VARIATIONS IN TIGHT AND GAP JUNCTIONS IN MAMMALIAN TISSUES

The fine structure and distribution of tight (zonula occludens) and gap junctions in epithelia of the rat pancreas, liver, adrenal cortex, epididymis, and duodenum, and in smooth muscle were examined in paraformaldehyde-glutaraldehyde-fixed, tracer-permeated (K-pyroantimonate and lanthanum), and freeze-fractured tissue preparations. While many pentalaminar and septilaminar foci seen in thin-section and tracer preparations can be recognized as corresponding to well-characterized freeze-fracture images of tight and gap junction membrane modifications, many others cannot be unequivocally categorized—nor can all freeze-etched aggregates of membrane particles. Generally, epithelia of exocrine glands (pancreas and liver) have moderate-sized tight junctions and large gap junctions, with many of their gap junctions basal to the junctional complex. In contrast, the adrenal cortex, a ductless gland, may not have a tight junction but does possess large gap junctions. Mucosal epithelia (epididymis and intestine) have extensive tight junctions, but their gap junctions are not as well developed as those of glandular tissue. Smooth muscle contains numerous small gap junctions The incidence, size, and configuration of the junctions we observed correlate well with the known functions of the junctions and of the tissues where they are found.     

THE FLUOROMETRIC DETERMINATION OF 5-METHOXYTRYPTAMINE IN MAMMALIAN TISSUES AND FLUIDS

Abstract— Using a sensitive and specific fluorometric procedure involving selective extraction, reaction of the extracts with o -phthalaldehyde (OP), separation of the OP derivatives by TLC, and determination of fluorescent characteristics and intensities, we have detected and measured 5-methoxytryptamine, (5-MT) in various central and peripheral tissues and fluids of the rat, dog, baboon, and man.
Distribution of 5-MT in peripheral tissues of the rat seemed to parallel that of 5-HT, with highest levels being found in the gastrointestinal (GI) tract and Harderian gland, regions that are rich in 5-HT and have been reported to contain systems capable of methylating 5-HT. 5-MT was detected in the lung, plasma, kidney, spleen, and heart of the rat. 5-MT was present in the CNS of all species examined. No marked interspecies differences were observed. In the rat CNS, the regional distribution of 5-MT did not parallel that of 5-HT indicating that the systems for the synthesis, uptake, or transport of 5-MT might be different than for 5-HT. Pretreatment of rats with iproniazid resulted in a 50% increase in whole brain 5-MT. Reserpine pretreatment had no effect, indicating that the storage or release mechanisms for 5-MT are different than for the conventional amine transmitters. 5-MT was detected in human CSF and urine but not in plasma. These data indicate that 5-MT, a compound with potent pharmacological properties, is more widely distributed in the mammalian body than had previously been supposed.     

CYTOCHEMICAL STUDIES OF MITOCHONDRIA : I. THE SEPARATION AND IDENTIFICATION OF A MEMBRANE FRACTION FROM ISOLATED MITOCHONDRIA

Mitochondria isolated from rat liver and suspended in 0.44 M sucrose were disrupted by treatment with 0.3 per cent Na deoxycholate. The treated suspension was fractionated by differential centrifugation into a number of fractions and the respective pellets were examined in sections in the electron microscope. One of these fractions was found to consist of apparently membrane-bound (vesicular) elements. The difference between interfaces and membranes was discussed and the material of this fraction was found to meet stated requirements identifying it as membranous. A detailed study of the disruption process undergone by mitochondria in the presence of Na deoxycholate showed that the elements of this fraction were derived from structural elements assumed to be mitochondrial membranes. The findings thus demonstrate that mitochondria do possess membranes as defined and that these membranes can be isolated in a relatively pure form.     

