Nonlysosomal, pre-Golgi degradation of unassembled asialoglycoprotein receptor subunits: a TLCK- and TPCK-sensitive cleavage within the ER.

The human asialoglycoprotein receptor subunit H2a is cotranslationally inserted into the ER membrane. When expressed together with subunit H1 in mouse fibroblasts part forms a hetero-oligomer that is transported to the cell surface, but when expressed alone it is all rapidly degraded. Degradation is insensitive to lysosomotropic agents and the undegraded precursor is last detected in the ER region of the cell. Small amounts of an intermediate 35-kD degradation product can be detected (Amara, J. F., G. Lederkremer, and H. F. Lodish. 1989. J. Cell Biol. 109:3315). We show here that the oligosaccharides on both precursor H2a and the 35-kD fragment are Man6-9GlcNAc2, structures typically found in pre-Golgi compartments. Subcellular fractionation shows that the intermediate degradation product does not cofractionate with the lysosomal enzyme beta-galactosidase, but is found in a part of the ER that contains ribosomes. Thus the intermediate degradation product is localized in the ER, indicating that the initial degradation event does take place in the ER. All degradation of H2a, including the initial endoproteolytic cleavage generating the 35-kD intermediate, is blocked by the protease inhibitors N-tosyl-L-lysine chloromethyl ketone and N-tosyl-L-phenylalanine chloromethyl ketone. These drugs do not inhibit ER-to-Golgi transport of H1. Depleting the cells of ATP or inhibiting protein synthesis allows the initial endoproteolytic cleavage to occur, but blocks further degradation of the 35-kD intermediate; thus we can convert all cellular H2 into the 35-kD intermediate. Approximately 50% of H2b, a splicing variant differing from H2a by a five amino acid deletion, can be transported to the cell surface, and the rest appears to be degraded by the same pathway as H2a, both when expressed alone in fibroblasts and together with H1 in HepG2 cells. Addition of N-tosyl-L-lysine chloromethyl ketone or N-tosyl-L-phenylalanine chloromethyl ketone blocks degradation of the approximately 50% that is not transported, but does not affect the fraction of H2b that moves to the Golgi region. Thus, a protein destined for degradation will not be transported to the Golgi region if degradation is inhibited.

Concanavalin A-induced redistribution of surface receptors in Acanthamoeba castellanii at different growth phases.

Concanavalin A (ConA)-induced redistribution of surface receptors has been studied in Acanthamoeba castellanii at different growth phases utilizing double fluorescent techniques and transmission electron microscopy. When the amoebae were incubated with 2 micrograms and 10 micrograms tetramethylrhodamine isothiocyanate (TRITC)-ConA/ml for 4 min and 15 min at 28 degrees C the staining pattern was characterized by various numbers of scattered aggregates of fluorescent ConA. Double labeling of the amoebae showed that the fluorescent aggregates represented internalized label, and the internalization was not preceded by any aggregation of ConA receptors on the cell surface as visualized by incubating with anti-ConA serum followed by fluorescein isothiocyanate-conjugated anti-IgG. Following exposure of the amoebae to 10 micrograms TRITC-ConA/ml for 4 min and 15 min at 28 degrees C intracellular accumulation of some of the fluorescent aggregates in cap-like structures occurred at the logarithmic and postlogarithmic growth phases but not at the early stationary growth phase. Electron microscopic observation of amoebae labeled with ferritin-conjugated ConA at 28 degrees C revealed a uniform surface labeling and an intracellular accumulation of the label in vesicular and tubular structures, and occasionally in cap-like structures. Surface capping of ConA receptors in Acanthamoeba was induced by treating the amoebae with ConA and anti-ConA serum at 0 degrees C followed by incubation at 28 degrees C. The formation of surface caps in Acanthamoeba showed growth-phase dependency, too. The visualization of the surface caps at the electron microscopic level was performed by indirect staining utilizing protein A-colloidal gold.(ABSTRACT TRUNCATED AT 250 WORDS)

Liver transplantation in familial amyloidotic polyneuropathy. Follow-up of the first 20 Swedish patients.

