Assembly of the ligand-binding conformation of Mr 46,000 mannose 6-phosphate-specific receptor takes place before reaching the Golgi complex.

The early steps in the biosynthesis of Mr 46,000 mannose 6-phosphate-specific receptor (MPR 46) have been studied by in vivo labeling of transfected BHK cells. The acquisition of phosphomannan-binding activity was compared with changes in protein structure and posttranslational modifications of MPR 46. Intramolecular disulfide bonds were formed before MPR 46 acquired a ligand-binding conformation. A conformational change that resulted in increased trypsin resistance, formation of highly immunogenic epitopes and assembly to noncovalently linked homodimers was observed almost simultaneously with the acquisition of ligand-binding activity. MPR 46 was shown to acquire ligand-binding activity before N-linked oligosaccharides were processed to complex-type forms. Maturation of the ligand-binding conformation was observed under conditions where transport to the Golgi was blocked by lowering the temperature to 16 degrees C, or by addition of brefeldin A or dinitrophenol to the medium at 37 degrees C. This suggests that receptor maturation and assembly take place before reaching the Golgi complex. The affinity towards phosphomannan-containing ligands was shown to be similar for the high-mannose and complex-glycosylated forms of MPR 46.

Differences in the endosomal distributions of the two mannose 6-phosphate receptors.

Multiple immunolabeling of cryosections was performed to compare the subcellular distributions of the two mannose 6-phosphate receptors (MPRs) involved in the intracellular targeting of lysosomal enzymes: the cation-dependent (CD) and cation-independent (CI) MPR. In two cell types, the human hepatoma cell line HepG2 and BHK cells double transfected with cDNA's encoding for the human CD-MPR and CI-MPR, we found the two receptors at the same sites: the trans-Golgi reticulum (TGR), endosomes, electron-dense cytoplasmic vesicles, and the plasma membrane. In the TGR the two receptors colocalized and were concentrated to the same extent in the same HA I-adaptor positive coated buds and vesicles. Endosomes were identified by the presence of exogenous tracers. The two MPR codistributed to the same endosomes, but semiquantitative analysis showed a relative enrichment of the CI-MPR in endosomes containing many internal vesicles. Two endosomal subcompartments were discerned, the central vacuole and the associated tubules and vesicles (ATV). We found an enrichment of CD-MPR over CI-MPR in the ATV. Lateral segregation of the two receptors within the plane of membranes was also detected on isolated organelles. Double immunolabeling for the CD-MPR and the asialoglycoprotein receptor, which mainly recycles between endosomes and the plasma membrane, revealed that these two receptors were concentrated in different subpopulations of endosomal ATV. The small GTP-binding protein rab4, which has been shown to mediate recycling from endosomes to the plasma membrane, was localized at the cytosolic face of many endosomal ATV. Quantitative analysis of double-immunolabeled cells revealed only a limited codistribution of the MPRs and rab4 in ATV. These data suggest that the two MPRs exit the TGR via the same coated vesicles, but that upon arrival in the endosomes CD-MPR is more rapidly than CI-MPR, segregated into ATV which probably are destined to recycle MPRs to TGR.

Secretogranins I and II: two tyrosine-sulfated secretory proteins common to a variety of cells secreting peptides by the regulated pathway.

We report on the biochemical and immunological properties as well as on the cellular and subcellular distribution of two proteins, called secretogranins I and II. These proteins specifically occur in a wide variety of endocrine and neuronal cells that package and sort regulatory peptides into secretory granules. Both secretogranins take the same intracellular route as the peptides and are also sorted into secretory granules. Secretogranins I and II are biochemically and immunologically distinct proteins and differ from chromogranin A. Yet, these three proteins are similar to each other in many respects and therefore constitute one class of proteins. A remarkable feature of this protein class is a very acidic pI, brought about by a high content of acidic amino acids as well as by phosphorylation on serine and sulfation on tyrosine and O-linked carbohydrate. As a result, this class of proteins has a high net negative charge even at the acidic pH of the trans Golgi cisternae. We discuss the possibility that this property of the proteins may point to a role in the packaging of regulatory peptides into secretory granules.

Effective diffusivities and mass fluxes in fungal biopellets.

