Transglutaminase 1-deficient recessive lamellar ichthyosis associated with a LINE-1 insertion in Jack Russell terrier dogs.

BACKGROUND: Congenital, nonepidermolytic cornification disorders phenotypically resembling human autosomal recessive ichthyosis have been described in purebred dog breeds, including Jack Russell terrier (JRT) dogs. One cause of gene mutation important to humans and dogs is transposon insertions. OBJECTIVES: To describe an autosomal recessive, severe nonepidermolytic ichthyosis resembling lamellar ichthyosis (LI) in JRT dogs due to insertion of a long interspersed nucleotide element (LINE-1) in the transglutaminase 1 (TGM1) gene. METHODS: Dogs were evaluated clinically, and skin samples were examined by light and electron microscopy. Phenotypic information and genotyping with a canine microsatellite marker suggested TGM1 to be a candidate gene. Genomic DNA samples and cDNA generated from epidermal RNA were examined. Consequences of the mutation were evaluated by Western blotting, quantitative reverse transcription-polymerase chain reaction (RT-PCR) and enzyme activity from cultured keratinocytes. RESULTS: Affected dogs had generalized severe hyperkeratosis. Histological examination defined laminated to compact hyperkeratosis without epidermolysis; ultrastructurally, cornified envelopes were thin. Affected dogs were homozygous for a 1980-bp insertion within intron 9 of TGM1. The sequence of the insertion was that of a canine LINE-1 element. Quantitative RT-PCR indicated a significant decrease in TGM1 mRNA in affected dogs compared with wild-type. TGM1 protein was markedly decreased on immunoblotting, and membrane-associated enzyme activity was diminished in affected dogs. CONCLUSIONS: Based on morphological and molecular features, this disease is homologous with TGM1-deficient LI in humans, clinically models LI better than the genetically modified mouse and represents its first spontaneous animal model. This is the first reported form of LI due to transposon insertion.

Activation of the redox-regulated molecular chaperone Hsp33--a two-step mechanism.

BACKGROUND: Hsp33 is a novel redox-regulated molecular chaperone. Hsp33 is present in the reducing environment of the cytosol and is, under normal conditions, inactive. The four highly conserved cysteines found in Hsp33 constitute a novel zinc binding motif. Upon exposure to oxidative stress, Hsp33's chaperone activity is turned on. This activation process is initiated by the formation of two intramolecular disulfide bonds. Recently, the 2.2 A crystal structure of Hsp33 has been solved, revealing that Hsp33 is present as a dimer in the structure (Vijayalakshmi et al., this issue, 367-375 [1]). RESULTS: We show here that oxidized, highly active Hsp33 is a dimer in solution. In contrast, reduced and inactive Hsp33 is monomeric. The incubation of reduced Hsp33 in H(2)O(2) leads to the simultaneous formation of two intramolecular disulfide bonds and the concomitant release of zinc. This concentration-independent step is followed by a concentration-dependent association reaction. The dimerization of Hsp33 requires highly temperature-sensitive structural rearrangements. This allows Hsp33's activation process to be greatly accelerated at heat shock temperatures. CONCLUSIONS: The regulation of Hsp33's chaperone function is highly sophisticated. On a transcriptional level, Hsp33 is under heat shock control. This increases the concentration of Hsp33 under heat and oxidative stress, a process that favors dimerization, a critical step in Hsp33's activation reaction. On a posttranslational level, Hsp33 is redox regulated. Dimerization of disulfide-bonded Hsp33 monomers leads to the formation of two extended, putative substrate binding sites. These sites might explain Hsp33's high and promiscuous affinity for unstructured protein folding intermediates.

The effects of thyroid hormones on the skin of beagle dogs.

