Comparison of three different lactic acid bacteria-fermented proteins on RAW 264.7 osteoclast and MC3T3-E1 osteoblast differentiation.

Osteoporosis is a state of bone weakening caused by an imbalance in osteoblast and osteoclast activity. In this study, the anti-osteoporotic effects of three proteins fermented by lactic acid bacteria (LAB) were assessed. Commercial proteins sodium caseinate (SC), whey protein isolate (WPI), and soy protein isolate (SPI) were fermented by LAB strains for 48 h. The fermented products (F-SC, F-WPI, and F-SPI, respectively) were used in an in vitro osteoclast and osteoblast-like cell model to assess their effects on bone health. Despite no difference in the results of TRAP staining of RANKL-induced osteoclastogenesis, F-WPI and F-SPI were effective in normalizing the altered gene expression of osteoclastogenesis markers such as TRAP, Nfatc1, RANK, and ATP6v0d. F-SPI was also effective in modulating osteoblasts by enhancing the expression of the osteoblastogenesis markers T1Col, Col2a, and OSX to levels higher than those in the SPI group, indicating that protein characteristics could be enhanced through bacterial fermentation. Moreover, these boosted effects of F-SPI may be involved with isoflavone-related metabolism during LAB-fermentation of SPI. These results demonstrate the potential of LAB-fermented proteins as dietary supplements to prevent bone loss. However, further understanding of its effects on balancing osteoblasts and osteoclasts and the underlying mechanisms is needed.

Effect of Probiotic-Fortified Infant Formula on Infant Gut Health and Microbiota Modulation.

Compared to infant formula, breast milk is the best source of nutrition for infants; it not only improves the neonatal intestinal function, but also regulates the immune system and gut microbiota composition. However, probiotic-fortified infant formula may further enhance the infant gut environment by overcoming the limitations of traditional infant formula. We investigated the probiotic formula administration for one month by comparing 118 Korean infants into the following three groups: infants in each group fed with breast milk (50), probiotic formula (35), or placebo formula-fed group (33). Probiotic formula improved stool consistency and defecation frequency compared to placebo formula-fed group. The probiotic formula helped maintaining the level of secretory immunoglobulin A (sIgA), which had remarkably decreased over time in placebo formula-fed infants (compared to weeks 0 and 4). Moreover, probiotic formula decreased the acidity of stool and considerably increased the butyrate concentration. Furthermore, the fecal microbiota of each group was evaluated at weeks 0 and 4. The microbial composition was distinct between each groups, and the abundance of health-promoting bacteria increased in the probiotic formula compared to the placebo formula-fed group. In summary, supplementation of probiotic infant formula can help optimize the infant gut environment, microbial composition, and metabolic activity of the microbiota, mimicking those of breast milk.

Screening for Lactic Acid Bacterial Strains as Probiotics Exhibiting Anti-inflammatory and Antioxidative Characteristic Via Immune Modulation in HaCaT Cell.

In this study, the basic probiotic characteristics and functional properties of lactic acid bacteria (LAB) were investigated using two in vitro models of inflammation induced by lipopolysaccharide (LPS) and H2O2. Fifteen strains were prescreened out of 60 LAB candidates based on their radical scavenging activity to determine the antioxidant capacity of the strains. The top 15 candidates were further investigated to evaluate their survival rate under low pH and bile salt conditions that mimic the intestinal environment. Three strains, Levilactobacillus brevis D70 (Levilact), Lactiplantibacillus pentosus S16 (Lactipla), and Limosilactobacillus fermentum MF10 (Limosilact), were capable of scavenging free radicals and survived under artificial intestinal conditions. Therefore, Levilact. brevis D70, Lactipla. pentosus S16, and Limosilact. fermentum MF10 were selected for further antioxidant, anti-inflammation, and mitochondrial activity examinations via cell models of inflammation and oxidative stress. Among the three strains, Limosilact. fermentum MF10 showed the highest anti-inflammatory activities by significantly downregulating the relative mRNA expression levels of inflammatory biomarkers such as interleukin 8 (IL-8) and interferon-gamma (IFN-γ) induced by LPS (P < 0.05). Moreover, Limosilact. fermentum MF10 was also capable of upregulating the gene expression levels of antioxidative mediator glutathione peroxidase 4 (GPX4) induced by reactive oxygen species (ROS) in both human HT-29 epithelial cells and human HaCaT keratinocytes. Limosilact. fermentum MF10 was also capable of regulating mitochondrial membrane potential (MMP), which plays a key role in the mitochondrial activity of HaCaT cells. As a result, Limosilact. fermentum MF10 showed the highest potential for probiotic properties and impacts the immune-related gut-skin axis by altering proinflammatory cytokines, antioxidative biomarkers, and MMP.

