Regulation of POU4F3 gene expression in hair cells by 5' DNA in mice.

The POU-domain transcription POU4F3 is expressed in the sensory cells of the inner ear. Expression begins shortly after commitment to the hair cell (HC) fate, and continues throughout life. It is required for terminal HC differentiation and survival. To explore regulation of the murine Pou4f3 gene, we linked enhanced green fluorescent protein (eGFP) to 8.5 kb of genomic sequence 5' to the start codon in transgenic mice. eGFP was uniformly present in all embryonic and neonatal HCs. Expression of eGFP was also observed in developing Merkel cells and olfactory neurons as well as adult inner and vestibular HCs, mimicking the normal expression pattern of POU4F3 protein, with the exception of adult outer HCs. Apparently ectopic expression was observed in developing inner ear neurons. On a Pou4f3 null background, the transgene produced expression in embryonic HCs which faded soon after birth both in vivo and in vitro. Pou4f3 null HCs treated with caspase 3 and 9 inhibitors survived longer than untreated HCs, but still showed reduced expression of eGFP. The results suggest the existence of separate enhancers for different HC types, as well as strong autoregulation of the Pou4f3 gene. Bioinformatic analysis of four divergent mammalian species revealed three highly conserved regions within the transgene: 400 bp immediately 5' to the Pou4f3 ATG, a short sequence at -1.3 kb, and a longer region at -8.2 to -8.5 kb. The latter contained E-box motifs that bind basic helix-loop-helix (bHLH) transcription factors, including motifs activated by ATOH1. Cotransfection of HEK293 or VOT-E36 cells with ATOH1 and the transgene as a reporter enhanced eGFP expression when compared with the transgene alone. Chromatin immunoprecipitation of the three highly conserved regions revealed binding of ATOH1 to the distal-most conserved region. The results are consistent with regulation of Pou4f3 in HCs by ATOH1 at a distal enhancer.

Adipokinetic hormone and the immune responses of locusts to infection.

Injections of Bacillus, or of blastospores from the entomopathogenic fungus, Metarhizium anisopliae, activate the prophenoloxidase (PPO) cascade, and coinjection of adipokinetic hormone-I (AKH) enhances and prolongs these responses. When injected concurrently with an immunizing dose of live bacteria, AKH suppresses the appearance of antimicrobial activity and, after a short delay, increases the growth of bacteria within the hemocoel. Injections of live Escherichia coli or Pseudomonas aeruginosa into locusts fail to activate PPO in the hemolymph, even when coinjected with AKH. The coinjection of bacteria and hormone is rarely lethal to the locust. However, if locusts are injected with AKH when they are infected with Metarhizium, they die more rapidly than if no AKH is administered.

B cell immunodeficiency fails to develop in CD4-deficient mice infected with BM5: murine AIDS as a multistep disease.

The immunodeficiency syndrome murine AIDS (MAIDS), caused by the BM5 retrovirus preparation, involves the activation, division, and subsequent anergy of the entire CD4(+) T cell population as well as extensive B cell hyperproliferation and hypergammaglobulinemia, resulting in splenomegaly and lymphadenopathy, followed many weeks later by death. The development of MAIDS requires CD4(+) T cells and MHC class II expression by the infected host, supporting a role for T-B interaction in disease development or progression. To explore this possibility, we examined development of MAIDS in mice deficient in CD4 (CD4 knockout), in which T-B interactions are compromised. We find that in CD4 knockout hosts, BM5 causes T cell immunodeficiency in the remaining T cells but has only a limited ability to induce B cell phenotypic changes, hyperproliferation, hypergammaglobulinemia, or splenomegaly. There is also delayed death of infected mice. This implies that CD4 dependent T-B interaction is needed to induce the B cell aspects of disease and supports a multistep mechanism of disease in which B cell changes follow and are caused by CD4(+) T cell effects.

Evidence that laminin-5 is a component of the tooth surface internal basal lamina, supporting epithelial cell adhesion.

Laminin-5 (Ln-5) is an extracellular matrix (ECM) glycoprotein found in epithelial basal laminae. We studied its expression on the surface of rat molars, in relationship to the location of the internal basal lamina (IBL) of the junctional epithelium (JE). In order to avoid disruption of the JE-tooth interface as much as possible, the surface of molars was prepared by mechanical removal of tissue debris and detergent/osmotic lysis of epithelial cell layers, and directly stained by immunohistochemistry, without sectioning. Antibodies to Ln-5 specifically stained a narrow band in the region of the cemento-enamel junction (CEJ), consistent with the expected location of the IBL. Western blotting of ECM material detergent--solubilized from the prepared tooth surfaces confirmed the molecular nature of Ln-5 identified by immunohistochemistry. By the use of a high-definition 3-D microscope, it appeared that Ln-5 coated the most apical part of the enamel and the most coronal portion of the cementum, on either side of the CEJ. In adhesion assays performed directly on tooth surfaces, epithelial cells adhered preferentially to the Ln-5 coated area of the tooth compared to the root surface, which is coated by other ECM components. Adhesion to the Ln-5 coated surface was specifically inhibited by a function-blocking monoclonal antibody to Ln-5. These results suggest that Ln-5 is a component of the IBL, and that it may be important in promoting adhesion of JE cells onto the tooth surface.

