Development and characterization of a new rice cultivar, 'Chikushi-kona 85', derived from a starch-branching enzyme IIb-deficient mutant line.

A new super-hard rice cultivar, 'Chikushi-kona 85', which was derived from a cross between 'Fukei 2032' and 'EM129', was developed via bulk method breeding. 'Chikushi-kona 85' showed a higher content of resistant starch than the normal non-glutinous rice cultivar, 'Nishihomare', and a higher grain yield than the first super-hard rice cultivar, 'EM10'. The amylopectin chain length of 'Chikushi-kona 85' and its progenitor line 'EM129' was longer than that of 'Nishihomare', and was similar to that of 'EM10'. This suggests that the starch property of 'Chikushi-kona 85' was inherited from 'EM129', which is a mutant line deficient in a starch branching enzyme similar to 'EM10'. Genetic analysis of 'Chikushi-kona 85' crossed with 'Nishihomare' also showed that the starch property of 'Chikushi-kona 85' was regulated by a single recessive gene. Consumption of processed cookies made from 'Chikushi-kona 85' flour showed a distinctive effect in controlling blood sugar levels in comparison to the normal non-glutinous rice cultivar 'Hinohikari'. These results show that 'Chikushi-kona 85' is a novel genetic source to develop new products made of rice, which could reduce calorie intake and contribute to additional health benefits.

Rice Endosperm Starch Phosphorylase (Pho1) Assembles with Disproportionating Enzyme (Dpe1) to Form a Protein Complex That Enhances Synthesis of Malto-oligosaccharides.

Starch synthesis in cereal grain endosperm is dependent on the concerted actions of many enzymes. The starch plastidial phosphorylase (Pho1) plays an important role in the initiation of starch synthesis and in the maturation of starch granule in developing rice seeds. Prior evidence has suggested that the rice enzyme, OsPho1, may have a physical/functional interaction with other starch biosynthetic enzymes. Pulldown experiments showed that OsPho1 as well as OsPho1 devoid of its L80 region, a peptide unique to higher plant phosphorylases, captures disproportionating enzyme (OsDpe1). Interaction of the latter enzyme form with OsDpe1 indicates that the putative regulatory L80 is not responsible for multienzyme assembly. This heterotypic enzyme complex, determined at a molar ratio of 1:1, was validated by reciprocal co-immunoprecipitation studies of native seed proteins and by co-elution chromatographic and co-migration electrophoretic patterns of these enzymes in rice seed extracts. The OsPho1-OsDpe1 complex utilized a broader range of substrates for enhanced synthesis of larger maltooligosaccharides than each individual enzyme and significantly elevated the substrate affinities of OsPho1 at 30 °C. Moreover, the assembly with OsDpe1 enables OsPho1 to utilize products of transglycosylation reactions involving G1 and G3, sugars that it cannot catalyze directly.

Development and evaluation of rice giant embryo mutants for high oil content originated from a high-yielding cultivar 'Mizuhochikara'.

Rice bran oil is a byproduct of the milling of rice (Oryza sativa L.). It offers various health benefits and has a beneficial fatty acid composition. To increase the amount of rice bran as a sink for triacylglycerol (TAG), we developed and characterized new breeding materials with giant embryos. To induce mutants, we treated fertilized egg cells of the high-yielding cultivar 'Mizuhochikara' with N-methyl-N-nitrosourea (MNU). By screening M2 seeds, we isolated four giant embryo mutant lines. Genetic analysis revealed that the causative loci in lines MGE12 and MGE13 were allelic to giant embryo (ge) on chromosome 7, and had base changes in the causal gene Os07g0603700. On the other hand, the causative loci in lines MGE8 and MGE14 were not allelic to ge, and both were newly mapped on chromosome 3. The TAG contents of all four mutant lines increased relative to their wild type, 'Mizuhochikara'. MGE13 was agronomically similar to 'Mizuhochikara' and would be useful for breeding for improved oil content.

Analysis of the Rice ADP-Glucose Transporter (OsBT1) Indicates the Presence of Regulatory Processes in the Amyloplast Stroma That Control ADP-Glucose Flux into Starch.

