Pathological features of oxalate nephrosis in a population of koalas (Phascolarctos cinereus) in South Australia.

The wild and captive koala population of the Mt Lofty Ranges in South Australia has a high level of renal dysfunction in which crystals consistent with calcium oxalate have been observed in the kidneys. This study aimed to describe the pathological features of the renal disease in this population, confirm the composition of renal crystals as calcium oxalate, and determine whether any age or sex predispositions exist for this disease. A total of 51 koalas (28 wild rescues, 23 captive) were examined at necropsy, of which 28 (55%) were found to have gross and/or histological evidence of oxalate nephrosis. Histopathological features included intratubular and interstitial inflammation, tubule dilation, glomerular atrophy, tubule loss, and cortical fibrosis. Calcium oxalate crystals were demonstrated using a combination of polarization microscopy, alizarin red S staining, infrared spectroscopy, and energy-dispersive X-ray analysis with scanning electron microscopy. Uric acid and phosphate deposits were also shown to be present but were associated with minimal histopathological changes. No significant differences were found between the numbers of affected captive and wild rescued koalas; also, there were no sex or age predispositions identified, but it was found that oxalate nephrosis may affect koalas <2 years of age. The findings of this study suggest that oxalate nephrosis is a leading disease in this koala population. Possible causes of this disease are currently under investigation.

Regulation of the murine alpha B-crystallin/small heat shock protein gene in cardiac muscle.

The murine alpha B-crystallin/small heat shock protein gene is expressed at high levels in the lens and at lower levels in the heart, skeletal muscle, and numerous other tissues. Previously we have found a skeletal-muscle-preferred enhancer at positions -427 to -259 of the alpha B-crystallin gene containing at least four cis-acting regulatory elements (alpha BE-1, alpha BE-2, alpha BE-3, and MRF, which has an E box). Here we show that in transgenic mice, the alpha B-crystallin enhancer directs the chloramphenicol acetyltransferase reporter gene driven by the alpha B-crystallin promoter specifically to myocardiocytes of the heart. The alpha B-crystallin enhancer was active in conjugation with the herpes simplex virus thymidine kinase promoter/human growth hormone reporter gene in transfected rat myocardiocytes. DNase I footprinting and site-specific mutagenesis experiments showed that alpha BE-1, alpha BE-2, alpha BE-3, MRF, and a novel, heart-specific element called alpha BE-4 are required for alpha B-crystallin enhancer activity in transfected myocardiocytes. By contrast, alpha BE-4 is not utilized for enhancer activity in transfected lens or skeletal muscle cell lines. Alpha BE-4 contains an overlapping heat shock sequence and a reverse CArG box [5'-GG(A/T)6CC-3']. Electrophoretic mobility shift assays with an antibody to serum response factor and a CArG-box-competing sequence from the c-fos promoter indicated that a cardiac-specific protein with DNA-binding and antigenic similarities to serum response factor binds to alpha BE-4 via the reverse CArG box; electrophoretic mobility shift assays and antibody experiments with anti-USF antiserum and heart nuclear extract also raised the possibility that the MRF E box utilizes USF or an antigenically related protein. We conclude that the activity of the alpha B-crystallin enhancer in the heart utilizes a reverse CArG box and an E-box-dependent pathway.

Dual roles for Pax-6: a transcriptional repressor of lens fiber cell-specific beta-crystallin genes.

