Two loci control phytoglycogen production in the monocellular green alga Chlamydomonas reinhardtii.

The STA8 locus of Chlamydomonas reinhardtii was identified in a genetic screen as a factor that controls starch biosynthesis. Mutations of STA8 cause a significant reduction in the amount of granular starch produced during nutrient limitation and accumulate phytoglycogen. The granules remaining in sta8 mutants are misshapen, and the abundance of amylose and long chains in amylopectin is altered. Mutations of the STA7 locus, which completely lack isoamylase activity, also cause accumulation of phytoglycogen, although sta8 and sta7 mutants differ in that there is a complete loss of granular starch in the latter. This is the first instance in which mutations of two different genetic elements in one plant species have been shown to cause phytoglycogen accumulation. An analytical procedure that allows assay of isoamylase in total extracts was developed and used to show that sta8 mutations cause a 65% reduction in the level of this activity. All other enzymes known to be involved in starch biosynthesis were shown to be unaffected in sta8 mutants. The same amount of total isoamylase activity (approximately) as that present in sta8 mutants was observed in heterozygous triploids containing two sta7 mutant alleles and one wild-type allele. This strain, however, accumulates normal levels of starch granules and lacks phytoglycogen. The total level of isoamylase activity, therefore, is not the major determinant of whether granule production is reduced and phytoglycogen accumulates. Instead, a qualitative property of the isoamylase that is affected by the sta8 mutation is likely to be the critical factor in phytoglycogen production.

Novel, starch-like polysaccharides are synthesized by an unbound form of granule-bound starch synthase in glycogen-accumulating mutants of Chlamydomonas reinhardtii.

In vascular plants, mutations leading to a defect in debranching enzyme lead to the simultaneous synthesis of glycogen-like material and normal starch. In Chlamydomonas reinhardtii comparable defects lead to the replacement of starch by phytoglycogen. Therefore, debranching was proposed to define a mandatory step for starch biosynthesis. We now report the characterization of small amounts of an insoluble, amylose-like material found in the mutant algae. This novel, starch-like material was shown to be entirely dependent on the presence of granule-bound starch synthase (GBSSI), the enzyme responsible for amylose synthesis in plants. However, enzyme activity assays, solubilization of proteins from the granule, and western blots all failed to detect GBSSI within the insoluble polysaccharide matrix. The glycogen-like polysaccharides produced in the absence of GBSSI were proved to be qualitatively and quantitatively identical to those produced in its presence. Therefore, we propose that GBSSI requires the presence of crystalline amylopectin for granule binding and that the synthesis of amylose-like material can proceed at low levels without the binding of GBSSI to the polysaccharide matrix. Our results confirm that amylopectin synthesis is completely blocked in debranching-enzyme-defective mutants of C. reinhardtii.

Physical and chemical transformations of cereal food during oral digestion in human subjects.

Chemical and physical transformations of solid food begin in the mouth, but the oral phase of digestion has rarely been studied. In the present study, twelve healthy volunteers masticated mouthfuls of either bread or spaghetti for a physiologically-determined time, and the levels of particle degradation and starch digestion before swallowing were compared for each food. The amounts of saliva moistening bread and spaghetti before swallowing were, respectively, 220 (SEM 12) v. 39 (SEM 6) g/kg fresh matter. Particle size reduction also differed since bread particles were highly degraded, showing a loss of structure, whereas spaghetti retained its physical structure, with rough and incomplete reduction of particle size. Starch hydrolysis was twice as high for bread as for spaghetti, mainly because of the release of high-molecular-mass alpha-glucans. The production of oligosaccharides was similar after mastication of the two foods, respectively 125 (SEM 8) and 92 (SEM 7) g/kg total starch. Starch hydrolysis, which clearly began in the mouth, depended on the initial structure of the food, as in the breakdown of solid food. These significant physical and chemical degradations of solid foods during oral digestion may influence the entire digestive process.

Starches from A to C. Chlamydomonas reinhardtii as a model microbial system to investigate the biosynthesis of the plant amylopectin crystal.

Wide-angle powder x-ray diffraction analysis was carried out on starch extracted from wild-type and mutant Chlamydomonas reinhardtii cells. Strains containing no defective starch synthases as well as mutants carrying a disrupted granule-bound starch synthase structural gene displayed the A type of diffraction pattern with a high degree of crystallinity. Mutants carrying a defect for the major soluble starch synthase (SSS), SSS II, were characterized by a switch to the B type of diffraction pattern with very low crystallinity. Mutant strains carrying SSS I as the only glucan elongation enzyme regained some of their crystallinity but switched to the C type of diffraction pattern. Differential scanning calorimetry analysis correlated tightly with the x-ray diffraction results. Together with the electron microscopy analyses, these results establish C. reinhardtii as a microbial model system displaying all aspects of cereal starch synthesis and structure. We further show that SSS II is the major enzyme involved in the synthesis of crystalline structures in starch and demonstrate that SSS I alone builds a new type of amylopectin structure.

Chemometric labeling of cereal tissues in multichannel fluorescence microscopy images using discriminant analysis.

