Invasive Front Grading and Epithelial-Mesenchymal Transition in Canine Oral and Cutaneous Squamous Cell Carcinomas.

Oral and cutaneous tissues are the most frequent origin in canine squamous cell carcinoma (SSC). In SCC, changes in adhesion molecule expression and transition from epithelial to mesenchymal phenotype are thought to be important in development of invasive behavior of neoplastic cells at the leading front of the tumor. We therefore investigated histological invasive front grading and epithelial-mesenchymal transition (EMT) in both oral SCCs and cutaneous SCCs. EMT was assessed by evaluating immunohistochemical expression of E-cadherin, ß-catenin, desmoglein, vimentin, and N-cadherin. Regardless of the anatomic location, invasive front grading resulted in higher histological grades than grading of the surface. Most oral SCCs were of significantly higher histologic grade than cutaneous SCCs ( P < .01). Expression of E-cadherin, ß-catenin, and desmoglein was significantly lower in oral SCC compared with cutaneous SCC ( P < .01). A significant association was found between invasive front grading and loss of E-cadherin, ß-catenin, and desmoglein ( P < .01). Also, vimentin-positive neoplastic cells had low immunoreactivity of these adhesion molecules, and a few of these neoplastic cells were positive for N-cadherin. These results suggest not only E-cadherin and ß-catenin but also desmoglein as markers for predicting biological behavior of canine SCC. Depending on their primary sites, EMT correlates with biological behavior and therefore histological grade of canine SCC. We suggest that combining invasive front grading with assessment of immunohistochemical expression of E-cadherin, ß-catenin, and desmoglein may allow more accurate prediction of biological behavior of canine SCCs.

Amyloid-Producing Odontogenic Tumors of the Facial Skin in Three Cats.

Amyloid-producing odontogenic tumors (APOTs) of the facial skin were diagnosed in 3 domestic cats. The neoplasms had the histopathological characteristics of the odontogenic tumor. The neoplastic cells were present in irregular islands, strands, and sheets. The peripheral neoplastic cells of the islands and strands were arranged in a palisading fashion, while the central cells were polyhedral to stellate and randomly arranged. Multiple spherules of homogeneous eosinophilic material were closely apposed to the neoplastic epithelial cells. The spherules stained with Congo red and produced an apple green birefringence under polarization microscopy, indicative of amyloid. Immunohistochemically, amyloid materials of the neoplasms reacted with polyclonal antibodies for ameloblastin, amelogenin, and sheathlin antibodies. Neoplastic epithelial cells also reacted with antiameloblastin, amelogenin, and sheathlin antibodies, with varied intensity. The histopathological and immunohistochemical characteristics of dermal neoplasms of the 3 cats were analogous to those of APOTs reported in the dog and the cat.

Diversity of Histologic Patterns and Expression of Cytoskeletal Proteins in Canine Skeletal Osteosarcoma.

Osteosarcoma (OS), the most common bone tumor, includes OS of the head (OSH) and appendicular OS (OSA). In dogs, it is classified into 6 histologic subtypes: osteoblastic, chondroblastic, fibroblastic, telangiectatic, giant cell, and poorly differentiated. This study investigated the significance of the histologic classification relevant to clinical outcome and the histologic and immunohistochemical relationships between pleomorphism and expression of cytoskeletal proteins in 60 cases each of OSH and OSA. Most neoplasms exhibited histologic diversity, and 64% of OS contained multiple subtypes. In addition to the above 6 subtypes, myxoid, round cell, and epithelioid subtypes were observed. Although the epithelioid subtypes were observed in only OSH, no significant difference in the frequency of other subtypes was observed. Also, no significant relevance was observed between the clinical outcome and histologic subtypes. Cytokeratin (CK) was expressed in both epithelioid and sarcomatoid tumor cells in various subtypes, and all CK-positive tumor cells also expressed vimentin. Vimentin and α-smooth muscle actin (SMA) were expressed in all subtypes. A few SMA-positive spindle-shaped tumor cells exhibited desmin expression. Glial fibrillary acidic protein-positive tumor cells were observed in many subtypes, and some of these cells showed neurofilament expression. Although OSH exhibited significantly stronger immunoreactivity for SMA than OSA, no significant difference in other cytoskeletal proteins was observed. Some tumor cells had cytoskeletal protein expression compatible with the corresponding histologic subtypes, such as CK in the epithelioid subtype and SMA in the fibroblastic subtype. Thus, canine skeletal OS is composed of pleomorphic and heterogenous tumor cells as is reflected in the diversity of histologic patterns and expression of cytoskeletal proteins.

