Amyloid fibril formation in the bovine mammary gland: an ultrastructural study

Bovine mammary secretory tissue was examined histologically to determine the origin of amyloid fibrils and their mode of deposition. Spherical bodies of amyloid fibrils found in alveolar lumina and epithelium were closely associated with epithelial and monocytoid cells. Small bundles of parallel fibrils were observed within and between alveolar epithelial cells, and large spherical bodies occasionally developed in these positions, protruding into the luminal space. Bundles of parallel fibrils at the periphery of amyloid bodies in the alveolar lumen appeared to develop from the apical epithelial plasma membrane or in the cytoplasm just within the cell border. Bundles of parallel amyloid fibrils were also observed in slight indentations in the plasma membrane of monocytoid cells. In some cases, the point of contact between single fibrils and the plasma membrane was not discerned, and fibrils appeared continuous with the cytoplasm. The alveolar lumina appeared to be the major site of amyloid body formation. It is suggested that epithelial and monocytoid cells elaborate a soluble precursor which polymerizes into fibrils at the plasma membrane and in the peripheral cytoplasm, or is secreted by the cell and polymerizes extracellularly.     

AN ELECTRON MICROSCOPE STUDY OF LYMPHATIC TISSUE IN RUNT DISEASE

The thymus, spleen, and lymph nodes were studied in runt disease induced by a graft of intravenously injected homologous splenic cells into newborn rats and mice. Adult Long-Evans cells (70 x 10⁶) were injected into Sprague-Dawley rats. Adult DBA cells (7 x 10⁶) were injected into C57BL/6 mice. Runted rats were sacrificed at 14 to 28 days of age; mice at 10 to 20 days. The thymic cortex is depleted of small lymphocytes. Those remaining are severely damaged and phagocytized. Evidence of damage includes swelling of mitochondria, myelin figure formation, margination of chromatin, and sharp angulation in nuclear contour. Large numbers of macrophages are present. Epithelial-reticular cells which envelop small cortical blood vessels are often retracted, with the result that the most peripheral layer in the thymic-blood barrier suffers abnormally large gaps. Lymphocytes of the periarterial lymphatic sheaths of spleen and of the cortex of lymph nodes are reduced in number and damaged. Vast numbers of plasma cells and many lymphocytes are evident throughout lymph nodes, in the periarterial lymphatic sheaths, and in the marginal zone and red pulp of the spleen. Plasma cells are of different sizes, the larger having dilated sacs of endoplasmic reticulum. Lymphocytes are small to medium in size. They contain, in varying quantity, ribosomes and smooth membrane-bounded cytoplasmic vesicles approximately 350 to 500 A in diameter. Most plasma cells and lymphocytes are damaged and many of these are phagocytized. Many lymphocytes in lymph nodes, however, show no evidence of damage. Reticular cells and other fixed cells of the connective tissues seldom appear affected. Thus, the major cell types reacting in runt disease are lymphocytes, plasma cells, and histiocytes or macrophages. It appears, therefore, that both the delayed and immediate types of sensitivity play a part in this disease.     

Postnatal development of dendritic reticulum cells and their immune complex trapping ability

The postnatal development of dendritic reticulum cells in the rat popliteal lymph nodes was electron microscopically investigated in relation to the appearance of immune complex trapping capacity. The popliteal lymph nodes of neonatal rat consisted of loosely arranged fibroblastic reticulum cells. In the following stage, the peripheral cortex and paracortex became distinguishable. The former was made up of an accumulation of small lymphocytes, scattered within a framework of reticulum cells. On te 28 th day, the first primary follicle appeared in the peripheral cortex. Simultaneously the immune complex could be trapped on the cytoplasmic membrane of reticulum cells, which were located in the central portion of the primary follicles. The early image of germinal centers appeared corresponding to immune complex trapping areas. In the well-developed secondary follicles, the immune complex trapping cells were mainly localized in the cap area. Their cytoplasmic membranes formed the dendritic processes, on which the distinct ability of trapping of the immune complex was recognized. It was demonstrated that the fibroblastic reticulum cells, forming the stroma of lymph nodes, were transformed into the typical dendritic reticulum cells with labyrinth structures in the cap area. Desmosomal junctions were often found, not only between the dendritic reticulum cells themselves, but also between the dendritic reticulum cells and lymphocytes. We suggest that the desmosomal junctions play a role as the channel for a transmission of immunological information.     

