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Biomedical subjects

T Nilsson

Publications and source records attributed to T Nilsson.

At least 145 records · Page 8Linked to original sources
PubMed1994

Kin recognition between medial Golgi enzymes in HeLa cells.

The medial Golgi enzymes, N-acetylglucosaminyltransferase I (NAGT I) and mannosidase II (Mann II), and the trans Golgi enzyme, beta-1,4-galactosyltransferase (GalT) were each retained in the endoplasmic reticulum (ER) by grafting on the cytoplasmic tail of the p33 invariant chain. Transient and stable expression of p33/NAGT I in HeLa cells caused relocation of endogenous Mann II to the ER and transient expression of p33/Mann II had a similar effect on endogenous NAGT I. Neither of these endogenous medial enzymes were affected by transient expression of p33/GalT. These data provide strong evidence for kin recognition between medial Golgi enzymes and suggest a role for them in the organization of the Golgi stack.

Amino Acid Sequence↗
PubMed1994

Simultaneous oscillations of cytoplasmic free Ca2+ concentration and Ins(1,4,5)P3 concentration in mouse pancreatic beta-cells.

Changes in the cytoplasmic free Ca2+ concentration ([Ca2+]i) in stimulated cells are often oscillatory, but the mechanisms that drive these oscillations are still a matter of controversy: different models of the generation of these [Ca2+]i oscillations make different assumptions as to whether oscillations in Ins(1,4,5)P3 concentration are necessary for this process. We have looked for changes in inositol polyphosphate levels that might occur in suspensions of murine pancreatic beta-cells when these cells are induced to display synchronized oscillations in [Ca2+]i by the sequential addition of glucose, an alpha 2-adrenergic stimulus and extracellular Ca2+. The intracellular level of Ins(1,4,5)P3 oscillated in a manner approximately in synchrony with changes in [Ca2+]i. Oscillations in the levels of Ins(1,4,5)P3 metabolites [Ins(1,3,4)P3 and inositol bisphosphates] were slightly delayed relative to the Ins(1,4,5)P3 oscillations, and the concentration of Ins(1,3,4,5,6)P5 remained approximately constant during the [Ca2+]i oscillations. These results demonstrate that [Ins(1,4,5)P3] and [Ca2+]i oscillate in synchrony in at least one type of cell. Whether such oscillations in intracellular [Ins(1,4,5)P3] provide a primary driving force for [Ca2+]i oscillations either in beta-cells or in other stimulated cells remains to be determined. Even if they do not, the [Ins(1,4,5)P3] oscillations will at least provide an amplifying influence on the [Ca2+]i changes.

Animals↗
PubMed1994

Toxic PCB congeners and organochlorine pesticides in Italian human milk.

Human milk from four major Italian cities was analyzed for individual congeners of polychlorinated biphenyls (PCB), DDT, DDE, hexachlorobenzene, and beta-hexachlorocyclohexane. Minimum and maximum concentrations in milk from individual mothers for most compounds ranged between one order of magnitude below and above the mean value of all mothers. Good agreements were found between results from pooled samples and mean values of individual samples. No statistically significant difference between cities was found and the levels in milk from Italian mothers did not differ significantly from published levels from other parts of the world. Principal component analyses revealed that the PCB congener distribution pattern was very similar in all mothers, independent of location. Average concentrations in milk from the four cities were 19(+/- 5) micrograms liter-1 total PCB or 3.2(+/- 0.8) ng liter-1 toxicity equivalents according to the most conservative TCDD toxicity equivalent factors of PCBs proposed in the literature, 70(+/- 18) micrograms liter-1 DDE, 4.5(+/- 1.2) micrograms liter-1 DDT, 5.6(+/- 1.9) micrograms liter-1 HCB, and 4.4(+/- 1.7) micrograms liter-1 beta-hexachlorocyclohexane.

