Minimally invasive right colectomy with transrectal natural orifice extraction: could this be the next step forward?

BACKGROUND: The transvaginal natural orifice specimen extraction (NOSE) approach for right-side colon surgery has been proven to exhibit favorable short-term outcomes. However, thus far, no study has reported the advantages of transrectal NOSE for right-side colon surgery. The aim of this study was to compare the technical feasibility, safety, and short-term outcomes of minimally invasive right hemicolectomy using the transrectal NOSE method and those of conventional mini-laparotomy specimen extraction. METHODS: A study was conducted on consecutive patients who had minimally invasive right hemicolectomy either for malignancy or benign disease at Chang Gung Memorial Hospital, Linkou, Taiwan, between January 2017 and December 2018. The patients were divided into two groups: conventional surgery with specimen extraction using mini-laparotomy and NOSE surgery. Surgical outcomes, including complications, postoperative short-term recovery, and pain intensity, were analyzed. RESULTS: We enrolled 297 patients (151 males, mean age 64.9 ± 12.8 years) who had minimally invasive right hemicolectomy. Of these 297 patients, 272 patients had conventional surgery with specimen extraction through mini-laparotomy and 25 patients had NOSE surgery (23 transrectal, 2 transvaginal). The diagnosis of colon disease did not differ significantly between the conventional and NOSE groups. Postoperative morbidity and mortality rates were comparable. The postoperative hospital stay was significantly (p = 0.004) shorter in the NOSE group (median 5 days, range 3-17 days) than in the conventional group (median 7 days, range 3-45 days). Postoperative pain was significantly (p = 0.026 on postoperative day 1 and p = 0.002 on postoperative day 2) greater in the conventional group than in the NOSE group. CONCLUSIONS: NOSE was associated with acceptable short-term surgical outcomes that were comparable to those of conventional surgery. NOSE results in less postoperative wound pain and a shorter hospital stay than conventional surgery. Larger studies are needed.

Heparin is essential for a single keratinocyte growth factor molecule to bind and form a complex with two molecules of the extracellular domain of its receptor.

Keratinocyte growth factor (KGF or FGF-7) is a member of the heparin binding fibroblast growth factor (FGF) family and is a paracrine mediator of proliferation and differentiation of a wide variety of epithelial cells. To examine the stoichiometry of complexes formed between KGF and its receptor, we have utilized a soluble variant of the extracellular region of the KGF receptor containing two tandem immunoglobulin-like loops, loops II and III (sKGFR). Ligand-receptor complexes were examined by size exclusion chromatography, light scattering, N-terminal protein sequencing, and sedimentation velocity. In the presence of low-molecular mass heparin ( approximately 3 kDa), we demonstrate the formation of complexes containing two molecules of sKGFR and one molecule of KGF. In the absence of heparin, we were unable to detect any KGF-sKGFR complexes using the above techniques, and additional studies in which sedimentation equilibrium was used show that the binding is very weak (Kd >/= 70 microM). Furthermore, using heparin fragments of defined size, we demonstrate that a heparin octamer or decamer can promote formation of a 2:1 complex, while a hexamer does not. Utilizing the highly purified proteins and defined conditions described in this study, we find that heparin is obligatory for formation of a KGF-sKGFR complex. Finally, 32D cells, which appear to lack low-affinity FGF binding sites, were transfected with a KGFR-erythropoeitin receptor chimera and were found to require heparin to achieve maximal KGF stimulation. Our data are consistent with the previously described concept that cell- or matrix-associated heparan sulfate proteoglycans (HSPGs) and FGF ligands participate in a concerted mechanism that facilitates FGFR dimerization and signal transduction in vivo.

Human keratinocyte growth factor recombinantly expressed in Chinese hamster ovary cells: isolation of isoforms and characterization of post-translational modifications.

