Use of 2D and co-culture cell models to assess the toxicity of zein nanoparticles loading insect repellents icaridin and geraniol.

After the latest dengue and Zika outbreaks, the fight against mosquito vectors has become an emerging area of research. One tool for this combat is repellents; however, these products are composed of different toxic agents. Botanical compounds with repellent potential are an alternative; however these compounds are highly volatile. Thus, the present study aimed to synthesize zein-based polymeric nanoparticles as an efficient carrier system for the sustained release of the repellents icaridin and geraniol and evaluate the toxicity of these nanorepellents comparing two different cell models. In vitro tests were carried out due to current Brazilian legislation prohibiting animal testing for cosmetics (current classification of repellents). The cytotoxicity and genotoxicity of the nanoparticles were evaluated in 2D and co-culture cell models (A549/lung epithelium, HaCaT/keratinocytes, HT-29/intestinal epithelium, and THP-1/peripheral blood monocytes). Cell viability by mitochondrial activity, cell membrane integrity, damage to genetic material, and expression of genes involved in the allergic/inflammatory system were evaluated. The results of cytotoxicity evaluation showed cell viability above 70% in both cell models. No differences were observed in genotoxicity assessment between cells exposed to nanorepellents and controls. In contrast, gene expression analysis showed increased cytokine expression for the emulsion compounds in 2D cell cultures compared to co-cultures. These findings open perspectives that zein-based nanorepellents have potential applications due to the reduced toxicity observed when the compounds are encapsulated and emerge as an alternative for arbovirus control. In addition, the study demonstrated that depending on the analysis, different results might be observed when comparing 2D and co-culture cell models to evaluate the toxicity of new nanosystems.

Comparing human and mouse salivary glands: A practice guide for salivary researchers.

Mice are a widely utilized in vivo model for translational salivary gland research but must be used with caution. Specifically, mouse salivary glands are similar in many ways to human salivary glands (i.e., in terms of their anatomy, histology, and physiology) and are both readily available and relatively easy and affordable to maintain. However, there are some significant differences between the two organisms, and by extension, the salivary glands derived from them must be taken into account for translational studies. The current review details pertinent similarities and differences between human and mouse salivary glands and offers practical guidelines for using both for research purposes.

L1 Peptide-Conjugated Fibrin Hydrogels Promote Salivary Gland Regeneration.

Hyposalivation contributes to dental caries, periodontitis, and microbial infections. Additionally, it impairs activities of daily living (e.g., speaking, chewing, and swallowing). Treatments for hyposalivation are currently limited to medications (e.g., the muscarinic receptor agonists pilocarpine and cevimeline) that induce saliva secretion from residual acinar cells and the use of saliva substitutes. However, given that these therapies provide only temporary relief, the development of alternative treatments to restore gland function is essential. Previous studies demonstrated that laminin 1 (L1) is critical for intact salivary cell cluster formation and organization. However, the full L1 sequence is not suitable for clinical applications, as each protein domain may contribute to unwanted effects, such as degradation, tumorigenesis, and immune responses that, when compounded, outweigh the potential benefits provided by their sum. Although the L1 peptides YIGSR and A99 linked to fibrin hydrogels (FHs) promote intact salivary epithelial formation in vitro, little is known about their role during salivary gland regeneration in vivo. Therefore, the goal of this study was to demonstrate whether L1 peptides conjugated to FHs promote tissue regeneration in a wound-healing model of mouse submandibular glands (mSMGs). Our results suggest that YIGSR-A99 peptides, chemically conjugated to FHs and applied to wounded mSMGs in vivo, formed new organized salivary tissue. In contrast, wounded mSMGs treated with FHs alone or in the absence of a scaffold showed disorganized collagen formation and poor tissue healing. Together these studies indicate that damaged salivary gland tissue can grow and differentiate when treated with FHs containing L1 peptides.

Stem Cell-Soluble Signals Enhance Multilumen Formation in SMG Cell Clusters.

