Search PubMed⌕ Search

Biomedical subjects

J Kok

Publications and source records attributed to J Kok.

107 records · Page 6Linked to original sources
PubMed1989

Identification of a gene required for maturation of an extracellular lactococcal serine proteinase.

Directly upstream of the Lactococcus lactis subsp. cremoris Wg2 proteinase gene is an oppositely directed open reading frame (ORF1). The complete nucleotide sequence of ORF1, encoding a 33-kilodalton protein, was determined. A protein of approximately 32 kilodaltons was synthesized when ORF1 was expressed in Escherichia coli by using a T7 RNA polymerase-specific promoter. L. lactis subsp. lactis MG1363 transformants carrying the proteinase gene but lacking ORF1 were phenotypically proteinase deficient, unlike transformants carrying both the proteinase gene and ORF1. Synthesis and secretion of proteinase antigen by L. lactis could be detected with proteinase-directed monoclonal antibodies regardless of whether ORF1 was present. The requirement of ORF1 for proteinase activation was reflected in a reduction in the molecular weight of the secreted proteinase. Furthermore, deletion of the 130 C-terminal amino acids of the Wg2 proteinase prevented attachment of the enzyme to lactococcal cells.

Amino Acid Sequence↗
PubMed1989

The role of tears in preventing protein deposition on contact lenses.

Recently the presence of a coating inhibitory factor was described in human tears which can prevent the binding of proteins to a solid phase. In these earlier studies depositions of lactoferrin and IgG onto plastic was studied. In the study described here, peroxidase conjugated albumin was used as a model to investigate the effect of human tears on the binding of proteins to plastic surfaces. We investigated the question whether contact lens deposits are associated with decreased levels of this factor in the tearfilm of contact lens wearers. The mean value of the coating inhibiting activity in the tears of the controls did not differ significantly from contact lens wearers without deposits. The contact lens wearers with deposits on their lenses, had a lower mean coating inhibiting activity than the contact lens wearers without deposits. No significant difference was seen in coating inhibiting activity in tears obtained from individuals before and after they had worn lenses for three weeks. Also no difference was observed in the coating inhibiting activity on the eyes of persons who were wearing a lens in one eye only. The results of our study show that a too low activity of the coating inhibiting factor in tears is associated with deposits on contact lenses.

Adult↗
PubMed1988

Genetics of proteinases of lactic acid bacteria.

Because it is essential for good growth with concomitant rapid acid production, and for the production of flavorous peptides and amino acids, the proteolytic ability of lactic acid bacteria is of crucial importance for reliable dairy product quality. In view of this importance, considerable research has been carried out to characterize the enzymes involved. The intensified genetic research in the lactic acid streptococci and the development of gene cloning systems for these organisms have resulted in a rapid increase of genetic data on lactic streptococcal proteinases. By now, an evaluation of the biochemical, immunological and genetic data seems to be feasible. These data are discussed and integrated into a working model tentatively explaining some of the characteristics of the proteolytic systems of lactic acid streptococci.

Base Sequence↗
PubMed1988

Nucleotide sequence of the cell wall proteinase gene of Streptococcus cremoris Wg2.

A 6.5-kilobase HindIII fragment that specifies the proteolytic activity of Streptococcus cremoris Wg2 was sequenced entirely. The nucleotide sequence revealed two open reading frames (ORFs), a small ORF1 with 295 codons and a large ORF2 containing 1,772 codons. For both ORFs, there was no stop codon on the HindIII fragment. A partially overlapping PstI fragment was used to locate the translation stop of the large ORF2. The entire ORF2 contained 1,902 coding triplets, followed by an apparently rho-independent terminator sequence. The inferred amino acid sequence would result in a protein of 200 kilodaltons. Both ORFs have their putative transcription and translation signals in a 345-base-pair ClaI fragment. ORF2 is preceded by a promoter region containing a 15-base-pair complementary direct repeat. Both the truncated 33- and the 200-kilodalton proteins have a signal peptide-like N-terminal amino acid sequence. The protein specified by ORF2 contained regions of extensive homology with serine proteases of the subtilisin family. Specifically, amino acid sequences involved in the formation of the active site (viz., Asp-32, His-64, and Ser-221 of the subtilisins) are well conserved in the S. cremoris Wg2 proteinase. The homologous sequences are separated by nonhomologous regions which contain several inserts, most notably a sequence of approximately 200 amino acids between the His and Ser residues of the active site.

