N-Linked glycan site occupancy impacts the distribution of a potassium channel in the cell body and outgrowths of neuronal-derived cells.

BACKGROUND: Vacancy of occupied N-glycosylation sites of glycoproteins is quite disruptive to a multicellular organism, as underlined by congenital disorders of glycosylation. Since a neuronal component is typically associated with this disease, we evaluated the impact of N-glycosylation processing of a neuronal voltage gated potassium channel, Kv3.1b, expressed in a neuronal-derived cell line, B35 neuroblastoma cells. METHODS: Total internal reflection fluorescence and differential interference contrast microscopy measurements of live B35 cells expressing wild type and glycosylation mutant Kv3.1b proteins were used to evaluate the distribution of the various forms of the Kv3.1b protein in the cell body and outgrowths. Cell adhesion assays were also employed. RESULTS: Microscopy images revealed that occupancy of both N-glycosylation sites of Kv3.1b had relatively similar amounts of Kv3.1b in the outgrowth and cell body while vacancy of one or both sites led to increased accumulation of Kv3.1b in the cell body. Further both the fully glycosylated and partially glycosylated N229Q Kv3.1b proteins formed higher density particles in outgrowths compared to cell body. Cellular assays demonstrated that the distinct spatial arrangements altered cell adhesion properties. CONCLUSIONS: Our findings provide direct evidence that occupancy of the N-glycosylation sites of Kv3.1b contributes significantly to its lateral heterogeneity in membranes of neuronal-derived cells, and in turn alters cellular properties. GENERAL SIGNIFICANCE: Our study demonstrates that N-glycans of Kv3.1b contain information regarding the association, clustering, and distribution of Kv3.1b in the cell membrane, and furthermore that decreased occupancy caused by congenital disorders of glycosylation may alter the biological activity of Kv3.1b.

Importance of glycosylation on function of a potassium channel in neuroblastoma cells.

The Kv3.1 glycoprotein, a voltage-gated potassium channel, is expressed throughout the central nervous system. The role of N-glycans attached to the Kv3.1 glycoprotein on conducting and non-conducting functions of the Kv3.1 channel are quite limiting. Glycosylated (wild type), partially glycosylated (N220Q and N229Q), and unglycosylated (N220Q/N229Q) Kv3.1 proteins were expressed and characterized in a cultured neuronal-derived cell model, B35 neuroblastoma cells. Western blots, whole cell current recordings, and wound healing assays were employed to provide evidence that the conducting and non-conducting properties of the Kv3.1 channel were modified by N-glycans of the Kv3.1 glycoprotein. Electrophoretic migration of the various Kv3.1 proteins treated with PNGase F and neuraminidase verified that the glycosylation sites were occupied and that the N-glycans could be sialylated, respectively. The unglycosylated channel favored a different whole cell current pattern than the glycoform. Further the outward ionic currents of the unglycosylated channel had slower activation and deactivation rates than those of the glycosylated Kv3.1 channel. These kinetic parameters of the partially glycosylated Kv3.1 channels were also slowed. B35 cells expressing glycosylated Kv3.1 protein migrated faster than those expressing partially glycosylated and much faster than those expressing the unglycosylated Kv3.1 protein. These results have demonstrated that N-glycans of the Kv3.1 glycoprotein enhance outward ionic current kinetics, and neuronal migration. It is speculated that physiological changes which lead to a reduction in N-glycan attachment to proteins will alter the functions of the Kv3.1 channel.

Single nucleotide polymorphisms for integrative mapping in the Turkey (Meleagris gallopavo).

When multiple genetic maps exist for a species, integration of these maps requires a set of common markers be genotyped across the individual mapping populations. In the turkey, three genetic maps based on separate mapping populations are available. In this study, SNP-based markers were developed for integrating the cDNA/RFLP-based map (1) with microsatellite markers of the second-generation turkey genome map (2). Forty-eight primer sets were designed and tested and 33 (69%) correctly amplified turkey genomic DNA by PCR. Putative SNPs were detected in 20 (61%) of the amplified gene fragments, and 10 SNP markers were subsequently genotyped by PCR/RFLP for segregation analysis. Eight SNP markers were incorporated into the turkey genetic map.

A comparative genetic map of the turkey genome.

Genetic markers (microsatellites and SNPs) were used to create and compare maps of the turkey and chicken genomes. A physical map of the chicken genome was built by comparing sequences of turkey markers with the chicken whole-genome sequence by BLAST analysis. A genetic linkage map of the turkey genome (Meleagris gallopavo) was developed by segregation analysis of genetic markers within the University of Minnesota/Nicholas Turkey Breeding Farms (UMN/NTBF) resource population. This linkage map of the turkey genome includes 314 loci arranged into 29 linkage groups. An additional 40 markers are tentatively placed within linkage groups based on two-point LOD scores and 16 markers remain unlinked. Total map distance contained within linkage groups is 2,011 cM with the longest linkage group (47 loci) measuring 413.3 cM. Average marker interval over the 29 linkage groups was 6.4 cM. All but one turkey linkage group could be aligned with the physical map of the chicken genome. The present genetic map of the turkey provides a comparative framework for future genomic studies.