It has been demonstrated previously that Pax-6, a paired domain (PD)/homeodomain (HD) transcription factor critical for eye development, contributes to the activation of the alphaB-, alphaA-, delta1-, and zeta-crystallin genes in the lens. Here we have examined the possibility that the inverse relationship between the expression of Pax-6 and beta-crystallin genes within the developing chicken lens reflects a negative regulatory role of Pax-6. Cotransfection of a plasmid containing the betaB1-crystallin promoter fused to the chloramphenicol acetyltransferase reporter gene and a plasmid containing the full-length mouse Pax-6 coding sequences into primary embryonic chicken lens epithelial cells or fibroblasts repressed the activity of this promoter by as much as 90%. Pax-6 constructs lacking the C-terminal activation domain repressed betaB1-crystallin promoter activity as effectively as the full-length protein, but the PD alone or Pax-6 (5a), a splice variant with an altered PD affecting its DNA binding specificity, did not. DNase footprinting analysis revealed that truncated Pax-6 (PD+HD) binds to three regions (-183 to -152, -120 to -48, and -30 to +1) of the betaB1-crystallin promoter. Earlier experiments showed that the betaB1-crystallin promoter sequence from -120 to -48 contains a cis element (PL2 at -90 to -76) that stimulates the activity of a heterologous promoter in lens cells but not in fibroblasts. In the present study, we show by electrophoretic mobility shift assay and cotransfection that Pax-6 binds to PL2 and represses its ability to activate promoter activity; moreover, mutation of PL2 eliminated binding by Pax-6. Taken together, our data indicate that Pax-6 (via its PD and HD) represses the betaB1-crystallin promoter by direct interaction with the PL2 element. We thus suggest that the relatively high concentration of Pax-6 contributes to the absence of betaB1-crystallin gene expression in lens epithelial cells and that diminishing amounts of Pax-6 in lens fiber cells during development allow activation of this gene.

Lens-preferred activity of the -1809/+46 mouse alpha A-crystallin promoter in stably integrated chromatin.

The alpha A-crystallin gene is expressed in a highly lens preferred manner. Here we show that the mouse alpha A-crystallin -1809/+46 promoter fragment displays lens-preferred activity in transgenic mice and in stably transfected lens cells. These findings are in contrast to the lack of activity of this promoter previously reported in transiently transfected lens cells. Our current findings suggest that the -1809/+46 mouse alpha A-crystallin promoter functions in a lens preferred manner when stably integrated into chromatin.

The chicken beta A4- and beta B1-crystallin-encoding genes are tightly linked.

Analysis of the 5' flanking region of the chicken beta B1-crystallin-encoding gene (beta B1-cry) revealed regions of sequence homology with the bovine beta A4-crystallin-encoding gene (beta A4-cry). Subsequently, the chicken beta A4-cry cDNA sequence was determined, and it was demonstrated that beta A4- and beta B1-cry are linked head to head in the chicken chromosome with 2147 nucleotides (nt) of intergenic spacer. Chicken beta A4-cry contains six exons, with the first exon being noncoding. Chicken beta A4-cry is the smallest beta-cry ever described, due to the small size of its introns which range in length from 68 to 96 nt. While three polymorphisms were noted between some cDNA clones and the genomic sequence, Southern blot analysis demonstrated that beta A4-cry exists as a single copy in the chicken genome. Northern blot analysis indicated that beta A4-cry is a lens-specific transcript which is expressed at higher levels in the embryo than in the adult. The beta A4-cry mRNA is present at 400-fold lower levels than the beta B1-cry mRNA in the 14-day embryonic chicken lens, and at 2000-fold lower levels than the beta B1-cry mRNA in the adult lens. These results are consistent with the idea that the beta-cry family was once clustered in the chromosome as the gamma-cry family is today, and raises the possibility that the relatively low expression of beta A4-cry is mechanistically linked to the high expression of beta B1-cry in the chicken lens.

Differential use of the regulatory elements of the alpha B-crystallin enhancer in cultured murine lung (MLg), lens (alpha TN4-1) and muscle (C2C12) cells.

The mouse alpha B-crystallin-encoding gene (alpha B-cry) is highly expressed in the lens and expressed to lesser extents in other tissues. Here, we investigated alpha B-cry expression in mouse-lung-derived MLg cells. Two sizes of MLg alpha B-cry transcripts comigrated with alpha B-cry transcripts contained in total and poly(A)+RNA from mouse lung, with preference for the larger species in the MLg cells. Expression of both alpha B-cry promoter/cat reporter gene constructs and alpha B-cry enhancer (nt -427/-259)/herpes simplex virus (HSV) thymidine kinase promoter (ptk)/human growth hormone reporter gene (hGH) constructs was studied in transfected MLg cells and the results compared with those obtained from alpha TN4-1 lens and C2C12 muscle cells. The alpha B-cry enhancer increased activity of the endogenous and tk promoters approx. 2-fold in the MLg cells, in contrast to its 3-7-fold effect in alpha TN4-1 cells and 17-20-fold effect in C2C12 myotubes. Site-specific mutagenesis of the previously identified enhancer control elements, alpha B-E-1 (nt -407 to -397), alpha BE-2 (-360 to -327) and MRF (-300 to -288), decreased enhancer strength in transfected MLg cells. DNase I footprinting showed that MLg nuclear proteins occupy only alpha BE-1 and alpha BE-2. Previous data have shown that lens cells use alpha BE-1, alpha BE-2 and alpha BE-3, while muscle cells use, in addition, the muscle regulatory factor-binding site (MRF). Thus, the present experiments correlate tissue-specific enhancer strength and the number of control elements utilized.

