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Biomedical subjects

A Gruss

Publications and source records attributed to A Gruss.

At least 37 records · Page 2Linked to original sources
PubMed1999

Gene replacement with linear DNA in electroporated wild-type Escherichia coli.

Gene replacement using linear double-stranded DNA fragments in wild-type Escherichia coli transformation is generally inefficient due to exonucleolytic degradation of incoming DNA. Recombination-proficient strains, in which the exonucleolytic activity of RecBCD is inactivated, have been used as transformation recipients to overcome this difficulty. Here we report that gene replacements using linear double-stranded donor DNA can be achieved in wild-type E.coli if electrocompetent cells are used. Using a plasmid target, we obtained 10(2)-10(3) gene replacement events/microgram linear DNA. Using an independent chromosomal target, approximately 60 gene replacement events/microgram linear DNA were obtained. The presence of Chi sites on the linear DNA, which are known to block DNA degradation and stimulate recombination in E.coli, had no effect on gene replacement efficiency in either case. RecBCD-mediated exonucleolytic activity was found to be diminished in electroporated cells. Electrotransformation thus provides a simple way to perform gene replacements in many E.coli strains.

Bacteriophage T4↗
PubMed1999

Effects of metabolic flux on stress response pathways in Lactococcus lactis.

Studies of cellular responses to stress conditions such as heat, oxygen or starvation have revealed the existence of numerous specific or interactive response pathways. We previously observed in Lactococcus lactis that inactivation of the recA gene renders the lactococcal strain sensitive not only to DNA-damaging agents but also to oxygen and heat. To further examine the stress response pathways in L. lactis, we isolated thermoresistant insertional mutants (Trm) of the recA strain. Eighteen independent trm mutations were identified and characterized. We found that mutations map in only seven genes, implicated in purine metabolism (deoB, guaA and tktA), phosphate uptake (pstB and pstS), mRNA stability (pnpA) and in one uncharacterized gene (trmA). All the trm mutations, with the exception of trmA, confer multiple stress resistance to the cell. Some of the mutations confer improved heat stress resistance not only in the recA but also in the wild-type context. Our results reveal that cellular metabolic pathways are intimately related to stress response and that the flux of particular metabolites, notably guanine and phosphate, may be implicated in stress response in lactococci.

ATP-Binding Cassette Transporters↗
PubMed1999

Results of immunochemotherapy with interleukin-2, interferon-alpha2 and 5-fluorouracil in the treatment of metastatic renal cell cancer.

OBJECTIVE: In patients with advanced metastatic renal cell carcinoma (RCC) seen at a single institution, the toxicity and long-term clinical effects of a combination therapy with recombinant interleukin-2 (rIL-2), recombinant interferon-alpha2 (rIFN-alpha2) and 5-fluorouracil (5-FU) were evaluated. METHOD: From August 1992 through August 1997, 47 consecutive patients (38 men) with metastatic RCC were treated using rIL-2 and rIFN-alpha2 subcutaneously in combination with intravenous 5-FU. An average of 2. 4 cycles/patient (range 1-9) was administered. RESULTS: Toxicity grades II and III (World Health Organization) were observed in 24 and 17 patients, respectively. We achieved 9 major responses (7 complete responses (CR) and 2 partial responses (PR)) for an objective response rate of 19.1% (95% confidence interval 9.1-33.3%). A further 13 patients (27.7%) had a stabilization of disease. After a mean follow-up of 17.9 (2-53) months, 4 patients are alive with no evidence of disease. The 1- and 3-year survival probability was 70 and 37%, respectively. In an univariate analysis, two prognostic factors were correlated with disease outcome: Karnofsky performance index (p = 0.01) and the presence of bone metastases (p = 0.023). CONCLUSION: This triple-drug combination therapy was effective in the treatment of progressive RCC in almost every fifth patient.

Antineoplastic Agents↗
PubMed1998

Identification of the lactococcal exonuclease/recombinase and its modulation by the putative Chi sequence.

