Recurrence of konzo in southern Tanzania: rehabilitation and prevention using the wetting method.

There have been four konzo outbreaks in Tanzania from 1985 to 2002/2003 with a total of 363 cases of konzo. Every outbreak of konzo resulted from large cyanogen intakes from bitter cassava during drought, which caused food shortages and led to people using short-cut methods of cassava processing. Rehabilitation of the 214 konzo subjects from the two most recent outbreaks of konzo in southern Tanzania was carried out by screening konzo subjects and included provision of crutches and wheel chairs. The wetting method was taught to 216 women activists from the konzo-prone villages, in the first large scale community based intervention to reduce cyanogen intake. Using cassava cyanide kits, the average total cyanide content was reduced by the wetting method about 4-fold, in agreement with previous studies. This model to help prevent konzo requires the widespread education of women activists to use the wetting method.

Konzo and continuing cyanide intoxication from cassava in Mozambique.

In Mozambique, epidemics of the cassava-associated paralytic disease, konzo, have been reported in association with drought or war: over 1100 cases in 1981, over 600 cases in 1992-1993, and over 100 cases in 2005. Smaller epidemics and sporadic cases have also been reported. Large epidemics have occurred at times of agricultural crisis, during the cassava harvest, when the population has been dependent on a diet of insufficiently processed bitter cassava. Konzo mostly affects women of child-bearing age and children over 2 years of age. When measured, serum or urinary thiocyanate concentrations, indicative of cyanide poisoning, have been high in konzo patients during epidemics and in succeeding years. Monitoring of urinary thiocyanate concentrations in schoolchildren in konzo areas has shown persistently high concentrations at the time of the cassava harvest. Inorganic sulphate concentrations have been low during and soon after epidemics. Programmes to prevent konzo have focused on distributing less toxic varieties of cassava and disseminating new processing methods, such as grating and the flour wetting method. Attention should be given to the wider question of agricultural development and food security in the regions of Africa where dependence on bitter cassava results in chronic cyanide intoxication and persistent and emerging konzo.

Simple method for determination of thiocyanate in urine.

BACKGROUND: It would be useful to develop a simple kit method for determination of thiocyanate in urine, which could be used to monitor cyanide overload in cassava-consuming populations. METHODS: The method was based on the quantitative oxidation of thiocyanate in acid permanganate at room temperature in a closed vial with liberation of HCN, which reacted with a picrate paper. For semiquantitative analysis in the field, the colored picrate paper was matched with a color chart prepared using known amounts of KSCN. In the laboratory, a more accurate result was obtained by elution of the colored complex in water and measurement of the absorbance at 510 nm. Over the range 0-100 mg/L, there was a linear relationship given by the equation: thiocyanate content (mg/L) = 78 x absorbance. RESULTS: The picrate thiocyanate method gave no interference with urine samples containing protein at less than 7 g/L, 21 amino acids, histamine, glucose, NaCl, urea, blood, and linamarin. For 53 urine samples analyzed by an accurate column method and the thiocyanate picrate method, a regression line gave very good agreement (r(2) = 1. 000). Quantitative recoveries of thiocyanate added to urine samples were obtained with the picrate method. CONCLUSIONS: A simple picrate kit for determination of thiocyanate in urine was developed and is available free of charge for workers in developing countries.

Cyanogenic potential of cassava flour: field trial in Mozambique of a simple kit.

The cyanogenic potential (ppm HCN equivalents) of 80 samples of cassava flour (obtained from the Mujocojo and Terrene-A areas of Nampula Province and the markets of Nampula City in Mozambique) were determined using a new simple kit, based on the use of picric acid paper (Egan et al., 1997). The kit is compact, requires only a small amount of water and is very simple to use in the field. Comparison with the results of a semi-quantitative method shows a mean deviation between the two methods of 20% (SD 12%). All samples fitted a single population distribution with a mean value of 45 ppm HCN equivalents (SD 37). Two maxima were observed in the distribution curve at 11-20 and 41-50 ppm. Five samples exceeded 100 ppm with two values of 200 ppm. The WHO safe level for cyanogens in cassava flour is 10 ppm. The lowest levels (2 and 6 ppm) were obtained from cassava flour prepared from sweet cassava. Over 76 samples the mean value of the cyanogenic potential of cassava flour produced by heap fermentation is only one half as large as that produced by sun-drying (P < 0.005). Interventions needed to reduce cyanogen levels are (1) improvements in processing methods, such as replacement of sun-drying by heap fermentation, (2) introduction of additional vegetables, pulses and fruit to alleviate the monotonous cassava diet of the people and (3) introduction of high-yielding, disease-resistant, low-cyanide cultivars.