Dietary aluminium and renal failure in the koala (Phascolarctos cinereus).

The study investigated the link between the potentially nephrotoxic levels of aluminium ingested in the natural diet of eucalypt leaves by koalas in the Adelaide Hills, South Australia, and the high incidence of renal failure in koalas within this habitat. Routine histology of kidney specimens revealed no pathologies at the light microscopic level and contrasted sharply with the clinical signs of renal failure. However staining with solochrome azurine and Perl's Prussian blue showed aluminium was present in some proximal convoluted tubules in all specimens. Aluminium was also found in bone samples. The presence of aluminium in bone and kidney tissues was confirmed using electron dispersive x-ray analysis with transmission and scanning electron microscopy. Ultrastructural changes, including a decrease in lysosomal numbers, were seen in proximal convoluted tubules and these changes were shown to coincide with the presence of aluminium. No aluminium was found in koalas that died from causes other than renal failure. It was concluded that renal failure in the koalas of the Adelaide Hills is characterised by the presence of aluminium in the kidneys and bone and it is probably related to the high levels of aluminium in their restricted diet of eucalypt leaves. However, it is not known if the presence of aluminium is the cause or effect of the renal failure. The study is the first account where aluminium ingested as part of the natural diet of mammals has been shown to accumulate in the animal and be implicated with nephrotoxicity.

GM crops and the rat digestive tract: a critical review.

The aim of this review is to examine the relationship between genetically modified (GM) crops and health, based on histopathological investigations of the digestive tract in rats. We reviewed published long-term feeding studies of crops containing one or more of three specific traits: herbicide tolerance via the EPSPS gene and insect resistance via cry1Ab or cry3Bb1 genes. These genes are commonly found in commercialised GM crops. Our search found 21 studies for nine (19%) out of the 47 crops approved for human and/or animal consumption. We could find no studies on the other 38 (81%) approved crops. Fourteen out of the 21 studies (67%) were general health assessments of the GM crop on rat health. Most of these studies (76%) were performed after the crop had been approved for human and/or animal consumption, with half of these being published at least nine years after approval. Our review also discovered an inconsistency in methodology and a lack of defined criteria for outcomes that would be considered toxicologically or pathologically significant. In addition, there was a lack of transparency in the methods and results, which made comparisons between the studies difficult. The evidence reviewed here demonstrates an incomplete picture regarding the toxicity (and safety) of GM products consumed by humans and animals. Therefore, each GM product should be assessed on merit, with appropriate studies performed to indicate the level of safety associated with them. Detailed guidelines should be developed which will allow for the generation of comparable and reproducible studies. This will establish a foundation for evidence-based guidelines, to better determine if GM food is safe for human and animal consumption.

Parathyroid morphology of the brush-tail possum, Trichosurus vulpecula.

BACKGROUND: Reviews of the comparative anatomy of the parathyroid glands contain very little or no information on this endocrine gland in marsupials. This paper is the initial report of a broader investigation of the parathyroid glands in marsupials and monotremes. METHODS: Nineteen possums (Trichosurus vulpecula) were used in the study of the anatomy, histology, and ultrastructure of the parathyroid glands. Tissue sections were examined using routine light and electron microscopic techniques. RESULTS: Parathyroid III is larger than parathyroid IV. The former occurs in the vicinity of the carotid bifurcation; the latter is found in the mediastinum, and in 67% of samples it was observed to be associated with thymic tissue. In parathyroid III specimens, 12.5% had follicles, 25% had cysts, and 16% contained thymic tissue. Ultrastructurally, the majority of specimens consisted of principal cells; no oxyphil cells were seen. Light and dark cells were only seen in glands that had been fixed by perfusion. Junctions between thymic and parathyroid tissues showed intervening epithelial reticular cells, but a basal lamina was not always apparent. In three specimens, cells that resembled water-clear cells were present. It is proposed that these unusual cells may reflect a state of impaired health in these possums. CONCLUSIONS: Except for the presence of vacuolated cells labelled water-clear cells, the histology of the parathyroid gland in the possum shows many similarities to that in many eutherians.

