ULTRASTRUCTURAL STUDIES ON THE LYMPHATIC ANCHORING FILAMENTS

The fine structure of the lymphatic capillary and the surrounding tissue areas was investigated. Instead of a continuous basal lamina (basement membrane) surrounding the capillary wall, these observations revealed the occurrence of numerous fine filaments that insert on the outer leaflet of the trilaminar unit membrane of the lymphatic endothelium. These filaments appear as individual units, or they are aggregated into bundles that are disposed parallel to the long axis of the lymphatic capillary wall and extend for long distances into the adjoining connective tissue area among the collagen fibers and connective tissue cells. The filaments measure about 100 A in width and have a hollow profile. They exhibit an irregular beaded pattern along their long axis and are densely stained with uranyl and lead. These filaments are similar to the microfibrils of the extracellular space and the filaments observed in the peripheral mantle of the elastic fibers. Infrequently, connections between these various elements are observed, suggesting that the lymphatic anchoring filaments may also contribute to the filamentous units of the extracellular space. It is suggested that these lymphatic anchoring filaments connect the small lymphatics to the surrounding tissues and represent the binding mechansim that is responsible for maintaining the firm attachment of the lymphatic capillary wall to the adjoining collagen fibers and cells of the connective tissue area.     

比较几种组织化学显色法在肺内毛细淋巴管的应用

目的 为肺内毛细血管和毛细淋巴管的鉴别提供更好的技术方法。方法 采用5’-核苷酸酶一碱性磷酸酶双重显色法（5′-Nase-ALP）、针对基底膜的免疫组化显色法及特殊染色法。结果 5′-Nase-ALP显色法能够较客观、较清楚地区分肺内毛细血管和毛细淋巴管。结论 针对基底膜的免疫组化显色法及特殊染色法不够准确或受器官结构特点限制,但对于部分实质性或空腔性器官及肿瘤淋巴管的研究仍不为是一种客观、可靠、经济、实用的方法,值得进一步尝试推广。     

ULTRASTRUCTURAL STUDIES ON THE PERMEABILITY OF THE MESOTHELIUM TO HORSERADISH PEROXIDASE

Peritoneal mesothelium was exposed for 2–60 min to solutions of horseradish peroxidase by incubation in vitro, or after intraperitoneal injection in vivo. Peroxidase was localized, with the electron microscope in the intercellular clefts of the mesothelium, often along their entire lengths, in vesicles adjoining or contiguous with the clefts, and along the peritoneal and basal surfaces of the cell, and also in intracytoplasmic vacuoles. The intercellular junctions of peroxidase-treated mesothelium did not differ from those of controls: open and closed junctions were present in both groups. Intercellular localization was also obtained when the mesothelium was exposed to peroxidase during or after fixation. Although intracellular absorption of peroxidase and its incorporation into larger vacuoles were observed, there was no clearcut evidence of vesicular transport across the mesothelium in these experiments. These findings are consistent with physiologic data which postulate that mesothelial transport can be accounted for, at least in part, by passive diffusion through a system of pores, and they suggest that these pores are located in the intercellular clefts.     

PERMEABILITY OF MUSCLE CAPILLARIES TO EXOGENOUS MYOGLOBIN

Whale skeletal muscle myoglobin (mol wt 17,800; molecular dimensions 25 x 34 x 42 Å) was used as a probe molecule for the pore systems of muscle capillaries. Diaphragms of Wistar-Furth rats were fixed in situ at intervals up to 4 h after the intravenous injection of the tracer, and myoglobin was localized in the tissue by a peroxidase reaction. Gel filtration of plasma samples proved that myoglobin molecules remained in circulation in native monomeric form. At 30–35 s postinjection, the tracer marked ~75% of the plasmalemmal vesicles on the blood front of the endothelium, 15% of those located inside and none of those on the tissue front. At 45 s, the labeling of vesicles in the inner group reached 60% but remained nil for those on the tissue front. Marked vesicles appeared on the latter past 45 s and their frequency increased to ~80% by 60–75 s, concomitantly with the appearance of myoglobin in the pericapillary spaces. Significant regional heterogeneity in initial labeling was found in the different segments of the endothelium (i.e., perinuclear cytoplasm, organelle region, cell periphery, and parajunctional zone). Up to 60 s, the intercellular junctions and spaces of the endothelium were free of myoglobin reaction product; thereafter, the latter was detected in the distal part of the intercellular spaces in concentration generally equal to or lower than that prevailing in the adjacent pericapillary space. The findings indicate that myoglobin molecules cross the endothelium of muscle capillaries primarily via plasmalemmal vesicles. Since a molecule of this size is supposed to exit through both pore systems, our results confirm the earlier conclusion that the plasmalemmal vesicles represent the large pore system; in addition, they suggest that the same structures are, at least in part, the structural equivalent of the small pore system of this type of capillaries.     

