Organization of ‘special’ parenchymal cells underlying the midgut in some Barnacles

The layer of cells immediately underlying the midgut in the Balanidae contains unusual structural features which distinguish it from the remaining parenchyma. The cells, in general, are expanded at their distal end (away from the midgut), narrow at the proximal end (towards the midgut) and are referred to in this study as morphologically polarized cells. The narrow portion contains numerous aligned microtubules which pass through holes in perpendicularly oriented membranous sacs. This portion of the cell continues toward the midgut between layers of muscle across hemolymphatic space and terminates within infolding at the basal end of the intestinal epithelial cells. The distal, expanded portion of the cell contains many dense, lamellar bodies. S.E.M. X-ray spectra of the lamellar bodies show predominant peaks for zinc and phosphorus. Some functional implications of the organization and location of these cells are discussed.     

Fine structure of the midgut epithelium in the developing brown shrimp,Penaeus aztecus

The midgut epithelium of larval and early postlarval brown shrimp has been studied with light and electron microscopy. Ultrastructurally the features of the midgut do not change during these stages of development. On the basis of electron density, two epithelial cell types can be distinguished, and these are referred to as light and dark cells. The dark cells contain more rough endoplasmic reticulum and more free ribosomes than the light cells. Mitochondria in the dark cells have a matrix which is less electron dense than the mitochondrial matrix of the light cells. Both cell types have a microvillous border with a surface coat. The microvilli lack microfilaments within their core, and a terminal web is not differentiated in the stages examined. Tubular smooth endoplasmic reticulum is abundant in the basal portions of the cells. Electron dense, membrane bound vesicles are consistently seen in association with the Golgi apparatus, apical cell surface, and gut lumen and therefore are believed to be secretory granules. Cells in the anterior portion of the midgut often contain very large lipid droplets in the cytoplasm.     

Cell cycle and positional constraints on FtsZ localization and the initiation of cell division in Caulobacter crescentus

Swarmer cells of Caulobacter crescentus are devoid of the cell division initiation protein FtsZ and do not replicate DNA. FtsZ is synthesized during the differentiation of swarmer cells into replicating stalked cells. We show that FtsZ first localizes at the incipient stalked pole in differentiating swarmer cells. FtsZ subsequently localizes at the mid-cell early in the cell cycle. In an effort to understand whether Z-ring formation and cell constriction are driven solely by the cell cycle-regulated increase in FtsZ concentration, FtsZ was artificially expressed in swarmer cells at a level equivalent to that found in predivisional cells. Immunofluorescence microscopy showed that, in these swarmer cells, simply increasing FtsZ concentration was not sufficient for Z-ring formation; Z-ring formation took place only in stalked cells. Expression of FtsZ in swarmer cells did not alter the timing of cell constriction initiation during the cell cycle but, instead, caused additional constrictions and a delay in cell separation. These additional constrictions were confined to sites close to the original mid-cell constriction. These results suggest that the timing and placement of Z-rings is tightly coupled to an early cell cycle event and that cell constriction is not solely dependent on a threshold level of FtsZ.     

An in vivo study on the synchronizing effect of hydroxyurea

The effect of hydroxyurea (HU; 0.5 mg/g body wt) on L 1210 ascites tumor cells has been studied using various cell kinetic methods. In contrast to the general assumption that HU blocks cells at the G1/S boundary [J. Brachet (1985) Molecular Cytology, Vol. I, p. 266, Academic Press, New York], the present results show that the cells are not held at G1/S but enter S at about the normal rate and are accumulated in early S phase due to a dose-dependent inhibiting effect of HU on DNA synthesis. Partial synchronization of the cells demonstrated by a distinct mitotic peak 10 h after HU application is not due to a G1/S block of the cells and their subsequent synchronous passage through the cycle after release from the block but is due to rather complex mechanisms of action of HU: a differential cytocidal effect and an effect on the passage of the cells through the cycle, both depending on the position of the cells throughout the cycle. HU kills S-phase cells, mainly cells in early S phase; i.e., a great portion of the cells "accumulated" in early S phase is killed by the drug, while G1-phase cells are almost not affected by the lethal effect of HU. These G1-phase cells pass through the cycle more rapidly after cessation of the HU effect. The same is true for the surviving cells accumulated in early S phase, while part of the cells in the remaining S phase are delayed in their passage through the cycle. This causes partial synchronization, since a great portion of all cells that survive HU treatment reach mitosis at the same time.     