Mass transport within biological aggregates is a key process that can determine overall turnover rates in submerged cultivations. A parameter commonly used for its description is the effective diffusion coefficient D(eff), which is highly dependent on biomass density and structure. Different approaches have been used to estimate or measure D(eff), yet the data still shows broad scattering. This study provides experimental data on effective diffusivities of oxygen within fungal pellets. A correlation is found with the hyphal gradient (dh/dr), which is a morphological parameter describing the structure of the pellet periphery. Furthermore, the dependency of D(eff) on fluid dynamic conditions at the pellet is investigated. The comparison of the results with data from literature clearly demonstrates the influence of the experimental methodology applied for determination of D(eff). Moreover, it is shown that while diffusion limitation of whole pellets is mainly a function of size, the influence of advection in the outer zone of pellets that is supplied with oxygen is actually rather high. Thus, it is concluded that the effective diffusion coefficient might not be sufficient for the description of mass transport within the pellet periphery for a broad range of realistic fluid dynamic conditions during cultivation. Nevertheless, although actual mass transport rates inside pellets are unknown, mass fluxes can be calculated on the basis of spatially resolved data of oxygen and biomass distribution within the pellet.

[Radiotherapy : an option for refractory salivary fistulas]. / Radiatio : Therapieoption bei therapierefraktärer Speichelfistel.

A 45-year-old patient presented with refractory salivary fistula, attributed to multiple surgery and Botulinum toxin, following lateral parotidectomy. He underwent fractionated radiotherapy of the remaining parotid gland including the fistula opening (total dose of 30 Gy) at our clinic. In time, fistula secretion could be inhibited completely. Although the indication for radiotherapy for such fistulas is rare since Botulinum toxin has been in use, it should still be considered in refractory disease courses.

Optically controlled interparticle distance tuning and welding of single gold nanoparticle pairs by photochemical metal deposition.

We report on the in-situ controlled tuning of the particle gap in single pairs of gold nanodisks by photochemical metal deposition. The optically induced growth of nanodisk dimers fabricated by electron beam lithography leads to a decrease of the interparticle gap width down to 0 nm. Due to the increasing particle size and stronger plasmonic coupling, a smooth redshift of the localized surface plasmon (LSP) resonances is observed in such particle pairs during the growth process. The interparticle gap width, and hence the LSP resonance, can be tuned to any desired spectral position. The experimental results we obtain with this nanoscale fabrication technique are well described by the so-called plasmon ruler equation. Consequently, both the changes in particle diameter as well as in gap width can be characterized in-situ via far-field read-out of the optical properties of the dimers.

A comparison of three-field and four-field techniques in different clinical target volumes in prostate cancer irradiation using dose volume histograms: a prospective three-dimensional analysis.

The purpose of the current study was to quantitatively assess differences between irradiation techniques on normal tissue exposure in different clinical target volumes (CTVs) in irradiation of prostate cancer. 14 patients with prostate cancer undergoing external beam radiotherapy were investigated. The prostate and prostate + proximal/entire seminal vesicles were delineated as CTVs. A three-field and two different four-field plans were generated and compared concerning rectum, bladder and femoral head dose-volume histograms (DVHs). The exposure of the rectum exposed to 40-60 Gy was significantly lower for all CTVs with the three-field technique compared with both four-field techniques. The exposure of the rectum to 70 Gy was significantly lower for all CTVs with the weighted four-field technique compared with the unweighted four-field and three-field techniques. The weighted four-field technique was worst in bladder dose sparing for the three CTVs. Comparing the three-field and the unweighted four-field technique for irradiation of the prostate and prostate + entire seminal vesicles, no technique provided a clear advantage or disadvantage in bladder dose sparing. For irradiation of the prostate + proximal seminal vesicles the unweighted four-field technique provided the best bladder dose sparing. Concerning the exposure of the femoral heads, the three-field technique was significantly worse for the three CTVs compared with both four-field techniques. No difference was found between the unweighted and the weighted four-field techniques. In conclusion, none of the studied techniques consistently proved superior in different CTVs in prostate cancer irradiation with respect to sparing all organs at risk. The absolute differences between the three techniques were small and the clinical relevance of these findings is uncertain.

Concurrent low-dose cisplatin and thoracic radiotherapy in patients with inoperable stage III non-small cell lung cancer: a phase II trial with special reference to the hemoglobin level as prognostic parameter.

