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A convenient small-scale laboratory method that can be used to simultaneously analyze multiple samples was developed to rapidly assess suitability of corn for nixtamalization. This new 100 g method was developed based on a previously reported 500 g laboratory process that has been shown to mimic the industrial nixtamalization process. The two methods were compared for nixtamal moisture, dry matter loss, degree of pericarp removal, and gelatinization properties of the cooked corn. The heating and cooling profiles of the 100 g method were developed using the 500 g method, by monitoring temperature every 30 s during cooking and steeping. Nixtamalization was conducted with a 1:4 corn/water ratio, with 1% lime. A response surface central composite design was used to model a wide range of processing conditions for the two methods: cook temperature (80–95°C), cook time (3–40 min), and steep time (2–12 h). Parameter estimates and response surfaces were compared, and predictive models were fitted. The response surface models for the two methods were not significantly different for nixtamal moisture, dry matter loss, and gelatinization enthalpy; there was an overlap of the 90% Bonferroni confidence intervals (P< 0.05, r2> 0.7). The bench-top 100 g nixtamalization process can successfully mimic the 500 g method over a wide range of processing conditions.

Recent articles have documented the importance of cultural competency in surgery. Surgical residency programs have used the Objective Structured Clinical Examinations or cultural standardized patient examinations as a training tool. Past studies evaluating cultural competency have noted the importance of including an observational (control) arm, which would allow for a more objective assessment of a resident’s competency in this area. The purpose of our article is to present the results of a follow-up study to a pilot cultural standardized patient examination for surgery residents.

The effects of dough moisture, mixing time, and cooking time on uncooked and cooked elbow macaroni by means of starch pasting and macaroni textural characteristics were investigated. In conventional elbow macaroni production, cooking time was found to have significant contributions to cooked macaroni starch pasting properties, indicating that degree of starch cook dependent on cooking time was the main influence on cooked macaroni starch pasting phenomena. Dough moisture also showed some significant (P< 0.05) relationships with cooked macaroni starch pasting properties; however, mixing time did not show significant effect. Cooked macaroni starch pasting properties showed significantly (P< 0.05) high correlations with cooked macaroni firmness and stickiness. Cooking time was the only major variable contributing to variations in cooked elbow macaroni starch and consequently in pasting and texture characteristics. Cooking time was highly related to firmness and stickiness of cooked elbow macaroni (P< 0.0001, R2= 0.8148; P< 0.0001, R2= 0.6215, respectively). In addition, dough moisture had a slight significant (P< 0.05) effect on cooked elbow macaroni firmness and stickiness. Cooked elbow macaroni firmness and stickiness were found to be highly correlated (P= 0.0001, R2= 0.8459). Increases in firmness increased cooked elbow macaroni stickiness. As a result, when elbow macaroni was cooked for shorter times, firmer and stickier macaroni was obtained.

Grain processors would benefit from information about the production environment and the influences of the sorghum [Sorghum bicolor(L.) Moench] hybrid on food‐grade flour properties. The objective of this study was to determine the effects of environment and hybrid on rapid‐visco‐analysis (RVA) flour properties of commercially available food‐grade sorghum. A randomized complete block experiment was planted in 12 environments, which included the 2004 and 2005 growing seasons and irrigated and dryland water regimes in eastern, central, and west central Nebraska, and a dryland, low‐N environment in eastern Nebraska. The environment accounted for 71–85% of the total variation in RVA parameters, while the hybrid accounted for 11–23% and the environment‐by‐hybrid interaction, 1–3%. Unfortunately, the results of this experiment suggest that it is difficult to predict the effect that environment will have on resulting sorghum‐flour parameters. Although of secondary importance in terms of total variation in sorghum‐flour RVA properties, the choice of hybrid predictably and significantly contributes to sorghum‐starch viscosity properties. Food‐grade hybrids were grouped based on viscosity properties into those best suited for dry‐mill and alkaline‐cooked products (Asgrow Orbit; Sorghum Partners NK1486) and those best suited for porridge, consumable alcohol, and ethanol production (Kelly Green Seeds KG6902; NC+ Hybrids 7W92; Asgrow Eclipse; and Fontanelle W‐1000). These results were consistent with those previously reported for grain density.

