Further characterization of embryonic tendon fibroblasts and the use of immunoferritin techniques to study collagen biosynthesis

Morphological studies were carried out on fibroblasts from chick embryo tendons, cells which have been used in a number of recent studies on collagen biosynthesis. The cells were relatively rich in endoplasmic reticulum and contained a well-developed Golgi complex comprised of small vesicles, stacked membranes, and large vacuoles. Techniques were then devised for preparing cell fragments which were penetrated by ferritin-antibody conjuates but which retained the essential morphological features of the cells. Finally, the new procedures were employed to develop further information as to how collagen is synthesized. As reported elsewhere, preliminary studies with ferritin-labeled antibodies showed that prolyl hydroxylase was found in the endoplasmic reticulum of freshly isolated fibroblasts and that procollagen is found in both the cisternae of the endoplasmic reticulum and the large Golgi vacuoles. In the experiments described here, the cells were manipulated so that amino acids continued to be incorporated into polypeptide chains but assembly of the molecule was not completed because hydroxylation of prolyl and lysyl residues was prevented. The results indicated that these manipulations produced no change in the distribution of prolyl hydroxylase. Examination of the cells with ferritin conjugated to antibodies which reacted with protocollagen, the unhydroxylated form of procollagen, demonstrated that protocollagen was retained in the cisternae of the endoplasmic reticulum during inhibition of the prolyl and lysyl hydroxylases. Assays for prolyl hydroxylase with an immunologic technique demonstrated that although the enzyme is found within the endoplasmic reticulum, it is not secreted along with procollagen. The observations provided further evidence for a special role for prolyl hydroxylase in the control of collagen biosynthesis.     

Further evidence for the dispersion of the human fibrillar collagen genes.

Recombinant DNA probes specific for the human pro alpha 1(II) and pro alpha 1(III) collagen chains have been used for the chromosomal localization of the two genes. Restriction endonuclease analysis of DNA from human-rodent hybrid cell lines in conjunction with in situ hybridization of human metaphasic chromosomes have shown that the gene coding for the pro alpha 1 chain of type II collagen (COL2A1) is located on chromosome 12 in the segment 12q131----12q132. Likewise, the gene coding for the pro alpha 1 chain of type III collagen (COL3A1) was assigned to the segment 2q31----2q323 of chromosome 2.     

Further evidence of a role for abscisic acid in conversion of somatic embryos ofDaucus carota

Summary Somatic embryogenesis has been shown to be an imperfect recapitulation of stages involved to form embryos from vegetative tissues. Although abscisic acid has been implicated in normalizing development, studies that specifically investigate conversion (vegetative leaf initiation) in somatic embryos are lacking. This report documents a follow-up of a study that implicated abscisic acid as a vital factor in allowing embryos ofDaucus carota to progress to the plantlet stage. Abscisic acid was determined to enhance conversion at doses ranging from 1 to 50 µM. Younger embryo stages were more responsive to abscisic acid application with regards to plantlet recovery. Pulses of abscisic acid were shown to elicit more rapid response with younger embryo stages, indicating more plastic development. Fluridone, an abscisic acid synthesis inhibitor, was shown to dramatically reduce conversion, even at low doses (<5µM). When abscisic acid was applied concurrently with fluridone, partial restoration of conversion was observed. Histologically, fluridone was seen to cause pronounced vacuolation in the shoot apical notch which resulted in the loss of meristematic cells, negating conversion capacity. Quantitation of total cytoplasmic area showed that abscisic acid reduced vacuolar intrusion into the apical notch, while fluridone caused a significant increase in vacuolation of cells in this region. This report documents further evidence of a role for abscisic acid in plantlet establishment from somatic embryos ofDaucus carota.     

Observations on the different substrate behavior of tropocollagen molecules in solution and intermolecularly cross-linked tropocollagen within insoluble polymeric collagen fibrils.