PURPOSE: To evaluate the efficacy of concurrent radiochemotherapy in patients with stage III non-small cell lung cancer (NSCLC), and to examine the effect of hemoglobin levels on survival of those patients. The negative impact of anemia on survival has been noticed for other cancer sites including the head and neck, and the uterine cervix, but it has been rarely described in NSCLC cancer patients treated with radiotherapy. METHODS: From April 1995 through March 2002, 56 patients with inoperable stage III non-small lung cancer were treated with radiotherapy consisting of 60 Gy (50 Gy+10 Gy boost) given in 30 fractions of 2 Gy daily, 5 days a week, over a period of 6 weeks, and concurrent low-dose daily chemotherapy (CHT) consisting of 6 mg/m(2) of cisplatin given Mondays-Fridays during weeks 1-2 and 5-6. All patients had stage III disease and ages ranged from 39 to 81 years old (median 63.9 years). RESULTS: The 2-year and 3-year survival rates were 34% and 16%, respectively. Patients with a pretreatment hemoglobin level superior or equal to 11.6 g/dl had a 2-year survival rate of 52% as compared to 15.5% for patients with a pretreatment hemoglobin level inferior to 11.6 g/dl (p=0.0075). Patients with higher KI (>70%) showed better survival rates than those with lower KI. Surprisingly, patients in stage IIIA did not survive significantly longer than those in stage IIIB. Hematological toxicity (grade > or =2) prevailed (25%), followed by esophageal (5.4%) and bronchopulmonary (2%) toxicity. Only three patients experienced acute grade 3 hematological toxicity. Because of acute toxic effects, irradiation was interrupted in 8 patients (14.3%) for 7-13 days (median 7.5 days). Late high-grade (> or =3) toxicity was not found. No grade 4 toxicity or treatment-related deaths were observed during this study. CONCLUSION: Our data show that concurrent radiotherapy with daily low dose cisplatin is well tolerated, and shows survival rates comparable to more aggressive treatment regimens. A combination of this chemotherapy with accelerated hyperfractionated radiotherapy might improve the results in the future. Furthermore, we could show that the hemoglobin levels prior to therapy have an influence on the prognosis, where lower levels were associated with worse outcome. Further trials should consider supplementation with erythropoietin.

[The Ahmed glaucoma valve. Medium term results]. / Die "Ahmed glaucoma valve" Mittelfristige Ergebnisse.

BACKGROUND: In complicated glaucoma, when classical filtrating surgery would be ineffective, aqueous shunts may be used. Complications due to hypotonia are reduced by valved systems, such as the Ahmed glaucoma valve (AGV). METHOD: In a retrospective case control study, 28 patients with complicated glaucoma were included. In addition to the clinical examination, we examined the size and function of the filtering area using ultrasound. RESULTS: The medium term follow-up was 25+/-16 months, the preoperative intraocular pressure (IOP) 35.5 mmHg+/-10.3 while 17 eyes were pseudophakic and nine aphakic. In the first weeks after AGV implantation, the mean IOP was 6.3+/-2.5 mmHg. In nine eyes, the pressure was less than 5 mmHg and five developed a temporary choroidal detachment. At the last visit, IOP was regulated in 22 eyes (82.1%). There was no correlation between IOP regulation and the size of the filtering bleb or the increase in the latter by digital pressure. CONCLUSION: In the management of complicated glaucoma, if there is a high risk of failure due to conjunctival scarring, AGV implantation can be used as a save procedure with a success rate comparable to other glaucoma implants.

[Development of cell content and shedding of Prototheca spp. in milk from infected udder quarters of cows]. / Entwicklung von Zellgehalt und Ausscheidung von Prototheca spp. in der Milch infizierter Euterviertel von Kühen.

