Fine structural studies on the reabsorption of colloid and fusion of colloid droplets in thyroid glands of TSH-treated mice

Summary The mechanism of the luminal colloid reabsorption and the fate of reabsorbed colloid droplets were studied ultracytochemically in epithelial cells of thyroid cells of TSH-treated mice. The luminal colloid is reabsorbed by micropinocytosis as well as phagocytosis into the follicle epithelial cell. Almost all the pinocytotic pits and vesicles are coated and often closely associated with actin filaments demonstrated by use of heavy meromyosin (HMM). This suggests the involvement of the actin filament system in making and transporting coated vesicles for micropinocytosis of the luminal colloid. Freeze-fracture images show aggregates of intramembrane particles on the P-face of the small depressions corresponding to the initial site for coated pits.The reabsorbed colloid droplets fuse with one another and with lysosomes. At the initial stage of this fusion, the limiting membranes of adjoining droplets fuse in a limited area to become pentalaminar, and then become trilaminar. Eventually, the membranes at the fusion point disappear, and the contents of both droplets become continuous. Freeze-fracture images reveal the disappearance of the intramembrane particles at the initial site where the fusion occurs.Examination of thin-sectioned tissue treated by rapid-freeze substitution fixation, shows clearly delineated cell organelles, and the rounded mitochondria have a characteristically high electron-dense matrix. Just beneath the limiting membrane of each colloid droplet, there always exists a low electron-dense layer about 10 nm thickness. The lysosomes are sometimes seen wrapped around the colloid droplet.This study was supported by grants (No. 56370002, No. 00544016) from the Japan Ministry of Education     

The tight junctions of renal tubules in the cortex and outer medulla

Summary Quantitative aspects of tight junction morphology were systematically studied in the cortical and outer medullary segments of the distal urinary tubules of rat, hamster, rabbit, cat, dog and the primitve primate Tupaia belangeri.Only minor differences in junctional architecture were found between straight and convoluted portions of the distal tubule. In contrast, the collecting duct in cortex and outer medulla, in all species, exhibits the most elaborate tight junctions observed along the uriniferous tubule.The present and previous findings from this laboratory indicate that increasing tightness of the junctional complexes is apparent along the course of the nephron in all species studied.The proposed relationship between quantitative aspects of the zonula occludens and presently available values for transepithelial electrical resistance was re-examined for the renal tubules. It was found that for the mammalian kidney a satisfactory correlation exists between the tight junction morphology and presently known functional parameters. This relationship is the more evident the more additional dimensional characteristics of the intercellular clefts are taken into consideration.It may therefore be concluded that, at least for the mammalian kidney, the assumption of differences in the molecular organization of the tight junctions is not needed to explain so far unresolved discrepancies between tubular morphology and function.Parts of these findings were presented at the 72nd Meeting of the Anatomical Society, Aachen; April 1977 (see Verh. Anat. Ges. 72:229–234 [1978])Supported by the Deutsche Forschungsgemeinschaft     

The membrane systems in different fibre types of the triceps surae muscle of cat

Summary The volume and surface area of mitochondria and sarcoplasmic reticulum in fast and slow twitch fibres of the cat triceps surae muscle were determined from thin sections. The width of the Z-line and the array of glycogen granules identified fast and slow twitch fibres.The relative volume occupied by mitochondria was largest in slow twitch gastrocnemius fibres. Fast twitch fibres showed the greatest scatter of mitochondrial content. This corresponds with the fact that motor units of the fast twitch type differ most with respect to resistance to fatigue.The relative volume of the sarcoplasmic reticulum was twice as large in fast as in slow twitch fibres. The volume fraction occupied by longitudinal tubules of the reticulum was the same in fast and slow twitch gastrocnemius fibres but was only half as large in the slow twitch soleus fibres. This difference may be related to post-tetanic potentiation: this property is present in all gastrocnemius fibres but is absent in soleus fibres.The specific tetanic force is 3 to 5 times smaller in slow twitch gastrocnemius than in slow twitch soleus fibres or fast gastrocnemius fibres. There was, however, no detectable morphological difference that might be related to this difference in force.Freeze fractures demonstrated directly that, in soleus fibres, terminal cisternae and longitudinal tubules of the reticulum were scarce as compared to gastrocnemius fibres. The plasma membranes of some gastrocnemius fibres displayed square arrays of 60-nm particles; these arrays were absent in other gastrocnemius fibres and in all soleus fibres. They probably characterize plasma membranes of fast twitch fibres.This study was supported by grants from the Danish Medical Research Council. I wish to thank Mrs. M. Bjærg for valuable technical help     