Keratinocyte growth factor (KGF) is a member of the fibroblast growth factor family that acts specifically on epithelial cells in a paracrine mode. We employed a mammalian expression system to synthesize recombinant human KGF and isolated two preparations, KGF-a and KGF-b, from medium conditioned by Chinese hamster ovary cells. On an SDS-PAGE gel, KGF-a migrates as two bands near 25-29 kDa and contains both N- and O-linked sugar moieties attached near the N-terminus. Detailed structural characterization confirms that KGF-a contains a single amino acid sequence predicted from cDNA sequence and the molecule has two intramolecular disulfide bridges, Cys1-Cys15 and Cys102-Cys106. An additional Cys at position 40 is free and resides in a solvent-inaccessible environment. Mass spectrometric analyses of KGF-a peptides verify the occurrence of several post-translational modifications in the molecule, including partial oxidation at Met28, partial sulfation at Tyr27, and glycosylation at Asn14 and Thr22. The Asn-linked carbohydrate structures are heterogeneous, which include biantennary, triantennary, and tetraantennary structures with none or up to four sialic acids attached to various structures, while the Thr-linked carbohydrates contain typical mucin-type structures. KGF-b is an N-terminally truncated form of KGF-a posttranslationally processed at Arg23 and is not glycosylated. Both KGF-a and KGF-b forms are capable of stimulating DNA synthesis in quiescent Balb/MK mouse epidermal keratinocytes.

Selective deamidation of recombinant human stem cell factor during in vitro aging: isolation and characterization of the aspartyl and isoaspartyl homodimers and heterodimers.

During in vitro aging, deamidation of recombinant human stem cell factor produced in Escherichia. coli was detected by HPLC analysis and by the release of soluble ammonia. The deamidation rate is very slow in buffers at low pH or at low temperatures; however, the rate is significantly accelerated in alkaline buffers such as sodium bicarbonate in combination with elevated temperatures. HPLC isolation of various deamidated forms followed by peptide mapping and mass spectrometric analyses revealed that the deamidation involves Asn10 in the sequence -T9NNV- near the N-terminus of the protein. Following peptide mapping analysis, significant amounts of aspartyl and isoaspartyl peptides were identified, indicating the conversion of asparagine into both aspartate and isoaspartate residues. As a result of spontaneous association-dissociation of stem cell factor dimer, a total of five deamidated forms, including two homodimers and three heterodimers, were detected and isolated. Cell proliferation assays showed that two rhSCF heterodimeric species, derived from dimerization between isoaspartyl and other stem cell factor monomers, retain only approximately half of the biological activity. The homodimer with isoaspartic acid in place of Asn10 is 50-fold less potent, while the aspartyl homodimer, either isolated during deamidation experiments or recombinantly prepared by site-directed mutagenesis (e.g., N10D and N10D/N11D variants), exhibits higher activity than the standard molecule. In comparison, synthetic N10A and N10E variants, though missing the deamidation site, are significantly less active. All these variants lacking the Asn10 deamidation site are relatively more stable than those containing the asparagine residue. The results indicate that the biological function and chemical stability of stem cell factor are influenced by the nature of the residue at position 10.

The majority of stem cell factor exists as monomer under physiological conditions. Implications for dimerization mediating biological activity.

Soluble Escherichia coli-derived recombinant human stem cell factor (rhSCF) forms a non-covalently associated dimer. We have determined a dimer association constant (Ka) of 2-4 x 10(8) M-1, using sedimentation equilibrium and size exclusion chromatography. SCF has been shown previously to be present at concentrations of approximately 3.3 ng/ml in human serum. Based on the dimerization Ka, greater than 90% of the circulating SCF would be in the monomeric form. When 125I-rhSCF was added to human serum and the serum analyzed by size exclusion chromatography, 72-49% of rhSCF was monomer when the total SCF concentration was in the range of 10-100 ng/ml, consistent with the Ka determination. Three SCF variants, SCF(F63C), SCF (V49L,F63L), and SCF(A165C), were recombinantly expressed in Escherichia coli, purified, and characterized. The dimer Ka values, biophysical properties, and biological activities of these variants were studied. Dimerization-defective variants SCF(F63C)S-CH2CONH2 and SCF(V49L,F63L) showed substantially reduced mitogenic activity, while the activity of the Cys165-Cys165 disulfide-linked SCF(A165C) dimer was 10-fold higher than that of wild type rhSCF. The results suggest a correlation between dimerization affinity and biological activity, consistent with a model in which SCF dimerization mediates dimerization of its receptor, Kit, and subsequent signal transduction.

