A study of macromolecular diffusion through native porcine mucus.

A diffusion chamber technique based on time-lag analysis for the estimation of effective diffusion coefficients of radiolabelled macromolecules of varying molecular weights through native mucus gel is reported. For all solutes studied, a reduction in effective diffusion coefficients was observed with a retardation of solute flux in both aqueous and mucus layers. Over the molecular weight range of solutes investigated (126-186,000 Daltons), a consistent effect of molecular weight was evident with regard to the retarding effect of mucus. No apparent or absolute molecular weight cut-off for macromolecular transfer was exhibited. However, at high molecular weights (greater than 30,000 Daltons) the retardation was greatly enhanced. The results confirm that mucus can be regarded as a gel with finite pores, but that it does not constitute an absolute barrier to even high molecular weight solutes.     

Molecular weight of human alpha2-H-ferroglobulin subunits. Comparison with molecular weight of ferritin subunits]

alpha2 H globulin, a glycoferroprotein, was first demonstrated in the sera of patients with malignant diseases. This protein was isolated from cancerous human liver, and compared with ferritin, a ferroprotein showing some identical properties (presence of iron, high molecular weight, common antigenic determinants). However, physicochemical differences were observed between these two proteins. The study of protein dissociation was performed by polyacrylamide gel electrophoresis in sodium dodecyl sulfate after reduction by mercaptoethanol. A similar molecular weight of 19 000 is obtained for subunits of these two proteins. This value agrees well with the results obtained by other authors for ferritin.     

Darstellung und physikochemische Charakterisierung der Peptidketten des Tabakmosaikvirus

Summary Highly purified tobacco mosaic virus was split by different methods, the ribonucleic acid removed and the size of the protein sub-unit determined by sedimentation and diffusion. The value for the molecular weight of a polypeptide chain of the tobacco mosaic virus was calculated to be 17–18 000.     

Molekulargewichtsbestimmung von Kalbsherzmyosin

Summary Sedimentation and diffusion coefficients, partial specific volume, intrinsic viscosity, ATP-ase activity and molecular weight of cardiac myosin from calf hearts were determined. The values agree well with the results ofMueller et al.⁴ for cardiac myosin of the dog, and are not essentially different from values reported for skeletal myosins.¹      

Chymotrypsin-like and trypsin-like protease activities in the sea urchin (Hemicentrotus pulcherrimus) egg

Proteolytic activities in extracts of sea urchin eggs were examined using SDS (sodium dodecyl sulphate)-polyacrylamide gels. In the unfertilized eggs, proteases were detected as bands corresponding to the molecular weights of 40 kD and 26 kD on the gelatin gel, and 35 kD and 30 kD on the casein gel. Using various protease inhibitors, it was found that 40 kD, 30 kD, and 26 kD are chymotrypsin-like proteases and that 35 kD is a trypsin-like protease. The activity of the 40 kD chymotrypsin-like protease was found to be almost completely lost after insemination.     

Chymotrypsin-like and trypsin-like protease activities in the sea urchin (Hemicentrotus pulcherrimus) egg.

Proteolytic activities in extracts of sea urchin eggs were examined using SDS (sodium dodecyl sulphate)-polyacrylamide gels. In the unfertilized eggs, proteases were detected as bands corresponding to the molecular weights of 40 kD and 26 kD on the gelatin gel, and 35 kD and 30 kD on the casein gel. Using various protease inhibitors, it was found that 40 kD, 30 kD, and 26 kD are chymotrypsin-like proteases and that 35 kD is a trypsin-like protease. The activity of the 40 kD chymotrypsin-like protease was found to be almost completely lost after insemination.     

Immunochemical evidence for the specific binding of putrescine to plasma proteins including fibronectin

Human plasma contains proteins capable of binding 14C putrescine by the action of Ca++ activated transglutaminase. These proteins have molecular weights from 32 to 220 K and above. One of these (with a molecular weight of 220 K) has been identified as fibronectin by the use of an antifibronectin antiserum. Evidence for a protein with a molecular weight identical to that of fibronectin has been obtained on PAGE analysis of the precipitate formed on incubating human serum with antipolyamine antiserum.     

