scholarly journals Purification and characterization of goblet-cell mucin of high Mr from the small intestine of sheep

1985 ◽  
Vol 229 (2) ◽  
pp. 419-428 ◽  
Author(s):  
T K S Mukkur ◽  
D L Watson ◽  
K S Saini ◽  
A K Lascelles

Crude soluble mucus from sheep small intestine was freed of nearly all the nucleic acid contaminants by precipitation with protamine sulphate and treatment with nucleases. After removal of non-covalently bound proteins by equilibrium density-gradient centrifugation in CsCl, a high-Mr glycoprotein was isolated by repeated h.p.l.c. from the partially purified mucin. The high degree of purity of the high-Mr mucin was borne out by (a) the observation of a single boundary on analytical ultracentrifugation in the presence of 5M-guanidinium chloride and (b) the observation of apparent monodispersity on sedimentation-equilibrium analysis. The Mr of the highly purified mucin, determined by sedimentation equilibrium, was 5.0 (+/- 0.1) X 10(6) and was concentration-independent. Finally, only goblet cells and the mucus blanket lining the intestinal epithelial cells were immunofluorescent when guinea-pig anti-(highly purified mucin) serum was used in an indirect immunofluorescence assay. The above antiserum reacted with apparently equal strength with goblet cells and with free mucin in abomasum, caecum and colon. The chemical composition of the glycoprotein was 66% carbohydrate and 34% protein, 45% of the latter being composed of valine and threonine. The glycoprotein migrated anodally on immunoelectrophoresis and contained 7.1% (w/w) sulphate. Neutral hexoses accounted for nearly half of the total carbohydrate content, followed by galactosamine and glucosamine. Whereas fucose and sialic acid were present in only small amounts, uronic acid was not detectable in the highly purified mucus glycoprotein.

1986 ◽  
Vol 250 (3) ◽  
pp. C430-C441 ◽  
Author(s):  
C. N. Conteas ◽  
A. A. McDonough ◽  
T. R. Kozlowski ◽  
C. B. Hensley ◽  
R. L. Wood ◽  
...  

Recent subcellular fractionation studies have raised the possibility that Na+-K+-ATPase might be present in both the apical and the basal-lateral membranes of exocrine gland acinar cells. Analytical fractionation and immunofluorescence microscopy studies of rat parotid glands were performed to confirm this interpretation. The distributions of biochemical markers after analyses based on differential sedimentation, equilibrium density-gradient centrifugation, and partitioning in an aqueous polymer two-phase system defined a total of 15 physically and biochemically distinct membrane populations. Among these populations, it was possible to select one (designated population i) with the characteristics expected of acinar cell basal-lateral plasma membranes. It contained Na+-K+-ATPase enriched 33-fold, and gamma-glutamyl transpeptidase enriched 23-fold with respect to the initial homogenate. A second population (designated population c) had the characteristics expected of acinar cell apical plasma membranes; it contained Na+-K+-ATPase enriched 28-fold, and gamma-glutamyl transpeptidase enriched 53-fold with respect to the initial homogenate. Although the identification of population c remains provisional, immunofluorescence studies verified that Na+-K+-ATPase is present in both the apical and the basal-lateral acinar cell plasma membranes. In view of these results, it is likely that the apical Na+-K+-ATPase would participate in series with basal-lateral sodium- and chloride-entry pathways in driving the secretory electrolyte fluxes.


1978 ◽  
Vol 24 (10) ◽  
pp. 1783-1787 ◽  
Author(s):  
P Vihko

Abstract The principal enzyme of human prostatic acid phosphatase [orthophosphoric monoester phosphohydrolase (acid optimum), EC 3.1.3.2], which had been highly purified by affinity chromatography, isoelectric focusing, and gel filtrations, was shown to be homogeneous at pH 5.0 by sedimentation equilibrium analysis. The amino acid composition was determined and the sedimentation coefficient of the native molecule measured. The relative molecular mass was 89,000 at pH 5.0, as measured by analytical ultracentrifugation. The Km-value of the enzyme for p-nitrophenyl phosphate as substrate is 1.8-10(-4) mol/liter. I also examined substrate speificity, different inhibitors, and the effects of pH, temperature, and serum on the enzyme activity.


