Deficient assembly and function of gap junctions in Trf1, a trafficking mutant of the human liver-derived cell line HuH-7.

The Trf1 cell line, selected from the human hepatoma cell line HuH-7, manifests altered trafficking of various plasma membrane proteins. In particular, there is a striking loss of State 2 asialoglycoprotein receptors. This cell line is shown here to also manifest defects in function and assembly of gap junctions comprising connexin43 (Cx43). No alteration of Cx43 expression or phosphorylation was apparent. Nevertheless, immunostaining of Cx43 revealed that fewer and smaller gap junctions were present at appositional membrane areas in Trf1 cells as compared with parental HuH-7. This correlated with a significant attenuation in gap junction-mediated communication between Trf1 cells as demonstrated by markedly decreased dye transfer and their reduced ability to propagate mechanically evoked Ca(2+) waves. Isoelectric focusing (IEF) of Cx43 in HuH-7 cells indicated that the pIs of this protein were significantly lower than that predicted from its amino acid sequence; no differences in pI were evident in Cx43 from Trf1 cells and the HuH-7 cell line. The effects of the Trf1 mutation on assembly and function of gap junctions indicate that this mutation influences trafficking of Cx43. Connexins differ in several respects from other membrane proteins thus far analyzed in Trf1 mutants: gap junctions localize exclusively to the lateral cell surface; they are not glycoproteins; and they do not play a role in endocytic pathways. The disruption of trafficking of Cx43 by this mutation suggests that the Trf1 phenotype is a defect at a common point along the trafficking pathway of cell-surface proteins, irrespective of their ultimate destination on the cell surface or their glycosylation profile.

Importance of early recognition and treatment of deformational plagiocephaly with orthotic cranioplasty.

OBJECTIVES: Infants with positional plagiocephaly often exhibit complex multistructural asymmetries that affect the face and skull base as well as the cranial vault. Dynamic Orthotic Cranioplasty (DOC) was developed as a nonsurgical alternative for the treatment of positional plagiocephaly. The effectiveness of DOC has been discussed elsewhere. The purpose of this study was to assess the influence of factors such as entrance age, treatment time, and initial severity on the effectiveness of correction. METHODS: The study sample consisted of 258 children with cranial vault asymmetry (CVA) treated prior to 1 year of age. In addition, 246 patients (92%) exhibited concurrent skull base (SBA) and orbitotragial depth (OTDA) asymmetries. All patients had been diagnosed with nonsynostotic plagiocephaly, did not have other contributing medical conditions, were compliant with DOC protocol, and had complete anthropometric measurements at entrance and exit from treatment. RESULTS: Mean age at start of treatment was 6.5 (+/-1.9) months (range, 2.8 to 11.0 months), with an average treatment time of 4.1 (+/-2.2) months. The effects of the treatment variables were analyzed using three-way analysis of variance. As expected, initial severity was significantly associated with the amount of correction (p = .0001). However, treatment time was not significant (p > .05). Most importantly, the analysis revealed that, having accounted for initial severity, entrance age had a statistically significant effect [F(1,254) = 8.36, p = .0042] on the correction of CVA. Similar results were identified for both the SBA [F(1,254) = 5.53, p = .0195] and the OTDA [F(1,254) = 5.22, p = .0231] asymmetries. CONCLUSIONS: These findings support clinical observations that earlier intervention results in significantly improved treatment of plagiocephaly, independent of the severity of the presenting asymmetries.

Event-related potentials in schizophrenic patients during a degraded stimulus version of the visual continuous performance task.

Previous studies of the auditory P300 event-related potential (ERP) have reported smaller amplitudes in chronic schizophrenics but similar consistencies have not been observed with visual P300s. This study examined P300s in symptomatically stable, medicated, chronic schizophrenics (n = 14) and normal controls (n = 14) performing a visual continuous performance task utilizing degraded stimuli to burden encoding processes. Performance analysis found slower response times, fewer target detections and more false alarms in patients than in controls. Analysis of ERPs showed P300 amplitudes of schizophrenics to be significantly smaller than those of controls and, unlike controls, schizophrenics failed to exhibit significant target vs. non-target P300 amplitude differences. Discriminant analysis indicated target and non-target midline (Fz, Cz, Pz) P300 amplitudes together correctly classified all patients and controls. Exploratory topographic analysis indicated that P300 amplitudes were not asymmetrical in patients, as has been observed with auditory P300s, and, unlike the performance measures, the P300s did not correlate with the patient's positive or negative symptom ratings. The implications of these findings are described in relation to attentional disturbances and trait versus state issues in schizophrenics.

Treatment of craniofacial asymmetry with dynamic orthotic cranioplasty.

