Production of safe therapeutic feeds from contaminated water supplies.

We have assessed the effectiveness of using an osmotic sachet for safe rehydration of therapeutic milk from contaminated water supplies in a poor urban area of Bangladesh. 35 women were given sachets to hydrate in their homes and comparison of the hydration water and the reconstituted milk showed that the high bacterial contamination reported in the domestic water supply was removed by use of the sachet. The mean rehydration time was 4.5 h (range 3.4-5.5). This rehydration method could have a role in the preparation of therapeutic feeds where microbiological contamination of the environment is a serious problem and availability of adequate water is not a limiting factor.

Poor nutrition is a serious problem in children with cerebral palsy in Palawan, the Philippines.

Children with cerebral palsy (CP) in developed countries have poor nutritional status; however there is little data from developing countries. In Palawan, in the Philippines, the nutritional status of 31 children with CP was compared to that of their siblings (n = 20) and a control group of neighbourhood children (n = 64), matched for age and sex. The children's weights, heights and armspans were measured. The heights of children with CP could not be measured and were estimated from their armspans using an equation relating height to armspan in siblings and controls. Haemoglobin levels of the study cases and siblings were measured. Siblings and controls had similar nutritional status. The children with CP had extremely poor nutritional status, and had significantly smaller weights for height, heights for age and weights for age than siblings or controls. Haemoglobin levels were not significantly different between the children with CP and their siblings. The nutritional status of children with quadriplegic CP was much poorer than that of similar children in the USA. The severity of malnutrition in children with CP is likely to be detrimental to their development, and a nutritional component should be incorporated into rehabilitation programmes. Also, there is a need to examine the nutritional status of children with CP in other developing countries.

Nutritional status and age at menarche in a rural area of Bangladesh.

The age at menarche and its association with nutritional status in a rural area of Bangladesh was determined. A cross-sectional study was conducted in four villages of Rupganj Thana of Narayanganj district. Data was collected through October to December 1996 using a pre-tested structured questionnaire interview schedule, and nutritional status was measured by weight, height, body mass index (BMI) and physical examination. Data were obtained on 436 adolescent girls aged 10-17 years. Among them, 165 (37.8%) girls had commenced menarche. The mean age at menarche as determined by retrospective recall was 13 years SD 0.89 (n = 165). The median age at menarche determined by the status quo method was 13.0. Among the adolescents 60.1% were thin (BMI < 5th centile WHO recommended reference) and 48.2% were stunted (< 3rd centile NCHS/WHO). The mean weight and BMI were significantly higher among the menstruating girls of 13, 14 and 15 years (p < 0.01) than non-menstruating girls. The mean height was found to be significantly higher at 11-14 years among the menstruating girls (p < 0.05). A lower prevalence of angular stomatitis was found among the menstruating adolescent girls compared with the non-menstruating girls, 36.4% versus 46.5%, although this was statistically non-significant (odds ratio = 0.66, 95% CI 0.43-1.00). For glossitis, no significant difference was found. Among the menstruating girls 12.1% were suffering from menorrhagia and 31.5% from dysmenorrhoea. We conclude that the age of menarche among this rural Bangladeshi community is not as delayed as expected. Not surprisingly, menarche is associated with better nutritional status. The surveyed population had extremely high rates of undernutrition which suggests that adolescents in this and similar situations require specific intervention programmes to improve their nutritional status.

Effects of memantine on recombinant rat NMDA receptors expressed in HEK 293 cells.