2005 ◽  
Vol 60 (11-12) ◽  
pp. 927-931 ◽  
Author(s):  
Joachim Behlke ◽  
Gertraud Koellner ◽  
Agnieszka Bzowsk

The influence of phosphate, ionic strength, temperature and enzyme concentration on the oligomeric structure of calf spleen purine nucleoside phosphorylase (PNP) in solution was studied by analytical ultracentrifugation methods. Sedimentation equilibrium analysis used to directly determine the enzyme molecular mass revealed a trimeric molecule with Mr = (90.6 ± 2.1) kDa, regardless the conditions investigated: protein concentration in the range 0.02 -1.0 mg/ml, presence of up to 100 mm phosphate and up to 200 mm NaCl, temperature in the range 4D25 °C. The sedimentation coefficient (6.04 ± 0.02) S, together with the diffusion coefficient (6.15 ± 0.11) 10-7 cm2/s, both values obtained from the classic sedimentation velocity method at 1.0 mg/ml PNP concentration in 20 mm Hepes, pH 7.0, yielded a molecular mass of (90.2 ± 1.6) kDa as expected for the trimeric enzyme molecule. Moreover, as shown by active enzyme sedimentation, calf spleen PNP remained trimeric even at low protein concentrations (1 μg/ml). Hence in solution, similar like in the crystalline state, calf spleen PNP is a homotrimer and previous suggestions for dissociation of this enzyme into more active monomers, upon dilution of the enzyme or addition of phosphate, are incorrect.


Author(s):  
А.А. Коваленко ◽  
Г.П. Титова ◽  
В.К. Хугаева

Оперативное лечение различных заболеваний кишечника сопровождается осложнениями в виде нарушений микроциркуляции в области анастомоза кишки. Ранее нами показана способность лимфостимуляторов пептидной природы восстанавливать нарушенную микроциркуляцию, что послужило основой для настоящего исследования. Цель работы - оценка влияния стимуляции лимфотока в стенке кишки на процессы восстановления микроциркуляции, структуры и функции тонкой кишки в области оперативного вмешательства. Методика. В экспериментах на наркотизированных крысах (хлоралгидрат в дозе 0,6 г/кг в 0,9% растворе NaCl) моделировали различные поражения тонкой кишки (наложение лигатуры, перевязка 1-3 брыжеечных артерий, перекрут петли кишки вокруг оси брыжейки, сочетание нескольких видов повреждений). Резекция поврежденного участка через 1 сут. с последующим созданием тонкокишечного анастомоза завершалась орошением операционного поля раствором пептида-стимулятора лимфотока (40 мкг/кг массы животного в 1 мл 0,9% раствора NaCl). На 7-е сут. после операции проводили гистологическое исследование фрагмента кишки в области анастомоза. Результаты. На 7-е сут. после резекции у выживших животных (летальность вследствие кишечной непроходимости составляла 30%) имеют место морфологические признаки острых сосудистых нарушений стенки кишки, изменений кровеносных и лимфатических микрососудов, интерстициальный отек всех слоев стенки кишки, дилатация просвета кишки, повреждение всасывающего эпителия ворсин с истончением щеточной каемки клеток, морфологические признаки гиперфункции бокаловидных клеток. Использование лимфостимулятора пептидной природы после операции увеличивало выживаемость животных на 24%. У части животных отмечалось уменьшение расширения просвета кишки, у других практически полная его нормализация. Восстанавливалась форма кишечных ворсин и распределение бокаловидных клеток. Отсутствовали признаки внутриклеточного и межмышечного отека. Отмечено умеренное полнокровие венул. Заключение. Использование лимфостимулятора при хирургическом лечении кишечной непроходимости увеличивает выживаемость животных на 24% по сравнению с контролем, способствует более раннему восстановлению структуры и функции тонкой кишки. Полученные результаты свидетельствуют о перспективности использования стимуляции лимфотока при операциях на кишечнике. Surgical treatment of bowel diseases is associated with complications that cause microcirculatory disturbances in the anastomosis area and may lead to a fatal outcome. This study was based on our previous finding that peptide-type lymphatic stimulators are able to restore impaired microcirculation. The aim of this work was stimulating the lymph flow in the intestinal wall to facilitate recovery of microcirculation, structure and function of the small intestine in the area of surgical intervention. Methods. In experiments on anesthetized rats (0.6 g/kg chloral hydrate in 0.9% NaCl), various small bowel lesions were modeled (bowel ligation, ligation of 1-3 mesenteric arteries, gut torsion, combination of several lesion types). In 24 h, the damaged area was resected, and a small intestine anastomosis was creased. The surgery was completed with irrigation of the operative field with a solution of lymph flow stimulating peptide (40 мg/kg body weight in 1 ml of 0.9% NaCl). A gut fragment from the anastomosis area was examined histologically on day 7 after the surgery. Results. On the 7th day after removing the intestinal obstruction, the surviving animals (lethality 30%) had morphological signs of acute vascular disorders in the intestinal wall; changes in blood and lymphatic microvessels; interstitial edema of all intestinal wall layers; dilatation of the intestinal lumen; damage to the absorptive epithelium of villi with thinning of the brush border, and hyperfunction of mucous (goblet) cells. The use of the peptide after surgery increased the survival rate of animals by 24% and provided a smaller dilatation of the intestinal lumen in some animals. In other animals, the lumen recovered. The shape of intestinal villi and distribution of goblet cells were restored. Signs of intracellular and intermuscular edema were absent. Moderate venular congestion was noticed. Conclusion. Using the lymphatic stimulator in surgical treatment of intestinal obstruction increases the survival rate of animals by 24% compared to the control, facilitates earlier restoration of the small intestine structure and function. The obtained results indicated the effectiveness of lymphatic stimulation in intestinal surgery.