Amino Acid Sequence↗
PubMed1988

Deletion analysis of the proteinase gene of Streptococcus cremoris Wg2.

The Streptococcus cremoris Wg2 proteinase gene, cloned in S. lactis, specified a proteinase which exhibited the same specificity toward casein as did the proteinase isolated from the original host. Although the cloned gene lacked the last 130 codons, the proteinase still specifically degraded beta-casein. Deletion of the C-terminal 343 amino acids from the proteinase did not influence this specificity. Cell-free transcription-translation studies of plasmids carrying deletion derivatives of the proteinase gene showed that the 100-kilodalton C-terminally truncated proteinase still exhibited proteolytic activity. Crossed immunoelectrophoresis revealed that proteins A and B identified in the proteolytic system of S. cremoris Wg2 are both encoded by the proteinase gene. A working model based on integration of available genetic, immunological, and biochemical data is presented to explain this result.

Amino Acid Sequence↗
PubMed1987

Cell Wall-Associated Proteases of Streptococcus cremoris Wg2.

Two components of the proteolytic system, proteins A and B (J. Hugenholtz, F. Exterkate, and W. N. Konings, Appl. Environ. Microbiol. 48:1105-1110, 1984), have been studied in Streptococcus cremoris Wg2 by immunological methods. The components could not be separated by standard chromatography techniques because both proteins had almost identical molecular weights (about 140,000) and isoelectric points (pH 4.5). Specific antibodies were raised against proteins A and B by excision of the different immunoprecipitates from crossed immunoelectrophoresis gels. With these antibodies, protein A or B was removed from solutions containing both proteins. The purified proteins A and B possessed proteolytic activity and were inhibited by the serine protease inhibitor phenylmethylsulfonyl fluoride. Each of these proteins accounted for approximately 50% of the total proteolytic activity isolated from S. cremoris Wg2. The specific antibodies against the proteases were also used for immuno-gold labeling studies. The proteases were clearly seen to be located at the outside of the cell wall. The proteases had the same location when the genetic information coding for the proteases was cloned in Streptococcus lactis and Bacillus subtilis.

Journal Article↗
PubMed1985

Cloning and expression of a Streptococcus cremoris proteinase in Bacillus subtilis and Streptococcus lactis.

Previously, curing experiments suggested that plasmid pWV05 (17.5 megadaltons [Md]) of Streptococcus cremoris Wg2 specifies proteolytic activity. A restriction enzyme map of pWV05 was constructed, the entire plasmid was subcloned in Escherichia coli with plasmids pBR329 and pACYC184. A 4.3-Md HindIII fragment could not be cloned in an uninterrupted way in E. coli but could be cloned in two parts. Both fragments showed homology with the 9-Md proteinase plasmid of S. cremoris HP. The 4.3-Md HindIII fragment was successfully cloned in Bacillus subtilis on plasmid pGKV2 (3.1 Md). Crossed immunoelectrophoresis of extracts of B. subtilis carrying the recombinant plasmid (pGKV500; 7.4 Md) showed that the fragment specifies two proteins of the proteolytic system of S. cremoris Wg2. PGKV500 was introduced in a proteinase-deficient Streptococcus lactis strain via protoplast transformation. Both proteins were also present in cell-free extracts of S. lactis(pGKV500). In S. lactis, pGKV500 enables the cells to grow normally in milk with rapid acid production, indicating that the 4.3-Md HindIII fragment of plasmid pWV05 specifies the proteolytic activity of S. cremoris Wg2.

Animals↗
PubMed1985

Construction of cloning, promoter-screening, and terminator-screening shuttle vectors for Bacillus subtilis and Streptococcus lactis.

Shuttle vectors have been constructed which are suitable both for the selection of regulatory sequences and for gene cloning in Bacillus subtilis and Streptococcus lactis. The promoter screening vectors contain a promoterless chloramphenicol acetyltransferase gene; the insertion of suitable DNA fragments upstream of the gene restored the enzyme activity. With a related set of vectors, transcription termination signals can be selected.

Acetyltransferases↗
PubMed1984

Construction of plasmid cloning vectors for lactic streptococci which also replicate in Bacillus subtilis and Escherichia coli.

