Production of recombinant human tektin 1, 2, and 4 and in vitro assembly of human tektin 1.

Proteins predicted to be composed of large stretches of coiled-coil structure have often proven difficult to crystallize for structural determination. We have successfully applied EPR spectroscopic techniques to the study of the structure and assembly of full-length human vimentin assembled into native 11 nm filaments, in physiologic solution, circumventing the limitations of crystallizing shorter peptide sequences. Tektins are a small family of highly alpha helical filamentous proteins found in the doublet microtubules of cilia and related structures. Tektins exhibit several similarities to intermediate filaments (IFs): moderate molecular weight, highly alpha helical, hypothesized to be coiled-coil, and homo- and heteromeric assembly into long smooth filaments. In this report, we show the application of IF research methodologies to the study of tektin structure and assembly. To begin in vitro studies, expression constructs for human tektins 1, 2, and 4 were synthesized. Recombinant tektins were produced in E. coli and purified by chromatography. Preparations of tektin 1 successfully formed filaments. The recombinant human tektin 1 was used to produce antibodies which recognized an antigen in mouse testes, most likely present in sperm flagella. Finally, we report the creation of seven mutants to analyze predictions of coiled-coil structure in the rod 1A domain of tektin 1. Although this region is predicted to be coiled-coil, our EPR analysis does not reflect the parallel, in register, coiled-coil structure as demonstrated in vimentin and kinesin. These results document that tektin can be successfully expressed and assembled in vitro, and that SDSL EPR techniques can be used for structural analysis.

Distribution and structure of efferent synapses in the chicken retina.

The visual system of birds includes an efferent projection from a visual area, the isthmo-optic nucleus in the midbrain, back to the retina. Using a combination of anterograde labeling of efferent fibers, reconstruction of dye-filled neurons, NADPH-diaphorase staining, and transmission electron microscopy, we have examined the distribution of efferent fibers and their synaptic structures in the chicken retina. We show that efferent fibers terminate strictly within the ventral retina. In two completely mapped retinas, only 2 fibers from a total of 15,359 terminated in the dorsal retina. The major synapse made by each efferent fiber is with a single efferent target amacrine cell (TC). This synapse consists of 5-25 boutons of 2 microm diameter, each with multiple active zones, pressed into the TC soma or synapsing with a basketwork of rudimentary TC dendrites in the inner nuclear layer (INL). This basketwork, which is sheathed by Muller cell processes, defines a private neuropil in the INL within which TCs were also seen to receive input from retinal neurons. In addition to the major synapse, efferent fibers typically produce several very thin processes that terminate nearby in single small boutons and for which the soma of a local amacrine cell is one of the likely postsynaptic partners. A minority of efferent fibers also give rise to a thicker process, terminating in a strongly diaphorase-positive ball about 5 microm in diameter.

Digital image capture and quantification of subtle lens opacities in rodents.

A rapid, sensitive, and cost-effective method is reported for the subjective and objective documentation of subtle opacities in lenses of unanesthetized transgenic mice or selenite-injected rat pups as models for cataract formation. Animal eyes were dilated with eye drops and the animal was positioned in front of a Nikon FS2 photo slit lamp. Slit-lamp observations were recorded using a Canon Optura Pi digital video recorder. High-quality images of opacifying lenses were captured from the video and quantified using densitometry at progressive stages of opacification. In mice, targeted genomic deletion of the proteins CP49 (a lens-specific filament) or Six5 (a model for myotonic dystrophy) resulted in subtle cataracts that were easily recorded and quantified using this instrumentation. In rats, the early progressive changes leading to a dense nuclear opacity caused by selenite injection were easily documented using this instrumentation. Low-cost components combined with a conventional slit-lamp ophthalmoscope were used to capture high-quality images of selected stages of cataract formation for quantitative analysis using commercial software.

Development- and differentiation-dependent reorganization of intermediate filaments in fiber cells.

