Immunotherapy of renal cell carcinoma by intratumoral administration of an IL-2 cDNA/DMRIE/DOPE lipid complex.

Intratumoral administration of cytokine genes in order to achieve paracrine secretion of immunostimulatory cytokines and to create tumor vaccines in situ can avoid difficulties associated with the production of autologous and allogeneic vaccines, and toxicity related to systemic administration of cytokines. In this review, we summarize our experience with intratumoral administration of IL-2 cDNA/DMRIE/DOPE lipid complex in patients with metastatic renal cell carcinoma. An objective response rate of 14% was achieved in a phase I/II clinical trial and was confirmed in the low-risk subgroup of a phase II study. The achieved objective clinical responses (PR/CR) were long lasting. Application of PCR and immunohistochemistry in post-treatment tumor biopsies detected the IL-2 plasmid in addition to increased IL-2 expression in tumor cells and CD8 infiltration. Clinical trials employing higher doses of the plasmid in renal cell carcinoma patients with limited disease are ongoing.

Advances in systemic therapy of small cell cancer of the lung.

Over the last 20 years, progress in the therapy of small cell lung cancer has been painfully slow. Despite dramatic initial responses to chemotherapy, most patients relapse quickly with an overall 5-year survival of about 5%. Recent trials however offer some hope at changing this picture. Combining standard regimens with newer agents has doubled median survival in some cases. The use of novel targeted agents holds the promise of significantly increasing the survival in this disease, with manageable toxicity. This review outlines current treatment strategies, summarizes recent clinical trials and offers a view of what the next 5 years may hold for the treatment of small cell lung cancer.

Production of recombinant human type I procollagen trimers using a four-gene expression system in the yeast Saccharomyces cerevisiae.

The expression of stable recombinant human collagen requires an expression system capable of post-translational modifications and assembly of the procollagen polypeptides. Two genes were expressed in the yeast Saccharomyces cerevisiae to produce both propeptide chains that constitute human type I procollagen. Two additional genes were expressed coding for the subunits of prolyl hydroxylase, an enzyme that post-translationally modifies procollagen and that confers heat (thermal) stability to the triple helical conformation of the collagen molecule. Type I procollagen was produced as a stable heterotrimeric helix similar to type I procollagen produced in tissue culture. A key requirement for glutamate was identified as a medium supplement to obtain high expression levels of type I procollagen as heat-stable heterotrimers in Saccharomyces. Expression of these four genes was sufficient for correct assembly and processing of type I procollagen in a eucaryotic system that does not produce collagen.

Production of recombinant human type I procollagen homotrimer in the mammary gland of transgenic mice.

The large scale production of recombinant collagen for use in biomaterials requires an efficient expression system capable of processing a large (> 400 Kd) multisubunit protein requiring post-translational modifications. To investigate whether the mammary gland of transgenic animals fulfills these requirements, transgenic mice were generated containing the alpha S1-casein mammary gland-specific promoter operatively linked to 37 Kb of the human alpha 1(I) procollagen structural gene and 3' flanking region. The frequency of transgenic lines established was 12%. High levels of soluble triple helical homotrimeric [(alpha 1)3] type I procollagen were detected (up to 8 mg/ml) exclusively in the milk of six out of 9 lines of lactating transgenic mice. The transgene-derived human procollagen chains underwent efficient assembly into a triple helical structure. Although proline or lysine hydroxylation has never been described for any milk protein, procollagen was detected with these post-translational modifications. The procollagen was stable in milk; minimal degradation was observed. These results show that the mammary gland is capable of expressing a large procollagen gene construct, efficiently assembling the individual polypeptide chains into a stable triple helix, and secreting the intact molecule into the milk.

Asymmetric mutation around the recombination break point of immunoglobulin class switch sequences on extrachromosomal substrates.

Junctions at class switch recombination sites in the genome are characterized by a unique sequence feature. Nucleotide substitutions and small deletions are common on either of the two sides of the switch junction, but not on both together. We have previously reported an extrachromosomal substrate assay system for analyzing the recombination of class switch sequences. Here we have sequenced nine junctions on each side of the break point and compared these to 17 recombination junctions of control substrates from the same cells. Five of the nine switch recombination junctions have nucleotide substitutions and deletions, with multiple nucleotide changes being more common. Furthermore, mutations were found only on a single side of the junction, just as for the recombination of switch sequences in the genome. In contrast, only one of 17 control substrate junctions had a mutation, and this was a single nucleotide insertion. This difference is highly significant (P < 0.00007) and indicates that the fundamental recombination mechanism is likely to be similar for switch sequences in the chromosome and on minichromosome substrates.