Saliva plays a major role in maintaining oral health. Patients with salivary hypofunction exhibit difficulty in chewing and swallowing foods, tooth decay, periodontal disease, and microbial infections. At this time, treatments for hyposalivation are limited to medications (e.g., muscarinic receptor agonists: pilocarpine and cevimeline) that induce saliva secretion from residual acinar cells as well as artificial salivary substitutes. Therefore, advancement of restorative treatments is necessary to improve the quality of life in these patients. Our previous studies indicated that salivary cells are able to form polarized 3-dimensional structures when grown on growth factor-reduced Matrigel. This basement membrane is rich in laminin-III (L1), which plays a critical role in salivary gland formation. Mitotically inactive feeder layers have been used previously to support the growth of many different cell types, as they provide factors necessary for cell growth and organization. The goal of this study was to improve salivary gland cell differentiation in primary cultures by using a combination of L1 and a feeder layer of human hair follicle-derived mesenchymal stem cells (hHF-MSCs). Our results indicated that the direct contact of mouse submandibular (mSMG) cell clusters and hHF-MSCs was not required for mSMG cells to form acinar and ductal structures. However, the hHF-MSC conditioned medium enhanced cell organization and multilumen formation, indicating that soluble signals secreted by hHF-MSCs play a role in promoting these features.

The 5'-flanking region of the RP58 coding sequence shows prominent promoter activity in multipolar cells in the subventricular zone during corticogenesis.

Pyramidal neurons of the neocortex are produced from progenitor cells located in the neocortical ventricular zone (VZ) and subventricular zone (SVZ) during embryogenesis. RP58 is a transcriptional repressor that is strongly expressed in the developing brain and plays an essential role in corticogenesis. The expression of RP58 is strictly regulated in a time-dependent and spatially restricted manner. It is maximally expressed in E15-16 embryonic cerebral cortex, localized specifically to the cortical plate and SVZ of the neocortex, hippocampus, and parts of amygdala during brain development, and found in glutamatergic but not GABAergic neurons. Identification of the promoter activity underlying specific expression patterns provides important clues to their mechanisms of action. Here, we show that the RP58 gene promoter is activated prominently in multipolar migrating cells, the first in vivo analysis of RP58 promoter activity in the brain. The 5.3 kb 5'-flanking genomic DNA of the RP58 coding region demonstrates promoter activity in neurons both in vitro and in vivo. This promoter is highly responsive to the transcription factor neurogenin2 (Ngn2), which is a direct upstream activator of RP58 expression. Using in utero electroporation, we demonstrate that RP58 gene promoter activity is first detected in a subpopulation of pin-like VZ cells, then prominently activated in migrating multipolar cells in the multipolar cell accumulation zone (MAZ) located just above the VZ. In dissociated primary cultured cortical neurons, RP58 promoter activity mimics in vivo expression patterns from a molecular standpoint that RP58 is expressed in a fraction of Sox2-positive progenitor cells, Ngn2-positive neuronal committed cells, and Tuj1-positive young neurons, but not in Dlx2-positive GABAergic neurons. Finally, we show that Cre recombinase expression under the control of the RP58 gene promoter is a feasible tool for conditional gene switching in post-mitotic multipolar migrating young neurons in the developing cerebral cortex.

Effects of short-term folic acid and/or riboflavin supplementation on serum folate and plasma total homocysteine concentrations in young Japanese male subjects.

OBJECTIVE: To investigate the effects of short-term folic acid and/or riboflavin supplementation on serum folate and plasma plasma total homocysteine (tHcy) concentrations in young Japanese male subjects. DESIGN: In a double blind, randomized controlled trial. INTERVENTION: Subjects were randomly assigned to one of four groups and received a placebo (control group), 800 microg/day folic acid (FA group), 8.4 mg/day riboflavin (R group), or both (FAR group) for 2 weeks. SETTING: Tokyo, Japan. SUBJECTS: In total, 32 healthy male volunteers aged 20-29 years. RESULTS: At the end of the 2 week supplementation period, the tHcy concentration decreased significantly in the FA group. Serum folate concentrations had increased between 2.7 and 2.0-fold in the FA and FAR groups, respectively, but the mean within-group changes in serum folate and plasma tHcy concentrations did not differ between these two groups. At the end of the study, alanine amino transferase was decreased in the R and FAR groups, while alanine amino transferase was increased in the FA group. CONCLUSION: Supplementation with folic acid, 800 microg/day, for 2 weeks, increased the serum and red blood cell folate concentrations and decreased the plasma tHcy concentrations in healthy young male subjects. Riboflavin supplementation may have blunted the effect of folic acid, which resulted in a diminished reduction of tHcy in our subjects.

Role of genetic factors (CETP gene Taq I B polymorphism and Apo A-I gene Msp I polymorphism) in serum HDL-C levels in women.

