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Camptothecin possesses broad antitumor spectra on various cancers. In spite of its marked tumor-suppressing potency, camptothecin is too hydrophobic to be solved in water and therefore not currently in clinical use. CPT-11 (irinotecan) is one of the hydrophilic analogues of camptothecin and widely prescribed. However, its water solubility is still low and furthermore evokes severe diarrhea. Therefore, we designed and synthesized novel highly hydrophilic camptothecin derivatives by conjugating SN38 with branched glycerol trimer (SN38-BGL), which we have been developing as a unique strategy to endow hydrophobic molecule with much hydrophilicity, to maximize the benefit of CPT-11 and minimize the adverse effects. The SN38-BGLs exhibited equivalent or slightly stronger tumor-suppressing effects in murine xenograft human lung cancer models compared to CPT-11. However, neither early- nor late-onset diarrhea was observed when SN38-BGL was administered. Heights of villi in jejunum and ileum were bigger than those from CPT-11-treated mice, indicating that SN38-BGL is less harmful than CPT-11. Ex vivo digestion by liver microsome did not yield SN38 but a couple of other molecules against our expectations, which suggests the involvement of other active metabolites than SN38 and may explain the differences. Hence, SN38-BGLs can be a novel hydrophilic camptothecin derivative without causing severe diarrhea.

Podoplanin (aggrus) is highly expressed in several types of cancers, including malignant pleural mesothelioma (MPM). Previously, we developed a rat anti-human podoplanin mAb, NZ-1, and a rat-human chimeric anti-human podoplanin antibody, NZ-8, derived from NZ-1, which induced antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity against podoplanin-positive MPM cell lines. In this study, we showed the antitumor effect of NZ-1, NZ-8, and NZ-12, a novel rat-human chimeric anti-human podoplanin antibody derived from NZ-1, in an MPM orthotopic xenograft SCID mouse model. Treatment with NZ-1 and rat NK (CD161a(+) ) cells inhibited the growth of tumors and the production of pleural effusion in NCI-H290/PDPN or NCI-H226 orthotopic xenograft mouse models. NZ-8 and human natural killer (NK) (CD56(+) ) cells also inhibited tumor growth and pleural effusion in MPM orthotopic xenograft mice. Furthermore, NZ-12 induced potent ADCC mediated by human MNC, compared with either NZ-1 or NZ-8. Antitumor effects were observed following treatment with NZ-12 and human NK (CD56(+) ) cells in MPM orthotopic xenograft mice. In addition, combined immunotherapy using the ADCC activity of NZ-12 mediated by human NK (CD56(+) ) cells with pemetrexed, led to enhanced antitumor effects in MPM orthotopic xenograft mice. These results strongly suggest that combination therapy with podoplanin-targeting immunotherapy using both NZ-12 and pemetrexed might provide an efficacious therapeutic strategy for the treatment of MPM.

Fibroblast growth factor 2 (FGF-2) is a potent mitogen for mesenchymal cells, and a local application of recombinant human FGF-2 (rhFGF-2) in a gelatin hydrogel has been reported to accelerate bone union in our animal studies and preparatory dose-escalation trial on patients with surgical osteotomy. We have performed a randomized, double-blind, placebo-controlled trial in which patients with fresh tibial shaft fractures of transverse or short oblique type were randomly assigned to three groups receiving a single injection of the gelatin hydrogel containing either placebo or 0.8 mg (low-dosage group) or 2.4 mg (high-dosage group) of rhFGF-2 into the fracture gap at the end of an intramedullary nailing surgery. Of 194 consecutive patients over 2 years, 85 met the eligibility criteria, and 70 (24 in the placebo group and 23 each in low- and high-dosage groups) completed the 24-week study. The cumulative percentages of patients with radiographic bone union were higher in the rhFGF-2-treated groups (p = .031 and .009 in low- and high-dosage group, respectively) compared with the placebo group, although there was no significant difference between low- and high-dosage groups (p = .776). At 24 weeks, 4, 1, and 0 patients in the placebo, low-dosage, and high-dosage groups, respectively, continued to show delayed union. No patient underwent a secondary intervention, and the time to full weight bearing without pain was not significantly different among the three groups (p = .567). There also was no significant difference in the profiles of adverse events among the groups. In conclusion, a local application of the rhFGF-2 hydrogel accelerated healing of tibial shaft fractures with a safety profile. © 2010 American Society for Bone and Mineral Research.