喉淋巴管的几种组织化学显色法比较观察

目的为喉毛细血管和毛细淋巴管的鉴别提供更好的技术方法。方法采用间接注射法,5′-核苷酸酶-碱性磷酸酶双重显色法（5′-Nase-ALPase）,针对基底膜的免疫组化显色法。结果酶组化染色结果显示毛细血管和血管显示有清楚的蓝色轮廓,而附近的毛细淋巴管和淋巴管则呈棕色轮廓,两者易于鉴别。免疫组化染色结果显示毛细血管和血管显示清楚的棕色轮廓,而附近的毛细淋巴管和淋巴管轮廓极其浅淡,两者亦可鉴别。结论酶组化和免疫组化显色法是喉内区别毛细淋巴管和毛细血管的一种可靠且特异的方法。     

脂质体通透性与脂多型性关系的研究

本文以TPE和TPE/DOPE(1:1.mol:mol)制成包裹荧光分子calcein的脂质体,通过测量荧光强度随扫描温度的变化,探讨了脂质体通透性与脂多型性之间的关系.结果表明,在不发生双层相(L)变成六角形Ⅱ相(H)相转变时,脂质体悬液的荧光强度不增加;当发生该转变时,脂质体悬液的荧光强度开始增加;完成该相转变后,脂质体悬液的荧光强度仍继续增加.据此,我们认为:脂质体的通透性与脂的多型性密切相关,当发生L→HⅡ相转变时,脂质体的通透性增加.由于荧光强度的变化对相变非常敏感,我们建议用测量脂质体荧光强度随温度的变化来监测脂质体稀悬液中脂的多型性.     

MORPHOMETRIC DATA ON THE ENDOTHELIUM OF BLOOD CAPILLARIES

Local differentiations within the endothelium of both muscular (diaphragm, myocardium) and visceral (pancreas, jejunal villi) capillaries have been studied in rats on sectioned and freeze-cleaved preparations. Four distinct parts have been recognized in the endothelial cells of all these vessels on the basis of subcellular components present in each part and on the basis of variations in the local frequency of plasmalemmal vesicles: (a) the parajunctional zone, (b) the peripheral zone, (c) the organelle region, and (d) the nuclear region. Our data indicate that ~16, ~7.0, and 8.5% of the endothelial cytoplasmic volume (in the peripheral zone) is accounted for by vesicles, their content, and their membranes, respectively. The average density of vesicular openings per µm² is 78 in diaphragm, 89 in myocardium, 25 in pancreas, and 10 in jejunal mucosa capillaries. The frequency of fenestrae is 1.7 times as high in jejunal (26/µm²) as in pancreatic capillaries (15/µm²), the corresponding fractional areas being ~9.5 and ~6%, respectively, of the endothelial surface. Intercellular spaces occupy a relatively small area (~0.08 to 0.2%) of the inner endothelial surface.     

STUDIES ON PERMEABILITY OF MEMBRANES : VII. CONDUCTIVITY OF ELECTROLYTES WITHIN THE MEMBRANE.

Two methods of measuring the electrical conductivity of the dried collodion membrane in contact with an electrolyte solution are described and the results of such measurements with different electrolytes in different ranges of concentration recorded. Some of the difficulties encountered in making these measurements are outlined. Of special interest was the fact that each membrane with each electrolyte showed a maximum level of resistance at a certain point in the dilution scale, a level which was not surpassed by further dilution. It is believed that this level was fixed by the collodion itself rather than by the contiguous electrolyte solution. Its existence limited the results available for reasonable interpretation. In relatively concentrated solutions the conductivity was shown to be approximately proportional to the concentration. With different electrolytes in the same concentration it was shown that the conductivities varied much more than in simple solutions without a membrane and that they fell in the order HCl > KCl > NaCl > LiCl. A method was described whereby the electrolyte content of a membrane in contact with different chloride solutions could be determined. It was shown that a membrane saturated with either 0.5 N HCl or 0.5 N KCl had practically the same total electrolyte content whereas the same membrane in contact with 0.5 N LiCl contained only half the quantity. These results were used in interpreting the conductivity data, the evidence presented strongly suggesting that two factors are operative in causing the widely divergent conductivities recorded with different electrolytes. The first factor depended on the quantity of electrolyte which can enter the membrane pores, a quantity dependent on the size of the pores and the volume of the larger of the two hydrated ions of the electrolyte. This factor was the chief one in determining the difference in conductivity between KCl and LiCl. The second factor was concerned with differences in the mobility of the various cations within the membrane brought about by friction between the moving ions and the pore walls. With KCl and HCl the quantity of electrolytes entering the membrane was in each case the same, being determined by the size of the larger Cl^- ion. The widely different conductivity values were explained as due to the changes in the mobility of the two cations within the membrane pores.     

