines IARC-28 and IARC-19 derived from IARC-20 show different patterns. IARC-19 begins uridine excretion in early log growth and the specific activity continues to decrease at about 2% of the rate observed during exponential growth at confluence. The IARC-28 cells also begin excretion in early log growth but pyrimidine biosynthesis stops at about midlog. This method may prove to be an additional aid in recognizing and differentiating transformed cells in culture that do not exhibit the transformed phenotype.     

Developmental potential and behavior of tetraploid cells in the mouse embryo

Tetraploid (4n) mouse embryos die at variable developmental stages. By examining 4n embryos from F2 hybrid and outbred mice, we show that 4n developmental potential is influenced by genetic background. The imprinted inactivation of an X chromosome-linked eGFP transgene in extraembryonic tissues occurred correctly in 4n embryos. A decrease of the cleavage rate in 4n preimplantation embryos compared to diploid (2n) embryos was revealed by real-time imaging, using a histone H2b:eGFP reporter. It has previously been known that mouse chimeras produced by the combination of diploid (2n) embryos with embryonic stem (ES) cells result in mixtures of the two components in epiblast-derived tissues. In contrast, the use of 4n host embryos with ES cells restricts 4n cells from the embryonic regions of chimeras, resulting in mice that are believed to be completely ES-derived. Using H2b:eGFP transgenic mice and ES cells, the behavior of 4n cells was determined at single cell resolution in 4n:2n injection and aggregation chimeras. We found a significant contribution of 4n cells to the embryonic ectoderm at gastrulation in every chimera analyzed. We show that the transition of the embryonic regions from a chimeric tissue to a predominantly 2n tissue occurs after gastrulation and that tetraploid cells may persist to midgestation. These findings suggest that the results of previously published tetraploid complementation assays may be influenced by the presence of tetraploid cells in the otherwise diploid embryonic regions.     

Synaptic connections of cone bipolar cells that express the neurokinin 1 receptor in the rabbit retina

We have investigated and further characterized, in the rabbit retina, the synaptic connectivity of the ON-type cone bipolar cells that are immunoreactive for an antibody against the neurokinin-1 receptor (NK1R). NK1R-immunoreactive bipolar cell axons terminate in stratum 4 of the inner plexiform layer. The axons of NK1R-positive bipolar cells receive synaptic inputs from amacrine cells through conventional synapses and from putative AII amacrine cells via gap junctions. The major outputs from NK1R-positive bipolar cells make contacts with amacrine cell processes. The most frequent postsynaptic dyads comprise two amacrine cell processes. Double-labeling experiments with antibodies against NK1R and either calretinin or glycine have demonstrated that NK1R-immunoreactive bipolar cells form gap junctions with AII amacrine cells. Thus, NK1R-positive cone bipolar cells, together with calbindin-positive cone bipolar cells, may play an important role in transferring rod signals to the ON-type ganglion cells of the cone pathway in the rabbit retina.I.-B. Kim and M.R. Park contributed equally to this work.This work was supported by the Ministry of Science and Technology of Korea (grant no. M1-0108-00-0059; Neurobiology Support Grant).