Parathyroids and ultimobranchial bodies in monotremes.

Only scant information is available in the scientific literature on the parathyroids and ultimobranchial bodies in the primitive mammals, the echidna (Tachyglossus aculeatus) and platypus (Ornithorhynchus anatinus). The major aim of this paper is to describe the morphology of the monotreme parathyroid gland and to compare it with parathyroids in mammals and reptiles. The gross anatomy and light microscopic structure of the ultimobranchial body, thymus, and thyroid are also given. Animals were dissected and routine light and electron microscopic techniques used to examine the microscopic morphology. The locations of parathyroid hormone, calcitonin and calcitonin gene-related peptide in tissue sections were identified by immunostaining. Monotremes have one pair of parathyroid glands located in the thorax and they are often associated with thymic tissue but never with the thyroid which is also present in the mediastinum. Ultimobranchial bodies are ventrolateral to the commencement of the trachea. Thymic lobules with Hassall's corpuscles are scattered in the fibrofatty tissue of the mediastinum and the ventral surface of the pericardium. Histologically, principal cells, water-clear cells, and non-secretory cells were identified in the parathyroid glands. Principal cells showed polarity and had microlamellar projections that formed intercellular canaliculi. Non-secretory cells had features similar to those of thymic epithelial reticular cells. Immunostaining of parathyroid hormone showed a diffuse distribution in parathyroid principal cells and none in ultimobranchial bodies. Identification of the ultimobranchial bodies was confirmed by immunostaining. The monotreme parathyroid gland, ultimobranchial bodies and thyroid show reptilian as well as mammalian features.

Cervical lymph nodes and mast cells in the marsupial Sminthopsis crassicaudata.

A study of the cervical lymph nodes from the fat-tailed dunnart, Smithopsis crassicaudata, revealed the nodes were pigmented with lipofuscin and contained many large cells which were identified as mast cells from their ultrastructure and histochemical staining properties. It is believed that the very high density of mast cells in the cervical lymph nodes contributed to an increase in size of these organs compared to other animals. Very high levels of histamine (90 micrograms/g) were found in the nodes. Cervical lymph nodes with these unusual features were found not only in healthy, umprimed laboratory bred adults, but also in pouch young, wild caught animals, and adults of the closely related species, Sminthopsis macroura. A comparison of the histochemical and ultrastructural characteristics of mast cells from various organs of adult S. crassicaudata was also made. Mast cells from lymph node, skin, tongue, salivary glands, intestinal mucosa, and spleen showed slight variations in staining and structure.

Phosphorylation of the budgerigar fledgling disease virus major capsid protein VP1.

The structural proteins of the budgerigar fledgling disease virus, the first known nonmammalian polyomavirus, were analyzed by isoelectric focusing and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The major capsid protein VP1 was found to be composed of at least five distinct species having isoelectric points ranging from pH 6.45 to 5.85. By analogy with the murine polyomavirus, these species apparently result from different modifications of an initial translation product. Primary chicken embryo cells were infected in the presence of 32Pi to determine whether the virus structural proteins were modified by phosphorylation. SDS-PAGE of the purified virus structural proteins demonstrated that VP1 (along with both minor capsid proteins) was phosphorylated. Two-dimensional analysis of the radiolabeled virus showed phosphorylation of only the two most acidic isoelectric species of VP1, indicating that this posttranslational modification contributes to VP1 species heterogeneity. Phosphoamino acid analysis of 32P-labeled VP1 revealed that phosphoserine is the only phosphoamino acid present in the VP1 protein.

Mutations in the putative calcium-binding domain of polyomavirus VP1 affect capsid assembly.