Studies of RecBCD-Chi interactions in Escherichia coli have served as a model to understand recombination events in bacteria. However, the existence of similar interactions has not been demonstrated in bacteria unrelated to E. coli. We developed an in vivo model to examine components of dsDNA break repair in various microorganisms. Here, we identify the major exonuclease in Lactococcus lactis, a Gram-positive organism evolutionarily distant from E. coli, and provide evidence for exonuclease-Chi interactions. Insertional mutants of L. lactis, screened as exonuclease-deficient, affected a single locus and resulted in UV sensitivity and recombination deficiency. The cloned lactococcal genes (called rexAB) restored UV resistance, recombination proficiency, and the capacity to degrade linear DNA, to an E. coli recBCD mutant. In this context, DNA degradation is specifically blocked by the putative lactococcal Chi site (5'-GCGCGTG-3'), but not by the E. coli Chi (5'-GCTGGTGG-3') site. RexAB-mediated recombination was shown to be stimulated approximately 27-fold by lactococcal Chi. Our results reveal that RexAB fulfills the biological roles of RecBCD and indicate that its activity is modulated by a short DNA sequence. We speculate that exonuclease/recombinase enzymes whose activities are modulated by short DNA sequences are widespread among bacteria.

Base Sequence↗
PubMed1998

Identification of the Chi site of Haemophilus influenzae as several sequences related to the Escherichia coli Chi site.

The Escherichia coli Chi site 5'-GCTGGTGG-3' modulates the activity of the powerful dsDNA exonuclease and helicase RecBCD. Genome sequence analyses revealed that Chi is frequent on the chromosome and oriented with respect to replication on the E. coli genome. Chi is also present much more frequently than predicted statistically for a random 8-mer sequence. Although it is assumed that Chi is ubiquitous, there is virtually no proof that its features are conserved in other microorganisms. We therefore identified and analysed the Chi sequence of an organism for which the full genome sequence was available, Haemophilus influenzae. The biological test we used is based on our finding that rolling circle plasmids provide a specific substrate for RecBCD analogues in different microorganisms. Unexpectedly, several related sequences, corresponding to 5'-GNTGGTGG-3' and 5'-G(G/C)TGGAGG-3', showed Chi activity. As in E. coli, the H. influenzae Chi sites are frequent on the genome, which is in keeping with the need for frequent Chi sites for dsDNA break repair of chromosomal DNA. Although statistically over-represented, this feature is less marked than that of the E. coli Chi site. In contrast to E. coli, the H. influenzae Chi motifs are only slightly oriented with respect to the replication strand. Thus, although Chi appears to have a highly conserved biological role in attenuating exonuclease activity, its sequence characteristics and statistical representation on the genome may differ according to the particular features of the host.

Base Sequence↗
PubMed1998

A nine-residue synthetic propeptide enhances secretion efficiency of heterologous proteins in Lactococcus lactis.

Lactococcus lactis, a gram-positive organism widely used in the food industry, is a potential candidate for the secretion of biologically useful proteins. We examined the secretion efficiency and capacity of L. lactis by using the Staphylococcus aureus nuclease (Nuc) as a heterologous model protein. When expressed in L. lactis from an efficient lactococcal promoter and its native signal peptide, only approximately 60% of total Nuc was present in a secreted form at approximately 5 mg per liter. The remaining 40% was found in a cell-associated precursor form. The secretion efficiency was reduced further to approximately 30% by the deletion of 17 residues of the Nuc native propeptide (resulting in NucT). We identified a modification which improved secretion efficiency of both native Nuc and NucT. A 9-residue synthetic propeptide, LEISSTCDA, which adds two negative charges at the +2 and +8 positions, was fused immediately after the signal peptide cleavage site. In the case of Nuc, secretion efficiency was increased to approximately 80% by LEISSTCDA insertion without altering the signal peptide cleavage site, and the yield was increased two- to fourfold (up to approximately 20 mg per liter). The improvement of NucT secretion efficiency was even more marked and rose from 30 to 90%. Similarly, the secretion efficiency of a third protein, the alpha-amylase of Bacillus stearothermophilus, was also improved by LEISSTCDA. These data indicate that the LEISSTCDA synthetic propeptide improves secretion of different heterologous proteins in L. lactis.