Amino-acid sequence of a trypsin/chymotrypsin inhibitor from giant taro (Alocasia macrorrhiza).

Giant taro (Alocasia macrorrhiza) contains a protein which inhibits both trypsin and chymotrypsin. This trypsin/chymotrypsin inhibitor exists as a dimer of two identical monomers each with slight polymorphism and is an attractive candidate for conferring insect resistance in transgenic plants. The 184 amino-acid sequence (molecular mass of 19774 Da for the Met-24, Glu-50 form) has been determined and is compared with those of other Kunitz-type trypsin, chymotrypsin and subtilisin inhibitors. There appears to be greater 'homology' between the giant taro inhibitor and those inhibitors from other monocotyledons than inhibitors from dicotyledons. The P1 loop region is different from that of other Kunitz-type inhibitors and contains a sequence Leu-Ala-Phe-Phe-Pro at residues 56-60. This section of sequence differs only by a Leu/Ile replacement to a tight binding inhibitor of neutrophil elastase, recently produced by genetic engineering. The most likely candidate for the P1 residue in the giant taro trypsin/chymotrypsin inhibitor is Leu-56.

Comparison of amino acid sequences of the trypsin inhibitors from taro (Colocasia esculenta), giant taro (Alocasia macrorrhiza) and giant swamp taro (Cyrtosperma chamissonis).

The amino acid sequences of the trypsin inhibitors from taro Colocasia esculenta var. esculenta and giant swamp taro Cyrtosperma chamissonis have been determined and are compared with the protein sequence of the trypsin/chymotrypsin inhibitor from giant taro Alocasia macrorrhiza. Both inhibitors display polymorphism and there is evidence of two components in the giant swamp taro. The positional identity between the proteins is highest at 73-75% for the comparison of the giant taro (GT) with the polymorphic forms of the taro (T) inhibitors and lowest at 56-58% for the pairs of taro and giant swamp taro (GST) proteins. The comparisons show that the inhibitors from T and GT are more related to each other than to GST, which supports their taxonomic classification into different tribes. Location of the P1 site for the trypsin inhibitors of aroids is different from that of other Kunitz-type inhibitors and could be at Leu56.

1H NMR studies of insulin: histidine residues, metal binding, and dissociation in alkaline solution.

The shifts of the H2 histidine B5 and B10 resonances of 2-Zn insulin hexamer were followed in 2H2O by 1H NMR spectroscopy at 270 MHz from pH 9.85 to 7. The two resonances present at high pH, previously assigned to H2 histidine B5 and B10 residues, moved slightly downfield and split into four resonances at pH 8.95 and also at pH 7. By use of a paramagnetic broadening probe (Mn2+) and the addition of Zn2+ to metal-free insulin, it was deduced that the four resonances arose from histidines B10 and B5 in two different magnetic environments, probably either bound to Zn2+ or not bound to Zn2+. The pK' values of the B5 and B10 histidines were determined in 60% 2H2O-40% dioxan, in which insulin was soluble throughout the pH range, to be 7.1 and 6.8, respectively at 37 degrees C. Studies at higher pH indicated that at a concentration level suitable for 1H NMR (approximately 1 mM) at 37 degrees C in 2H2O the 2-Zn hexamer was largely dissociated to dimer at pH 10.3 and to monomer at pH 10.8. Addition of paramagnetic shift probe Ni2+ to metal-free insulin caused changes to the spectrum similar to those produced on addition of diamagnetic Zn2+. Addition of Co2+ gave a different result, but there was no paramagnetic shift of the H2 histidine B10 resonance, probably because of rapid exchange at the binding site. Addition of Cd2+ and of Cd2+ and Ca2+ produced changes that were similar to each other but were different from those observed on addition of Zn2+, probably due to the binding of Cd2+ and Ca2+ at glutamate B13.

1H n.m.r. studies of insulin. Reversible transformation of 2-zinc to 4-zinc insulin hexamer.