BACKGROUND AND AIM: Plasma high density lipoprotein cholesterol (HDL-C) levels are determined by a variety of environmental and genetic factors. The cholesteryl ester transfer protein (CETP) and apolipoprotein A-I (Apo A-I) are considered to be associated with HDL-C metabolism. The aim of this study was to investigate the relationship between the CETP gene Taq I B and Apo A-I gene Msp I polymorphisms and plasma lipid levels taking into account environmental factors, and to determine the combined effects of these polymorphisms on HDL-C levels in Japanese women. METHODS AND RESULTS: The study involved 270 Japanese women aged 30-69 years. We found a significant association between the CETP genotypes and HDL-C levels (p=0.0020), which were also associated with the Apo A-I gene (M1) polymorphism. Stepwise multiple regression analysis revealed that both the CETP Taq I B and Apo A-I gene (M1) genotypes were independent predictive variables. The strength of the association between the Apo A-I (M1) subgroup and HDL-C levels was reduced in the subjects with a high Body Mass Index (BMI). The combination of genotypes provided more detailed information about HDL-C levels. The "high risk" combination of the M1+ (M1+/+) and B1B1 genotypes was associated with the lowest HDL-C level (1.52+/-0.36 mmol/L), and the "low risk" combination of the M1- (M1+/- or M1-/-) and B2B2 genotypes was associated with the highest HDL-C levels (2.06+/-0.34 mmol/L). CONCLUSIONS: Our results suggest that the combination of the two polymorphisms influences HDL-C levels in women, and that the association between genetic factors and HDL-C levels is altered by environmental factors. They may also help to detect individuals with low HDL-C levels at high risk for coronary artery syndrome.

Effects of tomato juice consumption on plasma and lipoprotein carotenoid concentrations and the susceptibility of low density lipoprotein to oxidative modification.

Effects of tomato juice supplementation on the carotenoid concentration in lipoprotein fractions and the oxidative susceptibility of LDL were investigated in 31 healthy Japanese female students. These subjects were randomized to one of three treatment groups; Control, Low and High. The Control, Low and High groups consumed 480 g of a control drink, 160 g of tomato juice plus 320 g of the control drink, and 480 g of tomato juice, providing 0, 15 and 45 mg of lycopene, respectively, for one menstrual cycle. The ingestion of tomato juice, rich in lycopene but having little beta-carotene, increased both lycopene and beta-carotene. Sixty-nine percent of lycopene in plasma was distributed in the LDL fraction and 24% in the HDL fraction. In the Low group, the lycopene concentration increased 160% each in the VLDL+IDL, LDL and HDL fractions (p<0.01). In the High group, the lycopene concentration increased 270% each in the VLDL+IDL and LDL fractions, and 330% in the HDL fraction (p<0.01). Beta-carotene also increased 120% and 180% in LDL fractions of the Low and the High groups, respectively. Despite these carotenoid increases in LDL, the lag time before oxidation was not prolonged as compared with that of the Control group. The propagation rate decreased significantly after consumption in the High group. Multiple regression analysis showed a positive correlation between lag time changes and changes in the alpha-tocopherol concentration per triglyceride in LDL, and a negative correlation between propagation rate changes and changes in the lycopene concentration per phospholipid in LDL. These data suggest that alpha-tocopherol is a major determinant in protecting LDL from oxidation, while lycopene from tomato juice supplementaion may contribute to protect phospholipid in LDI, from oxidation. Thus, oral intake of lycopene might be beneficial for ameliorating atherosclerosis.

Diesel exhaust-induced airway hyperresponsiveness in c-Ha-ras transgenic mice.

Recently the quantity of diesel exhaust (DE) emissions, which contain a variety of chemicals and can induce pulmonary carcinoma in animals, has been increasing in Japan. To assess the toxicity of DE, we evaluated airway hyperresponsiveness after exposure to DE in the rasH2 (CB6F1-TgHras2) mouse, which carries c-Ha-ras genes and shows marked sensitivity to treatment with various genotoxic carcinogens such as methylnitrosourea and dimethylbenzanthracene. We exposed rasH2 mice (n=18) and their nontransgenic littermates (n=19) to room air or 3 mg/m(3) DE for 4 weeks, measured their respiratory resistance (Rrs) during inhalation of acetylcholine (ACh; 0.005, 0.01, 0.02, 0.04, 0.08, 0.16, 0.31, 0.63, 1.28, 2.5, 5, or 10 mg/ml) for 2 min, and calculated the provocative ACh concentration needed to cause a 50% increase (PC(150)) in Rrs. At all doses of ACh, Rrs was significantly higher (P<0.05) in rasH2 mice exposed to DE than in those exposed to room air. In addition, Rrs in the DE-exposed rasH2 animals was significantly higher (P<0.05) at 0.16, 0.31, and 0.63 mg/ml ACh than in DE-exposed nontransgenic littermates. The PC(150) (mean+/-standard error) of DE-exposed rasH2 mice was 3.4+/-1.9 mg/ml, that in rasH2 mice exposed to room air was 10.6+/-2.5 mg/ml, and that in DE-exposed nontransgenic animals was 10.9+/-3.7 mg/ml. In conclusion, DE causes airway hyperresponsiveness in rasH2 mice and may induce the expression of c-Ha-ras genes.

