High Molecular Weight Molecules Research Articles

A General Equation for Correlating the Thermophysical Properties of n-Paraffins, n-Olefins, and Other Homologous Series. 3. Asymptotic Behavior Correlations for Thermal and Transport Properties

In this final paper of the series, asymptotic behavior correlations (ABCs) are presented for thermal properties (ideal-gas enthalpy and free energy of formation, ideal-gas heat capacity, enthalpy of vaporization, and liquid heat capacity) and for transport properties (liquid viscosity, thermal conductivity, and surface tension). The theoretical basis for asymptotic behavior of these properties is discussed. The correlations were developed using literature data for n-paraffins and n-olefins (1-alkenes) and give accurate and consistent predictions. They are preferable to existing correlations in most instances. For specific heat, viscosity, thermal conductivity, and surface tension, comparisons are made with high molecular weight, linear polyethylenes and other long-chain molecules.

Amplification of PFGE-separated chromosome-specific DNA by degenerate oligonucleotide-primed PCR (DOP-PCR)

Specific probes for DNA molecules of high molecular weight can be efficiently and rapidly prepared by the combination of pulsed-field gel electrophoresis (PFGE) with degenerate oligonucleotide-primed PCR (DOP-PCR). More than 10 mg of a specific probe were amplified from only 1 ng of a PFGE-separated DNA fragment of a red alga, Cyanidioschyzon merolae. The probe hybridized specifically to the fragment which was used as a template for DOP-PCR.

Cell-cell communication in nematode embryos: differences between Cephalobus spec. and Caenorhabditis elegans

During early nematode embryogenesis a series of asymmetric cleavages in the germ line generates several somatic founder cells and a primordial germ cell. We have found previously that the two soil nematodes Cephalobus spec. and Caenorhabditis elegans express considerable differences in the order of events and spatial arrangement of cells during early embryogenesis. With the help of microinjected fluorescent marker dyes, we show here that these dissimilarities partner major differences in the pattern of intercellular communication. Whilst in C. elegans all early blastomeres become dye-coupled simultaneously, in Cephalobus communication is established progressively in the sequence in which cells are born. In addition, in Cephalobus but not C. elegans, sequential lucifer yellow accumulation indicates stepwise changes in the state of early blastomeres: if injected into the uncleaved zygote, for example, the dye becomes equally distributed to all cells at first but rapidly accumulates in a single blastomere in the 4-cell stage. We speculate that such a redistribution mechanism may be involved in the differential segregation of cytoplasmic components to individual blastomeres. The most dramatic difference between the two species was found with respect to the transfer of high molecular weight molecules. In contrast to C. elegans, in Cephalobus not only small lucifer dyes but also high molecular weight dextrans can diffuse along specific pathways between early somatic cells indicating the presence of large communication channels. However, a transfer of dextran into or out of germ line cells never takes place. The origin of these channels as midbodies of previous mitoses and their potential role for normal development is discussed. Tissue-specific dye-coupling compartments in the slow developing Cephalobus are established in the same order but at a considerably earlier developmental stage than in C. elegans suggesting that this process may depend more on parameters like available time for transcription rather than the number of cell cycles passed through.

Fossil fuel characterization using laser desorption mass spectrometry: Applications and limitations