Persistence of infradian body weight cycles in castrated dormice (Glis glis)

Summary Male dormice were castrated at 2 phases of their infradian body weight cycles. No consistent changes were found in cycle period, amplitude, or absolute weights of the dormice following castration. Unlike other mammals, body weights of dormice appear unaffected by castration. Although both body weight and reproductive condition vary on an infradian basis, the changes in body weight appear to be programmed independently from changes in gonadal function.Acknowledgments. I would like to thank R. Melnyk, N. Mrosovsky, and J. D. Hallonquist for much helpful criticism and K. Lang and J. Kenyon for invaluable technical help. Research support was provided by a National Sciences and Engineering Research Council of Canada grant to N. Mrosovsky.     

Compatible solutes of halophilic eubacteria: molecular principles,water-solute interaction,stress protection

Compatible solutes are best described as organic osmolytes responsible for osmotic balance and at the same time compatible with the cells' metabolism. A comprehensive survey (using HPLC and NMR methods) on halophilic/halotolerant eubacteria has revealed the full diversity of compatible solutes employed in nature. Molecular principles derived from the spectrum of compounds found in the bacterial world may be summarized as follows. Compatible solutes are polar, highly soluble molecules and uncharged at physiological pH. With the exception of proline (a proteinogenic amino acid) they are characterized as amino acid derivatives of the following types: betaines, ectoines, N-acetylated diamino acids and N-derivatized carboxamides of glutamine. Using nearinfrared spectroscopy we have also been able to demonstrate that compatible solutes are strong water-structure formers and as such probably excluded from the hydration shell of proteins. This preferential exclusion probably explains their function as effective stabilizers of the hydration shell of native proteins (protection against heating freezing and drying). Hence these typical products of halophilic eubacteria have a considerable potential as stabilizing/protecting agents on both molecular and whole-cell level. Thorough understanding of common structural principles and fundamental water-solute interactions will ultimately enable us to design novel highly efficient stress protectants and stabilizers of biomolecules.     

A comparative study of proteoglycans from bovine lung,trachea, tracheal mucosa,and aorta

Summary Proteoglycans were isolated from bovine lung, trachea, tracheal mucosa, and aorta by dissociative extraction with 4M guanidinium hydrochloride. Fractionation of these tissue extracts by cesium chloride density centrifugation and gel chromatography allowed the isolation from each extract of a high molecular weight fraction consisting mainly of proteochondroitin sulfate and hyaluronic acid.Acknowledgment. This work was supported by a National Heart, Lung, and Blood Institute grant to K. Ehrlich (HL21617).     

Molecular weight of lipovitellins during egg development in the crustacean amphipod Orchestia gammarella (Pallas)]

Egg lipovitellins of Orchestia gammarella tested by electrophoresis on gels of different acrylamide concentrations, following the procedure of Hedrick and Smith (1968), display a migration pattern identical to that of proteins with molecular weights of congruent to 3.2 x 10(5) (lipovitellin I) and congruent to 5.5 x 10(5) (lipovitellin II) respectively. Since their molecular weight remains constant during embryogenesis, the changes of their relative mobility in disc gels indicates alterations in their ionic charges.     

Butanol extracts from myelin fragments. III. SDS-urea gel electrophoretic studies

Summary The butanol extracts from myelin fragments showing 5-HT binding capacity were studied by SDS-urea gel electrophoresis. 5 main bands were observed and their molecular weights were determined by the method of Ferguson relationship, and revealed that these extracts contained proteolipid protein, DM-20 and basic proteins. Furthermore, the reconstituted fraction with crude basic proteins and lipids showed the saturable binding capacity for C¹⁴·5-HT.Acknowledgment. We thank Prof. T. Iwamoto for his encouragement during the course of this study.     

Guinea pig liver cytosol has an L-threonine deaminase activity distinct from L-serine deaminase]

Using sodium sulfate precipitation, "Sephadex G200" gel filtration and polyacrylamide gel electrophoresis, a L-threonine desaminase was demonstrated in the Guinea-Pig liver cytosol. This enzyme was separated from the guinea pig liver L-serine desaminase possessing an auxiliary activity on L-threonine substrate described by us in a previous work. The optimals for pH (7,1) and temperature (+ 55 degrees C) and the apparent molecular weight (134,000 + 20,000) were established.     

