Marked depletion of the water-channel protein, AQP5, in the canine nictitating membrane glands might contribute to the development of KCS.

The objectives of this study were to investigate the normal histological localization of aquaporin (AQP) 5 protein in the lacrimal and nictitating membrane glands and to compare this localization in healthy and keratoconjunctivitis sicca (KCS) dogs. Lacrimal and nictitating membrane glands of 5 healthy Beagles and nictitating membrane glands of 5 KCS dogs (3 Beagles and 2 mongrel dogs: 0-13 years) were used for the present study. The owners of the KCS dogs did not consent to perform biopsies of the lacrimal glands. The localization and distribution of AQP5 protein were investigated by an immunohistochemical technique. In immunohistochemical staining, AQP5 was localized in the apical site of acinar epithelial and ductal epithelial cells from both the lacrimal and nictitating membrane glands in healthy dogs. However, AQP5 was not detected in the 5 KCS dogs. These results for immunohistochemical AQP5 localization might correlate with the deficiency in tear secretion found in KCS dogs.

Occurrence of prohormone convertase-like substances in the neural complex cells of the ascidian Halocynthia roretzi.

Our previous study on the distribution of adrenocorticotropin (ACTH)-like substances in the neural complex (cerebral ganglion, dorsal strand, and neural gland) of an ascidian Halocynthia roretzi revealed that some of the cells in the cerebral ganglion and the cells scattered along the dorsal strand were immunopositive with antiserum against ACTH. In order to ascertain whether these cells are equipped with prohormone convertases, we performed immunohistochemical studies on the neural complex by using antisera against PC1 and PC2. A considerable number of cells around the dorsal strand and a few cells in the neural ganglion were immunopositive with PC1 and/or PC2 antibodies. Immunoelectron microscopic study demonstrated that some granulated cells situated in the cerebral ganglion and along the dorsal strand contained PC1- or PC2-like substances within their secretory granules. Western blot analysis revealed the presence of 66-kDa PC1-like and 70-kDa PC2-like substances in the neural complex. Moreover, immunostaining of consecutive sections showed that the majority of the cells containing PC1- and/or PC2-like substances corresponded to the cells immunoreactive with antisera against ACTH and CLIP but not to those immunoreactive with an antiserum against PRL. Cells belonging to the neural gland neither contained electron-dense granules nor showed immunoreactivity with any antisera employed in this experiment. The possibility that some of the cells situated in the cerebral ganglion and along the dorsal strand are progenitors of vertebrate adenohypophyseal cells is discussed.

Induction of gamete release by gonadotropin-releasing hormone in a protochordate, Ciona intestinalis.

Gonadotropin-releasing hormone (GnRH) of vertebrates is now believed to have multiple functions in addition to its role as a hypophysiotropic hormone, as originally defined. Recently, it has been shown that GnRH occurs also in the ascidians, which are considered ancestral chordates. Here the author shows that GnRH induces spawning of gametes from mature individuals of Ciona intestinalis. Ciona accumulates mature gametes in the gonoducts and maintains them until spawning is triggered by a photoperiodic cue(s). Injection of synthetic tunicate GnRH-I or -II into various sites of mature individuals effectively induced gamete release (spawning), although the former was more potent. Gamete release often occurred on a larger scale than in spontaneous spawning. However, moderate gamete release, similar to spontaneous spawning, was often triggered by exogenous tunicate GnRH. GnRH in vivo apparently is released from the GnRH-containing neurons that are distributed from the region of the cerebral ganglion to the proximal part of the ovary along the dorsal strand within the blood sinus; this indicates that both forms of tunicate GnRH may be the actual inducers of spawning. It is suggested that, in the ancestral chordate, GnRH neurons release GnRH prior to the spawning and the released GnRH acts directly on the epithelium of gonoducts or functions as a neuromodulator of other neurons innervating the gonoducts to induce spawning.

Prolactin-like immunoreactivity in the granules of neural complex cells in the ascidian Halocynthia roretzi.

Electron-microscopic studies of the neural complex (neural gland, dorsal strand, and cerebral ganglion) of an ascidian, Halocynthia roretzi, were performed, paying particular attention to the secretory systems. We found that cells scattered along the dorsal strand and neural cells in the cerebral ganglion contained electron-dense secretory granules of variable size. Immunoelectron-microscopic studies with an antiserum to bullfrog prolactin revealed that the secretory granules (100-250 nm in diameter) of some granulated cells contained a prolactin-like substance. Cells belonging to the neural gland and dorsal strand neither contained electron-dense granules nor showed immunoreactivity. The possibility that cells in the cerebral ganglion and those along the dorsal strand are phylogenetic progenitors of vertebrate adenohypophyseal cells is discussed.

Test cell migration and tunic formation during post-hatching development of the larva of the ascidian, Ciona intestinalis.

Morphological changes in the tunic layers and migration of the test cells during swimming period in the larva of the ascidian, Ciona intestinalis, were observed by light and electron microscopy. The swimming period was divided into three stages. In stage 1, further formation of juvenile tunic layer started only in the larval trunk and neck region. In stage 2, the layer became swollen in the ventral and dorsal sides of the neck region and in stage 3, the swelling expanded backward. Concomitantly with these changes, the outermost larval tunic layer (outer cuticular layer), which had been formed before hatching, also swelled in the neck region in stage 2 and formed two humps in stage 3, although the layer did not change in the tail region during the swimming period. Test cells that were present over the entire larval tunic layer in stage 1 began to move from the surface of the fin toward that of the side of the body in stage 2, and finally gathered to form six bands running radially from the anterior end to the posterior end of the trunk region and aligned along the lateral sides of body in the tail region in stage 3. In electron microscopic observations, pseudopodia protruding from the test cells invaded the larval tunic, following which they extended proximate to the juvenile tunic in the trunk region. In the tail region, which had no juvenile tunic layer as that described, the pseudopodia invaded and remained adjacent to the surface of the epidermis or the sensory cilia protruded from the epidermis. Metamorphosis of the larvae, further tunic formation, degradation of adhesive papilla, attachment of larva to the substratum and tail resorption commenced after these morphological changes occurred. The possible role of the test cells in metamorphosis is discussed.