Selection and Characterization of Probiotic Bacteria Exhibiting Antiadipogenic Potential in 3T3-L1 Preadipocytes.

Abnormal adipocyte growth, distinguished by an increase in cell numbers and cellular differentiation, is regarded as a major pathological characteristic of obesity. Thus, inhibition of adipogenic differentiation in adipocytes could prevent obesity. Recently, certain probiotic stains have been reported to regulate lipid metabolism in vitro and/or in vivo. In this backdrop, this study aimed to investigate basic probiotic properties and potential antiobesity characteristics of mouse 3T3-L1 preadipocytes. Six lactic acid bacteria (LAB) strains were prescreened for their cholesterol-lowering activity, antioxidant activity, and survival at low pH and in a solution containing bile salts. These six strains were investigated for antiadipogenic activity by employing 3T3-L1 mouse preadipocytes. 3T3-L1 cells were treated with selected strains during the differentiation process. Lactobacillus johnsonii 3121 and Lactobacillus rhamnosus 86 were found to be more capable of reducing triglyceride and lipid accumulation, as compared to control group, which are fully differentiated 3T3-L1 adipocytes. These strains also inhibited adipocyte differentiation by downregulating the adipogenic transcription factor in 3T3-L1 adipocytes. Taken together, these results indicate that L. johnsonni 3121 and L. rhamnosus 86 could potentially act as probiotic bacteria and prevent fat accumulation by regulating adipogenesis-related markers.

Antiobesity Effect of Novel Probiotic Strains in a Mouse Model of High-Fat Diet-Induced Obesity.

Obesity is one of the major causes of the development of metabolic diseases, particularly cardiovascular diseases and type-2 diabetes mellitus. Increased lipid accumulation and abnormal adipocyte growth, which is an increase in cell numbers and differentiation, have been documented as major pathological characteristics of obesity. Thus, the inhibition of adipogenic differentiation prevents and suppresses obesity. Recently, specific probiotic strains have been known to regulate lipid metabolism in vitro and/or in vivo. Previously, we demonstrated that Lactobacillus johnsonni 3121 and Lactobacillus rhamnosus 86 could act as novel probiotic strains and reduce cholesterol levels. Moreover, both strains significantly reduced lipid accumulation and inhibited adipocyte differentiation by downregulating the adipogenic transcription factor in 3T3-L1 adipocytes. Therefore, L. johnsonni 3121 and L. rhamnosus 86 were selected for in vivo evaluation of their anti-obesity effects using a high-fat diet-induced obese mouse model. Daily oral administration of L. johnsonni 3121 and L. rhamnosus 86 for 12 weeks significantly improved serum lipid profile and downregulated the expression of genes related to adipogenesis and lipogenesis in epididymal white adipose tissue of high-fat diet fed obese mice (p < 0.05). Fecal analysis also suggested that the two probiotic strains could normalize the altered obesity-related gut microbiota in high-fat diet-fed obese mice. These results collectively demonstrate that oral administration of L. johnsonni 3121 and L. rhamnosus 86 could prevent obesity, thereby improving metabolic health.

Anti-inflammatory and Anti-osteoporotic Potential of Lactobacillus plantarum A41 and L. fermentum SRK414 as Probiotics.

This study involves an investigation on the probiotic properties of lactic acid bacteria and their potential applications in an in vitro model of lipopolysaccharide (LPS)-stimulated inflammation and dexamethasone-induced osteoporosis. Nine strains were pre-screened from 485 lactic acid bacteria based on their survival at a low pH and in a solution containing bile salts. All candidates were capable of surviving in an environment with low pH and with bile salts and could successfully colonize the intestine. Furthermore, their functional properties, such as anti-oxidation and anti-inflammation, were evaluated. Of the nine probiotic candidates, Lactobacillus plantarum A41 and L. fermentum SRK414 exhibited the highest anti-oxidative capacity. Moreover, only L. plantarum A41 and L. fermentum SRK414 could increase gut barrier function by upregulating the mRNA expression of tight junction proteins and inhibit the expression of inflammatory mediators induced by LPS-stimulated inflammation. Interestingly, these two strains were also capable of regulating several bone metabolism-related markers playing a role in bone homeostasis and osteoblast differentiation. In brief, L. plantarum A41 and L. fermentum SRK414 exhibited high probiotic potential and potentially impact immune-related bone health by modulating pro-inflammatory cytokines and bone metabolism-related markers.