We have investigated polyketone (PK)/polyamide 6 (PA) polymer alloys having enhanced Charpy impact energy greater than that of polycarbonate (PC) as a result of moisture absorption. From the results of differential scanning calorimetry (DSC), Raman spectroscopy, and transmission electron microscope (TEM) observation of the polymer alloys, it was found that PK-rich and PA-rich phases exist at the nanometer level in the polymer alloys; however, a microscopic interaction phase formed between the two phases. 3D-TEM observations, electron energy loss spectroscopy, and small-angle X-ray scattering measurements revealed that a co-continuous nanolayer formed from the PA-rich phase and lamella network of the PK-rich phase. Moreover, the interaction and mobility of PK and PA molecular chains were investigated by using a 13C cross polarization/magic angle sample spinning NMR technique. It was found that moisture absorption markedly enhances the mobility of PA molecular chains in PK/PA alloys. This suggests that the wet (moisture-absorbed) PA phase of PK/PA alloys allows quick deformation upon impact stimulation. On the other hand, the results of Charpy impact tests showed that the total impact energy (Etotal) of the wet polymer alloy was much higher than that of the dry one. An examination of the load-displacement curves revealed that the wet samples showed a pronounced increase in displacement compared with the dry ones. From these results, it was concluded that the lamella network of the PK-rich phase sustains the maximum stress and that the large displacement of the PA-rich phase increases the impact energy. © 2010 Wiley Periodicals, Inc. J Appl Polym Sci, 2010

Nagata et al. recently developed high-impact polyketone/polyamide-6 (PK/PA) alloys and investigated their mechanical properties (Nagata et al. Polym. Prep. Jpn. 2006, 55, 4286−4287). This paper presents the results of investigations of their morphology, such as the domain size of PA and PK, features of crystalline phase of PK (the long period and thickness of lamellae are deduced), and the change in crystalline form of PA in the PK/PA alloys, using solid-state nuclear magnetic resonance (NMR), small-angle X-ray scattering (SAXS), and transmission electron microscopy (TEM). Solid-state 13C and 15N NMR results revealed that the γ crystalline phase of PA changes into a thermodynamically stable α crystalline phase after alloying with PK. The difference in the long period of lamellae of PK/PA alloys between dry and wet conditions is discussed. We conclude that the characteristic morphology of PK and humidity of the amorphous phase of PA in the wet condition are very important factors enabling the PK/PA alloys ...

The Izod impact strength of two kinds of ternary composites was investigated. One consisted of polypropylene (PP), the triblock copolymer polystyrene-block-poly(ethylene butene)-block-polystyrene (SEBS), and calcium carbonate (CaCO3) particles, and the other consisted of PP, carboxylated SEBS (C-SEBS), and CaCO3 particles. The mean size of the CaCO3 particles was about 160 nm. According to scanning electron microscopy observations, the composite with SEBS showed a morphology in which SEBS domains and CaCO3 particles were independently dispersed in the PP matrix. On the other hand, the composite with C-SEBS showed a morphology in which CaCO3 particles were encapsulated by C-SEBS; that is, a core–shell structure was formed. The Izod impact strength of the composite with SEBS was higher than that of the composite with C-SEBS and the PP/SEBS and PP/C-SEBS binary blends. According to observations of the fractured surface, the stress-whitened area was larger in the composite with SEBS than in the composite with C-SEBS and the PP/SEBS and PP/C-SEBS binary blends. The toughening mechanism of the composite, using nanometer-sized CaCO3 particles in combination with SEBS, was examined. © 2009 Wiley Periodicals, Inc. J Appl Polym Sci, 2009

