Efficient and specific ribozyme-mediated reduction of bovine alpha-lactalbumin expression in double transgenic mice.

Transgenic mice carrying a bovine alpha-lactalbumin (alpha-lac) specific ribozyme gene under the transcriptional control of the mouse mammary tumor virus long terminal repeat were generated and cross-bred with animals that highly express a bovine alpha-lac transgene (0.4 mg of alpha-lac/ml(-1) of milk). The ribozyme contains the hammerhead catalytic domain, flanked by 12-nt sequences complementary to the 3' untranslated region of bovine alpha-lac transcript. High-level expression of the ribozyme gene was detected by Northern blot analysis in the mammary gland of 7-8 day lactating transgenic mice, from 3 of 12 lines analyzed. Heterozygous expression of the ribozyme resulted in a reduction in the levels of the target mRNA to 78, 58, and 50% of that observed in the nonribozyme transgenic littermate controls for three independent lines. The ribozyme-mediated reduction in the levels of the bovine protein paralleled that observed for the mRNA, and was positively correlated with the level of expression of the ribozyme. In nonribozyme expressing transgenic mice, the level of bovine alpha-lac mRNA and protein was not affected. The specificity of this activity is demonstrated by the absence of a reduction in the levels of the endogenous murine alpha-lac mRNA or protein. These results demonstrate the feasibility of ribozyme-mediated down-regulation of highly-expressed transcripts in transgenic animals.

Association study of calcitonin gene-related polypeptide-alpha (CALCA) gene polymorphism with migraine

Migraine is a neurological disorder that is associated with increased levels of calcitonin gene-related peptide (CGRP) in plasma. CGRP, being one of the mediators of neurogenic inflammation and a phenomenon implicated in the pathogenesis of migraine headache, is thus suggested to have an important role in migraine pathophysiology. Polymorphisms of the CALCA gene have been linked to Parkinson's disease, ovarian cancer and essential hypertension, suggesting a functional role for these polymorphisms. Given the strong evidence linking CGRP and migraine, it is hypothesised that polymorphisms in the CALCA gene may play a role in migraine pathogenesis. Seemingly non functional intronic polymorphisms are capable of disrupting normal RNA processing or introducing a splice site in the transcript. A 16 bp deletion in the first intron of the CALCA gene has been reported to be a good match for the binding site for a transcription factor expressed strongly in neural crest derived cells, AP-2. This deletion also eliminates an intron splicing enhancer (ISE) that may potentially cause exon skipping. This study investigated the role of the 16 bp intronic deletion in the CALCA gene in migraineurs and matched control individuals. Six hundred individuals were genotyped for the deletion by polymerase chain reaction followed by fragment analysis on the 3130 Genetic Analyser. The results of this study showed no significant association between the intronic 16 bp deletion in the CALCA gene and migraine in the tested Australian Caucasian population. However, given the evidence linking CGRP and migraine, further investigation of variants with this gene may be warranted.

Adaptive evolution of primate TRIM5 alpha, a gene restricting HIV-1 infection

Recent studies showed that nonhuman primate TRIM5 alpha can efficiently block HIV-1 infection in human cell lines. It can also restrict other retroviruses, therefore, suggested as a general defender against retrovirus infection. Here, we present an evolutionary analysis of TRIM5 alpha in primates. Our results demonstrated that TRIM5a has been evolving rapidly in primates, which is likely caused by Darwinian positive selection. The SPRY domain of TRM5 alpha, which may be responsible for recognition of incoming viral capsids showed higher nonsynonymous/synonymous substitution ratios than the non-SPRY domain, indicating that the adaptive evolution of TRIM5a ill primates might be an innate strategy developed in defending retrovirus infection during primate evolution. In addition, the comparative protein sequence analysis suggested that the amino acid substitution pattern at a single site (344R/Q/P) located in the SPRY domain may explain the differences in Susceptibilities of HIV-1 infection in diverse primate species. (c) 2005 Elsevier B.V. All rights reserved.