Calcium ions appear to play a major role in maintaining the structural integrity of the polyomavirus and are likely involved in the processes of viral uncoating and assembly. Previous studies demonstrated that a VP1 fragment extending from Pro-232 to Asp-364 has calcium-binding capabilities. This fragment contains an amino acid stretch from Asp-266 to Glu-277 which is quite similar in sequence to the amino acids that make up the calcium-binding EF hand structures found in many proteins. To assess the contribution of this domain to polyomavirus structural integrity, the effects of mutations in this region were examined by transfecting mutated viral DNA into susceptible cells. Immunofluorescence studies indicated that although viral protein synthesis occurred normally, infective viral progeny were not produced in cells transfected with polyomavirus genomes encoding either a VP1 molecule lacking amino acids Thr-262 through Gly-276 or a VP1 molecule containing a mutation of Asp-266 to Ala. VP1 molecules containing the deletion mutation were unable to bind 45Ca in an in vitro assay. Upon expression in Escherichia coli and purification by immunoaffinity chromatography, wild-type VP1 was isolated as pentameric, capsomere-like structures which could be induced to form capsid-like structures upon addition of CaCl2, consistent with previous studies. However, although VP1 containing the point mutation was isolated as pentamers which were indistinguishable from wild-type VP1 pentamers, addition of CaCl2 did not result in their assembly into capsid-like structures. Immunogold labeling and electron microscopy studies of transfected mammalian cells provided in vivo evidence that a mutation in this region affects the process of viral assembly.

alpha B-crystallin TATA sequence mutations: lens-preference for the proximal TATA box and the distal TATA-like sequence in transgenic mice.

The mouse alpha B-crystallin promoter is active in lens (preferentially), heart and skeletal muscle, and contains a proximal (-28/-22) and distal (-76/-69) TATA sequence. The present investigation explores by site-specific mutagenesis of alpha B-crystallin promoter-chloramphenicol acetyltransferase (cat) reporter gene constructs the function of these two potential TATA boxes in transfected lens cells and transgenic mice. Unexpectedly, mutagenesis of either or both TATA sequences had no effect on promoter activity in transfected lens cells. By contrast, in transgenic mice mutagenesis of the proximal, distal or both TATA sequences preferentially reduced promoter activity in the lens, with minimal effect in the heart or muscle. 5' RACE analysis of lens and muscle RNA of transgenic mice showed that elimination of the proximal TATA box led to transcription initiation at position -48. This upstream initiation site was apparently not due to the utilization of the distal TATA sequence, since the transgene carrying mutations in both TATA sequences also initiated at position -48. The preferential function of the distal TATA sequence in the lens is probably due to the binding of a transcription factor unrelated to transcription initiation, while the preferential lens function of the proximal TATA box appears to involve transcription initiation.

Spatial and temporal activity of the alpha B-crystallin/small heat shock protein gene promoter in transgenic mice.

In order to study the spatial and temporal activity of the mouse alpha B-crystallin/small heat shock gene promoter during embryogenesis, we generated mice harboring a transgene consisting of approximately 4 kbp of alpha B-crystallin promoter sequence fused to the Escherichia coli lacZ reporter gene. beta-galactosidase activity was first observed in the heart rudiment of 8.5 days post coitum (d.p.c.) embryos. An identical expression pattern was obtained for the endogenous alpha B-crystallin gene by whole mount in situ hybridization. At 9.5 d.p.c., beta-galactosidase activity was detected in the lens placode, in the myotome of the somites, in Rathke's pouch (future anterior pituitary), and in some regions of oral ectoderm. We also examined the stress inducibility of the alpha B-crystallin promoter in vivo. Injection of sodium arsenite into mice resulted in increased endogenous alpha B-crystallin expression in the adrenal gland and possibly the liver. Our results indicate that visualization of beta-galactosidase activity provides an accurate reflection of endogenous alpha B-crystallin expression and demonstrate that the complex developmental pattern of mouse alpha B-crystallin gene expression is regulated at the transcriptional level. This expression pattern, coupled with the present literature which addresses functions of the protein, suggests a role for the alpha B-crystallin/small heat shock protein in intermediate filament turnover and cellular remodeling which occur during normal development and differentiation.

Pediatric dental education and community service: a combined approach.