Chondrocytes produce several local regulatory factors such as basic fibroblast growth factor (bFGF), transforming growth factor-beta (TGF-beta) and insulin-like growth factor-I (IGF-I). In this study, we examined the effect of bFGF on the expressions of both mRNA and protein of the growth factors synthesized by chondrocytes. Treatment of chondrocytes with bFGF (1-100 ng/ml) stimulated the mRNA expression of bFGF and TGF-beta up to 121-604% and 130-220% at 12 h compared with the controls, respectively. On the other hand, the treatment of chondrocytes with bFGF (1-100 ng/ml) suppressed IGF-I mRNA expression to 79-47% at 12 h compared with the controls. An enzyme-linked immunosorbent assay (ELISA) revealed that the treatment of chondrocytes with bFGF (1-100 ng/ml) also enhanced the production of TGF-beta proteins in the chondrocytes up to 299-508% at 24 h compared with controls. We conclude that bFGF influenced the local expression of growth factors by chondrocytes, suggesting autoregulation of growth factor expression during chondrogenesis.

Cartilage graft is considered to be useful in repairing chondral or osteochondral defects. One method of the cartilage graft is achieved by autologous chondrocyte transplantation following cell culture. However, chondrocytes change their phenotype during culture. We used costal chondrocytes cultured over agarose (suspension culture) as a source of graft materials. The suspension-cultured chondrocytes formed aggregate in culture. We first examined the expressions of cartilage-specific matrices of cultured chondrocytes after two weeks in culture. The chondrocytes cultured over agarose expressed more type II collagen mRNA than those cultured on plastic dishes did after two weeks in culture. Safranin O staining showed the presence of glycosaminoglycans in the chondrocyte culture over agarose, while glycosaminoglycans were not observed in the culture on plastic dishes. We then examined the changes of rat articular osteochondral defects after transplantation of suspension-cultured chondrocytes. The aggregate of suspension-cultured chondrocytes was easily picked up with forceps and transplanted in the osteochondral defects. The defects were filled with safranin O-stained hyaline cartilage tissue two weeks after chondrocyte transplantation. On the contrary, the fibrous materials, which were not stained with safranin O, were observed in the control defects. These results suggest that the suspension-cultured chondrocytes are useful for autologous cartilage grafts by preserving chondrocyte phenotype.

Thyroid hormone plays a role in skeletal maturation. The present study was undertaken to investigate the effect of thyroid hormone on growth and maturation of the epiphyseal cartilage in the rat femur organ culture system. The femora increased in both weight and size over time in culture in the absence of serum. The growth rate was suppressed by thyroxine treatment. Thyroxine induced chondrocyte hypertrophy in the area adjacent to the articular surface in the femoral condyle. In this area, the expression of type X collagen, a marker of chondrocyte terminal differentiation, was detected after 21 days in culture by immunohistochemistry and reverse transcription/polymerase chain reaction. These data suggest that thyroxine suppresses cartilage growth by stimulating chondrocyte terminal differentiation.

1,25-dihydroxyvitamin D3 (1,25(OH)2D3) is thought to be an important systemic factor in the fracture repair process, but the mechanism of action of 1,25(OH)2D3 has not been clearly defined. In this study, the role of 1,25(OH)2D3 in the fracture repair process was analyzed in a rat closed femoral fracture model. The plasma concentration of 1,25(OH)2D3 rapidly decreased on day 3 and continued to decrease to 10 days after fracture. We assessed whether this decrease was based on the accelerated degradation or retardation of the synthesis rate of 1,25(OH)2D3, from 25(OH)D3. After radiolabeled 3H-1,25(OH)2D3 or 3H-25(OH)D3 was injected i.v. into fractured or control (unfractured) rats, the concentrations of 25(OH)D3 and 1,25(OH)2D3 metabolites were measured by HPLC. The plasma concentrations of these radiolabeled metabolites in fractured group were similar to those in control rats early after operation. However, radioactivity in the femurs of fractured rats was higher than that of the control group. Furthermore, the radioactivity was concentrated in the callus of the fractured group analyzed by autoradiography. 1,25(OH)2D3 receptor gene expression was detected early after fracture and, additionally, both in the soft and hard callus on days 7 and 13 after fracture. These results showed that the rapid disappearance of 1,25(OH)2D3 in the early stages after fracture was not due to either increased degradation or decreased synthesis of 1,25(OH)2D3, but rather to increased consumption. Further, these results suggest the possibility that plasma 1,25(OH)2D3 becomes localized in the callus and may regulate cellular events in the process of fracture healing.