RNA:DNA complex formation upon transcription of immunoglobulin switch regions: implications for the mechanism and regulation of class switch recombination.

Central the regulation and mechanism of class switch recombination is the understanding of the relationship between transcription and DNA recombination. We demonstrated previously, using mini-chromosome substrates, that physiologically oriented transcription is required for recombination to occur between switch regions. In this report, we demonstrate the formation of an RNA:DNA complex under in vitro transcription conditions for these same and other switch DNA fragments. We find that cell-free transcription of repetitive murine switch regions (Smu, S gamma 2b and S gamma 3) leads to altered DNA mobility on agarose gels. These altered mobilities are resistant to RNase A but sensitive to RNase H. Transcription in the presence of labeled ribonucleotides demonstrates the stable physical association of the RNA with the DNA. Importantly, complex formation only occurs upon transcription in the physiologic orientation. Reaban and Griffin [1990 Nature, 348, 342-344] found an RNA:DNA hybrid structure that was limited to an atypical 143 nucleotide purine region within a 2.3 kb S alpha segment. Here we demonstrate RNA:DNA hybrid formation in more typical switch sequences (lacking the atypical 143 nucleotide purine tract) from a variety of switch regions that are only 60-70% purine on the non-template strand. These results suggest a general model involving an RNA:DNA complex as an intermediate during class switch recombination.

Stapled stricturoplasty for Crohn's disease. A new technique.

PURPOSE: Stricturoplasty was originally used to treat multiple fibrotic strictures of tuberculosis. As the pendulum of treatment of Crohn's disease swung toward conservatism and bowel preservation, stricturoplasty was performed in Crohn's disease. Stricturoplasty can be used when there is limited, well-localized disease and should be avoided in the presence of grossly inflamed or infected material. We describe a new technique of stricturoplasty. METHODS: Typically stricturoplasty is performed in a manner similar to a Heineke-Mikulicz pyloroplasty. A stapled stricturoplasty technique has been previously described, but in actuality these are more similar to a bypass procedure. Our technique uses a stapled, open technique similar to the triangulating method of bowel anastomosis. This was performed in one patient to correct six strictures. RESULTS: Our patient did well postoperatively and developed no significant complications. He has no evidence of recurrent strictures. CONCLUSION: We describe a stapled stricturoplasty technique that is truly a stricturoplasty because the bowel lumen is increased; it is similar to the triangulating method of end-to-end stapled bowel anastomosis. It is safe, efficient, and effective. Additionally, it allows radiographic location of the stricturoplasty site, thus allowing determination of effectiveness of the procedure as well as recurrence.

Strand specificity in the transcriptional targeting of recombination at immunoglobulin switch sequences.

B-lymphocyte-specific class switch recombination is known to occur between pairs of 2- to 10-kb switch regions located immediately upstream of the immunoglobulin constant heavy-chain genes. Others have shown that the recombination is temporally correlated with the induction of transcription at the targeted switch regions. To determine whether this temporal correlation is due to a mechanistic linkage, we have developed an extrachromosomal recombination assay that closely recapitulates DNA deletional class switch recombination. In this assay, the rate of recombination is measured between 24 and 48 hr posttransfection. We find that recombinants are generated in a switch sequence-dependent manner. Recombination occurs with a predominance within B-cell lines representative of the mature B-cell stage and within a subset of pre-B-cell lines. Transcription stimulates the switch sequence-dependent recombination. Importantly, transcription activates recombination only when directed in the physiologic orientation but has no effect when directed in the nonphysiologic orientation.

Early postoperative feeding.

