Hypothalamic hamartoma, cerebellar hypoplasia, facial dysmorphism and very atypical combination of polydactyly: is it a new variant of oro-facio-digital syndrome?

We describe a newborn with multiple congenital anomalies consistent with an oro-facio-digital syndrome (OFDS). These are a group of inherited syndromes that have in common anomalies of the tongue (bifid or lobulated tongue with hamartomas), the face (median cleft lip) and the digits (brachydactyly, polydactyly, clinodactyly and/or syndactyly). OFDS has been classified into 11 types. The case described in this paper had manifestations overlapping with OFDS II (Mohr) and OFDS IV (Mohr-Majewski) and OFDS VI (Varadi). We propose that the present patient has a new variation of the OFDS due to the co-existence of the very atypical combination of polydactyly, cerebellar hypoplasia, hypothalamic hamartoma and classical facial findings of OFDS.     

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-[¹⁴C]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.Cereal grains contribute a significant portion of worldwide starch production. Unlike other plant tissue, starch biosynthesis in the endosperm storage organ of cereal grains is unique in its dependence on two ADP-Glc pyrophosphorylase (AGPase) isoforms (Denyer et al., 1996; Thorbjørnsen et al., 1996; Sikka et al., 2001), a major cytosolic enzyme and a minor plastidial one, to generate ADP-glucose (ADPglc), the sugar nucleotide utilized by starch synthases in the amyloplast (Cakir et al., 2015). The majority of ADPglc in cereal endosperm is generated in the cytosol from AGPase (Tuncel and Okita, 2013) as well as by Suc synthase (Tuncel and Okita, 2013; Bahaji et al., 2014) and subsequently transported into amyloplasts by the BRITTLE-1 (BT1) protein located at the plastid envelope (Cao et al., 1995; Shannon et al., 1998).The Bt1 gene, first identified in maize (Zea mays; Mangelsdorf, 1926) and isolated by Sullivan et al. (1991), encodes a major amyloplast membrane protein ranging from 39 to 44 kD (Cao et al., 1995). The BT1 protein and its homologs belong to the mitochondrial carrier family (Sullivan et al., 1991; Haferkamp, 2007), which has a diverse range of substrates (Patron et al., 2004; Leroch et al., 2005; Kirchberger et al., 2008). The assignment of BT1 protein as the ADPglc transporter in cereal endosperms was first proposed by Sullivan et al. (1991), and then it was characterized based on the increased ADPglc levels and reduced ADPglc import rate in endosperms of BT1-deficient maize and barley (Hordeum vulgare) mutants (Tobias et al., 1992; Shannon et al., 1996, 1998; Patron et al., 2004). Biochemical transport studies of the maize BT1 showed that it imported ADPglc by counter exchanging with ADP (Kirchberger et al., 2007). The wheat (Triticum aestivum) BT1 homolog also transports ADPglc but has similar affinities for ADP and AMP as the counter-exchange substrate (Bowsher et al., 2007).Evidence from previous studies by our laboratory (Sakulsingharoj et al., 2004; Nagai et al., 2009) suggested the potential role of BT1 as well as other downstream processes as a rate-limiting step in starch biosynthesis in the transgenic rice (Oryza sativa) GlgC (CS8) lines overexpressing an up-regulated AGPase (Escherichia coli glgC-TM). In GlgC (CS8) rice lines, grain weights (starch) are elevated up to 15% compared with wild-type plants, indicating that the AGPase-catalyzed reaction is a rate-limiting step in starch biosynthesis under normal conditions. When transgenic GlgC (CS8) plants were grown under elevated CO₂ levels, no further increases in grain weight were evident compared with those grown at ambient CO₂. As Suc levels are elevated in leaf blades, leaf sheaths, culms (Rowland-Bamford et al., 1990), and peduncle exudates (Chen et al., 1994) in rice plants grown under elevated CO₂, developing GlgC (CS8) grains were unable to convert the increased levels of sugars into starch. This lack of increase indicated that the AGPase-catalyzed reaction (ADPglc synthesis) was no longer rate limiting and that one or more downstream processes regulated carbon flux from source tissues in developing GlgC (CS8) endosperm (Sakulsingharoj et al., 2004). This view is also supported by a subsequent metabolite study in which several GlgC (CS8) lines were found to contain up to 46% higher ADPglc levels than wild-type plants (Nagai et al., 2009). As this increase in ADPglc levels was nearly 3-fold higher than the increase in grain weight, starch biosynthesis is saturated with respect to ADPglc levels and carbon flow into starch is restricted by one or more downstream steps. Potential events that may limit the utilization of ADPglc in starch in GlgC (CS8) lines are the import of this sugar nucleotide via the BT1 transporter into amyloplasts and/or the utilization of ADPglc by starch synthases. Mutant analysis of the two major starch synthases indicated no significant impact on grain weight when one of these starch synthases was nonfunctional, suggesting that this enzyme activity, contributed by multiple enzyme isoforms, is present at excessive levels (Fujita et al., 2006, 2007). Therefore, we suspected that BT1 is the likely candidate limiting carbon flow into starch in GlgC (CS8) endosperms.The aim of this study was to investigate the role of BT1 in mediating the transport of ADPglc into amyloplast and to determine whether this transport activity is rate limiting in rice endosperm. In order to address these questions, we show that BT1 is the major transporter of ADPglc by analysis of the EM1093 rice line, which contains a mutation at the shrunken3 (shr3) locus and, specifically, in the OsBt1-1 gene. Second, we assessed the impact of the expression of the maize ZmBt1 gene in wild-type and GlgC (CS8) seeds to determine the potential limiting role of BT1 transport activity on starch biosynthesis. Our results indicate that BT1 is essential for starch synthesis but is not rate limiting and that one or more stroma-localized processes limit maximum carbon flow into starch.     

