Structural and functional characterization of band 3 from Southeast Asian ovalocytes.

To determine why deletion of the nine amino acids joining the membrane and cytoplasmic domains of band 3 from Southeast Asian ovalocytes (SAO) renders the erythrocytes rigid, we compared the structural and functional properties of SAO and normal band 3. Calorimetric data, inhibitor binding studies, and anion transport assays all reveal that the membrane-spanning domain of SAO band 3 is denatured, while proteolysis studies and circular dichroism spectroscopy suggest the mutant domain retains much secondary structure. It is concluded that the transmembrane helices of SAO band 3 are dissociated and randomized but not unfolded. The cytoplasmic domain of SAO band 3 was shown to be structurally and functionally normal based on (i) calorimetric properties, (ii) native conformational change, (iii) ability to form an intersubunit disulfide bond, (iv) affinity and capacity for binding ankyrin and protein 4.1, and (v) kinetics of association with ankyrin. However, both normal and mutant isoforms of band 3 in SAO cells were found to adhere nonspecifically to the spectrin skeleton. Further, when SAO cells were osmotically swollen, the detergent extractability of band 3 became normal. We propose that much of band 3 is nonspecifically entrapped in the spectrin network in SAO cells and that this nonspecific adhesion may be responsible for the rigidity of the SAO erythrocyte.     

Molecular demonstration of SLC4A1 gene deletion in two Mexican patients with Southeast Asian ovalocytosis

We describe the finding of two Mexican patients with a specific 27-bp deletion in the solute carrier family 4 gene (SLC4A1delta27) (also known as the band 3 gene found on chromosome 17q21-q22), characteristic of Southeast Asian ovalocytosis (SAO). The patients were asymptomatic, and the initial diagnosis was made by microscopic observation of the presence of typical stomatocytic ovalocytes. The gene deletion was confirmed by PCR and DNA sequencing. Both patients were heterozygous for the deletion. One patient is from Tabasco state, in southeastern Mexico, a malaria-endemic zone. The other patient is from Mexico City, which is not a malaria-endemic area. Their families have no non-Mexican ancestors and their previous generations were born in Mexico. Both patients carry the HLA-B*3501 subtype, characteristic of Amerindians and Asian populations. Familial and HLA data led us to conclude that these two patients are the first report of SLC4A1delta27 in Amerindians. The nucleotide analysis showing a perfect match sequence between Southeast Asian and Mexican patients suggests, but does not prove, that the Mexican gene is not a de novo mutation. Instead, this gene might be the result of migration of individuals with Asian ancestry into the Mexican gene pool. We are looking for other families with the mutation to detect, by HLA analysis, the ancient ethnic origin of these patients.     

Dominant-negative effect of Southeast Asian ovalocytosis anion exchanger 1 in compound heterozygous distal renal tubular acidosis

The human chloride/bicarbonate AE1 (anion exchanger) is a dimeric glycoprotein expressed in the red blood cell membrane,and expressed as an N-terminal (Delta1-65) truncated form, kAE1(kidney AE1), in the basolateral membrane of alpha-intercalated cells in the distal nephron. Mutations in AE1 can cause SAO (Southeast Asian ovalocytosis) or dRTA (distal renal tubular acidosis), an inherited kidney disease resulting in impaired acid secretion. The dominant SAO mutation (Delta400-408) that results in an inactive transporter and altered erythrocyte shape occurs in manydRTA families, but does not itself result in dRTA. Compound heterozygotes of four dRTA mutations (R602H, G701D, DeltaV850 and A858D) with SAO exhibit dRTA and abnormal red blood cell properties. Co-expression of kAE1 and kAE1 SAO with the dRTAmutantswas studied in polarized epithelial MDCK(Madin-Darbycanine kidney) cells. Like SAO, the G701D and DeltaV850 mutants were predominantly retained intracellularly, whereas the R602H and A858D mutants could traffic to the basolateral membrane. When co-expressed in transfected cells, kAE1 WT (wild-type)and kAE1 SAO could interact with the dRTA mutants. MDCK cells co-expressing kAE1 SAO with kAE1 WT, kAE1 R602Hor kAE1 A858D showed a decrease in cell-surface expression of the co-expressed proteins. When co-expressed, kAE1 WT colocalized with the kAE1 R602H, kAE1 G701D, kAE1 DeltaV850 and kAE1 A858D mutants at the basolateral membrane, whereaskAE1 SAO co-localized with kAE1 WT, kAE1 R602H, kAE1 G701D, kAE1 DeltaV850 and kAE1 A858D in MDCK cells. The decrease in cell-surface expression of the dRTAmutants as a result of the interaction with kAE1 SAO would account for the impaired expression of functional kAE1 at the basolateral membrane of alpha-intercalated cells, resulting in dRTA in compound heterozygous patients.     