Amino Acid Sequence↗
PubMed1998

An export-specific reporter designed for gram-positive bacteria: application to Lactococcus lactis.

The identification of exported proteins by fusion studies, while well developed for gram-negative bacteria, is limited for gram-positive bacteria, in part due to drawbacks of available export reporters. In this work, we demonstrate the export specificity and use of the Staphylococcus aureus secreted nuclease (Nuc) as a reporter for gram-positive bacteria. Nuc devoid of its export signal (called delta(SP)Nuc) was used to create two fusions whose locations could be differentiated. Nuclease activity was shown to require an extracellular location in Lactococcus lactis, thus demonstrating the suitability of delta(SP)Nuc to report protein export. The shuttle vector pFUN was designed to construct delta(SP)Nuc translational fusions whose expression signals are provided by inserted DNA. The capacity of delta(SP)Nuc to reveal and identify exported proteins was tested by generating an L. lactis genomic library in pFUN and by screening for Nuc activity directly in L. lactis. All delta(SP)Nuc fusions displaying a strong Nuc+ phenotype contained a classical or a lipoprotein-type signal peptide or single or multiple transmembrane stretches. The function of some of the predicted signals was confirmed by cell fractionation studies. The fusions analyzed included long (up to 455-amino-acid) segments of the exported proteins, all previously unknown in L. lactis. Homology searches indicate that several of them may be implicated in different cell surface functions, such as nutrient uptake, peptidoglycan assembly, environmental sensing, and protein folding. Our results with L. lactis show that delta(SP)Nuc is well suited to report both protein export and membrane protein topology.

Amino Acid Sequence↗
PubMed1997

[Immunochemotherapy of metastatic renal cell carcinoma with interleukin 2, interferon-alpha and 5-fluorouracil].

Interleukin-2 (IL-2) and interferon-alpha (IFN-alpha) were both administered subcutaneously (SC) in combination with intravenously (IV) applied 5-fluorouracil (5-FU) for the treatment of patients with metastasized renal cell carcinoma (RCC). The therapy protocol consisted of a treatment cycle of 8 weeks, which could be carried out in an outpatient regimen. The IFN-alpha was given in each of the 8 weeks (6-9 MU/m2 once to three times weekly SC) combined sequentially with IL-2 (5-20 MU/m2 three times weekly SC for 4 weeks) and 5-FU (750 mg/m2 IV weekly for 4 weeks). Among the 30 consecutive patients treated, in 2 cases a complete, and in 9 cases a partial, remission was achieved in patients with mostly lung and skeletal metastases, with an overall objective response rate of 37%. Mean response duration was 8 months (range 3-18 months). A stable state of the disease lasting 3-18 months was observed in 10 cases. The side effects were only slight and corresponded to toxicity grade I (n = 2), grade II (n = 22) and grade III (n = 6), according to the WHO classification. In conclusion, this triple-drug biochemotherapy demonstrated significant clinical effectiveness comparable with that of an aggressive IL-2 treatment regimen (applied IV), but without its high toxicity.

Adult↗
PubMed1997

Characterization of Lactococcus lactis UV-sensitive mutants obtained by ISS1 transposition.

