Receptor-Mediated Endocytosis of a Manganese Complex of Transferrin into Neuroblastoma (SHSY5Y) Cells in Culture

Abstract: Exposure to manganese compounds often occurs as the result of industrial production or mining. Although manganese appears in traces in animal and human tissue and is essential to certain biological processes, it is also toxic. In humans and animals, toxicity is mainly associated with the nervous system. The mechanism underlying behavioral and biochemical alterations observed after manganese intoxication is not fully understood. We have shown that the manganese present in serum after exposure to manganese oxide is bound to transferrin as trivalent manganic ion. In this study of manganese uptake and storage we used a clone of human neuroblastoma cells (SHSY5Y). These cells differentiate and express catechol-aminergic properties. Saturation binding analysis of the transferrin-manganese complex to the cells revealed a single class of binding sites, with an apparent K _D of 13 ± 1 n M and a density of 11, 000 ± 2, 000 binding sites per cell. The complex was internalized in a temperature-dependent way and reached saturation after 2 h when ∼2% of the added manganese had been internalized. About 80% of the internalized manganese was found in ferritin after 24 h of exposure. The results demonstrate that the transferrin receptor on SHSY5Y cells can bind and internalize a manganese-transferrin complex as efficiently as an iron-transferrin complex, although a saturation of the manganese uptake was achieved.     

Microbial Delignification with White Rot Fungi Improves Forage Digestibility

Three wild-type white rot fungi and two cellulase-less mutants developed from Phanerochaete chrysosporium K-3 (formerly Sporotrichum pulverulentum) were tested for their ability to delignify grass cell walls and improve biodegradation by rumen microorganisms. Fungal-treated and control stems of Bermuda grass were analyzed for their content of ester- and ether-linked aromatics by using alkali extraction and gas chromatography, for in vitro dry weight digestion and production of volatile fatty acids in in vitro fermentations with mixed ruminal microorganisms, for loss of lignin and other aromatics from specific cell wall types by using microspectrophotometry, and for structural changes before and after in vitro degradation by rumen microorganisms by using transmission electron microscopy. P. chrysosporium K-3 and Ceriporiopsis subvermispora FP 90031-sp produced the greatest losses in lignin and improved the biodegradation of Bermuda grass over that of untreated control substrate. However, C. subvermispora removed the most lignin and significantly improved biodegradation over all other treatments. Phellinus pini RAB-83-19 and cellulase-less mutants 3113 and 85118 developed from P. chrysosporium K-3 did not improve the biodegradation of Bermuda grass lignocellulose. Results indicated that C. subvermispora extensively removed ester-linked p-coumaric and ferulic acids and also removed the greatest amount of non-ester-linked aromatics from plant cell walls. Microscopic observations further indicated that C. subvermispora removed esters from parenchyma cell walls as well as esters and lignin from the more recalcitrant cell walls (i.e., sclerenchyma and vascular tissues). C. subvermispora improved in vitro digestion and volatile fatty acid production by ruminal microorganisms by about 80%, while dry matter loss due to fungi was about 20% greater than loss in untreated control stems. The chemical and structural studies used identified sites of specific fungal attack and suggested mechanisms whereby improvement occurred.     

Z → B transition in poly[d(G-m5C)2] induced by interaction with 4′,6-diamidino-2-phenylindole

The Z form of poly[d(G-m⁵C)₂], in presence of Mg²⁺ ion, is found to be transformed into B form upon interaction with 4′,6-diamidino-2-phenylindole (DAPI). The Z → B transformation is complete at a mixing ratio of about 0.07 DAPI per DNA base pairs, i.e., each DAPI molecule may be related to the conversion of 6–7 base pairs. An interaction between DAPI and poly[d(G-m⁵C)₂] in its Z form at low drug: DNA ratios is suggested from optical dichroism and time-resolved luminescence anisotropy results. The spectroscopic behaviour of DAPI indicates that the Z conformation of DNA does not provide normal binding sites for DAPI, such as groove or intercalation sites, but that the initial association may be of external nature. © 1993 John Wiley & Sons, Inc.     

Refinement of the structure of human basic fibroblast growth factor at 1.6 A resolution and analysis of presumed heparin binding sites by selenate substitution.

The three-dimensional structure of human basic fibroblast growth factor has been refined to a crystallographic residual of 16.1% at 1.6 A resolution. The structure has a Kunitz-type fold and is composed of 12 antiparallel beta-strands, 6 of which form a beta-barrel. One bound sulfate ion has been identified in the model, hydrogen bonded to the side chains of Asn 27, Arg 120, and Lys 125. The side chain of Arg 120 has two conformations, both of which permit hydrogen bonds to the sulfate. This sulfate binding site has been suggested as the binding site for heparin (Eriksson, A.E., Cousens, L.S., Weaver, L.H., & Matthews, B.W., 1991, Proc. Natl. Acad. Sci. USA 88, 3441-3445). Two beta-mercaptoethanol (BME) molecules are also included in the model, each forming a disulfide bond to the S gamma atoms of Cys 69 and Cys 92, respectively. The side chain of Cys 92 has two conformations of which only one can bind BME. Therefore the BME molecule is half occupied at this site. The locations of possible sulfate binding sites on the protein were examined by replacing the ammonium sulfate in the crystallization medium with ammonium selenate. Diffraction data were measured to 2.2 A resolution and the structure refined to an R-factor of 13.8%. The binding of the more electron-dense selenate ion was identified at two positions. One position was identical to the sulfate binding site identified previously. The second selenate binding site, which is of lower occupancy, is situated 5.6 A from the first. This ion is hydrogen bonded by the side chain of Lys 135 and Arg 120. Thus the side chain of Arg 120 binds two selenate ions simultaneously. It is suggested that the observed second selenate binding site should also be considered as a possible binding site for heparin, or that both selenate binding sites might simultaneously contribute to the binding of heparin.     

