Purification and Characterization of a Population of EGFP-Expressing Cells from the Developing Pancreas of a Neurogenin3/EGFP Transgenic Mouse.

Neurogenin 3 (ngn3) is a basic helix loop helix transcription factor that is transiently expressed in the developing mouse pancreas with peak expression around E15. In mice lacking the ngn3 gene the endocrine cells of the pancreas fail to develop suggesting that the ngn3-positive cell may represent a progenitor cell for the endocrine pancreas. In order to purify and characterize this cell in detail we have generated a transgenic mouse, in which the ngn3 promoter drives expression of enhanced green fluorescent protein (EGFP). In the E15.5 embryo EGFP was expressed in the dorsal and ventral pancreas, the duodenum, and lower intestine as well as in the brain. This pattern of expression was in keeping with the known expression profile of the endogenous ngn3 gene. Within the pancreas EGFP was localized in close proximity to cells that stained positive for ngn3, insulin, and glucagon, but was absent from regions of the pancreas that stained positive for amylase. EGFP was also present in the pancreas at E18.5, although there was no detectable expression of ngn3. At this stage EGFP did not colocalize with any of the hormones or exocrine markers. EGFP(+) cells were FACS purified (96%) from the E15 pancreas yielding approximately 10,000 cells or 1.6% of the total pancreatic cells from one litter. RT/PCR analysis confirmed that the purified cells expressed EGFP, ngn3, insulin, glucagon, somatostatin and pancreatic polypeptide. The ability to purify ngn3(+) cells provides an invaluable source of material for charactering in detail their properties.

Enhanced expression of a furin-cleavable proinsulin.

Cell engineering or gene therapy may represent an alternative to current methods of treating diabetes mellitus. Cells could be engineered to secrete insulin ex vivo for transplantation or the insulin gene could be administered directly by injection into muscle. A problem has been that non-neuroendocrine cells lack the endoproteases (PC3/1 and PC2) that are responsible for the processing of proinsulin to insulin. This can be surmounted by engineering the paired basic amino acid processing sites within proinsulin to sites that would be recognized by the ubiquitously expressed protease, furin. However, in every study to date, the expression of the furin-cleavable construct was greatly reduced relative to that of the unmodified proinsulin construct. We investigated possible causes for this, including mRNA stability, the presence of additional CpG islands, and the amino acid substitutions within furin-cleavable proinsulin. Several furin-cleavable rat proinsulin I cDNAs were engineered and used to transfect human HEK293, rat L6 and mouse C(2)C(12) cell lines. The stability of wild-type and furin-cleavable proinsulin mRNA in transfected C(2)C(12) cells was measured by RT-PCR. Comparison of the decay rates in the presence of actinomycin D showed no significant difference between the two species of mRNA. A furin-cleavable proinsulin cDNA was created to contain the same distribution of CpG islands as wild-type proinsulin. Comparison of insulin-like immunoreactivity in all three cell lines transfected with either this construct or a widely used furin-cleavable proinsulin containing additional CpG islands showed that the presence of the extra CpG islands had no effect. Studies to examine amino acid substitutions used to create furin consensus sequences showed that the addition of basic residues at the C-peptide/A-chain junction was responsible for the reduced production of furin-cleavable proinsulin. Using this information, we engineered a cDNA for furin-cleavable rat proinsulin I that was efficiently processed to mature insulin and expressed at the same level as wild-type proinsulin.

Para-aural abscessation following traumatic ear canal separation in a dog.

Traumatic ear canal separation is rare in animals, with only eight dogs and one cat reported with the condition in the English language literature. Para-aural abscessation occurred in six of these nine animals. Diagnosis was made on otoscopic observation of a shortened, abruptly ending external ear canal that was free from advanced disease. Radiographs in those cases which have been described showed a disruption of the normal air opacity of the affected ear canal. Drainage, by creating a separate opening for the horizontal ear canal, or total ear canal ablation and lateral bulla osteotomy (TECA/LBO), have led to resolution of the clinical signs. This report adds a further case to the literature in which TECA/LBO was employed successfully.

Tearing of the dura mater in three dogs.