Familial amyloidotic polyneuropathy (FAP) is an inherited fatal form of amyloidosis caused by mutant transthyretin. The disease is characterized by progressive peripheral and autonomic neuropathy. Most of the transthyretin is produced by the liver, and we have shown previously that the metabolic deficiency can be corrected by liver transplantation. In the present study, the clinical results from the first 20 patients who underwent liver transplantation for FAP in Sweden are evaluated. Three of the patients suffered from renal failure and underwent a simultaneous kidney transplantation. Fourteen of the 20 patients (70%) are alive 10-52 months after transplantation. The patients' nutritional status at the time of transplantation had a significant impact on mortality and morbidity (P < 0.007). Long-standing disease was another negative prognostic factor (P < 0.02). One year after transplantation, the nutritional status had improved (P < 0.02). Improvements were also noted in walking capacity and for gastrointestinal and urogenital symptoms. The results show that liver transplantation offers an effective means to treat patients with FAP. The procedure should preferably be performed before the nutritional status is poor and advanced organ dysfunction has developed.

Glutamate-induced currents reveal three functionally distinct NMDA receptor populations in rat dorsal horn - effects of peripheral nerve lesion and inflammation.

The importance of the N-methyl-D-aspartate (NMDA) receptor in various painful conditions is well established. The effects of peripheral nerve lesion or joint inflammation, as models of different pain states, on NMDA receptor-mediated currents and NMDA receptor subunit mRNA expression were therefore studied in acutely dissociated neurones from the rat spinal cord dorsal horn. In the neuronal population from control rats, all four NR2 subunits and both NR1 splice variants assayed were detected. A majority of neurones expressed mRNA for more than one NR2 subunit, and some neurones expressed all four NR2 subunits as well as both NR1 splice variants. The NR2B subunit was the most commonly expressed, while the NR2C was the rarest. Following nerve lesion, fewer neurones expressed NR2A compared to the control. The dose-response curve for glutamate-evoked NMDA receptor-mediated currents in the neurones was best described by a three-component fit, suggesting that three functionally distinct NMDA receptor populations are present in the dorsal horn. Minor changes in the dose-response curve after nerve lesion could not be ascribed with certainty to the lesion. Changes in other parameters of NMDA receptor-mediated currents were observed neither after nerve lesion nor after joint inflammation. In summary, the present work demonstrates that single dorsal horn neurones express mRNA for several NMDA receptor subunits. The glutamate dose-response curves indicate that there are three major types of NMDA receptors present in dorsal horn neurones. We also report a reduced expression of NR2A following peripheral nerve lesion.

Secretory pattern of vasopressin in plasma and cerebrospinal fluid of patients with dementia and of two control groups.

Since the description of its antidiuretic effect in 1913, a variety of functions have been attributed to vasopressin, one of the most controversial throughout the years probably being its effect on memory processes. In an attempt to study the actual secretory rhythm of vasopressin in humans with demonstrated impaired memory, the plasma and cerebrospinal fluid levels of the peptide have been examined during 24 h in a group of patients with dementia, and their values compared with two healthy control groups of young and elderly volunteers. Patients with dementia had higher circulating levels of vasopressin in plasma than the healthy participants and the differences were statistically significant when compared with the healthy elderly (p = 0.003). This difference is not age-related because both groups were in the same age range. A possible explanation could be the higher plasma osmolality measured in the patients with dementia, despite the fact that their levels were within the normal ranges. The different results could not be attributed to changes in electrolytes or blood pressure because these parameters were similar in all groups (p = NS). But more interesting, perhaps, is the secretory pattern found in all three groups. The pattern is biphasic, with two significant peaks: at 16.00 h (p = 0.032) and at night (p = 0.002). This pattern was similar in all cases and in all groups. The total nocturnal secretion of vasopressin is higher than the diurnal secretion (p = 0.02) only in the plasma because the cerebrospinal fluid values were higher during the day.(ABSTRACT TRUNCATED AT 250 WORDS)

Perinatal development of galanin-like immunoreactivity in chromaffin tissues of the rabbit.