Projectin is an invertebrate connectin (titin): isolation from crayfish claw muscle and localization in crayfish claw muscle and insect flight muscle.

A filamentous protein was isolated from crayfish claw muscle. This protein had physiochemical properties very similar to vertebrate skeletal muscle connectin (titin), although its apparent molecular mass (approximately 1200 kDa) was considerably lower than that of connectin (approximately 3000 kDa). Polyclonal as well as monoclonal antibodies against chicken skeletal muscle connectin reacted with the 1200 kDa protein from crayfish claw muscle. Conversely, polyclonal antibodies against crayfish 1200 kDa protein cross-reacted with chicken connectin. Circular dichroic spectra indicated the abundance of beta-sheet structure (approximately 60%). Low-angle shadowed images showed filamentous structures (0.2-0.5 microns) by electron microscopy. Proteolysis of the 1200 kDa protein by alpha-chymotrypsin or V8 protease rapidly resulted in formation of 1000 kDa or 1100 and 800 kDa peptides. The amino acid composition was very similar to those of vertebrate connectins and of honeybee flight muscle projectin. Based on the molecular weight and amino acid composition, the 1200 kDa protein is regarded to be crayfish projectin. Immunofluorescence and immunoelectron microscopy revealed that crayfish projectin was localized in the A/I junction area and A-band except for its centre region in crayfish claw muscles. Polyclonal antibodies against crayfish claw muscle projectin reacted with 1200 kDa projectin of honeybee and beetle flight muscle. A monoclonal antibody against chicken skeletal muscle connectin also reacted with honeybee and beetle projectin. Immunoelectron microscopic observations revealed that anti-crayfish projectin antibodies bound the connecting filaments linking the Z-line and the thick filaments up to the M-line of honeybee muscle sarcomere. Anti-crayfish projectin antibodies bound the I-band region near the Z-line of beetle flight muscle. It is concluded that the 1200 kDa projectin from crayfish claw muscle is an invertebrate connectin (titin). Recent work with locust flight muscle mini-titin (Nave & Weber, 1990) is in good agreement with the present study, except that the isolated mini-titin estimated as 600 kDa appears to be a proteolytic product (approximately 1100 kDa) of the parent molecule (approximately 1200 kDa).

Formation of amyloid-like fibrils in COS cells overexpressing part of the Alzheimer amyloid protein precursor.

A pathological hallmark of Alzheimer's disease is the deposition of amyloid fibrils in the brain. The principal component of the amyloid fibril is beta/A4 protein, which is derived from a large membrane-bound glycoprotein, Alzheimer amyloid protein precursor (APP). Although the deposition of amyloid is thought to result from the aberrant processing of APP, the detailed molecular mechanisms of amyloidogenesis remain unclear. A C-terminal fragment of APP which spans the beta/A4 and cytoplasmic domains has a tendency to self-aggregate. In an attempt to establish a cultured-cell model for amyloid fibril formation, we have transfected COS-1 cells with complementary DNA encoding the C-terminal 100 residues of APP. In the perinuclear regions of a small population of DNA-transfected cells, we observed inclusion-like deposits which showed a strong immunohistochemical reaction towards an anti-C-terminal APP antibody or an anti-beta/A4 amyloid core-specific antibody. Electron microscope observations of the inclusion-carrying cells revealed an accumulation of amyloid-like fibrils of 8-22 nm diameter near and on the nuclear membrane. The fibrils showed a beaded or helical structure, and reacted positively with the anti-C-terminus antibody by immunoelectron microscopy. These results suggest that the formation of amyloid fibrils is an inherent characteristic of the C-terminal peptide of APP. The present system provides a suitable model for the molecular dissection of the process of brain amyloidogenesis.

Ultrastructure of the thread cells in the slime gland of Japanese hagfishes, Paramyxine atami and Eptatretus burgeri.

The thread cells in the slime gland of Japanese hagfishes, Paramyxine atami and Eptatretus burgeri were studied by light and electron microscopy. The mature thread cells are large elements (180 times 80 mu) filled with an intricately coiled thread, approximately 2 mu in diameter. The protein nature of the thread has been confirmed by histochemical examination. In the initial stage of growth, the thread consists of a bundle of distinctly parallel filaments approximately 90-120 A in diameter and a centrally located tubular component approximately 230-260 A in diameter which occurs singly or occasionally as a double and triple structure. The developing thread displays thin filaments, approximately 30-60 A in diameter. The thin filaments are composed of fine fibrous structures, subfilaments, approximately 10-30 A in diameter. On the outer surface of the thread a coating is apparent, giving it a fluffy appearance. Polysomal clusters consisting of five or six ribosomes are predominant. Fine fibrous structures are also found among the threads; they seem to have a spatial relationship with the polysomes and resemble the subfilament constituents of the thin filaments. From these results, it may be suggested that the fine fibrous structures synthesized by polysomes, twist together and coalesce into a thread. The problem of the polysome size and the molecular weight of the fibrous protein synthesized is discussed.