Silicon in rat liver organelles: Electron probe microanalysis

Summary Electron probe microanalysis of unfixed freeze-substituted rat liver tissue embedded in Spurr's low viscosity epoxy resin demonstrated the occurrence of Si as well as P, S, and Cl in the nucleus, nucleolus, mitochondria, and rough endoplasmic reticulum. Chemical analysis confirmed that the Si in the organelles did not originate from instrumental contaminants. This suggests that Si may be involved in the biochemistry of these subcellular organelles.Supported by Grant GM-08229-12-13 from the National Institutes of Health, USPHS.We are grateful to the Kevex Corporation and Mr. Glenn W. Meyer, Sales Engineer, for the use of the Kevex X-ray spectrometer; we wish to thank as well the Perkin-Elmer Corporation and their Western Branch Manager, Mr. Michael E. Mullen and Senior Microscopist, Mr. Minoru Shinorhara, for use of and assistance with the Hitachi HU 12 A transmission electron microscope. We also wish to acknowledge Mary Louise Chiappino for her technical assistance in preparing the thin sections, the final micrographs and the X-ray photographs, and Darlene Lum for technical assistance in the laboratory.     

Freeze-fracture study of the junctional complexes of human and rabbit thyroid follicles

Summary Zonulae occludentes, gap junctions and desmosomes have been demonstrated in replicas of freeze-fractured follicular cells of normal human and rabbit thyroid glands. The zonulae occludentes between the human follicular cells are composed of two to eight strands, which completely separate the intercellular space from the follicular lumen. Four to twelve or more strands are visible between the follicular cells of the rabbit thyroid gland.In the meshes of the zonulae occludentes as well as below them, gap junctions are present. They are numerous on the fracture faces of the human follicular cell membranes, but infrequent in those of the rabbit.Aggregates of particles related to desmosomes are found in the deeper meshes of the zonulae occludentes or close to them.     

Rapid responses to high temperature and desiccation but not to low temperature in the freeze tolerant sub-Antarctic caterpillar Pringleophaga marioni (Lepidoptera,Tineidae)

A broad definition of rapid cold hardening (RCH) is that it is the process whereby insects increase their survival of a sub-zero temperature after a brief (h) pre-exposure to a less severe low temperature. The effects of various pre-treatments on survival of two h at -7.9 degrees C were investigated in the freeze tolerant sub-Antarctic caterpillar Pringleophaga marioni (Lepidoptera: Tineidae), the first time RCH has been investigated in a freeze tolerant arthropod. All caterpillars froze when exposed to -7.9 degrees C, and none of the low temperature pre-treatments (-5, 0, 5 and 15 degrees C, as well as -5 degrees C and 0 degrees C with a delay before freezing) nor slow cooling (0.1 degrees C/min) elicited any improvement in survival of -7.9 degrees C as compared to controls. However, high temperature treatments (25, 30 and 35 degrees C), desiccation and acclimation for 5 days at 0 degrees C did result in significant increases in survival of the test temperature, possibly as a result of heat shock protein production. Haemolymph osmolality was elevated only by the 35 degrees C pre-treatment. It is suggested that the unpredictable environment of Marion Island means that P. marioni must always be physiologically prepared to survive cold snaps, and that this year-round cold hardiness therefore supersedes a rapid cold hardening response.     

