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J R Klein

Publications and source records attributed to J R Klein.

At least 73 records · Page 4Linked to original sources
PubMed1994

A novel marker of murine bone marrow hematopoietic stem cells that is expressed on peripheral T cells and is associated with a functionally important molecule on activated cytotoxic T lymphocytes.

A MAb (R2/60) has been isolated that defines a novel lymphocyte marker of murine T cells. The determinant recognized by MAb R2/60 is present on a subset of bone marrow (BM) hematopoietic stem cells, on adult thymocytes, on peripheral T cells (both resting and activated), and on murine T cell tumor lines, although it is not expressed on mature B cells. In immunoprecipitation studies using radiolabeled membrane lysates from adult thymocytes, MAb R2/60 precipitated a 44-kDa membrane-bound dimer. Functionally, MAb R2/60 mediated antigen-independent cell lysis by activated CTLs, and by CTL clones, when bridged to Fc receptor-bearing target cells; however, binding of MAb R2/60 to effector cells prior to cytotoxic assays did not inhibit target cell lysis by CTLs, suggesting that the R2/60 determinant is involved in transmembrane signaling to already activated CTLs, but that it is not involved in target cell adhesion or antigen recognition. Moreover, direct stimulation of T cells by MAb R2/60 in the absence of additional stimuli did not induce cell proliferation, further implying that the R2/60 determinant is functionally involved in the effector rather than the inductive phase of the T cell response.

Animals↗
PubMed1994

Phenotype and TCR gamma delta variable gene repertoire of intestinal intraepithelial lymphocytes in wild mice (Mus musculus domesticus): abundance of V gamma 1 transcripts and extensive delta gene diversity.

In order to study murine intestinal intraepithelial lymphocytes (IEL) independent of factors imparted by conditions of laboratory housing and breeding, and to provide a basis for comparison of IEL studies between inbred and outbred mouse populations, IEL from the domestic house mouse, Mus musculus domesticus, were analyzed by flow cytometric analyses using mAbs to murine lymphocyte markers, and by polymerase chain reaction to study the TCR gamma and delta V gene repertoires. The majority of IEL in wild mice were CD3+, CD8+CD4- T cells. CD4+CD8- also were present in IEL isolates from wild mice, although at low numbers. Among IEL, but not T cells from the spleen or lymph nodes, there was a notable lack of Thy-1 expression, a preponderance of CD8 alpha alpha + T cells, and a relatively high ratio (3:1) of TCR gamma delta + T cells over TCR alpha beta + T cells, suggesting that some IEL in wild mice may develop via an extrathymic pathway similar to that described for laboratory mice. Analyses of the IEL gamma and delta variable genes revealed rearrangements of three of six V region gamma genes (V gamma 1, V gamma 2, and V gamma 5), with an abundance of V gamma 1 transcripts as determined by Northern blot analyses. For the delta gene, rearrangement of five of seven V region elements had occurred (V delta 2, V delta 3, V delta 4, V delta 5, and V delta 6).(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1994

Cloning and nucleotide sequence analysis of pepV, a carnosinase gene from Lactobacillus delbrueckii subsp. lactis DSM 7290, and partial characterization of the enzyme.

Cell extracts of Lactobacillus delbrueckii subsp. lactis DSM 7290 were found to exhibit unique peptolytic ability against unusual beta-alanyl-dipeptides. In order to clone the gene encoding this activity, designated pepV, a gene library of strain DSM 7290 genomic DNA, prepared in the low-copy-number plasmid pLG339, was screened for heterologous expression in Escherichia coli. Recombinant clones harbouring pepV were identified by their ability to allow the utilization of carnosine (beta-alanyl-histidine) as a source of histidine by the E. coli mutant strain UK197 (pepD, hisG). Complementation was observed in a colony harbouring a recombinant plasmid (pKV101), carrying pepV. A 2.4 kb fragment containing pepV was subcloned and its nucleotide sequence revealed an open reading frame (ORF) of 1413 nucleotides, corresponding to a protein with predicted molecular mass of 51998 Da. A single transcription initiation site 71 bp upstream of the ATG translational start codon was identified by primer extension. No significant homology was detected between pepV or its deduced amino acid sequence with any entry in the databases. The only similarity was found in a region conserved in the ArgE/DapE/CPG2/YscS family of proteins. This observation, and protease inhibitor studies, indicated that pepV is of the metalloprotease type. A second ORF present in the sequenced fragment showed extensive homology to a variety of amino acid permeases from E. coli and Saccharomyces cerevisiae.

Amino Acid Sequence↗
PubMed1994

Cloning, heterologous expression, and sequencing of a novel proline iminopeptidase gene, pepI, from Lactobacillus delbrueckii subsp. lactis DSM 7290.