Isolation and characterization of human tissue kallikrein produced in Escherichia coli: biochemical comparison to the enzymatically inactive prokallikrein and methionyl kallikrein.

This report describes bacterial expression, isolation, and characterization of human tissue kallikrein recombinantly produced in Escherichia coli. Successful production of enzymatically active recombinant human kallikrein requires the following processes: expression, solubilization and refolding of prokallikrein, thermolysin activation, and chromatographic separation. All experimental data confirmed that bacterially derived human kallikrein is properly folded and exhibits expected biochemical functions. As confirmed by SDS-PAGE and reverse-phase HPLC, recombinant kallikrein is apparently pure and is devoid of reduced or other partially folded kallikrein forms. Recombinant kallikrein behaves as a monomeric molecule in solution and exhibits full enzymatic activity in hydrolyzing peptide substrates. The molecule can bind to aprotinin to form kallikrein-inhibitor complex at a 1:1 molar ratio. Peptide mapping analysis derived from pepsin digestion of recombinant kallikrein assigned five disulfide bonds which match those of porcine kallikrein predicted from X-ray structure. Peptides containing unpaired cysteines or mispaired disulfide bonds were not detected. Both properly folded prokallikrein and methionyl kallikrein, containing a propeptide and an initiator methionine at their N-termini, respectively, were also produced and isolated. These two molecules are structurally similar to recombinant kallikrein, but are not enzymatically active.

Purification and characterization of human tissue prokallikrein and kallikrein isoforms expressed in Chinese hamster ovary cells.

We report here the expression of recombinant human prokallikrein and kallikrein in engineered Chinese hamster ovary cells transfected with a human genomic gene encoding preprokallikrein. At high expression levels, recombinant prokallikrein, an inactive proenzyme form, is predominantly secreted into the culture medium. Upon chromatographic separations, the inactive prokallikrein as well as the mature kallikrein after thermolysin activation of the proenzyme can be prepared to apparent purity. Both prokallikrein and kallikrein can be further separated into two distinct high- and low-molecular-weight isoforms. Kallikrein preparations are fully active in standard kallikrein activity assays such as esterase activity and kinin release from kininogen. Both kallikrein and prokallikrein display multiple molecular forms with differences in both molecular sizes and charges. The structural differences in high- and low-molecular-weight kallikreins or prokallikreins were found to be due to glycosylation, with the high-molecular-weight species glycosylated at three Asn-linked sites and the low-molecular-weight species at two of the three Asn-linked sites. The multiply charged kallikrein isoforms are derived from different numbers of sialic acids attached at the detected Asn-linked carbohydrates. In comparison with kallikrein, prokallikrein appears to show a significant decrease in the magnitude of near uv-circular dichroism bands, suggesting a change in local conformation. This conformational change correlates with the loss of activity in proenzyme due to the presence of propeptide.

In vitro methionine oxidation of Escherichia coli-derived human stem cell factor: effects on the molecular structure, biological activity, and dimerization.

