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WstępRak jelita grubego (colorectal cancer – CRC) jest jednym z najczęstszych nowotworów na świecie – odpowiada za 10% rocznej globalnej zapadalności na raka. Przez długi czas może pozostać bezobjawowy i mimo postępu medycyny zbyt często zostaje zdiagnozowany za późno. Białko YKL-40 jest czynnikiem wzrostu stymulującym migrację komórek śródbłonka, odgrywającym rolę w stanach zapalnych oraz nowotworowych. Celem pracy była ocena znaczenia YKL-40 jako biomarkera w CRC oraz określenie powiązań stężeń YKL-40 w surowicy u chorych na CRC z wybranymi parametrami kliniczno-patologicznymi.Materiał i metodyW badaniu prospektywnym, obejmującym 133 osoby powyżej 50 roku życia, oznaczono stężenie białka YKL-40 w surowicy metodą ELISA. Pacjentów podzielono na dwie grupy: 91 osób chorujących na CRC oraz 42 osoby zdrowe. W analizie statystycznej wykorzystano testy t-Studenta dla danych niezależnych, U Manna-Whitneya i regresję logistyczną.WynikiStężenie YKL-40 w surowicy było znacznie wyższe u chorych na CRC (163 ± 36 μg/l) niż u osób zdrowych (54 ± 20 μg/l; p < 0,0001). U osób z CRC nie wykazano zależności między parametrami klinicznymi, tj. płcią, wiekiem i wskaźnikiem masy ciała (body mass index – BMI), a stężeniem białka YKL-40 w surowicy. U chorych z CRC stężenie YKL-40 w surowicy statystycznie istotnie różniło się we wczesnym i średniozaawansowanym stadium nowotworowym.WnioskiBiałko YKL-40 wydaje się obiecującym biomarkerem CRC. Jego stężenie w surowicy jest skorelowane ze stopniem zaawansowania nowotworu zależnie od głębokości nacieku w stosunku do błony podśluzowej i może być czynnikiem rokowniczo niepomyślnym dla tego nowotworu.

Duplication of the gastrointestinal tract is a rare congenital malformation that can occur throughout the length of the digestive tract. The duplication of the colon on a long section connected with the light of the digestive tract is a relatively rare form of gastrointestinal tract duplication. This defect is a difficult diagnostic and therapeutic problem due to non-characteristic symptoms. Imaging diagnostics allows to correctly recognize gastrointestinal duplication in only 25% of cases. The authors present the case of a girl who was hospitalized many times at the Department of Pediatric Surgery due to recurrent abdominal pain of various types character. Variable clinical symptoms and diagnostic difficulties have caused delay in making a diagnosis of tubular double colon. The final diagnosis was made only after two computed tomography, which was performed due to the rapidly deteriorating and unclear patients condition. The postoperative period after resection of the double intestine proceeded with a wide spectrum of complications that required long-term hospitalization. Duplication of the colon is a difficult diagnostic problem. Non-characteristic symptoms and unclear imaging results delay the diagnosis and expose the patient to complications.

Rectal prolapse is the partial or complete protrusion of the rectal wall into the anal canal. The most common etiology consists in the insufficiency of the diaphragm of the lesser pelvis and anal sphincter apparatus. Methods of surgical treatment involve perineal or abdominal approach surgical procedures. The aim of the study was to present the method of surgical rectal prolapse treatment, according to Mikulicz's procedure by means of the perineal approach, based on our own experience and literature review.The study group comprised 16 patients, including 14 women and 2 men, aged between 38 and 82 years admitted to the department, due to rectal prolapse, during the period between 2000 and 2012. Nine female patients, aged between 68 and 82 years (mean age-76.3 years) with fullthickness rectal prolapse underwent surgery by means of Mikulicz's method with levator muscle and external anal sphincter plasty. The most common comorbidities amongst patients operated by means of Mikulicz's method included cardiovascular and metabolic diseases.Mean hospitalization was 14.4 days (ranging between 12 and 17 days). Despite advanced age and poor general condition of the patients, complications during the perioperative period were not observed. Good early and late functional results were achieved. The degree of anal sphincter continence was determined 6-8 weeks after surgery showing significant improvement, as compared to results obtained prior to surgery. One case of recurrence consisting in mucosal prolapse was noted, being treated surgically by means of Whitehead's method. Good treatment results were observed.Transperineal rectosigmoidectomy using Mikulicz's method with levator muscle and external anal sphincter plasty seems to be an effective, minimally invasive and relatively safe procedure that does not require general anesthesia. It is recommended in case of patients with significant comorbidities and high surgical risk.