Biochemical and immunohistochemical characterization of the amyloid in canine amyloid-producing odontogenic tumor.

The amyloid of canine amyloid-producing odontogenic tumor (APOT) was evaluated biochemically and immunohistochemically. The N-terminal amino-acid sequence of purified amyloid protein from a canine APOT was strikingly similar to the sequence in both rat ameloblastin and porcine sheathlin. Immunohistochemically, the amyloid in APOT from 9 dogs was strongly reactive with anti-rat ameloblastin, anti-porcine sheathlin, and anti-canine APOT amyloid and weakly reactive with anti-porcine amelogenin but negative for antibodies to cytokeratins, vimentin, desmin, alpha-smooth muscle actin, amyloid A, glial fibrillary acidic protein, or S100 protein. The neoplastic epithelial cells of APOT were focally reactive with antibodies to ameloblastin, sheathlin, amelogenin, and canine APOT amyloid. The similarity in amino-acid sequence of the amyloid protein of canine APOT to that of enamel proteins, such as ameloblastin, sheathlin, and amelogenin, and the expression of these antigens in both APOT amyloid and in the neoplastic cells suggest that the amyloid of canine APOT is derived from enamel proteins secreted by ameloblasts.

Pleomorphic adenoma of the salivary gland in two dogs.

Pleomorphic adenomas of the salivary gland were diagnosed in two dogs. The tumours were single, firm and well circumscribed, with a smooth cut surface. Metastatic tumours were not detected. Histopathological examination revealed that the tumours contained multiple cysts lined with luminal epithelial cells and myoepithelial cells, and mucinous, myxochondroid and cartilaginous tissues. Immunohistochemical examination demonstrated labelling of luminal epithelial cells and myoepithelial cells, and mucinous, myxochondroid and cartilaginous tissues with antibodies to cytokeratin LU-5, AE1/AE3, CK-14, CALP, a-SMA, vimentin, GFAP, and S-100. Labelling for GFAP indicated stromal transformation into myxoid and chondroid tissues.

Organization of a gene family developmentally regulated during Dictyostelium discoideum spore germination.

mRNA specific to cDNA clone pLK109 is present in Dictyostelium discoideum spores, increases about two- to threefold at 0.5 to 1 h during spore germination, and then rapidly decreases. The mRNA is not detectable in vegetative cells or in early multicellular development on filters, but is present late during development, approximately at the time of sporulation. 109 mRNA in spores is 700 nucleotides in length but this is processed during germination by shortening of the poly(A) tail to about 600 nucleotides at 1 to 1.5 hours. pLK109 is a member of a multigene family containing three separate genes, and we have isolated and sequenced all of them. All three sequences code for deduced proteins of 127 amino acid residues, with only a few amino acid differences among them. Gene 1 represents the "transcribed" gene, since all 33 cDNAs we isolated are identical with the cDNA pLK109 and the coding region of this gene. Other open reading frames are in close proximity to each of the 109 sequences. About 200 base-pairs 3' to the gene 1 109 sequence is an open reading frame in the opposite orientation. Gene 2 fragment contains a sequence that codes for a protein similar to trypanosome alpha-tubulin 728 base-pairs 5' to the 109 sequence. Gene 3 fragment possesses two additional putative coding regions, one 5' and another 3' to the 109 gene. There is a remarkable similarity between the 5' upstream regions of all three genes. Each possesses a normal Dictyostelium TATA box and the usual T stretch. In addition, there are many other portions of about 400 to 500 base-pairs of the 5' regions that are either identical for long stretches or very similar.

Neuroendocrine carcinoma in the nasal cavity of ten dogs.