The effect of oxidation of the methionine residues of Escherichia coli-derived recombinant human stem cell factor (huSCF) to methionine sulfoxide on the structure and activity of SCF was examined. Oxidation was performed using hydrogen peroxide under acidic conditions (pH 5.0). The kinetics of oxidation of the individual methionine residues was determined by quantitation of oxidized and unoxidized methionine-containing peptides, using RP-HPLC of Asp-N endoproteinase digests. The initial oxidation rates for Met159, Met-1, Met27, Met36, and Met48 were 0.11 min-1, 0.098 min-1, 0.033 min-1, 0.0063 min-1, and 0.00035 min-1, respectively, when SCF was incubated in 0.5% H2O2 at room temperature. Although oxidation of these methionines does not affect the secondary structure of SCF, the oxidation of Met36 and Met48 affects the local structure as indicated by CD and fluorescence spectroscopy. The 295-nm Trp peak in the near-UV CD is decreased upon oxidation of Met36, and lost completely following the oxidation of Met48, indicating that the Trp44 environment is becoming significantly less rigid than it is in native SCF. Consistent with this result, the fluorescence spectra revealed that Trp44 becomes more solvent exposed as the methionines are oxidized, with the hydrophobicity of the Trp44 environment decreasing significantly. The oxidations of Met36 and Met48 decrease biological activity by 40% and 60%, respectively, while increasing the dissociation rate constant of SCF dimer by two- and threefold. These results imply that the oxidation of Met36 and Met48 affects SCF dimerization and tertiary structure, and decreases biological activity.

Spontaneous dissociation-association of monomers of the human-stem-cell-factor dimer.

In its native state, recombinant human-stem-cell-factor (SCF) dimer can spontaneously and rapidly undergo hybridization when two different SCF dimer species are incubated together. SCF species differing in molecular charge, e.g., a wild-type SCF form and a variant with Asp at position 10 instead of Asn, were used in the hybridization studies; the original species and newly formed dimer hybrid can be separated and quantified by cationic-exchange h.p.l.c. The hybridization reaches an equilibrium where the ratio of hybrid dimer to each of the original species is 2. Kinetic studies of the initial rate of hybridization enable a rate constant for monomer dissociation to be determined. This rate constant is influenced by pH, temperature and salt concentration. The pH and salt effects suggest that salt bridges between charged amino acids at the monomer-monomer interface may be present. From the temperature effects, the activation energy for monomer dissociation was determined to be 85.6 kJ/mol, which is typical for oligomeric proteins. Heavily glycosylated recombinant SCF from Chinese-hamster ovary cells exchanged equally well with the bacterially derived non-glycosylated SCF, indicating that the attached carbohydrate moieties had no effect on monomer exchange.

The McKusick-Kaufman hydrometrocolpos-polydactyly syndrome--a case report.

Hydrometrocolpos is a rare congenital anomaly and serious life threatening condition in the newborn infant due to its long-term compression sequelae and associated congenital anomalies. Prenatal diagnosis of hydrometrocolpos by sonogram allows appropriate management during the prenatal and neonatal period. The combination of hydrometrocolpos and polydactyly is the cardinal hallmark feature of McKusick-Kaufman Syndrome. We present a case of congenital hydrometrocolpos due to vaginal atresia combined with polydactyly of both feet, mild atrial septum defect, bilateral hydronephrosis, fetal ascites and polyhydramnios. Pathogenesis and treatment of hydrometrocolpos and its associated congenital anomalies are discussed.

Comparison of N-linked oligosaccharides of recombinant human tissue kallikrein produced by Chinese hamster ovary cells on microcarrier beads and in serum-free suspension culture.

Glycosylation heterogeneity in recombinant human tissue kallikrein (r-HuTK) produced by Chinese hamster ovary (CHO) cells from microcarrier culture and from a serum-free suspension cell recycle process has been compared. Significant differences in the degree of sialylation were observed in glycoform distribution and oligosaccharide heterogeneity. High-performance liquid chromatography with a pellicular anion-exchange column under low pH eluant conditions was used to characterize the number and types of N-linked complex type oligosaccharides present. The oligosaccharides were released by N-glycanase and, after reduction, were resolved into a number of peaks containing one, two, three, and four sialic acids with an additional subfractionation based on the nature of the antennary structure. The microcarrier process resulted in a reduced amount of sialylated oligosaccharide species as compared to the suspension cell process. Removal of sialic acid followed by chromatography of the asialooligosaccharides under high pH anion-exchange conditions indicated that the same antennary structures were present but in slightly different relative amounts. The oligosaccharide profiles are indicative of a highly complex array of microheterogeneity present, encompassing mono-, di-, tri-, and tetrasialylated complex type oligosaccharides.