The gene for proline iminopeptidase from Lactobacillus delbrueckii subsp. lactis DSM 7290 coding for an enzyme that hydrolyses the synthetic substrate L-prolyl-beta-naphthylamide (Pro-beta NA) was cloned in Escherichia coli. An enzymic plate assay was used to screen for positive clones. The gene, designated pepI, was subcloned into vector pUC18 and sequenced. The nucleotide sequence revealed an 882 bp open reading frame encoding 294 amino acids, coding for an enzyme with a calculated molecular mass of 32883 Da. By cloning under control of the lac promoter the peptidase was highly expressed. Sequence analysis showed that pepI is of a new sequence type, distinct from all peptidases so far sequenced. Amino acid homology to the active site of a Pseudomonas putida esterase and inhibitor studies of the enzyme imply involvement of a serine residue in catalysis.

Amino Acid Sequence↗
PubMed1994

Phenotypic and functional heterogeneity of murine intestinal intraepithelial lymphocytes defined by cell density: implications for route of differentiation and responsiveness to proliferation induction.

The phenotype and function of murine intestinal intraepithelial lymphocytes (IEL) was studied in a Percoll-fractionated preparation that consisted of low-density cells which migrated to the 40-50% Percoll interface (IEL-40), medium-density cells which migrated to the 50-55% interface (IEL-50), and high-density cells which migrated to the 55-70% interface (IEL-55). IEL-40 and IEL-50 cells, the subsets phenotypically most similar to mature IEL, consisted of CD3+ T cells that included CD4- CD8+ and CD4+ CD8+ cells; CD4+ CD8- cells were present only in the IEL-50 fraction. T-cell receptor (TcR)alpha beta and TcR gamma delta cells were present in both IEL-40 and IEL-50 fractions. In contrast, most IEL-55 were CD3-, heat-stable antigen (HSA)+ cells that were not B cells; some IEL-55 cells were CD3lo HSA- or CD3lo HSA+ suggesting that IEL-55 are immature T cells. TcR alpha beta but not TcR gamma delta was expressed in the IEL-55 fraction. All three IEL fractions consisted of both CD8 alpha alpha and CD8 alpha beta cells. There was considerable functional heterogeneity between the three IEL fractions such that CD3-induced proliferation was greatest for IEL-50 cells and least for IEL-55 cells; that activity correlated with the proportion of Thy-1+ cells within the fractions. Both IEL-40 and IEL-50 fractions contained activated cytotoxic T lymphocytes (CTL) that were 8-16-fold more lytic than IEL-55 cells. That IEL-55 cells may be precursors of some IEL-40 and IEL-50 cells was demonstrated by a shift in cell density and by an increase in proportions of cells expressing markers of IEL-40 and IEL-50 cells following in vitro stimulation via CD3. The relevance of these findings to differences in functional activities reported for murine IEL is discussed.

Animals↗
PubMed1993

Cloning, DNA sequence analysis and partial characterization of pepN, a lysyl aminopeptidase from Lactobacillus delbrückii ssp. lactis DSM7290.

In cell extracts of Lactobacillus delbrückii ssp. lactis DSM7290 a peptidase with the ability to hydrolyse Phe-beta-naphthylamide (Phe-beta-NA) and His-beta-NA could be detected. Escherichia coli lacking the enzyme activity in an enzymic plate assay was used to screen high-copy-number and low-copy-number plasmid libraries of size-fractionated Lactobacillus DNA. Clones with the desired phenotype were detected, and the gene, designated pepN, was further subcloned and sequenced. A large open reading frame of 2529 nucleotides is predicted to encode a protein of 843 amino acids (95358 Da). Comparison of the pepN gene from Lb. delbrückii ssp. lactis DSM7290 indicates that it is homologous to genes of the family of Zn(2+)-metallohydrolases and PepN shows identity with the active centre Zn(2+)-binding motif of these enzymes. The substrate Lys-beta-NA is more effectively cleaved than Phe-beta-NA or His-beta-NA which were used for screening in E. coli. The cloned pepN gene was efficiently overexpressed in E. coli and subcloning of the gene in Lactobacillus casei resulted in a moderate overexpression of approximately 20-fold. The pepN gene product was purified from the pepN-deficient E. coli strain CM89, using the substrate Lys-p-nitroanilide (Lys-NH-Ph) in the assay procedure. In a four-step procedure including streptomycin sulfate precipitation, anion-exchange chromatography and gel filtration the peptidase was purified to electrophoretic homogeneity.