Wound healing is an important biological process of crucial value for organisms survival and retention of its proper functions. The recognition of molecular mechanisms of these phenomenon is still under investigation. The transition of mesenchymal fibroblasts to myofibroblasts is a key point in wound healing. The contraction ability of myofibroblast enables the shrinkage of a wound and closes its edges. Alpha smooth muscle actin (alpha-SMA), one of six actin isoforms, is a marker of compeletely differentiated myofibroblast. The regulation of differentiation process depends on many growth factors (especially TGF beta 1), the level of active thymosin beta 4, extracellular matrix proteins--including fibronectin, and also on specificity of microenvironment. Thymosin beta 4 is responsible for maintenance of pool of monomeric actin and actin filaments depolymerization. It can also act as a transcription factor, migration stimulator and immunomodulator, so this protein deserves for more attention in wound healing research field.

Chymosin (EC 3.4.23.4) and rennet, the latter containing about 85% chymosin and 15% pepsin, have been compared according to their coagulation properties with native micelles of different sizes or monomeric caseins as substrate. The casein micelles were separated on columns of controlled-pore glass (CPG-10/3000), which fractionates particles of up to 300 nm diameter. The results show that the coagulation time varies with the micelle size. The effect, which is more pronounced with chymosin than with rennet, appears to be related to the availability of kappa-casein. Therefore the largest micelles, with a lower kappa-casein content, showed longer coagulation times than medium size micelles. In the region of the smallest micelles this time increases again, probably due to an increased beta-casein content. Addition of monomeric kappa-casein decreased the coagulation time with both rennet and chymosin, but alpha s1-and beta-casein had the opposite effect. When isolated monomeric caseins were treated alone with rennet or chymosin, kappa-casein caused turbidity, but alpha s1-and beta-casein did not. Centrifugation experiments with micelles after monomeric casein addition showed that a limited amount of the added casein was able to join the micelle. This was confirmed by chromatographic studies.

A phosphoryl-enzyme intermediate as part of the mechanism of phosphoglycerate kinase has been suggested for the rabbit muscle enzyme (6) and the yeast enzyme (7,8). ATP in the binary enzyme-substrate complexes appeared to phosphorylate these enzymes and ADP-ATP exchange activities were observed (6,7,8). The present report shows, however, that highly purified yeast enzyme cannot be phosphorylated by ATP. On the other hand ADP-ATP exchange activity was obtained but this was proportional to trace amounts of adenylate kinase activity, which was found to contaminate the enzyme preparations. Thus a Ping Pong mechanism as an alternative to a mechanism including a ternary complex between the enzyme and its two substrates appears very improbable. Whether the enzyme or the phosphoryl-group-accepting substrate is responsible for the primary nucleophilic attack occurring in the ternary complex is still an open question, however. Yeast phosphoglycerate kinase appears to have no ATPase activity.

Yeast phosphoglycerate kinase is irreversibly inactivated upon incubation with 5'-[p-(fluorosulfonyl)-benzoyl]-1-N6-ethenoadenosine (5'-FSB epsilon A), an analogue to the nucleotide substrate. Marked protection against inactivation occurs with MgATP, ATP, MgADP, ADP, and 3-phosphoglycerate, suggesting that a part of the catalytic center is modified. The time dependence of the inactivation is characterized by a nonlinear kinetic profile. Curve fitting of various models for ligand binding to the enzyme suggested a two-site model. Modification of one of the sites appears to protect the catalytically essential site from modification. Stoichiometric studies show that the relationship between moles of 5'-FSB epsilon A incorporated per mole of enzyme and the residual enzymatic activity also shows nonlinear behavior. An extrapolated value of 1.5 mol of bound label/mol of enzyme corresponds to complete inactivation. The apparent overall pseudo first-order rate constant for the reaction between phosphoglycerate kinase and 5'-FSB epsilon A, as well as the separate rate constants for the modification, exhibit saturation behavior with respect to the concentration of 5'-FSB epsilon A, indicative of a rapid reversible binding of the reagent to the enzyme prior to modification.