Bacterial collagenase was used to compare the extent of digestion of tropocollagen monomers in solution and in reconstituted fibrils with that of tropocollagen molecules intermolecularly cross-linked within insoluble polymeric collagen fibrils obtained from mature tendons at given time-intervals. The extent of digestion of tropocollagen monomers in solution was directly proportional to the enzyme concentration (a range of enzyme substrate molar ratios 1:200 to 1:10 was used). The extent of digestion of polymeric collagen was followed by measuring the solubilization of fluorescent peptides from fluorescent-labelled insoluble polymeric collagen fibrils. The extent of digestion of tropocollagen within polymeric collagen was linear over a very small range of enzyme concentrations, when the enzyme/substrate ratio in the reaction mixture was less than 1:400 on a molecular basis. The behavior of tropocollagen in the form of reconstituted collagen fibrils, which had been matured at 37 degrees C for 8 weeks, was intermediate between the behaviour of solutions of tropocollagen and insoluble polymeric collagen fibrils. The significance of the results is discussed in terms of the structure of polymeric collagen fibrils and the protection against enzymic attack provided by tropocollagen molecules on the circumference of the fibril. The results suggest that assays of collagenase activities based on tropocollagen as substrate cannot be directly related to the ability of these enzymes to degrade mature insoluble collagen fibrils.     

Isolation and characterization of a precursor form of M collagen from embryonic chicken cartilage

A disulfide-cross-linked collagen has been extracted with neutral salt solutions from organ cultures of embryonic chick sternal cartilage. This collagen, which we term pM collagen, is presumed to be the native extracellular precursor molecule to disulfide-cross-linked collagen fragments recently described. Cleavage of pM collagen under native conditions with pepsin gives rise to the collagen fragments M1 and M2, which had also been isolated from pepsin extracts of chick hyaline cartilage [K. von der Mark, M. van Menxel & H. Wiedemann (1982) Eur. J. Biochem. 124, 57-62]. Native pM collagen was purified by DEAE-cellulose chromatography and agarose gel filtration. On agarose and following polyacrylamide gel electrophoresis, the unreduced molecule migrates with an apparent Mr of 300 000. Reduction of disulfide bridges produces two subunits with Mr 80 000 (pMa) and 60 000 (pMb) when compared with collagen standards. Cyanogen bromide cleavage of pMa and pMb, excised from dodecyl sulfate gels, resulted in different peptide maps, indicating that both components are genetically distinct polypeptide chains. The occasional appearance of the unreduced pM collagen as a doublet band on dodecyl sulfate gels and the observation that pMa and pMb occur in non-stoichiometric ratios suggests that pMa and pMb form separate native molecules, although their incorporation into a single pM molecule cannot be excluded. Native pM collagen was completely digested with bacterial collagenase, and contained hydroxyproline and proline in a ratio of 1.15:1, indicating the absence of significant non-collagenous domains. Thus it represents, despite several pepsinlabile sites, more likely a largely triplehelical, processed form of collagen rather than a procollagen-like molecule containing globular domains. Processing of pM collagen to M1 and M2 fragments or other intermediate forms was not observed in cartilage organ culture or in chondrocyte cell cultures within 18 h.     

Changes induced by ozone and ultraviolet light in type I collagen. Bovine Achilles tendon collagen versus rat tail tendon collagen

High-molecular-mass aggregates were made soluble from insoluble collagens of bovine Achilles tendon and rat tail tendon by limited thermal hydrolysis. These polymeric collagen aggregates were cross-linked by 390-nm-fluorescent 3-hydroxy-pyridinium residues (excited at 325 nm) in the former tendon and by unknown non-fluorescent residues in the latter. With the solubilized insoluble-collagens from both tendons, as well as with acid-soluble collagen from rat tail tendon, other 350-385-nm fluorescence intensities (excited at 300 nm) were found to be higher in monomeric chains than in dimeric and polymeric chains. Low levels of ozone inhibited fibril formation of acid-soluble collagen particularly from young rat tail tendon, reacting with tyrosine residues and the 350-385-nm fluorophores. Aldehyde groups, involved in cross-linking, were not effectively modified by ozone. beta-Components (alpha-chain dimers) were not efficiently dissociated even by higher doses of ozone compared to gamma-components (alpha-chain trimers). Polymeric chain aggregates from bovine Achilles tendon collagen, whose 3-hydroxy-pyridinium cross-links are cleaved by ozone, were more readily dissociated by ozone than those from rat tail tendon collagen. Ultraviolet (300-nm) light, which destroyed the 350-385-nm fluorophores, inhibited fibril formation less effectively than ultraviolet (275-nm) light, which is absorbed by tyrosine residues, and did not dissociate collagen polymers from rat tail tendon. On the other hand, ultraviolet (320-nm) light, absorbed by 3-hydroxy-pyridinium cross-links which were rapidly photolyzed, partially dissociated polymeric collagen aggregates from bovine Achilles tendon after subsequent heating.     