1H n.m.r. studies at 270 MHz were made of the transformation of 2 Zn insulin hexamer to 4 Zn hexamer produced by the addition of anions (thiocyanate ion). Four separate H2 histidine resonances were observed for the B5 and B10 histidines in 2 Zn hexamer at pH 7 and 9 and four separate resonances also occurred in the 4 Zn hexamer. The observation of these resonances and others from phenylalanine, tyrosine and leucine residues showed that the 2 Zn to 4 Zn transformation probably occurred in solution in a similar manner to that observed in the crystal. Furthermore as occurred in the crystal, it was found that in solution the transformation was reversible (on removal of thiocyanate) and that 2 Cd insulin was unable to undergo the transformation. Des-Phe-Bl-insulin did not undergo the transformation. Addition of SCN- to Zn-free insulin (mainly dimer) produced only a small transformation, consistent with the idea that Zn2+ promotes formation of hexamer from dimer but probably does not otherwise affect the transformation.

1H n.m.r. studies of insulin. Assignment of resonances and properties of tyrosine residues.

The assignment of the aromatic 1H n.m.r. resonances of the four tyrosine residues of bovine 2-zinc insulin is reported, based on double resonance techniques, use of Hahn spin echo pulse sequences and examination of specific derivatives nitrated at tyrosines A14 and A19 as well as des-(B26-B30)-insulin. Titration curves of the four tyrosine residues show that residues A14 and B16 have normal pK' values of 10.3-10.6 in solution, consistent with their accessibility to solvent in monomer and dimer in the crystal. Tyrosine residues A19 and B26 have pK' values of 11.4 and exhibit other features in their titration curves that are consistent with limited accessibility to solvent and a nonpolar environment. The meta protons of residues B16 and B26 both observe the titration of a nearby tyrosine residue, probably A19. Interpretation of the n.m.r. data obtained in solution is consistent with the crystallographic data for the monomer and dimer obtained on insulin crystals [Blundell, Dodson, Hodgkin & Mercola (1972) Adv. Protein Chem. 26, 279-402].

Analyses of vegetables from the highlands of Papua New Guinea.

Ten vegetable foods of the highlands were analysed for crude protein, amino acids and trypsin inhibitor. Pandanus nut, lima bean, fern, greens, pitpit and choko had good to acceptable crude protein contents and the proteins of fern, pitpit, Rungia klossii and Indian mustard were of good quality. Lima beans had a very high content of trypsin inhibitor and all others had small or zero amounts, including fern, which is used at pig feasts and hence does not contribute to pig bel.

Digestibility of proteins of the histological components of cooked and raw rice.

The aleurone layer, grain coat and embryo which constitute rice bran are rich in vitamins, lipids, protein and lysine compared with the endosperm (milled rice). A method was developed to measure the in vitro protein digestibility of raw and cooked brown rice and their histological components. The protein digestibility of cooked endosperm by the in vitro method agreed with that of other workers using in vivo techniques. The protein digestibility of the aleurone layer and grain coat from raw rice was only 25% but increased to 65% from cooked rice, due to disruption of the cellulosic cell walls at 100 degrees, which was shown by electron microscopy. The decreased protein digestibility due to cooking was not the result of formation of epsilon-lysyl-gamma-glutamyl isopeptide cross-links, but may be due to formation of a cystine-rich core that is resistant to proteases. The protein digestibility of cooked brown rice was approximately the same as that of cooked milled rice, hence it is advantageous for those for whom rice is a staple food to consume brown rather than milled rice.

Conformational differences between various myoglobin ligated states as monitored by 1H NMR spectroscopy.

In paramagnetic metmyoglobin, cyanomyoglobin (CNMb), and deoxymyoglobin, His-36 has a high pK (approximately 8), and the NMR titration behavior of the H-2 resonance is perturbed, due to the presence at low pH of a hydrogen bond with Glu-38, which is broken at high pH. The His-36 H-4 resonance shows no shift with pK approximately 8 because of two opposing chemical shift effects but monitors the titration of nearby Glu-36 (pK = 5.6). In diamagnetic derivatives [(carbon monoxy)myoglobin (COMb) and oxymyoglobin (oxyMb)], the titration behavior of His-36 H-2 and H-4 resonances is normalized (pK approximately 6.8). The very slight alkaline Bohr effect in sperm whale myoglobin (Mb) is interpreted in terms of the pK change of His-36 from deoxyMb to oxyMb and compensating pK changes in the opposite direction of other unspecified groups. In sperm whale COMb at 40 degrees C, the distal histidine (His-64) and His-97 have pK values of 5.0 and 5.9. The meso proton resonances remote from these groups do not show a titration shift, but the nearby gamma-meso proton (pK = 5.3) responds to titration of both histidines, and the upfield Val-68 methyl at -2.3 ppm (pK = 4.7) witnesses the titration of nearby His-64. At 20 degrees C, the latter resonance is reduced in size, and a second resonance occurs at -2.8 ppm, which is insensitive to pH and, hence, more remote from His-64. Both resonances arise from two conformations of Val-68 in slow equilibrium. In oxyMb at 20 degrees C, only the latter resonance is observed, presumably because of the steric restrictions imposed by the hydrogen bond between ligand and His-64 in oxyMb, which is not present in COMb. In oxyMb the pK of His-97 (5.6) is similar to that of the meso proton resonances (5.5) and to the pK of other pH-dependent processes, including the very small acid Bohr effect. It is likely that these processes are controlled by the titration of His-97.(ABSTRACT TRUNCATED AT 250 WORDS)