A kinetic study of the effects of SO4(2-) in the activity of phosphoglycerate kinase (ATP: 3-phospho-D-glycerate 1-phosphotransferase, EC 2.7.2.3) is presented. SO4(2-) behaves both as an activator and inhibitor of the reaction. Activation does not appear to affect binding of one or the other of the two substrates to the catalytic centre. As an inhibitor SO4(2-) competes with both the substrates. Thus, each substrate can constrict SO4(2-) from the inhibitor binding site, probably the catalytic centre. Under these conditions activation becomes more and more evident. There appear to exist at least two SO4(2-) binding sites, one which earlier has been defined as an anion binding site, and a second, being the catalytic centre. The former seems to have a higher affinity for SO4(2-) than the latter.

The fractionation by size of casein micelles from bovine skim milk was performed by chromatography on controlled-pore glass granules (CPG-10/3000). Acid precipitation of the fractionated proteins in combination with polyacrylamide gel electrophoresis gave no indication for monomeric caseins in the whey fractions. A factor besides low temperature appears necessary for the dissociation of, for example, β-casein from casein micelles. The casein composition was studied by DEAE-cellulose chromatography. In bulk skim milk the α s -, β- and κ-caseins were shown to occur in the following relative amounts: 52, 33 and 15%, respectively. The distribution varies with the size of the micelle. In large and medium size micelles the α s 1 - casein content is almost constant; β-casein and κ-casein appear to be complementary so that the κ-casein content increases with the decrease in the size of the micelle. In small micelles the relative β-casein content is about 50%, α s 1 - casein is only about 33%. We suggest that β-casein plays a special role as initiator of micelle formation, and that α s 1 - casein stabilizes the structure of the larger micelles.

SUMMARYCasein micelles fractionated on controlled pore glass (CPG-10/3000) were shown to be stable by recycling experiments. Only minor effects on the size distribution of the casein micelles were found after heating skim-milk to 100 °C for 10 min, or freeze-drying skim-milk at – 70 °C followed by resuspension in the synthetic milk serum of Jenness & Koops (1962). The heating caused some whey proteins (β-lactoglobulin) to enter the micelle fractions while the freeze-drying caused some of the largest micelles to disrupt. In colloidal calcium phosphate-free skim-milk prepared according to Pyne & McGann (1960) all the micelles appeared to dissociate into monomeric caseins.

When 1-anilino-8-naphthalenesulfonate (ANS) interacts with phosphoglycerate kinase (ATP:3-phospho-D-glycerate 1-phosphotransferase, EC 2.7.2.3) its fluorescence is enhanced and a blue shift occurs. There is evidence that ANS binds to the site of the nucleotide substrate. The work described herein shows that when various anion inhibitors are added to the ANS-enzyme solution, de-enhancement of the fluorescence occurs. Extrapolation to infinite anion concentration shows that pyruvate ions are the most effective quenchers (ca. 90%) and nitrate ions the least effective, sulfate and phosphate ions being intermediate. The results are consistent with earlier enzymes kinetic findings suggesting that pyruvate ions and ANS, both competing with the nucleotide substrate, are able to bind to the enzyme simultaneously and that sulfate, phosphate and nitrate ions can, to various extents, affect the properties at the active centre of phosphoglycerate kinase via conformational changes without sharing ligands with the nucleotide substrate.

Three electrophoretic components of phosphoglycerate kinase have been isolated from baker's yeast. The isoionic point of the major component is 7.18 at 10 degrees C. Corresponding values for the minor ones are 6.91 and 7.48, respectively. There is a difference of one charge-unit between the isomers 1 and 2, and between the isomers 2 and 3. The release of component 3 from the yeast cells appears in contrast to the isomers 1 and 2 to be promoted by an organic solvent, thus suggesting this component to be bound to the cell-membrane. The amino-terminal amino acid residue appears to be N-acetylated serine in each of the three cases. The carboxyl-terminal ends seem to be identical also with -(Ala, Leu, Val, Lys)- Ala-Lys as the ultimate sequence. From the circular dichroism spectra the contents of alpha-helix and beta-structure were estimated to 15 and 40-50%, respectively. Factors have been determined for transformation and comparison of the specific activities as determined under the various conditions used at different laboratories.

The interaction between 1-anilino-8-naphthalenesulfonate (ANS) and yeast phosphoglycerate kinase (ATP:3-phospho-D-glycerate 1-phosphotransferase, EC 2.7.2.3) and the use of ANS as a probe for studying the structure and function of phosphoglycerate kinase has been investigated. The interaction has been studied by kinetic methods, equilibrium dialysis, and fluorometric titrations. ANS inhibits the activity of the enzyme. More than one inhibitor site exists. ANS is competitive with MgATP and noncompetitive with 3-phosphoglycerate at the first detected inhibitor binding site. The Ki value is 1-2 mM. Several ANS molecules bind to the enzyme. By fluorometric titrations the first detected site has a dissociation constant that is in the same range as Ki or bigger. When ANS interacts with phosphoglycerate kinase its fluorescence is increased and a blue shift occurs. ANS appears to bind to a strongly hydrophobic site. The fluorescence is sensitive to the addition of substrates. ADP, ATP, or combinations of Mg2+ and nucleotide decreases the fluorescence as does free Mg2+. 3-Phosphoglycerate, on the other hand, increases the fluorescence giving evidence for conformational changes upon 3-phosphoglycerate binding.

