Biomedical subjects

Peter Prehm

Publications and source records attributed to Peter Prehm.

8 recordsLinked to original sources

PubMed2006

Biosynthesis of hyaluronan: direction of chain elongation.

The mechanism of hyaluronan biosynthesis in vertebrates had been proposed to occur at the reducing end of growing chains. This mechanism was questioned because a recombinant synthase appeared to add new monosaccharides to the non-reducing end. I reinvestigated this problem with membranes from the eukaryotic B6 cell line. The membranes were incubated with UDP-[3H]GlcNAc and UDP-[14C]GlcA to yield differentially labelled reducing terminal and non-reducing terminal domains. Digestion of the product with a mixture of the exoglycosidases beta-glucuronidase and beta-N-acetylglucosaminidase truncated the hyaluronan chain strictly from the non-reducing end. The change in 3H/14C ratio of the remaining hyaluronan fraction, during the course of exoglycosidase digestion, confirmed the original results that the native eukaryotic synthase extended hyaluronan at the reducing end. This mechanism demands that the UDP-hyaluronan terminus is bound to the active site within the synthase and should compete with the substrates for binding. Accordingly, increasing substrate concentrations enhanced hyaluronan release from the synthase. A model is proposed that explains the direction of chain elongation at the reducing end by the native synthase and at the non-reducing end by the recombinant synthase based on a loss of binding affinity of the synthase towards the growing UDP-hyaluronan chain.

Acetylglucosaminidase↗

PubMed2006

Hyaluronate and its receptors in bone marrow.

Cell-cell and cell-matrix interactions, which are mediated by cell adhesion molecules, play a fundamental role during many cellular processes including growth, differentiation, cell migration and cancer metastasis. One molecule playing a major role in these processes is the CD44 surface receptor, which is expressed in a wide range of cells including many cells of the hemopoietic system, where it mediates the interaction with its major ligand, hyaluronate. However, little is known about CD44 and hyaluronate in bone marrow and this was investigated immunohistochemically in trephine biopsies and in cultivated human bone marrow stromal cells. In biopsy specimens, patches of hyaluronate deposition were detected in the extracellular matrix (ECM). However, most of the areas of the ECM were devoid of hyaluronate. Single mast cells and lymphocytes scattered throughout the marrow were CD44 immunopositive. Marrow-derived stromal cells (MDSC) expanded in cell culture were immunopositive for CD44, hyaluronate synthase, and hyaluronate. Hence, a marked difference between CD44 immunolocalisation and hyaluronate deposition can be observed between in situ and under cell culture conditions. Since in normal marrow in situ the number of CD44 immunopositive cells was low, interactions of CD44 and hyaluronate would appear to not to play a major role in cell adhesion in the normal bone marrow.

Bone Marrow↗

PubMed2005

Inhibitors of hyaluronan export prevent proteoglycan loss from osteoarthritic cartilage.

OBJECTIVE: Osteoarthritis (OA) is characterized by cartilage erosion, proteolysis of aggrecan and collagen, and disturbed synthesis rates of aggrecan and hyaluronan by chondrocytes. The hypothesis is tested that hyaluronan overproduction contributes to aggrecan loss from osteoarthritic cartilage. METHODS: Human chondrocytes or bovine cartilage explants were incubated with interleukin 1beta (IL-1beta) to induce upregulation of hyaluronan and downregulation of aggrecan. OA was induced by injection of iodoacetate into the synovial cavity in rat knees. Hyaluronan export was inhibited by ATP-binding cassette transporter inhibitors such as the multidrug resistance (MDR) inhibitors valspodar or verapamil. The concentration of aggrecan was measured in cell culture media or visualized histochemically in cartilage tissue sections. RESULTS: Valspodar inhibited hyaluronan export from human chondrocytes in cell culture selectively without reducing aggrecan secretion. Valspodar and other MDR inhibitors prevented loss of aggrecan from osteoarthritic cartilage explants in culture. Verapamil prevented loss of aggrecan from cartilage in osteoarthritic rat knees. CONCLUSION: Hyaluronan is synthesized at plasma membranes and exported out of the cell. We recently identified an ATP-binding cassette transport system that is responsible for hyaluronan export. A number of ATP-binding cassette transport inhibitors are known and are in use clinically. These inhibitors were used here to inhibit hyaluronan export and to prevent aggrecan loss from arthritic cartilage. New drugs for treatment of arthritis are suggested by these studies.

Aggrecans↗

PubMed2004

Inhibition of hyaluronan export from human fibroblasts by inhibitors of multidrug resistance transporters.

In a previous report we described the export of hyaluronan from Streptococcus pyogenes by an ABC transporter. Extending these findings a sequence homology search against human proteins revealed a strong homology to the multidrug resistance transporter ABC-B (MDR-1) and ABC-C (MRP 5). Using several inhibitors directed against these and other transporters, a decreased hyaluronan production in cell culture as well as in hyaluronan synthase activity in purified membrane fractions was observed. The inhibitory capacity (IC(50) concentrations) was compared the with reported IC(50)- or the K(i)-concentrations for individual transporters. These analyses revealed that hyaluronan is synthesized within the cytoplasm of mammalian cells and actively secreted into the pericellular space by energy dependent transport proteins. While inhibition of several transport proteins resulted in a decrease of hyaluronan export, inhibition of the MRP5 transporter was the most effective one to decrease hyaluronan in the cell culture supernatant indicating that hyaluronan export is one physiological role of this transport protein.