Detection and quantitation of recombinant granulocyte colony-stimulating factor charge isoforms: comparative analysis by cationic-exchange chromatography, isoelectric focusing gel electrophoresis, and peptide mapping.

Routine quantitation of recombinant human granulocyte colony-stimulating factor charge isoforms in the purified protein product requires development of a reliable analytical method. In this report, isoelectric focusing gel electrophoresis, peptide mapping, and cation-exchange high-performance liquid chromatography are compared and evaluated in the analysis of charge isomers that may be present in the recombinant factor. Due to a lack of sensitivity and reliability, isoelectric focusing gel electrophoresis and peptide mapping are not recommended. However, peptide mapping can distinguish aberrant peptides with differences in charges and provide separation for subsequent structural characterization. By this approach, an N-terminally blocked formylmethionyl species was identified to be the minor charge isoform in the purified preparations of recombinant human granulocyte colony-stimulating factor. In contrast to electrophoresis and peptide mapping, a strong cationic-exchange chromatographic procedure was found to be the most selective, sensitive, and reproducible analytical method. The sensitivity and reliability of the method were evaluated and validated using the formylmethionyl isoform and several deamidated analogs (Gln----Glu) made by site-directed mutagenesis. Recombinant human granulocyte colony-stimulating factor preparations contain a very low to undetectable level of the formylmethionine isoform and have no detectable deamidated isoforms.

Reversibility of acid denaturation of recombinant interferon-gamma.

It has been shown that interferon-gamma (IFN-gamma) loses activity after acid treatment and this property can be used to distinguish it from other types of interferons. Therefore, reversibility of acid denaturation of IFN-gamma was examined using the recombinant human protein. The fluorescence spectra showed that conformation of the protein is similar before and after acid treatment, suggesting reversibility of the acid denaturation. The antiviral activity of the protein was also identical in the same treatment. However, the antiviral activity was significantly reduced when it was determined by directly diluting the acidic samples into the assay medium containing high salts and serum proteins. Similar results were obtained with the recombinant murine IFN-gamma. This observation demonstrates that acid denaturation of the IFN-gamma is dependent on the way the protein is renatured, and hence that the difference in response to acid treatment between IFN-gamma and other interferons is quantitative rather than qualitative.

The effect of C-terminal processing on the activity of human interferon-gamma.

Homogeneous recombinant human interferon-gamma (IFN-gamma) obtained from Escherichia coli (E. coli) was treated with a protease-containing fraction prepared from mechanically lysed E. coli cells. Polyacrylamide gel electrophoresis of the resulting product revealed two major components of molecular weight less than that of intact IFN-gamma. These were purified by ion exchange chromatography in the presence of 7 M urea and shown to have intact IFN-gamma N-terminal sequences, suggesting that they resulted via C-terminal cleavages of IFN-gamma. Amino acid analysis indicated that 4 C-terminal residues of IFN-gamma were lacking in one, and 15 in the other. The species lacking 4 C-terminal residues had activities virtually indistinguishable from those of IFN-gamma in antiviral and growth inhibitory assays using Encepharomyocarditis-treated HeLa or T98G cells and in a macrophage activation assay using macrophage-like U937 cells. The species lacking 15 C-terminal residues had markedly decreased activities in each of these assays, and had decreased binding affinity for IFN-gamma cell surface receptors. These observations define the C-terminal residues important for IFN-gamma's biological activity--information which should be useful in designing analogs of IFN-gamma with enhanced or altered biological activities.