Further evidence for a direct cytoplasmatic pathway for Rb transport from the external solution to the xylem in barley seedlings

Summary The relation between the amount of Rb-ions present in the cytoplasm and the rate of upward translocation of Rb was investigated in young intact barley plants.A positive correlation between the rate of transport of Rb to the shoot and the cytoplasmatic Rb-content became apparent when the latter was varied experimentally by manipulating either the external Ca- or the outward Rb-concentration.These experiments provided no evidence of competition between root accumulation of Rb and upward Rb-transfer for a limited amount of Rb-ions entering the root system.     

Further characterization of the vesicular stomatitis virus temperature-sensitive O45 mutant: intracellular conversion of the glycoprotein to a soluble form.

Reexamination of the viral products of tsO45, a glycoprotein mutant of vesicular stomatitis virus, showed that at 39 degrees C there was a conversion of the glycoprotein (G) to a truncated, soluble form, Gs, which subsequently appeared in the extracellular medium. The half-life for this intracellular conversion and extracellular appearance was about 2 h at 39 degrees C. Gs was precipitated by a monoclonal antibody to the ektodomain but not by an antipeptide serum made against the first 15 amino acids at the carboxy terminus of G. Gs was also resistant to endoglycosidase H digestion. On the basis of pulse-chase experiments, the generation of Gs most probably occurred in the rough endoplasmic reticulum. This additional phenotype of the tsO45 mutant provides another approach for studying the generation and subsequent transport of a secreted protein in fibroblast cells.     

The helix-coil transformation for tropocollagen solutions and its relationship to transformations involving the crystalline form of the protein

Collagen molecules can exist, in the aggregate crystalline state (C) and in solutions, as tropocollagen helices (H) or random coils (RC). We have compared the role of temperature, salt type, and salt concentration, C₈, on the transformations: C → RC (I), H → RC (II), and H → C (III) under near-isoelectric conditions. Processes I and II occur in the direction from ordered to disordered form on increasing temperature, while process III occurs in the direction crystal → dissolved helices on lowering temperature. The order of both anions and cations for increasing the stability of the dissolved random coiled form is similar for processes I and II, but the order of the anions for increasing the stability of the dissolved helices, according to process III, is reversed with respect to the Hofmeister series. T_I and T_II are greatly and continuously depressed by some salts on increasing C₈ while, for other salts, they are at first slightly depressed and then raised. T_III, instead, is at first raised and then lowered on increasing C₈. For a given salt the field of stability of each form is determined, on a pseudo-phase diagram, by the relative positions of the T_I versus C₈, the T_II versus C₈, and the T_III versus C₈, curves. The similarity of the effect of temperature, salt type, and concentration on processes I and II supports the contention that both processes are controlled by the interaction of the random coiled form with the salt solution. The phenomenon of crystallization by increasing temperature is complicated by large time effects and, often by lack of reversibility. Assuming, nevertheless, that there is an underlying equilibrium process, two possible interpretations are proposed. The peculiar trend of the T_III versus C₈ curve is justified on the basis that it reflects the usual change from salting in to salting out on increasing C₈, allowing for the fact that the dissolved phase is stable in the low-temperature range. The reversal of the order of anions for process III can be most simply justified in terms of the slight cat ionic character of the tropocollagen units. Accordingly, the dissolved helices will be more; stable of the crystalline form depending upon the occurrence of a net charge different from zero on the protein, as well as upon low ionic strength and low temperature. Minute alterations of KCl concentrations are able to cause isothermal precipitation of fibers and probably constitute a controlling factor for the fibrogenesis in vivo.     