Previous studies showed that efforts to further elevate starch synthesis in rice (Oryza sativa) seeds overproducing ADP-glucose (ADPglc) were prevented by processes downstream of ADPglc synthesis. Here, we identified the major ADPglc transporter by studying the shrunken3 locus of the EM1093 rice line, which harbors a mutation in the BRITTLE1 (BT1) adenylate transporter (OsBt1) gene. Despite containing elevated ADPglc levels (approximately 10-fold) compared with the wild-type, EM1093 grains are small and shriveled due to the reduction in the amounts and size of starch granules. Increases in ADPglc levels in EM1093 were due to their poor uptake of ADP-[(14)C]glc by amyloplasts. To assess the potential role of BT1 as a rate-determining step in starch biosynthesis, the maize ZmBt1 gene was overexpressed in the wild-type and the GlgC (CS8) transgenic line expressing a bacterial glgC-TM gene. ADPglc transport assays indicated that transgenic lines expressing ZmBT1 alone or combined with GlgC exhibited higher rates of transport (approximately 2-fold), with the GlgC (CS8) and GlgC/ZmBT1 (CS8/AT5) lines showing elevated ADPglc levels in amyloplasts. These increases, however, did not lead to further enhancement in seed weights even when these plant lines were grown under elevated CO2. Overall, our results indicate that rice lines with enhanced ADPglc synthesis and import into amyloplasts reveal additional barriers within the stroma that restrict maximum carbon flow into starch.

The plastidial starch phosphorylase from rice endosperm: catalytic properties at low temperature.

MAIN CONCLUSION: Consistent with its essential role in starch biosynthesis at low temperatures, the plastidial starch phosphorylase from rice endosperm is highly active at low temperature. Moreover, contrary to results on other higher plant phosphorylases, the L80 peptide, a domain unique to plant phosphorylases and not present in orthologous phosphorylases from other organisms, is not involved in enzyme catalysis. Starch phosphorylase (Pho) is an essential enzyme in starch synthesis in developing rice endosperm as the enzyme plays a critical role in both the early and maturation phases of starch granule formation especially at low temperature. In this study, we demonstrated that the rice Pho1 maintains substantial enzyme activity at low temperature (<20 °C) and its substrate affinities for branched α-glucans and glucose-1-phosphate were significantly increased at the lower reaction temperatures. Under sub-saturating substrate conditions, OsPho1 displayed higher catalytic activities at 18 °C than at optimal 36 °C, supporting the prominent role of the enzyme in starch synthesis at low temperature. Removal of the highly charged 80-amino acid sequence L80 peptide, a region found exclusively in the plastidial Pho1 of higher plants, did not significantly alter the catalytic and regulatory properties of OsPho1 but did affect heat stability. Our kinetic results support the low temperature biosynthetic role of OsPho1 in rice endosperm and indicate that its L80 region is unlikely to have a direct enzymatic role but provides stability of the enzyme under heat stress.

Development of formulae for estimating amylose content, amylopectin chain length distribution, and resistant starch content based on the iodine absorption curve of rice starch.

Not only amylose but also amylopectin greatly affects the gelatinization properties of rice starch and the quality of cooked rice grains. We here characterized the starches of 32 rice cultivars and evaluated the relationship between their iodine absorption curve, apparent amylose content (AAC), pasting property, resistant starch (RS) content, and chain length distribution of amylopectin. We found that the iodine absorption curve differed among the various sample rice cultivars. Using the wavelength at which absorbance becomes maximum on iodine staining of starch (λmax), we propose a novel index, "new λmax" (AAC/(λmax of sample rice starches-λmax of glutinous rice starch)). We developed the novel estimation formulae for AAC, RS contents, and amylopectin fractions with the use of λmax and "new λmax." These formulae would lead to the improved method for estimating starch properties using an easy and rapid iodine colorimetric method.

Influence of single-nucleotide polymorphisms in the gene encoding granule-bound starch synthase I on amylose content in Vietnamese rice cultivars.

Amylose content is one of the most important factors influencing the physical and chemical properties of starch in rice. Analysis of 352 Vietnamese rice cultivars revealed a wide range of variation in apparent amylose content and the expression level of granule-bound starch synthase. On the basis of single-nucleotide polymorphisms (SNP) at the splicing donor site of the first intron and in the coding region of the granule-bound starch synthase I gene, Waxy gene, alleles can be classified into seven groups that reflect differences in apparent amylose content. The very low and low apparent amylose content levels were tightly associated with a G to T in the first intron whereas intermediate and high amylose was associated with a T genotype at SNP in exon 10. The correlation between the combination of T genotype at SNP in the first intron, C in exon 6, or C in exon 10 was predominant among low amylose rice varieties. Our analysis confirmed the existence of Wx (op) allele in Vietnamese rice germplasm. The results of this study suggest that the low amylose properties of Vietnamese local rice germplasm are attributable to spontaneous mutations at exons, and not at the splicing donor site.