We have analyzed the perinatal development of galanin-like immunoreactivity (GAL-LI) and catecholamines (CA) in the paraaortal paraganglia (PGGL) and adrenal glands. In the PGGL, the tissue content of GAL-LI was highest on the day of birth and decreased postnatally. The fetal levels were lower than at birth. In contrast, the content of CA in the PGGL increased with age. In the adrenal glands, the contents of both GAL-LI and CA also increased with age. During the first postnatal week the contents of both GAL-LI and CA in the PGGL were markedly higher than in the adrenal glands. Chromatographic analysis of GAL-LI in extracts of fetal and postnatal rabbit PGGL, respectively, indicated that most of the GAL-LI from both age groups co-eluted with synthetic porcine GAL. An additional, apparently more polar, component was also detected at both ages, which may represent a differently processed form of the peptide. The high content of GAL-LI in the PGGL at birth may reflect an enhanced synthesis associated with birth.

Effects of acute stress on the contents of catecholamines and neuropeptides in chromaffin tissues of the newborn rabbit.

The neuropeptides enkephalin (ENK), galanin (GAL) and neuropeptide Y (NPY) are abundantly expressed in the paraaortic body (PAB) and adrenal glands of the newborn rabbit. To examine whether these neuropeptides are affected by acute stress, we exposed neonatal rabbits to asphyxia, insulin-induced hypoglycemia, and reserpine. Asphyxia, caused by rebreathing for 60 min in an airtight box, reduced the content of catecholamines (CAs) in the adrenal glands and increased ENK-like immunoreactivity (-LI) in the PAB. Insulin-induced hypoglycemia reduced the content of CAs as well as ENK-LI in the adrenal glands. Reserpine caused a marked depletion of the CAs both in the PAB and in the adrenal glands. In contrast, reserpine did not cause any change in the contents of the neuropeptides in either organ. These data indicate that tissue levels of the neuropeptides GAL-LI and NPY-LI, coexisting with CA in the PAB and the adrenal glands, are not biochemically affected by asphyxia, hypoglycemia or reserpine, whereas tissue levels of ENK-LI are reduced by hypoglycemia and, to some extent, are increased by asphyxia. Furthermore, even the CAs in the PAB were unaffected by asphyxia and hypoglycemia. Also, while reserpine reduces CA content, peptide levels are unaffected.

Changes in enkephalin and neuropeptide Y-like immunoreactivity in rabbit chromaffin tissues during perinatal development.

Enkephalin-like immunoreactivity (ENK-LI), neuropeptide Y (NPY)-LI and dopamine-beta-hydroxylase (DBH)-LI were found within the chromaffin cells of both the paraaortic body and the adrenal medulla of the newborn rabbit using immunohistochemistry. Cells positive to DBH-LI were abundant in both the paraaortic body and the adrenal medulla. ENK-LI positive cells were frequent in the paraaortic body, but more sparse in the adrenal medulla. A few cells staining for NPY-LI could be detected in both organs. Some nerve fibers within these organs also contained substance P-LI and calcitonin-gene related peptide-LI. The tissue contents of ENK-LI and NPY-LI, as measured by radioimmunoassay, increased after birth in the adrenal glands and were significantly higher than the fetal levels from 1 week of age. In the paraaortic body the lowest content of ENK-LI was found around birth, whereas the content of NPY-LI was highest at that time. With advancing postnatal age, the content of ENK-LI increased, whereas the content of NPY-LI decreased. At each age, there was a higher content of ENK-LI as compared to NPY-LI in both organs. This indicates that the synthesis of ENK-LI and NPY-LI in the paraaortic body is differently regulated during perinatal development.

Abdominal vessel enhancement with an ultrasmall, superparamagnetic iron oxide blood pool agent: evaluation of dose and echo time dependence at different field strengths.