Molecular modeling of the effects of mutant alleles on chalcone synthase protein structure

Chalcone synthase (CHS) catalyzes the first committed step in flavonoid biosynthesis, a major pathway of plant secondary metabolism. An allelic series for the Arabidopsis CHS locus, tt4, was previously characterized at the gene, protein, and end-product levels. In an effort to deduce the molecular basis for the observed phenotypes, homology models were generated for five of the tt4 proteins based on the crystal structure of CHS2 from Medicago. Molecular dynamics simulations provided insights into how even those substitutions that are not in close spatial proximity to key functional residues may still alter the architecture and dynamic movement of the enzyme, with dramatic effects on enzyme function. Simulations carried out at different temperatures pointed to optimized positioning of key residues in the active site or dimerization domain, rather than enhancement of overall structure, as underlying the higher activity of two temperature-sensitive variants at lower temperatures. Extending this type of analysis to account for protein–protein interactions may offer additional insights into the mechanisms by which single amino-acid substitutions can affect diverse aspects of protein function.Electronic Supplementary Material Supplementary material is available in the online version of this article at and is accessible for authorized users     

Population structure of the Southeast Asian river catfish Mystus nemurus

A total of 143 microsatellites were isolated from Mystus nemurus using a 5' anchored polymerase chain reaction technique or the random amplified hybridization microsatellite method, the first set of microsatellite markers developed for the Southeast Asian river catfish. Twenty polymorphic microsatellite loci were used as markers for population characterization of M. nemurus from six different geographical locations in Malaysia (Perak, Kedah, Johor, UPM, Sarawak and Terengganu). The number of alleles per locus ranged from 2 to 11 with 6.3 as the average number of alleles per locus. Characterization of the populations showed relatively high levels of genetic variation compared with previous studies using allozyme markers. The highest genetic similarity was found between Perak and Kedah, while the highest genetic distance was found between Terengganu and Kedah. The majority of clustering was in accordance with geographical locations and the histories of the populations. Microsatellite analysis indicated that the Sarawak population might be genetically closer to the Peninsular Malaysian populations than has been previously shown by other molecular marker studies.     

Molecular design of PhoE porin and its functional consequences

The three-dimensional structure of PhoE porin from Escherichia coli, negatively stained with uranyl acetate, has been determined by electron crystallographic techniques to a resolution of about 18 A. The structure shows that PhoE porin consists of trimeric stain-filled channels as the basic unit. The trimeric channels converge as they transverse the membrane but they do not merge. Our three-dimensional structure of PhoE porin indicates that there is a short, narrower segment of channel, which extends beyond the visible strain-filled portion of the channel. The map of glucose-embedded PhoE porin in projection normal to the membrane has also been determined to a resolution of 6.5 A. The projected map shows trimeric ring-like structures, which are presumably cylindrical domains of beta-sheet. At the 3-fold symmetry axis of the trimer, there is a low density region, which is suggested to be a site of lipopolysaccharide that is required for channel and bacteriophage receptor activities. The structural model of the PhoE monomer consists of a flattened cylinder with a large water-filled vestibule about 35 A long with an elliptically shaped opening that is 27 A along the major axis and 18 A along the minor axis. The vestibule has a narrower extension about 10 A long with an average diameter of about 10 A. The vestibule wall is formed by beta-sheet, which may have a large fraction of the beta-strands oriented normal to membrane. Our structural model provides a clue as to how the surface charges on the outer membrane may regulate the permeation of ionic solutes through the channel.     

