Co-operative mineralization and protein self-assembly in amelogenesis: silica mineralization and assembly of recombinant amelogenins in vitro

An amorphous silica mineralization technique was used to produce inorganic/protein composites to elucidate the structure and mechanism of formation of amelogenin assemblies, which may play an important role in regulating enamel structure during the initial stages of amelogenesis. Full-length recombinant amelogenins from mouse (rM179) and pig (rP172) were investigated along with key degradation products (rM166 and native P148) lacking the hydrophilic C terminus found in parent molecules. The resulting products were examined using transmission electron microscopy and/or small-angle X-ray scattering. Using protein concentrations of 0.1–3 mg ml⁻¹, large monodisperse spheres of remarkably similar mean diameters were observed using rM179 (124 ± 4 nm) and rP172 (126 ± 7 nm). These spheres also exhibited 'internal structure', comprising nearly spherical monodisperse particles of ≈ 20 nm in diameter. In the presence of rM166, P148, and bovine serum albumin (control), large unstructured and randomly shaped particles (250–1000 nm) were observed. Without added protein, large dense spherical particles of silica (mean ≈ 500 nm) lacking internal structure were produced. These findings demonstrate that full-length amelogenins have the ability to form higher-order structures, whereas amelogenins that lack the hydrophilic C terminus do not. The results also suggest that full-length amelogenin can guide the formation of organized mineralized structures through co-operative interactions between assembling protein and forming mineral.     

Paediatric dacryocystorhinostomy

Of 258 cases of dacryocystorhinostomy performed on children in the period September 1981 to September 1991, 130 were for simple, unresolved congenital nasolacrimal duct obstruction. Other indications for surgery included punctal agenesis, lacrimal fistula, post-traumatic and post-inflammatory canalicular obstruction. Of 177 children without canalicular pathology, 171 (96%) were relieved of symptoms with one operation, without canalicular intubation. Of 81 cases with canalicular disease, 55 of 70 (79%) who underwent DCR plus canalicular intubation, and 10 of 11 who underwent DCR plus Lester-Jones tube, were substantially improved with one operation. No child required peroperative or postoperative blood transfusion. Dacryocystorhinostomy in childhood, in experienced surgical hands, is a safe procedure, achieving relief of symptoms in most cases, particularly in the absence of canalicular disease.     

The effect of supplementation with fish oil during pregnancy on breast milk immunoglobulin A, soluble CD14, cytokine levels and fatty acid composition

BACKGROUND: Breast milk contains many immunomodulatory factors (soluble CD14 (sCD14), IgA and cytokines) with the potential to influence infant immune development. OBJECTIVE: To determine if changes in breast milk omega-3 polyunsaturated fatty acid (n-3 PUFA) composition as a result of maternal dietary fish oil supplementation during pregnancy can modify levels of these immunological parameters in breast milk. METHOD: In a randomized controlled trial, 83 atopic women received either 4 g fish oil capsules (containing 3.7 g n-3 PUFA) (n = 40) or 4 g olive oil capsules (n = 43) from 20 weeks gestation until delivery. Breast milk was collected 3 days post-partum and fatty acids were analysed by gas liquid chromatography and IgA, sCD14 and cytokines (IL-5, IL-6, IL-10, TNF-alpha and IFN-gamma) were quantitated by ELISA or time resolved fluorescence (TRF). RESULTS: Omega-3 docosahexaenoic acid (DHA; 22:6n-3) and eicosapentaenoic acid (EPA; 20:5n-3) levels were significantly higher (P < 0.001) in breast milk from women supplemented with fish oil (n = 33, DHA mean 1.15%, SD 0.47% and EPA mean 0.16%, SD 0.07%) than in samples from the control group (n = 40, DHA mean 0.50%, SD 0.17% and EPA mean 0.05%, SD 0.02%). Breast milk arachidonic acid (AA; 20:4n-6) levels were significantly lower (P = 0.045) in the fish oil group (mean 0.55%, SD 0.12%) compared with the control group (mean 0.61%, SD 0.14%). Breast milk IgA was positively correlated with DHA (P = 0.046) and 22:5n-3 (P = 0.003), but inversely correlated with linoleic acid (LA; 18:2n-6) (P=0.034). Levels of sCD14 were also positively correlated with 22:5n-3 (P=0.009). Cytokines involved in IgA synthesis (IL-10 and IL-6) were also significantly correlated with both IgA and n-3 PUFA levels, although there were no differences in the levels of breast milk IgA, sCD14 or cytokines between study groups. CONCLUSION: Supplementation with fish oil during pregnancy significantly alters early post-partum breast milk fatty acid composition. omega-3 PUFA levels were positively associated with IgA and sCD14 levels, suggesting a relationship between fatty acid status and mucosal immune function.     