PURPOSE: To define the remodeling of lens fiber cell intermediate filaments (IF) that occurs with both development and differentiation. METHODS: Prenatal and postnatal mice were probed for the IF proteins phakosin, filensin, and vimentin, using light microscope immunocytochemical methodology. RESULTS: The pattern of vimentin accumulation in elongating fiber cells changed with development. Early in development vimentin first emerged predominantly as focal accumulations in the basal region of both epithelial and primary fiber cells. A light diffuse cytoplasmic staining was also noted. Later in embryonic development, and through maturity, vimentin in fiber cells was predominantly associated with the plasma membrane with no anterior-posterior polarity. Phakosin and filensin were first detected in the very latest stages of primary fiber elongation and continued to accumulate well after cells had completed elongation. Initially, these proteins accumulated in the anterior half of the fiber cells and were cytoplasmic in distribution. After P13, the pattern of initial distribution in differentiating fiber cells changed to a predominantly plasma membrane localization. Neither beaded filament protein showed focal basal accumulations. In mature lenses, all three proteins ultimately disappeared from the nuclear fiber cells. CONCLUSIONS: Beaded filament protein accumulation lags significantly behind both primary and secondary fiber cell elongation, suggesting a functional role subsequent to elongation. The subcellular distribution of vimentin and the beaded filament proteins showed marked differences within the cell, with differentiation, and with development. The differences in time of initial synthesis and in distribution of these IF proteins may bear on hypotheses about the role of IFs in fiber cell elongation and in structural-functional polarity of the fiber cell.

Evolutionary attempts at 4 eyes in vertebrates.

PURPOSE: To understand and compare the optical, histological, and ecological differences among 4 vertebrate species that have had evolutionary attempts toward 4 eyes. METHODS: An evolutionary attempt at 4 eyes in defined as the duplication or one or more structures integral to the refraction or interpretation of the visible spectrum for that animal. We reviewed and compared the known optics, histology, and ecology of each of these vertebrate species with attempts at 4 eyes including Anableps anableps, Dialomnus fuscus, Mnierpes macrocephalus, and Bathylychnops exilis. These animals have developed portions of ancillary eyes that have diverged from the primary globe in 3 different patterns. At least 1 specimen of each of those vertebrate species known to have 4 eyes was examined histologically and compared to the animal's ecology and current cladistic relationship. RESULTS: A anabteps has 2 distinct optical systems in each eye: an upper one for aerial vision and a lower system for aquatic vision. These systems feature separate retinae and an asymmetric lens to achieve focus in the aerial and aquatic vision, but only 1 optic nerve per eye. The visual system is split horizontally to function optimally in a "prone" position in the water. D fuscus is a terrestrial feeder and has a vertically (almost perpendicular to the long axis of the fish) divided cornea using pigment and a condensation of collagen as the divider, a single pupil, and a divided retina. The split cornea allows for the fish to remain vertical with 1 cornea in air and 1 cornea in water. M macrocephalus is probably closely related to D fuscus with a similar split cornea. B exilis is a mesopelagic inhabitant living at approximately 200 to 1,000 m and has an ancillary globe that "buds" off the primary globe. This secondary globe is directed inferiorly toward the ocean floor as compared to the primary globe, which is directed 35 degrees superiorly from the horizontal. Adult species of B exilis have 2 additional scleral bodies suspected to be lenses. If so, these structures would be capable of focusing light from the inferior field onto the superior retina, presumably adding to the panoramic inferior visual field. There are other mesopelagic species, including Styleophorus chordatus, Opisthoproctus grimaldii, Scopelarchus gantheri (or guentheri), Dolichopteryx binocularis, Benthalbella infans, and Evermannella indica, that have other unusual ocular mechanisms, such as retinal diverticulae and lens pads capable of reflection, but do not meet the definition of multiple eyes, as defined for purposes of this work. CONCLUSIONS: D fuscus and M macrocephalus are terrestrial feeders requiring aquatic and aerial vision, and hence have a split cornea for this purpose, and they probably use their anterior corneae for terrestrial vision. A anableps swims at the surface with combined aerial and aquatic vision for feeding and protection from predators. B exilis is a mesopelagic feeder requiring a binocular visual field in the horizontal meridian and above, and simultaneously is a bottom scavenger using an ancillary globe and perhaps scleral lenses for recognition of bioluminescent detritus. Although 2 of these models are related (D fuscus and M macrocephalus), these 4 fish represent 3 separate, distinct, and unrelated convergent evolutionary attempts toward 4 eyes in vertebrates satisfying the ecological needs of each. The 3 different models are unrelated evolutionarily and are found in 3 separate orders.