traditional gas-phase methods. The application and limitations of these techniques to model compounds and fossil-derived materials is discussed in this study. LD and MALDI mass spectrometry are increasingly used to detect intact molecular species, such as proteins with masses from 1000 to 100,000 amu and beyond.' MALDI is also being used for a few high molecular weight polymers.2 A good example, related to coal-type systems, is the report on lignin mass spectrometry by MALDI.3 Here the mass spectrum shows a wide molecular distribution of several hundred to larger than 16000, with the center of gravity of the distribution around 2600. The results are interpreted in terms of oligomeric lignin molecules. Thus, if there are indeed large molecular species in a polymeric content in fossil fuel extracts, MALDI is an attractive technique. We have previously reported laser desorption of coal extracts that show only low molecular weight ions (<ZOO0 amu).4-6 We compare LD with fast atom bombardment and desorption chemical ionization mass spectrometry, two other soft ionization techniques. All of these techniques produce similar data that differ only in minor details. We have used our two TOF mass spectrometers for coal analysis by MALDI, have carefully analyzed our data and the instrumental conditions and conclude that the bulk of coal material produces only low molecular weight ions (up to 2000 amu). In agreement with these reports, Hanky showed LD and MALDI data of pyridine extracts which produced a distribution of ions between 150 and 1500.7 In addition, field ionization mass spectrometry has found similar pattern in the low mass region.8 Recently, Herod et. aI9-'* have interpreted their LD and MALDI data in terms of high mass species being desorbed. We have presented alternate interpretations for these results.6 In our hands, LD or MALDI of coals and extracts do not show any reproducible ion intensity over mass 2000. The conditions whereby large molecular ions can be desorbed intact are very specialized. This paper will describe the scope and limitations of laser desorption and matrix-assisted laser desorption in time-of-flight mass spectrometers as applied to high molecular weight molecules, such as proteins and polymer systems. EXPERIMENTAL The coals used in this study are the Argonne Premium Coal Samples. The procedures for the pyridine extract of the Argonne Premium Coals have been reported previousl~.~ The laser desorption mass spectra and matrix-assisted laser desorption mass spectra were recorded on a linear time-of-flight mass spectrometer constructed at Argonne and a Kratos Kompact MALDI 111 linear/reflectron time-of-flight mass spectrometer. The spectra were produced by exposing the samples distributed as thin layer on a stainless steel sample holder to laser pulses from either a Nd:YAG or a

Confirmation by mass spectrometry of a trisulfide variant in methionyl human growth hormone biosynthesized in Escherichia coli.

A sulfur-containing compound found in acid hydrolysates of proteins was identified 30 years ago as a trisulfide: bis-(2-amino-2-carboxyethyl) trisulfide (cysteine2S3). At that time, studies concerning the chemistry of sulfur-transferring enzyme systems suggested that cysteine2S3 also existed in biological systems. Two decades later, a cystine trisulfide structure was postulated in the regulator protein molecule for the activation of delta-aminolevulinate synthetase. Recently, a trisulfide bond was reported to occur in the minor loop disulfide at Cys182-Cys189 in human growth hormone. We have detected a trisulfide structure in methionyl human growth hormone in the major loop disulfide Cys53-Cys165. The development of mass spectral analyses of high molecular weight molecules, such as proteins, led to the eventual identification of the modification. A tandem mass spectral analysis on a Sciex electrospray instrument localized an addition of 32 Da to the Cys53-Cys165 fragment. Elemental composition was determined by accurate mass measurement obtained by peak matching to a synthetic peptide and established that an extra sulfur atom was involved.

Catenin-dependent and -independent Functions of Vascular Endothelial Cadherin

Vascular endothelial cadherin (VE-cadherin, cadherin-5, or 7B4) is an endothelial specific cadherin that regulates cell to cell junction organization in this cell type. Cadherin linkage to intracellular catenins was found to be required for their adhesive properties and for localization at cell to cell junctions. We constructed a mutant form of VE-cadherin lacking the last 82 amino acids of the cytoplasmic domain. Surprisingly, despite any detectable association of this truncated VE-cadherin to catenin-cytoskeletal complex, the molecule was able to cluster at cell-cell contacts in a manner similar to wild type VE-cadherin. Truncated VE-cadherin was also able to promote calcium-dependent cell to cell aggregation and to partially inhibit cell detachment and migration from a confluent monolayer. In contrast, intercellular junction permeability to high molecular weight molecules was severely impaired by truncation of VE-cadherin cytoplasmic domain. These results suggest that the VE-cadherin extracellular domain is enough for early steps of cell adhesion and recognition. However, interaction of VE-cadherin with the cytoskeleton is necessary to provide strength and cohesion to the junction. The data also suggest that cadherin functional regulation might not be identical among the members of the family.