Basic fibroblast growth factor is a potent mitogen for chondrocytes and influences the protein synthesis of their extracellular matrix in vitro. To investigate its effect on normal developing articular cartilage in vivo, we injected basic fibroblast growth factor once into the knee joints of 4-week-old rats. Phosphate buffered saline was similarly injected into the contralateral knee joints as controls. A histological analysis showed that an injection of basic fibroblast growth factor induced enlargement of the articular cartilage area, especially in the condylar ridge region on day 7 after the injection. The extent of the enlargement was dose-dependent. The localization and amount of proliferating cells in the articular cartilage were analyzed immunohistochemically by the detection of proliferating cell nuclear antigen. On day 1 after the injection, the number of cells positive for proliferating cell nuclear antigen increased significantly in the joints that were injected compared with the controls, and Northern blot analysis showed that the level of messenger RNA for alpha 1(II) procollagen was lower in these joints than in the controls. The message in the joints that had been injected increased on day 7, and it was greater than that in the controls. This suggests that proliferating chondrocytes in developing articular cartilage respond to basic fibroblast growth factor with a resulting proliferation of chondrocytes followed by enlargement of cartilage.

IFO, one of the oxazaphosphorines, has great antitumor activity against a variety of neoplasms. However, urotoxicity has also been reported as one of the doselimiting factors of IFO. Mesna has been found effective in protecting the bladder from the renal toxicity of the oxazaphosphorines including IFO. Therefore, we used mesna in the chemotherapy of nine patients with malignant bone and soft tissue tumors in order to prevent the side effects of IFO. When we administered IFO without mesna, hemorrhagic cystitis was observed in all patient. The patients were given higher doses of IFO with mesna than those given IFO alone. However, hemorrhagic systitis was not observed in any patient given IFO with mesna.Six out of nine patients had primary or metastatic lesions in which chemotherapeutic effects could be evaluated on CT or MRI. Of these patients complete response in one case and the partial responses in two cases were observed. It should be noted that the case with a complete response was that of a synovial sarcoma, which usually does not respond well to chemotherapy.Based on the present study, we conclude that high-dose IFO therapy with mesna is safe and is likely to be effective for treating synovial sarcoma.

We performed a statistical analysis of patients who had interlocking wedge osteotomy for osteoarthritis of the knee in Kyushu University during 1980 to 88. Follow-up of patients was greater than five years and arthroscopic findings at the time of surgery were also evaluated.The result of the osteotomy was excellent with the JOA score increasing from sixty to eighty-six. Arthroscopic findings at surgery did not influence the result. Age at the time of operation, preoperative JOA score, post-operative mechanical axis, pain at follow-up and follow-up period did influence the result statistically.

We report on our experience of surgical treatment of a dysplasia epiphysealis hemimelica case. The patient was a 2 year 8 month old boy. On physical examination a nut sized, painless bony hard mass was palpable on the medial aspect of the right knee, which showed a valgus deformity. His right foot was planovalgus anad the head of the talus was palpable. A full range of motion was present at the knee joint, but plantar flexion of the right ankle was limited. The plain x-ray showed some accessory calcification centers on the inner aspect of the epiphysis of the femoral medial condyle, distal tibia, talus and navicular. We performed surgical treatment using excision of the mass for correcting the valgus deformity of the knee. Four months after operation, there was no reccurence of the valgus deformity. We review the literature and discuss the surgical treatment of this condition.