ATP-Binding Cassette Transporters↗

PubMed2004

Hyaluronan release from Streptococcus pyogenes: export by an ABC transporter.

Capsular hyaluronan of Streptococcus pyogenes is synthesized at the protoplast membrane. It is widely assumed that hyaluronan is exported by the synthase itself and that no additional protein is required for transfer through plasma membranes. However, we produced an insertional mutation that reduced the mucoid phenotype, hyaluronan production, and capsule formation. Nucleotide sequence analysis of the insertion site identified a gene coding for a protein with an ATP-binding cassette (ABC) that belonged to an ABC transporter system and was located next to the hyaluronan synthesis genes. The mucoid phenotype was reconstituted by complementation with DNA encoding the ABC transporter system. These results indicated that an ABC transporter was required for efficient capsule production.

ATP-Binding Cassette Transporters↗

PubMed2003

Supramolecular organization in streptococcal pericellular capsules is based on hyaluronan tertiary structures.

Supramolecular organization involving a polyanionic glycan in a bacterial capsule (hyaluronan, HA, in streptococcal capsules) is revealed, for the first time, by electron-histochemical methodology previously used to elucidate ultrastructure in extracellular matrix. Capsular HA filaments thereby revealed closely resemble aligned linear structures demonstrated by similar technology in HA solution. These parallel arrays, spontaneously formed, are based on HA tertiary structures (beta sheet-like) which are stabilized by hydrophobic and hydrogen bonds. HA tertiary structures in aqueous solutions resist shear stress as shown by rheo-NMR. Thus, supramolecular HA wrapping covering many cells probably stabilizes chains of bacteria. Streptococci possibly templated the ordered structures since eukaryotic B6 cell HA did not produce similarly organized envelopes. Supramolecular organization in streptococcal and pneumococcal capsules are compared. Their glycans are very similar but the potential for HA-like tertiary structures is not present in the pneumococcal type 3 polysaccharide and chains of cells are not formed to the same extent by pneumococci. We suggest that the streptococcal capsule exemplifies a simple extracellular matrix analogous to those in animal connective tissues, which contain glycans (chondroitin, keratan, and dermochondan sulfates) of the HA family, capable of undergoing aggregation to similar tertiary structures.

Bacterial Capsules↗

PubMed2003

Extracellular matrix components in nasal polyposis.

OBJECTIVE: Macroscopically, tissue hydration in nasal polyps seems to be a phenomenon of unknown etiology. As the macromolecular composition of extracellular matrix (ECM) determines to a large extent tissue hydration, which in turn determines tissue volume, we investigated ECM components in nasal polyps (NP) in comparison to nasal mucosa of the inferior turbinate (TM) and sinus mucosa from patients with chronic sinusitis without polyps (CS). MATERIAL AND METHODS: The following parameters were determined: (i) the dry weight of freeze-dried NP, TM and CS; (ii) the total protein content (Bio-Rad Protein Assay) of the tissue; (iii) the quality of proteins, using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE); (iv) the amount of albumin, using nephelometry; and (v) the amount of hyaluronic acid, using a chemical method: deaminative hydrolysis with carbazole. RESULTS: In 20 NP we found a significantly elevated total protein content compared to TM (n = 20) and CS (n = 15), referred to 0.1 g dry of tissue. In SDS-PAGE of NP (n = 20) a protein band at approximately 70 kDa, representing albumin, dominated, in comparison to TM. The amount of albumin was significantly increased in NP compared to CS and TM. In contrast, the amount of the glycosaminoglycan, hyaluronic acid, was not elevated in NP or CS. CONCLUSION: Albumin was significantly increased in NP and CS, possibly as a result of inflammatory plasma exudation mechanisms. Hyaluronic acid seems to play no role in the tissue hydration of NP.

Albumins↗

PubMed2002

Expression of hyaluronate and hyaluronate synthase in human primary tumours and their metastases in scid mice.

Hyaluronate and hyaluronate synthase expression were examined in primary tumours and if present in metastatic deposits of human breast, colon, ovarian and small-cell lung cancer cell lines transplanted into scid mice using biotinylated hyaluronectin and immunohistochemical staining of hyaluronate synthase. Very intensive hyaluronate and hyaluronate synthase expression could be observed in peripheral areas of tumours derived from highly metastatic cell lines (HT29, MCF-7). Even smaller lung metastases of up to 15 cells showed typically a focal binding of hyaluronectin predominantly at the host-tumour interface of the metastases, indicating that increased expression is closely correlated with the degree of invasiveness and metastatic potential of malignant tumours.

Animals↗