A dominant connexin43 mutant does not have dominant effects on gap junction coupling in astrocytes

Dominant mutations in GJA1, the gene encoding the gap junction protein connexin43 (Cx43), cause oculodentodigital dysplasia (ODDD), a syndrome affecting multiple tissues, including the central nervous system (CNS). We investigated the effects of the G60S mutant, which causes a similar, dominant phenotype in mice (Gja1(Jrt/+)). Astrocytes in acute brain slices from Gja1(Jrt/+) mice transfer sulforhodamine-B comparably to that in their wild-type (WT) littermates. Further, astrocytes and cardiomyocytes cultured from Gja1(Jrt/+) mice showed a comparable transfer of lucifer yellow to those from WT mice. In transfected cells, the G60S mutant formed gap junction (GJ) plaques but not functional channels. In co-transfected cells, the G60S mutant co-immunoprecipitated with WT Cx43, but did not diminish GJ coupling as measured by dual patch clamp. Thus, whereas G60S has dominant effects, it did not appreciably reduce GJ coupling.     

Molecular basis for the polyamine-ompF porin interactions: inhibitor and mutant studies

By testing the sensitivity of Escherichia coli OmpF porin to various natural and synthetic polyamines of different lengths, charge and other molecular characteristics, we were able to identify the molecular properties required for compounds to act as inhibitors of OmpF in the nanomolar range. Inhibitors require at least two amine groups to be effective. For diamines, the optimum length of the hydrocarbon spacer was found to be of eight to ten methylene groups. Triamine molecules based on a 12-carbon motif were found to be more effective that spermidine, an eight-carbon trivalent derivative. But differences in inhibition efficiencies were also found for trivalent compounds depending on the relative position of the internal secondary amine group with respect to the terminal groups. Finally, quaternary ammonium derivatives had no effect, suggesting that the nature of the terminal amine is important for the interaction. From these observations, we deduce that inhibition efficiency in the nanomolar range requires a 12-carbon chain triamine with terminal primary amine groups and replacement of the eighth methylene by a secondary amine. The need for this type of molecular architecture suggests that inhibition is governed by interactions between specific amine groups and protein residues, and that this is not simply due to the accumulation of charges into the pore. Together with previous observations from site-directed mutagenesis studies and inspection of the crystal structure of OmpF, these results allowed us to propose three residues (D113, D121 and Y294) as putative sites of interaction between the channel and spermine. Alanine substitution at each of these three residues resulted in a loss of inhibition by spermine, while mutations of only D113 and D121 affected inhibition by spermidine. Based on these observations, we suggest a model for the molecular determinants involved in the porin-polyamine interaction.     