Polymorphisms of the estrogen receptor alpha (ESR1) gene and the risk of Alzheimer's disease in a southern Chinese community

Background: Alzheimer's disease (AD) is a neurodegenerative disease with a higher prevalence in women. Expression of estrogen receptor 1 (ESR1) gene has been identified throughout the brain. Owing to the putative neuroprotective effects of estrogen, estro

Structure-activity studies on position 14 of human alpha-calcitonin gene-related peptide

A structure-activity study was performed to examine the role of position 14 of human alpha-calcitonin gene-related peptide (h-alpha-CGRP) in activating the CGRP receptor. Interestingly, position 14 of h-alpha-CGRP contains a glycyl residue and is part of an alpha-helix spanning residues 8-18. Analogues [Ala(14)]-h-alpha-CGRP, [Aib(14)]-h-alpha-CGRP, [Asp(14)]-h-alpha-CGRP, [Asn(14)]-h-alpha-CGRP, and [Pro(14)]-h-alpha-CGRP were synthesized by solid phase peptide methodology and purified by RP-HPLC. Secondary structure was measured by circular dichroism spectroscopy. Agonist activities were determined as the analogues' ability to stimulate amylase secretion from guinea pig pancreatic acini and to relax precontracted porcine coronary arteries. Analogues [Ala(1)4]-h-alpha-CGRP, [Aib(14)]-h-alpha-CGRP, [Asp(14)]-h-alpha-CGRP, and [Asn(14)]-h-alpha-CGRP, all containing residues with a high helical propensity in position 14, were potent full agonists compared to h-alpha-CGRP in both tissues. Interestingly, replacement of Gly(14) of h-alpha-CGRP with these residues did not substantially increase the helical content of these analogues. [Pro(14)]-h-alpha-CGRP, predictably, has significantly lower helical content and is a 20-fold less potent agonist on coronary artery, known to contain CGRP-1 receptor subtypes, and an antagonist on pancreatic acini, known to contain CGRP-2 receptor subtypes. In conclusion, the residue in position 14 plays a structural role in stabilizing the alpha-helix spanning residues 8-18. The alpha-helix is crucial for maintaining highly potent agonist effects of h-alpha-CGRP at CGRP receptors. The wide variety of functional groups that can be tolerated in position 14 with no substantial modification of agonist effects suggests the residue in this position is not in contact with the CGRP receptor. [Pro(14)]-h-alpha-CGRP may be a useful pharmacological tool to distinguish between CGRP-1 and CGRP-2 receptor subtypes.

Effects of bovine alpha-lactalbumin and casein glycomacropeptide-enriched infant formulae on faecal microbiota in healthy term infants

Objective: Certain milk factors may promote the growth of a host-friendly gastrointestinal microbiota, for example, one that is predominated by bifidobacteria, a perceived healthpromoting genus. This may explain why breast-fed infants experience fewer intestinal infections than their formula-fed counterparts who are believed to have a more diverse microbiota, which is similar to that of adults. The effects of formulas supplemented with 2 such ingredients from bovine milk, a-lactalbumin (alpha-lac) and casein glycomacropeptide (GMP), on gut flora were investigated in this study. Patients and Methods: Six-week-old (4-8 wk), healthy term infants were randomised to a standard infant formula or 1 of 2 test formulae enriched in alpha-Jac with higher or lower GMP until 6 months. Faecal bacteriology was determined by the culture-independent procedure fluorescence in situ hybridisation. Results: There was a large fluctuation of bacterial counts within groups with no statistically significant differences between groups. Although all groups showed a. predominance of bifidobacteria, breast-fed infants had a small temporary increase in counts. Other bacterial levels varied in formula-fed groups, which overall showed an adult-like faecal microflora. Conclusions: It can be speculated that a prebiotic effect for alpha-lac and GMP is achieved only with low starting populations of beneficial microbiota (eg, infants not initially breast-fed.

rRNA probes used to quantify the effects of glycomacropeptide and alpha-lactalbumin supplementation on the predominant groups of intestinal bacteria of infant rhesus monkeys challenged with enteropathogenic Escherichia coli

Objectives: Certain milk factors may help to promote the growth of a host-friendly colonic microflora (e.g. bifidobacteria, lactobacilli) and explain why breast-fed infants experience fewer and milder intestinal infections than those who are formula-fed. The effects of supplementation of formula with two such milk factors was investigated in this study. Materials and Methods: Infant rhesus macaques were breastfed, fed control formula, or formula supplemented with glycomacropeptide (GMP) or alpha-lactalburnin (alpha-LA) from birth to 5 months of age. Blood was drawn monthly and rectal swabs were collected weekly. At 4.5 months of age, 10(8) colonyforming units of enteropathogenic E.coli O127, strain 2349/68 (EPEC) was given orally and the response to infection assessed. The bacteriology of rectal swabs pre- and post-infection was determined by culture independent fluorescence in situ hybridization. Results: Post-challenge, breast-fed infants and infants fed alpha-LA-supplemented formula had no diarrhea, whilst those infants fed GMP-supplemented formula had intermittent diarrhea. In infants fed control formula the diarrhea was acute. Conclusions: Supplementation of infant formula with appropriate milk proteins may be useful for improving the infant's ability to resist acute infection caused by E.coli.