Increased micronucleus,nucleoplasmic bridge,nuclear bud frequency and oxidative DNA damage associated with prolactin levels and pituitary adenoma diameters in patients with prolactinoma

Prolactinoma is the most common pituitary tumor. Most pituitary tumors are benign, but they often are clinically signi?cant. We investigated cytokinesis-block micronucleus cytome (CBMN cyt) assay parameters and oxidative DNA damage in patients with prolactinoma to assess the relations among age, prolactin level, pituitary adenoma diameter and 8-hydroxy-2’-deoxyguanosine (8-OHdG) level in patients with prolactinoma. We investigated 27 patients diagnosed with prolactinoma and 20 age- and sex-matched healthy controls. We measured CBMN cyt parameters and plasma 8-OHdG levels in peripheral blood lymphocytes of patients with prolactinoma and controls. The frequencies of micronucleus (MN), nucleoplasmic bridge, nuclear bud, apoptotic and necrotic cells, and plasma 8-OHdG levels in patients with prolactinoma were significantly greater than controls. MN frequency was correlated positively with age, prolactin levels and pituitary adenoma diameters in patients with prolactinoma. The increased chromosomal and oxidative DNA damage, and the positive correlation between MN frequency, prolactin levels and pituitary adenoma diameters may be associated with increased risk of cancer in patients with prolactinoma, because increased MN frequency is a predictor of cancer risk.     

In vitro inhibition of cytosolic carbonic anhydrases I and II by some new dihydroxycoumarin compounds

A new series of 6, 7-dihydroxy-3-(methylphenyl) chromenones, including three new derivatives, i.e. 6,7-dihydroxy-3-(2-methylphenyl)-2H-chromen-2-one (OPC); 6,7-dihydroxy-3-(3-methylphenyl)-2H-chromen-2-one (MPC); 6,7-dihydroxy-3-(4-methylphenyl)-2H-chromen-2-one (PPC) and one previously described, namely 6,7-dihydroxy-3-phenyl-2H-chromen-2-one (DPC), were synthesized. These compounds were investigated as inhibitors of human carbonic anhydrase I (hCA-I) and human carbonic anhydrase II (hCA-II) which had been purified from human erythrocytes on an affinity gel comprised of L-tyrosine-sulfonamide-Sepharose 4B.     

Lavandula stoechas L. subsp. stoechas,a New Herbal Source for Ursolic Acid: Quantitative Analysis,Purification and Bioactivity Studies

In this study, methanol, ethanol, methanol-dichloromethane (1 : 1, v/v), acetone, ethyl acetate, diethyl ether, and chloroform extracts of lavender (Lavandula stoechas L. subsp. stoechas) were prepared by maceration, and the ursolic acid contents in the extracts were determined quantitatively by HPLC analyses. The present results show that the methanol-dichloromethane (1 : 1, v/v) solvent system is the most efficient solvent system for the extraction of ursolic acid from the plant sample with the highest yield (2.22 g/100 g plant sample). In the present study, a new practical method for the isolation of ursolic acid from polar extracts was also demonstrated for the first time. The inhibition effects of the extracts and ursolic acid were also revealed on α-glycosidase, acetylcholinesterase, butyrylcholinesterase, and human carbonic anhydrase I and II enzymes by determining IC₅₀ values for the first time. The extracts and ursolic acid acted as potent antidiabetic agents by strongly inhibiting the α-glycosidase activity, whereas they were found to be very weak neuroprotective agents. In view of the present results, L. stoechas and its major metabolite, ursolic acid, can be recommended as a herbal source to control postprandial blood sugar levels and prevent diabetes by delaying the digestion of starch in food.     