Neuroendocrine (NE) carcinoma was diagnosed in 10 dogs. In six cases examined by cephalometric radiography and computerized tomography, a large mass was seen to fill the nasal cavity. Histopathologically, sheets, nests or ribbons of neoplastic cells were separated by delicate or thick fibrovascular stroma. The neoplastic cells were round, oval, or spindle-shaped; cytoplasmic granules and hyperchromatic nuclei with prominent nucleoli were present. Neoplastic cells were invariably immunohistochemically positive for cytokeratin (CK) AE1/AE3, neuron-specific enolase, chromogranin A and vasoactive intestinal polypeptide. Eight dogs were positive for S100 protein, seven for synaptophysin, five for protein gene product 9.5, two for somatostatin, and one for Leu-7. Immunolabelling gave negative results for CK 8, CK 19, calcitonin, calcitonin gene-related polypeptide, neurofilaments, serotonin, gastrin and glial fibrillary acidic protein. Ultrastructurally, the neoplastic cells contained a large number of round, membrane-bounded, densely-cored granules corresponding to neurosecretory granules. These observations were consistent with the neuroendocrine nature of the carcinomas.

The primary Sauve-Kapandji procedure--for treatment of comminuted distal radius and ulnar fractures.

We have performed primary Sauve-Kapandji procedures on four patients with severe open comminuted fractures of both the distal radius and ulna. The fragmented distal ulna was fixed to the sigmoid notch in order to stabilize the ulnar side of the carpus, and a proximal pseudoarthrosis was maintained for forearm rotation. All the distal radial fractures united without major complications. The mean wrist flexion/extension arc was 76 degrees , the mean pronation/supination arc was 135 degrees, and grip strength was 64% of the contralateral side. All patients returned to their work or daily activities within short time period without any additional surgical treatment, except for removal of implants in three patients. The primary Sauve-Kapandji procedure is effective for the reconstruction of severely combined distal radius and ulnar fractures.

Liver microsomal drug metabolism in ethanol-treated hamsters.

Administration of ethanol in drinking water to Syrian golden hamsters for 1-3 weeks caused alterations of microsomal cytochrome P-450-dependent monooxygenase activities in the liver accompanied by a slight elevation in cytochrome P-450 content. Ethanol treatment resulted in an increase in the activities for ethanol oxidation, aniline p-hydroxylation and dimethylnitrosamine N-demethylation. In particular, when dimethylnitrosamine was used as a substrate, the rate of formaldehyde formation was enhanced by 2- to 2.7-fold, while ethanol oxidation and aniline p-hydroxylation were increased by 1.5- to 2- and 1.2- to 1.3-fold, respectively. On the other hand, the activities of 7-ethoxycoumarin O-deethylase, benzphetamine N-demethylase and benzo[a]pyrene 3-hydroxylase were apparently decreased after ethanol treatment. These results for hamsters were significantly different from those reported for rats.

Initiation of fibroin biosynthesis. I. Isolation of nascent fibroin peptides from the posterior silk gland of Bombyx mori.

1. Peptidyl-tRNA was prepared from the posterior silk gland ribosomes of Bombyx mori on the fourth to fifth days of the fifth instar to explore the initiation process in fibroin biosynthesis. 2. The peptidyl-tRNA was hydrolyzed at an alkaline pH and the resulting nascent peptides were fractionated on Sephadex G-75 and Sephadex G-200 columns into twelve fractions. Each fraction was analyzed for amino acid composition. 3. The nascent peptides of smaller molecular size were rather rich in glutamic and aspartic acids. However, the amino acid composition of the nascent peptides gradually approached that of fibroin as their molecular size increased. 4. A comparison between the nascent peptides of smaller molecular size and the small subunit of fibroin was made in respect to amino acid composition and tryptic peptide map. Considerable similarity between these two proteins was observed. The implications of these results in relation to the initiation process in fibroin biosynthesis are discussed.

Pulmonary microsomal cytochrome P-450 from 3-methylcholanthrene-treated hamsters: purification, characterization, and metabolism of benzo(a)pyrene.