Cellular composition of various structural zones of the iliac and mesenteric lymph node of pregnant rats

The dynamic of cellular reactions demonstrates certain changes in functional activity of all structures of the node during pregnancy. A similar trend of processes in the iliac (regional for the uterus) and mesenteric lymph nodes has been defined. At early stages of pregnancy, lymph nodule are the most active, this is demonstrated as an increasing portion of lymphoblasts, macrophages and dividing cells. During this period, cell composition of the cortical plateau is relatively stable. For the paracortical zone of the mesenteric lymph nodes a rather significant decrease in the portion of middle lymphocytes and reticular cells is characteristic. There is not any significant change in the relative amount of the cells in the same functional zone of the iliac lymph nodes during the same period of pregnancy. The medullar cords demonstrate an increasing number of blast forms and young plasmocytes. However, as the pregnancy develops, the structure of the paracortical zone undergoes an essential change--progressively increases the portion of lymphoblasts and large lymphocytes. The blastic reaction in the mesenteric lymph nodes is proved to depend, to some extent, on that in the iliac lymph nodes of the same animal. Mature plasma cells become the dominating cellular element in the medullary cords. At the end of the pregnancy a relative amount of the reticular cells increases in all structural zones of the node.     

Investigation into the role of macrophages in the formation and degradation of beta2-microglobulin amyloid fibrils

Dialysis related amyloidosis is a serious complication of long-term hemodialysis in which beta(2)-microglobulin (beta(2)m) forms amyloid fibrils that deposit predominantly in cartilaginous tissues. How these fibrils form in vivo, however, is poorly understood. Here we perform a systematic investigation into the role of macrophages in the formation and degradation of beta(2)m amyloid fibrils, building on observations that macrophages are found in association with beta(2)m amyloid deposits in vivo and that these cells contain intra-lysosomal beta(2)m amyloid. In live cell imaging experiments we demonstrate that macrophages internalize monomeric beta(2)m, whereupon it is sorted to lysosomes. At lysosomal pH beta(2)m self-associates in vitro to form amyloid-like fibrils with an array of morphologies as visualized by atomic force microscopy. Cleavage of the monomeric protein by both macrophages and lysosomal proteases isolated from these cells results in the rapid degradation of the monomeric protein, preventing amyloid formation. Incubation of macrophages with preformed fibrils revealed that macrophages internalize amyloid-like fibrils formed extracellularly, but in marked contrast with the monomeric protein, the fibrils were not degraded within macrophage lysosomes. Correspondingly beta(2)m fibrils were highly resistant to degradation by high concentrations of lysosomal proteases isolated from macrophages. Despite their enormous degradative capacity, therefore, macrophage lysosomes cannot ameliorate dialysis-related amyloidosis by degrading pre-existing amyloid fibrils, but lysosomal proteases may play a protective role by eliminating amyloid precursors before beta(2)m fibrils can accumulate in what may represent an otherwise fibrillogenic environment.     

Immunolocalization of lipid peroxidation/advanced glycation end products in amyloid A amyloidosis

Chronic inflammation, superimposed by amyloid fibril deposition, is believed to trigger the cascade of oxidative stress response in the affected organs and tissues. We examined immunohistochemically the distribution of 4-hydroxy-2-nonenal (HNE) and N(epsilon)-(carboxymethyl)lysine (CML), markers of lipid peroxidation and advance glycation end products (AGE), respectively, in spleen sections and peritoneal macrophages (MPhi) from mice before and during AA amyloidosis. With time, both HNE and CML immunoreactivities increased significantly in MPhi and splenic reticuloendothelial cells, known to be associated with the clearance of serum amyloid A, the precursor of AA fibrils. HNE and CML were localized to the plasma membrane and the cytoplasmic compartment of MPhi and HNE only at the nuclear membrane. These markers were also colocalized bound to AA fibrils infiltrating the splenic sinus walls. Our results reinforce the notion that oxidative stress is an integral component of amyloidotic tissues. Both lipid peroxidation and AGE have been implicated in protein modification and amyloid fibril formation. The significance of HNE and CML associated with the monocytoid cells and implicated in SAA clearance and AA fibril formation, is discussed with the pathogenesis of AA fibrils.     