RATIONALE AND OBJECTIVES: The purpose of the study was to determine the dose and echo time dependence of abdominal vessel enhancement at magnetic resonance (MR) imaging after injection of a blood pool contrast agent at two field strengths. MATERIALS AND METHODS: Sixteen healthy volunteers received NC100150 Injection at three dose levels (1.0 mg, 2.5 mg, and 4.0 mg of iron per kilogram of body weight). Images of the aorta and inferior vena cava (IVC) were obtained at 0.5 or 1.5 T. Four sequences with varying echo times were used with each subject. Signal intensities were recorded from the aorta, IVC, vessel vicinity, air, and a marker outside the patient. Contrast-to-noise ratios (CNRs) were calculated for the vessels. Aortic delineation was subjectively evaluated. RESULTS: Images with the highest mean vessel signal intensities, subjectively assessed as satisfactory for aortic delineation, were obtained with 2.5-4.0 mg of iron per kilogram of body weight at both field strengths. The highest CNR was found with 4.0 mg of iron per kilogram of body weight at 1.5 T. An increase in echo time caused larger signal intensity loss at larger dose levels. The signal intensity from the IVC was higher than that of the aorta at all dose levels, echo times, and field strengths. CONCLUSION: NC100150 Injection is an efficient T1-reducing agent at both 0.5 and 1.5 T. A positive dose response for CNR of the aorta and IVC was seen at 1.5 T.

A cube model for the classification of work with hand tools and the formulation of functional requirements.

In an interdisciplinary research project, a model, visualized as a cube, was developed for the classification and analysis of work with hand tools and for communication of different ways of solving problems related to manual handling. The dimensions of the cube are demands of force, precision and time. Each dimension is divided into three levels of low, moderate and high demands respectively. Preliminary limits are proposed for acceptable and non-acceptable use situations and for situations that have to be investigated further. Using a case study of plate shears as a starting point, various measures of improving the position in the cube are discussed. The hand tool, the workplace, the work organization as well as the user of the hand tool are included in the analysis.

An approach to ergonomics evaluation of hand tools.

Based on current knowledge of cumulative trauma disorders in the hand and forearm, related to the use of hand tools, an analysis was undertaken of variables to be considered in ergonomics evaluation of hand tools. Measurement methods were developed and an evaluation station was implemented. Measurement methods are physical, physiological or psychophysical. They focus on the tool, and on the effect of typical use of the tool on the operator. The evaluation station serves as a resource in the development, selection, and testing of tools for a given purpose.

Liver transplantation for familial amyloidotic polyneuropathy (FAP): a single-center experience over 16 years.

Orthotopic liver transplantation (LTx) is currently the only available treatment that has been proven to halt the progress of familial amyloidotic polyneuropathy (FAP). The aim of this study was to assess mortality and symptomatic response to LTx for FAP. All 86 FAP patients transplanted at our hospital between April 1990 and November 2005 were included in the study. Five patients underwent retransplantation. The 1-, 3- and 5-year patient survival rates in patients transplanted during 1996-2005 were 94.6%, 92.3% and 92.3%, respectively, a significant difference from the rates of 76.7%, 66.7% and 66.7%, respectively, during 1990-1995 (p = 0.0003). Multivariate analysis revealed that the age at the time of LTx (>or=40 years), duration of the disease (>or=7 years) and modified body mass index (mBMI) (<600) were independent prognostic factors for patient survival. A halt in the progress of symptoms was noted in most patients, but only a minority experienced an improvement after LTx. To optimize the posttransplant prognosis, LTx should be performed in the early stages of the disease, and close post-LTx monitoring of heart function by echocardiography and of heart arrhythmia by Holter ECG is mandatory.

Analysis of proteins from membrane-enriched cerebellar preparations by two-dimensional gel electrophoresis and mass spectrometry.

Two-dimensional polyacrylamide gel electrophoresis and mass spectrometry is a powerful combination for the separation of complex protein mixtures in biological samples and the subsequent identification of individual polypeptides. We have used this approach to construct a database of proteins of the porcine cerebellum, with emphasis on membrane-bound proteins, as part of our studies on the structure and function of the central nervous system. We compared the ability of different solubilization conditions (using zwitterionic and nonionic detergents; urea and thiourea) to improve the resolution of high molecular weight and hydrophobic proteins, and found the combination of 3-[(3-cholamidopropyl)dimethylammonio]-1-propane-sulfonate (CHAPS), Tris, thiourea and urea to give the best results in our experiments. As a marker membrane protein, the NR1 subunit of the N-methyl D-aspartate receptor, a 120 kDa hydrophobic protein, was identified using a monoclonal antibody in combination with Western blotting. Sodium chloride treatment of the membrane preparation prior to solubilization caused further enrichment of membrane proteins. Fifty-six spots were identified using matrix-assisted laser desorption/ionization time-of-flight and nanoelectrospray mass spectrometry.