Prophylactic use of probiotic chocolate modulates intestinal physiological functions in constipated rats.

BACKGROUND: This study investigated the in vivo prophylactic effect of probiotic chocolate on constipation. Rats were administered chocolate containing 2.5 × 1010 CFU g-1 of probiotics daily for 4 weeks and treated with loperamide (5 mg kg-1 ) daily at the fourth week of treatment. RESULTS: Probiotic chocolate treatment significantly (P < 0.05) increased the intestinal motility, colon length, fecal moisture content and number of excreted fecal pellets in constipated rats. Moreover, quantitative real-time polymerase chain reaction data and histological images also revealed that both probiotic chocolate LYC and BB12 treatments were capable of upregulating the mRNA expression levels of colonic ZO-1, occludin and AQP8, leading to the maintenance of the defensive barrier function in the constipated rats compared with the negative controls. Interestingly, these treatments also modulated gut bacterial populations by increasing the abundance levels of Lactobacillus and Bifidobacterium, as well as reducing the abundance level of Enterobacteriaceae. CONCLUSION: The present study demonstrated that probiotic chocolate LYC and BB12 could potentially be used as alternative agents for prophylactic constipation. © 2018 Society of Chemical Industry.

Botulinum Toxin Treatment on Upper Limb Function in School Age Children With Bilateral Spastic Cerebral Palsy: One Year Follow-up.

OBJECTIVE: To prospectively investigate the long-term effects of botulinum toxin treatment on the upper limb function and performance of school age children with spastic bilateral cerebral palsy, who have limitations in performing activities of daily living and school activities, due to spasticity of the upper extremities. METHODS: Botulinum type A toxin (BoNT-A) was injected into 24 spastic upper limbs of 15 children. We used a Modified Ashworth Scale and a Modified Tardieu Scale for the evaluation of upper limb spasticity, and Quality of Upper Extremity Skills Test (QUEST), Canadian Occupational Performance Measure (COPM), and Test of Visual-Motor Skills-Revised (TVMS-R) for the evaluation of upper limb function and performance. RESULTS: Upper limb spasticity continuously decreased until the end of the one-year follow-up. Upper limb function on QUEST and COPM showed the best performance at 3 months and deteriorated slightly, but still showed a significantly better performance at 9 and 12 months than at pre-injection. In more functional nine subjects who could perform TVMS-R, the performance enhancement effects remained constant after 12 months, suggesting that the reduced spasticity led to the learning effect acquired by the repeated use of the affected upper limb. CONCLUSION: For school age children with bilateral spastic cerebral palsy whose upper limb functions are important, BoNT-A injections seem to be of help in the performance of school activities and activities of daily living.

Efficient production of transgenic mice by intracytoplasmic injection of streptolysin-O-treated spermatozoa.

Many methods for efficient production of transgenic animals for biomedical research have been developed. Despite great improvements in transgenesis rates resulting from the use of intracytoplasmic sperm injection (ICSI), the ICSI-based sperm-mediated gene-transfer (iSMGT) technique is still not optimal in terms of sperm permeabilization efficiency and subsequent development. Here, we demonstrate that streptolysin-O (SLO) can efficiently permeabilize mouse spermatozoa, leading to improved developmental competence and high transgenesis rates in iSMGT embryos and pups. In particular, the most efficient production of iSMGT-transgenic embryos resulted from pretreatment with 5 U/ml SLO for 30 min and co-incubation with 1.0 ng/µl of an EGFP expression vector. By incubating spermatozoa with Cy-3-labelled DNA, we found that fluorescence intensity was prominently detected in the head region of SLO-treated spermatozoa. In addition, blastocyst development rate and blastomere survival were greatly improved by iSMGT using SLO-treated spermatozoa (iSMGT-SLO) as compared to freeze-thawed spermatozoa. Consistent with this, a high proportion of transgenic offspring was obtained by iSMGT-SLO after transfer into foster mothers, reaching 10.6% of the number of oocytes used (42.3% among pups). Together with successful germline transmission of transgenes in all founders analyzed, our data strongly suggest that SLO makes spermatozoa amenable to exogenous DNA uptake, and that the iSMGT-SLO technique is an efficient method for production of transgenic animals for biomedical research.

Mammary gland-specific expression of biologically active human osteoprotegerin in transgenic mice.