Assignment of 1H NMR resonances of histidine and other aromatic residues in met-, cyano-, oxy-, and (carbon monoxy)myoglobins.

The resolved 1H NMR resonances of the aromatic region in the 270-MHz NMR spectrum of sperm whale, horse, and pig metmyoglobin (metMb) have been assigned, including the observable H-2 and H-4 histidine resonances, the tryptophan H-2 resonances, and upfield-shifted resonances from one tyrosine residue. The use of different Mb species, carboxymethylation, and matching of pK values allows the assignment of the H-4 resonances, which agree in only three cases out of seven with scalar-correlated two-dimensional NMR spectroscopy assignments by others. The conversion to hydroxymyoglobin at high pH involves rearrangements throughout the molecule and is observed by many assigned residues. In sperm whale ferric cyanomyoglobin, nine H-2 and eight H-4 histidine resonances have been assigned, including the His-97 H-2 resonance and tyrosine resonances from residues 103 and 146. The hyperfine-shifted resonances from heme and near-heme protons observe a shift with a pK = 5.3 +/- 0.3 (probably due to deprotonation of His-97, pK = 5.6) and another shift at pK = 10.8 +/- 0.3. The spectrum of high-spin ferrous sperm whale deoxymyoglobin is very similar to that of metMb, which allows the assignment of seven surface histidine H-2 and H-4 resonances and also resonances from the two tryptophan residues and one tyrosine. In diamagnetic sperm whale (carbon monoxy)myoglobin (COMb), 10 His H-2 and 11 His H-4 resonances are observed, and 8 H-2 and 9 H-4 resonances are assigned, including His-64 H-4, the distal histidine. This important resonance is not observed in sperm whale oxymyoglobin, which in general shows very similar titration curves to COMb. Histidine-36 shows unusual titration behavior in the paramagnetic derivatives but normal behavior in the diamagnetic derivatives, which is discussed in the accompanying paper [Bradbury, J. H., & Carver, J. A. (1984) Biochemistry (following paper in this issue)].

Determination of the structures of trisaccharides by 13C-n.m.r. spectroscopy.

A literature survey of the 13C-n.m.r. chemical-shift data for aqueous solutions of monosaccharides, disaccharides, oligosaccharides, and their methyl derivatives is reported. Analysis of these data reveals a set of empirical rules which may be used in the elucidation of the structure of trisaccharides of known monosaccharide composition, and an example is reported. However, it is not possible to extend the rules to tetrasaccharides and higher saccharides without additional chemical-shift data for related model compounds.

Leghemoglobin. Kinetic, nuclear magnetic resonance, and optical studies of pH dependence of oxygen and carbon monoxide binding.

The rate of dissociation of oxygen from soybean oxyleghemoglobin a increases about 5-fold between pH 4 and pH 7, with apparent pK = 5.46 and n = 1. The rate of dissociation of carbon monoxide from carbon monoxyleghemoglobin a and the rates of combination of oxygen and carbon monoxide with ferrous leghemoglobin a are all invariant in this range of pH. The optical spectrum of oxyleghemoglobin in the visible region and the resonances of the four heme meso protons (protons of the bridge carbon atoms) in the NMR spectrum of oxyleghemoglobin are also dependent on pH with pK near 5.5. We suggest that protonation of the imidazole of the distal histidine residue (His 61) leads to formation of a hydrogen bond to the bound oxygen molecule which affects the electronic configuration or conformation of the heme and decreases the rate of oxygen dissociation.