Nerve dependency of regeneration: the role of Distal-less and FGF signaling in amphibian limb regeneration.

Dlx-3, a homolog of Drosophila Dll, has been isolated from an axolotl blastema cDNA library, and its expression in developing and regenerating limbs characterized. The normal expression pattern, and the changes that occur during experimental treatments, indicate a correlation between Dlx-3 expression and the establishment of the outgrowth-permitting epidermis. Dlx-3 is expressed at high levels in a distal-to-proximal gradient in the epidermis of developing limb buds, and is upregulated in the apical ectodermal cap (AEC) during limb regeneration. Expression is maximal at the late bud stage of regeneration, coincident with the transition from the early phase of nerve dependency to the later phase of nerve independence. Dlx-3 expression in the epidermis is rapidly downregulated by denervation during the nerve-dependent phase and is unaffected by denervation during the nerve-independent phase. We investigated this relationship between nerves and Dlx-3 expression by implanting FGF-2 beads into regenerates that had been denervated at a nerve-dependent stage. Dlx-3 expression was maintained by FGF-2 after denervation, and regeneration progressed to completion. In addition, we detected FGF-2 protein in the AEC and in nerves, and observed that the level of expression in both tissues decreases dramatically in response to denervation. We conclude that both limb development and regeneration require a permissive epidermis, characterized by Dlx-3 and FGF expression, both of which are maintained by FGF through an autocrine loop. The transformation of the limb epidermis into a functional AEC that produces and responds to FGF autocatalytically, is presumed to be induced by FGF. Since nerves appear to be a source of this priming FGF, it is possible that a member of the FGF family of growth factors is the elusive neurotrophic factor of limb regeneration.

Concentration of boron and other elements in human foods and personal-care products.

The element boron is ubiquitous in the environment. Comparatively low concentrations of dietary boron affect several aspects of mineral metabolism in animals and human beings. Therefore, it is appropriate to determine precisely the concentration of boron in human foodstuffs and absorbed, inhaled, or ingested nonfood substances. In this article, we report the analyzed concentrations of boron and other elements in selected foods (animal products, water, condiments, confections, fruits, tuberized roots, vegetables, cereal grains, and spices) and personal-care products (analgesics, antibiotics, decongestants, antihistamines, dental hygiene products, gastric antacids, and laxatives). We conclude that daily intake of boron usually differs considerably between any two individuals for three main reasons. First, concentration of boron in water varies considerably according to geographic source. At some locations, boron in drinking water and water-based beverages may account for most of the total dietary boron intake. Second, individual food preference greatly influences daily intake of boron. Fruits, vegetables, tubers, and legumes have relatively much higher concentrations of boron than do cereal grains or animal tissues and fluids. Third, boron was determined to be a notable contaminant or major ingredient of many personal-care products.

Ascorbic acid: effect on ongoing iron absorption and status in iron-depleted young women.

The effect of ascorbic acid on iron retention from a diet with predicted low iron bioavailability (containing minimal meat and ascorbic acid) was investigated in iron-depleted premenopausal women. Eleven women were depleted of storage iron (indicated by serum ferritin) through a combination of diet (5.0 mg Fe/2000 kcal for 67-88 d) and phlebotomy. They then consumed a diet containing 13.7 mg Fe/2000 kcal, supplemented with placebo or ascorbic acid three times daily (1500 mg total) with meals for 5.5 wk. Ascorbic acid improved apparent iron absorption (balance method) [38 +/- 2% (means +/- SEM) vs 27 +/- 2%]. Ascorbic acid also improved hemoglobin, erythrocyte protoporphyrins, and serum iron but not hematocrit, serum ferritin, iron-binding capacity, or transferrin saturation. In iron-depleted women consuming a diet with predicted poor iron availability, ascorbic acid supplementation enhanced body iron retention for 5.5 wk.

Effect of dietary boron on mineral, estrogen, and testosterone metabolism in postmenopausal women.

A study was done to examine the effects of aluminum, magnesium, and boron on major mineral metabolism in postmenopausal women. This communication describes some of the effects of dietary boron on 12 women between the ages of 48 and 82 housed in a metabolic unit. A boron supplement of 3 mg/day markedly affected several indices of mineral metabolism of seven women consuming a low-magnesium diet and five women consuming a diet adequate in magnesium; the women had consumed a conventional diet supplying about 0.25 mg boron/day for 119 days. Boron supplementation markedly reduced the urinary excretion of calcium and magnesium; the depression seemed more marked when dietary magnesium was low. Boron supplementation depressed the urinary excretion of phosphorus by the low-magnesium, but not by the adequate-magnesium, women. Boron supplementation markedly elevated the serum concentrations of 17 beta-estradiol and testosterone; the elevation seemed more marked when dietary magnesium was low. Neither high dietary aluminum (1000 mg/day) nor an interaction between boron and aluminum affected the variables presented. The findings suggest that supplementation of a low-boron diet with an amount of boron commonly found in diets high in fruits and vegetables induces changes in postmenopausal women consistent with the prevention of calcium loss and bone demineralization.