Molecular basis of the functional specificities of phototropin 1 and 2

A blue-light photoreceptor in plants, phototropin, mediates phototropism, chloroplast relocation, stomatal opening, and leaf-flattening responses. Phototropin is divided into two functional moieties, the N-terminal photosensory and the C-terminal signaling moieties. Phototropin perceives light stimuli by the light, oxygen or voltage (LOV) domain in the N-terminus; the signal is then transduced intramolecularly to the C-terminal kinase domain. Two phototropins, phot1 and phot2, which have overlapping and distinct functions, exist in Arabidopsis thaliana. Phot1 mediates responses with higher sensitivity than phot2. Phot2 mediates specific responses, such as the chloroplast avoidance response and chloroplast dark positioning. To elucidate the molecular basis for the functional specificities of phot1 and phot2, we exchanged the N- and C-terminal moieties of phot1 and phot2, fused them to GFP and expressed them under the PHOT2 promoter in the phot1 phot2 mutant background. With respect to phototropism and other responses, the chimeric phototropin consisting of phot1 N-terminal and phot2 C-terminal moieties (P1n/2cG) was almost as sensitive as phot1; whereas the reverse combination (P2n/1cG) functioned with lower sensitivity. Hence, the N-terminal moiety mainly determined the sensitivity of the phototropins. Unexpectedly, both P1n/2cG and P2n/1cG mediated the chloroplast avoidance response, which is specific to phot2. Hence, chloroplast avoidance activity appeared to be suppressed specifically in the combination of N- and C-terminal moieties of phot1. Unlike the chloroplast avoidance response, chloroplast dark positioning was observed for P2G and P2n/1cG but not for P1G or P1n/2cG, suggesting that a specific structure in the N-terminal moiety of phot2 is required for this activity.     

Molecular phylogeny and evolutionary history of Southeast Asian macaques forming the M. silenus group

The 12 presently recognized taxa forming the Macaca silenus group represent the most diverse lineage within the genus Macaca. The present study was set up to clarify the phylogenetic relationships of the extant members of the M. silenus group and to explain their geographical distribution patterns seen today. A combined approach involving the analysis of one paternal (TSPY) and two maternal (cyt b and 12S-16S rRNA) molecular markers enabled us to resolve the phylogenetic relationships within this lineage. Our Y chromosomal marker is not informative enough to allow detailed conclusion. Based on our mitochondrial data, however, M. pagensis, endemic to the three southern Mentawai islands (Sipora, North- and South Pagai), split off early (2.4-2.6 mya) and represents a sister clade to the macaques from the northern Mentawai island of Siberut and from those of the Southeast Asian mainland, which diverged in a radiation-like splitting event about 1.5-1.7 mya. By combining our new results with available data on behavioural as well as climate and sea level changes in Southeast Asia during the Plio- and Pleistocene, we have developed two scenarios for the evolutionary history of this primate group, which may help explain the current geographical distribution of its members.     

The projected effects of climatic and vegetation changes on the distribution and diversity of Southeast Asian bats

Southeast‐Asia (SEA) constitutes a global biodiversity hotspot, but is exposed to extensive deforestation and faces numerous threats to its biodiversity. Climate change represents a major challenge to the survival and viability of species, and the potential consequences must be assessed to allow for mitigation. We project the effects of several climate change scenarios on bat diversity, and predict changes in range size for 171 bat species throughout SEA. We predict decreases in species richness in all areas with high species richness (>80 species) at 2050–2080, using bioclimatic IPCC scenarios A2 (a severe scenario, continuously increasing human population size, regional changes in economic growth) and B1 (the ‘greenest’ scenario, global population peaking mid‐century). We also predicted changes in species richness in scenarios that project vegetation changes in addition to climate change up to 2050. At 2050 and 2080, A2 and B1 scenarios incorporating changes in climatic factors predicted that 3–9% species would lose all currently suitable niche space. When considering total extents of species distribution in SEA (including possible range expansions), 2–6% of species may have no suitable niche space in 2050–2080. When potential vegetation and climate changes were combined only 1% of species showed no changes in their predicted ranges by 2050. Although some species are projected to expand ranges, this may be ecologically impossible due to potential barriers to dispersal, especially for species with poor dispersal ability. Only 1–13% of species showed no projected reductions in their current range under bioclimatic scenarios. An effective way to facilitate range shift for dispersal‐limited species is to improve landscape connectivity. If current trends in environmental change continue and species cannot expand their ranges into new areas, then the majority of bat species in SEA may show decreases in range size and increased extinction risk within the next century.     