Re‐programming of gene expression in the CS8 rice line over‐expressing ADPglucose pyrophosphorylase induces a suppressor of starch biosynthesis

The CS8 transgenic rice (Oryza sativa L.) lines expressing an up‐regulated glgC gene produced higher levels of ADPglucose (ADPglc), the substrate for starch synthases. However, the increase in grain weight was much less than the increase in ADPglc levels suggesting one or more downstream rate‐limiting steps. Endosperm starch levels were not further enhanced in double transgenic plants expressing both glgC and the maize brittle‐1 gene, the latter responsible for transport of ADPglc into the amyloplast. These studies demonstrate that critical processes within the amyloplast stroma restrict maximum carbon flow into starch. RNA‐seq analysis showed extensive re‐programming of gene expression in the CS8 with 2073 genes up‐regulated and 140 down‐regulated. One conspicuous gene, up‐regulated ~15‐fold, coded for a biochemically uncharacterized starch binding domain‐containing protein (SBDCP1) possessing a plastid transit peptide. Confocal microscopy and transmission electron microscopy analysis confirmed that SBDCP1 was located in the amyloplasts. Reciprocal immunoprecipitation and pull‐down assays indicated an interaction between SBDCP1 and starch synthase IIIa (SSIIIa), which was down‐regulated at the protein level in the CS8 line. Furthermore, binding by SBDCP1 inhibited SSIIIa starch polymerization activity in a non‐competitive manner. Surprisingly, artificial microRNA gene suppression of SBDCP1 restored protein expression levels of SSIIIa in the CS8 line resulting in starch with lower amylose content and increased amylopectin chains with a higher degree of polymerization. Collectively, our results support the involvement of additional non‐enzymatic factors such as SBDCP in starch biosynthesis.     

Purification and characterization of the CS2 pili of colonization factor antigen II produced by human enterotoxigenic Escherichia coli

A subtype (CS2) of the colonization factor antigen II (CFA/II) of human enterotoxigenic Escherichia coli (ETEC) was studied. Analysis revealed that CS2-possessing ETEC was predominant among isolates from traveller's diarrhea at Osaka, Japan. TH61 pili produced by a clinical strain (TH61) were purified as a native form to homogeneity by zone electrophoresis and successive column chromatographies on Sepharose 4B and Phenyl-Sepharose CL-4B. It was demonstrated by immunogold staining technique and bacterial agglutination test that antisera against the purified pili of strain TH61 recognized pili of both strain TH61 and strain #C91f, a control strain possessing only CS2 pili. This suggests that TH61 pili purified in this study are CS2 pili. Subunit (pilin) of the purified pili has a molecular weight of about 16,000. Strains bearing CS2 could attach to human jejunal epithelial cells, and this attachment was inhibited by pretreating the enterocytes with purified pili. These indicate that CS2 pili are a factor responsible for attachment of ETEC bearing CS2 to human intestinal cells.     

Review of wheat improvement for waterlogging tolerance in Australia and India: the importance of anaerobiosis and element toxicities associated with different soils

Background and Aims

The lack of knowledge about key traits in field environments is a major constraint to germplasm improvement and crop management because waterlogging-prone environments are highly diverse and complex, and the mechanisms of tolerance to waterlogging include a large range of traits. A model is proposed that waterlogging tolerance is a product of tolerance to anaerobiosis and high microelement concentrations. This is further evaluated with the aim of prioritizing traits required for waterlogging tolerance of wheat in the field.

Methods

Waterlogging tolerance mechanisms of wheat are evaluated in a range of diverse environments through a review of past research in Australia and India; this includes selected soils and plant data, including plant growth under waterlogged and drained conditions in different environments. Measurements focus on changes in redox potential and concentrations of diverse elements in soils and plants during waterlogging.

Key Results

(a) Waterlogging tolerance of wheat in one location often does not relate to another, and (b) element toxicities are often a major constraint in waterlogged environments. Important element toxicities in different soils during waterlogging include Mn, Fe, Na, Al and B. This is the first time that Al and B toxicities have been indicated for wheat in waterlogged soils in India. These results support and extend the well-known interactions of salinity/Na and waterlogging/hypoxia tolerance.

Conclusions

Diverse element toxicities (or deficiencies) that are exacerbated during waterlogging are proposed as a major reason why waterlogging tolerance at one site is often not replicated at another. Recommendations for germplasm improvement for waterlogging tolerance include use of inductively coupled plasma analyses of soils and plants.Key words: Waterlogging, microelements, toxicity, redox potential, wheat, anaerobiosis

‘No grain is ever produced without water, but too much water tends to spoil the grain and inundation is as injurious to growth as dearth of water.’ Narada Smriti XI, 19; circa 3000 bc.‘Waterlogging’ is defined as a condition of the soil where excess water limits gas diffusion; while ‘waterlogging tolerance’ is defined as survival or the maintenance of high growth rates, biomass accumulation or grain yield under waterlogging relative to non waterlogged (usually drained soil) conditions (Setter and Waters, 2003).