The rice endosperm ADP-glucose pyrophosphorylase large subunit is essential for optimal catalysis and allosteric regulation of the heterotetrameric enzyme.

Although an alternative pathway has been suggested, the prevailing view is that starch synthesis in cereal endosperm is controlled by the activity of the cytosolic isoform of ADPglucose pyrophosphorylase (AGPase). In rice, the cytosolic AGPase isoform is encoded by the OsAGPS2b and OsAGPL2 genes, which code for the small (S2b) and large (L2) subunits of the heterotetrameric enzyme, respectively. In this study, we isolated several allelic missense and nonsense OsAGPL2 mutants by N-methyl-N-nitrosourea (MNU) treatment of fertilized egg cells and by TILLING (Targeting Induced Local Lesions in Genomes). Interestingly, seeds from three of the missense mutants (two containing T139I and A171V) were severely shriveled and had seed weight and starch content comparable with the shriveled seeds from OsAGPL2 null mutants. Results from kinetic analysis of the purified recombinant enzymes revealed that the catalytic and allosteric regulatory properties of these mutant enzymes were significantly impaired. The missense heterotetramer enzymes and the S2b homotetramer had lower specific (catalytic) activities and affinities for the activator 3-phosphoglycerate (3-PGA). The missense heterotetramer enzymes showed more sensitivity to inhibition by the inhibitor inorganic phosphate (Pi) than the wild-type AGPase, while the S2b homotetramer was profoundly tolerant to Pi inhibition. Thus, our results provide definitive evidence that starch biosynthesis during rice endosperm development is controlled predominantly by the catalytic activity of the cytoplasmic AGPase and its allosteric regulation by the effectors. Moreover, our results show that the L2 subunit is essential for both catalysis and allosteric regulatory properties of the heterotetramer enzyme.

Improved palatability and bio-functionality of super-hard rice by soaking in a barley-koji miso suspension.

Cooked grains of ae rice cultivars are too hard and non-sticky due to the presence of long-chain amylopectin, and ae rice cultivars are therefore called ``super-hard rice'' and cannot be used as table rice. However, they are promising in terms of their bio-functionality such as preventing diabetes. Miso (soybean paste) is a yeast-fermented food, made from steamed soybeans, salt, and inoculated cereals known as koji, made from rice, barley, or soybeans.We investigated the effects of soaking ae mutant rice cultivars in a miso suspension. Their chemical components, physical properties, and enzyme activities were measured under different conditions (milled rice before or after soaking in a 5% barley-koji miso suspension). Rice grains cooked after soaking in the miso suspension were less hard and more sticky than those cooked after soaking in water. Rice grains cooked after soaking in a 5% barley-koji miso suspension maintained high amounts of resistant starch and dietary fiber, and were fortified with polyphenols and isoflavones. Palatable and bio-functional ae rice could therefore be produced by cooking after soaking in a 5% barley-koji miso suspension.

Hepatic steatosis with relation to increased expression of peroxisome proliferator-activated receptor-γ in insulin resistant mice.

We have isolated insulin resistant mice (ddY-H mice) which are spontaneously induced even if fed with the standard chow pellets. Since marked accumulation of triglycerides (TG) in liver was observed, the present study investigated causes of hepatic TG accumulation in ddY-H mice fed with the standard chow pellets. In ddY-H mice, hepatic TG content increased from seven-weeks of age, and further marked accumulation of TG was observed at 20-weeks of age. Histologically, fat droplets appeared in pericentral parenchymal cells of the liver from nine-weeks of age, and the size and number of droplets were increased in hepatic lobules at 15-weeks of age, suggesting hepatic steatosis was spontaneously induced. Although secretion of TG from liver to blood in ddY-H mice was not increased, fat absorption from the digestive tract was significantly enhanced. The mRNA expressions of peroxisome proliferator-activated receptor γ (PPARγ) involved in fat accumulation and fatty acid translocase (CD36) involved in the transportation of fatty acid into the liver were markedly increased. However, gene expressions of factors involved in lipogenesis, ß-oxidation of fatty acid and lipoprotein secretion were not changed. Pioglitazone (9 mg/kg), the PPARγ agonist, administered for six weeks deteriorated hepatic steatosis in ddY-H mice. Although pioglitazone did not affect gene expressions of PPARγ in the liver, CD36 and fat-specific protein 27 (fsp27), targets of PPARγ, were markedly elevated. These results suggest that, in the livers of ddY-H mice, hepatic steatosis is induced by increased incorporation of fatty acid into the liver via increased PPARγ expression.