1. The actions of the uncompetitive N-methyl-D-aspartate (NMDA) receptor antagonists, memantine (1-amino-3,5-dimethyladamantane) and (+)-MK-801 ((+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine maleate, dizocilpine), on recombinant NMDA receptors has been studied by use of the whole-cell patch clamp technique. 2. Human embryonic kidney (HEK) 293 cells were transiently transfected with different NMDA receptor subunit combinations (NR1a/NR2A, NR1a/NR2B and NR1a/NR2D). A mutant form of the green fluorescent protein (GFP) cotransfected with the NMDA receptor subunits to enable the visualization of transfected cells. 3. Memantine (0.3-30 microM) blocked L-glutamate (100 microM)-mediated currents in a concentration-dependent manner in NR1a/NR2A, NR1a/NR2B and NR1a/NR2D transfected cells with IC50 values (at -70 mV) of 0.93 +/- 0.15 microM, 0.82 +/- 0.12 microM and 0.47 +/- 0.06 microM (mean +/- s.c. mean), respectively. 4. The memantine-induced block was strongly voltage-dependent. Alteration of the holding potential from -70 mV to +60 mV resulted in an e-fold increase in the IC50 values per 30-33 mV change in membrane potential, for all 3 subunit combinations investigated. 5. The kinetics of the actions of memantine (30 microM) were investigated for the NR1a/2A combination, in 6 cells (13-15 determinations). At -70 mV, the block and recovery from block were both best described by two exponentials with time-constants of 201 +/- 23 ms (81 +/- 2%) and 3.9 +/- 0.6 s and 597 +/- 94 ms (18 +/- 1%) and 18.6 +/- 2.4 s, respectively. The predominant effect of depolarization was to increase the weight of the faster recovery time-constant. Kinetic analysis suggests that these results are consistent with previously proposed Markov models. 6. (+)-MK-801 was studied briefly for comparative purposes. (+)-MK-801 (200 nM) preferentially blocked NMDA receptor currents (at -70 mV) in NR1a/NR2A and NR1a/NR2B (82 +/- 10% and 93 +/- 2% depressions) compared to NR1a/NR2D (38 +/- 7%) transfected cells. (+)-MK-801 appeared to be less voltage-dependent than memantine on all three receptor combinations. 7. In conclusion, memantine was a voltage-dependent antagonist of recombinant rat NMDA receptors expressed in HEK 293 cells but showed little selectivity between the subunits investigated. Its actions on these recombinant receptor combinations are similar to its actions on native NMDA receptors.

An investigation of the membrane topology of the ionotropic glutamate receptor subunit GluR1 in a cell-free system.

We have utilized cell-free translation in rabbit-reticulocyte lysate supplemented with canine pancreatic microsomal membranes to study the processing and membrane topology of the rat ionotropic glutamate receptor subunit GluR1. In vitro-synthesized RNA encoding GluR1 was translated to yield a primary translation product with an apparent molecular mass of 99 kDa. In the presence of microsomal membranes this protein was processed to give a band of 107 kDa. Treatment with peptide-N-glycosidase F showed that this increase in molecular mass was due to N-linked glycosylation. Incubation of the processed receptor with proteinase K revealed the presence of a 68 kDa protease-resistant band which decreased to 56 kDa following deglycosylation. A deletion mutant (GluR1M1) lacking the predicted transmembrane domains was fully translocated across the microsomal membrane and protected from the action of the protease. The mutant and wild-type receptor could be immunoprecipitated by anti-peptide antibodies directed against the C-terminus. Following translocation of the wild-type and mutant receptor across the microsomal membrane and treatment with proteinase K the antibody binding to GluR1 was abolished, but was retained for GluR1M1. These data allow identification of the orientation of the N- and C-termini of GluR1 within the microsome; results which are consistent with an extracellular N-terminal and intracellular C-terminal localization following incorporation into the plasma membrane.

In vitro translation and membrane topology of rat recombinant mGluR1 alpha.

The structure and post-translational processing of the metabotropic glutamate receptor 1 alpha (mGluR1 alpha) was analysed by in vitro cell-free translation, protease protection and deglycosylation. We show that mGluR1 alpha can be synthesized in the rabbit-reticulocyte translation system to yield a predominant polypeptide product with an apparent molecular weight of 142 kDa. In the presence of dog-pancreatic microsomes this polypeptide was processed to an apparent molecular weight of 147 kDa. Treatment with the enzyme peptide-N-glycosidase F (PNGF) demonstrated that the increase in the apparent molecular weight of the processed translation product was due to N-linked glycosylation. Addition of the non-selective protease, proteinase K; resulted in the loss of this 147 kDa band and the appearance of a protected fragment of approx 92 kDa. A carboxy-terminal deletion mutant of mGluR1 alpha was almost completely protected from protease action. These data show that the amino terminal of mGluR1 alpha is translocated into the lumen of the endoplasmic reticulum and will consequently be located extracellularly when targeted to the plasma membrane. The data presented here on mGluR1 alpha indicates the potential of in vitro translation and protease protection in the study of the molecular structure and processing of glutamate receptors.

Autocatalytic maturation of the prohormone convertase PC2.