THE PENETRATION OF THE MEMBRANE OF BRAIN MITOCHONDRIA BY ANIONS

The permeability of the membrane of rat brain non-synaptosomal mitochondria, towards inorganic and substrate anions, was assessed by measuring the rate of swelling that occurred when mitochondria were suspended in an iso-osmotic solution of a permeant anion, in the presence of a permeant cation such as NH⁺₄ or K⁺ in the presence or absence of valinomycin. In NH⁺₄-phosphate swelling was higher than it was in KCI or K⁺-phosphate, which showed the prevalence of the mechanism of phosphate transport previously demonstrated in liver mitochondria. The entry of succinate and L-malate seemed to require the presence in the inner mitochondrial membrane of specific carriers. as previously postulated for liver mitochondria, but the rate of swelling of brain mitochondria was lower than that of liver organelles. In K⁺-succinate, in the presence of antimycin, added ATP induced swelling and this was attributable to the simultaneous permeation both of the anion and the cation. Fumarate did not penetrate into brain mitochondria. Practically no swelling was recorded in NH⁺₄ or K⁺-citrate, which indicated that this anion penetrated poorly into the isolated brain mitochondria even in the presence of malate. Swelling occurred in NH⁺₄-L-glutamate in the presence of rotenone, and the entry of this anion seemed to follow a gradient of concentration although the presence of a specific translocator in the inner mitochondrial membrane might be concerned. The entry of glutamate was independent of that of phosphate and N-ethylmaleimide appeared to be a specific inhibitor of this entry. Swelling in K⁺-L-glutamate, in the presence of rotenone, was enhanced by the addition of valinomycin or ATP but in the latter case when osmotic equilibrium was reached swelling was not reversed by oligomycin. In conclusion, the lesser extent of swelling of isolated brain mitochondria compared with liver mitochondria could be attributed to the heterogeneity of the populations of these organelles, each population possessing its own characteristics of membrane permeability. Observations of electron micrographs of brain mitochondria incubated in iso-osmotic substrate anions confirmed the heterogeneous rate of swelling of these particles.     

A DEMONSTRATION OF SEVERAL DEOXYRIBONUCLEASE ACTIVITIES IN MAMMALIAN CELL MITOCHONDRIA

Extracts of purified mitochondria from adult rabbit liver and kidney have been prepared by lysis with Triton X-100. Such extracts contain deoxyribonuclease activity demonstrable at alkaline pH. Studies utilizing the effects of substrate variation, differing ionic strength, nucleoside di- and triphosphates, and SH-group inhibitors reveal the existence of at least five distinguishable deoxyribonuclease activities in these extracts. Assay of lysosomal and mitochondrial enzyme markers indicates no significant lysosomal contamination of the mitochondrial extracts. Further studies also suggest that the alkaline deoxyribonuclease activity is specifically located in or in association with mitochondria.     

几种哺乳动物精子顶体膜囊泡形成的研究

在猪、绵羊、地鼠这几种哺乳动物的精子体外获能后,在顶体反应的超显微结构中观察到:精子顶体膜囊泡化呈现多种形态,但囊泡都是由精子顶体的双层外膜多位点自我融合而形成的,质膜并不参与囊泡化,这一结果与前人报道的不同。     

MITOTIC CONTROL IN ADULT MAMMALIAN TISSUES

Mitotic homeostasis: Mitotic control is maintained by the interaction of a tissue-specific mitosis-inhibiting chalone, which permeates the whole tissue, and a non-tissue-specific mitosis-promoting mesenchymal factor, which originates in the connective tissue and acts only on connective-tissue-adjacent cells. In the basal layer of the epidermis the mitotic rate is determined by the relative concentrations of these two substances; in the distal layers the chalone is dominant so that all cells must become post-mitotic, age, and die. Thus the perfect balance between cell gain and cell loss that is maintained equally in hypoplasia, normality, and hyperplasia is ensured by the fact that all cells forced distally by mitotic pressure enter a chalone concentration that is high enough to direct them into post-mitosis and so to their deaths. The mitotic rate of the basal epidermal cells and the ageing rate of the distal cells are both inversely related to the chalone concentration. A change in the mitotic rate is matched by an equal change in the ageing rate so that, within limits, epidermal thickness (or mass) remains constant. Epidermal thickness is determined by the tissue-specific ratio, mitotic rate: ageing rate; it is influenced by the mitotic rate only when this exceeds a certain critical level. Evidently all epithelial tissues, even when these form solid masses (e.g. liver hepato-cytes), have a similar control mechanism, the ‘basal cells’ being those that are connective-tissue-adjacent and the ‘distal cells' those that are not. Tissues that are not connective-tissue-based (e.g. erythrocytes and granulocytes) have specialized mechanisms involving differentiation from relatively undifferentiated stem cell populations, as also do the connective tissues themselves. Local tissue damage leads via local chalone loss to a temporarily and locally increased mitotic rate; chronic damage leads via chronic chalone loss to hyperplasia, the increase in tissue mass being limited by the reduced life-span of the post-mitotic cells. Compensatory hypertrophy When a tissue mass is so large (e.g. the hepatocytes) in relation to the total body mass that the escaping chalone forms a significant systemic concentration, extensive damage leads to compensatory hypertrophy. The reduced tissue mass (e.g. after partial hepatectomy) produces less chalone, leading to a reduced systemic concentration, and therefore a higher chalone loss from the surviving tissue. This results in a general mitotic response in that tissue, as the relative power of the mesenchymal factor increases, and thus to an increase in tissue mass. Growth ceases when the normal tissue mass is attained. When a large tissue suffers chronic damage (e.g. liver cirrhosis) the chronic chalone lack results in hypertrophy, which is limited by the reduced life-span of the post-mitotic cells. Tumour growth Mitotic control is lost when the chalone concentration falls so low that the ‘distal cells’ remain mitotic; cell gain then exceeds cell loss and a tumour appears. Such chalone loss is related to permanent membrane damage, which may be the central event in carcinogenesis. The evidence is that a tumour continues to produce and to respond to the chalone of its tissue of origin. As a tumour grows the systemic concentration of its chalone rises steadily so that there is an increasing mitotic inhibition, first, in the parent tissue, and second, in the tumour itself. Thus tumour growth may be described as an exponential process limited by an exponential retardation. This means that, if the host survives, the tumour growth will cease and the tumour mass will reach a plateau. This is a negative feedback mechanism which differs from compensatory hypertrophy only in that, at the plateau, the mass attained is greater than normal, and also in that, at any time, further cell damage may cause the tumour to ‘progress’. When this happens the new and higher plateau may be unattainable before the host is killed. Tumour growth is normally slower than would be expected if the mitotic advantage were the only factor involved; clearly tumour growth is usually inhibited by factors other than the chalone, in particular perhaps by the immune response to the altered cell membrane. It is an especial pleasure to acknowledge the constant help and encouragement that has been given by Johanna U. R. Deol.     