Voltage-gated Na+ channel EOIII-segment-like immunoreactivity in fish retinal ganglion cells.

Although single-channel and whole-cell patch-clamp recordings have demonstrated the presence of Na+ currents in retinal ganglion cell somata, it has not previously been reported that an anti-Na+-channel antiserum stains both retinal ganglion cell somata and proteins with molecular weights corresponding to complexes of alpha and beta subunits. We probed adult goldfish retinas for Na+ channel-like immunoreactivity with a polyclonal antibody directed against the EOIII segment of vertebrate voltage-gated Na+ channels. In vertical sections and whole mounts, this antibody consistently stained the somata, axons, and proximal dendrites of retinal ganglion cells. Some somata in the proximal third of the inner nuclear layer were also stained. In Western blots, this antibody specifically stained multiple protein bands from retina and optic nerve, all with apparent molecular weights between 200 and 315 kDa. The largest of these molecular weights agrees with that reported previously for complexes of alpha and beta subunits in mammalian neurons, including retinal ganglion cells. The intermediate and lowest molecular weights are consistent with the presence of multiple Na+ channel alpha subunits, either in individual proximal retinal neurons or in different morphological subtypes.

A juvenile-onset, progressive cataract locus on chromosome 3q21-q22 is associated with a missense mutation in the beaded filament structural protein-2.

Juvenile-onset cataracts are distinguished from congenital cataracts by the initial clarity of the lens at birth and the gradual development of lens opacity in the second and third decades of life. Genomewide linkage analysis in a multigenerational pedigree, segregating for autosomal dominant juvenile-onset cataracts, identified a locus in chromosome region 3q21.2-q22.3. Because of the proximity of the gene coding for lens beaded filament structural protein-2 (BFSP2) to this locus, we screened for mutations in the coding sequence of BFSP2. We observed a unique C-->T transition, one that was not observed in 200 normal chromosomes. We predicted that this led to a nonconservative R287W substitution in exon 4 that cosegregated with cataracts. This mutation alters an evolutionarily conserved arginine residue in the central rod domain of the intermediate filament. On consideration of the proposed function of BFSP2 in the lens cytoskeleton, it is likely that this alteration is the cause of cataracts in the members of the family we studied. This is the first example of a mutation in a noncrystallin structural gene that leads to a juvenile-onset, progressive cataract.

Autosomal-dominant congenital cataract associated with a deletion mutation in the human beaded filament protein gene BFSP2.

Congenital cataracts are a common major abnormality of the eye that frequently cause blindness in infants. At least one-third of all cases are familial; autosomal-dominant congenital cataract appears to be the most-common familial form in the Western world. Elsewhere, in family ADCC-3, we mapped an autosomal-dominant cataract gene to chromosome 3q21-q22, near the gene that encodes a lens-specific beaded filament protein gene, BFSP2. By sequencing the coding regions of BFSP2, we found that a deletion mutation, DeltaE233, is associated with cataracts in this family. This is the first report of an inherited cataract that is caused by a mutation in a cytoskeletal protein.

Mucous fistula refeeding in neonates with short bowel syndrome.

