Chronic suppurative otitis media in Zimbabwean school children: a cross-sectional study.

OBJECTIVE: Chronic suppurative otitis media is a major cause of disabling childhood hearing loss, especially in low-income countries. Estimates on its prevalence in sub-Saharan Africa range from the lowest to the highest in the world (less than one per cent to more than five per cent). However, the prevalence of chronic suppurative otitis media in Zimbabwe is largely unknown. This study aimed to determine the prevalence of paediatric chronic suppurative otitis media and other middle-ear pathology in rural Zimbabwe. METHOD: A cross-sectional study was performed in primary school children aged 4-13 years from the rural province of Mashonaland East. Participants underwent video otoscopy and tympanometry. RESULTS: Out of 451 examined children, two (0.4 per cent) had chronic suppurative otitis media. Acute otitis media was present in one (0.2 per cent), otitis media with effusion was present in five (1.1 per cent) and scarring was present in 69 (15.3 per cent). CONCLUSION: Chronic suppurative otitis media and otitis media sequelae were surprisingly uncommon in this sample of rural primary school children in Zimbabwe. More studies, preferably population-based, are needed to enable more precise estimates of chronic suppurative otitis media prevalence in Zimbabwe.

The anticarcinogenic conjugated fatty acid c9, t11-c18:2, or rumenic acid, in human milk: amounts and effects.

The conjugated linoleic acid (CLA) c9, t11-C18:2 has been detected in human milk by gas-liquid chromotography (GLC) using standards for tentative identification. The amounts found were: 5.5 mg/g fat, Australia, 1988; and 3.6 mg/g fat, Idaho, 1997. We also employed GLC and standards and confirmed the identity of coeluting peaks by mass spectrometry. Our values were 1.8+/-0.02 mg/g fat. CLA inhibits growth of several cancers, is antiatherosclerotic, partially overcomes catabolic responses to endotoxin injection, enhances weight gain and improves feed efficiency in rats, and modulates immune response and bone deposition. The dietary sources are ruminant-derived foods, particularly aged cheese.

Parenteral infusion of a lactating woman with intralipid: changes in milk and plasma fatty acids.

A nursing woman afflicted with short bowel syndrome received parenteral infusions of Intralipid. In the morning following 2 nights of infusion, samples of milk and blood were taken; additional samples were taken the morning after 1 and 2 nights of no infusion. The fatty acid composition of these samples was determined by gas chromatography. The Intralipid infusion contained 51.5% linoleic acid (C18:2). The C18:2 content of the milk was highest (14%) after each infusion and dropped to about 10% on days 1 and 2. Inverse changes were seen in stearic acid (C18:0). The C18:2 content of the plasma showed little change, remaining at about 23%. These results provide further direct evidence that the composition of milk fatty acids can be influenced by changes in the composition of external sources of fatty acids to the lactating mother.

Consumption of lipophilic contaminants in human milk by infants: quantities are usually incorrect.

We evaluated several reports on the detection of nitromusks (artificial perfumes) in human milk. The nitromusks found were separated by gas-liquid chromatography and identified by mass spectrometry. However, the quantities reported as being consumed by infants were often questionable (Jensen, 1995). The investigators did not always a) obtain a representative samples of milk, b) extract and quantify the fat by a recognized, accurate method, or c) determine the amount of milk, hence fat, consumed by the infant. Some investigators gave almost no data on the milk, except to say that a sample was obtained. Reports on some other contaminants in human milk were similarly deficient. We have published a description of recommended extraction procedures in a paper presenting a detailed protocol for the processing of milk so as to determine the actual amounts of lipophilic contaminants ingested by the nursing infant (Jensen et al. 1997).

Disturbance in the allocation of carbohydrates to regenerative organs in transgenic Nicotiana tabacum L.