The objective of this study was to identify and characterize different popped popcorn flake shapes, or polymorphisms, arising from a yellow butterfly popcorn hybrid (YP-213), and then to determine the impact of popcorn flake shape on composition and sensory characteristics. Kernels were popped using a microwave oven and visually sorted into three different polymorphisms depending on whether the appendages were expanded unilaterally, bilaterally, or multilaterally. When popped, 9.0 ± 3.1%, 71.2 ± 5.9%, and 12.3 ± 3.8% of kernels were expanded unilaterally, bilaterally, and multilaterally, respectively, while 7.6 ± 1.4% of kernels remained unpopped. Expansion volumes for unilaterally, bilaterally, and multilaterally expanded polymorphisms were 28.6 ± 3.84, 43.0 ± 0.84, and 53.5 ± 2.5 cm3/g, respectively. Unilateral popcorn flakes retained the most fat, saturated fat, and sodium, while multilaterally expanded flakes had the highest levels of protein, total carbohydrate, and popcorn-like aromatic pyrazines. Sensory evaluation revealed significant differences among polymorphisms for flavor and texture attributes, with the unilaterally expanded polymorphism receiving the highest overall product liking. These data show that different popcorn flake polymorphisms produced from a single hybrid of popcorn affect sensory and compositional profiles. More research is necessary to elucidate the factors that affect popcorn flake polymorphisms and support development of new varieties or techniques to produce the most desirable microwave popcorn.

Dent corn (Zea maysL.) and sorghum (Sorghum bicolorL. Moench) sample sets representative of commonly grown hybrids and diverse physical attributes were analyzed for alkaline cooking performance. The influence of kernel characteristics including hardness, density, starch properties (thermal, pasting, and crystallinity), starch content, protein content, and prolamin content on alkaline cooking performance was also determined. Corn nixtamal moisture content was lower for hard, dense kernels with high protein contents; sorghum nixtamal moisture content was lower for kernels with low moisture contents and low starch relative crystallinities. Statistically significant (P< 0.05) regression equations showed that corn nixtamal moisture content was influenced by TADD (tangential abrasive dehulling device) index, kernel moisture content, starch content, and protein content; sorghum nixtamal moisture content was influenced by starch relative crystallinity, kernel moisture content, and abrasive hardness index. Pericarp removal was not strongly correlated with kernel characterization tests. Location (environmental) and hybrid (genetic) factors influenced most kernel characteristics and nixtamalization processing variables.

Few studies have examined grain quality of food‐grade sorghum hybrids. The objective of this study was to determine the effects of environment and hybrid on grain quality of commercially available food‐grade sorghums. A randomized complete block experiment with three replications was planted in 12 environments, which included the 2004 and 2005 growing seasons and irrigated and dryland water regimes in eastern, central, and west central Nebraska and a dryland low‐N environment in eastern Nebraska. Environment accounted for 5 to 140 times greater variation in measured parameters than hybrid, and the hybrid × environment interaction accounted for less than 2% of the total variation. Grain yield and kernel mass varied, with low yields of 1.4 Mg ha−1and kernels weighing 9.5 g 1000 kernels−1in the low‐N 2004 environment, high grain yields of 10.5 Mg ha−1under irrigated conditions in central Nebraska in 2005, and kernels weighing 27.8 g 1000 kernels−1in the eastern Nebraska dryland 2005 environment. Harder grain was produced in 2005 than in 2004, with the west central and central 2005 environments having the lowest tangential abrasive dehulling device (TADD) removals of 14%. Non‐food‐grade hybrids produced higher grain yields and kernel mass than food‐grade hybrids. Grain hardness was greater for non‐food‐grade and medium maturity hybrids when environmental means were lower (i.e., softer) but showed little or no difference in hardness when environmental means were high. Nebraska production environments have the capability to produce high quality food‐grade sorghums for specific food uses to benefit both the producer and the food processor.