Lysozyme in the midgut of Manduca sexta during metamorphosis.

Low levels of lysozyme were found in the midgut epithelium of the tobacco hornworm, Manduca sexta, during the early part of the fifth larval stadium. This was observed in control insects as well as in bacterially challenged insects. No lysozyme was detected in the gut contents of either group of insects which were actively eating or in the early stages of metamorphosis. However, high levels of lysozyme activity were detected in homogenates of midgut tissue collected from insects later in the stadium. Immunocytochemical studies demonstrated that lysozyme accumulates in large apical vacuoles in regenerative cells of the midgut during the larval-pupal molt. These cells, initially scattered basally throughout the larval midgut epithelium, multiply and form a continuous cell layer underneath the larval midgut cells. At the larval/pupal ecdysis the larval midgut epithelium is sloughed off and the regenerative cells, now forming the single cell layer of the midgut, release the contents of their vacuoles into the midgut lumen. This release results in high lysozyme activity in the lumen of the pupal midgut and is thought to confer protection from bacterial infection. This is the first indication that the lysozyme gene may be developmentally regulated in a specific tissue in the absence of a bacterial infection.     

Two different thresholds of wingless signalling with distinct developmental consequences in the Drosophila midgut.

Drosophila wingless encodes a Wnt protein which mediates communication between cells. Although wingless protein is secreted from cells, there is debate as to what is the range of wingless action. We examined the function of wingless in the larval midgut, and found that wingless acts at two different thresholds to pattern this tissue. Low wingless levels are required to promote the development of copper cells, highly differentiated midgut cells of the larval midgut that are specified by the homeotic gene labial. High wingless levels repress copper cell development and allow differentiation of an alternative cell type, called large flat cells. These two developmental outcomes reflect labial expression, which is stimulated at low levels and repressed at high levels of wingless signalling. Thus, midgut cells respond differentially to distinct wingless thresholds in terms of both gene control and cellular differentiation.     

Characterization of microsome-associated tobacco BY-2 centrins

Centrin - higher plants - MTOCs - microtubules nucleation In most eukaryotic cells, the Ca(2+)-binding protein centrin is associated with structured microtubule-organizing centers (MTOCs) such as centrosomes. In these cells, centrin either forms centrosome-associated contractile fibers, or is involved in centrosome biogenesis. Our aim was to investigate the functions of centrin in higher plant cells which do not contain centrosome-like MTOCs. We have cloned two tobacco BY-2 centrin cDNAs and we show that higher plant centrins define a phylogenetic group of proteins distinct from centrosome-associated centrins. In addition, tobacco centrins were found primarily associated with microsomes and did not colocalize with gamma-tubulin, a known MTOC marker. While the overall level of centrin did not vary during the cell cycle, centrin was prominently detected at the cell plate during telophase. Our results suggest that in tobacco, the major portion of centrin is not MTOC-associated and could be involved in the formation of the cell plate during cytokinesis.     

The ultrastructural investigation of the midgut in the quill mite Syringophilopsis fringilla (Acari,Trombidiformes: Syringophilidae)

The midgut of the females of Syringophilopsis fringilla (Fritsch) composed of anterior midgut and excretory organ (=posterior midgut) was investigated by means of light and transmission electron microscopy. The anterior midgut includes the ventriculus and two pairs of midgut caeca. These organs are lined by a similar epithelium except for the region adjacent to the coxal glands. Four cell subtypes were distinguished in the epithelium of the anterior midgut. All of them evidently represent physiological states of a single cell type. The digestive cells are most abundant. These cells are rich in rough endoplasmic reticulum and participate both in secretion and intracellular digestion. They form macropinocytotic vesicles in the apical region and a lot of secondary lysosomes in the central cytoplasm. After accumulating various residual bodies and spherites, the digestive cells transform into the excretory cells. The latter can be either extruded into the gut lumen or bud off their apical region and enter a new digestive cycle. The secretory cells were not found in all specimens examined. They are characterized by the presence of dense membrane-bounded granules, 2–4 μm in diameter, as well as by an extensive rough endoplasmic reticulum and Golgi bodies. The ventricular wall adjacent to the coxal glands demonstrates features of transporting epithelia. The cells are characterized by irregularly branched apical processes and a high concentration of mitochondria. The main function of the excretory organ (posterior midgut) is the elimination of nitrogenous waste. Formation of guanine-containing granules in the cytoplasm of the epithelial cells was shown to be associated with Golgi activity. The excretory granules are released into the gut lumen by means of eccrine or apocrine secretion. Evacuation of the fecal masses occurs periodically. Mitotic figures have been observed occasionally in the epithelial cells of the anterior midgut.     