Female↗
PubMed1994

Oscillations in cytoplasmic free calcium concentration in human pancreatic islets from subjects with normal and impaired glucose tolerance.

Plasma insulin levels in healthy subjects oscillate and non-insulin-dependent diabetic patients display an irregular pattern of such oscillations. Since an increase in cytoplasmic free Ca2+ concentration ([Ca2+]i) in the pancreatic beta cell is the major stimulus for insulin release, this study was undertaken to investigate the dynamics of electrical activity, [Ca2+]i-changes and insulin release, in stimulated islets from subjects of varying glucose tolerance. In four patients it was possible to investigate more than one of these three parameters. Stimulation of pancreatic islets with glucose and tolbutamide sometimes resulted in the appearance of oscillations in [Ca2+]i, lasting 2-3 min. Such oscillations were observed even in some islets from patients with impaired glucose tolerance. In one islet from a diabetic patient there was no response to glucose, whereas that islet displayed [Ca2+]i-oscillations in response to tolbutamide, suggesting that sulphonylurea treatment can mimic the complex pattern of glucose-induced [Ca2+]i-oscillations. We also, for the first time, made patch-clamp recordings of membrane currents in beta-cells in situ in the islet. Stimulation with glucose and tolbutamide resulted in depolarization and appearance of action potentials. The islet preparations responded to stimulation with a number of different secretagogues with release of insulin. The present study shows that human islets can respond to stimulation with glucose and sulphonylurea with oscillations in [Ca2+]i, which is the signal probably underlying the oscillations in plasma insulin levels observed in healthy subjects. Interestingly, even subjects with impaired glucose tolerance had islets that responded with oscillations in [Ca2+]i upon glucose stimulation, although it is not known to what extent the response of these islets was representative of most islets in these patients.

Aged↗
PubMed1994

Retention and retrieval in the endoplasmic reticulum and the Golgi apparatus.

Resident proteins of the exocytic pathway contain at least two types of information in their primary sequence for determining their subcellular location. The first type of information is found at the carboxyl terminus of soluble proteins of the endoplasmic reticulum (ER) and in the cytoplasmic domain of some ER and Golgi membrane proteins. It acts as a retrieval signal, returning proteins that have left the compartment in which they reside. The second type of information has been found in the membrane-spanning domain of several ER and Golgi proteins and, though the mechanism by which it operates is still unclear, it acts as a retention signal, keeping the protein at a particular location within the organelle. The presence of both a retrieval signal and a retention signal in a trans-Golgi network resident protein suggests that more than one mechanism operates to ensure correct localization of resident proteins along the exocytic pathway.

Amino Acid Sequence↗
PubMed1994

Isolation of a matrix that binds medial Golgi enzymes.

Rat liver Golgi stacks were extracted with Triton X-100 at neutral pH. After centrifugation the low speed pellet contained two medial-Golgi enzymes, N-acetylglucosaminyltransferase I and mannosidase II, but no enzymes or markers from other parts of the Golgi apparatus. Both were present in the same structures which appeared, by electron microscopy, to be small remnants of cisternal membranes. The enzymes could be removed by treatment with low salt, leaving behind a salt pellet, which we term the matrix. Removal of salt caused specific re-binding of both enzymes to the matrix, with an apparent dissociation constant of 3 nM for mannosidase II. Re-binding was abolished by pretreatment of intact Golgi stacks with proteinase K, suggesting that the matrix was present between the cisternae.

Amino Acid Sequence↗
PubMed1994

The TGN38 glycoprotein contains two non-overlapping signals that mediate localization to the trans-Golgi network.