Native corn starch, plasticized with water, glycerol and stearic acid, was extruded in a conical twinscrew extruder and sheeted into 0.4–0.6 mm thick films. The effects of extrusion and plasticizers on gelatinization, as well as the molecular and structural changes, in thermoplastic starch were analyzed. The onset and peak gelatinization temperatures of extruded starch varied from 42–46°C and 52.9–56.9°C, respectively, depending on the glycerol content. The enthalpy of gelatinization of extruded thermoplastic starch in excess water varied from 3.6–7.6 Jg, which also increased with plasticizer content. Amyloselipid complexes were formed during extrusion, and their enthalpies depended on the initial stearic acid and moisture contents. Highperformance sizeexclusion chromatography HPSEC data revealed that the starch underwent fragmentation during extrusion even under highly plasticized conditions, but the degradation was not severe as compared to previous findings. The relative percentages of amylopectin and amylose in native starch were 76.9 and 23.1, respectively, which were changed to 71.3–76.6 and 23.4–28.7 in the extrudates. The average molecular weights of amylopectin and amylose in the extrudates ranged from 1.55×107–2.07×107and 4.35×105–7.39×105, respectively. On the other hand, the molecular weights of amylopectin and amylose in native corn starch were observed as 2.27×107and 4.68×105, respectively. Crosspolarization magical angle spinning CPMAS and highpower decoupling HPDEC nuclear magnetic resonance NMR spectra of thermoplastic starch revealed the characteristics of amylomaize starch, confirming HPSEC results that the amylopectin macromolecules underwent fragmentation into amyloselike fractions. In the extrudates, glycerol was found to be less mobile and entrained within the starch network.

It has long been recognized that limitations exist in the analytical methodology for amylose determination. This study was conducted to evaluate various amylose determination methods. Purified amylose and amylopectin fractions were obtained from corn, rice, wheat, and potato and then mixed in proportion to make 10, 20, 30, 50, and 80% amylose content starch samples for each source. These samples, considered amylose standards, were analyzed using differential scanning calorimetry (DSC), high-performance size-exclusion chromatography (HPSEC), and iodine binding procedures to generate standard curves for each of the methods. A single DSC standard equation for cereal starches was developed. The standard curve of potato starch was significantly different. Amylose standard curves prepared using the iodine binding method were also similar for the cereal starches, but different for potato starch. An iodine binding procedure using wavelengths at 620 nm and 510 nm increased the precision of the method. When HPSEC was used to determine % amylose, calculations based on dividing the injected starch mass by amylose peak mass, rather than calculations based on the apparent amylose/amylopectin ratio, decreased the inaccuracies associated with sample dispersion and made the generation of a cereal amylose standard curve possible. Amylose contents of pure starch, starch mixtures from different sources with different amylose ranges, and tortillas were measured using DSC, HPSEC, iodine binding, and the Megazyme amylose/amylopectin kit. All the methods were reproducible (±3.0%). Amylose contents measured by these methods were significantly different (P< 0.05). Amylose measurements using iodine binding, DSC, and Megazyme procedures were highly correlated (correlation coefficient >0.95). DSC and traditional iodine binding procedures likely overestimated true amylose contents as residual butanol in the amylose standards caused interference. The modified two-wavelength iodine binding procedure seemed to be the most precise and generally applicable method. Each amylose determination method has its benefits and limitations.

Starch granules undergo structural and morphological changes during food processing unit operations as they interact with other food ingredients. This study was conducted to isolate and characterize starch granules from corn masa. A proteolytic enzyme, thermolysin, was effective in separating and isolating starch granules from endosperm proteins present in masa. The efficiency of starch extraction using thermolysin was 74% (w/w), and subsequent analyses showed that the isolated granules were free of contaminants. Starch samples were characterized using light microscopy, SEM, DSC, and XRD. Starch granules isolated from masa had undergone internal structural changes and some granules (˜40%) lost birefringence during nixtamalization. These internal changes occurred, in most cases, without visible alterations in general granular morphology. Nixtamalized granules underwent changes mostly consistent with a “heat-moisture treatment” process.