This paper presents a novel, semiautomatic method for microscopic identification of multicomponent samples, which allows the identification, location, and percentage quantity of each component to be determined. The method involves applying discriminant analysis to a sequence of multichannel fluorescence microscopy images via a supervised learning approach; by selecting groups of pixels that are representative for each component type in a "known" sample, a computer is "taught" how to recognize the behavior (i.e., fluorescence emission) of the various components when illuminated under different spectral conditions. The identity, quantity, and location of these components in "unknown" samples (i.e., samples with the same component types but in different ratios or distributions) can then be investigated. The technique therefore enables semiautomatic quantitative fluorescence microscopy and has potential as a quality control tool. This work demonstrates the application of the technique to artificial and natural samples and critically discusses its quality, potential, and limitations.

Banana starch breakdown in the human small intestine studied by electron microscopy.

OBJECTIVE: To determine the origin of the poor digestibility of banana starch granules in the human small intestine. DESIGN: The subjects received the same experimental meal. SETTING: Nutrition Research Unit, Laënnec Hospital, CHU, Nantes. SUBJECTS: Six healthy young subjects. INTERVENTIONS: The digestion of raw green banana flour in the upper part of the gut was studied by the intubation technique. After ingestion of 30 g banana flour mixed with a complex meal, ileal samples were continuously collected during 14 h. In order to determine the structural nature of this resistant starch, the dried ileal samples were observed with scanning and transmission electron microscopy. Transmission electron microscopy was performed after treatment with periodic acid-thiosemicarbazide-silver nitrate. RESULTS: Banana starch proved very resistant to in vivo amylase hydrolysis since 84% of the starch ingested reached the terminal ileum. The microscopic observations showed that raw banana flour contained irregularly shaped dense starch granules with smooth surfaces. After their passage through the small intestine, starch granules appeared exocorroded, with porous surfaces, and some exhibited several irregular pits, crevices or holes by which the enzymes had penetrated and hydrolysed the inner part. Cell walls closely associated with starch granules could have hindered enzyme access to starch. CONCLUSIONS: Encapsulation could be partly responsible for the low digestibility of starch in banana flour, together with the intrinsic resistance of banana starch granules.

A role for sigma binding in the antipsychotic profile of BMY 14802?

BMY 14802 was identified as a potential antipsychotic drug in traditional model systems, and this identification was confirmed in modern behavioral and electrophysiological systems. The drug appears to be atypical as an antipsychotic in its lack of activity in models predictive of the potential to produce extrapyramidal side effects and tardive dyskinesia. Indeed, this suggestion is corroborated by clinical findings to date. The atypical profile of BMY 14802 extends to its neurochemical actions and appears to find its basis in regionally selective, indirect modulation of the dopamine system. Furthermore, BMY 14802 exhibits interactions with sigma binding sites in vitro and in vivo, a notion supported by data from neurophysiological, behavioral, and biochemical investigations. BMY 14802 also appears to be neuroprotective in some model systems and may have utility in the treatment of stroke (Boissard et al. 1991). BMY 14802 appears to interact with 5-HT1A receptors, but this interaction does not seem to contribute significantly to the potential antipsychotic actions of the drug. Moreover, the formation of active metabolites of BMY 14802 does not appear to occur in animals or humans to an extent of physiological or behavioral relevance. If clinically efficacious, BMY 14802 may treat the symptoms of schizophrenia by a mechanism novel for antipsychotic drugs: regionally selective, indirect modulation of dopaminergic systems by specific interaction at sigma sites.

Physical characteristics of starch granules and susceptibility to enzymatic degradation.

Starch, the most abundant component of the diet, is characterized by its variety as well as the versatility of its derivatives in foods. This paper is an overview of the main physical characteristics of the native starch granule. Three different levels of organization are presented: macromolecular structure, crystalline organization and ultrastructure. Starch consists of amylose and amylopectin. Amylose is an essentially linear polymer composed of alpha-1,4-linked D-anhydroglucose units (AGU); amylopectin is a branched polymer clustering a large amount of short linear chains by the linkage of alpha-1,6-bonds, constituting about 5% of the total glycosidic bonds. In the native starch granules, a large number of the macromolecular chains are organized in crystalline structures. Three forms have been found, the A, B and C patterns. So far only A and B starch crystals have been modelled. There is a variation in the susceptibility of the starch granules to enzymatic digestion. This is explained by variation in the morphology of the granules and their crystalline organization.

The influence of microflora on the breakdown of maize starch granules in the digestive tract of chicken.

Influence of the microflora on the extent of degradation of starch in the chicken digestive tract has been studied by scanning electron microscopy. Axenic (germfree), holoxenic (conventional), and monoxenic (only one germ) chickens inoculated with two strains of lactobacillus (strains 220 and 207) isolated from the crop of the cock were used in these experiments. In vitro starch granules were less hydrolyzed by strain 207 than by strain 220. A more extensive degradation was observed when starch was included in feed. This breakdown resulted in formation of cavities and pin holes in the starch granules. In vivo starch granules in the crop of holoxenic and monoxenic chickens were scarcely damaged and erosion was always superficial. In monoxenic 207 chicken as in holoxenic ones, bacteria were fixed on the crop wall. In feces, the pattern of the breakdown was dependent on the type of animal. These results demonstrated the role of the total microflora in the breakdown of starch and the specific intervention of the two strains of lactobacilli.