BACKGROUND/PURPOSE: Neonates with enterostomies commonly suffer from a functional short bowel syndrome (SBS) and have a greater risk of electrolyte and fluid loss with poor weight gain. The authors describe their experience with refeeding stoma effluent into the mucous fistula in neonates. METHODS: A 5-year (1993 to 1997) chart review of neonates with stoma effluent refeeding was undertaken. Demographics, medical history, surgical procedures, timing, and duration of refeedings were reviewed. Enteral and total parenteral nutritional (TPN) requirements, electrolyte, and acid-base disturbances were recorded. RESULTS: Six neonates (gestational ages of 27 to 38 weeks, birth weights of 533 to 3400 g) were identified with nutritional or electrolyte complications before the commencement of refeeding. Enterostomy indications included necrotizing enterocolitis (n = 2), intestinal atresia type 3b (n = 1), complications from ruptured omphalocoele (n = 1), congenital adhesive band obstruction (n = 1), and midgut volvulus after congenital diaphragmatic hernia repair (n = 1). Weight gain during refeeding ranged from 5 to 25 g/kg/d with duration of refeeding lasting 16 to 169 days (two neonates were refed at home) until reanastomoses were done 6 to 44 weeks after the original surgery. There were no complications, and TPN requirements were diminished or eliminated. CONCLUSION: This technique represents a simple and safe method, which lessens the need for TPN and electrolyte supplementation in neonates with enterostomies and SBS before reanastomosis.

Primary sequence, secondary structure, gene structure, and assembly properties suggests that the lens-specific cytoskeletal protein filensin represents a novel class of intermediate filament protein.

The ocular lens fiber cell assembles a novel cytoskeletal element, the Beaded Filament, from CP49 and filensin, two proteins expressed only in the differentiated lens fiber cell. We report the primary sequence, secondary structural analysis, gene structure and Yeast Two Hybrid interaction data for human filensin, and develop a consensus model of filensin from the human and previously reported bovine and chicken filensin sequences. This consensus model, combined with gene structure and Yeast Two Hybrid studies establish that filensin is a member of the Intermediate Filament family of proteins. Specifically, filensin exhibits (1) divergence at amino acid sequence motifs otherwise highly conserved among intermediate filament proteins, (2) a loss of 29 amino acids from the central rod domain which is unique among cytoplasmic intermediate filament proteins, (3) an absence of sequence identity with any existing class of intermediate filament protein, (4) a gene structure unique among intermediate filament family, (5) an inability to dimerize with representatives of Type I, II, and III intermediate filament proteins. Thus, at each level of analysis, we find that filensin is similar to the consensus model of intermediate filament proteins, supporting our conclusion that filensin's relatedness to the IF family is not the consequence of convergent evolution. However, filensin also shows unique or extreme distinctions from the consensus intermediate filament protein at each level of analysis, indicating that filensin constitutes a novel class of IF protein. Some of filensin's unique features are incompatible with current models of IF assembly. Analysis of filensin gene structure suggests that the 29 amino acid reduction in the central rod domain was not the result of a single splice site mutation, the mechanism suggested for the transition between nuclear lamins and cytoplasmic intermediate filament proteins.

Protection of a restriction enzyme from heat inactivation by [alpha]-crystallin.

PURPOSE: To determine whether the chaperone activity of human alpha-crystallin can protect a restriction enzyme from heat inactivation. METHODS: The restriction enzyme Nde I was heated in the presence or absence of purified bovine alpha-crystallin. Following heat treatment, the enzymatic activity of the heat treated samples was assayed by cleavage of plasmid DNA. The extent of digestion was monitored by agarose gel electrophoresis and visualization of DNA fragments by ethidium bromide staining. RESULTS: Heating of Nde I in the absence of alpha-crystallin resulted in inactivation. However, Nde I heated in the presence of alpha-crystallin remained active. Furthermore, an increased amount of alpha-crystallin provided a longer period of thermal protection. CONCLUSIONS: The chaperone activity and thermo-protective effect of alpha-crystallin extend to protection of enzymatic activity, not merely the protection from thermally induced aggregation/denaturation. In addition, inclusion of alpha-crystallin during some enzymatic reactions may be beneficial.

Histopathologic analysis of interval appendectomy specimens: support for the role of interval appendectomy.

