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Biomedical subjects

M Nijhuis

Publications and source records attributed to M Nijhuis.

24 records · Page 2Linked to original sources
PubMed1995

Rapid changes in human immunodeficiency virus type 1 RNA load and appearance of drug-resistant virus populations in persons treated with lamivudine (3TC).

The effect of the appearance of drug-resistant human immunodeficiency virus type 1 (HIV-1) on viral RNA load was studied in patients treated with the reverse transcriptase inhibitor lamivudine. During the first 12 weeks of treatment, HIV-1 RNA concentrations and amino acid changes in codon 184, causing high-level resistance to lamivudine, were determined in longitudinal serum samples from HIV-1 p24 antigen-positive and -negative patients. A marked decline in the amount of HIV-1 RNA (approximately 95% below baseline) and HIV-1 p24 antigen was observed within 2 weeks, followed by a rise that coincided with the appearance of lamivudine-resistant viruses in serum (isoleucine mutants initially, which were subsequently replaced by valine variants). After 12 weeks, a partial antiviral effect was observed despite the presence of a complete codon 184 mutant virus population in serum. This study shows that the rapid appearance of drug-resistant virus in serum is followed by an increase in viral RNA load.

CD4 Lymphocyte Count↗
PubMed1993

Engineering of the substrate-binding region of the subtilisin-like, cell-envelope proteinase of Lactococcus lactis.

The substrate-binding region of the cell-envelope proteinase of Lactococcus lactis strain SK11 was modelled, based on sequence homology of the catalytic domain with the serine proteinases subtilisin and thermitase. Substitutions, deletions and insertions were introduced, by site-directed and cassette mutagenesis of the prtP gene encoding this enzyme, based on sequence comparison both with subtilisin and with the homologous L.lactis strain Wg2 proteinase, which has different proteolytic properties. The engineered enzymes were investigated for thermal stability, proteolytic activity and cleavage specificity towards small chromogenic peptide substrates and the peptide alpha s1-casein(1-23). Mutations in the subtilisin-like substrate-binding region showed that Ser433 is the active site residue, and that residues 138 and 166 at either side of the binding cleft play an important role in substrate specificity, particularly when these residues and the substrate are oppositely charged. The K748T mutation in a different domain also affected specificity and stability, suggesting that this residue is in close proximity to the subtilisin-like domain and may form part of the substrate-binding site. Several mutant SK11 proteinases have novel properties not previously encountered in natural variants. Replacements of residues 137-139AKT along one side of the binding cleft produced the 137-139GPP mutant proteinase with reduced activity and narrowed specificity, and the 137-139GLA mutant with increased activity and broader specificity. Furthermore, the 137-139GDT mutant had a specificity towards alpha s1-casein(1-23) closely resembling that of L.lactis Wg2 proteinase. Mutants with an additional negative charge in the binding region were more stable towards autoproteolysis.

Amino Acid Sequence↗
PubMed1993

Integration and gene replacement in the Lactococcus lactis lac operon: induction of a cryptic phospho-beta-glucosidase in LacG-deficient strains.

Insertions, replacement mutations, and deletions were introduced via single or double crossover recombination into the lacE (enzyme IIlac) and lacG (phospho-beta-galactosidase) genes of the Lactococcus lactis chromosomal lacABCDFEGX operon. LacG production was abolished in strains missing the lacG gene or carrying multicopy insertions in the lacE gene that affected expression of the lacG gene. However, these LacG-deficient strains could still ferment lactose slowly and were found to contain an enzymatic activity that hydrolyzed the chromogenic substrate o-nitrophenyl-beta-D-galactopyranoside phosphate. Induction of this phospho-beta-glycohydrolase activity coincided with the appearance of a new 55-kDa protein cross-reacting with anti-LacG antibodies that had a size similar to that of LacG but a higher isoelectric point (pI 5.2) and was not found in wild-type cells during growth on lactose. Since the phospho-beta-glycohydrolase activity and this protein with a pI of 5.2 were highly induced in both mutant and wild-type cells during growth on cellobiose that is likely to be transported via a phosphoenolpyruvate-dependent phosphotransferase system, we propose that this induced activity is a phospho-beta-glucosidase that also hydrolyzes lactose-6-phosphate.

Biological Transport↗
PubMed1991

Engineering of the Lactococcus lactis serine proteinase by construction of hybrid enzymes.

Plasmids containing wild-type and hybrid proteinase genes were constructed from DNA fragments of the prtP genes of Lactococcus lactis strains Wg2 and SK11. These plasmids were introduced into the plasmid-free strain L. lactis MG1363. The serine proteinases produced by these L. lactis strains were isolated, and their cleavage specificity and rate towards alpha s1- and beta-casein was investigated. The catalytic properties of both the SK11 and Wg2 proteinases, which differ in 44 out of 1902 amino acid residues, could be changed dramatically by the reciprocal exchange of specific fragments between the two enzymes. As a result, various L. lactis strains were constructed having new proteolytic properties that differ from those of the parental strains. Furthermore, two segments in the proteinase could be identified that contribute significantly to the cleavage specificity towards casein; within these two segments, several amino acid residues were identified that are important for substrate cleavage rate and specificity. The results also indicate that the lactococcal proteinase has an additional domain involved in substrate binding compared with the related subtilisins. This suggests that the 200 kd L. lactis proteinase may be the representative of a new subclass of subtilisin-like enzymes.

Amino Acid Sequence↗