Functional properties of human vascular endothelial cadherin (7B4/cadherin-5), an endothelium-specific cadherin.

Human vascular endothelial cadherin (VE-cadherin, 7B4/cadherin-5) is an endothelial-specific cadherin localized at the intercellular junctions. To directly investigate the functional role of this molecule we cloned the full-length cDNA from human endothelial cells and transfected its coding region into Chinese hamster ovary cells. The product of the transfected cDNA had the same molecular weight as the natural VE-cadherin in human endothelial cells, and reacted with several VE-cadherin mouse monoclonal antibodies. Furthermore, it selectively concentrated at intercellular junctions, where it codistributed with alpha-catenin. VE-cadherin conferred adhesive properties to transfected cells. It mediated homophilic, calcium-dependent aggregation and cell-to-cell adhesion. In addition, it decreased intercellular permeability to high-molecular weight molecules and reduced cell migration rate across a wounded area. Thus, VE-cadherin may exert a relevant role in endothelial cell biology through control of the cohesion and organization of the intercellular junctions.

Derivatization of a new poly(ether urethane amide) containing chemically active sites

A novel poly(ether urethane amide) was synthesized by chain-extending the prepolymer with fumaric acid, resulting in a polymer exhibiting superior mechanical properties and containing chemically active derivatization sites, stemming from the fumaramide double bond. The derivatization of the active sites was performed via a Michael-like addition of amine-terminated poly(ethylene oxide) chains, which when performed in bulk, greatly affected the mechanical properties of the polymer, except in the case in which a relatively high molecular weight was grafted. Surface grafting of the polymer led to a significantly lower effect on its mechanical properties, which was minimal when a high molecular weight molecule was used.

Controls of the microbial loop: Nutrient limitation and enzyme production, location and control

A major controlling factor for bacterial growth is their ability to hydrolyze high molecular weight molecules too complex to be transported directly across the cell's membrane. The utility of such an extracellular enzyme hydrolysis system, location of the enzymes (free or attached), environmental controls of enzyme production, and implications of multiple hydrolysis-uptake systems are explored in relation to free-living oceanic bacteria and bacteria attached to rapidly sinking aggregates.

Biosynthetic radiolabelling of excretions-secretions of adult male Onchocerca gibsoni

Maintenance of adult male worms of the bovine filarial parasite Onchocerca gibsoni in vitro, in the presence of radioactive precursors, resulted in the time-dependent excretion-secretion of radiolabelled parasite macromolecules (ES). Molecules labelled with amino acids ([ 35S] methionine, pH] leucine) covered a wide range of molecular weights, whereas labelling with [ 3H] glucosamine produced predominantly molecules of high molecular weight. Many of the products were recognized by antibodies in two serum pools from humans infected with Onchocerca volvulus. O. gibsoni ES may therefore provide a substitute source of material for studies on the ES of the less readily available human parasite.

Biochemical and cytological characterization of DROP-1: a widely distributed proteoglycan in Drosophila

Using Drosophila testis as a source of antigen, 12 monoclonal antibodies were isolated that all recognize a set of three high molecular weight molecules present on Drosophila sperm and also in the fertilized egg. Among these antibodies, one is highly specific for sperm, while the remaining 11 detect epitopes present not only on sperm, but also in yolk spheres or in a punctate distribution in the egg. Here we cytologically and biochemically characterize the (common) antigens to five of these antibodies. Several biochemical properties suggest that these antibodies recognize a family of glycosaminoglycan-containing proteoglycans: (1) three diffuse, poorly focused high molecular weight bands, all in excess of 200,000 Da were observed on Western blots of denaturing SDS gels; (2) all three bands have a pI in the range of 3.0–3.5; (3) the molecules are strongly resistant to proteolysis; (4) mild periodate oxidation renders the molecules reactive towards the derivatizing agent digoxygenin-hydrazide, indicating the likely presence of saccharide moieties; (5) trifluoromethylsulfonic acid treatment, which removes saccharide moieties, shifts the pI to 7.0; (6) β-elimination increases electrophoretic mobility of the antigens on SDS gels; (7) nitrous acid treatment, which cleaves N-sulfated glycosaminoglycans, also increases the electrophoretic mobility of the antigens on SDS gels. We conclude that the antigens recognized by these antibodies are likely to be heparan sulfate proteoglycans. These results indicate that DROP-1 may represent a family of proteoglycans present during embryogenesis and later stages of development in Drosophila. DROP-1 represents the third proteoglycan to be characterized in Drosophila.