SPECIALIZED MEMBRANE JUNCTIONS BETWEEN NEURONS IN THE VERTEBRATE CEREBELLAR CORTEX

"Gap" junctions, the morphological correlate for low-resistance junctions, are demonstrated between some mossy fiber terminals and granule cell dendrites in some lower vertebrate cerebella (gymnotid and frog). Most of the gap junctions (GJs) seen in the gymnotid-fish cerebellum exhibit an asymmetrical configuration, the electron-opaque cytoplasmic material underlying the junction being more extensive in the dendritic than in the axonal side. In the frog cerebellum, the GJs have a symmetrical distribution of such electron-opaque material. In both species the GJs are encountered at the same synaptic interface as the conventional synaptic zone (CSZ), constituting "mixed synapses" in a morphological sense. The axonal surface covered by CSZs is larger than that covered by GJs. In mammalian cerebellum, GJs are observed only in the molecular layer, between perikarya, dendrites, or perikarya and dendrites of the inhibitory interneurons. These GJs are intermixed with attachment plates and intermediary junctions interpreted as simply adhesive. In the mammalian cerebellum, a new type of junction which resembles the septate junctions (SJs) of invertebrate epithelia is observed between axonal branches forming the tip of the brush of basket fibers around the initial segment of the Purkinje cell axon. It is suggested that such junctions may be modified forms of septate junctions. The physiological implications of the possible existence of high-resistance cross-bridges between basket cell terminals, which may compartmentalize the extracellular space and thus regulate extracellular current flow, must be considered.     

DEGENERATIVE CHANGES IN THE MITOCHONDRIA OF FLIGHT MUSCLE FROM AGING BLOWFLIES

Mitochondria from flight muscle of aging blowflies, Phormia regina, were examined morphologically and biochemically with the electron microscope. An age-dependent degeneration of the mitochondria that is characterized, in part, by the reorganization of the inner membrane into myelin-like whorls has been found. The concentric rings increase in size and number, eventually replacing the normal cristal conformation. Glycogen rosettes are frequently seen in the center of the whorl and may represent the intrusion into the mitochondria of the glycogen in the cytoplasmic matrix of the muscle. The degenerating mitochondria are not associated with lysosomal activity, as indicated by the absence of acid phosphatase. An intense acid phosphatase activity is noted, however, in the dyad, comprising elements of the T system and sarcoplasmic reticulurn. Cytochrome oxidase is active in the ultrastructurally intact portion of the mitochondrion but activity is not evident in that part of the mitochondrion that has undergone morphological change. Thus, the ultrastructural degradation of the mitochondria is correlated with a decrease in biochemical function. This suggests a correspondence between a decrease in the bioenergetic capacity of the flight muscle and a decline in the ability of the aged insect to fly.     

MEMBRANE DIFFERENTIATIONS IN FREEZE-FRACTURED MAMMALIAN SPERM

A correlated thin-sectioning and freeze-fracturing study has been made of guinea pig and rat spermatozoa. In sections, the cell membrane over the acrosome has a concanavalin A and ruthenium red reactive glycocalyx which exhibits an ordered pattern related to the lattice of crystalline domains within the plane of the membrane revealed by freeze-fracturing. The cleaved acrosomal membrane also shows a finer linear periodicity in some areas. The membrane over the equatorial segment of the guinea pig acrosome is marked by a palisade of oblique ridges not observed in the rat. The plasmalemma of the postacrosomal region is rich in membrane intercalated particles, many randomly dispersed, others clustered in rectilinear arrays. A particle-poor zone is found just anterior to the posterior ring. The fold of redundant nuclear envelope posterior to the ring has many nuclear pores in close hexagonal array. The nuclear envelope lining the implantation fossa is devoid of pores. When cleaved it has a particle-free central area surrounded by a broad zone of large, closely packed, hollow particles. The membrane of the mid-piece in the guinea pig (but not the rat) contains linear strands of 6–8-nm particles oriented circumferentially. The membrane investing the principal piece exhibits the usual randomly distributed particles but in addition, a double row of larger (9 nm) particles runs longitudinally within the membrane over outer dense fiber 1. In the corresponding position in thin sections a local thickening of the membrane is discernible. These observations form a basis for further studies on the functional correlates of these regional specializations of the sperm membrane.     