Wx/ae double-mutant brown rice prevents the rise in plasma lipid and glucose levels in mice.

A wx/ae double-mutant rice is generated by crossing waxy mutant and amylose-extender mutant in rice. Wx/ae brown rice contains highly beneficial nutrients for lipid and glucose metabolism, including resistant starch, dietary fiber, and γ-oryzanol, when compared to Koshihikari brown rice, the non-waxy japonica cultivar. To examine the effects of wx/ae brown rice on glucose and lipid metabolism, type 2 diabetic NSY/Hos mice were fed a high-fat diet containing 25% of wx/ae brown rice or Koshihikari brown rice for 10 weeks. The plasma total cholesterol, non-high-density lipoprotein cholesterol, triglyceride, and non-esterified fatty acid levels of the wx/ae group were significantly lower than those of the Koshihikari group. Moreover, the fasting blood glucose level and pathological score of glycosuria of the wx/ae group were also significantly lower than those of the Koshihikari group. These results indicate that wx/ae brown rice has the potential to prevent the rise in plasma lipid and glucose levels.

The OsGEN-L protein from Oryza sativa possesses Holliday junction resolvase activity as well as 5'-flap endonuclease activity.

OsGEN-L has a 5'-flap endonuclease activity and plays an essential role in rice microspore development. The Class 4 RAD2/XPG family nucleases, including OsGEN-L, were recently found to have resolving activity for the Holliday junction (HJ), the intermediate of DNA strand recombination. In this study, we performed a detailed characterization of OsGEN-L, as a structure-specific endonuclease. Highly purified OsGEN-L was prepared as the full-length protein for in vitro endonuclease assays using various structured DNAs, and the 5'-flap endonuclease activity, which is stimulated in a PCNA-dependent manner, was demonstrated. In addition, the in vitro HJ resolving activity of OsGEN-L represents the first such activity originating from plant cells. OsGEN-L cleaved HJ at symmetrically related sites of the branch point. However, the two branched strands seemed to be cleaved individually, and not cooperatively, by each OsGEN-L monomer protein. The substrate specificity suggests that OsGEN-L functions in multiple processes of DNA metabolism in rice cells.

RNA targeting to a specific ER sub-domain is required for efficient transport and packaging of α-globulins to the protein storage vacuole in developing rice endosperm.

Studies focusing on the targeting of RNAs that encode rice storage proteins, prolamines and glutelins to specific sub-domains of the endoplasmic reticulum (ER), as well as mis-localization studies of other storage protein RNAs, indicate a close relationship between the ER site of RNA translation and the final site of protein deposition in the endomembrane system in developing rice endosperm. In addition to prolamine and glutelin, rice accumulates smaller amounts of α-globulins, which are deposited together with glutelin in the protein storage vacuole (PSV). In situ RT-PCR analysis revealed that α-globulin RNAs are not distributed to the cisternal ER as expected for a PSV-localized protein, but instead are targeted to the protein body-ER (PB-ER) by a regulated process requiring cis-sorting sequences. Sequence alignments with putative maize δ-zein cis-localization elements identified several candidate regulatory sequences that may be responsible for PB-ER targeting. Immunocytochemical analysis confirmed the presence of α-globulin on the periphery of the prolamine protein bodies and packaging in Golgi-associated dense vesicles, as well as deposition and storage within peripheral regions of the PSV. Mis-targeting of α-globulin RNAs to the cisternal ER dramatically alters the spatial arrangement of α-globulin and glutelin within the PSV, with the accompanying presence of numerous small α-globulin particles in the cytoplasm. These results indicate that α-globulin RNA targeting to the PB-ER sub-domain is essential for efficient transport of α-globulins to the PSV and its spatial arrangement in the PSV. Such RNA localization prevents potential deleterious protein-protein interactions, in addition to performing a role in protein targeting.