Transgenic tobacco (Nicotiana tabacum L, cv. SR-1) expressing mannitol 1-phosphate dehydrogenase, MTLD, in chloroplasts and myo-inositol O-methyltransferase, IMT1, in the cytosol after crossing of lines which expressed these foreign genes separately has been analysed. Plants expressing both enzymes accumulated mannitol and D-ononitol in amounts comparable to those following single gene transfer and showed phenotypically normal growth during the vegetative stage. Induction of flowering for transgenovar and wild-type occurred at the same time, but during flowering the phenotype of the transformed plants changed. Compared to wild-type, transgenic plants were characterized by curled, smaller upper leaves and elongated stems during flowering; incomplete development of flower buds with shorter sepals and pedicels resulted in increased abortion. Flowers completing development were normal. The vegetative biomass of the transformed plants was slightly higher than that of wild-type. Concentrations of soluble sugars and potassium were lower than in wild-type only in the apical parts of the transgenic plants. Both enzymes, under control of the CaMV 35S promoter, promoted accumulation of mannitol and D-ononitol in the youngest leaves close to the vegetative meristem and in flowers, suggesting that their presence could signal lower sink demand leading to a decrease in carbon import to flowers and developing seed capsules. The interpretation here is that increases of inert carbohydrates in developing sinks interfere with metabolism, such as respiration or glycolysis. This interference may be less significant in source tissues during vegetative growth than in sink tissues during seed development.

Roles of sugar alcohols in osmotic stress adaptation. Replacement of glycerol by mannitol and sorbitol in yeast.

For many organisms there is a correlation between increases of metabolites and osmotic stress tolerance, but the mechanisms that cause this protection are not clear. To understand the role of polyols, genes for bacterial mannitol-1-P dehydrogenase and apple sorbitol-6-P dehydrogenase were introduced into a Saccharomyces cerevisiae mutant deficient in glycerol synthesis. Sorbitol and mannitol provided some protection, but less than that generated by a similar concentration of glycerol generated by glycerol-3-P dehydrogenase (GPD1). Reduced protection by polyols suggested that glycerol had specific functions for which mannitol and sorbitol could not substitute, and that the absolute amount of the accumulating osmoticum might not be crucial. The retention of glycerol and mannitol/sorbitol, respectively, was a major difference. During salt stress, cells retained more of the six-carbon polyols than glycerol. We suggest that the loss of >98% of the glycerol synthesized could provide a safety valve that dissipates reducing power, while a similar high intracellular concentration of retained polyols would be less protective. To understand the role of glycerol in salt tolerance, salt-tolerant suppressor mutants were isolated from the glycerol-deficient strain. One mutant, sr13, partially suppressed the salt-sensitive phenotype of the glycerol-deficient line, probably due to a doubling of [K(+)] accumulating during stress. We compare these results to the "osmotic adjustment" concept typically applied to accumulating metabolites in plants. The accumulation of polyols may have dual functions: facilitating osmotic adjustment and supporting redox control.

Lipids in human milk.

I have reviewed recent (March 1995-December 1997) papers on human milk lipids including many on fatty acid (FA) composition. The effects of maternal diets on the profiles are apparent. However, more data on the composition of milk lipids are needed. It is noteworthy that so few papers on milk FA composition have reported analyses using high-resolution gas-liquid chromatography columns. Two of these were on milk from women in North America. The diets in North America are varied and the number of analyses few. We do not have a reliable data base showing the ranges of biologically important acids. Except for the gangliosides, few new data on the other lipids appeared during this period.

The anticarcinogenic conjugated fatty acid, 9c, 11t-18:2, in human milk: confirmation of its presence.

The concentration of the anticarcinogenic fatty acid, 9c, 11t-18:2, in human milk was determined by gas-liquid chromatography (GLC). The mean concentration of 20 samples from 5 women taken at 1, 7, 14, and 21 days was: 0.18% +/- 0.02; range, 0.14-0.28%. Identity was confirmed by GLC-mass spectrometry (MS). Conjugated isomers other than 9c, 11t-18:2 were not detected. The amounts were not changed by supplementation of the maternal diet with fish oil beginning on day 1 after the milk sample was taken.

Chimeric Arabidopsis thaliana ribulose-1,5-bisphosphate carboxylase/oxygenase containing a pea small subunit protein is compromised in carbamylation.