Altered regulation of c-myc expression in adenovirus-transformed cells

Expression of the oncogenes c-myc, c-raski, and p53 is studied in normal primary mouse cultures and in two adenovirus-transformed mouse cell lines. In all cases oncogene expression is measured in cells arrested in G1 (or G0 for primary cells) by serum starvation and at different times after cell cycle traverse is stimulated by addition of high serum. For primary mouse cells, c-myc mRNA levels are found to increase four- to six-fold within 1 h of serum addition and then decline by 4 h to nearly the level observed in serum-starved cells. This level is maintained throughout the remainder of the cell cycle. The early induction of c-myc is dependent on serum concentration and is independent of cell density. These results confirm and extend previous observations for primary cells. By contrast, expression of c-raski does not vary at all through the cell cycle and p53 increases with time after mitogenic stimulation. In the adenovirus-transformed cell lines, the regulation of expression of c-myc with respect to the cell cycle is altered. There is an increase in c-myc in S phase cells which is dependent on cell density and the early induction in response to serum addition as seen in primary cells is absent. Expressions of c-raski and p53 are found to show similar profiles to those observed for primary cells.     

Morphological regional differences of epithelial cells along the midgut in Diatraea saccharalis Fabricius (Lepidoptera: Crambidae) larvae

The sugarcane borer, Diatraea saccharalis Fabricius, is a pest to sugarcane and many other crops. This work aims to characterize morphological variability in the epithelial cells (columnar, goblet and regenerative) along the midgut of D. saccharalis larvae. Fragments of the midgut (anterior, middle and posterior regions) were fixed and processed by light and scanning electron microscopy. There are both cytochemical and ultrastructural differences in the morphology of the epithelial cells, depending on their localization along the midgut. The apical surface of columnar cells shows an increase in both number and size of the apical protrusions from the anterior to the posterior midgut regions. There is an increase in the amount of PAS-positive (Periodic Acid-Schiff Reaction) granules detected in the cytoplasm of both the columnar and regenerative cells, from the anterior to the posterior region. The goblet cell apical surface is narrow in the anterior region, and enlarged in the posterior midgut; the chamber's cytoplasm extrusion are small and thin at the apical cavity surface, being thicker, longer and more numerous at the basal portion of the cavity. Our results suggest that the sugarcane borer midgut has two morphologically different regions, the anterior and the posterior; the middle region is a transitional region.     

Nuclear metabolic changes induced by tumor necrosis factor in Daudi lymphoma cells; a multiparametric analysis.

The effects of r-TNF alpha on cell cycle progression and DNA polymerase activity in Daudi lymphoma cells have been analyzed. Cytofluorimetric analysis of the cell cycle after 6 to 24 hr of treatment revealed both a decrease of BrdU incorporation per cell and a light inhibition of S phase as assessed by the analysis of the percentual distribution of cell cycle compartments. The reduction of BrdU incorporation can be related to the early decrease in the rate of DNA synthesis that follows r-TNF alpha treatment. These results suggest that one of the early events induced by r-TNF alpha at nuclear level is the slowering of DNA synthesis leading to a reduced cell cycle progression.     

Mode of estrogen action on cell proliferation in CAMA-1 cells: II. Sensitivity of G1 phase population