Scanning electron microscope observation of the dog mesenteric lymph node

Summary The perfused large mesenteric lymph node of the dog was observed under the scanning electron microscope.The lymph sinus contains reticulum cells which mostly are two-dimensionally formed stellate plates oriented in a uniform direction. Large round macrophages are loosely fixed by the reticulum cell processes. No intermediate type between both cells has been observed. Macrophages having a few long tentacle-like projections are densely covered by clubbed cytoplasmic processes. Smaller round cells, probably plasma cells and lymphocytes also remained in the sinus.The pulp of the node is built up by reticulum cells, much smaller than those in the sinus, and by densely packed round cells including a few macrophages.The trabeculae and the reticulum of the nodal parenchyme form a continuous structure.Cordial thanks are expressed to Dr. Toshihiro Ishii, Professor of Anatomy of the Tohoku University Medical School, for his valuable advice. Thanks are also due to the kind cooperation of Mr. Akira Kubotsu of the Central Research Laboratories, Kuraray Co. Ltd.     

Model for mediastinal lymph node metastasis produced by orthotopic intrapulmonary implantation of lung cancer cells in mice

This study is designed to establish a pulmonary tumor model to investigate the biology and therapy of lung cancer in mice. Current methods for forming a solitary intrapulmonary nodule and subsequent metastasis to mediastinal lymph nodes are not well defined. Lewis lung carcinoma cell (LLC) suspensions were orthotopically introduced into the lung parenchyma of C57/BL6 mice via a limited skin incision without thoracotomy followed by direct puncture through the intercostal space. The implantation process was performed within approximately 50 sec per mouse, and the operative mortality was less than 5%. Single pulmonary nodules developed at the implanted site in 93% of animals and subsequent mediastinal lymph nodes metastasis were observed in all mice that were succeeded to form a lung nodule after intrapulmonary implantation. The size of tumor nodule and the weight of mediastinal lymph node increased in a time-dependent manner. The mean survival time of mice implanted successfully with LLC cells was 21 +/- 2 days (range; 19-24 days). Histopathological analysis revealed that no metastatic tumor was detectable in the mediastinal lymph nodes on day 11, but metastatic foci at mediastinal lymph nodes were clearly observed on days 17 and 21 after implantation. Other metastases in distant organs or lymph nodes were not observed at 21 days after the implantation. Comparative studies with intrapleural and intravenous injections of LLC cells suggest that the mediastinal lymph node metastasis by intrapulmonary implantation is due to the release of tumor cells from the primary nodule, and not due to extrapulmonary leakage of cells. An intravenous administration of CDDP on day 1 after tumor implantation tended to suppress the primary tumor nodule and significantly inhibited the lymph node metastasis. Thus, a solitary pulmonary tumor nodule model with lymph node metastasis approximates clinical lung cancer, and may provide a useful basis for lung cancer research.     

Demonstration of the capacity of nacre to induce bone formation by human osteoblasts maintained in vitro.

Nacre implanted in vivo in bone is osteogenic suggesting that it may possess factor(s) which stimulate bone formation. The present study was undertaken to test the hypothesis that nacre can induce mineralization by human osteoblasts in vitro. Nacre chips were placed on a layer of first passage human osteoblasts. None of the chemical inducers generally required to obtain bone formation in vitro was added to the cultures. Osteoblasts proliferated and were clearly attracted by nacre chips to which they attached. Induction of mineralization appeared preferentially in bundles of osteoblasts surrounding the nacre chips. Three-dimensional nodules were formed by a dense osteoid matrix with cuboidal osteoblasts at the periphery and osteocytic-like cells in the center. These nodules contained foci with features of mineralized structures and bone-like structures, both radiodense to X-ray. Active osteoblasts (e.m.) with abundant rough endoplasmic reticulum, extrusion of collagen fibrils and budding of vesicles were observed. Matrix vesicles induced mineral deposition. Extracellular collagen fibrils appeared cross-banded and electrodense indicating mineralization. These results demonstrate that a complete sequence of bone formation is reproduced when human osteoblasts are cultured in the presence of nacre. This model provides a new approach to study the steps of osteoblastic differentiation and the mechanisms of induction of mineralization.     

Intraperitoneal amyloid formation by amyloid enhancing factor--rich macrophages in ascitic fluid.