Three-dimensional structure of the Z band in a normal mammalian skeletal muscle

The three-dimensional structure of the vertebrate skeletal muscle Z band reflects its function as the muscle component essential for tension transmission between successive sarcomeres. We have investigated this structure as well as that of the nearby I band in a normal, unstimulated mammalian skeletal muscle by tomographic three- dimensional reconstruction from electron micrograph tilt series of sectioned tissue. The three-dimensional Z band structure consists of interdigitating axial filaments from opposite sarcomeres connected every 18 +/- 12 nm (mean +/- SD) to one to four cross-connecting Z- filaments are observed to meet the axial filaments in a fourfold symmetric arrangement. The substantial variation in the spacing between cross-connecting Z-filament to axial filament connection points suggests that the structure of the Z band is not determined solely by the arrangement of alpha-actinin to actin-binding sites along the axial filament. The cross-connecting filaments bind to or form a "relaxed interconnecting body" halfway between the axial filaments. This filamentous body is parallel to the Z band axial filaments and is observed to play an essential role in generating the small square lattice pattern seen in electron micrographs of unstimulated muscle cross sections. This structure is absent in cross section of the Z band from muscles fixed in rigor or in tetanus, suggesting that the Z band lattice must undergo dynamic rearrangement concomitant with crossbridge binding in the A band.     

Molecular basis of the silkworm mutant rel causing red egg color and embryonic death

The coloration and hatchability of insect eggs can affect individual and population survival. However, few genetic loci have been documented to affect both traits, and the genes involved in regulating these two traits are unclear. The silkworm recessive mutant re^l shows both red egg color and embryo mortality. We studied the molecular basis of the re^l phenotype formation. Through genetic analysis, gene screening and sequencing, we found that two closely linked genes, BGIBMGA003497 (Bm-re) and BGIBMGA003697 (BmSema1a), control egg color and embryo mortality, respectively. Six base pairs of the Bm-re gene are deleted in its open reading frame, and BmSema1a is expressed at abnormally low levels in mutant re^l. BmSema1a gene function verification was performed using RNA interference and clustered randomly interspersed palindromic repeats (CRISPR)/CRISPR-associate protein 9. Deficiency of the BmSema1a gene can cause the death of silkworm embryos. This study revealed the molecular basis of silkworm re^l mutant formation and indicated that the Sema1a gene is essential for insect embryo development.     

Primary structure and differential expression of beta-amylase in normal and mutant barleys

The primary structure of barley endosperm beta-amylase, an enzyme which catalyses the liberation of maltose from 1,4-alpha-D-glucans, has been deduced from the nucleotide sequence of a cloned full-length cDNA. The mRNA is 1754 nucleotides long [excluding the poly(A) tail] and codes for a polypeptide of 535 amino acids with a relative molecular mass of 59,663. The deduced amino acid sequence was compared with the sequences of ten peptides obtained from the purified enzyme and unambiguous identification was obtained. The N-terminal region of the deduced sequence was identical to a 12-residue cyanogen-bromide-peptide sequence, indicating that beta-amylase is synthesized as the mature protein. A graphic matrix homology plot shows four glycine-rich repeats, each of 11 residues, preceding the C-terminus. Southern blotting of genomic DNA demonstrates that beta-amylase is encoded by a small gene family, while cDNA sequence analysis indicates the presence of at least two types of mRNA in the endosperm. Dot and northern blot analysis show that Hiproly barley contains greatly increased levels of beta-amylase mRNA compared to the normal cultivar Sundance, whereas Ris? mutant 1508 contains only trace amounts. These results correlate well with the deposition of beta-amylase during endosperm development in these lines. Low but similar amounts of beta-amylase mRNAs sequences were detected in leaves and shoots from normal and mutant barleys, demonstrating that the mutant lys3a (1508) and lysl (Hiproly) genes do not affect the expression of beta-amylase in these tissues.