Soybean [Glycine max(L.) Merr.] rotation enhances grain sorghum [Sorghum bicolor(L.) Moench] yield, but influence on grain quality has not been measured. The objective was to determine the effect of cropping sequence (CS) and soil amendment (SA) on grain yield and quality. Sorghum grain yield and quality, soil NO3–N and water were measured in a rotation study in 2003 and 2004 on a Sharpsburg silty clay loam (fine, smectitic, mesic Typic Argiudoll). Cropping sequences were continuous sorghum, and sorghum rotated with non‐nodulating and nodulating soybean. Soil amendments consisted of no amendment, manure (17–26 Mg dry matter ha−1yr−1), and N (84 kg ha−1yr−1). CS × SA interaction effects were found for most parameters. Rotation with non‐nodulating soybean without SA increased yield by 2.6 to 2.8 Mg ha−1over continuous sorghum without SA. Rotation without SA with nodulating soybean further increased yield by 1.7 to 1.8 Mg ha−1over rotation with non‐nodulating soybean. Grain N increased by 0.5 to 1.0, 2.5 to 5.0, and 3.3 to 4.9 g kg−1for N application to continuous sorghum and sorghum rotated with non‐nodulating and nodulating soybean, respectively. Tangential abrasive dehulling device (TADD) removal indicated that continuous sorghum without SA produced the softest grain with 43 to 44% TADD removal, and sorghum rotated with nodulating soybean with manure produced the hardest grain with 22 to 27% TADD removal. As food end‐use opportunities for sorghum grain evolve, use of crop rotation and SA application will be important to produce grain with desirable quality attributes.

A genetically and environmentally diverse collection of maize (Zea maizeL.) samples was evaluated for physical properties and grit yield to help develop a standard set of criteria to identify grain best suited for dry-milling. Application of principal component analysis (PCA) reduced a set of approximately 500 samples collected from six states to 154 maize hybrids. Selected maize hybrids were placed into seven groups according to their dry-milled grit yields. Regression analysis explained only 50% of the variability in dry-milling grit yield. Patterns of differences in the physical properties for the seven grit yield groups implied that the seven yield groups could be placed into two or three groups. Using two pattern recognition techniques for improving classification accuracy, quadratic discriminant analysis and the classification and regression tree (CART) model, dry-milled grit yield groups were predicted. The estimated correct classification rates were 69–80% when the samples were divided into three yield groups and 81–90% when samples were divided into two yield groups. The results indicated the comparable success of both techniques and the superiority of the decision tree algorithm to quadratic discriminant analysis by offering higher accuracy and clearer classification rules in differentiating among dry-milled grit yield groups.

Preservation of starch structure/properties, including structures formed during partial or complete cooking, are important when the impact of processing conditions is being studied. Two preservation techniques used to study changes in starch during thermal-mechanical processing are commonly cited in the literature: 1) rapid freezing followed by lyophilization, and 2) a dehydration procedure using alcohols. A comparative determination on how these methods affect various starch structures has not been widely reported. Corn starch samples were collected from the Rapid Visco-Analyser (RVA) at 3 min (swollen granules, 30°C), at the top of the pasting peak (gelatinized granules, 95°C), at the bottom of the trough (dispersed polymers, 95°C), and a completed RVA sample stored for 120 hr at 4°C (retrograded starch). Samples of masa were obtained by nixtamalizing corn. Differential scanning calorimetry (DSC) endotherms of starch and masa, and X-ray diffraction (XRD) patterns of masa were evaluated after being preserved by alcohol- or freeze-drying. No significant differences (P> 0.05) between methods were found for onset, end, and peak temperatures (°C), enthalpy (J/g) and % relative crystallinity in any of the samples analyzed. Liquid nitrogen freeze-drying and ethanol dehydration are both effective methods of preserving various starch systems for structural changes detectible by DSC and XRD; freeze-drying is generally less expensive and time-consuming.

A laboratory nixtamalization process was developed to imitate larger scale cooking/steeping conditions. Corn (45 kg) was cooked in a pilot plant gas-fired cook/steep tank and temperature was monitored every 30 sec. Cooling and heating rates were mimicked in the laboratory using a digital temperature programmable hot plate that adjusted grain-water-lime temperature changes at a specified rate. A Response Surface Central Composite Design was used to model pasting and thermal properties of nixtamal and masa as a function of cooking temperature (86–96°C), cooking time (20–40 min), and steeping time (3–11.77 hr). Nixtamal and masa moisture, dry matter loss, nixtamal and masa RVA peak temperature, shear thinning, nixtamal peak viscosity, masa final viscosity, nixtamal and masa DSC enthalpy peak and end temperatures, and nixtamal onset temperature were explained by the same regression terms for results obtained using both processes conditions. The intercept and slopes of the fitted models for the pilot plant and laboratory responses were not significantly different (P< 0.05). The laboratory method can be used to mimic larger scale processing over a wide range of nixtamalization conditions.