Although resident peritoneal cells from amyloidotic mice (amyloidotic peritoneal cells) are capable of processing the precursor protein of secondary amyloidosis, serum amyloid A (SAA) to amyloid fibrils, the peritoneum is a rare site for amyloid deposition. This is considered to be due to a deficiency of SAA in the peritoneum. To increase the supply of SAA to the peritoneum, ascitic fluid containing about the same protein constituents as in the serum was induced in mice. Amyloidotic peritoneal cells were packed in a microchamber which was shielded with filter membranes, and cultured in ascitic fluid supplemented with additional inflammatory factors. On the 7th day, Congo red-positive structures which showed green birefringence under polarized light were found inside and occasionally outside the chamber. By anti-AA or -SAA immunostaining, amyloid deposits and the cell surfaces of macrophages were positive. Immunologic depletion of T- and B-lymphocytes from the amyloidotic peritoneal cells did not adversely effect the amyloid formation in microchambers. These results suggest that either ascitic fluid containing sufficient amounts of SAA, or peritoneal macrophages with a high amyloid enhancing factor (AEF) activity are indispensable for AA amyloid fibrillogenesis in the peritoneum.     

Interdigitating reticulum cells in lymph nodes of Sézary syndrome. Freeze-fracture and ultrathin-section morphology

Lymph nodes with extensive leukemic infiltration from three patients with the Sézary syndrome were examined in ultrathin sections and in freeze-fracture replicas. Sézary cells (SC) and interdigitating reticulum cells (IDC) were the predominant cell types in the lymph nodes. Both were closely connected with each other by apparently interdigitating cytoplasmic processes. The projections between these cells were, in the main, processes from the IDC. In freeze-fracture replicas these cellular processes did not appear as interdigitations but were more bubble-like, and for this reason these cells are imprecisely described by the term "interdigitating." The SC were seen to possess only short cytoplasmic processes. The frequent polar grouping of cell organelles in SC in the region of the contact zone with IDC and the high organelle content of IDC ('activated IDC') could be the morphologic expression of intense interaction between IDC and SC. IDC displayed three features in freeze-fracture which are not specific to the Sézary syndrome, but should be applicable to IDC in general: (1) they exhibited an approximately equal density of intramembrane particles in both the E-face and the P-face, (2) some of the intramembrane particles in the P-face were assembled in clusters and (3) the surface showed bubble-like formations of the cytoplasmic processes. On the basis of these properties it was possible to distinguish IDC from macrophages and lymphocytes in freeze-fracture replicas.     

The paracortical area in dermatopathic lymphadenitis and other reactive conditions of the lymph node

We compared the paracortical area in 4 cases of dermatopathic lymphadenitis (DL) with the same area in 11 cases of various other reactive conditions of the lymph node by immuno- and enzymehistochemical techniques. In addition, electron microscopy was performed on three cases of DL. The paracortical nodules in DL proved to be composed of a variable number of dendritic, OKT6+ OKIa + ATPase+ cells, admixed with helper T-lymphocytes. All other lymph nodes studied lacked dendritic OKT6+ cells, whereas OKIa positivity was found in the cortical (follicular centers and mantle zones) and paracortical area (lymphocytes and scattered dendritic cells). Short incubation for ATPase revealed a paracortical, pericellular staining pattern in cases of DL, whereas in all other cases this staining pattern was observed only after long incubation times. On electron microscopy, three types of dendritic cells were found in DL, namely interdigitating reticulum cells ( IDRC ). Langerhans cells (LC) and macrophages. Intermediate forms between IDRC and LC, containing a few Birbeck granules and a well developed rough endoplasmic reticulum, were found. It is suggested that immunoreactivity for the monoclonal antibody OKT6 is restricted to cases of DL, and is due to the appearance of dendritic cells that have LC-characteristics. These cells either arrive from the skin along afferent lymph vessels, or are the result of a local transformation process of IDRC that acquire LC-characteristics, i.e. OKT6 immunoreactivity and Birbeck granules.     