A 76-bp deletion in the Mip gene causes autosomal dominant cataract in Hfi mice.

Hfi is a dominant cataract mutation where heterozygotes show hydropic lens fibers and homozygotes show total lens opacity. The Hfi locus was mapped to the distal part of mouse chromosome 10 close to the major intrinsic protein (Mip), which is expressed only in cell membranes of lens fibers. Molecular analysis of Mip revealed a 76-bp deletion that resulted in exon 2 skipping in Mip mRNA. In Hfi/Hfi this deletion resulted in a complete absence of the wildtype Mip. In contrast, Hfi/+ animals had the same amount of wildtype Mip as +/+. Results from pulse-chase expression studies excluded hetero-oligomerization of wildtype and mutant Mip as a possible mechanism for cataract formation in the Hfi/+. We propose that the cataract phenotype in the Hfi heterozygote mutant is due to a detrimental gain of function by the mutant Mip resulting in either cytotoxicity or disruption in processing of other proteins important for the lens. Cataract formation in the Hfi/Hfi mouse is probably a combined result of both the complete loss of wildtype Mip and a gain of function of the mutant Mip.

Overexpression of human H-ras transgene is responsible for tumors induced by chemical carcinogens in mice.

Level of human prototype H-ras transgene expression in tumors induced by chemical carcinogens (N-ethyl-N-nitrosourea and N-methyl-N-nitrosourea) was analyzed in human H-ras transgenic mice (CB6F1-TgrasH2 Jic mice). All forestomach tumors examined revealed about 2-fold overexpression of the human H-ras transgene with or without point mutation at codon 12 or codon 61. However, endogenous mouse H- and K-ras genes exhibited neither point mutation nor overexpression. These results suggested that increased levels of ras gene products in the cell played an important role in facilitating chemical carcinogenesis in transgenic mice.

Two-color allele-specific polymerase chain reaction (PCR-SSP) assay of the leptin receptor gene (Leprdb) for genotyping mouse diabetes mutation.

An allele specific polymerase chain reaction (PCR-SSP) assay for genotyping the mouse leptin receptor (Leprdb) mutation and its wild type (Lepr+) gene was developed using two different fluorescent dye-labeled primers. First, we determined the Leprdb and Lepr+ allele by PCR-SSP assay with usual dye-unlabeled primers. However this method requires two separate PCR reactions because the amplified products specific for each allele are almost the same size. We further developed a simple and reliable two-color PCR-SSP method that uses a color complementation strategy to distinguish the Leprdb and Lepr+ alleles. Leprdb/Leprdb, Leprdb/Lepr+ and Lepr+/Lepr+ of mice (5 each) were clearly genotyped by the two-color PCR-SSP. We also performed PCR-direct sequencing for the same samples and confirmed the accuracy of this method. This method makes it possible to reduce the number of PCR reactions because both alleles are amplified in the same reaction mixture.

Cilnidipine more highly attenuates cold pressor stress-induced platelet activation in hypertension than does amlodipine.

The clinical significance of N-type calcium channel blockade has not been fully examined. We here compared the effects of the N-type calcium channel blockers cilnidipine and amlodipine on the sympathetic nervous system and platelet function in hypertension under resting and stressed conditions. Thirty-two patients with hypertension (58+/-9 years) received cilnidipine or amlodipine for 4 weeks in this crossover study. On day 28 of each treatment, plasma levels of epinephrine (EP), norepinephrine (NEP), and beta-thromboglobulin (BTG), and EC50 of ADP-induced platelet aggregation (ADPE50) were determined at rest and after a cold pressor test. On day 29, the group receiving cilnidipine was switched to amlodipine treatment, and vice versa. At rest, the blood pressure, heart rates, EP, NEP, ADPEC50, and BTG, were similar in both treatments. After the cold pressor test, increases in EP (35+/-17 to 44+/-25 pg/ml; p<0.05) and BTG (40+/-13 to 49+/-22 ng/ml; p<0.01) and a decrease in ADPEC50 (32+/-26 to 27+/-24 micromol; p<0.05) were observed in the amlodipine treatment, but not in the cilnidipine treatment. In addition, the increase in NEP was significantly greater (p<0.05) in the amlodipine (276+/-78 to 318+/-87 pg/ml; p<0.01) than in the cilnidipine treatment (273+/-88 to 291+/-100 pg/ml; p<0.05). Cilnidipine more highly attenuates the activation of platelet function in response to cold pressor stress than does amlodipine. Attenuated activation of the sympathetic nervous system via N-type calcium channel blockade may contribute to this phenomenon.