A major form of pulmonary cytochrome P-450 (pulmonary P-450MC) was purified approximately 165-fold from lung microsomes of 3-methylcholanthrene (MC)-treated hamsters. The purified preparation contained 14.2 nmol of cytochrome P-450 (P-450) per mg protein and was essentially free from NADPH-cytochrome P-450 (cytochrome c)-reductase (NADPH-reductase) and epoxide hydrolase. Pulmonary P-450MC exhibits an absorption maximum at 446.5 nm in the difference spectrum of reduced hemoprotein-CO complex, and a low-spin state of ferric iron in the heme. By sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, the molecular weight of pulmonary P-450MC was estimated to be 56,000. In a reconstituted system, pulmonary P-450MC efficiently catalyzed benzo(a)pyrene (BP) hydroxylation, but showed low activities for 7-ethoxycoumarin O-deethylation and benzphetamine N-demethylation. In Ouchterlony double diffusion analysis, hamster pulmonary P-450MC reacted to the antibody prepared against rat hepatic P-450MC to form a faint precipitation line with a spur, indicating that the two P-450MCs have a common antigenic site but are not immunologically identical. When incubated with [14C]BP in a reconstituted system containing NADPH-reductase and epoxide hydrolase, hamster pulmonary P-450MC formed much higher amounts of BP diols, especially 7,8-diol, than were formed by rat pulmonary P-450MC.

Hamster cytochrome P-450 IA gene family, P-450IA1 and P-450IA2 in lung and liver: cDNA cloning and sequence analysis.

Two cDNA clones, 2C19 and 4C1, were isolated from a lung cDNA library of 3-methylcholanthrene (MC)-treated hamster by using rat P-450c cDNA as a probe. The cDNA determined from 2C19 and 4C1 was 2,916 bp long and contained an entire coding region for 524 amino acids with a molecular weight of 59,408. The deduced amino acid sequence showed a 85% identity with that of rat P-450c indicating 2C19 and 4C1 encode the hamster P-450IA1 protein. Another cDNA clone, designated H28, was isolated from a MC-induced hamster liver cDNA library by using the hamster lung 2C19 or 4C1 cDNA clone as a probe. H28 was 1,876 bp long and encoded a polypeptide of 513 amino acids with a molecular weight of 58,079. The N-terminal 20 residues deduced from nucleotide sequence of H28 were identical to those determined by sequence analysis of purified hamster hepatic P-450MCI. The high similarity of the nucleotide and deduced amino acid sequences between H28 and P-450IA2 of other species indicated that H28 encoded a P-450 protein which belongs to the P-450IA2 family. Northern blot analysis revealed that the mRNAs for hamster P-450IA1 and IA2 were about 2.9 and 1.9 kb long, respectively. Hamster P-450IA1 mRNA was induced to the same level in lungs as in livers by MC treatment, whereas hamster P-450IA2 mRNA was induced and expressed only in hamster liver.

Purification and characterization of cytochromes P-450 from liver microsomes of 3-methylcholanthrene-treated crab-eating monkeys.

Two forms of cytochrome P-450 (P-450MC1 and P-450MC2) were purified from liver microsomes of crab-eating monkeys (Macaca irus) treated with 3-methylcholanthrene (MC). Monkey P-450MC1 preparation had a specific content of 14.0 nmol/mg protein and showed a main protein band with a minimum molecular weight of 52,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Monkey P-450MC2 preparation had a specific content of 12.1 nmol/mg protein and a minimum molecular weight of 54,000. The carbon monoxide-reduced difference spectral peaks of monkey P-450MC1 and P-450MC2 were at 448 and 447 nm, respectively. In the reconstituted system, monkey P-450MC2 had high activities for benzo[a]pyrene 3-hydroxylation and 7-ethoxycoumarin O-deethylation. Monkey P-450MC1 had low activities toward these two substrates and a high activity for benzphetamine N-demethylation. Monkey P-450MC1 and P-450MC2 were detected by immunoblotting using an antibody prepared against rat cytochrome P-450c, which is a major form of cytochrome P-450 in liver microsomes of MC-treated rats. These results suggested that the molecular properties of cytochrome P-450 in liver microsomes of crab-eating monkeys treated with MC are similar to those in rats.

Cytochrome P-450 system dependent depression of delta-aminolevulinic acid dehydratase activity by bromobenzene in rats.