Amino Acid Sequence↗
PubMed1993

EnvC, a new lipoprotein of the cytoplasmic membrane of Escherichia coli.

A gene product with an apparent molecular mass of approximately 39,000 Da can be identified in the cytoplasmic membrane of Escherichia coli upon expression of cloned envC. In this communication we report that the product was labelled with [3H]glycerol and [3H]palmitic acid, and a precursor molecule of increased molecular mass was accumulated when cells were treated with globomycin, a specific inhibitor for the prolipoprotein signal peptidase. The same precursor molecule was encoded by an envC mutant gene, in which the cysteine residue in a pentapeptide sequence, Leu-Ile-Ala-Gly-Cys24 within the amino terminal region of EnvC, was replaced by tryptophane (Trp24). This protein was not labelled with [3H]glycerol. The results demonstrate that the envC gene product represents a new lipoprotein of the cytoplasmic membrane of E. coli.

Amino Acid Sequence↗
PubMed1993

Characterization and sequence analysis of a small cryptic plasmid from Lactobacillus curvatus LTH683 and its use for construction of new Lactobacillus cloning vectors.

Lactobacillus curvatus LTH683, a strain originally isolated from raw sausage, contains the single cryptic plasmid called pLC2. The sequence and genetic organization of the complete 2489-bp plasmid pLC2 was determined and used as the basis for construction of a series of vectors useful in Lactobacillus strains. The major parts of pLC2 nucleotide sequence could be aligned with other plasmids from gram-positive bacteria replicating by a rolling circle mechanism of replication (RCR). Direct evidence for a RCR mechanism was obtained by showing the accumulation of single-stranded plasmid intermediates in the presence of rifampicin. Three protein-coding sequences could be predicted and the corresponding proteins were detected after in vitro transcription/translation of pLC2 plasmid DNA. ORFs 1 and 3 showed minor homologies to plasmids of gram-positive bacteria. The replication protein coded by ORF2 and its corresponding target sequence, the plus origin, were similar to replication regions of other gram-positive bacteria plasmids like pLS1, pWV01, and pE194. Upstream of the ori+ site, in a noncoding region, which was nonessential for replication, strong homology to other Lactobacillus plasmids like pC30i1, pLP1, pLJ1, and pLAB1000 could be detected. A palindromic sequence predicted to be the minus origin of replication was localized there. Small vectors (3213 bp) suitable for cloning in lactobacilli were constructed based on a 1635-bp DNA fragment of pLC2, containing the region necessary for replication, marked with the chloramphenicol resistance gene and a multiple cloning site.

Amino Acid Sequence↗
PubMed1993

Cloning and sequence analysis of the X-prolyl-dipeptidyl-aminopeptidase gene (pepX) from Lactobacillus delbrückii ssp. lactis DSM7290.

Lactobacillus delbrückii ssp. lactis DSM7290 possesses an X-prolyl-dipeptidyl-aminopeptidase, designated PepX, which catalyses the hydrolytic removal of N-terminal dipeptidyl residues from peptides containing proline in the penultimate position. Using the specific substrate L-Ala-L-Pro-p-nitroanilide, PepX was purified by a four-step procedure including ammonium sulphate fractionation, hydrophobic interaction chromatography, ion exchange chromatography, and affinity chromatography. The N-terminus of the purified protein was sequenced. Screening of a gene library of chromosomal Lactobacillus delbrückii ssp. lactis DSM7290 DNA in the low-copy-number vector pLG339 resulted in the identification of the pepX gene in Escherichia coli using a specific plate assay with Gly-L-Pro-beta-naphthylamide as substrate. Nucleotide sequence analysis revealed an open reading frame of 2376 bp, coding for a protein of 792 amino acids with a molecular mass of 88449 Da.

Amino Acid Sequence↗
PubMed1993

T cell receptor delta gene repertoire and diversity of intestinal intraepithelial lymphocytes in athymic mice.

T cell receptor (TCR) delta gene rearrangements in intestinal intraepithelial lymphocytes (IEL) were studied in athymic radiation chimeras using polymerase chain reaction (PCR) and sequence analysis of DNAs spanning the variable (V), diversity (D), and junctional (J) genes. In both thymus-bearing and athymic mice, IEL delta gene rearrangements occurred for V delta 3, V delta 4, V delta 5 and V delta 6. V-D-J junctional-site sequence analyses of cloned DNAs from rearranged IEL delta genes in athymic mice revealed a predominance of in-frame rearrangements; junctional diversity consisting of nucleotide removal from V, D and/or J genes; N segment nucleotide insertions; and high overall gene diversity. Evaluation of PCR-amplified cDNAs made from IEL RNA indicated that all four rearranged V delta genes were expressed in IEL from athymic mice. The high diversity observed at the gene level also was present in amino acid sequences encoded by the V-D-J region of IEL delta genes in athymic mice. These data demonstrate that there is extensive diversity of rearranged delta genes in IEL which develop extrathymically, and suggest that the delta chain of IEL TCR-gamma delta+ T cells has the potential for interactions with polymorphic structures.