The effects of hypothyroidism on canine skin were determined by comparing morphologic, morphometric, and hair cycle differences in skin biopsy samples from 3 groups of age- and gender-matched Beagle dogs: (1) euthyroid dogs; (2) dogs made hypothyroid by administration of 131I; and (3) dogs made hypothyroid and maintained in a euthyroid state by treatment with synthetic thyroxine. After 10 months of observation, there was slower regrowth of hair 2 months after clipping in the untreated-hypothyroid dogs. Untreated-hypothyroid dogs had a greater number of follicles in telogen and fewer hair shafts (ie, a greater number of hairless telogen follicles) than did the control group. The control dogs had a greater number of telogen follicles but the same number of hair shafts as the treated-hypothyroid group. Treated-hypothyroid dogs had the greatest number of follicles in the growing stage of the hair cycle (anagen). This study suggests that, at least in Beagles, induced hypothyroidism does not affect the pelage as dramatically as has been described in naturally occurring disease. This is because normal Beagles retain hair shafts in follicles for long periods, and the alopecia of hypothyroidism appears to evolve slowly because of the prolongation of this haired telogen stage. The evaluation of thyroxine-treated hypothyroid dogs demonstrates that thyroid hormone supplementation of Beagle dogs with induced hypothyroidism stimulates hair growth.

Characterization of a new human diploid myeloid leukemia cell line (PLB-985) with granulocytic and monocytic differentiating capacity.

A new human diploid cell line, designated PLB-985, has been established from the peripheral blood of a patient with acute nonlymphocytic leukemia (ANLL). Cells of this line are capable of granulocytic and monocytic maturation in the presence of inducing agents. By morphology, the analysis of surface antigens, and cytochemical staining PLB-985 cells are myelomonoblasts. Transmission electron microscopy reveals them to be devoid of neutrophilic primary or secondary granules and to have an open chromatin pattern with frequent nucleoli. The modal karyotype of the line is 46,XX, with no consistent marker chromosomes or recognizable translocations. Myelomonoblasts of this line form colonies in soft agar and induce tumors (chloromas) in nude mice. Growth of the cells in the presence of dimethyl sulfoxide, cis-retinoic acid, or dibutyryl cyclic adenosine monophosphate results in granulocytic maturation as determined by morphology, histochemical staining characteristics, and incorporation of 35S-methionine into the neutrophil primary granule proteinases elastase and cathepsin G. The tumor-promoting phorbol ester phorbol myristate acetate induces PLB-985 cells to differentiate as monocytes. Cells grown in the presence of this agent rapidly become adherent to plastic, display markedly increased phagocytosis of latex particles, stain positively for alpha-naphthyl acetate esterase, and lose the ability to synthesize the neutrophilic proteinases. Induction of differentiation along either pathway is accompanied by a marked decrease in myc oncogene transcription.

Large-capacity separation of malignant cells and lymphocytes from the Furth mast cell tumor in a reorienting zonal rotor.

The Furth murine mastocytoma was adapted to the ascitic form and separated into fractions enriched with respect to lymphocytes and malignant cells by velocity sedimentation in the SZ-14 reorienting zonal rotor or in the isokinetic gradient. Lymphocytes were more highly purified (p less than 0.01) in the isokinetic gradient than in the zonal rotor, i.e., lymphocytes comprised 99.1% of the nucleated cells in the purest fraction from the isokinetic gradient and 80.1% of the nucleated cells in the purest fraction from the zonal rotor. Neoplastic mast cells were similarly purified by the two methods; they comprised 67.7 and 78.5% of the nucleated cells in the purest fractions from the isokinetic gradient and zonal rotor, respectively. Up to 160 million tumor cells can be purified in a single step with the reorienting zonal rotor, whereas 30 to 40 million cells per gradient approach the limit of the isokinetic gradient. After centrifugation in the zonal rotor, recovery was 85.6 +/- 12% (S.D.) of the cells layered over the gradient; and the separated tumor cells retained their ability to form tumors when transplanted into mice. The separation of large numbers of lymphocytes and malignant cells from the same tumor in the SZ-14 rotor should aid in the biochemical and immunological characterization of cancer.