A cDNA of pea (Pisum sativum L.) RbcS 3A, encoding a small subunit protein (S) of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), has been expressed in Arabidopsis thaliana under control of the cauliflower mosaic virus 35S promoter, and the transcript and mature S protein were detected. Specific antibodies revealed two protein spots for the four Arabidopsis S and one additional spot for pea S. Pea S in chimeric Rubisco amounted to 15 to 18% of all S, as judged by separation on two-dimensional isoelectric focusing/sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels from partially purified enzyme preparations and quantitation of silver-stained protein spots. The chimeric enzyme had 11 +/- 1% fewer carbamylated sites and a 11 +/- 1% lower carboxylase activity than wild-type Arabidopsis Rubisco. Whereas pea S expression, preprotein transport, and processing and assembly resulted in a stable holoenzyme, the chimeric enzyme was reproducibly catalytically less efficient. We suggest that the presence of, on average, one foreign S per holoenzyme is responsible for the altered activity. In addition, higher-plant Rubisco, unlike the cyanobacterial enzyme, seems to have evolved species-specific interactions between S and the large subunit protein that are involved in carbamylation of the active site.

Mutations in the small subunit of ribulose-1,5-bisphosphate carboxylase/ oxygenase increase the formation of the misfire product xylulose-1,5-bisphosphate.

The small subunit (S) increases the catalytic efficiency of ribulose-1,5-bisphosphate carboxylase/oxygenase (EC 4.1.1.39) by stabilizing the active sites generated by four large subunit (L) dimers. This stabilization appears to be due to an influence of S on the reaction intermediate 2,3-enediol, which is formed after the abstraction of a proton from the substrate ribulose-1,5-bisphosphate. We tested the functional significance of residues that are conserved among most species in the carboxy-terminal part of S and analyzed their influence on the kinetic parameters of Synechococcus holoenzymes. The replacements in S (F92S, Q99G, and P108L) resulted in catalytic activities ranging from 95 to 43% of wild type. The specificity factors for the three mutant enzymes were little affected (90-96% of wild type), but Km(CO2) values increased 0.5- to 2-fold. Mutant enzymes with replacements Q99G and P108L showed increased mis-protonation, relative to carboxylation, of the 2,3-enediol intermediate, forming 2 to 3 times more xylulose-1,5-bisphosphate per ribulose-1,5-bisphosphate utilized than wild-type or F92S enzymes. The results suggest that specific alterations of the L/S interfaces and of the hydrophobic core of S are transmitted to the active site by long-range interactions. S interactions with L may restrict the flexibility of active-site residues in L.

Increased resistance to oxidative stress in transgenic plants by targeting mannitol biosynthesis to chloroplasts.

To investigate the potential role of a polyol, mannitol, in oxidative stress protection, a bacterial mannitol-1-phosphate dehydrogenase gene was targeted to chloroplasts by the addition of an amino-terminal transit peptide. Transgenic tobacco (Nicotiana tabacum) lines accumulate mannitol at concentrations ranging from 2.5 to 7 mumol/g fresh weight. Line BS1-31 accumulated approximately 100 mM mannitol in chloroplasts and was identical to the wild type in phenotype and photosynthetic performance. The presence of mannitol in chloroplasts resulted in an increased resistance to methyl viologen (MV)-induced oxidative stress, documented by the increased retention of chlorophyll in transgenic leaf tissue following MV treatment. In the presence of MV, isolated mesophyll cells of BS1-31 exhibited higher CO2 fixation than the wild type. When the hydroxyl radical probe dimethyl sulfoxide was introduced into cells, the initial formation rate of methane sulfinic acid was significantly lower in cells containing mannitol in the chloroplast compartment than in wild-type cells, indicating an increased hydroxyl radical-scavenging capacity in BS1-31 tobacco. We suggest that the chloroplast location of mannitol can supplement endogenous radical-scavenging mechanisms and reduce oxidative damage of cells by hydroxyl radicals.

Mannitol Protects against Oxidation by Hydroxyl Radicals.