Amino Acid Sequence↗
PubMed1992

A rapid method for isolating murine intestine intraepithelial lymphocytes with high yield and purity.

Because murine intestine intraepithelial lymphocytes (IEL) are dispersed throughout the intestine epithelium, it is important that IEL extraction procedures result in lymphocyte preparations of sufficient purity for use in in vitro and in vivo experimental systems. Here, we describe an improved technique for isolating murine IEL consisting of a single 30 min extraction followed by multiple nylon wool filtrations and centrifugation through Percoll. This procedure yields a preparation of IEL with high overall recovery and purity yet takes only 2-2.5 h. Evaluation of individual steps in the extraction process indicated that nylon wool filtration, in particular multiple filtrations, and Percoll fractionation both were important for achieving highly-enriched IEL populations by removal of enterocytes and cellular debris, and demonstrated that multiple nylon wool filtration improved the overall IEL recovery. This procedure has several advantages for studies of murine IEL in that the resultant IEL population is ideal for phenotypic, functional, or molecular analyses. Moreover, this technique is effective for isolating IEL on a single-animal basis, thereby permitting analyses of IEL from individual mice rather than as pooled IEL obtained from several animals.

Animals↗
PubMed1992

Peripheral engraftment of fetal intestine into athymic mice sponsors T cell development: direct evidence for thymopoietic function of murine small intestine.

Adult athymic, lethally irradiated, F1-->parent bone marrow-reconstituted (AT x BM) mice were engrafted bilaterally with day 16-18 fetal intestine or fetal thymus into the kidney capsule and were studied for evidence of peripheral T cell repopulation of 1-12 wk postengraftment. Throughout that time period, both types of grafts were macroscopically and histologically characteristic of differentiated thymus or intestine tissues, respectively. Beginning at week 2 postengraftment, clusters of lymphocytes were present within intestine grafts, particularly in subepithelial regions and in areas below villus crypts. As determined by immunofluorescence staining and flow cytometric analyses, lymphocytes from spleen and lymph nodes of sham-engrafted mice (AT x BM-SHAM) were essentially void of T cells, whereas in AT x BM thymus-engrafted (AT x BM-THG) mice, which served as a positive control for T cell repopulation, normal levels of T cells were present in spleen and lymph nodes by week 3 postengraftment, and at times thereafter. Most striking, however, was the finding that T cell repopulation of the spleen and lymph nodes occurred in AT x BM fetal intestine-engrafted (AT x BM-FIG) mice beginning 3 wk postengraftment. Based on H-2 expression, peripheral T cells in AT x BM-FIG mice were of donor bone marrow origin, and consisted of CD3+, T cell receptor (TCR)-alpha/beta+ T cells with both CD4+8- and CD4-8+ subsets. Peripheral T cells in AT x BM-FIG mice were functionally mature, as demonstrated by their capacity to proliferate after stimulation of CD3 epsilon. Moreover, alloreactive cytotoxic T lymphocytes were generated in primary in vitro cultures of spleen cells from AT x BM-FIG and AT x BM-THG mice, though not in spleen cell cultures from AT x BM-SHAM mice. Histologic studies of engrafted tissues 3-4 wk postengraftment demonstrated that thymus leukemia (Tl) antigens were expressed on epithelial surfaces of intestine grafts, and that both TCR-alpha/beta+ and TCR-gamma/delta+ lymphocytes were present in intestine grafts. Collectively, these findings indicate that the murine small intestine has the capacity to initiate and regulate T cell development from bone marrow precursors, thus providing a mechanism by which extrathymic development of intestine lymphocytes occur.

Animals↗
PubMed1992

The promoter region of the Escherichia coli pepD gene: deletion analysis and control by phosphate concentration.

A series of deletions removing progressively larger parts of the 5' flanking region of the Escherichia coli pepD gene was constructed. After fusing the resulting promoter fragments to the chromosomal malPQ operon, their activities were determined by assaying for amylomaltase, the product of the malQ gene. Transcription from the pepD promoter region in exponentially growing cells was estimated to be about 5 times less efficient than transcription from the induced lac promoter. Approximately 115 bp preceding the translation start site of the pepD gene are important for regular promoter functioning, whereas the more distal sequences could be deleted without any significant effects. In bacterial cultures containing limiting amounts of inorganic phosphate, the rate of de novo synthesis of peptidase D, simultaneously with the derepression of alkaline phosphatase, increased about fivefold as a consequence of phosphate starvation. This regulation was shown to occur at the transcriptional level by the use of chromosomal pepD promoter-malPQ fusions. The inducibility by phosphate limitation was conserved in all of the deletion clones in which the pepD promoter region was still functional. As demonstrated by the use of phoB, R, and M mutants, the modulation of pepD expression is independent of the genetic system controlling the pho regulon.