Maize (Zea maysL.) is an important crop in Brazil, and concerns about grain quality are increasingly important with increasing exports and use of grain for specific end‐uses. A wide range of genotypes are grown and N application is required to produce high yields. The objectives of these studies were to: (i) determine N application effects on the kernel hardness and breakage susceptibility of a wide range of Brazilian genotypes ranging from dent to flint kernel types and (ii) determine relationships among kernel hardness and breakage susceptibility tests, yield and N and oil concentration. Three studies were conducted with a broad range of maize genotypes and N application rates of 0, 60, 120, and 180 kg ha−1Grain was harvested and yields corrected for water content, and grain was evaluated through a series of chemical and physical quality tests. Application of 180 kg ha−1N application increased grain yield by 747 to 1466 kg ha−1, increased grain N concentration by 0.9 to 2.4 g kg−1, and increased hardness to a lesser extent, while reducing breakage susceptibility by 1.9 to 6.9%. Genotype had a much larger influence on grain quality parameters than did N rate. The limited correlation between grain yield, grain N concentration, and grain oil concentration to kernel hardness suggests that development of further improved genotypes with high maize yields and excellent dry milling grain quality is feasible in Brazil. The large variation in grain yield and dry milling grain quality in intermediate kernel‐type (semident, semiflint) genotypes used in Brazil presents short‐term potential to select hybrids that produce both high yield and good dry milling grain quality.

Relationships between the structural properties of raw and extruded corn starches and their functionalities were investigated using mixtures of these starch types. Extruded starch had higher water absorption and water solubility indices, and produced lower RVA viscosity profiles when compared with raw starch. It also had no differential scanning calorimetry (DSC) endotherm. Gel cohesiveness and adhesiveness of both starch types were similar, while extruded starch gels were softer. Extruded starch produced lower Rapid Visco Analyser (RVA) viscosity profiles than raw starch due to starch degradation during extrusion. The raw and extruded starch components had negative interaction coefficients, thus RVA viscosity parameters were lowered as the fraction of extruded starch in the mixture increased. Starch degradation in the extruded starch was a likely significant factor associated with low viscosity profiles. Mixtures of raw and extruded starches could be commercially prepared to obtain finished starch products with a range of functional attributes.

The molecular behavior of amylose-lipid complexes was studied using differential scanning calorimetry. rapid viscoanalysis and texture analysis methods. Three amyloses were fractionated, one each from regular, 50% amylose and 70% amylose corn starches. High performance size exclusion chromatography, coupled with multiple angle laser light scattering, was used to determine amylose molecular weight profiles; fractions differed statistically (P>0.05) in their z-average molecular weights (Mz). Each amylose fraction was complexed with five different fatty acids. After 12 days of storage, amylose-lipid complexes had recrystallization percentages ranging from 42.7 to 98.2%. Cohesiveness (r = -0.84) and adhesiveness (r = -0.75) decreased with increasing Mz of amyloses (P>0.05). An inverse relationship was obtained between fatty acid chain length and percent recrystallization (r = -0.84, P>0.05). Percent recrystallization decreased when fatty acid chain length increased from C16:0 to C18:0. All complex samples, when adjusted to an equal total starch basis, had decreased viscosities when pasted compared to their native starch or amylose counterparts. Shear thinning of complexes increased with increasing molecular weight (Mz) of amyloses (r = 0.71, P>0.05). Lower recrystallization rates and decreased viscosity properties could be accomplished complexing by amylose and fatty acids.

The incidence of moniliformin (MON) producing Fusariumspp. in selected corn (Zea maysL.) samples from Mexico and the United States and the effects of alkaline cooking and the tortilla manufacturing processes on the reduction of MON were determined. The percentage of infected kernels with Fusariumspp. ranged from 0 to 22% in eight foodgrade corn samples, including six from Mexico and two from the United States. Complete (100%) reduction of MON was observed when a naturally contaminated corn sample containing 1.4 µg of MON/g of corn was used in a pilot-scale alkaline cooking and tortilla manufacturing process. In a companion laboratory-scale study, using a cultured corn sample containing 17.6 µg of MON/g of corn, a 71% reduction of the toxin was observed during the process. Alkaline cooking appeared to be an effective method for reduction of MON in corn.