It was the objective of this study to analyse shedding patterns and somatic cell counts in cows and quarters infected with Prototheca spp. and to evaluate two approaches to identify infected animals by somatic cell count (SCC) or by bacteriological analysis of pooled milk samples. Five lactating dairy cows, chronically infected with Prototheca spp. in at least one quarter were studied over 11 weeks to 13 months. Quarter milk samples and a pooled milk sample from 4 quarters were collected aseptically from all quarters of the cows on a weekly basis. Culture results of quarter milk and pooled samples were compared using cross tabulation. SCC of quarter milk samples and of pooled samples were related to the probability of detection in the infected quarters and cows, respectively. Shedding of Prototheca spp. was continuous in 2 of 8 quarters. In the other quarters negative samples were obtained sporadically or over a longer period (1 quarter). Overall, Prototheca spp. were isolated from 83.6% of quarter milk samples and 77.0% of pooled milk samples of infected quarters and cows. Somatic cell counts were higher in those samples from infected quarters that contained the algae than in negative samples (p < 0.0001). The same applied for composite samples from infected cows. Positive samples had higher SCC than negative samples. However, Prototheca spp. were also isolated from quarter milk and pooled samples with physiological SCC (i.e. < 10(5)/ml). Infected quarters that were dried off did not develop acute mastitis. However, drying off had no effect on the infection, i.e. samples collected at calving or 8 weeks after dry off still contained Prototheca spp. Results indicate that pre-selection of cows to be sampled for Prototheca spp. by SCC and the use of composite samples are probably inadequate in attempts to eradicate the disease. However, due to intermittent shedding of the algae in some cows, single herd sampling using quarter milk samples probably also fails to detect all infected cases. Therefore, continuous monitoring of problem cows with clinical mastitis or increased SCC in herds during eradication programs is recommended.

Comparative localization of mannose-6-phosphate receptor with 2,6sialyltransferase in HepG2 cells: an analysis by confocal double immunofluorescence microscopy.

Recent advances in confocal immunofluorescent microscopy have led to significant improvements in delineating membrane-bounded organelles. In this study using HepG2 cells we focused on two functionally distinct but closely apposed organelles that have been difficult to distinguish by conventional immunofluorescent microscopy, namely the Golgi apparatus, the trans Golgi network (TGN) and late endosomes. The following markers were used: for the Golgi apparatus beta 1,4galactosyltransferase (gal-T), for the TGN, 2, 6(N)sialytransferase (sia-T) and for late endosomes/TGN, the mannose-6-phosphate/insulin growth factor II receptor (CIMPR). In addition, that part of the TGN previously shown to contain CIMPR was also identified using antibodies to the gamma-chain of the HA-1 adaptor (Klumperman et al. J. Cell Biol. 121, 997-1010 (1993)). True colocalization of intracellular antigens was ascertained by double staining of gal-T using both monoclonal and polyclonal antibodies. As previously reported, our results revealed essentially complete colocalization of gal-T and sia-T in this cell line. While the compartments containing CIMPR appeared to overlap with those containing sia-T by conventional immunofluorescence, both compartments were clearly distinct by double-label confocal microscopy. Differences between these organelles became more evident following treatment with brefeldin A. Finally, HA-1 gamma-chain was also localized to structures that were close to but clearly different from the sia-T-containing compartment. Absence of colocalization of CIMPR or HA-1 gamma-chain with sia-T indicates that these markers are enriched in distinct domains of the trans Golgi network.

Lysosomal acid phosphatase is internalized via clathrin-coated pits.

The presence of lysosomal acid phosphatase (LAP) in coated pits at the plasma membrane was investigated by immunocytochemistry in thymidine kinase negative mouse L-cells (Ltk-) and baby hamster kidney (BHK) cells overexpressing human LAP (Ltk-LAP and BHK-LAP cells). Double immunogold labeling showed that at various stages of invaginating coated pits LAP colocalized with clathrin and plasma membrane adaptors (HA-2 adaptors). Quantitation of the immunogold label showed similar density of wild-type LAP in coated over non-coated areas of the plasma membrane, whereas an internalization-deficient, truncated mutant of LAP which lacks the cytoplasmic tail was less efficiently included into coated pits. Internalization of anti-LAP antibodies into endosomal vesicles was accompanied by rapid dissociation of the coat proteins as shown by an immunofluorescence assay. The role of clathrin-coated vesicles in internalization of LAP was further corroborated by microinjecting monoclonal antibodies against clathrin or HA-2 adaptors into BHK-LAP cells. Internalization of LAP as detected by an immunofluorescence assay was transiently blocked by microinjected antibodies against clathrin or HA-2 adaptors, whereas unrelated antibodies did not affect internalization. These data suggest that LAP is included into clathrin-coated pits of the plasma membrane for rapid internalization.