Dynamic Orthotic Cranioplasty (DOC) was developed to treat craniofacial deformities associated with positional plagiocephaly. This investigation describes the treatment of more than 750 patients with the DOC Band since 1988. All patients undergoing DOC treatment were fit with a custom fabricated orthosis made from a plaster impression taken from the infant's head. When the orthosis was applied, the corrective pressure was directed to hold growth at the calvarial prominences and redirect symmetrical growth. A detailed medical history was obtained and anthropometric measurements were taken at start, exit, 12, 18, and 24 months follow-up. This information was recorded in a database created in Microsoft Excel. Mean length of treatment was 4.3 months with an average entrance age of 6.9 months. Analysis of anthropometric data showed significant reduction in mean cranial vault, skull base, and facial asymmetries. Correction of the more difficult skull base was documented with computed tomography. Our anthropometric and clinical observations document complete or near complete correction of asymmetry for a wide variety of head shapes. Based on the results of this investigation, we are able to support the earlier claims of our pilot study, which concluded that DOC is effective in the treatment of positional plagiocephaly.

Localization of procollagen I in the lysosome/endosome system of human fibroblasts.

A significant amount of newly synthesized collagen is degraded intracellularly rather than secreted, but there is controversy about whether this process occurs in the lysosomes. We addressed this problem using confocal microscopy and immunofluorescence imaging to study the distribution of procollagen I in the Golgi and the lysosome/endosome system of cultured human fibroblasts. Cells were incubated under basal conditions and then permeabilized and exposed to fluorescently tagged probes for procollagen, Golgi markers (Helix pomatia binding protein or beta-coatamer protein), and lysosome/endosome markers (cathepsin B or LAMP-2). Strong signals for procollagen codistributed with the Golgi and lysosome/endosome markers. Of note, many structures were positive for procollagen and lysosome/endosome markers but not for Golgi markers. When cells were incubated with the proline analog cis-hydroxyproline, which inhibits correct triple helix formation and increases intracellular degradation, the amount of procollagen codistributing with the lysosome/endosome markers increased greatly. Similar results were obtained in I-cells, which do not have functioning lysosomal hydrolases. These findings strongly indicate that the lysosome/endosome system participates in the intracellular degradation of newly synthesized procollagen and that trafficking of procollagen to the lysosome/endosome system does not depend on the cells having active lysosomal hydrolases. We present a model that integrates our findings with other work and resolves inconsistencies in the literature. This model postulates the existence of three separate degradation paths for newly synthesized procollagen. In addition to the endosome/lysosome system, degradation also takes place in the proximal region of the secretory pathway such as the endoplasmic reticulum, cis-Golgi network, or cis-Golgi and in a distal region of the secretory pathway such as the trans-Golgi or trans-Golgi network.

Treatment of positional plagiocephaly with dynamic orthotic cranioplasty.

Dynamic orthotic cranioplasty (DOC) was developed to treat asymmetrical head shape of a nonsynostotic origin, which is defined by the term positional plagiocephaly. These positional deformations have been found to correlate with a number of environmental factors. Infants with positional plagiocephaly may exhibit complex multistructural asymmetry affecting the cranial vault, face, and skull base, or expression may be local in nature. Between 1988 and 1993, we performed DOC on 124 infants with positional plagiocephaly. Through clinical, anthropometric, radiographic, and statistical evaluation, we found that DOC corrects positional deformation of the cranial vault, skull base, and upper face, with no evident relapse following treatment. The design and the global approach to deformation address a wide spectrum of abnormal head shapes. The procedure is dynamic and customized, and it does not rely on passive growth alone for improvement.

Brefeldin A inhibits degradation as well as production and secretion of collagen in human lung fibroblasts.

Brefeldin A (BFA) inhibits protein secretion, collapses the Golgi complex into the endoplasmic reticulum (ER), causes redistribution of processing enzymes normally resident in the Golgi to the ER, and uncouples the proximal and distal regions of the secretory pathway. We used BFA to determine where intracellular degradation of newly synthesized collagen degradation occurs. In normal human fetal lung fibroblasts, BFA (50 ng/ml) completely blocked collagen secretion and reduced collagen production by two-thirds. In cells synthesizing collagen under normal conditions, intracellular degradation was about 16%; BFA (50 ng/ml) reduced degradation to less than 5%. In cells induced to synthesize structurally abnormal collagen (by incubation with the proline analog cis-hydroxyproline), degradation was approximately 33%; BFA reduced this level to less than 10%. When the y axes were scaled appropriately, the dose-response curves for collagen degradation +/- cis-hydroxyproline versus BFA concentration coincided. A pulse-chase experiment demonstrated that BFA did not inhibit hydroxylation of prolyl residues, a major posttranslational modification of collagen that occurs in the ER, and that inhibition of degradation was independent of inhibition of collagen synthesis. Immunofluorescence examination revealed that BFA redistributed Golgi glycoproteins to the ER. At the ultrastructural level, Golgi complex could not be found in fibroblasts exposed to BFA for 1 h; however, clusters of small vesicles were observed. A different structure, comprising one or two lamellae and resembling a partial Golgi complex, was observed in cells incubated with BFA for 6 h. This structure was adjacent to ER but far from the nucleus. In addition, the ER was devoid of ribosomes. The inhibition of intracellular collagen degradation by BFA indicates that collagen degradation does not occur in the ER. Rather, it suggests that collagen degradation occurs beyond the BFA block, perhaps in the trans-Golgi network.

Collagen degradation in I-cells is normal.