Osteoprotegerin (OPG) is a secreted glycoprotein that regulates bone resorption by inhibiting differentiation and activation of osteoclast, thereby potentially useful for the treatment of many bone diseases associated with increased bone loss. In this study, we designed a novel cDNA expression cassette by modifying the potent and mammary gland-specific goat ß-casein/hGH hybrid gene construct and examined human OPG (hOPG) cDNA expression in transgenic mice. Six transgenic mice all successfully expressed hOPG in their milk at the level of 0.06-2,000 µg/ml. An estimated molecular weight of the milk hOPG was 55 kDa in SDS-PAGE, which is the same as a naturally glycosylated monomer. This hOPG expression was highly specific to the mammary glands of transgenic mice. hOPG mRNA was not detected in any organs analyzed except mammary gland. Functional integrity of milk hOPG was evaluated by TRAP (tartrate-resistant acid phosphatase) activity assay in bone marrow cell cultures. OPG ligand (OPG-L) treatment increased TRAP activity by two fold but it was completely abolished by co-treatment with transgenic milk containing hOPG. Taken together, our novel cDNA expression cassette could direct an efficient expression of biologically active hOPG, a potential candidate pharmaceutical for bone diseases, only in the mammary gland of transgenic mice.

Nox4-dependent H2O2 production contributes to chronic glutamate toxicity in primary cortical neurons.

Reactive oxygen species (ROS) can trigger neuronal cell death and has been implicated in a variety of neurodegenerative diseases as well as brain ischemia. Here, we demonstrate that chronic (but not acute) glutamate toxicity in primary cortical neuronal cultures is associated with hydrogen peroxide (H(2)O(2)) accumulation in the culture medium and that neurotoxicity can be eliminated by external catalase treatment. Neuronal cultures in Ca(2+)-free medium or treated with BAPTA showed reduced glutamate-induced H(2)O(2) generation, indicating that H(2)O(2) generation is Ca(2+)-dependent. Pharmacological and genetic approaches revealed that NADPH oxidase plays a role in glutamate-induced H(2)O(2) generation and that activation of NMDA and AMPA receptors is involved in this H(2)O(2) generation. The Nox4 siRNA reduced NMDA-induced H(2)O(2) production by 54% and cytotoxicity in parallel, suggesting that Nox4-containing NADPH oxidase functions NMDA receptor-mediated H(2)O(2) production resulting in neurotoxicity. These findings suggest that the modulation of NADPH oxidase can be used as a new therapeutic strategy for glutamate-induced neuronal diseases.

Chronic glutamate toxicity in mouse cortical neuron culture.

Two pathways for glutamate toxicity have been described, receptor-mediated excitotoxicity and non-receptor mediated oxidative glutamate toxicity. Here, we show that two distinct forms of receptor-mediated primary cortical neuronal death exist, chronic and acute glutamate toxicity, and that these depend on exposure time. In vitro, neuronal sensitivity to chronic glutamate exposure increased as neurons matured and the initial plating medium contributed as well. In immature neurons, high concentrations of glutamate induced neuronal death. The chronic glutamate toxicity was independent of neuronal density, whereas increased density potentiated acute glutamate toxicity. Activation of ionotropic glutamate receptors (iGluRs) contributed to induction of chronic and acute glutamate toxicity at similar rates at DIV14. Inactivation of the metabotropic glutamate receptors (mGluRs) by AIDA increased neuronal sensitivity to chronic glutamate exposure but not to acute exposure. Neuronal death by acute toxicity was much faster than by chronic toxicity in which activation of mGluRs was involved. These results suggest that acute glutamate toxicity is quite different from chronic toxicity, in which activation of mGluRs is associated with resistance to glutamate toxicity.

Transient decrease in F-actin may be necessary for translocation of proteins into dendritic spines.

It remains poorly understood as to how newly synthesized proteins that are required to act at specific synapses are translocated into only selected subsets of potentiated dendritic spines. Here, we report that F-actin, a major component of the skeletal structure of dendritic spines, may contribute to the regulation of synaptic specificity of protein translocation. We found that the stabilization of F-actin blocked the translocation of GFP-CaMKII and inhibited the diffusion of 3-kDa dextran into spines (in 2-3 weeks cultures). Neuronal activation in hippocampal slices and cultured neurons led to an increase in the activation (decrease in the phosphorylation) of the actin depolymerization factor, cofilin, and a decrease in F-actin. Furthermore, the induction of long-term potentiation by tetanic stimulation induced local transient depolymerization of F-actin both in vivo and in hippocampal slices (8-10 weeks), and this local F-actin depolymerization was blocked by APV, a N-methyl-D-aspartate (NMDA) receptor antagonist. These results suggest that F-actin may play a role in synaptic specificity by allowing protein translocation into only potentiated spines, gated through its depolymerization, which is probably triggered by the activation of NMDA receptors.