Male Wistar rats (200-230 g) were treated with bromobenzene in soybean oil intraperitoneally (i.p.) (4 mmol/kg) once a day for 1 or 2 days while control rats received soybean oil alone. delta-Aminolevulinic acid dehydratase (ALA-D) activity was depressed to 80% and 43% in bone marrow after 24 h and 48 h, respectively. ALA-D activity was also depressed significantly in the liver after the administration of bromobenzene while the activity in peripheral erythrocytes was not altered. After the administration of bromobenzene, the concentration of reduced non-protein sulfhydryls in liver was the lowest at 24 h and increased thereafter. No significant change was observed in the activity of delta-aminolevulinate synthase in liver. The decrease of ALA-D activity was also reproducible in vitro. The 105 000 g supernatant fractions of rat bone marrow lyzates as ALA-D source were incubated with liver microsomes prepared from rats treated with phenobarbital. ALA-D activity was decreased by bromobenzene but no decrease was observed when the microsomes were preincubated with CO to inhibit cytochrome P-450. The effect of bromobenzene on ALA-D purified from rat erythroid cells was studied in incubations containing a reconstituted cytochrome P-450 system prepared from rat liver. The decrease of ALA-D activity was proportional to both the incubation time and to the concentration of P-450 while no decrease was detected when P-450 was inhibited by CO before the incubation.

The effect of immunosuppressive agents (FK-506, rapamycin) on renal P450 systems in rat models.

It is well known that cyclosporin, rapamycin and FK-506 (tacrolimus) are metabolized by the liver microsomal cytochrome P450 enzyme system. Although there have been reports of interaction between these drugs and the renal P450 enzyme system, differences among these immunosuppressants has not been comprehensively demonstrated. We have studied the individual capacities of these immunosuppressants to induce renal microsomal P450 enzymes similar to CYP2B4 and CYP4A2 by examining renal function in treated rats, and have correlated the results by means of biochemical, immunological and immunohistochemical assays of renal P450 enzymes. Cyclosporin caused impairment of renal function with an increase in renal-specific P450 content, but FK-506 and rapamycin did not. Laurate omega- and (omega-1)-hydroxylase activity increased in rats treated with rapamycin but decreased in those treated with FK-506. Prostaglandin A1 (PGA1) omega-hydroxylase activity increased in rats treated with FK-506 but was reduced by treatment with cyclosporin. Aminopyrine N-demethylase activity increased in rats treated with cyclosporin or FK-506, but not in those treated with rapamycin. Western-blot analysis revealed significant induction of P450, (similar to CYP2B4 of the rabbit P450 isozyme) in kidneys from rats treated with cyclosporin but not in those from rats receiving FK-506 or rapamycin. Histochemical studies clearly demonstrated a form of P450 such as CYP4A2 in the proximal tubules of rats treated with cyclosporin, but not in those of rats treated with FK-506 or rapamycin. These results show that although cyclosporin has a strong effect on renal P450 systems and induces such a system in kidney cortex (microsomal P450), FK-506 and rapamycin have no substantial effect on the induction of renal P450. These findings might clarify the nephrotoxicity induced by these immunosuppressive drugs.

Calcifying epithelial odontogenic tumours in small domesticated carnivores: histological, immunohistochemical and electron microscopical studies.

Histological, immunohistochemical and electron microscopical studies revealed one feline and four canine calcifying epithelial odontogenic tumours in 115 oral tumours over a 10-year period. The tumours consisted of islands and sheets of odontogenic epithelium of varying size within a stroma of fibrous connective tissues. The tumour cells were pleomorphic with variable amounts of eosinophilic cytoplasm and large hyperchromatic, polymorphic nuclei with prominent nucleoli. Clusters of keratinized tumour cells ("shadow cells") were frequently seen within the islands and sheets. The multiple spherules of homogeneous eosinophilic material stained positively with Congo red and Dylon stains and produced an apple green birefringence under polarization microscopy, indicative of amyloid. Mineralized foci were scattered throughout the tumour masses and in the homogeneous spherules. Immunohistochemically, the tumour cells reacted with anti-human keratin antibody, but not with anti-human vimentin or anti-chicken desmin antibodies. The homogeneous spherules did not react with anti-human keratin, anti-human vimentin, anti-chicken desmin, anti-amyloid A, anti-laminin or anti-human collagen (type I, III, IV) antibodies. Ultrastructurally, the cytoplasm of tumour cells was abundant and contained a large number of electron-dense bundles of tonofilaments. The homogeneous spherules consisted of fine filaments measuring about 10-12 nm in diameter.