Gene expression profiling of mouse postnatal cerebellar development.

Expression patterns of 1,869 genes were determined using adapter-tagged competitive PCR (ATAC-PCR) at 6 time points during mouse postnatal cerebellar development. The expression patterns were classified into 12 clusters that were further assembled into 3 groups by hierarchical cluster analysis. Among the 1,869 genes, 1,053 known genes were assigned to 90 functional categories. Statistically significant correlation between the clusters or groups of gene expression and the functional categories was ascertained. Genes involved in oncogenesis or protein synthesis were highly expressed during the earlier stages of development. Those responsible for brain functions such as neurotransmitter receptor and synapse components were more active during the later stages of development. Many other genes also showed expression patterns in accordance with literature information. The gene expression patterns and the inferred functions were in good agreement with anatomical as well as physiological observations made during the developmental process.

Vitamin E level changes in serum and red blood cells due to acute exhaustive exercise in collegiate women.

The purpose of this study was to investigate the change in vitamin E level in both serum and red blood cells (RBC) during exercise and to clarify the effect of vitamin E supplementation. Ten young sedentary female subjects received 200 mg D-alpha-tocopherol acetate daily for 1 wk after the initial exercise bout. After 1 wk of vitamin E supplementation, the same subjects repeated the same exercise. Before vitamin E supplementation, the alpha-tocopherol level in the serum (serum-alpha-tocopherol) did not change after exercise, but a significant decrease in the alpha-tocopherol level in RBC (RBC-alpha-tocopherol) was observed after exercise (p < 0.05). On the other hand, after vitamin E supplementation, the serum-alpha-tocopherol level decreased significantly after exercise (p < 0.05), while the RBC-alpha-tocopherol level was maintained after exercise. Furthermore, a negative correlation between the changes in serum- and RBC-alpha-tocopherol levels was observed only after vitamin E supplementation (r = 0.667, p < 0.05). The present results suggest that as RBC suffers oxidative stress, vitamin E in RBC is consumed to protect RBC from oxidative damage during exercise. These results also suggest that when there is a sufficient amount of vitamin E in the serum, vitamin E is shifted from the serum to RBC, resulting in a steady RBC-alpha-tocopherol level and a decrease in the serum-alpha-tocopherol level under oxidative stress such as exercise.

Correlation between gene functions and developmental expression patterns in the mouse cerebellum.

Quantitative changes of 419 gene transcripts during postnatal mouse cerebellar development were accurately determined with a novel polymerase chain reaction (PCR)-based technique. About 70% of the genes showed differences in expression levels, and the magnitude of difference was relatively small. By hierarchic cluster analysis of developmental expression patterns, the genes were categorized into 19 clusters, which were subsequently assembled into four major groups: group 1, with elevation of gene expression throughout the time course; group 2, with relatively unchanged levels; group 3, with transiently high expression at approximately 12 days; and group 4, with highest expression at approximately 4 days. Genes related to brain functions were segregated into several clusters of group 1 and group 3: the same clusters in which cerebellum-specific genes were also segregated. Genes for protein synthesis belonged to group 4. Genes with housekeeping functions belonged to group 2. Western blotting analysis of representative protein products of each group revealed correlation with the mRNA level for those belonging to group 1 and group 4, but not necessarily in the other groups. The close correlation of algorithmically categorized temporal expression patterns of genes with their functions will be useful for estimating the functions of thousands of novel genes.

Rapid induction of uterine tumors with p53 point mutations in heterozygous p53-deficient CBA mice given a single intraperitoneal administration of N-ethyl-N-nitrosourea.