The mammary cancer cell line CAMA-1 synchronized at the G1/S boundary by thymidine block or at the G1/M boundary by nocodazole was used to evaluate 1) the sensitivity of a specific cell cycle phase or phases to 17 beta-estradiol (E2), 2) the effect of E2 on cell cycle kinetics, and 3) the resultant E2 effect on cell proliferation. In synchronized G1/S cells, E2-induced 3H-thymidine uptake, which indicated a newly formed S population, was observed only when E2 was added during, but not after, thymidine synchronization. Synchronized G2/M cells, enriched by Percoll gradient centrifugation to approximately 90% mitotic cells, responded to E2 added immediately following selection; the total E2-treated population traversed the cycle faster and reached S phase approximately 4 hr earlier than cells not exposed to E2. When E2 was added during the last hour of synchronization (ie, at late G2 or G2/M), or for 1 hr during mitotic cell enrichment, a mixed response occurred: a small portion had an accelerated G1 exit, while the majority of cells behaved the same as controls not incubated with E2. When E2 addition was delayed until 2 hr, 7 hr, or 12 hr following cell selection, to allow many early G1 phase cells to miss E2 exposure, the response to E2 was again mixed. When E2 was added during the 16 hr of nocodazole synchronization, when cells were largely at S or possibly at early G2, it inhibited entry into S phase. The E2-induced increase or decrease of S phase cells in the nocodazole experiments also showed corresponding changes in mitotic index and cell number. These results showed that the early G1 phase and possibly the G2/M phase are sensitive to E2 stimulation, late G1, G1/S, or G2 are refractory; the E2 stimualtion of cell proliferation is due primarily to an increased proportion of G1 cells that traverse the cell cycle and a shortened G1 period, E2 does not facilitate faster cell division; and estrogen-induced cell proliferation or G1/S transition occurs only when very early G1 phase cells are exposed to estrogen. These results are consistent with the constant transition probability hypothesis, that is, E2 alters the probability of cells entering into DNA synthesis without significantly affecting the duration of other cell cycle phases. Results from this study provide new information for further studies aimed at elucidating E2-modulated G1 events related to tumor growth.     

Primary and continuous midgut cell cultures from Pseudaletia unipuncta (Lepidoptera: Noctuidae)

Midgut epithelial cells were isolated from fifth-instar Pseudaletia unipuncta larvae by collagenase treatment of midgut tissue, and cultured in TNM-FH medium. Long-term continuous culture and maintenance of midgut cells were achieved with P. unipuncta armyworm intestinal cells. Several cells lines were obtained from these P. unipuncta primary cultures, and they have been subcultured and maintained for over 24 mo. The three major midgut cell types were present in the cultures, including stem (regenerative), columnar, and goblet cells. In vitro morphogenesis and differentiation of columnar and goblet cells from stem cells were observed. There appeared to be a cycle of cell death of goblet and columnar cells followed by their replacement from stem cells every 7-8 wk. After approximately six passages, the cell density in T-flasks appeared to be somewhat constant, reaching 10(3)-10(4) cells per milliliter of medium. The columnar cells are round to rectangular in shape and possess a brush border, while the goblet cells have a classic flask-like shape with a central cavity. Peritrophic membrane-like secretions were observed in all the culture flasks. Infection of these cells with multiply embedded nucleopolyhedrovirus was confirmed, and we conclude that these midgut cells can be used as an in vitro model system to study early events in baculovirus infection.     

Alterations of cell cycle kinetics and vimentin expression in TPA-treated, asynchronous MPC-11 mouse plasmacytoma cells

Vimentin expression throughout the cell cycle has been analyzed at the single-cell level in asynchronously growing MPC-11 cells using multiparameter flow cytometry. We have previously shown that these cells normally lack detectable amounts of intermediate filament proteins. In the presence of the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA), cell proliferation ceases and large quantities of the intermediate filament protein vimentin are synthesized and accumulate in most of the cells. In the present study, the short-term effect of TPA on distribution of cells within the cell cycle was a depletion in early S phase followed by a depletion in mid- and late S phase. In parallel, the G1-phase fraction increased significantly. In addition, a delay in progression through G2/M phase was observed. These data strongly suggest an inhibition of progression of cells through the cell cycle in G1 phase as the primary event on cell cycle kinetics elicited by TPA. Vimentin accumulation could be detected by flow cytometry as early as 2 h after TPA addition; at this time, the percentage of vimentin-positive cells was highest in G2/M phase. Prolonged TPA treatment induced vimentin accumulation in cells of all cell cycle phases. However, even at later times, the G1-phase population consisted of two subpopulations with low and high vimentin content, respectively. The fraction of cells which displayed a higher level of vimentin probably represents those G1-phase cells which previously had undergone cell division in the presence of TPA. Our data indicate that TPA-induced vimentin synthesis is regulated in a cell cycle-dependent manner and is maximally induced in cells which have passed a putative cell cycle restriction point in G1 phase.     