Dental educators nationwide have expressed concern regarding the decreasing pediatric clinical experience available to undergraduate dental students. Some educators have suggested that dental programs should utilize extramural clinics and rotations to enhance current patient pools. This paper presents a successful clinical program that is designed to 1) augment the dental education of predoctoral dental students, and 2) provide dental care for an underserved pediatric dental population in an urban community.

Structure and alternate tissue-preferred transcription initiation of the mouse alpha B-crystallin/small heat shock protein gene.

We have determined the complete nucleotide sequence (-865 to +3515) of the murine alpha B-crystallin/small heat shock protein gene, a major soluble protein of the vertebrate eye lens. Its 3 exon/2 intron structure is identical to that of the rat, hamster and human gene, with the exons being much more conserved than the introns. Previous reports indicated that there are two sizes of alpha B-crystallin mRNA; a larger alpha B-crystallin mRNA predominates in the lung and brain and is also found in low levels in most other tissues (except in lens and liver), while a smaller alpha B-crystallin mRNA exists at a high level in the lens and in variable amounts elsewhere. Sequence analysis suggests that secondary structure in the 5' untranslated sequence of the longer mRNA has led to difficulty in mapping the transcription initiation site of the longer transcript. Here we provide evidence by primer extension, S1 nuclease protection, and PCR (polymerase chain reaction) experiments for a transcription initiation site in the murine lung and brain at position -474. We also detected the utilization of the -474 initiation site in lens and of the +1 site in lung and brain, indicating that the tissue preference for these sites is not absolute. In vitro transcription experiments revealed that cell-free HeLa nuclear extracts specifically initiate transcription at the -474 and +1 sites. alpha B-crystallin was immunocytochemically localized to the bronchioles of the lung. Thus, regulation of alpha B-crystallin/small heat shock protein expression involves the utilization of tissue-preferred transcription initiation sites.

Identification of amino acid sequences in the polyomavirus capsid proteins that serve as nuclear localization signals.

The molecular mechanism participating in the transport of newly synthesized proteins from the cytoplasm to the nucleus in mammalian cells is poorly understood. Recently, the nuclear localization signal sequences (NLS) of many nuclear proteins have been identified, and most have been found to be composed of a highly basic amino acid stretch. A genetic "subtractive" and a biochemical "additive" approach were used in our studies to identify the NLS's of the polyomavirus structural capsid proteins. An NLS was identified at the N-terminus (Ala1-Pro-Lys-Arg-Lys-Ser-Gly-Val-Ser-Lys-Cys11) of the major capsid protein VP1 and at the C-terminus (Glu307 -Glu-Asp-Gly-Pro-Glu-Lys-Lys-Lys-Arg-Arg-Leu318) of the VP2/VP3 minor capsid proteins.

The use of additive and subtractive approaches to examine the nuclear localization sequence of the polyomavirus major capsid protein VP1.

A nuclear localization signal (NLS) has been identified in the N-terminal (Ala1-Pro-Lys-Arg-Lys-Ser-Gly-Val-Ser-Lys-Cys11) amino acid sequence of the polyomavirus major capsid protein VP1. The importance of this amino acid sequence for nuclear transport of VP1 protein was demonstrated by a genetic "subtractive" study using the constructs pSG5VP1 (full-length VP1) and pSG5 delta 5'VP1 (truncated VP1, lacking amino acids Ala1-Cys11). These constructs were used to transfect COS-7 cells, and expression and intracellular localization of the VP1 protein was visualized by indirect immunofluorescence. These studies revealed that the full-length VP1 was expressed and localized in the nucleus, while the truncated VP1 protein was localized in the cytoplasm and not transported to the nucleus. These findings were substantiated by an "additive" approach using FITC-labeled conjugates of synthetic peptides homologous to the NLS of VP1 cross-linked to bovine serum albumin or immunoglobulin G. Both conjugates localized in the nucleus after microinjection into the cytoplasm of 3T6 cells. The importance of individual amino acids found in the basic sequence (Lys3-Arg-Lys5) of the NLS was also investigated. This was accomplished by synthesizing three additional peptides in which lysine-3 was substituted with threonine, arginine-4 was substituted with threonine, or lysine-5 was substituted with threonine. It was found that lysine-3 was crucial for nuclear transport, since substitution of this amino acid with threonine prevented nuclear localization of the microinjected, FITC-labeled conjugate.