TRANSPLANTATION AND POTENTIAL IMMORTALITY OF MAMMALIAN TISSUES

1. Serial transplantation of tumors made it possible in 1901 and following years to draw the conclusion that various mammalian tissues have potential immortality. Serial transplantations of normal tissues did not succeed at first, because the homoioreaction on the part of the lymphocytes and connective tissue of the host injures the transplant. 2. In continuation of these experiments we found that cartilage of the rat can be transplanted serially to other rats at least for a period of 3 years. At the end of that time great parts of the transplanted cartilage and perichondrium are alive. 3. Not only the cartilage of young rats can be homoiotransplanted, but also the cartilage of very old rats which are nearing the end of life. By using such animals we have been able to obtain cartilage and perichondrium approaching an age of 6 years which is almost double the average age of a rat. 4. We found that cartilage can be homoiotransplanted more readily than other tissues for the following reasons: (a) While in principle the homoioreaction towards cartilage is the same as against other tissues, cartilage elicits this reaction with less intensity; (b) cartilage is better able to resist the invasion of lymphocytes and connective tissue than the majority of other tissues; (c) a gradual adaptation between transplant and host seems to take place in the case of cartilage transplantation, as a result of which the lymphocytic reaction on the part of the host tissue decreases progressively the longer the cartilage is kept in the strange host. 5. At time of examination we not only found living transplanted cartilage tissue, but also perichondrial tissue, which in response to a stimulus apparently originating in the necrotic central cartilage, had been proliferating and replacing it. These results suggest that it may perhaps be possible under favorable conditions to keep cartilage alive indefinitely through serial transplantations. 6. At the same time these experiments permit the analysis of the factors which are favorable or unfavorable to the continued life of the transplants. Favorable factors are: (a) Well preserved perichondrium around transplant; (b) cellular newly formed perichondrial cartilage—though it is doubtful whether such young cartilage cells allow a state of stable equilibrium. Host connective tissue does not invade transplant under these conditions. Unfavorable factors are: (a) Cartilage differentiation and the production of paraplastic substances (hyaline capsules in parts of transplant far removed from vessels and sources of oxygen and food; (b) cartilage necrosis when a still greater distance from nourishment exists; (c) disturbance of equilibrium between host connective tissue and transplant due to above conditions, resulting in (d) attack by host connective tissue on transplanted cartilage, which is the chief danger in the preservation of the life of the whole transplant 7. It is pointed out that also in old age there exist similar problems of disturbances of tissue equilibria, due to degenerative changes in certain parenchymatous structures and to proliferative processes on the part of connective tissue and glia elements together with increase in paraplastic structures.     

THE ULTRASTRUCTURE OF THE NEUROMUSCULAR JUNCTIONS OF MAMMALIAN RED, WHITE, AND INTERMEDIATE SKELETAL MUSCLE FIBERS

Distinct ultrastructural differences exist at the neuromuscular junctions of red, white, and intermediate fibers of a mammalian twitch skeletal muscle (albino rat diaphragm). The primary criteria for recognizing the three fiber types are differences in fiber diameter, mitochondrial content, and width of the Z line. In the red fiber the neuromuscular relationship presents the least sarcoplasmic and axoplasmic surface at each contact. Points of contact are relatively discrete and separate, and axonal terminals are small and elliptical. The junctional folds are relatively shallow, sparse, and irregular in arrangement. Axoplasmic vesicles are moderate in number, and sarcoplasmic vesicles are sparse. In the white fiber long, flat axonal terminals present considerable axoplasmic surface. Vast sarcoplasmic surface area is created by long, branching, closely spaced junctional folds that may merge with folds at adjacent contacts to occupy a more continuous and widespread area. Axoplasmic and sarcoplasmic vesicles are numerous. Both axoplasmic and sarcoplasmic mitochondria of the white fiber usually contain intramitochondrial granules. The intermediate fiber has large axonal terminals that are associated with the most widely spaced and deepest junctional folds. In all three fiber types, the junctional sarcoplasm is rich in free ribosomes, cisternae of granular endoplasmic reticulum, and randomly distributed microtubules.