2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Michelle Cutajar ◽  
Fabrizio Andriulo ◽  
Megan R. Thomsett ◽  
Jonathan C. Moore ◽  
Benoit Couturaud ◽  
...  

AbstractThere is currently a pressing need for the development of novel bioinspired consolidants for waterlogged, archaeological wood. Bioinspired materials possess many advantages, such as biocompatibility and sustainability, which makes them ideal to use in this capacity. Based on this, a polyhydroxylated monomer was synthesised from α-pinene, a sustainable terpene feedstock derived from pine trees, and used to prepare a low molar mass polymer TPA5 through free radical polymerisation. This polymer was extensively characterised by NMR spectroscopy (chemical composition) and molecular hydrodynamics, primarily using analytical ultracentrifugation reinforced by gel filtration chromatography and viscometry, in order to investigate whether it would be suitable for wood consolidation purposes. Sedimentation equilibrium indicated a weight average molar mass Mw of (4.3 ± 0.2) kDa, with minimal concentration dependence. Further analysis with MULTISIG revealed a broad distribution of molar masses and this heterogeneity was further confirmed by sedimentation velocity. Conformation analyses with the Perrin P and viscosity increment ν universal hydrodynamic parameters indicated that the polymer had an elongated shape, with both factors giving consistent results and a consensus axial ratio of ~ 4.5. These collective properties—hydrogen bonding potential enhanced by an elongated shape, together with a small injectable molar mass—suggest this polymer is worthy of further consideration as a potential consolidant.


2010 ◽  
Vol 39 (7) ◽  
pp. 1538-1547
Author(s):  
Patrícia Regina de Souza Siqueira Campos ◽  
José Fernando Coelho da Silva ◽  
Hernán Maldonado Vásquez ◽  
Andréa Vittori ◽  
Martinho de Almeida e Silva

It was evaluated by the Cornell System carbohidrates fractions and nitrogenous compounds of the following grasses at the cutting ages of 14, 28, 42, and 56 days: nilo grass (Acroceras macrum), angola grass (Brachiaria purpurascens), aleman grass (Echinochloa polystachya), limpo grass (Hemarthria altíssima), setaria grass (Setaria anceps), tanner grass (Brachiaria arrecta), and tifton-85 grass (Cynodon spp). The experiment was carried out in a complete randomized block design, in a split plot arrangement in a way that the grasses were evaluated in the plots and the ages of cut in the split-plots. The age of cutting had an effect on the composition of the studied grasses. In most of the grasses, total carbohydrate levels, non-fibrous carbohydrates and A+B1 fraction carbohydrates increased linearly according to the age of cutting. The potentially degradable fraction of carbohydrates (fraction B2) showed a quadratic behavior according to the cutting ages for all grasses. The C fraction of the carbohydrates in tifton-85 grass linearly increased with the age but it did not increase significantly for the other grasses. In setaria grass, the intermediate levels of B2 and B3 nitrogenous fractions were high, which might represent a potential source of protein for ruminal degradation and for the small intestine. Except for setaria grass, all studied grasses show similar values of the A, B1, B2 and B3 nitrogenous fractions.


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