Isolation of sulphate transport defective mutants ofCandida utilis: Further evidence for a common transport system for sulphate,sulphite and thiosulphate

Selenate-resistant mutants ofCandida utilis were isolated. They did not take up sulphate while incorporation of an organic sulphur source, such asl-methionine, was similar to the wild-type strain. They grew poorly on sulphate, sulphite and thiosulphate and, as expected, grew well on methionine. Sulphite reductase activities of the mutants were similar to the wild type strain. The properties of these mutants support the view of a common transport system for sulphate, sulphite and thiosulphate.     

Intermediates in the limited proteolytic conversion of procollagen to collagen.

The conversion of chick bone procollagen to collagen proceeds in a stepwise fashion to produce a limited number of intermediates. Initial proteolytic cleavages remove NH2-terminal nonhelical extensions and yield an intermediate which remains disulfide-bonded via COOH-terminal extensions. Subsequent stepwise scission of one or two chains of the triple-stranded molecule in its COOH-terminal domain produces intermediates which can only be distinguished after dissociation of the noncovalently bonded alpha chains. A final cleavage in this region produces the collagen molecule and a disulfide-bonded triple-stranded fragment which represents the COOH-terminal domain. In all likelihood the endopeptidases which effect cleavage in the NH2- and COOH-terminal regions differ. More than two enzymes may be required for conversion of procollagen to collagen if the nonhelical domains are not released in an en bloc fashion.     

Glycoproteins from experimental granulation tissue and their effects on collagen synthesis in embryonic chick tendon cells

Buffer-soluble and pronase-liberated glycoproteins from experimental granulation tissue were fractionated by gel filtration and DEAE-cellulose chromatography. The age of the granuloma was reflected in the gel filtration pattern. Two glycoproteins were isolated, purified to homogeneity and analyzed for their carbohydrate and amino acid compositions.The collagen synthesis in embryonic chick tendon cells was measured in the presence of these fractions, which were found to be inhibiting even at 10^?6 M. These glycoproteins may be significant in the feedback regulation of the development of granulation tissue and fibrosis.     

Conversion of pepsinogen to pepsin. Further evidence for intramolecular and pepsin-catalyzed activation.

Exposure of pepsinogen to acid for less than 2 min yields a product with proteolytic activity. This activity is due to intramolecular and intermolecular formation of pepsin from pepsinogen. We find no evidence for intermolecular proteolytic activity in the zymogen. These conclusions are based upon two sets of experiments. First, chemical cleavage of pepsinogen during short activation is demonstrated by quantitative analysis of the NH2-terminal 2 residues of the pepsin and pepsinogen in an activation mixture. In addition, quantitative NH2-terminal analyses after activation under different conditions confirm our previous inference that the product of unimolecular pepsinogen activation is homogeneous whereas bimolecular activation produces a pepsin product with a variety of NH2 termini. Second, spectral changes which occur upon acidification of a pepsinogen solution and are reversed by neutralization are shown to be consistent with the chemical cleavage of pepsinogen during acidification. The first order rate constant for pepsinogen activation, calculated from these spectral experiments, agrees well with the value we had determined previously.     

Thermo-mechanical extrusion pretreatment for conversion of soybean hulls to fermentable sugars

Thermo-mechanical extrusion pretreatment for lignocellulosic biomass was investigated using soybean hulls as the substrate. The enzyme cocktail used to hydrolyze pretreated soybean hulls to fermentable sugars was optimized using response surface methodology (RSM). Structural changes in substrate and sugar yields from thermo-mechanical processing were compared with two traditional pretreatment methods that utilized dilute acid (1% sulfuric acid) and alkali (1% sodium hydroxide). Extrusion processing parameters (barrel temperature, in-barrel moisture, screw speed) and processing aids (starch, ethylene glycol) were studied with respect to reducing sugar and glucose yields. The conditions resulting in the highest cellulose to glucose conversion (95%) were screw speed 350 rpm, maximum barrel temperature 80 °C and in-barrel moisture content 40% wb. Compared with untreated soybean hulls, glucose yield from enzymatic hydrolysis of soybean hulls increased by 69.6%, 128.7% and 132.2%, respectively, when pretreated with dilute acid, alkali and extrusion.