A systematic, genome-wide, phenotype-driven mutagenesis programme for gene function studies in the mouse.

As the human genome project approaches completion, the challenge for mammalian geneticists is to develop approaches for the systematic determination of mammalian gene function. Mouse mutagenesis will be a key element of studies of gene function. Phenotype-driven approaches using the chemical mutagen ethylnitrosourea (ENU) represent a potentially efficient route for the generation of large numbers of mutant mice that can be screened for novel phenotypes. The advantage of this approach is that, in assessing gene function, no a priori assumptions are made about the genes involved in any pathway. Phenotype-driven mutagenesis is thus an effective method for the identification of novel genes and pathways. We have undertaken a genome-wide, phenotype-driven screen for dominant mutations in the mouse. We generated and screened over 26,000 mice, and recovered some 500 new mouse mutants. Our work, along with the programme reported in the accompanying paper, has led to a substantial increase in the mouse mutant resource and represents a first step towards systematic studies of gene function in mammalian genetics.

Recurring enteric peritonitis associated with non-perforating colon carcinoma.

Peritonitis of enteric origin may occur during treatment with peritoneal dialysis due to visceral perforation or injury or, in the absence of perforation, due to transmural migration of enteric bacteria across the bowel wall into the peritoneal cavity. To the best of our knowledge, peritonitis has not previously been reported associated with carcinomatous colon polyp in the absence of bowel wall perforation. We describe the case of a 31-year-old female who experienced recurring episodes of enteric peritonitis associated with a clinically occult adenocarcinoma of the colon, without having any other known risk factors for peritonitis. A 15 mm carcinomatous polyp was not visible on CT scan but was found at colonoscopy with polypectomy. She proceeded to transverse colectomy; the resected colon showed no evidence of bowel wall perforation. This case demonstrates that a non-perforating carcinomatous polyp of the colon may predispose to enteric peritonitis in the setting of peritoneal dialysis, and it emphasizes the importance of making an aggressive search for underlying pathology, in patients who present with recurring enteric peritonitis or unusual presentations of enteric peritonitis.

The oxidation of naphthalene and pyrene by cytochrome P450cam.

Mutants of the heme monooxygenase cytochrome P450cam in which Y96 had been replaced with hydrophobic residues, have been shown to oxidise naphthalene and pyrene with rates one to two orders of magnitude faster than the wild-type. Naphthalene was oxidised to 1- and 2-naphthol, probably via the 1,2-oxide intermediate. In the case of the Y96F mutant, naphthalene was oxidised at a rate comparable to camphor. Pyrene oxidation gave 1,6- and 1,8-pyrenequinone with no evidence for attack at the K-region, in contrast to mammalian enzymes. The results show that the Y96 residue plays a key role in controlling the substrate range of P450cam.

Alterations of the crossed parabigeminotectal projection induced by neonatal eye removal in rats.

Projections of the parabigeminal nucleus to the contralateral superior colliculus and dorsal lateral geniculate nucleus were examined in normal adult pigmented rats and in adult rats from which one or both eyes had been removed at birth. In normal rats the crossed parabigeminotectal projection is restricted to the superficial layers in anterior and medial areas of colliculus, regions innervated also by the lower temporal portion of the ipsilateral retina. In unilaterally enucleated animals the crossed parabigeminotectal projection to the "denervated" colliculus is expanded, as is the retinal projection from the ipsilateral eye. In addition, there is a crossed parabigeminal projection to the "denervated" dorsal lateral geniculate nucleus in these rats. In bilaterally enucleated animals the parabigeminotectal projection is expanded, but not as greatly as in unilateral enucleation cases; there is a crossed parabigeminothalamic projection in these animals as well. The corresponding termination patterns of the contralateral parabigeminal nucleus and the ipsilateral retina in the normal superior colliculus may indicate a functional and/or developmental interdependence between the projections from these two regions. The existence of an expanded parabigeminotectal projection in bilaterally enucleated rats shows that a sustained ipsilateral retinotectal projection is not necessary for the establishment of a crossed parabigeminotectal projection, but points to the possibility that ipsilateral retinal input may constrain the parabigeminal projection to terminate within certain boundaries. The even greater expansion of the projection from the parabigeminal nucleus to the colliculus which receives an expanded projection from the ipsilateral retina of unilaterally enucleated rats suggests that the functional organization of the ipsilateral retinotectal projection may be capable of restricting the size of the terminal field of the crossed parabigeminotectal projection.

Morphology of radial glia, ependymal cells, and periventricular neurons in the optic tectum of goldfish (Carassius auratus).

There are three populations of cells in the deep layers of the optic tectum in a normal adult goldfish: the periventricular neurons, the ependymal cells, and the radial glia. The characteristic morphological features which distinguish the three cell populations are examined at light and electron microscopic levels in the present work. A radial glial cell has a deeply invaginated nucleus located in a subependymal layer. Its cytoplasm contains mitochondria with 35-nm dark granules and 20-nm microtubules but no intermediate filaments. Its prominent radial process extends through the superficial tectal layers. In contrast, the processes of ependymal cell ramify and interweave within the ependymal region. The cytoplasm of an ependymal cell contains prominent bundles of intermediate filaments but not microtubules. Its soma lies at or near the ventricular surface. A periventricular neuron has a round nucleus and a smooth dendrite which extends toward the superficial tectal layers. Its cytoplasm contains microtubules and agranular mitochondria. Axosomatic and axodendritic synapses are found on periventricular neurons. The morphological characteristics of these cell types are considered in relation to previous descriptions of teleost tectal cytology and with regard to the atypical natures of the cytoskeletal elements of the ependymal and radial glial cells.

Differential distribution of phosphorylated and non-phosphorylated neurofilaments within the retina and optic nerve of hamsters.