The membrane-spanning and cytoplasmic domains of CD4 and CD8 were replaced by those of TGN38. After transient expression in HeLa cells, the location of the hybrid proteins was determined using immunofluorescence and quantitative immuno-electron microscopy, FACS analysis and metabolic labeling. The membrane-spanning domain was found to contain a signal that localized hybrid proteins to the TGN. This was in addition to the signal previously identified in the cytoplasmic domain (Bos, K., C. Wraight, and K. Stanley. 1993. EMBO (Eur. Mol. Biol. Organ) J. 12:2219-2228. Humphrey, J. S., P. J. Peters, L. C. Yuan, and J. S. Bonifacino. 1993. J. Cell Biol. 120:1123-1135. Wong, S. H., and W. Hong. 1993. J. Biol. Chem. 268:22853-22862). The different properties of these two signals suggest that each operates by a different mechanism.

Amino Acid Sequence↗
PubMed1994

Localization of the Lys, Asp, Glu, Leu tetrapeptide receptor to the Golgi complex and the intermediate compartment in mammalian cells.

The carboxyl-terminal Lys-Asp-Glu-Leu (KDEL), or a closely-related sequence, is important for ER localization of both lumenal as well as type II membrane proteins. This sequence functions as a retrieval signal at post-ER compartment(s), but the exact compartment(s) where the retrieval occurs remains unresolved. With an affinity-purified antibody against the carboxyl-terminal sequence of the mammalian KDEL receptor, we have investigated its subcellular localization using immunogold labeling on thawed cryosections of different tissues, such as mouse spermatids and rat pancreas, as well as HeLa, Vero, NRK, and mouse L cells. We show that rab1 is an excellent marker of the intermediate compartment, and we use this marker, as well as budding profiles of the mouse hepatitis virus (MHV) in cells infected with this virus, to identify this compartment. Our results demonstrate that the KDEL receptor is concentrated in the intermediate compartment, as well as in the Golgi stack. Lower but significant labeling was detected in the rough ER. In general, only small amounts of the receptor were detected on the trans side of the Golgi stack, including the trans-Golgi network (TGN) of normal cells and tissues. However, some stress conditions, such as infection with vaccinia virus or vesicular stomatitis virus, as well as 20 degrees C or 43 degrees C treatment, resulted in a significant shift of the distribution towards the trans-TGN side of the Golgi stack. This shift could be quantified in HeLa cells stably expressing a TGN marker. No significant labeling was detected in structures distal to the TGN under all conditions tested. After GTP gamma S treatment of permeabilized cells, the receptor was detected in the beta-COP-containing buds/vesicles that accumulate after this treatment, suggesting that these vesicles may transport the receptor between compartments. We propose that retrieval of KDEL-containing proteins occurs at multiple post-ER compartments up to the TGN along the exocytotic pathway, and that within this pathway, the amounts of the receptor in different compartments varies according to physiological conditions.

Animals↗
PubMed1994

Impaired nerve conduction in the carpal tunnel of platers and truck assemblers exposed to hand-arm vibration.

OBJECTIVES: The goal of this study was to assess the relative risks of sustaining impaired nerve conduction in the hands among vibration-exposed persons as opposed to nonvibration-exposed referents. METHODS: In a cross-sectional study design platers and truck assembly workers were contrasted to office workers. The 4-h frequency-weighted vibration was 4.6 m.s-2 and 1.0 m.s-2 for the platers and assemblers, respectively. The ergonomic factors were measured as forced grip time. The conduction velocity, distal latency time, and amplitude of the median nerve were measured for both hands. RESULTS: The sensory nerve conduction velocity was slower in the right hand than in the left. An increased risk of prolonged latency time was found for the platers and assemblers when contrasted to the office workers. The relative risks (rate ratios) of impaired nerve conduction for the carpal tunnel segment varied between 1.4 and 2.0 for the distal latency and between 0.9 and 1.7 for the nerve conduction velocity. The rate ratios were generally higher for the left-hand side than for the right-hand side. The risks were not proportional to either the weighted or unweighted vibration exposure. CONCLUSIONS: The contributions from vibration and ergonomic factors to impaired nerve conduction velocity were inseparable in this study. Ergonomic factors such as forceful gripping and extreme positions, apart from vibration exposure, may be strong determinants of impaired nerve conduction in the carpal tunnel area.