Characterization of a cysteine-free analog of recombinant human basic fibroblast growth factor.

Using oligo site-directed mutagenesis, we have modified our synthetic gene for human basic fibroblast growth factor (bFGF) to replace all four cysteine codons with serine codons. The corresponding protein was expressed in Escherichia coli and purified from inclusion bodies by solubilization in urea followed by a series of column chromatographies and a folding step. The resulting protein, having no cysteine residues, is unable to form either intramolecular or intermolecular disulfide bonds. The secondary and tertiary structures of the purified analog, as determined by circular dichroism and fluorescence spectroscopy, were identical within experimental error to recombinant bovine and human bFGF with unaltered amino acid sequences. Reflecting the similar conformation, the analog protein exhibited mitogenic activity on NIH 3T3 cells which was indistinguishable from the natural sequence molecule.

Role of single disulfide in recombinant human tumor necrosis factor-alpha.

Two analogs of tumor necrosis factor-alpha (TNF-alpha) were produced by in vitro site-directed mutagenesis. In these analogs, cysteine residues at positions 69 and 101, which form a disulfide bond, were changed to alanine or leucine. CD spectra showed that the analogs are apparently similar in secondary and tertiary structure to the natural sequence TNF-alpha. In addition, the molecular size of the analogs was identical to that of the natural sequence TNF-alpha as determined by gel filtration. However, fluorescence spectra and quenching indicated that the removal of the disulfide bond alters the local conformation around tryptophan residues. The cytolytic, macrophage activation, and lipogenic activities decreased in the order of the natural sequence TNF-alpha greater than the alanine analog greater than the leucine analog, suggesting that the surface involving the disulfide bond plays a role in these biological functions and the introduced modifications decrease the activity. Differential effect of the modifications was suggested in the antiviral activity, since in this assay only the leucine analog showed significantly lower activity.

Acid unfolding and self-association of recombinant Escherichia coli derived human interferon gamma.

The secondary and tertiary structure of recombinant human interferon gamma, determined by far- and near-UV circular dichroism, showed a transition from the native state to an unfolded state below pH 4.5. The acid unfolding was extensively studied at pH 3.5 as a function of NaCl concentration. Addition of 0.05-0.2 M NaCl to a pH 3.5 sample increased the amount of beta-sheet structure with no change in the amount of alpha-helix and also induced reversible self-association of interferon gamma to form large aggregates from the monomer. When samples at pH 3.5 were dialyzed against 0.1 M ammonium acetate (pH 6.9) to refold interferon gamma, the samples that contained NaCl in acid formed aggregates upon dialysis while those without NaCl formed a dimer apparently identical with the starting protein (i.e., before acid treatment). Thus, the self-association of interferon gamma in acid is closely correlated with its aggregation behavior in 0.1 M ammonium acetate after removal of acid.

Structure and activity of recombinant human interferon-gamma analogs.

We have prepared interferon-gamma (IFN-gamma) analogs to study the structural role of particular amino acids in relation to their effects on antiviral activity. Three IFN-gamma analogs were prepared on the basis of predicted secondary structure. In two of the analogs, [Gln25]IFN-gamma and [Thr45]IFN-gamma, changes were made at residue 25 (Asn to Gln) and at residue 45 (Met to Thr), respectively. [Gln25Lys78]IFN-gamma had two changes, at residue 25 (Asn to Gln) and residue 78 (Asn to Lys). Another analog, [Cys-Tyr-Cys]IFN-gamma, incorporated Cys-Tyr-Cys at the amino terminus. Comparison of the structure and activity of these analogs with that of the natural sequence protein suggested that residues 25 and 78 are at the protein surface and play an important role in antiviral activity. The residue at position 45 was found to be important for maintaining the protein structure, as assessed by circular dichroism spectroscopy. The addition of Cys-Tyr-Cys resulted in a small perturbation of protein structure and a small decrease in antiviral activity.