Lower Cell Layers Research Articles

Tight junctions of human keratinocytes in primary culture: A freeze-fracture study

Human skin explants obtained from 2- to 5-yr-old patients with harelips were cultured in NCTC 168 medium at 37 degrees C, in a humidified atmosphere containing 5% CO2 in air. After a 2-week incubation period, the newly grown cells were studied with special reference to tight junctions by freeze-fracture electron microscopy. Many completely formed tight junctions were observed between the uppermost living cells of migrating epithelium, and fragmented tight junctions were seen between the lower layer cells. The tight junctions in the uppermost cells developed so well that they formed a belt-like network consisting of two to six rows of strands. This observation may suggest that human keratinocytes have the ability to produce tight junctions perfectly enough to serve as a barrier, although no complete tight junctions were formed in situ.

Role of fibronectin in collagen deposition: Fab' to the gelatin-binding domain of fibronectin inhibits both fibronectin and collagen organization in fibroblast extracellular matrix.

We report the effect of Fab' (anti-60k) to a 60,000 mol wt gelatin binding domain of fibronectin (1981, J. Biol. Chem. 256:5583) on diploid fibroblast (IMR-90) extracellular fibronectin and collagen organization. Anti-60k Fab' did not inhibit IMR-90 attachment or proliferation in fibronectin-depleted medium. Fibroblasts cultured with preimmune Fab' deposited a dense extracellular network of fibronectin and collagen detectable by immunofluorescence, while anti-60k Fab' prevented extracellular collagen and fibronectin fibril deposition. Matrix fibronectin and collagen deposition remained decreased in cultures containing anti-60k Fab' until cells became bilayered or more dense, when fibronectin and collagen began to appear in lower cell layers. Anti-60k Fab' added to confluent cultures 24 h before fixation and staining had no effect on matrix fibronectin or collagen, so anti-60k Fab' did not simply block immunostaining. Confluent cultures grown in anti-60k Fab' and labeled for 24 h with [3H]proline incorporated identical amounts of [3H]proline and [3H]hydroxyproline, but [3H]hydroxyproline deposition in the cell layer was significantly decreased by anti-60k Fab' (P less than 0.01). Extracellular matrix collagen does not appear to form a scaffold for fibronectin deposition, as neither gelatin nor a gelatin-binding fragment of plasma fibronectin inhibited deposition of matrix fibronectin. Our results suggest that interstitial collagens and fibronectin interact to form a fibrillar component of the extracellular matrix, and that fibronectin is required for normal collagen organization and deposition by fibroblasts in vitro. Domain-specific antibodies to fibronectin are powerful tools to study the biological role of fibronectin in extracellular matrix organization and other processes.

Rat C-6 glioma cells maintained in an organ culture system: a study of kinetic parameters.

Rat C-6 glioma cells were grown on a sponge foam matrix in an organ culture system and the cell cycle parameters, including the growth fraction (GF), were assessed after autoradiography. The zones of growth consisted of a compact upper layer (UL) at the gaseous interface, a central necrotic layer and a deeper lower layer (LL) which invaded the matrix. The fraction of continuously labeled mitoses (FCLM) was similar in both the UL and LL cells. The derivatives of the FCLM curves obtained in three experiments gave an average modal TG2 of 5 hr. A mathematical model relating GF, TG2, TC and labeling index as a function of time, LI(t), was devised for cells in a steady state exposed continuously to tritiated thymidine and was applied to data obtained from UL cells. A mean GF of 9% (range: 8-10%) and a mean cell cycle time (TC') of 27 hr (range: 13-47 hr) were obtained. The mean TS was calculated to be 11 hr (range: 8-16 hr) by the method of grain counts per mitotic figure or grain index (GI). Knowledge of TS permitted alternative calculation of the cell cycle time from the equation Ts/TC = LI(0)/GF:this gave a mean cell cycle time (TC") of 29 hr (range: 20-45hr). Except for the GF, the cell kinetics were comparable to those of the same cell line grown in monolayer culture. The GF in the in vitro system described is in the lower range reported in some human malignant gliomas in vivo.

Preliminary Account of the Development of the Lesser Weever-Fish, Trachinus vipera.