Adult↗
PubMed1993

Cytochrome bo from E. coli does not exhibit the same proton transfer characteristics as the bovine cytochrome c oxidase during oxygen reduction.

The reaction where fully reduced cytochrome bo from E. coli partially reduces dioxygen has been characterized with respect to the kinetics of the associated proton uptake, and with respect to the pH- and D2O-sensitivity of the electron transfer reactions. A monophasic proton uptake with a rate constant of about 8 x 10(3) s-1 and a stoichiometry of 0.8 H+/bo were recorded, using the indicator dye, Cresol red, at pH 8.2. The electron transfer reactions were independent of pH in the range 6.0-9.5 and were not affected by exchanging H2O to D2O as solvent. Comparison of these results with those obtained in an earlier investigation of the bovine cytochrome c oxidase [(1992) Biochemistry 31, 11853-11859], indicates differences between the two oxidases with respect to the role of protons in oxygen reduction and/or the mechanism of proton uptake from the medium.

Animals↗
PubMed1993

Flow-flash study of the reaction between cytochrome bo and oxygen.

The reaction between reduced cytochrome bo from Escherichia coli and oxygen has been studied using flash photolysis of the CO complex of the reduced protein after rapid mixing with oxygen. Absorbance changes were monitored in the alpha and Soret spectral regions. Two kinetic phases taking place at catalytically competent rates could be detected. The apparent rate constant obtained for both the first and second phase showed a hyperbolic dependence on the oxygen concentration. For the first phase, we obtained limiting first- and second-order rate constants at saturating and low oxygen concentrations of 4.5 x 10(4) s-1 and 1.6 x 10(8) M-1 s-1, respectively. The corresponding values for the second phase were 5 x 10(3) s-1 and 1.7 x 10(7) M-1 s-1. The first phase accounted for 30% of the total absorbance change in the Soret band (430 nm) and 15% of the total absorbance change in the alpha band (555 nm). These reactions are followed by a very slow phase with a lifetime of about 1 s. We have also studied the interaction between the fully oxidized enzyme and hydrogen peroxide, and we have found that peroxide binding induces an absorbance increase in the alpha band and a red shift of the Soret band. A consideration of the magnitude of the absorbance changes taking place during the first phase suggests that this reaction includes at least partial oxidation of the low-spin cytochrome b.

Cytochrome b Group↗
PubMed1993

Viral and cellular factors influence the activity of the Epstein-Barr virus BCR2 and BWR1 promoters in cells of different phenotype.

Transformation of B-lymphocytes by Epstein-Barr virus (EBV) is characterized by the expression of six viral nuclear antigens (EBNA1 to EBNA6) which are encoded by messages derived from long primary transcripts initiated at one of two promoters located in the BamHI C (BCR2) and BamHI W (BWR1) regions of the viral genome. The BWR1 promoter is preferentially utilized during the initial phases of EBV infection, whereas the BCR2 promoter is almost invariably used in transformed lymphoblastoid cell lines (LCLs). In order to gain some insight into the molecular mechanisms underlying promoter usage we have analyzed the activity of reporter plasmids carrying different parts of the BWR1 and BCR2 regulatory sequences in EBV-negative and EBV-carrying B cell lines that, on the basis of their surface marker expression, are representative of different stages of B cell activation/differentiation. We show that: (i) there is an inverse correlation between the activity of BWR1 and oriP-containing BCR2 reporter plasmids in cell lines expressing a BL group I versus a group III phenotype, the BWR1 promoter being virtually inactive in group III cells; (ii) BCR2 reporter plasmids devoid of the oriP region are active in EBV-negative cell lines and EBV-positive cells expressing a group I or group II phenotype and virtually inactive in BL group III cells and LCLs, suggesting that cellular factors are required for activation of BCR2 promoter elements. These factors are lost upon progression to a group III phenotype); (iii) expression of EBNA2 is sufficient to activate reporter plasmids containing the proximal part of the BCR2 promoter in EBV negative cells, whereas coexpression of EBNA2 and EBNA1 is required to activate the promoter in oriP-containing plasmids; (iv) the 30-bp repeat region of oriP acts as a negative cis-element on downstream promoters but is transformed into a transcriptional enhancer by the concerted action of EBNA1 and cellular factors. There was a poor correlation between the activity of exogenous reporter plasmids and endogenous BWR1 and BCR2 promoters in phenotypically different EBV-positive cell lines. The presence of the appropriate trans-acting factors was not sufficient to activate the endogenous BWR1 and BCR2 promoters in BL cells expressing a group I phenotype.