Lentiviral transduction of murine oligodendrocytes in vivo.

Lentiviral vectors are used widely to direct efficient gene transfer in vivo. We examined cell-specific expression in adult murine white matter after stereotaxic microinjection of four lentiviral constructs. We synthesized vesicular stomatitis virus glycoprotein (VSV-G) pseudotyped lentiviruses with combinations of two promoters, cytomegalovirus (CMV) or myelin basic protein (MBP), and two reporter sequences, cytosolic enhanced green fluorescent protein (eGFP) or a plasma membrane-targeted eGFP (human lymphocyte-specific protein tyrosine kinase [Lck]-eGFP). For all constructs, intracerebral injections to lateral corpus callosum resulted in widespread GFP expression in forebrain white matter glial cells. Intense cellular GFP fluorescence was observed within 3 days after injection and lasted for at least 28 days. The CMV promoter directed GFP expression in multiple glial cell types, whereas the MBP promoter targeted GFP specifically to oligodendrocytes. Expression of the membrane-targeted Lck-eGFP construct distinctly labeled individual myelinating processes of oligodendrocytes. Lentiviral constructs expressing eGFP or Lck-eGFP under the MBP promoter provide excellent visualization of oligodendrocyte morphology in intact white matter, and may prove valuable for delivering additional genes of interest to oligodendrocytes in vivo.

Epac1-mediated Rap1 activation is not required for the production of nitric oxide in BV2, murine microglial cells.

This study demonstrates that cyclic AMP (cAMP) production is induced by lipopolysaccharide (LPS) stimulation and activates two different pathways in murine BV2 microglial cells. Two principal effector proteins for cAMP are protein kinase A (PKA) and cAMP-responsive guanine nucleotide exchange factor (Epac), a Rap GDP exchange factor. When cells were treated with various cAMP level modulators, nitric oxide (NO) production increased as the result of posttreatment with Type IV phosphodiesterase (PDE4) inhibitor, rolipram or dibutyryl-cAMP (dbcAMP), at 2 hr after LPS stimulation. Intracellular cAMP increased due to LPS stimulation and the cAMP modulators phosphorylate transcription factor CREB, which is enhanced in turn by posttreatment with dbcAMP. In contrast, the Epac-specific cAMP analog 8-(4-chloro-phenylthio)-2'-O-methyladenosine-3',5'-cyclic monophosphate (8CPT-2Me-cAMP) activates Rap1 in the BV2 cells, but does not induce PKA activation, as judged by CREB phosphorylation. NO production was enhanced by posttreatment with dbcAMP but not by treatment with 8CPT-2Me-cAMP. This suggests that LPS-stimulated NO production is mainly PKA-dependent and also that Epac1-mediated Rap1 activation is not required for the induction of NO production.

Transplantation of embryonic stem cells overexpressing Bcl-2 promotes functional recovery after transient cerebral ischemia.

The study tested the hypothesis that transplantation of embryonic stem (ES) cells into rat cortex after a severe focal ischemia would promote structural repair and functional recovery. Overexpression of the human anti-apoptotic gene bcl-2 in ES cells was tested for increasing survival and differentiation of transplanted cells and promoting functional benefits. Mouse ES cells, pretreated with retinoic acid to induce differentiation down neural lineages, were transplanted into the post-infarct brain cavity of adult rats 7 days after 2-h occlusion of the middle cerebral artery (MCA). Over 1-8 weeks after transplantation, the lesion cavity filled with ES cell-derived cells that expressed markers for neurons, astrocytes, oligodendrocytes, and endothelial cells. ES cell-derived neurons exhibited dendrite outgrowth and formed a neuropil. ES cell-transplanted animals exhibited enhanced functional recovery on neurological and behavioral tests, compared to control animals injected with adult mouse cortical cells or vehicle. Furthermore, transplantation with ES cells overexpressing Bcl-2 further increased the survival of transplanted ES cells, neuronal differentiation, and functional outcome. This study supports that ES cell transplantation and gene modification may have values for enhancing recovery after stroke.

Neurotrophin and GDNF family ligands promote survival and alter excitotoxic vulnerability of neurons derived from murine embryonic stem cells.