Summary. The egg of Trachinus is about 1.32 millim. in diameter, and contains from 20 to 30 small oil-globules, thus differing from the majority of floating fish eggs hitherto described. In the unfertilized egg a vitelline membrane is easily distinguishable, but afterwards this comes in close contact with the zona radiata, and often requires the action of reagents to show it properly. My observations appear to confirm those of Henneguy, that the invagination observed in optic section in the living egg is an inward folding of the lower layer cells of the epiblast, and that afterwards the alimentary tract is built up from this layer, together with material derived from the intermediary layer. This point cannot, however, be settled definitely without a careful examination of sections of this stage. My observations confirm those of Ryder as to the nature and persistence of the segmentation-cavity, and in this respect pelagic teleostean eggs seem to differ from all others hitherto described. Although the heart appears early on the fourth day, its venous end remains closely applied to the vitellus up to several days after hatching, and I have not been able to find any vascular system either in the embryo or in the vitellus up to 14 or 15 days after development begins, that is 4 or 5 days after hatching. In this respect the observations of Ryder and Kingsley and Conn agree with my own, although in non-pelagic teleostean eggs an elaborate circulatory system is developed both in the vitellus and in the embryo a considerable time before hatching. I have nothing new to record in the later stages of development. The liver and the pancreas arise as little pouches budded off from the mesenteron; the proctodeum arises late, but is well formed at the time of hatching. The stomodeum does not appear to develop until the embryo has left the shell. The young fish usually hatch out on the tenth or eleventh day after impregnation, the early ones with little pigment on the eyes and body, the later ones with the pigment much more developed.

Scientific Serials

THE Quarterly Journal of Microscopic Science may, at the present time, be looked upon as the representative of the most modern phase of biological thought. The current number contains articles of much more than ordinary importance. The first is by Mr. F. M. Balfour, being “A comparison of the early stages in the Development of Vertebrates.” The plate which accompanies the memoir is coloured in a particularly instructive manner, which illustrates the ultimate destination of the different elements of the cellular layers of the blastoderm. Mr. Balfour's observations are in favour of the blastopore becoming neither the mouth nor the anus of the adult animal, but of its cicatrix being a weak spot at which one or the other may subsequently be more easily formed than elsewhere. The gap between the observed structure of the developing amphibian and selachian is made more simple by the introduction of a hypothetical intermediate form in which the segmentation cavity is represented as if “it were sunk down so as to be completely within the lower layer cells,” a condition not quite easy to comprehend. Many other very important theoretical points are discussed in this particularly interesting paper.—The second paper is a reprint from the Privy Council Reports, of Dr. Klein's observations on the pathology of sheep-pox.—Mr. W. H. Jackson describes and figures a new Peritrichous Infusorian, named Cyclochaeta spongilla, found in a sponge from the river Chirwell.—Mr. A. A. W. Hubrecht of Leyden makes “some remarks about the minute anatomy of Mediterranean Nemertcans,” including notes on the dermal tissues, nervous system, &c., of species of Meckelia, Polia, Lineus, Ommatoplea, and Drepanophorus (n.g.)—Prof. Lankester publishes in full his observations read before the Linnean Society, “On some points in the structure of Amphioxus, and their bearing on the morphology of vertebrata.” The exact homology of the atrial chamber and of the perivisceral cavity in the Lancelet has been a fruitful source of discussion, and Prof. Lankester's study of the question throws considerable additional light on the subject. The conclusions to which his investigations lead are “first that the peritoneal cavity of the vertebrate is the same thing as the coelom of the worm and of Amphioxus; second, that the earlier vertebrate ancestors (represented in a degenerate form by Amphioxus) developed epipleura, which coalesced in the median line postorally to form an atrium; third, that whilst Amphioxus retains this atrium in functional activity, the other vertebrata have lost it by the coalescence of its outer and inner bounding wall, respectively epipleuia and somatopleura; fourth, that whilst the indications of the earlier historical steps of this process are suppressed in all craniate vertebrata at present investigated, yet the Elasmobranchs do continue to present to us an ontogenetic phase in which the somatopleura and the epipleura are widely separated; thus enclosing between them an epicoel (the atrium of amphioxus).”—Mr. F. R. Lewis writes on Nematode Haematozoa in the dog, closely allied to Filaria sanguinolenta, found in the walls of the aorta. These are figured, as are the parts of Amphiporous spectabilis and other Nemerteans, described by Dr. M'Intosh in considerable detail,—There is an admirable paper by Prof. Thiselton Dyer, containing a review of the various modes of sexual reproduction known among Thallophytes, with a sketch of the classification of that section of Cryptogams—including Algæ, Lichens, Fungi, and Characeæ—recently proposed by Prof. Sachs.