Once hypertensive, always hypertensive? A three year follow-up after stopping medication.

OBJECTIVE: To verify the existence of hypertension in a group of long-term pharmacologically treated hypertensives and to evaluate the possibility of discontinuing their medication. DESIGN: The diagnosis of hypertension was established when after a wash-out period (one month) the blood pressure measured at three consecutive examinations, with at least one week interval between them, was always higher than WHO's reference levels for the diagnosis of hypertension. Those who did not fulfil these criteria would continue, with regular controls, without medication as long as clinically indicated. The final evaluation was done after a three year follow-up. SETTING: The out-patient Hypertensive Unit of the Department of Geriatrics, Skellefteå Hospital, Sweden. PARTICIPANTS: 86 out-patients (33 males and 53 females) aged 68 to 82 years (mean 74) with long-term hypertension sent to our unit by general practitioners in our health district (population 80,000). RESULTS: 34 of the initial 86 patients required medication by the end of the wash-out period. The remaining 52, 16 males and 36 females, continued without medication and after the three year follow up 14 of them were still without it. There was a striking difference between males and females since a significantly higher number of males than females were free of medication at the end of the period (p < 0.001). In those who restarted pharmacological therapy, the period without medication lasted no longer than five months. CONCLUSIONS: Arterial hypertension can easily be over-represented as a diagnosis if not revised when clinically advisable or if established without accurate criteria. The possibility of stopping the antihypertensive medication in old patients is worth considering, particularly in male patients. The dangers of such strategy are practically minimal when regular controls are undertaken during the attempt.

Unfolded H2b asialoglycoprotein receptor subunit polypeptides are selectively degraded within the endoplasmic reticulum.

When expressed alone in fibroblasts, approximately 80% of newly made H2b subunits of the human asialoglycoprotein receptor are retained and degraded in the endoplasmic reticulum (ER), whereas about 20% reaches the plasma membrane (1). Thapsigargin, an inhibitor of the ER Ca2+ ATPase, blocks ER folding of the H1 (2) as well as of the H2b subunit, prevents maturation of H2b, and accelerates ER degradation of newly made H2b. The secretory pathway is normal in thapsigargin-treated cells, as monitored by maturation of the vesicular stomatitis virus G protein. The protease inhibitors TLCK and TPCK block the first step in ER degradation of H2, an endoproteolytic cleavage just exoplasmic to the membrane-spanning domain. In protease inhibitor-treated cells, the approximately 80% of H2b that would normally be degraded remains in the ER; as judged by migration on nonreducing SDS-polyacrylamide gel electrophoresis this H2b is improperly folded. Thus, incorrectly folded H2b is normally subjected to ER degradation. In the presence of thapsigargin H2b cannot fold properly and is degraded within the ER. The preferential ER degradation of misfolded or unfolded membrane proteins demonstrated here, functions as a step in ER quality control.

Endoplasmic reticulum degradation of a subunit of the asialoglycoprotein receptor in vitro. Vesicular transport from endoplasmic reticulum is unnecessary.

The H2a subunit of the human asialoglycoprotein receptor is rapidly degraded from the endoplasmic reticulum (ER) when expressed in CHO15B cells. We have reconstituted ER degradation of H2a in semipermeable cells. At least the initial step in degradation (a proteolytic cleavage inhibited by N alpha-p-tosyl-L-lysine chloromethyl ketone and L-1-tosylamido-2-phenylethyl chloromethyl ketone) can occur in vitro in the presence of guanosine 5'-3-O-(thio)triphosphate or in the absence of ATP and postnuclear supernatant, conditions that do not allow vesicular transport of subunit H1 from the ER to the Golgi. We conclude that vesicular transport from the ER is not required for ER degradation of H2a to occur and thus that it takes place in the ER itself.