Studies of cellular responses to DNA-damaging agents, mostly in Escherichia coli, have revealed numerous genes and pathways involved in DNA repair. However, other species, particularly those which exist under different environmental conditions than does E. coli, may have rather different responses. Here, we identify and characterize genes involved in DNA repair in a gram-positive plant and dairy bacterium, Lactococcus lactis. Lactococcal strain MG1363 was mutagenized with transposition vector pG+host9::ISS1, and 18 mutants sensitive to mitomycin and UV were isolated at 37 degrees C. DNA sequence analyses allowed the identification of 11 loci and showed that insertions are within genes implicated in DNA metabolism (polA, hexB, and deoB), cell envelope formation (gerC and dltD), various metabolic pathways (arcD, bglA, gidA, hgrP, metB, and proA), and, for seven mutants, nonidentified open reading frames. Seven mutants were chosen for further characterization. They were shown to be UV sensitive at 30 degrees C (the optimal growth temperature of L. lactis); three (gidA, polA, and uvs-75) were affected in their capacity to mediate homologous recombination. Our results indicate that UV resistance of the lactococcal strain can be attributed in part to DNA repair but also suggest that other factors, such as cell envelope composition, may be important in mediating resistance to mutagenic stress.

Bacterial Proteins↗
PubMed1997

Cell wall anchoring of the Streptococcus pyogenes M6 protein in various lactic acid bacteria.

The M6 protein from Streptococcus pyogenes is the best-characterized member of a family of cell envelope-associated proteins. Based on the observation that the C-terminal sorting signals of these proteins can drive cell wall anchoring of heterologous unanchored proteins, we have cloned and expressed the emm6 structural gene for the M6 protein in various lactic acid bacteria (LAB). The emm6 gene was successfully expressed from lactococcal promoters in several Lactococcus lactis strains, an animal-colonizing Lactobacillus fermentum strain, Lactobacillus sake, and Streptococcus salivarius subsp. thermophilus. The M6 protein was efficiently anchored to the cell wall in all strains tested. In lactobacilli, essentially all detectable M6 protein was cell wall associated. These results suggest the feasibility of using the C-terminal anchor moiety of M6 for protein surface display in LAB.

Antigens, Bacterial↗
PubMed1996

Lactococcus lactis and stress.

It is now generally recognized that cell growth conditions in nature are often suboptimal compared to controlled conditions provided in the laboratory. Natural stresses like starvation and acidity are generated by cell growth itself. Other stresses like temperature or osmotic shock, or oxygen, are imposed by the environment. It is now clear that defense mechanisms to withstand different stresses must be present in all organisms. The exploration of stress responses in lactic acid bacteria has just begun. Several stress response genes have been revealed through homologies with known genes in other organisms. While stress response genes appear to be highly conserved, however, their regulation may not be. Thus, search of the regulation of stress response in lactic acid bacteria may reveal new regulatory circuits. The first part of this report addresses the available information on stress response in Lactococcus lactis. Acid stress response may be particularly important in lactic acid bacteria, whose growth and transition to stationary phase is accompanied by the production of lactic acid, which results in acidification of the media, arrest of cell multiplication, and possible cell death. The second part of this report will focus on progress made in acid stress response, particularly in L. lactis and on factors which may affect its regulation. Acid tolerance is presently under study in L. lactis. Our results with strain MG1363 show that it survives a lethal challenge at pH 4.0 if adapted briefly (5 to 15 minutes) at a pH between 4.5 and 6.5. Adaptation requires protein synthesis, indicating that acid conditions induce expression of newly synthesized genes. These results show that L. lactis possesses an inducible response to acid stress in exponential phase. To identify possible regulatory genes involved in acid stress response, we determined low pH conditions in which MG1363 is unable to grow, and selected at 37 degrees C for transposition insertional mutants which were able to survive. About thirty mutants resistant to low pH conditions were characterized. The interrupted genes were identified by sequence homology with known genes. One insertion interrupts ahrC, the putative regulator of arginine metabolism; possibly, increased arginine catabolism in the mutant produces metabolites which increase the pH. Several other mutations putatively map at some step in the pathway of (p)ppGpp synthesis. Our results suggest that the stringent response pathway, which is involved in starvation and stationary phase survival, may also be implicated in acid pH tolerance.