In an effort to concentrate the source of wax, abrasive decortication of grain sorghum was used to separate whole kernels into bran and abraded kernel fractions. Abrasive decortication was accomplished using a tangential abrasive dehulling device (TADD). The variables studied were moisture addition with tempering time and abrasion time. The constituents of dry matter, starch, protein, ash and wax were measured for whole sorghum kernels. Mass of fractions of abraded kernels and bran were also measured subsequent to decortication. Chemical analysis was then performed to determine the amount of each constituent recovered in each fraction. The total matter recovered was determined using a summation of the amounts recovered in each fraction. The TADD method of this study recovered an average of over 94% of the total dry matter, total starch, total protein and total ash between the two fractions, while less than 85% of the total wax was recovered from any one sample. As longer abrasion times were used to remove the bran from the kernel, more starch was removed as well. The greatest wax concentration, 66·9±5·6%, in the bran fraction with a starch content of 5·5±0·8% occurred after abrading kernels at 10·4% moisture content for 80 s. In conclusion, using the TADD to concentrate wax to high levels with minimal starch contamination was not achieved.

The rates of collagen biosynthesis and the tissue concentrations of collagen in normal scar, hypertrophic scar and keloid were determined as a function of the duration of the lesions. The rate of collagen synthesis in normal scar was approximately constant between 6 months and 20 years after the initial wounding, but in both hypertrophic scar and keloid the rate was initially approximately twice that in normal scar, and 2–3 years after wounding it fell to approximately the same level as in normal scar. The tissue concentration of collagen appeared to be relatively constant in normal scar with respect to the time elapsed after wounding, but in both types of abnormal scar it was initially somewhat lower than normal scar and then rose to values higher than in normal scar after about 2–3 years. Since similar results were obtained for both types of abnormal scar in terms of the rates of collagen biosynthesis with respect to the time elapsed after wounding and also in terms of the tissue concentrations of collagen, this may indicate that the events in the pathogenesis of both abnormal scar types are similar. The results also raise the possibility that the formation of both types of abnormal scar may occur in two phases: an initial phase characterized by abnormally high rates of collagen biosynthesis, and a later phase characterized by essentially normal rates of collagen biosynthesis.

1. Confluent human skin fibroblasts maintained in a chemically defined medium incorporate l-[1-3H]fucose in a linear manner with time into non-diffusible macromolecules for up to 48h. Chromatographic analysis demonstrated that virtually all the macromolecule-associated3H was present as [3H]fucose. 2. Equilibrium CsCl-density-gradient centrifugation established that [3H]fucose-labelled macromolecules released into the medium were predominantly glycoproteins. Confirmation of this finding was provided by molecular-size analyses of the [3H]fucose-labelled material before and after trypsin digestion. 3. The [3H]fucose-labelled glycoproteins released into fibroblast culture medium were analysed by gel-filtration chromatography and sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. These techniques demonstrated that the major fucosylated glycoprotein had an apparent mol.wt. of 230000–250000; several minor labelled species were also detected. 4. Dual-labelling experiments with [3H]fucose and14C-labelled amino acids indicated that the major fucosylated glycoprotein was synthesized de novo by cultured fibroblasts. The non-collagenous nature of this glycoprotein was established by three independent methods. 5. Gel-filtration analysis before and after reduction with dithiothreitol showed that the major glycoprotein occurs as a disulphide-bonded dimer when analysed under denaturing conditions. Further experiments demonstrated that this glycoprotein was the predominant labelled species released into the medium when fibroblasts were incubated with [35S]cysteine. 6. The relationship between the major fucosylated glycoprotein and a glycoprotein, or group of glycoproteins, variously known as fibronectin, LETS protein, cell-surface protein etc., is discussed.