Effect of the method of breast feeding on breast engorgement, mastitis and infantile colic

This exploratory study compared the effect of two methods of breast feeding on breast engorgement, mastitis, infantile colic and duration of breast feeding. An opportunity sample of subjects was assigned either to the experimental group (prolonged emptying of one breast at each feed) (n = 150) or to the control group (both breasts equally drained at each feed) in = 152) and both groups were followed prospectively to 6 months after delivery. The experimental group had a lower incidence of breast engorgement in the first week (61.4% versus 74.3%; p < 0.02) and colic over the first 6 months (12% versus 23.4%; p < 0.02). There was no significant difference between the two groups in the incidence of mastitis over 6 months and the length of breast feeding (16.510.3 weeks versus 17.510 weeks experimental versus control group). The majority of mothers in the experimental group (63%) felt it necessary to offer the second breast at the end of a feed to satisfy their infant's hunger. The "perceived insufficient milk supply syndrome" was the main reason given for cessation of breast feeding in both groups. This study provides data to advise nursing mothers about these two methods of breast feeding. Breast engorgement, breast feeding methods, foremilk, hindmilk, infantile colic, mastitis
K Evans, Department of Nursing (ward 4C), Flinders Medical Centre, Bedford Park, South Australia 5042, Australia     

Biochemical characterization of recombinant mouse amelogenins: protein quantitation, proton absorption, and relative affinity for enamel crystals

Four recombinant mouse amelogenins, which varied by the presence or absence of the exon 4 encoded segment as well as the carboxyl-terminus were heterologously expressed and purified from bacteria. The rM193 and rM179 contain the carboxyl-terminus, whereas the rM180 and rM166 do not. The rM193 and rM180 contain the polypeptide segment encoded by exon 4 of the amelogenin gene. A precisely weighed sample of purified rM179 was quantified by Lowry, Bicinchoninic Acid and Bradford assays. It was determined that these protein quantification methods characteristically under or overestimate the amount of amelogenin. The calculated correction factors were: Lowry (x 1.35), BCA (x 1.96), and Bradford (x 0.78). Recombinant mouse amelogenin (rM179) was characterized with respect to its hydrogen ion binding properties. The protein absorbs 11.9 +/- 1.7 protons during a pH change from 8.0 to 5.0, suggesting that amelogenins buffer the enamel fluid in vivo. Crystal binding experiments were performed using rM193, rM180, rM179 and rM166. The carboxyl-terminus enhanced the binding of amelogenin to enamel crystals while the exon 4 encoded segment did not appreciably affect crystal binding.     

The effect of anti-CD4 on helper function of CD4,45RA+ versus CD4,45RO+ T cells.

Here we have investigated and compared the effects of anti-CD4 on helper function of CD4,45RA+ versus CD4,45RO+ T cells. Only CD4,45RO+ cells, but not CD4,45RA+ cells were able to promote B cell differentiation resulting in immunoglobulin production in vitro (IgM as well as IgG) which could be inhibited by anti-CD4 MoAbs (MAX.16H5 and T151). In pokeweed mitogen (PWM)-induced B cell proliferation a similar pattern of responsiveness was obtained. When we studied the anti-CD4 effects on cytokine production in T cells stimulated in mixed lymphocyte reaction (MLR) or by mitogens, we found that neither IL-2 nor IL-4 production was dramatically influenced by anti-CD4 in CD4,45RO+ cells. This led us to the conclusion that the inhibitory effect of anti-CD4 on B cell proliferation and immunoglobulin secretion was not due to inhibition of cytokine production. To clarify this point, we investigated the ability of anti-CD4 to inhibit conjugate formation between B and T cells. It was found that CD4,45RO+ T cells formed more conjugates than CD4,45RA+ cells, and that only the conjugate formation by CD4,45RO+ T cells was inhibited by anti-CD4. These results suggest that (i) anti-CD4 inhibits T helper functions primarily by affecting CD4,45RO+ cells, and (ii) this effect is probably mediated by contact inhibition in the early phase of T-B collaboration.