Hydroxyl radicals may be responsible for oxidative damage during drought or chilling stress. We have shown that the presence of mannitol in chloroplasts can protect plants against oxidative damage by hydroxyl radicals (B. Shen, R.G. Jensen, H.J. Bohnert [1997] Plant Physiol 113: 1177-1183). Here we identify one of the target enzymes that may be protected by mannitol. Isolated thylakoids in the presence of physiological concentrations of Fe2+ generated hydroxyl radicals that were detected by the conversion of phenylalanine into tyrosine. The activity of phosphoribulokinase (PRK), a thiol-regulated enzyme of the Calvin cycle, was reduced by 65% in illuminated thylakoids producing hydroxyl radicals. Mannitol (125 mM) and sodium formate (15 mM), both hydroxyl radical scavengers, and catalase (3000 units mL-1) prevented loss of PRK activity. In contrast, superoxide dismutase (300 units mL-1) and glycine betaine (125 mM) were not effective in protecting PRK against oxidative inactivation. Ribulose-1,5-bisphosphate carboxylase/oxygenase activity was not affected by hydroxyl radicals. We suggest that the stress-protective role of mannitol may be to shield susceptible thiol-regulated enzymes like PRK plus thioredoxin, ferredoxin, and glutathione from inactivation by hydroxyl radicals in plants.

Increased Salt and Drought Tolerance by D-Ononitol Production in Transgenic Nicotiana tabacum L.

A cDNA encoding myo-inositol O-methyltransferase (IMT1) has been transferred into Nicotiana tabacum cultivar SR1. During drought and salt stress, transformants (I5A) accumulated the methylated inositol D-ononitol in amounts exceeding 35 [mu]mol g-1 fresh weight In I5A, photosynthetic CO2 fixation was inhibited less during salt stress and drought, and the plants recovered faster than wild type. One day after rewatering drought-stressed plants, I5A photosynthesis had recovered 75% versus 57% recovery with cultivar SR1 plants. After 2.5 weeks of 250 mM NaCl in hydroponic solution, I5A fixed 4.9 [plus or minus] 1.4 [mu]mol CO2 m-2 s-1, whereas SR1 fixed 2.5 [plus or minus] 0.6 [mu]mol CO2 m-2 s-1. myo-Inositol, the substrate for IMT1, increases in tobacco under stress. Preconditioning of I5A plants in 50 mM NaCl increased D-ononitol amounts and resulted in increased protection when the plants were stressed subsequently with 150 mM NaCl. Pro, Suc, Fru, and Glc showed substantial diurnal fluctuations in amounts, but D-ononitol did not. Plant transformation resulting in stress-inducible, stable solute accumulation appears to provide better protection under drought and salt-stress conditions than strategies using osmotic adjustment by metabolites that are constitutively present.

Coordinate transcriptional induction of myo-inositol metabolism during environmental stress.

The pathway from glucose 6-phosphate (G 6-P) to myoinositol 1-phosphate (Ins 1-P) and myo-inositol (Ins) is essential for the synthesis of various metabolites. In the halophyte Mesembryanthemum crystallinum (common ice plant), two enzymes, myo-inositol O-methyltransferase (IMT1) and ononitol epimerase (OEP1), extend this pathway and lead to the accumulation of methylated inositols, D-ononitol and D-pinitol, which serve as osmoprotectants. This paper describes transcripts for the enzyme, Inps1, encoding myo-inositol 1-phosphate synthase (INPS1), from the ice plant. Two Inps-like sequences are present in the genome. The deduced amino acid sequences of the cloned transcript are 49.5% and 87-90%, respectively, identical to those of yeast and other higher plant sequences. Inps1 RNA amounts are upregulated at least fivefold and amounts of free Ins accumulate approximately 10-fold during salinity stress. Inps1 induction is by transcription, similar to the induction of Imt1. In contrast, Arabidopsis thaliana does not show upregulation of Inps1 or increased amounts of Ins when salt-stressed. The lack of Inps1 induction in Arabidopsis exemplifies differences in glycophytic and halophytic regulation of gene expression at the point of entry into a pathway that leads to osmoprotection. The stress-induced coordinate upregulation of this pathway and its extension by novel enzymes in the ice plant also highlights biochemical differences.

The lipids in human milk.

The summary will be limited to the areas that should be intensively investigated. The first is: determination of fatty acid profiles using modern methods on a world wide basis. We have no more than five or six papers in which my criterion was applied, one from Canada and the remainder from Europe with some data from Africa. Obviously, milk cannot be used as the gold standard on this meager data base. The second area is analysis of TG structure. These analyses are difficult, but structure is one of the factors controlling digestion. Data on the effects of maternal diet on structure would be useful. The third area is the role of primary or derived milk lipids as microbicidal agents. The fourth area is examination of globule parameters, i.e. number, size, volume, surface, and how they are affected by diet. There are many others which may interest the reader.