The fluorescence of dissolved organic matter in porewaters of marine sediments

The fluorescence of porewaters from marine sediment cores from six different areas was measured. In most cases, fluorescence was affected primarily by the diagenesis of organic carbon first through sulfate reduction and subsequently by methane generation. Typically, fluorescence, dissolved organic carbon (DOC), absorbance, alkalinity, and ammonium ion concentrations correlate quite well, increasing in the upper sections of anoxic sediments and co-varying in deeper sections of these cores. The good correlation of DOC with fluorescence in the three cores in which DOC was measured indicates that fluorescence can be used to make a first order estimate of DOC concentration in anoxic porewaters. Data are consistent with a model in which labile organic matter in the sediments is broken down by sulfur reducing bacteria to low molecular weight monomers. These monomers are either remineralized to CO 2 or polymerize to form dissolved, fluorescent, high molecular weight molecules. The few exceptions to this model involve hydrothermally generated hydrocarbons that are formed in situ in the Guaymas Basin or are horizontally advected along the decollement in the Nankai Trench.

Molecular karyotype of related clones of Entamoeba histolytica

The molecular karyotype of 3 clones derived from strain HM1:IMSS of Entamoeba histolytica was studied by transverse alternating field electrophoresis. 11–20 bands ranging between 0.3 and over 3 Mb were resolved. Hybridization with total DNA detected highly repetitive sequences in the slow-migrating molecules, while non-repetitive sequences were located in the intermediate and fast-migrating molecules, rDNA, tubulin, actin, cysteine proteases DNA fragments, and a variable DNA sequence (EhVR1) located the respective genes mainly in the 1.3–1.5-Mb region, although they differed in the three clones. Two-dimensional transverse alternating field electrophoresis showed that more than one high-molecular weight molecule may comigrate in a single DNA band. rDNA, and EhVR1 hybridized with slow-migrating bands in a characteristic ladder pattern. Most of the bands recognized by EhVR1 seems to be linear molecules, although exonuclease III-resistant bands also hybridized with EhVR1, suggesting the presence of circles.

Structural discrepancies in resistant starch obtained in vivo in humans and in vitro

In-vivo resistant starch (RS) was collected at the end of the human ileum by an intubation technique after ingestion of two partly resistant starches (retrograded (R) or complexed (C) high amylose maize starch). In-vitro RS fractions were prepared enzymatically from R and C according to three methods of RS determination in food. Physicochemical characteristics of these two different fractions were compared. The ileal RS appeared to consist of three fractions: a first population of high molecular weight α-glucans attributed to amorphous potentially digestible material, a second made of B-type retrograded amylose crystallites and a third containing oligosaccharides. The in-vitro RS fractions showed no high molecular weight molecules, due to more extensive hydrolysis in the in-vitro procedure. Therefore, none of the in-vitro RS determinations allowed the isolation of a fraction qualitatively similar to in-vivo RS.

