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J Lenz

Publications and source records attributed to J Lenz.

At least 19 recordsLinked to original sources
PubMed1999

High levels of IL-4 and IL-10 mRNA and low levels of IL-2, IL-9 and IFN-gamma mRNA in MuLV-induced lymphomas.

The expression of cytokines may influence the development of lymphoma in retrovirally infected animals in at least two ways: (1) cytokines in the tumor environment may stimulate the proliferation of tumor cells and/or (2) cytokines in the tumor environment may diminish the cell-mediated antitumor immune response. To evaluate these possibilities, a semiquantitative RT-PCR approach was utilized to permit a broad screening of cytokine mRNAs in a large number of tissue samples. Examination of MuLV-induced end-stage lymphomas revealed the absence of mRNA for cytokines known to stimulate the proliferation of T cells (i.e., IL-2, IL-9), the absence of mRNA for cytokines known to enhance cell-mediated antitumor immune responses (i.e., IL-2, IFNgamma), and the presence of mRNA for cytokines known to diminish such responses (i.e., IL-4, IL-10). Similar patterns of cytokine mRNA expression were detected in tumor-derived cell lines. Spleen and thymus from animals collected longitudinally during infection and from age-matched uninfected mice also demonstrated a similar pattern, except that IFNgamma mRNA was readily detectable. These findings do not support the hypothesis that the developing tumor depends on cytokines to provide proliferative signals. The findings suggest that cytokines in the immediate environment of the lymphoma support tumor development by acting to diminish an effective antitumor immune response.

Animals↗
PubMed1999

Many human endogenous retrovirus K (HERV-K) proviruses are unique to humans.

BACKGROUND: Endogenous retroviruses contribute to the evolution of the host genome and can be associated with disease. Human endogenous retrovirus K (HERV-K) is related to the mouse mammary tumor virus and is present in the genomes of humans, apes and cercopithecoids (Old World monkeys). It is unknown how long ago in primate evolution the full-length HERV-K proviruses that are in the human genome today were formed. RESULTS: Ten full-length HERV-K proviruses were cloned from the human genome. Using provirus-specific probes, eight of the ten were found to be present in a genetically diverse set of humans but not in other extant hominoids. Intact preintegration sites for each of these eight proviruses were present in the apes. A ninth provirus was detected in the human, chimpanzee, bonobo and gorilla genomes, but not in the orang-utan genome. The tenth was found only in humans, chimpanzees and bonobos. Complete sequencing of six of the human-specific proviruses showed that full-length open reading frames for the retroviral protein precursors Gag-Pro-Pol or Env were each present in multiple proviruses. CONCLUSIONS: At least eight full-length HERV-K genomes that are in the human germline today integrated after humans diverged from chimpanzees. All of the viral open reading frames and cis-acting sequences necessary for HERV-K replication must have been intact during the recent time when these proviruses formed. Multiple full-length open reading frames for all HERV-K proteins are present in the human genome today.

Animals↗
PubMed1999

Suppressor mutations within the core binding factor (CBF/AML1) binding site of a T-cell lymphomagenic retrovirus.

The transcriptional enhancer of the lymphomagenic mouse retrovirus SL3 contains a binding site for the transcription factor core binding factor (CBF; also called AML1, PEBP2, and SEF1). The SL3 CBF binding site is called the core. It differs from the core of the weakly lymphomagenic mouse retrovirus Akv by one nucleotide (the sequences are TGTGGTTAA and TGTGGTCAA, respectively). A mutant virus called SAA that was identical to SL3 except that its core was mutated to the Akv sequence was only moderately attenuated for lymphomagenicity. In most SAA-infected mice, tumor proviruses contained either reversions of the original mutation or one of two novel core sequences. In 20% of the SAA-infected mice, tumor proviruses retained the original SAA/Akv core mutation but acquired one of two additional mutations (underlined), TGCGGTCAA or TGTGGTCTA, that generated core elements called So and T*, respectively. We tested whether the novel base changes in the So and T* cores were suppressor mutations. SL3 mutants that contained So or T* cores in place of the wild-type sequence were generated. These viruses induced T-cell lymphomas in mice more quickly than SAA. Therefore, the mutations in the So and T* cores are indeed second-site suppressor mutations. The suppressor mutations increased CBF binding in vitro and transcriptional activity of the viral long terminal repeats (LTRs) in T lymphocytes to levels comparable to those of SL3. Thus, CBF binding was increased by any of three different nucleotide changes within the sequence of the SAA core. Increased CBF binding resulted in increased LTR transcriptional activity in T cells and in increased viral lymphomagenicity.