PURPOSE: Our intent was to show that immediate postoperative feeding of a regular diet after elective colorectal surgery was safe, tolerable, and feasible. METHODS: This was a nonrandomized, prospective study of 38 consecutive patients who underwent elective colorectal surgery over a three-month period. Our study parameters included operative length of time, intraoperative blood loss, need for transfusions, day to flatus or defecation, ability to tolerate a diet, placement of nasogastric tubes postoperatively, and length of hospital stay. These patients were compared with patients treated by different surgeons during the same time period but fed only after return of bowel sounds or flatus. RESULTS: Thirty-one of 36 patients eventually included in this study were able to tolerate our early feeding regimen. There were a total of seven other complications that were minor and unrelated to the early feeding. Patients who tolerated early feeding had shorter postoperative length of stays, 5.7 vs. 10.6 days. Patients who tolerated early feeding had shorter length of stays than patients treated in a more conventional manner, 5.7 vs. 8.0 days. Those who did not tolerate early feeding had longer procedures and greater intraoperative blood loss. CONCLUSIONS: Early postoperative feeding is safe and is tolerated by the majority of patients. Early feeding, if tolerated, decreases length of hospital stay and may decrease health care costs. Longer operative time and increased blood loss intraoperatively may indicate a more difficult procedure and identify those patients who will not tolerate early feeding.

Distinct roles for RAG-1 in the initiation of V(D)J recombination and in the resolution of coding ends.

Although RAG-1 and RAG-2 have been shown to be indispensible for V(D)J recombination, their exact role in this reaction remains unclear. Co-transfecting RAG-1 and RAG-2 expression vectors into NIH3T3 fibroblasts confers V(D)J recombination activity to these otherwise recombinationally inactive cells. In this report we have found that in transient transfections of mouse NIH3T3 fibroblasts with RAG-1 and RAG-2 and the appropriate recombination substrates, one RAG-1 expression vector, pRAG-1A, is capable of yielding both signal joints and coding joints, while another RAG-1 expression vector, pRAG-1B, yields only signal joints. The RAG-1 open reading frame for these two expression vectors is interchangeable, indicating that the inability to resolve coding joints is due to the 45-base pair difference found in the 5'-untranslated regions of these constructs. Differences in this region result in a 15-fold difference in gene expression when the luciferase coding region is substituted for the RAG-1 cDNA. This report provides evidence that RAG-1 may have a role in both the initiation of V(D)J recombination as well as the resolution of coding ends. The data also suggest that these RAG-1 activities may be dependent on different levels of RAG-1 expression.

The glucose transporter of Escherichia coli. Mutants with impaired translocation activity that retain phosphorylation activity.

The glucose transporter of the bacterial phosphotransferase system couples translocation with phosphorylation of the substrate in a 1:1 stoichiometry. It is a complex consisting of a transmembrane subunit (IIGlc) and a hydrophilic subunit (IIIGlc). Both subunits are transiently phosphorylated. IIIGlc is phosphorylated at a histidyl residue by the cytoplasmic phosphoryl carrier protein phospho-heat-stable phosphoryl carrier protein; IIGlc is phosphorylated at a cysteinyl residue by phospho-IIIGlc. The IIGlc subunit consists of two domains. The N-terminal hydrophobic domain is presumed to span the membrane several times; the C-terminal cytoplasmic domain includes the phosphorylation site. IIGlc phosphorylates glucose and methyl-alpha-D-glucopyranoside in transit across the inner membrane but can also phosphorylate intracellular glucose. Ten mutants resistant against extracellular toxic methyl-alpha-D-glucopyranoside yet capable of phosphorylating intracellular glucose were isolated. Strong impairment of transport activity in these mutants was accompanied by only a slight decrease of phosphorylation activity. Amino acid substitutions occurred at six sites that are clustered in three presumably hydrophilic loops in the transmembrane domain of IIGlc: M17T, M17I, G149S, K150E, S157F, H339Y, and D343G. We presume that the three polypeptide segments are directly involved in sugar translocation and/or binding but are of little importance for phosphorylation activity, folding, and membrane localization of IIGlc.

Insertion of the mannitol permease into the membrane of Escherichia coli. Possible involvement of an N-terminal amphiphilic sequence.