Antigens, Viral↗
PubMed1993

Cell phenotype-dependent control of Epstein-Barr virus latent membrane protein 1 gene regulatory sequences.

Growth transformation-associated Epstein-Barr virus-encoded genes are differentially regulated depending on the host cell phenotype. We have previously identified an LMP1 regulatory region (LRS) 5' of the LMP1 gene (+40 to -634) and analyzed its role in transcription initiation in the EBV-negative Burkitt's lymphoma line DG 75 (Fåhraeus et al., 1990a). In order to investigate the cell phenotype dependence of LMP1 gene regulation we have now compared the activity of positive and negative cis-acting LRS elements in cell lines of B lymphoid and epithelial cell origin in the presence and absence of the virus-encoded nuclear antigen EBNA2. Our results show that reporter plasmids that contain only the -54/+40 region of LRS are active in all tested cell lines. Furthermore, the previously identified negative cis-elements in the -144/-54 region were found to suppress promoter activity independent of the cell phenotype. EBNA2 was able to override the effect of the negative elements in all lines of B-cell origin, whereas it had no effect in epithelial lines. The positive effect of EBNA2 was mediated by cis-acting elements in the -214/-144-bp region. In all six tested cell lines of epithelial origin, reporter plasmids that carried the full-length LRS were active, independent of EBNA2. The LMP1 promoter in these constructs was activated by the concerted action of EBNA2-independent, positive elements in the -214/-144 and -324/-214 positions that counteracted the effect of the negative LRS elements.

Animals↗
PubMed1993

Overlapping distribution of two glycosyltransferases in the Golgi apparatus of HeLa cells.

Thin, frozen sections of a HeLa cell line were double labeled with specific antibodies to localize the trans-Golgi enzyme, beta 1,4 galactosyltransferase (GalT) and the medial enzyme, N-acetylglucosaminyltransferase I (NAGT I). The latter was detected by generating a HeLa cell line stably expressing a myc-tagged version of the endogenous protein. GalT was found in the trans-cisterna and trans-Golgi network but, contrary to expectation, NAGT I was found both in the medial- and trans-cisternae, overlapping the distribution of GalT. About one third of the NAGT I and half of the GalT were found in the shared, trans-cisterna. These data show that the differences between cisternae are determined not by different sets of enzymes but by different mixtures.

Base Sequence↗
PubMed1993

Retrieval of transmembrane proteins to the endoplasmic reticulum.

A COOH-terminal double lysine motif maintains type I transmembrane proteins in the ER. Proteins tagged with this motif, eg., CD8/E19 and CD4/E19, rapidly receive post-translational modifications characteristic of the intermediate compartment and partially colocalized to this organelle. These proteins also received modifications characteristic of the Golgi but much more slowly. Lectin staining localized these Golgi modified proteins to ER indicating that this motif is a retrieval signal. Differences in the subcellular distribution and rate of post-translational modification of CD8 maintained in the ER by sequences derived from a variety of ER resident proteins suggested that the efficiency of retrieval was dependent on the sequence context of the double lysine motif and that retrieval may be initiated from multiple positions along the exocytotic pathway.

3T3 Cells↗