Animals↗
PubMed1999

R region sequences in the long terminal repeat of a murine retrovirus specifically increase expression of unspliced RNAs.

A stem-loop structure at the 5' end of the R region of the long terminal repeat (LTR) of the murine leukemia virus SL3 and other type C mammalian retroviruses is important for maximum levels of expression of a reporter gene under the control of the viral LTR. This element, termed the R region stem-loop (RSL), has a small effect on transcriptional initiation and no effect on RNA polymerase processivity. Its major effect is on posttranscriptional processing of LTR-driven transcripts. Here we tested whether the RSL affected the production of RNAs from a full-length SL3 genome. Mutation of the RSL in the 5' LTR of SL3 reduced the cytoplasmic levels of full-length viral transcripts but not those of spliced, env mRNA transcripts. Thus, the RSL specifically affected the cytoplasmic levels of the unspliced viral RNA. To test further whether the effect was specific for unspliced transcripts, a system was devised in which the expression of a reporter gene under the control of the viral LTR was tested in the presence or absence of an intron. Mutation of the RSL resulted in only about a twofold decline in the level of reporter gene expression when the transcripts contained an intron. However, when the intron was removed, mutation of the RSL reduced expression of the reporter gene about 10- to 60-fold in various cell lines. The secondary structure of the RSL was essential for its activity on the intronless transcript. Thus, the RSL appears to be important for the cytoplasmic accumulation of unspliced viral RNA and unspliced RNA from chimeric transcription units under the control of the viral LTR.

Animals↗
PubMed1999

Selection of reversions and suppressors of a mutation in the CBF binding site of a lymphomagenic retrovirus.

The retrovirus SL3 induces T-cell lymphomas in mice. The transcriptional enhancer in the long terminal repeat (LTR) of SL3 contains two 72-bp repeats. Each repeat contains a binding site for the transcription factor CBF (also called AML1). The CBF binding sites are called core elements. SAA is a mutant that is identical to SL3 except for the presence of a single-base-pair substitution in each of the two core elements. This mutation significantly attenuates viral lymphomagenicity. Most lymphomas that occur in SAA-infected mice contain proviruses with reversions or second-site suppressor mutations within the core element. We examined the selective pressures that might account for the predominance of the reversions and suppressor mutations in tumor proviruses by analyzing when proviruses with altered core sequences became abundant during the course of lymphomagenesis. Altered core sequences were easily detected in thymus DNAs by 4 to 6 weeks after SAA infection of mice, well before lymphomas were grossly evident. This result is consistent with the hypothesis that viruses with the core sequence alterations emerged because they replicated more effectively in mice than SAA. The number of 72-bp tandem, repeats in the viral LTR was found to vary, presumably as a consequence of reverse transcriptase slippage during polymerization. Proviruses with two repeats predominated in the thymuses of SAA- and SL3-infected mice before lymphomas developed, although LTRs with one or three repeats were also present. This suggested that two was the optimal number of 72-bp repeats for viral replication. However, in lymphomas, proviruses with three or four repeats usually predominated. This suggested that a late step in the process of lymphomagenesis led to the abundance of proviruses with additional repeats. We hypothesize that proviruses with additional 72-bp repeats endowed the cells containing them with a selective growth advantage.

Animals↗
PubMed1998

Thermal stresses in metal-ceramic specimens for the ISO crack initiation test (three-point flexure bond test).

OBJECTIVES: At the October 1996 meeting of the ISO/TC106/SC 2/WG 1 working group, a special three-point flexure test for the characterization of the metal-ceramic bond was incorporated in the standard (ISO CD 9693). Due to the fabrication process, like real porcelain-fused-to-metal (PFM) restorations, the specimens contain thermal (eigen-) stresses upon which the load stresses are superimposed in the actual test. This study is devoted to the determination of these residual thermal stresses. METHODS: The residual thermal stresses in the specimen were calculated with the aid of the finite element method (FEM) using an especially fine mesh in the vicinity of the edge of initial debonding. Young's modulus, EM, of the alloy was varied within the interval 80 GPa < or = EM < or = 220 GPa which covers the spectrum of dental alloys. The analysis also allows the calculation of thermal stresses as a function of the difference delta alpha = alpha M - alpha C of the coefficients of thermal expansion of alloy and ceramic and the glass transition temperature theta G of porcelain. RESULTS: The thermal shear and normal stresses at the bond interface concentrate at the end of the ceramic veneer and practically vanish over about three quarters of the central part of the layer. The larger the Young's modulus, EM, of the alloy, the higher both stresses. SIGNIFICANCE: The results permit a deeper comprehension of the debonding process in the test: shear stress induced by loading increases the overall shear stress at the end of the bond interface, whereas load tensile stress is buffered by thermal compressive stress.