The binding of Mn ++ to 3-phosphoglycerate kinase, and to ADP and ATP, has been studied at pH 7.1 and 38 °. The influence of the concentrations of Mn ++ and of the various substrates on the rate of the enzymic reaction has also been investigated at the same conditions. The protein displays only a weak, nonspecific interaction with Mn ++ . By correlating the kinetic and binding data, it is possible to show that activation does not involve binding of Mn ++ to the enzyme but rather the formation of a Mn ++ -ATP complex. The significance of the results in relation to the general problem of the mechanism of metal-ion activation of enzymes is discussed.

The single cysteine residue present in phosphoglycerate kinase (ATP:3-phospho- d -glycerate 1-phosphotransferase, EC 2.7.2.3) was modified with p- hydroxymercuribenzoate , N- ethylmaleimide , iodoacetate, iodoacetamide, and 5,5′-dithiobis-(2-nitrobenzoic acid). The SH group is fairly unreactive in its native state with most of these reagents. After 10 h about 60, 45, 25 and 15% of the group has reacted with N-ethylmaleimide, iodoacetamide, iodoacetate and 5,5′-dithiobis(2-nitrobenzoic acid), respectively. The modified enzyme is fully active, and the stability of the enzyme appears unaffected. ATP4, MgATP2− and 3-P- glycerate , when used separately, did not protect the SH group from reaction. The kinetic parameters of MgATP2−, MgADP− and 3-P- glycerate , respectively, are identical for the modified and unmodified enzymes. Consequently, the SH group does not seem to be directly located in the active site region, and the modification does not appear to influence the substrate binding. Kinetic studies with the p- hydroxymercuribenzoate-treated enzyme show that Zn2+ is an uncompetitive inhibitor of ZnATP2−. A result demonstrating that the SH group is not a Zn2+ ligand as could earlier be assumed. The inhibitor constant is higher for the modified enzyme compared with the native enzyme, indicating that the modification reaction affects binding of free Zn2+ to its inhibitor site.

1. 1.|The Michaelis constants for the two substrates of phosphoglycerate kinase (ATP: d -3-phosphoglycerate i -phosphotransferase, EC 2.7.2.3), MgATP2− and 3-phosphoglycerate, are each independent of the concentration of the second substrate. Three prevalent mechanisms can describe this situation. 2. 2.|The Mg2+ complex of 3-phosphoglycerate does not appear to be in an active substrate form. 3. 3.|Mg2+ at high concentrations inhibits the enzyme non-competitively with respect to 3-phosphoglycerate. 4. 4.|High concentrations of Mg2+ change the kinetic relationships and non-linear Lineweaver-Burk plots are obtained for the two substrates. The curves can be approximated to two straight lines. The two intersection points with the abciss axis are independent of the concentration of the second substrate. 5. 5.|The data are interpreted in terms of two different binding sites for each substrate, the second set of sites being involved only at high Mg2+ concentrations. Various mechanisms for this effect are considered and it is suggested that these could also be important for other enzymes involving reaction with ATP.

A kinetic study for understanding how the phosphoglycerate kinase (ATP: 3-phospho-D-glycerate 1-phosphotransferase, EC 2.7.2.3) reaction discriminates between MgATP2- and ATP-4 is presented. The results show that in contrast to MgATP2-, ATP4- is competitive with 3-phospho-D-glycerate. ATP4- binds to the free enzyme as an inhibitor. When binding to the enzyme-MgATP2- (3-phospho-D-glycerate) complex, ATP4- acts as an activator.

ATP-stimulated DNA polymerase activity involving DNA polymerase I has been found to be present in cell extracts from wild type and recC mutant strains of Escherichia coli, but not in extracts from recB strain. The activity has been separated from recBC DNase by DEAE-cellulose ion exchange. It is suggested that recB-dependent factor is involved in the ATP-stimulation of polymerase. Evidence is provided that this stimulation may be due to the interaction of recB-dependent factor with DNA polymerase I.