The effects of alpha-lactalbumin and glycomacropeptide on the association of CaCo-2 cells by enteropathogenic Escherichia coli, Salmonella typhimurium and Shigella flexneri

Two milk components, alpha-lactalbumin (alpha-La) and glycomacropeptide (GMP) may inhibit intestinal infection/intoxification. (3)[H] thymidine-labeled enteropathogenic Escherichia coli (EPEC), Salmonella typhimurium (ATCC 6994) or Shigella flexneri (ATCC 9199) were introduced to CaCo-2 cultures and their association with CaCo-2 cells was assessed. Undigested, pepsin-digested and pepsin- and pancreatin-digested alpha-lactalbumin and glycomacropeptide inhibited association. Thus, milk supplemented with alpha-lactalbumin and glycomacropeptide might be effective in inhibiting associations of the pathogens EPEC, Salmonella typhimurium, and Shigella flexneri to intestinal cells.

A two-stage continuous culture system to study the effect of supplemental alpha-lactalbumin and glycomacropeptide on mixed cultures of human gut bacteria challenged with enteropathogenic Escherichia coli and Salmonella serotype Typhimurium

Aims: Certain milk factors may promote the growth of a gastrointestinal microflora predominated by bifidobacteria and may aid in overcoming enteric infections. This may explain why breast-fed infants experience fewer intestinal infections than their formula-fed counterparts. The effect of formula supplementation with two such factors was investigated in this study. Methods and Results: Infant faecal specimens were used to ferment formulae supplemented with glycomacropeptide (GMP) and alpha-lactalbumin (alpha-la) in a two-stage compound continuous culture model. At steady state, all fermenter vessels were inoculated with 5 ml of 0.1 M phosphate-buffered saline (pH 7.2) containing 10(8) CFU ml(-1) of either enteropathogenic Escherichia coli 2348/69 (O127:H6) or Salmonella serotype Typhimurium (DSMZ 5569). Bacteriology was determined by independent fluorescence in situ hybridization. Vessels that contained breast milk (BM), as well as alpha-la and GMP supplemented formula had stable total counts of bifidobacteria while lactobacilli increased significantly only in vessels with breast milk. Bacteroides, clostridia and E. coli decreased significantly in all three groups prior to pathogen addition. Escherichia coli counts decreased in vessels containing BM and alpha-la while Salmonella decreased significantly in all vessels containing BM, alpha-la and GMP. Acetate was the predominant acid. Significance and Impact of the Study: Supplementation of infant formulae with appropriate milk proteins may be useful in mimicking the beneficial bacteriological effects of breast milk.

The effect of proteolysis on the induction of cell death by monomeric alpha-lactalbumin.

α-Lactalbumin (α-la) is a major whey protein found in milk. Previous data suggested that α-la has antiproliferative effects in human adenocarcinoma cell lines such as Caco-2 and HT-29. However, the cell death inducing α-la was not a naturally occurring monomer but either a multimeric variant or an α-la:oleic acid complex (HAMLET/BAMLET). Proteolysis showed that both human and bovine α-la are susceptible to digestion. ELISA assays assessing cell death with the native undigested α-la fractions showed that undigested protein fractions did have a significant cell death effect on CaCo-2 cells. Bovine α-la was also more effective than human α-la. A reduction in activity corresponded with lower concentrations of the protein and partial digestion and fragmentation of the protein using trypsin and pepsin. This suggests that the tertiary structure is vital for the apoptotic effect.

Hydrolyzed alpha-lactalbumin as a source of protein to the exercising

This work describes the effect of feeding enzymatically hydrolyzed a-lactalbumin on blood sugar, albumin and fatty acids, muscular and hepatic glycogen of rats subjected to physical exercise. Three normoenergetic/normoproteic diets, containing either casein (C), alpha-lactalbumin (L) or alpha-lactalbumin hydrolyzate (H) were fed to thirty male Wistar rats for five weeks. During this period, half of the rats swam for 1 hr daily (T category) while the other half remained sedentary (S category). At the end of training, all rats were required to swim to exhaustion. The results showed that those rats of the T-category consuming diet H reached exhaustion with significantly higher concentrations of serum glucose ([H] 56.0 and [L] 32.3 mg/100ml), serum albumin ([H] 3.8 and [L] 2.1 mg/dl) and muscle glycogen ([H] 2.1 and [L] 0.6 mg/g), while no differences were observed between diets regarding the time of arrival to exhaustion. Results from diets C and L differed minimally. It was concluded that feeding the hydrolyzed protein may result in nutritional advantage to the exercising rat. (C) 1998 Elsevier B.V.