Tyrosine sulfation: a post-translational modification of proteins destined for secretion?

Protein sulfation was studied in germ-free rats by prolonged in vivo labeling with [35S]sulfate. Specific sets of sulfated proteins were observed in all tissues examined, in leucocytes, and in blood plasma. No protein sulfation was detected in erythrocytes. Analysis of the type of sulfate linkage showed that sulfated proteins secreted into the plasma contained predominantly tyrosine sulfate, whereas sulfated proteins found in tissues contained largely carbohydrate sulfate. This implies some kind of selection concerning the intracellular processing, secretion, turnover or re-uptake of sulfated proteins which is responsible for the enrichment of tyrosine-sulfated proteins in the plasma.

Antibodies against the cytoplasmic tail can differentiate between the quaternary forms of the Mr 46,000 mannose 6-phosphate receptor.

An antiserum against a peptide of the cytoplasmic tail of the Mr 46,000 mannose 6-phosphate receptor is described which recognizes preferentially the tetrameric versus the dimeric form of this receptor. This indicates that the conformation of the cytoplasmic tail, which harbours signals necessary for the trafficking of the receptor, depends on the quaternary structure of the receptor.

Immunohistochemical localization of secretogranin II in the rat cerebellum.

Secretogranin II (chromogranin C) is a peptide related to chromogranin A and secretogranin I (chromogranin B) which is secreted by a regulated pathway from both neurons and endocrine cells. In the present study we have determined by light microscopic immunocytochemistry its distribution in the cerebellum and in adjacent brain stem regions. Secretogranin II was found to be widely distributed throughout the gray matter of these regions. Highly immunoreactive structures in the cerebellar cortex included the majority of climbing fibers, a large number of mossy fibers, sparse varicose fibers in the molecular layer and a subpopulation of neuronal perikarya in the granule cell layer. The location and shape of these neurons are very similar to those of a novel type of cerebellar neurons which has been recently described. A moderate level of immunoreactivity was observed on fibers travelling among Purkinje cells and parallel to the pial surface in the Purkinje cell layer. A variable, but in general low, degree of immunoreactivity was also detectable in the perikarya of Purkinje cells. In the deep cerebellar nuclei a loose network of secretogranin II-positive fibers was visible. Neurons of the nuclei, however, were non-immunoreactive. A dense network of highly immunoreactive fibers was found throughout the brain stem regions adjacent to the cerebellum. Our results indicate that secretogranin II has in the cerebellum and adjacent regions a distribution more widespread than that of known regulatory peptides and suggest that the peptide-mediated signaling in the cerebellum plays a role more important that has been acknowledged so far.

Regulation of chromogranin B/secretogranin I and secretogranin II storage in GH4C1 cells.

GH4C1 cells are a rat pituitary tumor cell strain in which the level of cellular prolactin (PRL) and PRL-containing secretory granules can be regulated by hormone treatment. The chromogranins/secretogranins (Sg) are a family of secretory proteins which are widely distributed in the secretory granules of endocrine and neuronal cells. In the present study, we investigated in GH4C1 cell cultures the regulation of the cell content of the Sg by immunoblotting and the relationship between the storage of Sg I and Sg II and PRL by double immunocytochemistry. GH4C1 cells grown in the presence of gelded horse serum, a condition in which these cells contain a low level of secretory granules, contained low levels of PRL, Sg I, and Sg II. Treatment of GH4C1 cells with a combination of 17 beta-estradiol, insulin, and epidermal growth factor for 3 days, known to induce a marked increase in the number of secretory granules, increased the cell contents of PRL, Sg I, and Sg II. To determine whether the induction of PRL was morphologically associated with that of the Sg, the distribution of PRL and the Sg was determined by double immunofluorescence microscopy. After hormone treatment, 54% of cells showed positive PRL immunoreactivity, fluorescence being extranuclear and consistent with staining of the Golgi zone and secretory granules. Forty-six percent of PRL-positive cells stained coincidently for Sg I, while 72% of the PRL cells were also reactive with anti-Sg II. To determine whether PRL storage was associated with storage of at least one of the Sg, cells were stained with anti-PRL and anti-Sg I and anti-Sg II together. Eighty-six percent of PRL cells stained for one or the other of the Sg. Therefore, PRL storage in GH4C1 cell cultures is closely but not completely associated with the storage of Sg I and/or II.