The molecular and crystal structure of dextromoramide has been determined by X-ray methods. The crystals are orthorhombic, space group P212121 with unit cell dimensions a = 9.720(4) A; b = 12.226(3) A; c = 18.381(3) A. The structure was determined by direct methods and the model refined to an R-value of 0.036 for 1788 observed reflections. The mean e.s.d.'s in bond lengths and angles are 0.004 A and 0.3, respectively. The morpholine moiety is nearly in antiposition relative to the quaternary carbon atom C6, the pertinent angle C6 - C7 - C9 - N2 being - 159.4. This conformation is similar to that previously reported for the bitartrate of the title compound. The pyrrolidine ring has the envelope conformation and the amide group is strictly planar. The conformation of some acyclic analgetics are discussed.

Product inhibiton studies on yeast phosphoglycerate kinase (ATP:3-phospho-d-glycerate 1-phosphotransferase, EC 2.7.2.3) have been performed with 1,3-P2-glycerate. The results indicate that: 1. 1. The catalytic reaction can be affected via four substrate binding sites, two for MgATP2− and two for 3-P-glycerate. 2. 2. There is one catalytic centre per enzyme molecule. 3. 3. The catalytic reaction primarily occurs at the ‘first’ or ‘high affinity’ MgATP2− and 3-P-glycerate binding sites. The ‘second’ set of sub-sites for these substrates are located in a region for regulatin of the catalytic reaction. 4. 4. The products of the reaction, 1,3-P2-glycerate and ADP, are preferentially bound to the regulatory region. 5. 5. MgATP2− and 1,3-P2-glycerate are able to bind simultaneously to this region. When liganded with MgATP2− the apparent Ki value for 1,3-P2-glycerate increases from 3 μM to 20 μM.

(A) The effects of phosphate, chloride, nitrate, pyruvate, malate, succinate and glutamate ions on the kinetics of yeast phosphoglycerate kinase (ATP: 3-phospho-D-glycerate 1-phosphotransferase, EC 2.7.2.3) were studied with MgATP2- and 3-P-glycerate as variable substrates. Three types of patterns were obtained: (1) Nitrate, succinate, malate and glutamate ions, strictly noncompetitive versus both the substrates. (2) Phosphate and chloride ions, noncompetitive versus MgATP2- and mixed versus 3-P-glycerate. (3) Pyruvate ions, being very weak inhibitors, competitive with MgATP2- and noncompetitive with 3-P-glycerate. (B) Based on experiments with simultaneous inhibition by various combinations of two anions the following suggestions were made: The type 1 anions presumably bind to a site outside the active centre. These ions appear to bind to the enzyme independently of type 2. The latter also appears to include sulfate ions, which are competitive versus both the substrates as well as versus the phosphate and chloride ions. Sulfate and phosphate ions are electronically similar, but show different inhibition patterns, presumably due to various effects on the protein conformation. Type 3 inhibition exerted by pyruvate ions was shown earlier for 1-anilino-8-naphthalenesulfonate and salicylate ions, but as these two anions are supposed to bind to the adenine binding pocket of the catalytic centre, the results indicate that pyruvate ions might preferably compete with the nucleotide substrate for the polyphosphate binding site.

Casein micelles have been separated from skim milk by chromatography on CPG-10 3000 glass beads. Fractionation of the micelles according to size has been demonstrated. Poly-acrylamide gel electrophoresis of urea treated micelles reveals that different relative amounts of the major casein components occur in the various micelle fractions. No discernible dissociation of the micelles into monomeric caseins has been observed.

1 The commercially available crystalline phosphoglycerate kinase from yeast was found to be a mixture of enzymically active and inactive proteins. These different protein components can be separated by column electrophoresis. Besides a large fraction (about 50% of the material) without phosphoglycerate kinase activity three enzymically active electrophoretic components appeared. 2 Sedimentation and sedimentation equilibrium studies indicated that the proteins of the main active component and one of the two smaller active components are homogeneous and of the same size. The molecular weight was estimated to 44600 ± 1600. 3 The amino acid analyses indicated that the amino acid compositions of these two protein components are the same. From these analyses the nitrogen and sulphur contents were estimated to about 16.96% and 0.29%, respectively. E1%1cm at 280 nm is close to 0.50. The amino acid composition shows also that if the enzyme consists of subunits, these cannot all be identical. 4 The specific activity of the two similar protein components was estimated to 990 units/mg, when measured in the direction of 1,3-diphosphoglycerate formation.