The effect of a gastric mucus barrier on the dynamic response of a pH electrode

Hydrogen ion mobility in gastric mucus has been found to be reduced to a greater extent than that hitherto suspected, though at low pH (less than 4) and in buffer this mobility increases. Mucus, at an optimized pH, may therefore protect the gastric mucosa from acid digestion by providing a diffusion barrier.     

The trefoil protein TFF1 is bound to MUC5AC in humangastric mucosa

The trefoil protein TFF1 is expressed principally in the superficial cells of the gastric mucosa. It is a small protein and forms homo- and hetero-dimers via a disulphide bond through Cys58 which is located three amino acids from the C terminus. TFF1 is co-expressed with the secreted mucin MUC5AC in superficial cells of the gastric mucosa suggesting that it could be involved in the packaging or function of gastric mucus. We have previously shown that TFF1 co-sediments with mucin glycoproteins on caesium chloride gradients. To extend this observation we have now used gel filtration under physiological conditions, immunoprecipitation and Western transfer analysis to characterise the interaction of TFF1 with gastric mucin glycoproteins. We show that TFF1 co-elutes with MUC5AC but not MUC6 on gel filtration and that immunoprecipitation and Western transfer analysis confirms that TFF1 interacts with MUC5AC. We also demonstrate that the TFF1 dimer is the predominant molecular form bound to MUC5AC. Salt and chelators of divalent cations such as EDTA and EGTA disrupted the TFF1- MUC5AC interaction and increased the degradation of MUC5AC, whereas calcium increased the amount of TFF1 bound to MUC5AC. These data support the contention that TFF1 is pivotal in the packaging and function of human gastric mucusa.Received 24 March 2004; received after revision 14 May 2004; accepted 7 June 2004     

Plant aquaporin selectivity: where transport assays, computer simulations and physiology meet

Plants contain a large number of aquaporins with different selectivity. These channels generally conduct water, but some additionally conduct NH₃, CO₂ and/or H₂O₂. The experimental evidence and molecular basis for the transport of a given solute, the validation with molecular dynamics simulations and the physiological impact of the selectivity are reviewed here. The aromatic/arginine (ar/R) constriction is most important for solute selection, but the exact pore requirements for efficient conduction of small solutes remain difficult to predict. Yeast growth assays are valuable for screening substrate selectivity and are explicitly shown for hydrogen peroxide and methylamine, a transport analog of ammonia. Independent assays need to address the relevance of different substrates for each channel in its physiological context. This is emphasized by the fact that several plant NIP channels, which conduct several solutes, are specifically involved in the transport of metalloids, such as silicic acid, arsenite, or boric acid in planta.     

Influence of the enteric surface coat on the unidirectional flux of acetamide across the wall of rat small intestine

In vivo treatment of the jejunal mucosa with glycosidic enzymes seems to remove the enteric surface coat of the enterocyte. As a consequence, the mucosa-to-serosa unidirectional flux of acetamide increases remarkably. The glycocalyx probably represents a barrier to the diffusion of small hydrosoluble solutes.     

Protection of rat gastric mucosa against ethanol injury by the new synthetic prostaglandin MDL 646

Summary Oral administration to fasted rats of absolute ethanol produces extensive necrotic lesions of gastric mucosa as well as a massive leakage of proteins and mucus glycoproteins into gastric lumen. When the new synthetic prostaglandin MDL 646, belonging to the PGE₁ series, is administered intragastrically (2 or 10 g/kg) 30 min before ethanol administration, a significant protection of rat gastric mucosa against alcohol injury is observed.This work was supported in part by a contribution from Ministero della Pubblica Istruzione (MPI), Rome, Italy.     

Two-step purification of estradiol calf uterine cytosol receptor]

The use of two affinity chromatography methods: heparin-Ultrogel and estradiol 7 alpha-(CH2)3-poly (alanine-lysine)-agarose, allows us to purify the calf uterine estradiol cytosol receptor with a 20% recovery. Electrophoresis under denaturing conditions allows us to estimate its molecular weight as 70 000 dalton.