Effect of keratinocyte growth factor on the proliferation, clonogenic capacity and colony size of human epithelial tumour cells in vitro.

PURPOSE: The effect of recombinant human keratinocyte growth factor (rHuKGF) on the proliferation, clonogenic capacity and colony size of low-passage human epithelial tumour cells was tested in vitro. MATERIALS AND METHODS: Five tumour cell cultures derived from head and neck squamous cell carcinomas, three cultures derived from pleural effusions of carcinomas of different origin and normal human nasal epithelial cells were analysed in passages 2-4. Expression of FGF7 and its receptor (FGFR2) were determined by the RNase protection assay. Cells were incubated with rHuKGF (10-200 ng ml(-1)) 3 days before or immediately after plating for clonal growth in serum-depleted media. To determine cellular radiosensitivity, single doses of 1-8 Gy X-rays were applied. Colony formation as well as colony size, reflecting the number of cell divisions, was determined after 10-15 days of growth in rHuKGF-treated and control cells. RESULTS: Normal nasal epithelial cells showed a two- to threefold increase in the number of cell divisions due to rHuKGF-treatment. In tumour cell cultures, significant stimulation of proliferation occurred in only one of eight samples. Tumour cells expressed FGF7 mRNA and protein, and low levels of FGFR2 mRNA. The addition of rHuKGF to the medium of the tumour cell cultures influenced neither radiation-induced impairment of proliferation nor clonogenic cell survival. CONCLUSION: rHuKGF has been shown to ameliorate the radiation tolerance of normal epithelia. The minimum in vitro tumour cell response to rHuKGF compared with normal epithelial cells suggests a potential for selective protection of normal epithelia during radiotherapy. The low FGFR2 expression as well as the FGF7 expression in the tumour cells may contribute to their resistance to rHuKGF treatment.

Influence of clodronate on breast cancer cells in vitro.

UNLABELLED: One of the major therapies of bone metastasis is administration of clodronate. But the influence of clodronate alone on tumour cells is not quite clear. Radiotherapy and administration of clodronate increasingly are used in combination. The influence of clodronate on radiosensitivity of tumour cells is not known. METHODS: We used MDA-MB-435S and MCF-7 cells (breast cancer) in vitro and exposed the cells to clodronate in different concentrations and different short application times. In a second experiment we added graded doses of radiotherapy to the cell cultures. All experiments were done under standard conditions of the colony test. RESULTS: At different concentrations and different incubation times clodronate is able to reduce the cell survival of MDA-MB-435S cells, but not of MCF-7 cells. Even very low concentrations of clodronate in the cell culture medium are sufficient to reduce the tumour cell survival of MDA-MB-435S down to 59%. This reduction is time and concentration dependent. Using irradiation, clodronate has definitively no influence on the radiosensitivity of MDA-MB-435S cells in vitro, but the shoulder of the survival curve of MCF-7 cells is markedly reduced, demonstrating reduced repair of sublethal radiation damage.

Intake and fecal excretion of PCDDs, PCDFs, HCB and PCBs (138, 153, 180) in a breast-fed and a formula-fed infant.

Intake and fecal excretion of PCDDs, PCDFs, HCB and PCBs (IUPAC Nos. 138, 153, 180) were measured in a breast-fed and a formula-fed infant at the age of 1 and 5 months. As expected, the intake of these compounds was clearly higher in the breast-fed infant. In this baby an almost complete absorption was observed for lower chlorinated PCDDs and PCDFs and also for HCB and PCBs, whereas for hepta- and octachlorinated PCDDs and PCDFs fecal excretion was considerably higher (from 20% up to nearly 100% of the intake). Due to low concentrations in diet and feces of the formula-fed infant an evaluation was possible only for a few compounds at the age of 5 months. These values were in the same range when compared with those of the breast-fed infant. For collection of feces new cotton diapers were used which were pre-extracted in order to reduce the levels of polychlorinated compounds. Unexpectedly, after washing the tissue a much higher contamination was observed which made a calculation of fecal excretion rates in the formula-fed infant at the age of 1 month impossible.