Dental Bonding↗
PubMed1998

The secondary structure of the R region of a murine leukemia virus is important for stimulation of long terminal repeat-driven gene expression.

In addition to their role in reverse transcription, the R-region sequences of some retroviruses affect viral transcription. The first 28 nucleotides of the R region within the long terminal repeat (LTR) of the murine type C retrovirus SL3 were predicted to form a stem-loop structure. We tested whether this structure affected the transcriptional activity of the viral LTR. Mutations that altered either side of the stem and thus disrupted base pairing were generated. These decreased the level of expression of a reporter gene under the control of viral LTR sequences about 5-fold in transient expression assays and 10-fold in cells stably transformed with the LTR-reporter plasmids. We also generated a compensatory mutant in which both the ascending and descending sides of the stem were mutated such that the nucleotide sequence was different but the predicted secondary structure was maintained. Most of the activity of the wild-type SL3 element was restored in this mutant. Thus, the stem-loop structure was important for the maximum activity of the SL3 LTR. Primer extension analysis indicated that the stem-loop structure affected the levels of cytoplasmic RNA. Nuclear run-on assays indicated that deletion of the R region had a small effect on transcriptional initiation and no effect on RNA polymerase processivity. Thus, the main effect of the R-region element was on one or more steps that occurred after the template was transcribed by RNA polymerase. This finding implied that the main function of the R-region element involved RNA processing. R-region sequences of human immunodeficiency virus type 1 or mouse mammary tumor virus could not replace the SL3 element. R-region sequences from an avian reticuloendotheliosis virus partially substituted for the SL3 sequences. R-region sequences from Moloney murine leukemia virus or feline leukemia virus did function in place of the SL3 element. Thus, the R region element appears to be a general feature of the mammalian type C genus of retroviruses.

3T3 Cells↗
PubMed1998

CBF, Myb, and Ets binding sites are important for activity of the core I element of the murine retrovirus SL3-3 in T lymphocytes.

Transcriptional enhancers within the long terminal repeats of murine leukemia viruses are major determinants of the pathogenic properties of these viruses. Mutations were introduced into the adjacent binding sites for three transcription factors within the enhancer of the T-cell-lymphomagenic virus SL3-3. The sites that were tested were, in 5'-to-3' order, a binding site for core binding factor (CBF) called core II, a binding site for c-Myb, a site that binds members of the Ets family of factors, and a second CBF binding site called core I. Mutation of each site individually reduced transcriptional activity in T lymphocytes. However, mutation of the Myb and core I binding sites had larger effects than mutation of the Ets or core II site. The relative effects on transcription in T cells paralleled the effects of the same mutations on viral lymphomagenicity, consistent with the idea that the role of these sequences in viral lymphomagenicity is indeed to regulate transcription in T cells. Mutations were also introduced simultaneously into multiple sites in the SL3-3 enhancer. The inhibitory effects of these mutations indicated that the transcription factor in T cells that recognizes the core I element of SL3-3, presumably CBF, needed to synergize with one or more factors bound at the upstream sites to function. This was tested further by generating a multimer construct that contained five tandem core I elements linked to a basal long terminal repeat promoter. This construct was inactive in T cells. However, transcriptional activity was detected with a multimer construct in which the transcription factor binding sites upstream of the core were also present. These results are consistent with the hypothesis that CBF requires heterologous transcription factors bound at nearby sites to function in T cells.

Animals↗
PubMed1997

Estimation of membrane potential deltapsi in reconstituted plasma membrane vesicles using a numerical model of oxonol VI distribution.

A model of membrane potential-dependent distribution of oxonol VI to estimate the electrical potential difference deltapsi across Schizosaccharomyces pombe plasma membrane vesicles (PMV) has been developed. deltapsi was generated by the H+-ATPase reconstituted in the PMV. The model treatment was necessary since the usual calibration of the dye fluorescence changes by diffusion potentials (K+ + valinomycin) failed. The model allows for fitting of fluorescence changes at different vesicle and dye concentrations, yielding deltapsi in ATP-energized PMV of 80 mV. The described model treatment to estimate deltapsi may be applicable for other reconstituted membrane systems.