We report on a 51-year-old man complaining of dysphagia that was caused by ossification of the anterior longitudinal ligament of the upper cervical spine. The ossification was extirpated using the transoral approach which was an easy and safe method. The patient's symptoms were completely relieved after the removal of the bony mass and surrounding fibrous soft tissue.

A three-dimensional study of the ultrastructure of endothelial cells is helpful in understanding important endothelial functions such as vascular transport and cell permeability. For this purpose, in addition to serial sectioning electron microscopy and high-voltage electron microscopy, the quick-freeze, deep-etching technique also enables us to analyze structures at the molecular level by its high resolution and is useful for three-dimensional morphological studies. Some modifications on the conventional deep-etching method were made in this study to reduce the undesirable aggregation of proteins and salts during etching. Using this technique, we examined the rat aortic endothelium, particularly the membrane structures and cytoskeletons. The luminal surface of the endothelium was covered with a fine filamentous coat, which was anchored to the plasma membrane. In the cytoplasm, actin filaments were prominent and were oriented randomly or in a parallel fashion near the plasma membrane. Of the vesicles seen in the endothelium, some had basket coats of clathrin, and others had striped coats on the cytoplasmic membrane surface. These surface structures of the vesicles suggest the transport mechanism of the vesicles in association with the fine filaments attached to the vesicles.

Based on preclinical evidence in animal models, the present study examined the clinical efficacy and safety of recombinant human fibroblast growth factor-2 (rhFGF-2) to accelerate bone repair in a dose-escalation prospective trial. One of three dosages (200, 400 or 800 microg) of rhFGF-2 in a biodegradable gelatin hydrogel was injected during surgery into the osteotomy site of 59 knee osteoarthritis patients undergoing high tibial osteotomy, and 57 of them were monitored for 16 weeks. The rhFGF-2 dose dependently increased the percentage of patients with radiographic bone union, and decreased the average time needed for such union. The percentages of patients with an absence of pain and full-weight bearing were also greater in the higher dosage groups than in the low dosage group, especially in the clinically critical periods 6, 8, and 10 weeks. Neither blood chemistries nor clinical adverse events were associated with the rhFGF-2 dosages. We therefore conclude that the rhFGF-2 in gelatin hydrogel dose dependently accelerated radiographic bone union of a surgical osteotomy with a safety profile at least at the dosages used, suggesting the clinical efficacy of this agent for bone repair.

Osteoarthritis is characterized by marked osteophyte formation consisting of new cartilage and bone. Because several growth factors are known to be involved in chondrogenesis and osteogenesis, the expression of transforming growth factor-beta 1 and basic fibroblast growth factor in the osteophytes of the human femoral heads in osteoarthritis were examined. Transforming growth factor-beta 1 messenger ribonucleic acid was detected in the osteophytes by reverse transcription-polymerase chain reaction. All of the nine examined osteophytes expressed transforming growth factor-beta 1 messenger ribonucleic acid, whereas one of four osteoarthritic femoral heads and none of four osteonecrotic femoral heads expressed transforming growth factor-beta 1 messenger ribonucleic acid. The extent of transforming growth factor-beta 1 messenger ribonucleic acid expression varied among the osteophytes. Transforming growth factor-beta 1 or basic fibroblast growth factor was analyzed in osteophytes immunohistochemically. Transforming growth factor-beta 1 was localized in the superficial cells in the osteophyte cartilage, but it was scarcely detected in the superficial cells in the degenerative articular cartilage. Basic fibroblast growth factor was detected in the cells in the whole layer of osteophyte cartilage and in the articular cartilage. There was a difference in the localization, which suggests the different roles of transforming growth factor-beta 1 and basic fibroblast growth factor in bone and cartilage metabolism in osteophyte formation.