Embryonic stem (ES) cells are genetically manipulable pluripotential cells that can be differentiated in vitro into neurons, oligodendrocytes, and astrocytes. Given their potential utility as a source of replacement cells for the injured nervous system and the likelihood that transplantation interventions might include co-application of growth factors, we examined the effects of neurotrophin and GDNF family ligands on the survival and excitotoxic vulnerability of ES cell-derived neurons (ES neurons) grown in vitro. ES cells were differentiated down a neural lineage in vitro using the 4-/4+ protocol (Bain et al., Dev Biol 168:342-57, 1995). RT-PCR demonstrated expression of receptors for neurotrophins and GDNF family ligands in ES neural lineage cells. Neuronal expression of GFRalpha1, GFRalpha2, and ret was confirmed by immunocytochemistry. Exposure to 30-100 ng/ml GDNF or neurturin (NRTN) resulted in activation of ret. Addition of NT-3 and GDNF did not increase cell division but did increase the number of neurons in the cultures 7 days after plating. Pretreatment with NT-3 enhanced the vulnerability of ES neurons to NMDA-induced death (100 microM NMDA for 10 min) and enhanced the NMDA-induced increase in neuronal [Ca2+]i, but did not alter expression of NMDA receptor subunits NR2A or NR2B. In contrast, pretreatment with GDNF reduced the vulnerability of ES neurons to NMDA-induced death while modestly enhancing the NMDA-induced increase in neuronal [Ca2+]i. These findings demonstrate that the response of ES-derived neurons to neurotrophins and GDNF family ligands is largely similar to that of other cultured central neurons.

A proteasomal stress response: pre-treatment with proteasome inhibitors increases proteasome activity and reduces neuronal vulnerability to oxidative injury.

We report here that exposure to low concentrations of proteasome inhibitors (e.g. 10-100 nm MG-132, 0.1-3 nm epoxomicin or 10-30 nm clasto-lactacystin beta-lactone) resulted in an enhancement, rather than an inhibition, of proteasome activity in cultured neocortical neurons. Size-fractionation chromatography confirmed that the enhanced peptide cleavage activity was associated with proteasome-sized complexes. This sub toxic exposure reduced neuronal death caused by subsequent exposure to oxidative stress (100-200 microm H(2)O(2) for 30 min, 24-h exposure to 100 microm paraquat or 7.5 microm menadione), but did not alter vulnerability to excitotoxicity (5-min exposure to 30-100 microm NMDA or 24 exposure to 12 microm NMDA). Sub toxic proteasome inhibitor exposure caused an increase in levels of proteasome core subunit proteins and mRNAs, but not in levels of potentially cytoprotective heat shock proteins (hsp70, hsp90 and hsp40). The neuroprotective effects of proteasome inhibitor pre-treatment were blocked by coapplication of proteasome inhibitors during the oxidative insult. These findings support a model in which sublethal proteasome inhibition induces neurons to increase proteasome activity and promotes resistance to oxidative injury and suggests that enhancement of proteasome activity is a potential therapeutic target for diseases in which oxidative stress has been implicated.

Retinoic acid response element in HOXA-7 regulatory region affects the rate, not the formation of anterior boundary expression.

Since it is known that a 307 bp fragment of the position specific regulatory element of human HOXA-7 contains two (DR3 and DR5) retinoic acid response elements (RAREs) at its 3' end, we constructed several deletion constructs containing different numbers of RAREs and examined their effects in vitro and in vivo. The 5' deletion constructs, BM112 and OM213, retaining both RAREs, were highly responsible (about 8 fold induction) for RA in F9 teratocarcinoma cells, versus NM307 (4-5 fold). The construct NS218, with both RAREs deleted but retaining the 5' sequences lost RA responsibility completely, whereas NR271, with one RARE(DR5) deleted retained a 50% inducibility (2.5 fold). In vivo transgenic analysis revealed that the constructs NM307 and NR271, but not OM213 nor BM112, directed the position specific expression pattern. Sequence analysis revealed that HOXA-7 enhancer sequences, including the RARE repeat sequences, were well conserved in human, mouse and chick. Part of the RAREs overlaps with the CDX1 binding site, and sequences of the DR3 RAREs were identical in this species. Two GAGA binding sites were also found to be strictly conserved. Because OM213, which had one GAGA site disrupted but retaining both RAREs, did not direct anterior boundary formation in transgenic mice, these results suggest the importance of the 5' 94 bp region, including the GAGA binding site, in anterior boundary formation and the involvement of the RARE in the rate of expression not in anterior boundary formation.