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Biomedical subjects

K F Johnson

Publications and source records attributed to K F Johnson.

At least 19 recordsLinked to original sources
PubMed1999

Synthesis of oligosaccharides by bacterial enzymes.

Many human pathogens initiate disease by utilizing their microbial adhesin proteins to attach to glycoconjugates on host cell mucosal surfaces. Soluble oligosaccharides of identical or similar structure to these naturally occurring ligands can both prevent bacterial attachment as well as mediate the release of attached bacteria. Since it has not been possible to isolate large quantities of these compounds, we have developed enzyme-based technologies to synthesize several relevant human oligosaccharides. Using cloned bacterial glycosyltransferases, we can synthesize several hundred grams of these oligosaccharides at a time. The availability of these large quantities will allow these compounds to be tested as anti-adhesive pharmaceutical agents as well as lead to expanded practical applications.

Bacteria↗
PubMed1995

A determinant in the cytoplasmic tail of the cation-dependent mannose 6-phosphate receptor prevents trafficking to lysosomes.

The bovine cation-dependent mannose 6-phosphate receptor (CD-MPR) is a type 1 transmembrane protein that cycles between the trans-Golgi network, endosomes, and the plasma membrane. When the terminal 40 residues were deleted from the 67-amino acid cytoplasmic tail of the CD-MPR, the half-life of the receptor was drastically decreased and the mutant receptor was recovered in lysosomes. Analysis of additional cytoplasmic tail truncation mutants and alanine-scanning mutants implicated amino acids 34-39 as being critical for avoidance of lysosomal degradation. The cytoplasmic tail of the CD-MPR was partially effective in preventing the lysosomal membrane protein Lamp1 from entering lysosomes. Complete exclusion required both the CD-MPR cytoplasmic tail and transmembrane domain. The transmembrane domain alone had just a minor effect on the distribution of Lamp1. These findings indicate that the cytoplasmic tail of the CD-MPR contains a signal that prevents the receptor from trafficking to lysosomes. The transmembrane domain of the CD-MPR also contributes to this function.

Amino Acid Sequence↗
PubMed1992

A His-Leu-Leu sequence near the carboxyl terminus of the cytoplasmic domain of the cation-dependent mannose 6-phosphate receptor is necessary for the lysosomal enzyme sorting function.

The determinants on the cytoplasmic tail of the cation-dependent mannose 6-phosphate receptor (CD-MPR) required for lysosomal enzyme sorting have been analyzed. Mouse L cells deficient in the mannose 6-phosphate/insulin-like growth factor-II receptor were transfected with normal bovine CD-MPR cDNA or cDNAs containing mutations in the 67-amino acid cytoplasmic tail and assayed for their ability to target the lysosomal enzyme cathepsin D to lysosomes. Cells expressing the wild-type bovine CD-MPR sorted 67 +/- 2% of newly synthesized cathepsin D compared with the base-line value of 47 +/- 1%. The presence of mannose 6-phosphate in the medium did not affect the efficiency of cathepsin D sorting, indicating that the routing of the ligand-receptor complex is completely intracellular. Mutant receptors with the carboxyl-terminal His-Leu-Leu-Pro-Met67 residues deleted or replaced with alanines sorted cathepsin D below the base-line value. A mutant receptor with the outermost Pro-Met residues replaced with alanines sorted cathepsin D better than the wild-type receptor, indicating that the essential residues for sorting are the His-Leu-Leu sequence. Disruption of a putative casein kinase II phosphorylation site at Ser57 had no detectable effect on sorting. The mutant receptor with the five-amino acid deletion was able to bind to a phosphopentamannose affinity column, proving that its ligand binding site was grossly intact. Resialylation experiments showed that this mutant receptor recycled from the cell surface to the Golgi at a rate similar to the normal CD-MPR, indicating that the defect in sorting is at the level of the Golgi.

Amino Acid Sequence↗
PubMed1992

The cytoplasmic tail of the mannose 6-phosphate/insulin-like growth factor-II receptor has two signals for lysosomal enzyme sorting in the Golgi.

The mannose 6-phosphate/insulin-like growth factor-II (Man-6-P/IGF-II) receptor is known to cycle between the Golgi, endosomes, and the plasma membrane. In the Golgi the receptor binds newly synthesized lysosomal enzymes and transports them directly to an endosomal (prelysosomal) compartment without traversing the plasma membrane. Deletion of the carboxyl-terminal Leu-Leu-His-Val residues of the 163 amino acid cytoplasmic tail of the bovine Man-6-P/IGF-II receptor partially impaired this function, resulting in the diversion of a portion of the receptor-ligand complexes to the cell surface, where they were endocytosed. The same phenotype was observed when 134 residues of the cytoplasmic tail were deleted from the carboxyl terminus. Disruption of the Tyr24-Lys-Tyr-Ser-Lys-Val29 plasma membrane internalization signal alone had little effect on Golgi sorting, but when combined with either deletion resulted in a complete loss of this function. The mutant receptors retained the ability to recycle to the Golgi and bind cathepsin D. These results indicate that the cytoplasmic tail of the Man-6-P/IGF-II receptor contains two signals that contribute to Golgi sorting, presumably by interacting with the Golgi clathrin-coated pit adaptor proteins. The Leu-Leu-containing sequence represents a novel motif for mediating interaction with Golgi adaptor proteins.

Amino Acid Sequence↗
PubMed1991

Localization of the signal for rapid internalization of the bovine cation-independent mannose 6-phosphate/insulin-like growth factor-II receptor to amino acids 24-29 of the cytoplasmic tail.