Serum amyloid P colocalizes with apolipoproteins in human atheroma: functional implications

Serum amyloid P (SAP) is a common component of human amyloid deposits and has been identified in atherosclerotic lesions. We investigated the extent of the colocalization of SAP with apolipoprotein A-I (apoA-I), apoB, apoC-II, and apoE in human coronary arteries and explored potential roles for SAP in these regions, specifically the effect of SAP on the rate of formation and macrophage recognition of amyloid fibrils composed of apoC-II. Analysis of 42 human arterial sections by immunohistochemistry and double label fluorescence microscopy demonstrated that SAP and apoA-I, apoB, apoC-II, and apoE were increased significantly in atherosclerotic lesions compared with nonatherosclerotic segments. SAP colocalized with all four apolipoproteins to a similar extent, whereas plaque macrophages were found to correlate most strongly with apoC-II and apoB. In vitro studies showed that SAP accelerated the formation of amyloid fibrils by purified apoC-II. Furthermore, SAP strongly inhibited the phagocytosis of apoC-II amyloid fibrils by primary macrophages and macrophage cell lines and blocked the resultant production of reactive oxygen species. The ability of SAP to accelerate apoC-II amyloid fibril formation and inhibit macrophage recognition of apoC-II fibrils suggests that SAP may modulate the inflammatory response to amyloid fibrils in atherosclerosis.     

A Modified Dominici Technic for Autoradiography: Acidified Eosin-Orange and Alcoholic Toluidine Blue

Mouse lymph nodes were fixed in Bouin-Hollande solution, processed through paraffin embedding, sectioned and coated with Kodak NTB3 liquid emulsion. After development, the autoradiographs were stained 10 min in a 0.5% eosin Y-orange G mixture acidified to pH 4.4, differentiated in 0.5% acid alcohol and counterstained 10 sec with 0.25% toluidine blue O in 50% ethanol. The technic yielded distinct differentiation of neutrophils, eosinophils, blast cells, plasmacytes, mast cells, macrophages, reticular cells, histiocytes, lymphocytes and other lymphoid cells. The method also provided sensitive definition of the cytoplasmic and nuclear characteristics of all cell types.     

Cytoskeletal arrays in the cells of soybean root nodules: The role of actin microfilaments in the organisation of symbiosomes

Summary Within the infected cells of root nodules there is evidence of stratification and organisation of symbiosomes and other organelles. This organisation is likely to be important for the efficient exchange of nutrients and metabolites during functioning of the nodules. Using immunocytochemical labelling and confocal microscopy we have determined the organisation of cytoskeletal elements, micro tubules and actin microfilaments in soybean nodule cells, with a view to assessing their possible role in organelle distribution. Most microtubule arrays occurred in the cell cortex where they formed disorganised arrays in both uninfected and infected cells from mature nodules. In infected cells from developing nodules, parallel arrays of microtubules, transverse to the long axis of the cell, were observed. In incipient nodules, before release of rhizobia into the plant cells, the cells also had an array of microtubules which radiated from the nucleus into the cytoplasm. Three actin arrays were identified in the infected cells of mature nodules: an aster-like array which emanated from the surface of the nucleus, a cortical array which had an arrangement similar to that of the cortical microtubules, and, throughout the cytoplasm, an array of fine filaments which had a honeycomb arrangement consistent with a distribution between adjacent symbiosomes. Uninfected cells from mature nodules had only a random cortical array of actin filaments. In incipient nodules, the density of actin microfilaments associated with the nucleus and radiating through the cytoplasm was much less than that seen in mature infected cells. The cortical array of actin also differed, being composed of swirling configurations of filaments. After invasion of nodule cells by the rhizobia, the number of actin filaments emanating from the nucleus increased markedly and formed a network through the cytoplasm. Conversely, the cytoplasmic array in uninfected cells of developing nodules was identical to that in the cells of incipient nodules. The cytoplasmic network in infected cells of developing nodules is likely to be the precursor of the honeycomb array seen in mature nodule cells. We propose that this actin array plays a role in the spatial organisation of symbiosomes and that the microtubules are involved in the localisation of mitochondria and plastids at the cell periphery in the infected cells of root nodules.     

Pineal corpora arenacea produced by arachnoid cells in the bat Myotis blythi oxygnathus

There are corpora arenacea among the cell layers of the arachnoid on the dorsal surface of the pineal organ of the bat (Myotis blythi oxygnathus). The pineal arachnoid consists of electron lucent cells connected by cell injunctions to flat sheets and sandwiched on both sides by electron-dense cell rows. Among the superficial cell layers, collagen fibrils form loose bundles. In the electron-lucent cells, pinocytotic vesicles, rough surfaced endoplasmic reticulum, active Golgi areas and granular vesicles of various sizes can be found. Electron dense cells display fewer cytoplasmic organelles than the light ones. Lying between and below the hemispheres and cerebellum the pineal arachnoid does not contact the dura mater directly, therefore it continues on its both sides into arachnoid trabeculae. Corpora arenacea occur in lacunar enlargements of the arachnoid, first of all in the thickened dorsal portion of the pineal leptomeninx. The acervuli are insulated by collagen fibrils and exhibit concentric layers of various density. Needle-shaped structures resembling hydroxyapatite crystals were found in these concentric layers. There was no sign of formation of acervuli in the pinealocytes or elsewhere in the pineal nervous tissue proper. These findings confirm that view that corpora arenacea can be produced by the pineal arachnoid. The formation of acervuli is accompanied by secretory and resorptive phenomena of arachnoid cells.     