Base Sequence↗
PubMed1992

Repopulation kinetics of intestinal intraepithelial lymphocytes in murine bone marrow radiation chimeras.

The kinetics of lymphoid cell repopulation of murine intestinal intraepithelial lymphocytes (IEL) from BM stem cells were studied in F1----parent radiation chimeras and were compared with T cell repopulation of the thymus and the spleen. T cells arising from donor bone marrow were present in the gut epithelium as early as day 5 postreconstitution in radiation chimeras. By day 7 postreconstitution, and at times thereafter, the IEL consisted of 70-95% CD3+ donor bone marrow-derived T cells, most of which were CD8+ cells with variable Thy-1 expression. CD4+8- IEL also were detected between days 7 and 14 postreconstitution; however, the CD4+8+ IEL subset did not appear within the gut epithelium until several weeks later and was followed by a progressive increase in the intensity of CD8 expression on CD4+8+ IEL. Functionally mature T cell receptor gamma/delta + and alpha/beta + IEL were present throughout repopulation of the gut epithelium. In contrast to the IEL, T cells were not detected in the thymus or the spleen until days 14 and 21 postreconstitution, respectively, and evidence of T cell function in the spleen was not detected until day 21 postreconstitution. These findings have implications for human bone marrow transplantation in that they demonstrate that T cell repopulation of the gut epithelium begins prior to T cell repopulation of the thymus and the spleen, and indicate that even in the presence of a thymus some IEL proceed through an extrathymic developmental pathway.

Animals↗
PubMed1991

Molecular analysis and nucleotide sequence of the envCD operon of Escherichia coli.

The chromosomal DNA insert in plasmid pJK131, which complements the phenotypic defects associated with a mutation in the envC gene of Escherichia coli strain PM61, was sequenced. The analysis of the nucleotide sequence revealed two open reading frames (ORFs) coding for the proteins EnvC (41,281 daltons) and EnvD (104,415 daltons). The envC gene product is synthesized as a pre-protein and, after cleavage of a signal peptide, the mature protein is incorporated into the cytoplasmic membrane. The detection of a common transcript for both ORFs indicated the existence of an envCD operon. Deletion analysis and the generation of frameshifts demonstrated that simultaneous expression of both genes is required to complement the defects in strain PM61. Overproduction of EnvC protein appears to be lethal to Escherichia coli. The envD gene, however, could be cloned and expressed at high levels under control of the tac promoter without deleterious effects on the host.

Amino Acid Sequence↗
PubMed1991

Proliferative properties of murine intestinal intraepithelial lymphocytes (IEL): IEL expressing TCR alpha beta or TCR tau delta are largely unresponsive to proliferative signals mediated via conventional stimulation of the CD3-TCR complex.

Murine intestinal intraepithelial lymphocytes (IEL) were studied for their capacity to proliferate in vitro following stimulation of the T cell receptor (TCR)-associated CD3 epsilon molecule, or upon direct stimulation of the TCR complex itself. Although IEL consisted primarily of CD3+ T cells which included activated cytotoxic T lymphocytes as demonstrated in CD3- and TCR-mediated redirected cytotoxic assays, IEL displayed minimal proliferative responses following stimulation with anti-CD3, anti-TCR alpha beta, or anti-TCR tau delta monoclonal antibodies under soluble conditions, or under conditions which effect membrane cross-linking, including the addition of accessory cells to IEL cultures. The lack of proliferation induction could not be overcome by stimulation of IEL in the presence of T cell-dependent cytokines, phorbol ester, or interleukin-4. Moreover, unlike splenic T cells, stimulation of IEL failed to result in expression of interleukin-2 receptor, further demonstrating an inability of IEL to respond to exogenous proliferative signals. This study is the first to examine the proliferative potential of murine IEL following direct CD3 or TCR stimulation. The findings described here: (i) identify an important functional distinction between intestinal IEL and other peripheral alpha beta or tau delta T cells which generally respond well to proliferative signals mediated through the CD3-TCR complex, and (ii) demonstrate that on murine IEL the CD3-TCR complex can discriminate signals of lytic activity from those of cell proliferation.

Animals↗