The treatment of appendiceal abscess is controversial. For patients initially treated "conservatively" with antibiotics with or without drainage, the role of interval appendectomy is an area of considerable debate. Without interval appendectomy, the true risks of recurrent disease and missed pathological findings are uncertain, and large, long-term, prospective studies are unavailable. To evaluate the role of interval appendectomy, the authors reviewed the histopathologic specimens from patients with presumed appendiceal abscess treated by interval appendectomy. Over a 7-year period, 162 children presented with a clinical diagnosis of perforated appendicitis. Eighteen patients had localized abscesses treated conservatively, followed by interval appendectomy. Standard histopathologic sections of 17 of the 18 appendices were examined by one pathologist who was blinded to the clinical data and to the interpretation of the original pathologist. Of the 11 boys and seven girls (mean age, 7.4 +/- 3.4 years), eight underwent percutaneous drainage and one underwent operative drainage. All received intravenous antibiotics for a mean of 8.6 +/- 3.2 days with a hospital stay of 10.4 +/- 8.3 days. Interval appendectomy was performed at a mean of 92.7 +/- 20.7 days after initial admission, with discharge at a mean of 2 +/- 1.3 days after surgery. There were no complications or deaths. Histopathologic review showed normal appendix (n = 4), normal appendix with mild serositis (n = 6), normal appendix with unsuspected resolved Meckel's diverticulitis (n = 1), appendiceal duplication (n = 1), granulomatous appendicitis (n = 3), and acute appendicitis (n = 2). All appendices had patent lumens, and 15 were documented to be present to the tip. There was no correlation between the histopathologic findings and the interval between abscess and interval appendectomy. Interval appendectomy was performed with no morbidity and a short hospital stay. Two patients had histopathologic recurrent acute appendicitis, five had unsuspected pathological findings (appendiceal duplication, Meckel's diverticulitis, granulomatous inflammation), and none of the appendices had an obliterated lumen, suggesting that all patients were at long-term risk for recurrent disease. These data support the role of interval appendectomy in cases of perforated appendicitis treated conservatively.

Intratracheal granuloma formation: a late complication of Marlex mesh splinting for tracheomalacia.

External splinting of the trachea has been used alone or in combination with aortopexy for the treatment of severe tracheomalacia. The authors describe the case of a 12-year-old boy who had a Marlex mesh splint placed because of life-threatening primary tracheomalacia at 6 months of age. He presented at 12 years of age with a 5-month history of shortness of breath on exertion, dry cough, and audible wheeze. Radiological and endoscopic examinations showed near-complete obstruction of the orifice of the right mainstem bronchus by a large polypoid granuloma. Initially the patient was treated with endoscopic resection on two occasions, but the granuloma and bronchial obstruction recurred each time. He underwent a right thoracotomy, which showed that the lower edge of the mesh had eroded through the trachea wall and was acting as a nidus for granuloma formation. After removal of the mesh, the resulting defect at the site of erosion of the trachea was closed with a pericardial patch. The postoperative course was uncomplicated, and the patient remains well 2 years after surgery. External splinting of the trachea has been shown to be effective in the treatment of complicated tracheomalacia, but one must be aware of the potential long-term complications, as demonstrated in this case.

Pediatric laparoscopic splenectomy using the lateral approach.

Laparoscopic splenectomy in children has been shown to be safe, to reduce postoperative pain and hospital stay, and to accelerate return to full activities. We describe our experience with a four-port "lateral" approach in 18 patients. Patients were placed in the lateral decubitus position and the table was flexed to separate the left subcostal margin and iliac crest. The camera port was inserted at the umbilicus and additional ports were placed in the epigastrium and left lower quadrant. After mobilization of the splenic flexure a port was inserted in the left flank below the 12th rib for elevation of the spleen. A 30 degrees laparoscope was used and the splenic vessels were controlled with an endo-GIA and/or clips. The spleens were placed in a bag, morcellated, and extracted through a port site. Eight females and 10 males with a median age of 12.5 years (5-17 years) and weight of 55.5 kg (17-124 kg) underwent splenectomy of idiopathic thrombocytopenia purpora (10), spherocytosis (6), elliptocytosis (1), and Hodgkin's disease (1). The median operating time was 160 min (90-300 min) and median blood loss was 105 ml (5-350 ml). Accessory spleens were removed in four cases. Three patients required extensions of a port site to remove large spleens which could not be placed in a bag. The sole complication was a transient pancreatitis with associated pleural effusion. The median postoperative hospital stay was 2 days (1-11 days) and time to full activities was 8 days (3-25 days). The lateral approach affords excellent visualization of the splenic vessels, pancreas, and accessory spleens. This approach is safe and reliable and is our preferred approach for laparoscopic splenectomy in children.