Microsatellite loci for genetic mapping in the turkey (Meleagris gallopavo).

New microsatellite loci for the turkey (Meleagris gallopavo) were developed from two small insert DNA libraries. Polymorphism at these new loci was examined in domestic birds and two resource populations designed for genetic linkage mapping. The majority of loci (152 of 168) was polymorphic in domestic turkeys and informative in two mapping resource populations and thus will be useful for genetic linkage mapping.

Engagement in activities revealing the body and psychosocial adjustment in adults with a trans-tibial prosthesis.

The purpose of this study was to examine the effects of the appearance of a prosthesis on social behaviour, social discomfort and psychological well-being in eleven amputees taking delivery of a prosthesis with a silicone cover. Two new scales were developed: the 'Engagement in everyday activities involving revealing the body' (EEARB); and the 'Discomfort-Engagement in everyday activities involving revealing the body' (Discomfort-EEARB) scales. The psychometric properties of these scales were determined using a sample of 101 able-bodied adults. The Hospital Anxiety and Depression Scale and the Rosenberg Self-Esteem Scale were also used to measure psychological well-being in the amputee sample. The EEARB and Discomfort-EEARB proved to have good reliability and validity. Comparison of amputees' scores prior to receiving the silicone cosmesis with those of the able-bodied adults revealed significant behavioural limitations and social discomfort, associated with low self-esteem, anxiety and depression. There was a significant increase in amputees' scores three months afier taking delivery of their prosthesis, indicating that amputees reported engaging in more activities which involved revealing their body, and that they would feel more comfortable in situations which involved revealing the body. As the amputee sample available was small and self-selected, it is not possible to generalise these findings to the amputee population as a whole. However, since there is little previous research investigating the effects of the appearance of the prosthesis, these findings demonstrate the need for further research in this area.

A study of androgen biosynthesis by the human testis in vitro.

The biosynthetic pathways in human testicular tissue have been studied extensively in our laboratory without the use of radioisotopes. Experiments were conducted with normal testicular tissue from patients undergoing orchiectomy for prostatic cancer. These studies have shown that the preferred pathway of testosterone biosynthesis is influenced by the nature and concentration of cofactor added to the incubation medium. Four enzymes are involved in the transformation of pregnenolone to testosterone, that is, 3 beta-hydroxysteroid dehydrogenase, 17 alpha-hydroxylase, C17-C20 lyase and 17 beta-hydroxysteroid oxidoreductase. Our studies show that the 4-ene pathway predominates in the biosynthesis of testosterone from pregnenolone. Analysis of several samples of human testicular vein blood supports the contention that 4-androsten-3,17-dione is the immediate precursor of testosterone.

In vitro studies on the effect of cofactors on the 5 alpha-reductase and 3 alpha- and 3 beta-hydroxysteroid reductase activities in the hyperplastic human prostate.

Many studies have intimated that the accumulation and hence elevation of dihydrotestosterone (DHT) in the human prostate may be the primary factor in the development of benign prostatic hyperplasia (BPH). This accumulation has been explained in terms of an increase in the 5 alpha-reductase enzymatic activity which converts testosterone to DHT and a decrease in the relative activities of the 3 alpha-HSORred and 3 beta-HSORred enzymes. To investigate this hypothesis further, the activities of these two enzymes were studied in the presence and absence of NADPH in benign hyperplastic tissue and in normal peripheral (NPR) and benign hyperplastic periurethral (BPH) tissue taken from the same prostate. The results of these studies demonstrate a several fold increase in the activities of 3 alpha-HSORred and 3 beta-HSORred in the presence of NADPH in the hyperplastic human prostate. This increase in the activities of these two enzymes is found to the same degree in normal peripheral and benign hyperplastic periurethral tissue taken from the same prostate. There was no difference in percent increase in 3 alpha- and 3 beta-diol formation from DHT with NADPH in normal peripheral versus benign hyperplastic periurethral prostatic tissue. In subsequent experiments, Vmax/Km, as an index of the enzymatic capacity of the 3 alpha-HSORred and 3 beta-HSORred enzymes, was determined in both NPR and BPH tissue in media fortified with one mM NADPH. This quotient was found to be essentially the same in NPR and BPH tissue for both the 3 alpha-HSORred and the 3 beta-HSORred. Subsequently, the Vmax/Km value for the 5 alpha-reductase in BPH tissue was found to be equal to the combined Vmax/Km values of the 3 alpha-HSORred and 3 beta-HSORred. The reverse reaction or the conversion of 5 alpha-androstane-3 alpha,17 beta-diol and 5 alpha-androstane-3 beta,17 beta-diol is completely blocked in a medium containing one mM NADPH. These studies suggest that the concentration of DHT in prostatic tissue is dependent on the level of NADPH necessary for the 3 alpha-HSORred and 3 beta-HSORred enzymes to convert DHT to its respective diols.

