Absorption of zinc and retention of calcium: Dose-dependent inhibition by phytate

The dose-dependent inhibitory effect of sodium phytate (myo-inositol-hexaphosphate) on absorption of zinc and retention of calcium was studied in man. No systematic study of this dose-response effect has been reported to this time. Forty subjects were served meals containing white wheat rolls without/with additions of phytate. Ten subjects were given test meals containing one or two of the studied levels of phytate and in addition all subjects were served meals to which no phytate was added. The zinc content was 3.1 mg (47 micromol) and the calcium content 266 mg (6.6 mmol). The rolls were labelled extrinsically with radioisotopes, 65Zn and 47Ca, and whole-body retention of both minerals was measured. Totally 105 meals were served, 36 meals in which no phytate was added and 9-10 meals on each level of phytate. The zinc absorption in meals to which either 0, 25, 50, 75, 100, 140, 175 or 250 mg of phytate-P (0, 134, 269, 403, 538, 753, 941 or 1344 micromol phytate) had been added was 22%, 16%, 14%, 11%, 7%, 7%, 7% and 6%, respectively (mean values). The addition of 50 mg phytate-P or more significantly decreased zinc absorption (p=0.01) as compared to absorption from the test meals with no added phytate. The calcium retention at day 7 in the same meals was 31%, 28%, 27%, 26%, 22%, 19%, 14% and 11% (mean values). The addition of 100 mg phytate-P or more significantly decreased calcium retention (p=0.03) compared to the test meals with no added phytate. It was concluded that the inhibitory effect of phytate on the absorption of zinc and the retention of calcium was dose dependent.     

Structural and Functional Characterization of an Anesthetic Binding Site in the Second Cysteine-Rich Domain of Protein Kinase Cδ?

Elucidating the principles governing anesthetic-protein interactions requires structural determinations at high resolutions not yet achieved with ion channels. Protein kinase C (PKC) activity is modulated by general anesthetics. We solved the structure of the phorbol-binding domain (C1B) of PKCδ complexed with an ether (methoxymethylcycloprane) and with an alcohol (cyclopropylmethanol) at 1.36-Å resolution. The cyclopropane rings of both agents displace a single water molecule in a surface pocket adjacent to the phorbol-binding site, making van der Waals contacts with the backbone and/or side chains of residues Asn-237 to Ser-240. Surprisingly, two water molecules anchored in a hydrogen-bonded chain between Thr-242 and Lys-260 impart elasticity to one side of the binding pocket. The cyclopropane ring takes part in π-acceptor hydrogen bonds with the amide of Met-239. There is a crucial hydrogen bond between the oxygen atoms of the anesthetics and the hydroxyl of Tyr-236. A Tyr-236-Phe mutation results in loss of binding. Thus, both van der Waals interactions and hydrogen-bonding are essential for binding to occur. Ethanol failed to bind because it is too short to benefit from both interactions. Cyclopropylmethanol inhibited phorbol-ester-induced PKCδ activity, but failed to do so in PKCδ containing the Tyr-236-Phe mutation.     

Short-term effect of different teaching methods on nasopharyngeal carcinoma for general practitioners in Jakarta, Indonesia

In Indonesia, Nasopharyngeal Carcinoma (NPC) is the most frequent cancer of the head and neck region. At first presentation in the hospital most patients already have advanced NPC. Our previous study showed that general practitioners (GPs) working in Yogyakarta, Indonesia lack the knowledge necessary for early detection of NPC. By providing training on early symptoms of NPC we hope that the diagnosis and referral will occur at an earlier stage. Here we assess the current NPC knowledge levels of GPs in Jakarta, evaluate improvement after training, compare the effectiveness of two training formats, and estimate the loss of recall over a two week period.

Methods

Two Indonesian GPs visited 31 Primary Health Care Centres (PHCCs) and provided a lecture on NPC. The alternative format consisted of a symposium at the Universitas Indonesia, Jakarta, presented by local head and neck surgeons, with all GPs in the region being invited. To evaluate the effect of both formats a questionnaire was conducted before and after.

Results

The lecture in the PHCCs was attended by 130 GPs. Sixty-six GPs attended the training in the university hospital and 40 GPs attended both. Pre training the NPC knowledge level was poor with an average of 1.6 symptoms being correctly identified out of a potential maximum of 12, this was increased to 4.9 post training (p<0.0001). GPs attending the PHCC course recorded a greater increase in correct symptoms than those attending the symposium (3.8 vs. 2.8; p = 0.01). After a two week period the knowledge levels had declined slightly from 5.5 correctly identified symptoms to 4.2 (p = 0.25).

Conclusion

These results confirm our findings regarding GPs insufficient knowledge of NPC. Lectures in the PHCC and a symposium have both been proven to be effective training tools in the education of GPs.     

SEA domain autoproteolysis accelerated by conformational strain: mechanistic aspects