1. The glycosylation of hydroxylysine during the biosynthesis of procollagen by embryonic chick tendon and cartilage cells was examined. When free and membrane-bound ribosomes isolated from cells labelled for 4min with [14C]lysine were assayed for hydroxy[14C]lysine and hydroxy[14C]lysine glycosides, it was found that hydroxylation took place only on membrane-bound ribosomes and that some synthesis of galactosylhydroxy[14C]lysine and glucosylgalactosylhydroxy[14C]lysine had occurred on the nascent peptides. 2. Assays of subcellular fractions isolated from tendon and cartilage cells labelled for 2h with [14C]lysine demonstrated that the glycosylation of procollagen polypeptides began in the rough endoplasmic reticulum. 14C-labelled polypeptides present in the smooth endoplasmic reticulum and Golgi fractions were glycosylated to extents almost identical with the respective secreted procollagens. 3. Assays specific for collagen galactosyltransferase and collagen glucosyltransferase are described, using as substrate chemically treated bovine anterior-lens-capsule collagen. 4. When homogenates were assayed for the collagen glycosyltransferase activities, addition of Triton X-100 (0.01%, w/v) was found to stimulate enzyme activities by up to 45%, suggesting that the enzymes were probably membrane-bound. 5. Assays of subcellular fractions obtained by differential centrifugation for collagen galactosyltransferase activity indicated the specific activity to be highest in the microsomal fractions. Similar results were obtained for collagen glucosyltransferase activity. 6. When submicrosomal fractions obtained by discontinuous-sucrose-density-gradient-centrifugation procedures were assayed for these enzymic activities, the collagen galactosyltransferase was found to be distributed in the approximate ratio 7:3 between rough and smooth endoplasmic reticulum of both cell types. Similar determinations of collagen glucosyltransferase indicated a distribution in the approximate ratio 3:2 between rough and smooth microsomal fractions. 7. Assays of subcellular fractions for the plasma-membrane marker 5′-nucleotidase revealed a distribution markedly different from the distributions obtained for the collagen glycosyltransferase. 8. The studies described here demonstrate that glycosylation occurs early in the intracellular processing of procollagen polypeptides rather than at the plasma membrane, as was previously suggested.

Embryonic-chick tendon poly(A)-containing RNA was translated in the wheat-germ and mRNA-dependent rabbit reticulocyte-lysate systems. The ability of each system to synthesize polypeptides similar to pro-α chains of collagen was tested on the bases of electrophoretic mobility and susceptibility to highly purified bacterial collagenase. Very small amounts of polypeptides in the size range of pro-α chains were synthesized in the wheat-germ system, whereas efficient synthesis of two polypeptides similar to pro-α1 and pro-α2 chains was achieved in the reticulocyte lysate. The collagenous nature of the major high-molecular-weight products synthesized was demonstrated by their susceptibility to collagenase and ability to act as a substrate for purified collagen proline hydroxylase. Determinations of the relative amounts of these translation products suggest that the 2:1 ratio of pro-α1 and pro-α2 chains found in type I procollagen is reflected in proportional amounts of translatable mRNA for pro-α1 and pro-α2 chains. Comparisons of the electrophoretic mobilities of hydroxylated and unhydroxylated reticulocyte-lysate translation products were made with appropriate standards of hydroxylated and unhydroxylated procollagen polypeptides. The results suggest that, in common with a number of secreted proteins, procollagen is synthesized as pre-pro molecules consistent with the ‘Signal Hypothesis’.

Cells were isolated from the major arteries of 17-day chick embryos by digestion of the tissue with collagenase and trypsin. The cells, when examined immediately after isolation, exhibited a high degree of viability and they were shown to synthesize and secrete procollagen at a high and constant rate for several hours when incubated in suspension in modified Krebs medium. Continuous labelling of the cells with [14C]proline demonstrated a lag of about 30min between the time at which the synthesis of non-diffusible peptide-bound hydroxy[14C]proline became linear and the time at which its secretion into the medium became linear. This lag time compares with that of 18min observed for freshly isolated matrix-free cells from embryonic-chick tendon, which synthesize and secrete the same type of collagen. Gel-filtration chromatography and polyacrylamide-gel electrophoresis indicated that the collagenous polypeptides secreted into the medium were in the precursor form, known as procollagen, and that the constituent pro-α-chains were linked by interchain disulphide bonds and were also in a triple-helical conformation. Characterization of the secreted procollagen by gel-filtration chromatography, polyacrylamide-gel electrophoresis, DEAE-agarose chromatography, and polyacrylamide-gel electrophoresis of peptides obtained by CNBr cleavage, indicated that the predominant form was type-I procollagen. This work extends the range of freshly isolated matrix-free cell systems, which have been characterized for use in studies on the biosynthesis and secretion of procollagen, and it indicates differences in the rates of secretion of procollagen in different cell types secreting the same type of procollagen.