To investigate the sensitivity of heterozygous p53-deficient CBA mice to carcinogens, 20 female mice [p53(+/-)] and 20 wild-type littermates [p53(+/+)] were given an intraperitoneal injection of 120 mg/kg body wt of N-ethyl-N-nitrosourea (ENU) and were maintained without any other treatment for a further 26 weeks. Histopathology showed that uterine tumors (endometrial polyps and stromal sarcomas) and lung adenomas were induced in both p53(+/-) and p53(+/+) mice. The incidence of uterine tumors and lung adenomas (94% and 81%, respectively) in p53(+/-) mice was significantly greater than that in p53 (+/+) mice (37% and 42%, respectively). Malignant lymphomas were only induced in p53(+/-) mice, at an incidence of 31%. Concerning uterine tumors and preneoplastic lesions, there were endometrial stromal sarcomas and atypical hyperplasias of the endometrial gland in 90% and 63%, respectively, of p53(+/-) mice, with significantly greater incidences than in p53(+/+) mice. Gene analysis revealed GCG-->GTG point mutations in codon 135 of exon 5 of the p53 allele in all of the uterine endometrial stromal sarcomas examined. Our results suggest that female p53(+/-) CBA mice are very susceptible to uterine carcinogenesis, providing a useful model for ENU-induced uterine tumors.

Gene mapping of SEZ group genes and determination of pentylenetetrazol susceptible quantitative trait loci in the mouse chromosome.

Gene mapping of the newly discovered SEZ genes (seizure-related genes) in the mouse was performed by linkage analysis. SEZ6 was on chromosome 11, SEZ12 on chromosome 16, SEZ15 on chromosome 3 and SEZ17 (PTZ17) on chromosome 18. The mouse chromosomal locus related to high susceptibility to pentylenetetrazol (PTZ) was also determined by linkage analysis using the recombinant inbred mouse, BXD (C57BLxDBA). A significant level of PTZ susceptibility was found on chromosome 2. Chromosomal loci of the newly discovered SEZ genes were not coincident with the significant chromosomal loci to PTZ susceptibility. Since epilepsy is assumed to be a disease syndrome which is probably manifested by abnormal expression of multifocal genes, determination of the role of each chromosomal locus in the provocation of seizure activity is important.

Gene expression in mouse cerebellum during its development.

Genes expressed during the cerebellar development of the mouse were identified in 3'-directed cDNA libraries prepared from the postnatal day 4, day 12, and week 6 cerebellar tissues. Among about 5500 clones selected randomly from each library, there were approximately 3500 distinct species. A total of 7728 species were identified in the three libraries, 1346 of which were known genes in the GenBank, 3041 EST-matching genes, and 3341 new genes. Relative expression levels at the three postnatal stages were quantitated by adapter-tagged competitive PCR for 130 known genes that appeared six times or more in one of the libraries. Genes for ribosomal proteins and some cytoskeletal and nuclear proteins were abundantly expressed at the early stage, coincidently with extensive proliferation of granule cells as the major cerebellar component. Genes related to brain functions, including those for mitochondrial activities and some ion channel systems, were more active at a later stage when the majority of granule cells were engaged in axon extension and synapse formation or the cerebellum had reached maturity. Compared to these stage-specifically expressed genes, genes for transcriptional regulation, signal transduction, protein modification, and basic cellular functions, in general, were not abundantly expressed at any stage of development.

The transcription factor Sp3 interacts with promoter elements of the lens specific MIP gene.

PURPOSE: To characterize the cis regulatory elements and their interaction with transcription factors responsible for the lens specific expression of the MIP gene, which encodes the Major Intrinsic Protein of the lens fiber membranes. METHODS: Study interaction of factors present in newborn mouse lens nuclear extracts with DNA fragments corresponding to mouse MIP gene 5' flanking sequence by electrophoresis mobility shift assay (EMSA) and DNase I footprinting. RESULTS: We found a high degree of identity in the first 100 bp of 5' flanking sequence of mice and humans, however, a lower degree of conservation is observed further upstream. We have found by DNase I footprinting analysis that lens specific factors may interact with the first 100 bp of 5' flanking sequence. A domain containing an E box, conserved in mouse and human, may interact with a lens specific factor. However, general factors may interact with a NF-1 binding site. An overlapping GC and CT box is present in the mouse MIP gene. In the human MIP gene GC and CT boxes are found in different domains of the MIP gene promoter. Both CT boxes interact with factors present in lens nuclear extracts including Sp3. They are able to interact with purified Sp1but not with Sp1 present in mouse lens nuclear extracts. CONCLUSIONS: The transcription factor Sp3 may play an important role in regulating MIP gene expression in the lens.