Cell Membrane↗
PubMed1997

The feline leukemia virus long terminal repeat contains a potent genetic determinant of T-cell lymphomagenicity.

Feline leukemia virus (FeLV) is an important pathogen of domestic cats. The most common type of malignancy associated with FeLV is T-cell lymphoma. SL3-3 (SL3) is a potent T-cell lymphomagenic murine leukemia virus. Transcriptional enhancer sequences within the long terminal repeats (LTRs) of SL3 and other murine retroviruses are crucial genetic determinants of the pathogenicities of these viruses. The LTR enhancer sequences of FeLV contain identical binding sites for some of the transcription factors that are known to affect the lymphomagenicity of SL3. To test whether the FeLV LTR contains a genetic determinant of lymphomagenicity, a recombinant virus that contained the U3 region of a naturally occurring FeLV isolate, LC-FeLV, linked to the remainder of the genome of SL3 was generated. When inoculated into mice, the recombinant virus induced T-cell lymphomas nearly as quickly as SL3. Moreover, the U3 sequences of LC-FeLV were found to have about half as much transcriptional activity in T lymphocytes as the corresponding sequences of SL3. This level of activity was severalfold higher than that of the LTR of weakly leukemogenic Akv virus. Thus, the FeLV LTR contains a potent genetic determinant of T-cell lymphomagenicity. Presumably, it is adapted to be recognized by transcription factors present in T cells of cats, and this yields a relatively high level of transcription that allows the enhancer to drive the requisite steps in the process of lymphomagenesis.

3T3 Cells↗
PubMed1997

Importance of a c-Myb binding site for lymphomagenesis by the retrovirus SL3-3.

All murine leukemia viruses (MuLVs) and related type C retroviruses contain a highly conserved binding site for the Ets family of transcription factors within the enhancer sequences in the viral long terminal repeats (LTRs). The T-cell lymphomagenic MuLV SL3-3 (SL3-3) also contains a c-Myb binding site adjacent to the Ets site. The presence of this Myb site distinguishes SL3 from most other MuLVs. We tested the importance of these two sites for the lymphomagenicity of SL3-3. Mutation of the Ets site had little effect on viral pathogenicity, as it only slightly extended the latency period to disease onset. In contrast, mutation of the Myb site strongly inhibited pathogenicity, as only a minority of the inoculated mice developed tumors in the two mouse strains that were tested. All tumors that were induced by either mutant appeared to be lymphomas, and no evidence for reversion of either mutation was detected. The effects of the Ets and Myb site mutations on transcriptional activity of the SL3 LTR were tested by inserting the viral enhancer sequences into a plasmid containing the promoter region of the c-myc gene linked to a reporter gene. Mutation the Myb site almost eliminated enhancer activity in T lymphocytes, while mutation of the Ets site had smaller effects. Thus, the effects of the enhancer mutations on transcriptional activity in T cells paralleled their effects on viral lymphomagenicity. The absence of the c-Myb site in the LTR enhancer of the weakly lymphomagenic MuLV, Akv, likely contributes to the low pathogenicity of this virus relative to SL3-3. However, Moloney MuLV also lacks the Myb site in its LTR, although it induces T-cell lymphomas with a potency similar to that of SL3-3. Thus, it appears that SL3-3 and Moloney MuLV evolved genetic determinants of T-cell lymphomagenicity that are, at least in part, distinct.

Animals↗
PubMed1996

Transcriptional activation of a retrovirus enhancer by CBF (AML1) requires a second factor: evidence for cooperativity with c-Myb.

Transcriptional enhancer sequences within the long terminal repeats (LTRs) of murine leukemia viruses are the primary genetic determinants of the tissue specificity and potency of the oncogenic potential of these retroviruses. SL3-3 (SL3) is a murine leukemia virus that induces T-cell lymphomas. The LTR enhancer of this virus contains two binding sites for the transcription factor CBF (also called AML1 and PEBP2) that flank binding sites for c-Myb and the Ets family of factors. Using cotransfection assays in P19 cells, we report here that CBF and c-Myb cooperatively stimulate transcription from the SL3 LTR. By itself, c-Myb had no stimulatory effect on transcription. However, when cotransfected with a cDNA encoding one form of the alpha subunit of CBF called CBFalpha2-451, a level of transactivation higher than that seen with CBFalpha2-451 alone was detected. The negative regulatory domain near the carboxyl terminus of c-Myb did not affect this activity. Electrophoretic mobility shift assays indicated that CBF and c-Myb bind to DNA independently. Therefore, it appears that the cooperative stimulation of transcription by these factors occurs at a step in the process of transcription after the two factors are bound to the enhancer. Sequences near the carboxyl terminus of CBFalpha2-451 were important for cooperativity with c-Myb, consistent with previous reports that this region contains an activation domain. However, CBFalpha2-451 failed to activate transcription from a version of the SL3 LTR in which the enhancer was replaced with five tandem CBF-binding sites. Thus, it appears that transcriptional activation of the SL3 enhancer by CBF requires that an appropriate heterologous transcription factor be bound to a neighboring site in the regulatory sequences.