PC2 is a member of the eukaryotic family of subtilisin-like proteases, which is thought to participate in the processing of prohormones and proneuropeptides in neuroendocrine cells. PC2 is synthesized as a 69-kDa prepropolypeptide. The NH2-terminal signal sequence is removed during segregation within the endoplasmic reticulum, where glycosylation occurs to generate a 75-kDa propolypeptide. A combination of site-directed mutagenesis and a cell-free translation/translocation system from Xenopus eggs was used to investigate the processing of the pro-PC2 precursor. The 75-kDa polypeptide underwent slow cleavage after the sequence Arg-Lys-Lys-Arg84 to generate a 68-kDa mature enzyme. Cleavage was blocked when the tetrabasic sequence was deleted (PC2M3) or when the active site Asp142 was changed to Asn (PC2M4). This latter observation suggested that cleavage of the 75-kDa propolypeptide to the mature 68-kDa enzyme was autocatalytic. Incubation of the PC2M4 mutant with the wild type PC2 precursor resulted in cleavage of both the wild type polypeptide and the catalytically inactive PC2M4 mutant. This indicates that cleavage could occur through an intermolecular reaction. The results also demonstrate that the novel Xenopus egg extract translation/translocation system represents a powerful cell-free method for studying proteolytic processing of propolypeptides.

Induction of LTP in the hippocampus needs synaptic activation of glutamate metabotropic receptors.

Understanding the mechanisms of long-term potentiation (LTP) should provide insights into the molecular basis of learning and memory in vertebrates. Ionotropic glutamate receptors play a central role in LTP; AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate) receptors and NMDA (N-methyl-D-aspartate) receptors mediate synaptic responses that are enhanced in LTP and, in addition, NMDA receptors are necessary for the induction of LTP in most pathways. There is also circumstantial evidence that metabotropic glutamate receptors (mGluRs) may be involved in LTP because the specific mGluR agonist aminocyclopentane dicarboxylate can augment tetanus-induced LTP2 and, under certain circumstances, can itself induce a slow-onset potentiation. But the absence of any effective mGluR antagonist has prevented the determination of whether mGluRs are involved in the induction of tetanus-induced LTP. We report here that (RS)-alpha-methyl-4-carboxyphenylglycine is a specific mGluR antagonist in the hippocampus and have used this compound to examine the nature of the involvement of mGluRs in LTP. We show that synaptic activation of mGluRs is necessary for the induction of both NMDA receptor-dependent and NMDA receptor-independent forms of LTP in the hippocampus.

A member of the eukaryotic subtilisin family (PC3) has the enzymic properties of the type 1 proinsulin-converting endopeptidase.

PC3, a mammalian homologue of the yeast subtilisin-like proteinase Kex2, was expressed in Xenopus oocytes and its activity was characterized. PC3 cleaved human proinsulin at one of the two dibasic sites (KTRR32 but not LQKR65). The specificity, inhibitor profile, pH optimum (5.5) and Ca(2+)-dependence (K0.5 = 2.5-3 mM) paralleled those of the insulin-granule type 1 endopeptidase activity, suggesting a role for PC3 in the conversion of prohormones.

Site-directed mutagenesis and expression of PC2 in microinjected Xenopus oocytes.

The biosynthesis and post-translational maturation of PC2, a neuroendocrine-specific Kex2-like endoprotease, following expression in Xenopus oocytes is described. The initial translation product was a 75-kDa membrane-associated protein which was released from the oocytes as a glycosylated 71-kDa protein. During extended chase periods, the extracellular 71-kDa protein was converted to a mature 68-kDa product. A deletion mutant lacking a putative COOH-terminal amphipathic helix was still membrane-associated, suggesting that this domain was not essential for attachment of PC2 to membranes. Two putative proregion cleavage site mutants were also constructed. Conversion of the 75-kDa peptide to the 71-kDa peptide involved cleavage at the sequence Lys-Arg-Arg-Arg (amino acids 78-81), since mutation of this sequence to Lys-Val-Arg-Leu resulted in the secretion of the 75-kDa peptide. Extracellular conversion of the 71-kDa peptide to the 68-kDa peptide involved cleavage at the sequence Arg-Lys-Lys-Arg (amino acids 106-109), since deletion of this tetrabasic sequence resulted in secretion of the 71-kDa peptide without further conversion to the 68-kDa form. Finally, a mutation which changed a catalytically important Asp to Asn did not affect processing of proPC2. These results may be relevant to our understanding of mechanisms in the intracellular sorting and maturation of proPC2 in neuroendocrine cells.