Characteristics of pregelatinized ae mutant rice flours prepared by boiling after preroasting.

As ae mutant rice, such as EM10, lacks the starch branching enzyme IIb, its amylopectin contains more long-chain glucans than that of ordinary Indica and Japonica rice grains. Although boiled grains of ae rice cultivars are too hard and nonsticky for table rice, they are promising in terms of biofunctionality, such as prevention of diabetes. The present paper investigates the characterization of a novel group of four ae mutant rice cultivars (EM72, EM145, EM174, and EM189). They were subjected to the evaluation for their main chemical components, physical properties, and enzyme activities at different grain conditions (raw milled rice, roasted rice, boiled rice, and rice boiled after preroasting). These mutant rice grains are characterized by high apparent amylose, high protein and high glucose contents, high pasting temperature, high α-amylase activities, high resistant starch, and low degree of gelatinization. A novel method was developed to maintain the high resistant starch contents of gelatinized rice grains. Rice boild after preroasting showed a higher ratio of resistant starch and a lower amount of glucose than ordinary boiled rice. It became possible to produce high-quality and biofunctional pregelatinized rice flours by boiling with frozen fruits, such as tomatoes, after rice grains had been preroasted. These ae mutants were found to be suitable materials for rice/fruit or rice/vegetable products to serve as palatable, low-glucose, and high resistant starch rice products.

Genetic analysis of cysteine-poor prolamin polypeptides reduced in the endosperm of the rice esp1 mutant.

The esp1 mutant CM21 specifically exhibits reduced levels of cysteine-poor (CysP) prolamin bands with pIs of 6.65, 6.95, 7.10, and 7.35 in rice seed. Matrix-assisted laser desorption-ionization time-of-flight mass spectrometry (MALDI-TOF MS) analysis demonstrated that the bands with pIs 6.65, 6.95, and 7.35 are encoded by different structural genes. These results suggest that the Esp1 locus encodes a regulatory factor involved in the synthesis and/or accumulation of CysP prolamin molecules. Isoelectric focusing (IEF) analysis of CysP prolamins in chromosome substitution lines showed that structural genes for bands with pI values of 6.95, 7.10, and 7.35, which are reduced in esp1 mutant lines, are located as a gene cluster in the 44.2 cM region on chromosome 5.

Crystal structure of the branching enzyme I (BEI) from Oryza sativa L with implications for catalysis and substrate binding.

Starch-branching enzyme catalyzes the cleavage of α-1, 4-linkages and the subsequent transfer of α-1,4 glucan to form an α-1,6 branch point in amylopectin. Sequence analysis of the rice-branching enzyme I (BEI) indicated a modular structure in which the central α-amylase domain is flanked on each side by the N-terminal carbohydrate-binding module 48 and the α-amylase C-domain. We determined the crystal structure of BEI at a resolution of 1.9 Å by molecular replacement using the Escherichia coli glycogen BE as a search model. Despite three modular structures, BEI is roughly ellipsoidal in shape with two globular domains that form a prominent groove which is proposed to serve as the α-polyglucan-binding site. Amino acid residues Asp344 and Glu399, which are postulated to play an essential role in catalysis as a nucleophile and a general acid/base, respectively, are located at a central cleft in the groove. Moreover, structural comparison revealed that in BEI, extended loop structures cause a narrowing of the substrate-binding site, whereas shortened loop structures make a larger space at the corresponding subsite in the Klebsiella pneumoniae pullulanase. This structural difference might be attributed to distinct catalytic reactions, transglycosylation and hydrolysis, respectively, by BEI and pullulanase.

A novel factor FLOURY ENDOSPERM2 is involved in regulation of rice grain size and starch quality.