Experimental Trichinella infection in seals

The susceptibility of seals to infection with Trichinella nativa and the cold tolerant characteristics of muscle larvae in seal meat were evaluated. Two grey seals, Halichoerus grypus, were inoculated with 5000 (100 larvae/kg) T. nativa larvae and two grey seals with 50000 (1000 larvae/kg). One seal from each dose group and two control seals were killed at 5 and 10 weeks post-inoculation (p.i.). At 5 weeks p.i., infection was established in both low and high dose seals with mean larval densities of 68 and 472 larvae per gram (lpg), respectively, using eight different muscles for analyses. At 10 weeks p.i., mean larval densities were 531 and 2649 lpg, respectively, suggesting an extended persistence of intestinal worms. In seals with high larval density infections, the distribution of larvae in various muscles was uniform, but in one seal with a low larval density infection, predilection sites of larvae included muscle groups with a relative high blood flow, i.e. diaphragm, intercostal and rear flipper muscles. Trichinella-specific antibody levels, as measured by ELISA, increased during the 10 week experimental period. Infected seal muscle was stored at 5, -5 and -18 degrees C for 1, 4 and 8 weeks. Muscle larvae released from stored seal muscle by artificial digestion were inoculated into mice to assess viability and infectivity. Larvae from seal muscle 10 weeks p.i. tolerated -18 degrees C for 8 weeks but larvae from seal muscle 5 weeks p.i. tolerated only 1 week at -18 degrees C, supporting the hypothesis that freeze tolerance increases with the age of the host-parasite tissue complex. The expressed susceptibility to infection, extended production of larvae, antibody response and freeze tolerance of T. nativa in seals are new findings from the first experimental Trichinella infection in any marine mammal and suggest that pinnipeds (phocids, otariiids or walrus) may acquire Trichinella infection by scavenging even small amounts of infected tissue left by hunters or predators.     

Are gap junction membrane plaques implicated in intercellular vesicle transfer?

Gap junction channels are concentrated in specialised plaques of plasma membrane where cells are in close apposition. In this communication evidence is provided showing that these specialised regions of membrane also provide a site for vesicular transfer between cells. Vesicle distribution in eye lenses was found to generally reflect the reported distribution of gap junction membrane plaques. In certain areas of the lens gap junction membrane plaques and vesicles could be seen to form combined, complex structures. Ultrastructure of the vesicle and gap junction membrane plaque complexes was consistent with the vesicles moving through membrane plaques from one lens fibre cell to the next. To investigate whether transport of substances was consistent with intercellular vesicle transfer, transport of various markers was investigated. Time course experiments showing the rate of uptake of various markers into the lens did not show dramatic differences for molecules smaller or larger then gap junction pores formed by connexons. While considered as a primary intercellular transport mechanism in the lens, connexon pores were not the sole agent mediating the observed transport. Other reported mechanisms of intercellular transport in the lens can only account for the movement of relatively small molecules. Vesicular transport may therefore be a major form of transport into the outer lens layers for larger molecules. Implicit in these observations is a new hypothesis for intercellular vesicle movement via gap junction membrane plaques. Intercellular vesicle movement could possibly provide a path for large molecules associated with intact vesicles to be transported into the eye lens tissue.     

Molecular characterization and sequencing of antifreeze proteins from larvae of the beetle Dendroides canadensis

The deduced amino acid sequences of antifreeze proteins (AFPs) from larvae of the beetle Dendroides canadensis were determined from both complementary DNAs (cDNAs) and from peptide sequencing. These consisted of proteins with a 25-residue signal peptide and mature proteins 83 (Dendroides antifreeze protein; DAFP-1) or 84 (DAFP-2) amino acids in length which differed at only two positions. Peptide sequencing yielded sequences which overlapped exactly with those of the deduced cDNA sequences of DAFP-1 and DAFP-2, while the partial sequence of another AFP (DAFP-3) matched 21 of 28 residues. Seven 12- or 13-mer repeating units are present in these antifreeze proteins with a consensus sequence consisting of: Cys-Thr-X₃-Ser-X₅-X₆-Cys-X₈-X₉-Ala-X₁₁-Thr-X₁₃, where X₃ and X₁₁ tend toward charged residues, X₅ tends toward threonine or serine, X₆ toward asparagine or aspartate, X₉ toward asparagine or lysine, and X₁₃ toward alanine in the 13-mers. The most interesting feature of these proteins is that throughout the length of the mature antifreeze proteins every sixth residue is a cysteine. These sequences are not similar to any of the known fish AFPs, but they are similar to AFPs from the beetle Tenebrio molitor. Accepted: 14 November 1997