Alzheimer-like plaque formation by human macrophages is reduced by fibrillation inhibitors and lovastatin

The cerebral deposition of Abeta-peptide as amyloid fibrils and plaques represents a hallmark characteristic of Alzheimer's disease (AD). AD plaques are defined by their green birefringence after Congo red staining, their spherulite-like superstructure and their association with specific secondary components. Here we show that primary human macrophages promote the formation of amyloid plaques that correspond in all aforementioned characteristics to typical amyloid plaques from diseased tissues: they consist of aggregated Abeta-peptide, they reveal the typical 'Maltese cross" structure and they are associated with the secondary components glycosaminoglycanes, apolipoprotein E (apoE) and the raft lipids cholesterol and sphingomyelin. Plaque formation can be impaired in this cell system by addition of small molecules, such as Congo red, melantonine and lovastatin, suggesting potential applications for the study of cellular amyloid formation and for the identification or validation of drug candidates.     

Structure of the non-lymphoid cells during the postnatal development of the rat lymph nodes

This study describes the postnatal development of the nonlymphoid cells with special reference to the fibroblastic reticulum cells (FRCs) and interdigitating cells (IDCs). The first lymphocytes of the neonatal lymph nodes are located in the developing deep cortex units (DCUs) identified by the Gomori's technique for reticulin fibres. Ultrastructural studies demonstrate that FRCs form the stroma of the DCUs. By light and electron microscopy, it is demonstrated that FRCs occupy the outer cortex in the following stages of development of the lymph nodes. Thus, FRCs form the stroma of the primary follicles and, later, are transformed in follicular dendritic cells (FDCs) of the germinal centres. Immature or pro-IDCs appear as migrating elements in the deep cortex of lymph nodes of the neonatal rats. The ultrastructure of the pro-IDCs resembles that of the mature IDCs but not that of the phagocytic cells. Pro-IDCs are transformed into mature IDCs whose cytoplasmic expansions contact lymphocytes via tight junctions. Some of these lymphocytes are likely apposed to FRCs of the DCUs. No cells containing Birbeck granules were found in the parenchyma of the lymph nodes during the postnatal development. The role of these nonlymphoid cells is discussed with respect to the immunologic function of mammalian lymph nodes.     

Ultrastructure and functioning of the transport system of the leguminous root nodule

Summary The structure of the vascular tissues of nitrogen-fixing nodules of 27 genera of legumes and some non-legumes has been investigated by light microscopy. Pisum and Trifolium nodules have been examined by electron microscopy.Attention is directed to the presence of a pericycle in the vascular bundles of the nodules. In 7 of the legumes the pericycle cells possess a wall labyrinth consisting of branched filiform protuberances. The ultrastructure of the pericycle cell cytoplasm is described: its most striking feature is its abundant rough endoplasmic reticulum. These cells surround the xylem and phloem of the bundles, and are in turn surrounded by a layer of endodermal cells with Casparian strips. The pericycle cells develop their wall labyrinth in the levels of the nodule at which the bacterial tissue becomes pigmented; in nodule senescence their cytoplasm is disrupted level with the breakdown of the bacterial tissue.A pathway for symplastic lateral transfer of assimilates exists, from the sieve elements through the pericycle, endodermis and cortex to the bacterial tissue. The apoplast within the endodermis consists largely of the pericycle wall labyrinth and the xylem. The ultrastructure of the Casparian strip resembles that of roots.Intact, detached nodules can be induced to bleed a fluid from their severed vascular tissue. This fluid is exceptionally rich in organic nitrogen, particularly amides, but does not appear to contain sugars. Comparison between its amino acid composition and that of other parts of the nodule suggests that an active uptake or secretion of nitrogenous compounds precedes export from the nodule. Special functions are suggested for the nodule endodermis and the pericycle cells in this export process.