Renal calculi in spinal cord-injured patient: association with reflux, bladder stones, and foley catheter drainage.

Renal units associated with reflux in the spinal cord-injured (SCI) patient have a greater incidence of kidney stones developing than in the nonrefluxing units. It is logical to assume that SCI patients with persistent reflux and bladder stones treated with cystolitholapaxy would be at even higher risk for kidney stones developing. Of the 898 SCI patients studied (1,793 renal units), in 14.8 percent kidney stones developed. Kidney stones occurred in 161 of 1,517 (10.6%) of nonrefluxing units. Of the 276 renal units with reflux, in 104 (37.7%) an ipsilateral stone developed. Of the 198 patients in whom a kidney stone formed, 56.6 percent managed their bladder with a Foley catheter whereas only 28 percent of 700 patients in whom a stone did not form used a Foley catheter. On evaluating 261 patients (520 renal units) with bladder stones treated with cystolitholapaxy, 62.5 percent of these patients were managed with a Foley catheter. The association between cystolitholapaxy treatment, reflux, and the formation of kidney stones was (1) in 22 of 111 (19.8%) refluxing units, an ipsilateral stone formed; (2) in 70 of 409 (17.1%) units that were nonrefluxing, stones developed; and (3) overall, in 92 (17.7%) renal units, kidney stone developed. We concluded that kidney stone development is significantly increased in the SCI patient with reflux and/or Foley catheter drainage. From these data cystolitholapaxy treatment in the presence of reflux does not appear to increase the development of kidney stones.

Bladder hypocompliance in the spinal cord injury population.

To determine the incidence and effects of low bladder compliance on the upper urinary tracts in spinal cord injury patients, we evaluated the last 254 patients during a 3-year period who underwent a complete urodynamic study, along with an excretory urogram and/or renal ultrasound. Compliance was determined at 100 cc volume. A compliance number of 20 or less was considered low. Of the 254 patients 43 (17 per cent) had low compliant bladders by our definition. Hydronephrosis was present in 56 of the 84 renal units (64 per cent) and reflux was present in 39 (46 per cent). These results are in sharp contrast to the normal compliant group in which 21 per cent of 418 renal units had hydronephrosis and only 6 per cent had reflux. Of the 254 patients over-all 44 had lower motor neuron lesions as determined by no bulbocavernosus reflex as well as a denervated external sphincter; 22 of these 44 patients (50 per cent) had low compliant bladders. Conversely, among the 210 patients with suprasacral lesions only 21 (10 per cent) had low compliant bladders. In conclusion, the development of a low compliant bladder in spinal cord injury patients is not an uncommon event and places the upper urinary tracts at great risk. All attempts must be directed toward maintaining low detrusor pressures, and these patients must have adequate and frequent followup evaluation.

Characterization of a new G6PD variant: G6PD Titusville.

We describe a new glucose-6-phosphate dehydrogenase mutant, G-6-PD Titusville. The propositus is a 7-month-old black male infant with a transient hemolytic episode. The mutant enzyme is characterized by abnormal electrophoretic mobility, thermolability, Km for NADP, abnormal deamino NADP use and a decreased sensitivity to inhibition by NADPH. G-6-PD activity of hemolysate, as measured under optimal in vitro conditions, was not initially decreased, whereas fibroblasts, granulocytes, and platelets showed a markedly decreased level of enzyme activity. These properties identify G6PD Titusville as a unique variant of this X-linked, housekeeping enzyme. We conclude that although the propositus with G6PD Titusville had a transient hemolytic episode, we cannot be certain whether this association was a causative one.

A home-centered instructional communication strategy for severely handicapped children.

Family involvement is an essential element of language intervention with severely handicapped children for several reasons. First, the parent-child interaction is the focus of normal language development, and can be a powerful impetus in language learning for handicapped children. Second, limited generalization and maintenance of skills often occur when they are acquired in environments that do not also teach the appropriate use of skills. Third, parents can be successful intervention agents and may generalize their skills to other interactions with their child. Training conducted in the home must be compatible with that environment: it should involve only those skills that are of immediate use in the home. The Instructional Communication Strategy described herein represents such a program. It is a synthesis of training strategies used with normal and handicapped children, and is applicable regardless of child's level of functioning, age, or handicapping condition. This training model involves considerable modification in the role of speech-language pathologists dealing with the severely handicapped. The professional's skills are best utilized for assessment, program development, monitoring progress, and training specialized skills. The parents provide most of the direct training; however, professionals are also utilized for the child's maximum benefit.