Gene structure and cDNA sequence identify the beaded filament protein CP49 as a highly divergent type I intermediate filament protein.

The fiber cell of the vertebrate ocular lens assembles a cytoskeletal structure, the beaded filament, which contains two proteins unique to the fiber cell: CP49 (phakinin) and CP115/CP95 (filensin). We report here the complete primary sequence and gene structure for human CP49. These data show that CP49 is a member of the intermediate filament family, but highly unusual in several regards. 1) CP49 primary sequence does not permit unambiguous assignment to any existing class of intermediate filament protein, but exhibits a gene structure that is identical to the Type I cytokeratins. 2) CP49 essentially lacks one of the three major domains that characterize all intermediate filament proteins, the carboxyl-terminal tail domain. 3) CP49 shows substitutions at 3 of 4 residues in the otherwise highly conserved intermediate filament protein motif LNDR. Notably, this divergence includes an Arg to Cys substitution that has only been observed in the mutant human cytokeratin K14, a mutation shown to cause the skin blistering seen in the genetic disorder Dowling-Meara epidermolysis bullosa simplex.

One-stage versus two-stage Soave pull-through for Hirschsprung's disease in the first year of life.

Several investigators have reported good results after a one-stage Soave procedure without a stoma for infants with Hirschsprung's disease. The authors reviewed their concurrent experience with the one- and two-stage approaches, comparing the two groups with respect to rate of complications and clinical outcome. Over a 3-year period, 36 infants with colonic Hirschsprung's disease presenting in the first year of life were treated with a Soave pull-through. Thirteen had a one-stage pull-through, and 23 had a two-stage procedure using an initial stoma. There was no difference with respect to median age at time of diagnosis, median follow-up period, length of aganglionosis, or male:female ratio between the groups. The incidences of major complications such as small bowel obstruction, segmental or acquired aganglionosis, anastomotic leak, and malabsorption were equal between the two groups. However, 13% of the two-stage patients required revision of the stoma. All major complications in the one-stage group were in those who weighed less than 4 kg at the time of surgery. Minor complications such as wound infection, perianal excoriation, and need for repeated dilatation were similar between the groups, but minor stoma-related complications (prolapse or retraction) occurred in 26% of the two-stage infants. When complications were stratified using a more sophisticated scale of severity, no significant difference was found between the groups. The overall complication rate was 1.5 events per patient in the one-stage group and 2.0 events per patient in the two-stage group. This small difference was related to the presence of a stoma in the two-stage group. Overall, 10 of 12 survivors in the one-stage group and 22 of 23 in the two-stage group were doing well, with normal bowel function noted on long-term follow-up (mean period, of 14 and 19 months, respectively). Both one- and two-stage approaches were associated with a significant complication rate, although long-term outcome was excellent in both groups. The higher complication rate in the two-stage group was attributable to the presence of a stoma. For small infants, it may be beneficial to delay the one-stage pull-through until weight exceeds 4 kg.

Filensin is proteolytically processed during lens fiber cell differentiation by multiple independent pathways.