Animals↗
PubMed1996

[Liberation of neopterin after lung resecting interventions with and without bronchial carcinoma].

Neopterin has been suggested as a marker for the activation of macrophages, as indicated in several malignant tumors. We therefore studied 63 patients undergoing thoracic surgery, including lung tissue resection. These patients were prospectively monitored regarding the release of neopterin and other mediators of cellular immune reaction (PMN-elastase, CRP, sIL-2-R, PGE2). Our study revealed that surgical interventions in lung adenocarcinomas resulted in a marked (P < or = 0.01) and long-lasting (5 days) release of neopterin and sIL-2-R postoperatively. In contrast, the so-called inflammatory mediators (CRP, PMN-elastase) were only transiently increased. When comparing these findings in lung carcinoma with operations on benign tumors of the lung, on the basis of our results we conclude that the carcinoma per se obviously induces a distinct immune reaction. This, however, may be caused by surgical manipulation of the tumor during tissue resection. No prognostic value has been found so far for the release of neopterin regarding survival and/or recurrence of tumor.

Adenocarcinoma↗
PubMed1995

Bond strength of metal-ceramic systems in three-point flexure bond test.

This study deals with a three-point flexure test for the metal-ceramic bond involving geometrically simple specimens (alloy strips partly coated with ceramic) that can be fabricated with reasonable expenditure and sufficient reproducibility. The calculation of the stress distribution in such specimens with the aid of the finite-element method (FEM) is presented. The aim of this numerical analysis is: to investigate the stress distribution in a ceramometallic specimen with dimensions that, in a large number of experiments, have proven to lead to debonding at one end of the ceramic veneer instead of a crack in the middle of the veneer; and to assign a bond strength to the measured critical bending force that takes into account the influence of the Young's modulus of the alloy as well as a possible deviation of the thickness of the metal substrate from the standard value. Bond strength values of a variety of metal-ceramic combinations are demonstrated. These experimental results demonstrate the reproducibility of the test method as well as its sensitivity to diverse parameters. The presented method is proposed as an alternative to another flexure bond test nominated for international standard. In this test ceramometallic strips are bent over a rod to a 90 degrees angle of the specimen ends, subsequently flattened, and the fracture surface visually inspected for adherence of the ceramic to the alloy substrate along the predominant part of the middle third of the specimen. It is clear that such a test can at most deliver qualitative results.

Biocompatible Materials↗
PubMed1995

Long terminal repeat enhancer core sequences in proviruses adjacent to c-myc in T-cell lymphomas induced by a murine retrovirus.

The transcriptional enhancer in the long terminal repeat (LTR) of the T-lymphomagenic retrovirus SL3-3 differs from that of the nonleukemogenic virus Akv at several sites, including a single base pair difference in an element termed the enhancer core. Mutation of this T-A base pair to the C-G C-G sequence found in Akv significantly attenuated the leukemogenicity of SL3-3. Thus, this difference is important for viral leukemogenicity. Since Akv is an endogenous virus, this suggests that the C-G in its core is an adaptation to being minimally pathogenic. Most tumors that occurred in mice inoculated with the mutant virus, called SAA, contained proviruses with reversion or potential suppressor mutations in the enhancer core. We also found that the 72-bp tandem repeats constituting the viral enhancer could vary in number. Most tumors contained mixtures of proviruses with various numbers of 72-bp units, usually between one and four. Variation in repeat number was most likely due to recombination events involving template misalignment during viral replication. Thus, two processes during viral replication, misincorporation and recombination, combined to alter LTR enhancer structure and generate more pathogenic variants from the mutant virus. In SAA-induced tumors, enhancers of proviruses adjacent to c-myc had the largest number of core reversion or suppressor mutations of all of the viral enhancers in those tumors. This observation was consistent with the hypothesis that one function of the LTR enhancers in leukemogenesis is to activate proto-oncogenes such as c-myc.

3T3 Cells↗