Tearing of the thoracolumbar dura mater was diagnosed after myelography in three large breed dogs. All three dogs lost the ability to walk suddenly, after a period of vigorous running or struggling. Radiographic changes suggested that the tearing of the dura was associated with intervertebral disc injury in two dogs. These dogs regained the ability to walk after medical treatment. Postmortem findings suggested that the tearing of the dura mater in the third dog was associated with nerve root injury and spinal cord haemorrhage after an episode of violent struggling.

Idiopathic multifocal osteopathy in four Scottish terriers (1991-1996).

This case series describes four Scottish terriers with an osteopathic condition, characterized by multifocal absence of bone in the skull, cervical spine, and proximal radii, ulnae, and femora. All dogs were affected clinically; two dogs were euthanized due to progression of the disease, one died acutely, and one was euthanized for an oral melanoma. Histopathology in one case was characterized by osteoclastic osteolysis and replacement of bone with fibrous tissue. This disease has some characteristics of human osteolysis syndromes. Three of the dogs were related through pedigree analysis, and the pedigree of the other dog was not available. The name, idiopathic multifocal osteopathy, is used to describe a new disease in dogs, found particularly in Scottish terriers.

Synovial fluid interleukin 6, tumor necrosis factor, and nitric oxide values in dogs with osteoarthritis secondary to cranial cruciate ligament rupture.

OBJECTIVE: To measure and compare values of interleukin 6 (IL-6), tumor necrosis factor (TNF), and nitric oxide (NO) metabolites in synovial fluid from canine joints with osteoarthritis (OA) secondary to naturally acquired cranial cruciate ligament (CCL) rupture and experimental CCL transection. ANIMALS: 57 dogs (clinical group) with OA secondary to CCL rupture; 5 dogs (experimental group) with OA secondary to CCL transection; 19 control dogs with normal joints. PROCEDURE: Joints were radiographed and graded for seventy of OA. Synovial fluid was collected from dogs: at surgery from the clinical group, at 90 days after surgery from the experimental group, and at necropsy from the control group. Activities of IL-6 and TNF, as well as concentration of the NO metabolites (NO2-/NO3-) were measured, and results were reported as mean +/- SEM. RESULTS: IL-6 activity in dogs of the clinical (290 +/- 40 U/ml) and experimental (494 +/- 165 U/ml) groups was greater than that in control dogs (6 +/- 1.6 U/ml; P < 0.05). The TNF values in dogs of the clinical (3.0 +/- 0.5 pg/ml) and experimental (2.0 +/- 0.9 pg/ml) groups were lower than those in control dogs (8.6 +/- 2.3 pg/ml; P < 0.05). The IL-6 values were negatively associated with radiographic score of OA and were positively associated with age (R2 = 26.5%, P < 0.05). CONCLUSION: Dogs with OA secondary to naturally acquired CCL rupture and experimental CCL transection had significantly different alterations in synovial fluid IL-6 and TNF values. The decrease in IL-6 activity with advancing OA was independent of the increase in IL-6 activity with aging. CLINICAL RELEVANCE: IL-6 and TNF may be involved in pathogenesis of OA secondary to naturally acquired and experimentally induced CCL rupture.

Use of the Wagner apparatus to lengthen the antebrachium of a growing dog.

A six-month-old male golden retriever was presented with a shortened radius and ulna and valgus deformity of the carpus secondary to premature closure of the distal radial and ulnar physes. Osteotomies of the radius and ulna were performed and the antebrachium was lengthened over eight weeks using a Wagner apparatus. This external fixation distractor is used in humans for limb-lengthening procedures. In this case, it was used to correct the angular deformity and to lengthen the limb.

Isolation of a human von Willebrand factor cDNA from the hybrid endothelial cell line EA.hy926.

A permanent human hybrid endothelial cell line (EA.hy926) was shown to produce the von Willebrand factor, a protein of 250,000 relative mass (Mr) which was secreted into the medium as a 220,000 Mr protein. A cDNA library was constructed in lambda gt11 using mRNA from these hybrid cells. Several von Willebrand factor cDNA clones were isolated from this library using a synthetic oligodeoxyribonucleotide as a hybridization probe. These cDNA clones were used to analyze the von Willebrand factor gene in normal individuals and in cultured cells.

Use of a BamHI polymorphism in the factor IX gene for the determination of hemophilia B carrier status.