The in vivo membrane assembly of the mannitol permease, the mannitol Enzyme II (IImtl) of the Escherichia coli phosphotransferase system, has been studied employing molecular genetic approaches. Removal of the N-terminal amphiphilic leader of the permease and replacement with a short hydrophobic sequence resulted in an inactive protein unable to transport mannitol into the cell or catalyze either phosphoenol-pyruvate-dependent or mannitol 1-phosphate-dependent mannitol phosphorylation in vitro. The altered protein (68 kDa) was quantitatively cleaved by an endogenous protease to a membrane-associated 39-kDa fragment and a soluble 28-kDa fragment as revealed by Western blot analyses. Overproduction of the wild-type plasmid-encoded protein also led to cleavage, but repression of the synthesis of the plasmid-encoded enzyme by inclusion of glucose in the growth medium prevented cleavage. Several mtlA-phoA gene fusions encoding fused proteins with N-terminal regions derived from the mannitol permease and C-terminal regions derived from the mature portion of alkaline phosphatase were constructed. In the first fusion protein, F13, the N-terminal 13-aminoacyl residue amphiphilic leader sequence of the mannitol permease replaced the hydrophobic leader sequence of alkaline phosphatase. The resultant fusion protein was inefficiently translocated across the cytoplasmic membrane and became peripherally associated with both the inner and outer membranes, presumably via the noncleavable N-terminal amphiphilic sequence. The second fusion protein, F53, in which the N-terminal 53 residues of the mannitol permease were fused to alkaline phosphatase, was efficiently translocated across the cytoplasmic membrane and was largely found anchored to the inner membrane with the catalytic domain of alkaline phosphatase facing the periplasm. This 53-aminoacyl residue sequence included the amphiphilic leader sequence and a single hydrophobic, potentially transmembrane, segment. Analyses of other MtlA-PhoA fusion proteins led to the suggestion that internal amphiphilic segments may function to facilitate initiation of polypeptide trans-membrane translocation. The dependence of IImtl insertion on the N-terminal amphiphilic leader sequence was substantiated employing site-specific mutagenesis. The N-terminal sequence of the native permease is Met-Ser-Ser-Asp-Ile-Lys-Ile-Lys-Val-Gln-Ser-Phe-Gly.... The following point mutants were isolated, sequenced, and examined regarding the effects of the mutations on insertion of IImtl into the membrane: 1) S3P; 2) D4P; 3) D4L; 4) D4R; 5) D4H; 6) I5N; 7) K6P; and 8) K8P.(ABSTRACT TRUNCATED AT 400 WORDS)

Properties of a Tn5 insertion mutant defective in the structural gene (fruA) of the fructose-specific phosphotransferase system of Rhodobacter capsulatus and cloning of the fru regulon.

In photosynthetic bacteria such as members of the genera Rhodospirillum, Rhodopseudomonas, and Rhodobacter a single sugar, fructose, is transported by the phosphotransferase system-catalyzed group translocation mechanism. Previous studies indicated that syntheses of the three fructose catabolic enzymes, the integral membrane enzyme II, the peripheral membrane enzyme I, and the soluble fructose-1-phosphate kinase, are coordinately induced. To characterize the genetic apparatus encoding these enzymes, a Tn5 insertion mutation specifically resulting in a fructose-negative, glucose-positive phenotype was isolated in Rhodobacter capsulatus. The mutant was totally lacking in fructose fermentation, fructose uptake in vivo, phosphoenolpyruvate-dependent fructose phosphorylation in vitro, and fructose 1-phosphate-dependent fructose transphosphorylation in vitro. Extraction of the membrane fraction of wild-type cells with butanol and urea resulted in the preparation of active enzyme II free of contaminating enzyme I activity. This preparation was used to show that the activity of enzyme I was entirely membrane associated in the parent but largely soluble in the mutant, suggesting the presence of an enzyme I-enzyme II complex in the membranes of wild-type cells. The uninduced mutant exhibited measurable activities of both enzyme I and fructose-1-phosphate kinase, which were increased threefold when it was grown in the presence of fructose. Both activities were about 100-fold inducible in the parental strain. Although the Tn5 insertion mutation was polar on enzyme I expression, fructose-1-phosphate kinase activity was enhanced, relative to the parental strain. ATP-dependent fructokinase activity was low, but twofold inducible and comparable in the two strains. A second fru::Tn5 mutant and a chemically induced mutant selected on the basis of xylitol resistance showed pleiotropic loss of enzyme I, enzyme II, and fructose-1-phosphate kinase. These mutants were used to clone the fru regulon by complementing the negative phenotype with a wild-type cosmid bank.