There is evidence that lysosomal proteases mediate the intracellular degradation of structurally abnormal collagen. I-Cell disease (Mucolipidosis II) is characterized by marked deficiency of many lysosomal hydrolases, including the collagenolytic enzyme cathepsin B. The experiments reported here tested the hypothesis that degradation of abnormal collagen would be severely impaired in I-cells. Skin fibroblasts from 3 patients with I-cell disease were incubated with and without cis-hydroxyproline, a proline analog that causes structural abnormalities in collagen, and [14C]proline. The amount of [14C]hydroxyproline in a low molecular weight fraction relative to total [14C]hydroxyproline was used as a measure of intracellular collagen degradation. Levels of degradation were significantly higher in I-cells exposed to cis-hydroxyproline than in cells incubated without the analog. Similar data were obtained for normal human fetal lung fibroblasts incubated under the same conditions. Degradation of [125I]-epidermal growth factor was used to assess the functionality of the lysosomal pathway for protein degradation, and it was much lower in I-cells than in normal cells. It can be concluded that a completely functional complement of lysosomal enzymes is not necessary for structurally abnormal collagen to be degraded intracellularly; the data suggest that a nonlysosomal pathway exists.

Ammonium chloride inhibits basal degradation of newly synthesized collagen in human fetal lung fibroblasts.

The objective of this work was to characterize basal degradation of newly synthesized collagen in human fetal lung fibroblasts. Analysis of 22 separate determinations showed that in cells incubated under normal conditions, the level of intracellular degradation was normally distributed with a mean of 15.2% and a standard deviation of 2.6%. Within each experiment, however, the uncertainty (standard deviation) in determining degradation was very small, usually less than 1.5%. Consideration of the large variation between experiments and the ability of our analytic technique to detect small, but "statistically significant," differences between groups within the same experiment led us to formulate two criteria for determining whether degradation measured in cultures exposed to some agent differs in a "biologically significant" way from degradation measured in control cultures. These criteria were used to evaluate the effects of the following proteinase inhibitors on basal degradation: NH4Cl, which increases the pH of subcellular compartments that are normally acidic; and leupeptin and Na-p-tosyl-L-lysine chloromethyl ketone (TLCK), which are inhibitors of lysosomal cathepsins (B and L) that degrade collagen. NH4Cl (16 mM) lowered degradation to an extent that was both statistically and biologically significant, but neither leupeptin nor TLCK affected degradation. The effect of NH4Cl on degradation was independent of its inhibitory effects on production of collagen, protein, and ATP. These results suggest that basal degradation occurs in, or beyond, an acidic (i.e., NH4Cl-sensitive) but nonlysosomal compartment of the cell, and that NH4Cl inhibits processing within, or transport to, that compartment. This is the first report of an agent that inhibits basal degradation of newly synthesized collagen in soft tissue fibroblasts.

Psychological normality and abnormality in persistent headache patients.

One hundred and three patients referred to a neurological outpatient clinic were examined to assess the relationship between persistent headache, not due to significant physical illness, and emotional disturbance. Overall, the patients showed slightly more evidence of emotional disturbance than a general practice population but less than psychiatric outpatients. Thus, with cut-off points of 4/5 and 9/10 on the General Health Questionnaire (GHQ 28) the whole group had 52% or 20% of psychiatric 'cases' respectively. On the Crown-Crisp Experiential Index the 70 females had mean total scores of 37.19 +/- 11.11 and the 33 males had scores of 31.79 +/- 11.36. In addition the childhood experiences measured by the Parental Bonding Instrument appeared to be normal. Seven patients had significant depressive illness, according to the Levine-Pilowsky Depression Questionnaire. Statistically significant differences in psychological state did not emerge between the diagnoses of cluster headache, classical migraine, common migraine, tension headache or combined headache. However, negative correlations were found between the duration of illness and measures of anxiety. It is concluded that although the emotional state contributes to the development of pain and headache in some patients, there are others in whom comparable headaches are unlikely to be due to emotional factors. Selection effects are held to be important and some of the emotional changes will vary at different phases of a chronic disorder. A new symptom may initially cause anxiety but when a condition persists some patients will be increasingly concerned or depressed whilst others develop tolerance for the situation.

Effect of surveillance on the number of hysterectomies in the province of Saskatchewan.

In 1972 the College of Physicians and Surgeons of Saskatchewan appointed a committee to study hysterectomies because the Saskatchewan Department of Health had data showing that the annual number of hysterectomies carried out in the province had increased by 72.1 per cent between 1964 and 1971, whereas the number of women over 15 years of age had increased by 7.6 per cent. The committee compiled a list of indications for hysterectomy. Any hysterectomy carried out for one of these reasons was classified as justified, and the remainder as unjustified. Five hospitals were reviewed in 1970 and a further two in 1973. In 1974, all seven hospitals were reviewed again. In these hospitals, the average proportion of unjustified hysterectomies had dropped from 23.7 per cent at the time of the first review to 7.8 per cent in 1974. The total number of hysterectomies in the province dropped by 32.8 per cent between 1970 and 1974.