A BamHI polymorphism has been identified in the human factor IX gene. This polymorphism, which occurs in approximately 6% of X chromosomes, has been used to determine the carrier status of a female in a family with a history of hemophilia B. This family was uninformative for the previously reported TaqI and Xmnl polymorphisms in the factor IX gene.

Characterization of an almost full-length cDNA coding for human blood coagulation factor X.

A human liver cDNA library was screened by colony hybridization with a bovine factor X cDNA probe. Three of the positive plasmids contained overlapping DNA that coded for most of human factor X mRNA. DNA sequence analysis of these three clones allowed the prediction of the complete amino acid sequence of plasma factor X. From these studies, we predict that human factor X is synthesized as a single polypeptide chain precursor in which the light and heavy chains of plasma factor X are linked by the tripeptide Arg-Lys-Arg. The cDNA sequence also predicts that human factor X is synthesized as a preproprotein having an amino-terminal leader peptide of at least 28 amino acid residues. A comparison of the amino acid sequences of human and bovine factor X shows high sequence identity around the calcium-binding regions and catalytic regions but low sequence identity around the nonfunctional regions.

Histone deacetylase from HeLa cells: properties of the high molecular weight complex.

In previous work [Hay, C. W., & Candido, E. P. M. (1983) J. Biol. Chem. 258, 3726-3734], we have shown that the histone deacetylase from HeLa cell nuclei is associated with a high molecular weight, nuclease-resistant complex. This complex was found to contain a variety of non-histone proteins, and indirect evidence for the importance of protein-protein interactions in the maintenance of its structure was obtained. In the present report, we examine the effects of beta-mercaptoethanol and neocuproine on the deacetylase complex and present data on the level of histone acetylation and the presence of satellite DNA sequences in this material. HeLa cell histone deacetylase complex partially dissociates in 10 mM beta-mercaptoethanol, resulting in a loss of non-histone proteins. The presence of 10 mM beta-mercaptoethanol during the micrococcal nuclease digestion of HeLa cell nuclei results in a greatly reduced yield of histone deacetylase complex, with a correspondingly large increase in the production of small oligonucleosomes and mononucleosomes. Histone deacetylase activity on endogenous labeled histone within the complex is strongly inhibited by either 1 or 10 mM beta-mercaptoethanol or 3 mM neocuproine. This loss of histone deacetylase activity does not seem to be due to an inactivation of the enzyme but appears to be a consequence of the disruption of the structure of the deacetylase complex itself. Histone H4 in the deacetylase complex prepared from HeLa cell nuclei by micrococcal nuclease digestion was more highly acetylated than H4 in bulk nucleosomes. Restriction enzyme analysis of the DNA associated with the histone deacetylase complex revealed neither an enrichment nor a depletion of major satellite sequences in this material.

Histone deacetylase. Association with a nuclease resistant, high molecular weight fraction of HeLa cell chromatin.

The chromatin-bound histone deacetylase of HeLa cells has been studied using endogenous [3H]acetyl-labeled polynucleosomes containing the enzyme, prepared in the presence of 40 mM butyrate. Histone deacetylase was assayed upon removal of the butyrate, and it was found that active enzyme is found only in association with a high molecular weight complex. This deacetylase-containing complex is relatively resistant to digestion with micrococcal nuclease. No activity is found on mononucleosomes or oligonucleosomes. Up to 90% of labeled acetyl groups are removed from histone deacetylase complexes incubated in the absence of butyrate. Free histones are a poor substrate under these conditions, but histones in mononucleosomes are deacetylated when they are incubated with histone deacetylase complex. Histone deacetylase remains bound to this complex in 1-2 M NaCl and does not dissociate from it during its reaction with acetylated core histones. Under typical nuclease digestion conditions, the histone deacetylase complex contains DNA with a size distribution of 5-11 kilobase pairs and a variety of nonhistone proteins. Comparison of the protein composition of histone deacetylase complexes with that of nuclear matrix preparations shows some similarities. Taken together, the results on the chromatographic behavior, the DNA fragment sizes, and the protein composition of the deacetylase complex suggest that protein-protein interactions may be important in maintaining its structure and also in the binding of the deacetylase itself to the complex.