The signal for the rapid internalization of the cation-independent mannose 6-phosphate/insulin-like growth factor-II receptor has been previously localized to the inner half of the 163-amino acid cytoplasmic tail, including tyrosine 24 and tyrosine 26 (Lobel, P., Fujimoto, K., Ye, R. D., Griffiths, G., and Kornfeld, S. (1989) Cell 57, 787-796). To define this signal more precisely, we generated a series of truncation and substitution mutants and analyzed them for their ability to mediate the endocytosis of extracellular lysosomal enzymes. Mutant receptors containing cytoplasmic domains of 29 or more amino acids functioned normally in endocytosis whereas a mutant receptor with a 28-amino acid cytoplasmic domain was internalized extremely slowly. Alanine scanning of the region between amino acids 19 and 30 identified tyrosine 26 and valine 29 as the most important residues for rapid receptor internalization. Tyrosine 24 and lysine 28 also contributed to the signal while the other amino acids were not critical. The tyrosine residues could be substituted with phenylalanines with no loss of activity, indicating the requirement for an aromatic residue in these positions rather than tyrosine specifically. Conservative substitutions of arginine or histidine for lysine 28 also preserved the internalization signal. These results indicate that the sequence Tyr-Lys-Tyr-Ser-Lys-Val serves as the internalization signal for the mannose 6-phosphate/insulin-like growth factor-II receptor. The crucial elements of this sequence are present in the cytoplasmic tails of a number of other membrane receptors and proteins known to undergo rapid internalization.

Amino Acid Sequence↗
PubMed1991

Synthesis and processing of lysosomal alpha-fucosidase in cultured human fibroblasts.

The lysosomal enzyme alpha-L-fucosidase from human skin fibroblasts is synthesized as a 53 kDa glycosylated precursor which is then proteolytically processed to a 50 kDa mature form. This was confirmed by pulse-chase labeling studies with chase times up to 72 h. In fibroblasts treated with 1-deoxymannojirimycin to prevent trimming of high mannose oligosaccharides, endoglycosidase H (endo H) treatment completely deglycosylated and reduced the size of immunoprecipitated alpha-fucosidase by 4-5 kDa, suggesting the presence of two oligosaccharide units. Endoglycosidase H and endo F studies on untreated alpha-fucosidase suggested the presence of one complex-type and one high mannose-type unit, and that the final processing from 53 to 50 kDa did not involve the removal of carbohydrate. Processing was inhibited by the thiol proteinase inhibitor Ep-459, but not by Ep-475 or leupeptin. Since Ep-459 treatment increased both alpha-fucosidase activity (3-fold) and the amount of immunoprecipitable alpha-fucosidase protein in normal human skin fibroblasts, this suggests a role for cysteine-like proteinases either directly or indirectly in lysosomal hydrolase processing and turnover. Subcellular fractionation studies revealed that the proteolytic processing of the 53 kDa precursor to the 50 kDa mature form occurred in the lysosome, or some other dense organelle.

Cell Compartmentation↗
PubMed1990

Cation-dependent mannose 6-phosphate receptor contains two internalization signals in its cytoplasmic domain.

The signals required for rapid internalization of the bovine cation-dependent mannose 6-phosphate receptor have been localized to two distinct regions of the 67-amino acid cytoplasmic domain. One signal includes phenylalanine 13 and phenylalanine 18, while the other involves tyrosine 45. The former signal is more potent than the latter, but both must be present for the maximal rate of receptor internalization. Each signal shares similarities with the known internalization signals of other recycling receptors.

Amino Acid Sequence↗
PubMed1988

Vendor support for computer systems.

As hospital data processing systems have matured, they have also become more complex. As a result, the level of vendor support has become almost as important as the system's functional features. Although much has been written about the "do's and don'ts" of acquiring hospital information systems, relatively little is said about the request for qualifications (RFQ)--a document detailing how much support vendors will provide to implement and maintain the system. The RFQ should be used to obtain knowledge about the vendors, their systems, their users, and the way they support their users. Six key points should be considered when developing RFQs: Ask vendors to list and briefly describe their current systems and those under development. Ask how long they have been in the hospital information systems business and how many new users they have added each year. Ask about their user retention rates. To address system integration, ask the vendors to briefly describe their approach to passing data between their systems and those from other vendors. Ask them to describe the installation and implementation process. Find out what user support and technical support the vendors provide.

Commerce↗
PubMed1987

Integrated system brings hospital data together.

Healthcare industry changes during the 1980s--increased competition and alterations in the Medicare payment methodology--place new and more complex demands on a hospital's information systems, which often fall short of meeting those demands. These systems were designed for financial reporting, billing, or providing clinical data, and few of them are capable of linking with other unrelated systems. Today's hospital manager needs timely and simultaneous access to data from a variety of sources within the hospital. All the elements to accomplish this are collected somewhere in the hospital, but finding them and bringing them together is difficult. The key to the efficient management and use of data bases is in understanding the fundamental concept of relational data bases, which is the capability of linking or joining separate data files through a common data element in each file. In this way, data files may be integrated into a "related" data base. Any number of separate files, or tables, may exist within a "relational" data base as long as a series of threads links them. A strategic management information data base includes the information necessary to analyze, understand, and manage the hospital's markets, products, resources, and profitability. The major components of this information system are the case mix and cost accounting, budgeting, and modeling systems. The case mix and cost accounting factors involve managing concrete pieces of data, whereas the budgeting and modeling factors manipulate data to create a scenario. The strategic management information data base is the foundation of a hospital's decision support system, which is rapidly moving into the category of a necessary tool of the hospital manager's trade.

Computer Systems↗