The cryptic Streptococcus cremoris Wg2 plasmid pWV01 (1.5 megadaltons) was genetically marked with the chloramphenicol resistance (Cmr) gene from pC194. The recombinant plasmid (pGK1, 2.4 megadaltons) replicated and expressed Cmr in Bacillus subtilis. From this plasmid an insertion-inactivation vector was constructed by inserting the erythromycin resistance (Emr) gene from pE194 cop-6. This plasmid (pGK12, 2.9 megadaltons) contained a unique BclI site in the Emr gene and unique ClaI and HpaII sites outside both resistance genes. It was stably maintained in B. subtilis at a copy number of approximately 5. pGK12 also transformed Escherichia coli competent cells to Cmr and Emr. The copy number in E. coli was about 60. Moreover, pGK12 transformed protoplasts of Streptococcus lactis. In this host both resistance genes are expressed. pGK12 is stably maintained in S. lactis at a copy number of 3.

Bacillus subtilis↗
PubMed1982

Screening for syphilis among homosexual men in bars and saunas in Amsterdam.

Serological screening of 1627 homosexual men visiting two saunas and two bars in Amsterdam showed that 32.2% were seroreactive for syphilis. The 18-month study period ended in June 1981. Follow-up was possible in 91% (224/245) of subjects with both positive TPHA and VDRL test results; 76 cases of previously undetected syphilis were identified among 75 of the 224 men (incidence rate 4.6%). The rate of previously undetected syphilis declined appreciably during the study period. The cost of identifying one new case of syphilis was Df1577 (pounds 120 or US $ 220).

Adolescent↗
PubMed1981

Pyruvate dehydrogenase deficiency restricted to brain.

We studied a child with a rapidly progressive neurologic disorder, with psychomotor retardation, hypotonia, seizures, and respiratory disturbances. Laboratory studied showed elevated levels of lactate and pyruvate in cerebrospinal fluid (CSF), without notable elevated levels in serum. In liver, muscle, leukocytes, and cultured fibroblasts we found no abnormality in pyruvate oxidation; biochemical studies of a brain biopsy showed an isolated deficiency of pyruvate dehydrogenase complex in brain tissue with the morphologic picture of progressive poliodystrophy with hypomyelination.

Brain Diseases↗
PubMedDate not supplied

From DNA sequence to application: possibilities and complications.

The development of sophisticated genetic tools during the past 15 years have facilitated a tremendous increase of fundamental and application-oriented knowledge of lactic acid bacteria (LAB) and their bacteriophages. This knowledge relates both to the assignments of open reading frames (ORF's) and the function of non-coding DNA sequences. Comparison of the complete nucleotide sequences of several LAB bacteriophages has revealed that their chromosomes have a fixed, modular structure, each module having a set of genes involved in a specific phase of the bacteriophage life cycle. LAB bacteriophage genes and DNA sequences have been used for the construction of temperature-inducible gene expression systems, gene-integration systems, and bacteriophage defence systems. The function of several LAB open reading frames and transcriptional units have been identified and characterized in detail. Many of these could find practical applications, such as induced lysis of LAB to enhance cheese ripening and re-routing of carbon fluxes for the production of a specific amino acid enantiomer. More knowledge has also become available concerning the function and structure of non-coding DNA positioned at or in the vicinity of promoters. In several cases the mRNA produced from this DNA contains a transcriptional terminator-antiterminator pair, in which the antiterminator can be stabilized either by uncharged tRNA or by interaction with a regulatory protein, thus preventing formation of the terminator so that mRNA elongation can proceed. Evidence has accumulated showing that also in LAB carbon catabolite repression in LAB is mediated by specific DNA elements in the vicinity of promoters governing the transcription of catabolic operons. Although some biological barriers have yet to be solved, the vast body of scientific information presently available allows the construction of tailor-made genetically modified LAB. Today, it appears that societal constraints rather than biological hurdles impede the use of genetically modified LAB.

Bacteriophages↗
PubMedDate not supplied

Anchoring of proteins to lactic acid bacteria.

The anchoring of proteins to the cell surface of lactic acid bacteria (LAB) using genetic techniques is an exciting and emerging research area that holds great promise for a wide variety of biotechnological applications. This paper reviews five different types of anchoring domains that have been explored for their efficiency in attaching hybrid proteins to the cell membrane or cell wall of LAB. The most exploited anchoring regions are those with the LPXTG box that bind the proteins in a covalent way to the cell wall. In recent years, two new modes of cell wall protein anchoring have been studied and these may provide new approaches in surface display. The important progress that is being made with cell surface display of chimaeric proteins in the areas of vaccine development and enzyme- or whole-cell immobilisation is highlighted.

Bacterial Proteins↗