Retinae and optic nerve sections from adult hamsters were reacted with antibodies against phosphorylated (P) or non-phosphorylated (NP) neurofilament proteins. NP epitopes were observed within ganglion cell bodies and extended into the proximal portion of the optic nerve. P epitopes became prominent within axons as they approached the optic disc and remained throughout the optic nerve. This spatial distribution appears similar to the pattern of myelination within this axonal population.

Kinetics of cell proliferation in the halved tectum of adult goldfish.

The time course of post-operative cell proliferation was studied in the halved optic tectum of adult goldfish using autoradiographic methods. At varying intervals after bilateral optic nerve crush and unilateral lesion of the caudal hemitectum, fish were injected with [3H]thymidine and allowed to survive for designated times before sacrifice. Comparisons of labeled cell numbers in different laminar regions on the intact and operated sides of the tecta were made. When short post-injection survival times were employed, there was a bimodal increase in proliferation on both tectal sides with increases occurring by 5 and between 20 and 35 days after surgery (dpo). More labeled cells were seen on the lesioned side in the outer tectal zone in fish injected at 25 and 35 dpo. Within their inner tectal zones these same fish showed more labeled cells in the periependymal layer on the lesioned side. With extended post-injection survival there was a general decline in numbers of labeled cells on both tectal sides. The only labeled cells which persisted in force were those of the periependymal layer; equal numbers were seen on both operated and intact sides. On the basis of these observations we conclude that the cell proliferation which occurs in this situation may be attributed primarily to the effects of optic nerve regeneration; the tectal surgery per se does not induce massive cell proliferation which might numerically reconstitute the halved tectum.

Henoch-Schönlein purpura: simultaneous demonstration of IgA deposits in involved skin, intestine, and kidney.

Recent reports indicate that circulating IgA immune complexes may play a primary role in the pathogenesis of Henoch-Schönlein vasculitis and are responsible for the granular deposits of IgA seen in biopsy specimens of skin and kidney. A patient had classic Henoch Schönlein syndrome, including hematuria, purpura, and abdominal pain; tissue taken simultaneously from the small intestine, skin, and kidney was examined by light immunofluorescent, and electron microscopy. Granular deposits of IgA were found in small-vessel walls of the intestinal tissue and skin, and in the glomerular mesangium. This provides further support for the notion that IgA deposits produce tissue injury in intestine, skin, and kidney in Henoch-Schönlein syndrome.

Comparison of the Difco EZ Coil rapid detection system and Petrifilm test kit-HEC for detection of Escherichia coli O157:H7 in fresh and frozen ground beef.

The Difco EZ Coli Rapid Detection System was compared to the 3M Petrifilm method for detection of Escherichia coli O157:H7 in raw ground beef. Raw meatballs (25 g) were inoculated with 10 to 15 cells of Escherichia coli O157:H7, stored for various times and at different temperatures, and then stomached for 2 min in 225 ml of EZ Coli enrichment broth, which was then incubated at 42 degrees C for 18 to 24 h. A 1-ml sample of the enrichment broth was loaded into the top of the detector tips and the remaining EZ Coli broth held at 35 degrees C before streaking onto MacConkey sorbitol agar and tryptic soy agar with yeast extract. A duplicate set of meatballs were tested using the 3M Petrifilm Test Kit-HEC for hemorrhagic Escherichia coli O157:H7. In this method raw meatballs (25 g) were enriched for 6 h in modified EC broth containing novobiocin at 37 degrees C prior to inoculation of the Petrifilm E. coli Count Plates, which were incubated at 42 degrees C for 18 h. The immunoblot ELISA was performed following this incubation. Presumptive positive isolates from both methods were confirmed using Oxoid E. coli Latex Agglutination and Difco Pasco ID Tripanels. Both methods permitted detection of 10 to 15 cells of E. coli O157:H7 per ml (i) immediately following inoculation, (ii) after 3 days of refrigerated storage at 8 degrees C, and (iii) after 30 days in frozen storage at -20 degrees C. The Difco EZ Coli Detection System proved to be a simpler and faster screening method with identification of negative and presumptive positive samples within 15 to 18 h.

Growth of retinal ganglion cell axons following optic nerve crush in adult hamsters.

Regrowth of retinal ganglion cell axons was examined 2 to 60 days after intraorbital optic nerve crush lesions in adult hamsters. Anterograde axonal transport of intraocularly injected wheat germ agglutinin-horseradish peroxidase conjugate was used to label the axons after specific postinjury time periods. Labeled axons were present in the region of the optic nerve lying between the eye and the crush site at all times, but their numbers appeared to decrease with increasing survival time. Labeled axons were first detected in the segment of optic nerve lying distal to the crush site 1 week after injury and had extended as far as 2.3 mm beyond the crush site by 60 days postinjury, growing at a rate similar to that at which the collateral branches of developing ganglion cell axons extend into their targets. Although most axons failed to regrow after these lesions, the slow reextention exhibited by members of a small population of axons indicates that the degenerating adult mammalian optic nerve provides an adequate environment for a particular mode of regrowth by injured axons of the central nervous system.

Growth of optic tract axons in nerve grafts in hamsters.

Segments of peripheral nerve, transplanted to the brain or spinal cord, recently have been shown to support regeneration of axons from a variety of central neurons. However, long-tract axons, injured at considerable distances from their cell bodies, have proven refractory to such regenerative support. This report presents evidence for successful, although similarly limited, growth of retinal ganglion cell axons into peripheral nerve grafts placed in the optic tract of adult hamsters. The demonstration of such growth allows the possibility that the primary visual pathways may serve as an advantageous model system in which to study the mechanism of graft-effected regeneration of long-tract axons in the adult mammalian central nervous system.