Bilateral femurs of 12-week-old female Sprague-Dawley rats were fractured, and the fractured femurs were harvested 36 h, 3, 7, 10, and 14 days after the fracture. Localization of cell proliferation in the fracture calluses was investigated using immunohistochemistry with antiproliferating cell nuclear antigen (PCNA) monoclonal antibodies. Thirty-six hours after the fracture, many PCNA-positive cells were observed in the whole callus. The change was not limited to mesenchymal cells at the fracture site where the inflammatory reaction had occurred, but extended in the periosteum along almost the entire femoral diaphysis where intramembranous ossification was initiated. On day 3, periosteal cells or premature osteoblasts in the newly formed trabecular bone during intramembranous ossification still displayed intense staining. On day 7, many premature chondrocytes and proliferating chondrocytes were PCNA positive. Endochondral ossification appeared on days 10 and 14, and the premature osteoblasts and endothelial cells in the endochondral ossification front were stained with anti-PCNA antibodies. Quantification of PCNA-positive cells was carried out using an image analysis computer system, obtaining a PCNA score for each cellular event. The highest score was observed in the periosteum early after the fracture near the fracture site. Immunohistochemistry using anti-PCNA antibodies showed that the distribution of proliferating cells and the degree of cell proliferation varied according to the time lag after the fracture, suggesting the existence of local regulatory factors such as growth factors, and that significant cell proliferation was observed at the beginning of each cellular event.

We examined the effect of growth hormone on local growth factor mRNA expression in male Sprague-Dawley rats. Repetitive systemic administration of growth hormone (0.4 IU every 4 h) increased the expression of IGF-I mRNA up to 2.8-fold in costal cartilage tissue compared with controls. Basic FGF (bFGF) mRNA expression gradually increased up to 15.5-fold compared with pre-injection samples, where the mRNA expression was 5.3-times greater than vehicle-injected controls. TGF-β mRNA showed little changes. Moreover, one μg/ml of growth hormone enhanced the expression of bFGF mRNA in costal chondrocytes in culture. We conclude that growth hormone increased the local expression of bFGF, as well as that of IGF-I, in cartilage, and suggest that bFGF is directly regulated by growth hormone within a local area.

The membrane specializations of the fresh unfixed kidney cortex of adult and neonatal ICR mice were examined by using rapid freezing replica methods. In proximal tubular cells, numerous apical intracellular tubules exhibited helical patterns on the E face with a pitch of about 12 nm. This regular pattern was often continuous with similar striped indentations on the edge of the vacuoles connecting with the tubules. On the luminal surface (ES) of these vacuoles, membrane surface particles were arranged regularly in striped patterns with a center-to-center spacing of about 12 nm. We could not identify differentiations on the PF or PS of the same membrane systems. Another membrane specialization was a plaque or patch of clear pits in tilted lattice alignments on the P face of the large vacuoles with a center-to-center spacing of about 20 nm. This type of specialization was often observed in the neonatal mice proximal tubular cells. These membrane specializations may indicate the active membrane functions in the proximal tubules and suggest the functional continuity and structural relationship of these apical endocytic membrane systems. © 1993 Wiley-Liss, Inc.

Chondrogenesis can occur during a bone repair process, which is related to several growth factors. Transforming growth factor beta 1 (TGF-beta 1) downregulates the expression of type II collagen by chondrocytes in vitro, but injection of TGF-beta 1 into the periosteum in vivo increases type II collagen mRNA levels and initiates chondrogenesis. We examined the effect of TGF-beta 1 on collagen gene expression in a bovine periosteum-derived cell culture system to evaluate its direct effect on the periosteum. Cultured cells expressed alkaline phosphatase and collagen pro alpha 1(I) and pro alpha 1(II) mRNAs. A low level of type II collagen synthesis was demonstrated by immunoprecipitation. TGF-beta 1 had no effect on periosteal cell proliferation. Expression of collagen pro alpha 1(I) mRNA did not change with TGF-beta 1 treatment, but alkaline phosphatase mRNA showed a dose-dependent decrease. Expression of collagen pro alpha 1(II) mRNA was stimulated 2.7-fold by TGF-beta 1. TGF-beta 1 also caused a 2.6-fold increase in type II collagen synthesis by immunoprecipitation. These findings indicate that TGF-beta 1 is an enhancer of the expression of the chondrocyte phenotype of the periosteal cells and suggest that TGF-beta 1 is important in initiating and promoting cartilage formation in vivo.