Rice (Oryza sativa) endosperm accumulates a massive amount of storage starch and storage proteins during seed development. However, little is known about the regulatory system involved in the production of storage substances. The rice flo2 mutation resulted in reduced grain size and starch quality. Map-based cloning identified FLOURY ENDOSPERM2 (FLO2), a member of a novel gene family conserved in plants, as the gene responsible for the rice flo2 mutation. FLO2 harbors a tetratricopeptide repeat motif, considered to mediate a protein-protein interactions. FLO2 was abundantly expressed in developing seeds coincident with production of storage starch and protein, as well as in leaves, while abundant expression of its homologs was observed only in leaves. The flo2 mutation decreased expression of genes involved in production of storage starch and storage proteins in the endosperm. Differences between cultivars in their responsiveness of FLO2 expression during high-temperature stress indicated that FLO2 may be involved in heat tolerance during seed development. Overexpression of FLO2 enlarged the size of grains significantly. These results suggest that FLO2 plays a pivotal regulatory role in rice grain size and starch quality by affecting storage substance accumulation in the endosperm.

Protein disulfide isomerase like 1-1 participates in the maturation of proglutelin within the endoplasmic reticulum in rice endosperm.

The rice esp2 mutation was previously characterized by the abnormal accumulation of elevated levels of proglutelin and the absence of an endosperm-specific protein disulfide isomerase like (PDIL1-1). Here we show that Esp2 is the structural gene for PDIL1-1 and that this lumenal chaperone is asymmetrically distributed within the cortical endoplasmic reticulum (ER) and largely restricted to the cisternal ER. Temporal studies indicate that PDIL1-1 is essential for the maturation of proglutelin only when its rate of synthesis significantly exceeds its export from the ER, a condition resulting in its build up in the ER lumen and the induction of ER quality control processes which lower glutelin levels as well as those of the other storage proteins. As proglutelin is initially synthesized on the cisternal ER, its deposition within prolamine protein bodies in esp2 suggests that PDIL1-1 helps retain proglutelin in the cisternal ER lumen until it attains competence for ER export and, thereby, indirectly preventing heterotypic interactions with prolamine polypeptides.

Palatable and bio-functional wheat/rice products developed from pre-germinated brown rice of super-hard cultivar EM10.

It became possible to produce high-quality and bio-functional wheat/rice bread and wheat/rice noodles by blending, pre-germinated and cooked brown rice of a super-hard cultivar with wheat flour. Super-hard rice (SHR) is not suitable for table rice because of its low palatability. Nevertheless, it was found to be suitable as a blending material for bread-making or noodle-making due to its hard texture and high content of resistant starch. We developed a novel rapid germination method to improve the quality and to save the time needed for germination. By blending pre-germinated and cooked SHR (30% w/w on a dry basis) as a rice gel with wheat flour (70% w/w on a dry basis), the bread became very soft and any hardening after bread-making was markedly retarded. Similarly, blending pre-germinated and cooked SHR as cooked a rice gel with wheat flour gave high-quality noodles with a similar texture to that of durum semolina noodles. The resistant starch of the SHR-blended bread and noodles was also markedly increased. White waxy rice (9%) soaked and cooked with the pre-germinated brown rice of SHR (21%) produced a rice gel that was very useful as a material for bread-making and noodle-making by blending with wheat flour (70%) to prepare soft, tasty and bio-functional wheat/rice bread and wheat/rice noodles.

Structure, physical, and digestive properties of starch from wx ae double-mutant rice.

Amylopectin is the principal component of starch. The amylose extender (ae) gene encodes the starch-branching enzyme IIb, which is critical in determining the fine structure of endosperm starch. To determine the relationship between the fine structure of amylopectin and its physical properties, rice mutant lines defective in the ae function with altered fine structure of amylopectin and in combination with the waxy (wx) background were selected for comparative studies with primary wild-type and ae starches. The ae mutant endosperms accumulated a high amylose content starch with long amylopectin chains. The ae and wx ae starches showed no significant difference in the unit chain-length distribution of amylopectin and starch granule morphology. The wx ae starch displayed a higher pasting temperature and higher peak viscosity. The gelatinization peak temperatures of the wx, ae, and wx ae starches were 2.2, 13.1, and 17.1 degrees C higher, respectively, than that of the wild-type starch, and the wx ae starch showed a retrogradation peak with a shorter cooling period than that of ae starch. The raw ae and wx ae starches were almost indigestible by alpha-amylase in vitro. Rats fed the wx ae starch showed slowly increasing blood glucose at a lower level than the rats fed the wx or wild-type starch. These results indicate that the primary structure of the rice wx ae amylopectin with enriched long chains changes the granular structure of the starch, including its crystal structure, and results in resistance to in vitro or in vivo degradation.