A subclass of SEA (sea urchin sperm protein, enterokinase, and agrin) domain proteins undergoes autoproteolysis between glycine and serine in a conserved G^− 1S^+ 1VVV motif to generate stable heterodimers. Autoproteolysis has been suggested to involve only the intramolecular catalytic action of the conserved serine hydroxyl in combination with conformational strain of the glycine-serine peptide bond. We conducted a number of experiments and simulations on the SEA domain from the MUC1 mucin to test this mechanism. Alanine-scanning mutagenesis of polar residues in the vicinity of the cleavage site demonstrates that only the nucleophile at position + 1 is required for efficient proteolysis. Molecular modeling shows that an uncleaved trans peptide is incompatible with the native heterodimeric structure, resulting in disruption of secondary structure elements and distortion of the scissile peptide bond. Insertion of glycine residues (to obtain G_nG^− 1S^+ 1VVV motifs) appears to relieve strain, and autoproteolysis is 100 times slower in a 1G (n = 1) mutant and not measurable in 2G and 4G mutants. Removal of the catalytic serine hydroxyl hampers cleavage considerably, but measurable autoproteolysis of this S1098A mutant still proceeds in the presence of strain alone. The uncleaved SEA precursor populates interconverting partially folded conformations, and autoproteolysis coincides with adoption of proper β-sheet secondary structure and completed folding. Molecular dynamics simulations of the precursor show that the serine hydroxyl and the preceding glycine carbonyl carbon can be in van der Waals contact at the same time as the scissile peptide bond becomes strained. These observations are all consistent with autoproteolysis accelerated by N → O acyl shift and conformational strain imposed upon protein folding in a reaction for which the free-energy barrier is decreased by substrate destabilization rather than by transition-state stabilization. The energetics of this coupled folding and autoproteolysis mechanism is accounted for in an accompanying article.     

SEA domain autoproteolysis accelerated by conformational strain: energetic aspects

A subclass of proteins with the SEA (sea urchin sperm protein, enterokinase, and agrin) domain fold exists as heterodimers generated by autoproteolytic cleavage within a characteristic G^− 1S^+ 1VVV sequence. Autoproteolysis occurs by a nucleophilic attack of the serine hydroxyl on the vicinal glycine carbonyl followed by an N → O acyl shift and hydrolysis of the resulting ester. The reaction has been suggested to be accelerated by the straining of the scissile peptide bond upon protein folding. In an accompanying article, we report the mechanism; in this article, we provide further key evidence and account for the energetics of coupled protein folding and autoproteolysis. Cleavage of the GPR116 domain and that of the MUC1 SEA domain occur with half-life (t_½) values of 12 and 18 min, respectively, with lowering of the free energy of the activation barrier by ∼ 10 kcal mol^− 1 compared with uncatalyzed hydrolysis. The free energies of unfolding of the GPR116 and MUC1 SEA domains were measured to ∼ 11 and ∼ 15 kcal mol^− 1, respectively, but ∼ 7 kcal mol^− 1 of conformational energy is partitioned as strain over the scissile peptide bond in the precursor to catalyze autoproteolysis by substrate destabilization. A straining energy of ∼ 7 kcal mol^− 1 was measured by using both a pre-equilibrium model to analyze stability and cleavage kinetics data obtained with the GPR116 SEA domain destabilized by core mutations or urea addition, as well as the difference in thermodynamic stabilities of the MUC1 SEA precursor mutant S1098A (with a G^− 1A^+ 1VVV motif) and the wild-type protein. The results imply that cleavage by N → O acyl shift alone would proceed with a t_½ of ∼ 2.3 years, which is too slow to be biochemically effective. A subsequent review of structural data on other self-cleaving proteins suggests that conformational strain of the scissile peptide bond may be a common mechanism of autoproteolysis.     

Stimulated efflux of amino acids and glutathione from cultured hippocampal slices by omission of extracellular calcium: likely involvement of connexin hemichannels

Omission of extracellular Ca(2+) for 15 min from the incubation medium of cultured hippocampal slices stimulated the efflux of glutathione, phosphoethanolamine, hypotaurine, and taurine. The efflux was reduced by several blockers of gap junctions, i.e. carbenoxolone, flufenamic acid, and endothelin-1, and by the connexin43 hemichannel blocking peptide Gap26 but was unchanged by the P2X(7) receptor inhibitor oxidized ATP, a pannexin1 hemichannel blocking peptide and an inactive analogue of carbenoxolone. Pretreatment of the slices with the neurotoxin N-methyl-d -aspartate left the efflux by Ca(2+) omission unchanged, indicating that the stimulated efflux primarily originated from glia. Elevated glutamate efflux was detected when Ca(2+) omission was combined with the glutamate uptake blocker l-trans-pyrrolidine-2,4-dicarboxylate and when both Ca(2+) and Mg(2+) were omitted from the medium. Omission of Ca(2+) for 15 min alone did not induce delayed toxicity, but in combination with blocked glutamate uptake, significant cell death was observed 24 h later. Our results indicate that omission of extracellular Ca(2+) stimulates efflux of glutathione and specific amino acids including glutamate via opening of glial hemichannels. This type of efflux may have protective functions via glutathione efflux but can aggravate toxicity in situations when glutamate reuptake is impaired, such as following a stroke.     

Precision and accuracy of indole-3-acetic acid analyses performed with the 2-methylindolo-α-pyrone fluorescence assay and with a high performance liquid chromatography technique with spectrofluorimetric detection, exemplified on pine tissue (Pinus sylvestris L.)

The precision and accuracy of two versions of the 2-methylindolo-α-pyrone (2-MIP) assay and of a high performance liquid chromatography (HPLC) technique was assessed when applied to analysis of indole-3-acetic acid (IAA) in pine ( Pinus sylvestris ) tissue. In one version of the 2-MIP assay total fluorescence at wavelengths > 450 nm was recorded, whereas in the other version fluorescence spectra were recorded and analyzed. Such analysis revealed the occurrence in some samples of fluorescent contaminants that impaired both precision and accuracy. The precision and accuracy of the 2-MIP assay could be improved by additional sample purification, and the over-all accuracy of the assay was finally verified, at a high level of sample purity, by successive approximation. The precision of the assay was then 14%. Successive approximation also verified that the HPLC technique was accurate at a less advanced stage of sample purity than the 2-MIP assay, and had a precision of 14%.