The cytoplasmic surface of plasmalemmal vesicles in aortic endothelial cells was examined in quick-freeze, deep-etching replicas. In addition to the clathrin-coated vesicles, striped patterns were observed over the cytoplasmic surface membranes of small vesicles (60-80 nm in diameter) in the unfixed specimens. These patterns were more clearly visible in saponin-extracted specimens; the stripes were composed of several ridges or strands of 6-10 nm in width, and some were crossed. These were distinct from the characteristic pentagons or hexagons of the clathrin-coated vesicles. This striped structure was enhanced by treatment with myosin subfragment-1 or phalloidin, thereby indicating a possible relation to actin filaments. Patchy plaques with similar striped patterns appeared on the cytoplasmic surface of the plasmalemma proper and also on the cytoplasmic vacuole membranes together with clathrin. These striped structures may be involved in the formation and transport of so-called "uncoated" vesicles.

SUMMARY A simple new method of deep-etching, ‘upside-down etching’, has been developed in an attempt to obtain clearer images or replicas, especially of fresh, untreated biological tissues. During etching, specimens were kept facing downwards and shaken periodically so that non-volatile materials in the subliming ice could fall out of the frozen tissue. This improvement leads to a considerable reduction of sedimented debris of unetchable materials on ‘true’ membrane surfaces, as well as in the cytoplasm exposed by deep-etching.

Previous rapid-freeze, deep-etch replica studies have revealed the differences between heart and liver gap junctions; cytoplasmic surfaces of in situ and phenylmethylsulfnoyl fluoride (PMSF)-unproteolyzed isolated cardiac gap junctions (MW 47 kD) have a particulate substructure, which is absent both in the proteolyzed heart junctions (MW 29 kD) and in the liver junctions isolated with PMSF (MW 28 kD). The present deep-etch replica studies of gap junction cytoplasmic surface (CS) membranes in several tissues of rats and mice were performed to examine whether or not this difference between liver and heart is typical of variations in gap junction proteins from tissue to tissue. In surface mucous cells of the stomach, intestinal epithelial cells, and kidney tubule cells, these epithelial gap junctions always showed smooth cytoplasmic surfaces, similar to the liver gap junctions. In contrast, in the atrial myocardium, aortic endothelium, and the ciliary process, cytoplasmic surface membranes of the gap junctions consistently revelaed particulate patterns. Close examinations disclosed that those granular structures were not merely attached to the membrane surface, but they also protruded from the membrane interior as an integral component of gap junctions particles. Furthermore, in the pregnant rat uterus at term, cytoplasmic surface membrances of myometrial smooth muscle gap junctions were particulate, but those of endometrial epithelium were smooth. The present observations strongly suggest that tissue specificity exists in cytoplasmic surface structures of gap junctions between the “true” epithelial and the noneepithelial tissues: the nonepithelial gap junctions contain the additional cytoplasmic surface domain that is absent in the gap junctions of “true” epithelial origin.

We examined the cytoplasmic surface structures of the plasmalemmal vesicles in various tissues using quick-freeze, deep-etching replicas, and identified three types of cytoplasmic surface specializations. In addition to the baskets of clathrin-coated vesicles, stripe-surfaced and bumpy-surfaced vesicles could be identified. The striped patterns were clearly observed on the vesicles in endothelial cells and fibroblasts of various tissues, while in epithelial cells such as hepatocytes, renal proximal tubule cells, and superficial cells of the urinary bladder, instead of the striped patterns, rough-surfaced vesicles could be identified. These differences in 'uncoated' vesicles may suggest that there is the tissue specificity of the cytoplasmic surface structures of plasmalemmal vesicles.