Popliteal lymph node enlargement induced by procainamide

The ability of procainamide (PA) to induce primary local politeal lymph node (PLN) reactions has been investigated. We employed an in vitro drug-metabolizing system, based on the hypothesis that the negative result for PA in a PLN assay (PLNA) was due to insufficient metabolizing metabolizing activity at the reaction site. PA was incubated previously in vitro with the S-9 mixture derived from rat liver. The reactants were ultrafiltered in order to eliminate high molecular-weight molecules, and then the low molecular-weight fraction was subcutaneously (s.c.) injected into the hind footfad of mice. PLN reactions wer eassessed by weighing the popliteal lymph node of the injected side. The reactants of more than 5 mg of PA and S-9 mixture induced PLN enlargement in C3H/He mice 8 days after injection. BALB/c and C57BL/6 mice were also susceptible to PLN reaction in response to the reactants of PA (10 mg) and S-9 mixture. PLN reactions to PA were induced 2 days after the injection and sustained until 18 days. Contact of PA with the S-9 mixture for 30 min at 37°C was sufficient to induce PLN enlargement. However, contact of PA with 24 h reduced the peak reaction. On the other hand, PA which had not been incubated with teh S-9 mixture and acetylprocainamide (acetylPA0 gave little or no reaction. Local PNA has not been considered to be suitable for the detection of drugs with teh potential to induce immune disoreders on cases whre a metabolite contributes to the adverse reactions. However, the employment of an in vitro drug-metabolizing system may overcome that defect.

Permeability of the macula densa basement membrane area to high molecular weight molecules.

To investigate the permeability properties of the basement membrane beneath macula densa cells and between extraglomerular mesangial cells, thick ascending limbs with attached glomeruli were dissected from rabbit kidney and incubated in a Ringers solution containing either horseradish peroxidase (HRP) (1 mg/ml; molecular weight approximately 40,000) or native or cationic ferritin (both at 5 mg/ml molecular weight approximately 450,000) for 5 and 20 min at room temperature. Tubules were processed for electron microscopy. At both time points, HRP reaction product fully permeated the matrix material between extraglomerular mesangial cells, the basement membrane underneath the macula densa, and also was occasionally located in intercellular spaces and intracellular vesicles within macula densa cells. Similar results were obtained with native and cationic ferritin. In separate experiments, thick ascending limbs with attached glomeruli were perfused for 20 min at room temperature with a Ringer solution containing HRP (1 mg/ml). HRP was found in tubulovesicular bodies within the apical cytoplasm of macula densa cells again indicating that these cells exhibit endocytotic activity. However, HRP did not gain access to intercellular spaces indicating that the apical junctional complex was impermeable to HRP. These results demonstrate that the macula densa basement membrane and matrix material between extraglomerular mesangial cells is permeable to high molecular weight molecules and suggest unhindered diffusion of water and solutes within this area.

Analysis of the intracellular maturation of the herpes simplex virus type 1 glycoprotein gH in infected and transfected cells

We have expressed the HSV-1 glycoprotein, gH, in transiently transfected COS-1 cells. The expressed protein was retained intracellularly, contained unprocessed carbohydrate, and was unrecognized by the monoclonal antibody, LP11. In addition, the protein was aggregated. These properties suggest that unlike other HSV glycoproteins, gH is misfolded in transfected cells. Pulse-chase studies of HSV-1-infected cells indicate that the kinetics of processing of gH are comparable to those of gB, gC, and gD. Rescue studies suggest that gH may interact with another protein during maturation in infected cells. However, we were unable to detect any stable interaction, although analysis of gH on neutral sucrose gradients shortly after synthesis indicated a possible transient association with a high molecular weight molecule or complex. The processing and cell surface expression of gH were also analyzed in HSV-1 virus mutants lacking gB, gC, or gD. Our results indicate that the maturation and cell surface transport of gH did not require the presence of these HSV-1 glycoproteins. In addition, three truncation mutants were constructed by linker insertion mutagenesis. Each of the three truncated proteins was synthesized, but the proteins were aggregated, contained only endo H-sensitive carbohydrate, and none were secreted.