1. Subcellular fractions of freshly isolated matrix-free embryonic chick tendon and sternal cartilage cells have been characterized by chemical analysis, electron microscopy and the location of specific marker enzymes. These data indicate the fractions to be of a high degree of purity comparable with those obtained from other tissues, e.g. liver and kidney. 2. When homogenates were assayed for protocollagen prolyl hydroxylase and protocollagen lysyl hydroxylase activities, addition of Triton X-100 (0.1%, w/v) was found to stimulate enzyme activities by up to 60% suggesting that the enzymes were probably membrane-bound. 3. Assay of subcellular fractions obtained by differential centrifugation for protocollagen prolyl hydroxylase activity indicated the specific activity to be highest in the microsomal fraction. Similar results were obtained for protocollagen lysyl hydroxylase activity. 4. Submicrosomal fractions obtained by discontinuous sucrose-gradient centrifugation were assayed for the two enzymes and protocollagen prolyl hydroxylase and protocollagen lysyl hydroxylase were found to be associated almost exclusively with the rough endoplasmic reticulum fraction in both tendon and cartilage cells.

Scorbutic guinea pigs were wounded and the influence of administering ascorbic acid 6 days later was studied with respect to cellular morphology, ribosomal distribution and protein synthesis. Electron-microscopic studies revealed that the dilated endoplasmic reticulum observed in the fibroblasts of scorbutic wound tissue had reverted to a normal configuration 24h after intraperitoneal injection of 100mg of ascorbate. Quantitative determination of the distribution of free and membrane-bound ribosomes indicated a significant increase in membrane-bound ribosomes in wound tissue from ascorbate-supplemented (recovery) animals. Sucrose-density-gradient centrifugation indicated a significant increase in the proportion of large membrane-bound polyribosomes in the range 300–350S and a concomitant decrease in 80S monoribosomes in the ribosome sedimentation profile of recovery tissue. Determination of the synthesis of non-diffusible [3H]hydroxyproline in scorbutic and recovery wounds showed a 3–4-fold stimulation in peptidyl-proline hydroxylation in recovery tissues. Studies carried out in which scorbutic and recovery tissues were incubated with [14C]leucine indicated that general protein synthesis, as measured by14C incorporated into non-diffusible material/μg of DNA, was unaltered by ascorbate supplementation. Similar studies of [3H]proline incorporation suggested that in recovery tissues there was a small but significant increase in [3H]proline incorporated/μg of DNA, which probably represents an increase in protocollagen synthesis. This observation correlates well with the increase seen in recovery tissues of large polyribosomes on which collagen precursor polypeptides are known to be synthesized. Preliminary characterization of the repair collagen synthesized by recovery animals showed it to be a typical Type I collagen having the chain composition (α1)2α2. The extent of glycosylation of the hydroxylysine of the newly synthesized collagen was greater than that reported for either normal guinea-pig dermal collagen or dermal scar collagen.

This investigation has sought to determine the significance of the wide range of extractable collagen fractions which appear to exist in growing connective tissues and to determine their position in the process of fibrogenesis. Carrageenin granulomata were induced in guinea pigs and, after injection of 14C-glycine, this tissue and skin from the same animal were subjected to successive extractions with neutral salt solutions of increasing ionic strength, citrate buffer pH 3.6, and to gelatinization. The specific activity of these fractions was determined at various time intervals. At 8 hours it was found that the specific activity decreased with increasing ionic strength of the neutral salts and was still lower in the citrate extracts and gelatin. At 36 hours the situation was almost completely reversed except that the citrate extract and gelatin still had the lowest activities. The data from skin were more clear cut than that from the granuloma and the reasons for this are discussed. It is concluded that at any given time in developing connective tissue, there is a continuous spectrum of collagen aggregates of varying degrees of strength of cross-linkage, dependent upon the time that has elapsed since their constituent molecules were synthesized. The various extraction media used remove a particular cross-section of these aggregates depending upon their disaggregating power. These extracts will thus be biologically heterogeneous. The fraction extracted with 0.14 M NaCl will contain the collagen molecules most recently synthesized and in this respect can be considered the earliest form of extracellular collagen.