Heat-shock gene expression and cell cycle changes during mammalian embryonic development

Synchronized regulation of cell division during gastrulation is essential for the regional proliferation of cells and pattern formation of the early CNS. The neural plate and neuroectoderm cells are a rapidly dividing and differentiating population of cells with a unique and rapid heat-shock response. Heat shock and the heat-shock genes were studied during neural plate development in a whole rat embryo culture system at 9.5-11.5 days. A lethal heat shock can cause cell death and severe developmental defects to the forebrain and eye during organogenesis. Heat shock can also result in acquired thermotolerance whereby cell progression is delayed at the G1/S and S/G2 boundaries of the cell cycle. This delay in cell cycle progression caused an overall lengthening of the cell cycle time of at least 2 hr. The heat shock genes may therefore function as cell cycle regulators in neuroectoderm induction and differentiation. The kinetics and expression of the hsp genes were examined in neuroectodermal cells by flow cytometry and Northern analysis. The levels of hsp mRNA 27, 71, 73, and 88 were identified following exposure at 42°C (nonlethal), 43deg;C (lethal) and 42deg;/43deg;C (thermotolerant) heat shock. Examination of hsp gene expression in the neural plate showed tight regulation in the cell cycle phases. Hsp 88 expression was enhanced at Go and hsp71 induction at G2 + M of the cell cycle. Cells exposed to a thermotolerant heat shock of 42deg;C induced hsp71 mRNA expression in all phases of the cell cycle with the mRNA levels of hsp27, 73, and 88 increased but relatively constant. Following a lethal heat shock, dramatic changes in hsp expression were seen especially enhanced hsp71 induction in late S phase. The regulated expression of hsps during the cell cycle at various phases could play a unique and important role in the fate and recovery of neuroectoderm cells during early mammalian embryo development. © 1993Wiley-Liss, Inc.     

Light and electron microscopical study of two types of endocrines cell in the midgut of the adult worker honeybee (apis mellifera)

The occurrence, development and ultrastructure of two types of gut endocrine cell have been studied in the midgut of adult honeybees. These cells, one of a basal granular type and one of a vesicular type, are evenly distributed throughout the posterior three-quarters of the midgut. Each crypt complex contains one of each cell type, both of which may be derived from the same stem cells as the enterocytes. They already contain their respective secretory product while still in the nidus. Both reach the midgut lumen by a narrow apex and are therefore of the open type. The granular cells release their secretory granules at the cell base in a typical endocrine way. In young vesicular cells the secretory vesicles are released at the cell base and in the intercellular spaces. Old cells are still filled with vesicles when they are shed in the midgut lumen. This seems to indicate that these cells have both an endocrine (or paracrine) and an exocrine function, the latter apparently by holocrinc release.     

Trypsin-like activity in the vaginal epithelial cells of the rat.

Rat vaginal epithelial cells have trypsin-like activity as shown by the formation of a colored product when the cells are incubated with alpha-N-methyl alpha-N-toxyl-L-lysine beta-naphthol ester and hexazotized pararosanilin. This enzyme activity in vaginal smears is maximal at proestrus, i.e., the day in the 5-day estrus cycle when plasma estrogen is maximal. Only the rounded nucleated epithelial cells present at late diestrus, proestrus and early estrus demonstrate the trypsin-like enzyme activity. These are the cells that stain blue in the Papanicolaou method. Preincubation of cell suspensions with the serine protease inhibitor, p-nitrophenyl p-guanidino benzoate, prevented the enzyme staining reaction, further demonstrating the trypsin-like nature of the cellular enzyme. The advantages of this enzyme staining technique over the fibrin plate method for the demonstration of trypsin-like enzymes in cells are increased resolution and ability to show trypsin inhibitor effects.     

Caffeine inhibition of cytokinesis: Ultrastructure of cell plate formation/degradation

Summary Caffeine is a potent inhibitor of cell plate formation in dividing plant cells. Previous studies living cells reveal that the drug always permits the cell plate to arise and grow normally until about 80% complete, but then causes it to break down. In the present investigation we examine this formation/degradation cycle at the ultrastructure level. Our results show that during the formation phase the caffeine treated plate is indistinguishable from untreated controls. Phragmoplast microtubules arise and align in the interzone, Golgi vesicles are produced and aggregate in a line that defines the young cell plate, and considerable fusion of these vesicles occurs to form islands of plate material. However, under the influence of caffeine these islands do not fuse to form the enlarged lamellar expanses characteristic of maturing cell plates. Instead, the partially fused material reverts to small vesicles which appear to become resorbed by the cellular membrane systems. The resorption process continues leaving no evidence of the previously developing plate, although occasionally we observe a stub of fused vesicles attached to the parent wall. Following cell plate disintegration the reformed nuclei move close together and occupy the central region of the cell. These observations focus attention on the consolidation phase of cell plate formation as the one being maximally affected by caffeine.Dedicated to the memory of Professor Oswald Kiermayer