PERMEABILITY OF INTESTINAL CAPILLARIES : Pathway followed by Dextrans and Glycogens

The pathway followed by macromolecules across the wall of visceral capillaries has been studied by using a set of tracers of graded sizes, ranging in diameter from 100 A (ferritin) to 300 A (glycogen). Polysaccharide particles, i.e. dextran 75 (mol wt ~75,000; diam ~125 A), dextran 250 (mol wt 250,000; diam ~225 A), shellfish glycogen (diam ~200 A) and rabbit liver glycogen (diam ~300 A), are well tolerated by Wistar-Furth rats and give no vascular reactions ascribable to histamine release. Good definition and high contrast of the tracer particles were obtained in a one-step fixation—in block staining of the tissues by a mixture containing aldehydes, OsO₄ and lead citrate in phosphate or arsenate buffer, pH 7.4, followed by lead staining of sections. The glycogens and dextrans used move out of the plasma through the fenestrae and channels of the endothelium relatively fast (3–7 min) and create in the pericapillary spaces transient (2–5 min) concentration gradients centered on the fenestrated sectors of the capillary walls. The tracers also gained access to the plasmalemmal vesicles, first on the blood front and subsequently on the tissue front of the endothelium. The particles are temporarily retained by the basement membrane. No probe moved through the intercellular junctions. It is concluded that, in visceral capillaries, the fenestrae, channels, and plasmalemmal vesicles, viewed as related parts in a system of dynamic structures, are the structural equivalent of the large pore system.     

THE EFFECT OF LIGHT ON THE PERMEABILITY OF PARAMECIUM

1. The permeability of Paramecium to NH₄OH is greater when the cells are exposed to light than it is when they are in darkness. 2. This change can be demonstrated in cells exposed to monochromatic red light, though it is small. It becomes greater as the wave lengths shorten, and is greatest in the near ultra-violet. 3. The permeability increases as the duration of exposure to light is prolonged. 4. These experiments demonstrate the necessity of controlling the illumination when using Paramecium in physiological tests.     

STUDIES ON PERMEABILITY OF MEMBRANES : V. THE DIFFUSION OF NON-ELECTROLYTES THROUGH THE DRIED COLLODION MEMBRANE.

A study has been made of the relative rates at which various organic non-electrolytes diffuse through the dried collodion membrane. It was found that acetone and urea pass through the membrane many times more rapidly than glycerine and that glycerine in its turn diffuses much faster than glucose. It was also demonstrated that the rate of diffusion varies directly with the difference in concentration between the solutions on the two sides of the membrane. It was shown that the presence of glycerine on the two sides of the membrane did not appreciably affect the rate of diffusion of acetone. In a study of the changes going on during the establishment of the stationary diffusion gradient with glucose experiments were described which strongly suggested that many of the membrane channels may gradually become clogged up with glucose molecules so that the diffusion rate decreases from day to day until the stationary gradient is finally reached. In explaining the various experimental data the conception of the collodion membrane as a sieve with pores approximating in smallness the size of individual molecules was utilized. The large differences in the diffusion rates between different substances were then referred to differences in molecular size, the relatively large molecules of glycerine and glucose being unable to pass through many of the smaller pores available for urea and acetone. From the data available it was possible to estimate that 98 per cent of the pore area distributed among holes large enough for the diffusion of acetone was unavailable for the passage of glycerine and that only 0.3 per cent of the pore area available for acetone could be utilized by glucose. In trying to correlate the ratio between the diffusion rates of two different substances with the characteristic concentration potential (Co P) given by the same membrane it was found (1) that with the acetone-glycerine ratio there is no correspondence (2) that with the acetone-glucose ratio a suggestive relation exists and (3) that with the glycerine-glucose ratio a definite correspondence can be shown, the higher ratios being obtained only with membranes giving high CO P values. A rational explanation for these facts was proposed.     