Stimulation of glutathione absorption in rat small intestine by α ‐adrenergic agonists

The alpha-adrenergic agonist, phenylephrine (1.6 microM), caused a threefold stimulation of glutathione (GSH) transport from the lumen into the vasculature in isolated, vascularly perfused rat small intestine. Stimulation of GSH transport by phenylephrine was blocked by the alpha-adrenergic antagonists, prazosin or phentolamine. Norepinephrine and epinephrine (both alpha and beta agonists) also stimulated GSH absorption but not to the same extent as phenylephrine. Isoproterenol, a strict beta-adrenergic agonist, had no effect on the rate of GSH absorption. Under physiological luminal GSH concentrations, phenylephrine stimulated GSH efflux from the lumen, accumulation in the intestinal mucosa, and transport into the mesenteric vasculature. Phenylephrine did not stimulate the transport of polyethylene glycol, a high molecular weight molecule, and stimulated uptake of cysteine and glycine by 30%. This suggests that the effect of phenylephrine on GSH transport is not due to enhanced bulk flow through paracellular pathways. Studies with isolated small intestinal epithelial cells showed that phenylephrine also stimulated the release of GSH from the cells. Oral administration of phenylephrine with GSH caused a two- to fivefold transient increase in plasma GSH concentrations in rats. Phenylephrine alone or with the amino acid constituents of GSH caused no increase in plasma GSH concentration. Thus, absorption of dietary GSH is under hormonal regulation. The physiological importance of this regulation is not known, although such regulation may function to control utilization of dietary GSH for detoxication and may have therapeutic benefits for individuals with deficient GSH or increased risk of oxidative or chemically induced injury.

Acceleration of yeast autolysis by chemical methods for production of intracellular enzymes

Known methods for the acceleration of yeast autolysis have been investigated and new methods have been developed. It is shown that autolysis can be induced by plasmolysis with a number of solvents. The efficiency of this treatment is dependent on the nature of the solvent, its concentration and the duration of the treatment. Plasmolysis generally does not cause release of molecules of high molecular weight (MW) such as enzymes. However, addition of water initiates autolysis and the enzyme carboxypeptidase Y (MW 64000), for example, is released. The rate of this process is very dependent on pH; at the optimal pH (around 8.0) essentially complete autolysis is achieved within 20 h using the best solvents. Control of pH through the process is required. Straight-chain alcohols of medium chain length, i.e. C(6)-C(9) appear to function efficiently in amounts of only 1.2 ml/100 g yeast. In amounts of 2.5-10 ml solvent/100 g yeast trichloroethane, chloroform and in particular ether also provide efficient plasmolysis. Furthermore, it was shown that treatment of an aqueous suspension of yeast cells with a variety of non-ionic as well as ionic detergents caused autolysis. The influence of pH corresponds to that observed with organic solvents, i.e. a pH around 8.0 is optimal. This autolysis process was most efficient when the compressed yeast had been initially plasmolysed by treatment with sodium chloride followed by addition of water. The inexpensive detergents Triton X-100 and N-lauroylsarcosine appeared to be among the most efficient. The methods described in this paper are inexpensive and can be employed on a large scale. In addition, cell debris is easily removed, which is very important for subsequent down-stream processing. In the alternative method using physical breakage by homogenization this step is highly problematic.

Binder-substrate interactions in wet granulation. 2: The effect of binder molecular weight

The interactions during wet granulation between microcrystalline cellulose and aqueous solutions of two molecular weight grades of two polymer binders, have been monitored using an instrumented mixer torque rheometer. The rheological behaviour indicated that different molecular weight grades of the same polymer exhibited different behaviour at equivalent viscosity. This observation could only partly be explained using traditionally considered theories relating binder surface tension to granule strength. An examination of polymer adsorption phenomena gave results consistent with earlier data indicating a different degree of interaction between the two polymer types, and possible preferential adsorption of high molecular weight molecules. A model to describe particle: binder behaviour during wet massing has been suggested to explain the observed differences in rheological behaviour.