Filensin is a lens-specific intermediate filament protein, expressed in the lens fiber cells but not the lens epithelium. Using antibodies to filensin and the other lens intermediate filament proteins, vimentin and CP49, the codistribution of filensin with CP49 and independence of this network from the vimentin network was confirmed. Monoclonal and polyclonal antibodies to peptides and specific subdomains of filensin were used to follow changes in the subcellular distribution of filensin during bovine lens fiber cell differentiation. Filensin is shown to be extensively processed during lens fiber cell differentiation to give protein fragments derived from distinct protein domains, one corresponding to the N-terminal non-alpha-helical/and rod domain and the other to the C-terminal non-alpha-helical tail domain. Immunoblotting analysis using anti-filensin peptide polyclonal antibodies suggested that the two fragment sets arose separately. Residues 331 to 430 in filensin have been identified as an important region in the processing pathway(s). Our results clarify previous confusion in the literature regarding the processing of filensin which arose because of the similar relative electrophoretic mobilities by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of the different fragment sets. The predicted secondary structure characteristics of the different domains of filensin suggests different functions for the two fragment sets to give filensin a dual role in the lens. This suggestion is supported by the subtly different subcellular distributions in the peripheral and mature fiber cells of the two filensin fragment sets.

Vimentin and CP49/filensin form distinct networks in the lens which are independently modulated during lens fibre cell differentiation.

The cells of the eye lens contain the type III intermediate filament protein vimentin, as well as two other intermediate filament proteins, CP49 and filensin. These two proteins appear to be unique to the differentiated lens fibre cell. Immunoblotting and confocal microscopy were used to describe changes which occur in these three intermediate filament proteins and the networks they form during fibre cell differentiation and maturation. The vimentin network was present in both epithelial cells and some fibre cells. Fibre cells were vimentin positive up to a specific point 2-3 mm in from the lens capsule where the vimentin signal was drastically reduced. The CP49/filensin network was not present in the undifferentiated epithelial cells but emerged in the differentiating fibre cells. This latter network exhibited a principally plasma membrane localization in younger fibre cells but became more cytoplasmic in older fibre cells. This change also occurred at a distinct point in fibre cell differentiation, much earlier than the observed loss of the vimentin network. The subcellular changes in the distributions of these cytoskeletal networks were correlated to the loss of the fibre cell nucleus, another feature of fibre cell differentiation. No correlation was found to changes in the vimentin network but nuclear loss did coincide with changes in the CP49/filensin network. Concomitant with nuclear pyknosis, there were also changes in the nuclear lamina as well as infringement of the nuclear compartment by CP49, as shown by confocal microscopy. This study demonstrates vimentin and the CP49/filensin network to be independent in the lens but both networks undergo dramatic changes in subcellular distribution during the differentiation/maturation of the fibre cell. Only changes in the CP49/filensin network can be correlated to nuclear loss. Thus in the lens, unlike mammalian erythropoiesis which is also characterized by nuclear loss, the vimentin network does not appear linked to nuclear retention.

Chromosomal locations of the genes for the beaded filament proteins CP 115 and CP 47.

We have used the polymerase chain reaction (PCR) to amplify CP 115 and CP 47 encoding sequences from human lens cDNA samples. DNA sequence and northern blot analysis were used to confirm human origin. From the determined cDNA sequences, human-specific oligonucleotides were synthesized and assessed for the ability to amplify human genomic DNA. After empirically selecting a primer pair for each gene able to amplify human genomic DNA, and optimizing PCR conditions for human specificity, we used the PCR to screen a panel of mouse/human somatic cell hybrid DNA samples. Amplification of CP 115 or CP 47 sequences in each of the somatic cell hybrid samples was correlated with the presence/absence of human genomic DNA sequences encoding the respective gene sequences. From our results, we conclude that the gene for human CP 115 resides on chromosome 20 and the gene for human CP 47 on chromosome 3. Further mapping using somatic cell lines carrying derivatives of human chromosome 3 localize the gene for CP 47 to 3q21-25. We propose LIFL-H (Lens Intermediate Filament Like-Heavy) for CP 115 and LIFL-L (Lens Intermediate Filament Like-Light) for CP 47 as the gene symbols for these loci.