STUDIES ON PERMEABILITY OF MEMBRANES : VIII. THE BEHAVIOR OF THE DRIED COLLODION MEMBRANE TOWARD BIVALENT CATIONS.

A study of the behavior of the dried collodion membrane toward the bivalent calcium ion showed that: 1. There is almost no potential difference established across a membrane separating two calcium chloride solutions of 0.1 and 0.01 N concentrations. 2. The transfer numbers of chlorine and calcium, as measured in electrical transfer experiments, are both close to 0.5. 3. A sample of membrane in equilibrium with a solution of calcium chloride has an extremely high electrical resistance, greater than is observed with solutions of the chlorides of any of the monovalent cations. 4. The total electrolyte content of a membrane in equilibrium with a solution of calcium chloride was only 20 per cent of that observed when the solution was lithium chloride and 10 per cent of that found when the solution was potassium chloride. In explaining these various results it is supposed that (1), (2) and (3) are all the result of (4), that is, of the inability of the calcium ion to penetrate any but the largest of the membrane pores. As the total quantity of electrolyte able to penetrate the membrane is very small the electrical conductivity must also be very small. Moreover, the few larger pores that are large enough to transport the hydrated calcium ion are too large to exert any appreciable effect in decreasing the mobility of the anion. Thus the membrane has no effect in modifying the potentials established across concentration chains with CaCl₂ and the transfer numbers determined experimentally are what one would expect if no membrane were present.     

STUDIES ON PERMEABILITY OF MEMBRANES : I. INTRODUCTION AND THE DIFFUSION OF IONS ACROSS THE DRIED COLLODION MEMBRANE.

The theoretical aspects of the problem of sieve-like membranes are developed. The method of preparing the dried collodion membrane is described, and the method of defining the property of a particular membrane is given. It consists of the measurement of the Co P, that is the P.D. between an 0.1 and an 0.01 M KCl solution separated by the membrane. Co P is in the best dried membranes 50 to 53 millvolts, the theoretically possible maximum value being 55 millivolts. Diffusion experiments have been carried out with several arrangements, one of which is, for example, the diffusion of 0.1 M KNO₃ against 0.1 M NaCl across the membrane. The amount of K⁺ diffusing after a certain period was in membranes with a sufficiently high Co P (about 50 millivolts or more) on the average ten times as much as the amount of diffused Cl^-. In membranes with a lower Co P the ratio was much smaller, down almost to the proportion of 1:1 which holds for the mobility of these two ions in a free aqueous solution. When higher concentrations were used, e.g. 0.5 M solution, the difference of the rate of diffusion for K⁺ and Cl^- was much smaller even in the best membranes, corresponding to the fact that the P.D. of two KCl solutions whose concentrations are 10:1 is much smaller in higher ranges of concentration than in lower ones. These observations are confirmed by experiments arranged in other ways. It has been shown that, in general, the diffusion of an anion is much slower than the one of a cation across the dried collodion membrane. The ratio of the two diffusion coefficients would be expected to be calculable in connection with the potential difference of such a membrane when interposed between these solutions. The next problem is to show in how far this can be confirmed quantitatively.     

STUDIES ON THE PERMEABILITY OF MEMBRANES : III. ELECTRIC TRANSFER EXPERIMENTS WITH THE DRIED COLLODION MEMBRANE.

The transfer numbers of the ions of electrolytes in the dried collodion membrane, as determined in a previous paper indirectly from the E.M.F. of concentration chains, can also be determined directly by electrical transfer experiments. It is shown that the difficulties involved in such experiments can be overcome. The transfer numbers obtained by the two methods are in satisfactory agreement. The experimental results obtained in the transfer experiments furnish an additional argument in favor of maintaining the theory that the electromotive effects observed in varying concentrations of different electrolytes with the dried collodion membrane may be referred to differences in the mobilities of the anions and cations within the membrane. As was shown by the method of the previous paper, the transfer number depends largely on concentration. There are some minor discrepancies between the values of the transfer numbers obtained by the two methods which, as yet, cannot be completely explained.