Phylogenetic diversification of immunoglobulin genes and the antibody repertoire

Immunoglobulins are encoded by a large multigene system that undergoes somatic rearrangement and additional genetic change during the development of immunoglobulin-producing cells. Inducible antibody and antibody-like responses are found in all vertebrates. However, immunoglobulin possessing disulfide-bonded heavy and light chains and domain-type organization has been described only in representatives of the jawed vertebrates. High degrees of nucleotide and predicted amino acid sequence identity are evident when the segmental elements that constitute the immunoglobulin gene loci in phylogenetically divergent vertebrates are compared. However, the organization of gene loci and the manner in which the independent elements recombine (and diversify) vary markedly among different taxa. One striking pattern of gene organization is the "cluster type" that appears to be restricted to the chondrichthyes (cartilaginous fishes) and limits segmental rearrangement to closely linked elements. This type of gene organization is associated with both heavy- and light-chain gene loci. In some cases, the clusters are "joined" or "partially joined" in the germ line, in effect predetermining or partially predetermining, respectively, the encoded specificities (the assumption being that these are expressed) of the individual loci. By relating the sequences of transcribed gene products to their respective germ-line genes, it is evident that, in some cases, joined-type genes are expressed. This raises a question about the existence and/or nature of allelic exclusion in these species. The extensive variation in gene organization found throughout the vertebrate species may relate directly to the role of intersegmental (V<==>D<==>J) distances in the commitment of the individual antibody-producing cell to a particular genetic specificity. Thus, the evolution of this locus, perhaps more so than that of others, may reflect the interrelationships between genetic organization and function.      

Refined structure of bovine carbonic anhydrase III at 2.0 Å resolution

The three-dimensional structure of bovine carbonic anhydrase III (BCA III) from red skeletal muscle cells has been determined by molecular replacement methods. The structure has been refined at 2.0 Å resolution by both constrained and restrained structure-factor least squares refinement. The current crystallographic R-value is 19.2% and 121 solvent molecules have so far been found associated with the protein. The structure is highly similar to the refined structure of human carbonic anhydrase II. Some differences in amino acid sequence and structure between the two isoenzymes are discussed. In BCA III, Lys 64 and Arg 91 (His 64 and Ile 91 in HCA II) are both pointing out from the active site cavity forming salt bridges with Glu 4 and Asp 72 (His 4 and Asp 72 in HCA II), respectively. However, Arg 67 and Phe 198 (Asn 67 and Leu 198 in HCA II) are oriented towards the zinc ion and significantly reduce the volume of the active site cavity. Phe 198 particularly reduces the size of the substrate binding region at the “deep water” position at the bottom of the cavity and we sugest that this is one of the major reasons for the differences in catalytic properties of isoenzyme III as compared to isozyme II. © 1993 Wiley-Liss, Inc.     

An attempt to predict long-term effects of atmospheric nitrogen deposition on the yield of Norway spruce stands (Picea abies (L.) Karst.) in southwestern Sweden

Long-term field experiments in Norway spruce stands on fertile sites (site indices 27–35 m) in southwestern Sweden were analysed with respect to volume increment. Three treatments were included (0=No fertilization, N = Fertilization with N, NP = Fertilization with N and P).Volume growth was monitored for 18 years in 10 blocks. No significant differences in annual volume increment between the treatments were detected. Volume increments in the N treatment were 97%, 99% and 107% as high as those in the 0 treatment for the periods 1–5, 6–10 and 11–15 years after the first fertilization. Corresponding values for the NP treatment were 104%, 108% and 110%, indicating that P has a small positive effect.The amount of N-fertilization would correspond to an annual N deposition of 20 kg ha^-1 during the next 30 years in southwestern Sweden. For this period, the results imply that this N deposition would not affect the growth of Norway spruce stands on fertile sites.     

Inhibition enzyme-linked immunosorbent assay for detection of Pseudomonas fluorescens on meat surfaces.

An inhibition enzyme-linked immunosorbent assay was developed for Pseudomonas fluorescens enumeration of meat surfaces. The assay detected contamination levels as low as 3 x 10(5) bacteria per ml and could be completed within 4 h. It could be used as a framework for a test system for quantifying P. fluorescens spoilage in meat products.     

Muscle-specific enzyme activity patterns of the capillary bed of human oro-facial,masticatory and limb muscles

Enzyme-histochemical methods were used to analyse the activities of alkaline phosphatase (AP), dipeptidylpeptidase IV (DPP IV) and adenosine triphosphatase (ATPase) in capillaries of four different human oro-facial muscles, the major and minor zygomatic, the orbicularis oris and buccinator, one masticatory, the masseter and two limb muscles, the biceps brachii and first dorsal interosseus muscles. In all muscles, except for the orbicularis oris, the majority of the capillaries lacked enzyme activity. Therefore, none of these enzymes seems to be reliable as a general marker for human muscle capillaries. In general, the capillaries of the limb muscles and the major and minor zygomatic and the buccinator, were similar in their staining pattern for AP and ATPase, but differed in DPP IV staining. The orbicularis oris muscle differed from the other muscles by showing the largest proportion of capillaries with AP and ATPase activity. The masseter muscle had the largest proportion of capillaries stained for DPP IV. The muscle specific differences in enzyme activity of the capillaries are in agreement with our previous findings of specific differences between limb, oro-facial and masticatory muscles with respect to capillary supply and composition of fibre types and myosins. The results reflect functional specialization of the capillary bed of human muscles.