Adaptation, Physiological↗
PubMed1996

Efficient insertional mutagenesis in lactococci and other gram-positive bacteria.

In lactococci, the study of chromosomal genes and their regulation is limited by the lack of an efficient transposon mutagenesis system. We associated the insertion sequence ISS1 with the thermosensitive replicon pG+ host to generate a mutagenic tool that can be used even in poorly transformable strains. ISS1 transposition is random in different lactococcal strains as well as in Enterococcus faecalis and Streptococcus thermophilus. High-frequency random insertion (of about 1%) obtained with this system in Lactococcus lactis allows efficient mutagenesis, with typically one insertion per cell. After ISS1 replicative transposition, the chromosome contains duplicated ISS1 sequences flanking pG+ host. This structure allows cloning of the interrupted gene. In addition, efficient excision of the plasmid leaves a single ISS1 copy at the mutated site, thus generating a stable mutant strain with no foreign markers. Mutants obtained by this transposition system are food grade and can thus be used in fermentation processes.

Base Sequence↗
PubMed1996

Chemotherapy in advanced seminoma and the role of postcytostatic retroperitoneal lymph node dissection.

Between 1979 and 1992, 79 patients with seminoma were treated at our institution. Nineteen of these patients with advanced seminoma were treated with cisplatin-based chemotherapy (stage IIA, n = 2; stage IIB, n = 6; stage IIC, n = 2; stage III, n = 5 [2 with primary extragonadal tumor site]; relapse, n = 5 [1 with previously stage III seminoma]). One patient died of progressive disease 3 months after treatment with chemotherapy and retroperitoneal lymph node dissection (RPLND) which had led initially to a complete clinical response. Another patient (62 years old) died of an acute heart failure due to a sepsis caused by chemotherapy. Treatment of the other 17 patients was successful. Ten patients with a residual retroperitoneal mass after inductive chemotherapy underwent a RPLND. In 1 case viable seminoma (diameter of the residual mass 5 cm), in 4 cases necrotic tumor tissue, and in 5 cases fibrosis was diagnosed histopathologically. The 2 patients with extragonadal seminoma showed complete responses after surgery and chemotherapy. In conclusion, in patients with advanced seminoma, inductive cytostatic therapy seems to be the best treatment regimen. Residual retroperitoneal masses after chemotherapy with a diameter of > 3 cm should be treated with RPLND.

Adult↗
PubMed1996

Immunochemotherapy for metastatic renal cell carcinoma using a regimen of interleukin-2, interferon-alpha and 5-fluorouracil.

PURPOSE: Promising results of recent clinical trials with a triple drug bio-chemotherapy regimen encouraged its use in patients with renal cell carcinoma. MATERIALS AND METHODS: In a phase II study of patients with metastatic renal cell carcinoma the efficacy and toxicity of a treatment regimen were evaluated using interleukin-2 and interferon-alpha 2 subcutaneously in combination with intravenous 5-fluorouracil. The treatment protocol consisted of an 8-week cycle given on an outpatient basis, with 6 to 9 MU/m2. interferon-alpha given 1 to 3 times a week during the 8 weeks, and sequentially combined with 5 to 20 MU/m2. interleukin-2, 3 times a week for 4 weeks and 750 mg./m.2 5-fluorouracil once a week for 4 weeks. RESULTS: Among 25 consecutive men and 9 women treated 3 (9%) had a complete and 10 (29%) had a partial remission (overall objective response rate 38%). Median response duration (complete plus partial) was 12.5 months (range 3 to 20+). Stable disease lasting 3 to 24+ months was noted in 12 patients (35%). There were only minor side effects, for a maximum toxicity grade of I in 3 patients, II in 25 and III in 6 according to the World Health Organization classification. There were no dose limiting toxicities and no treatment related deaths. CONCLUSIONS: Triple drug immunochemotherapy resulted in a significant clinical effect comparable to an aggressive intravenous interleukin-2 treatment regimen but without significant toxicity.

Adult↗