Vitamin D regulated keratinocyte differentiation

The epidermis is the largest organ in the body. It is comprised primarily of keratinocytes which are arranged in layers that recapitulates their programmed life cycle. Proliferating keratinocytes are on the bottom-the stratum basale. As keratinocytes leave the stratum basale they begin to differentiate, culminating in the enucleated stratum corneum which has the major role of permeability barrier. Calcium and the active metabolite of vitamin D, 1,25(OH)(2)D(3), play important roles in this differentiation process. The epidermis has a gradient of calcium with lowest concentrations in the stratum basale, and highest concentrations in the stratum granulosum where proteins critical for barrier function are produced. Vitamin D is made in different layers of the epidermis, but 1,25(OH)(2)D(3) is made primarily in the stratum basale. Together calcium and 1,25(OH)(2)D(3) regulate the ordered differentiation process by the sequential turning on and off the genes producing the elements required for differentiation as well as activating those enzymes involved in differentiation. Animal models in which the sensing mechanism for calcium, the receptor for 1,25(OH)(2)D(3), or the enzyme producing 1,25(OH)(2)D(3) have been rendered inoperative demonstrate the importance of these mechanisms for the differentiation process, although each animal model has its own phenotype. This review will examine the mechanisms by which calcium and 1,25(OH)(2)D(3) interact to control epidermal differentiation.     

Amperometric determination of lactate with novel trienzyme/poly(carbamoyl) sulfonate hydrogel-based sensor

A novel trienzyme sensor for the amperometric determination of lactate was constructed by immobilizing salicylate hydroxylase (SHL, E.C. 1.14.13.1), l-lactate dehydrogenase (LDH, E.C. 1.1.1.27), and pyruvate oxidase (PyOD, E.C. 1.2.3.3) on a Clark-type oxygen electrode. The enzymes were entrapped by a poly(carbamoyl) sulfonate (PCS) hydrogel on a Teflon membrane. LDH catalyzes the specific dehydrogenation of lactate consuming NAD(+). SHL catalyzes the irreversible decarboxylation and the hydroxylation of salicylate in the presence of oxygen and NADH produced by LDH. PyOD decarboxylates pyruvate using oxygen and phosphate. SHL and PyOD force the equilibrium of dehydrogenation of lactate by LDH to the product side by consuming NADH and pyruvate, respectively. Dissolved oxygen acts as an essential material for both PyOD and SHL during their respective enzymatic reactions. Therefore, an amplified signal, caused by the consumptions of dissolved oxygen by the two enzymes, was observed in the measurement of lactate. Regeneration of cofactor was found in the trienzyme system. A Teflon membrane was used to fabricate the sensor in order to avoid interferences. The sensor has a fast response (2s) and short recovery times (2 min). The total test time for a measurement by using this lactate sensor (4 min) was faster than using a commercial lactate testing kit (up to 10 min). The sensor has a linear range between 10 and 400 microM lactate, with a detection limit of 4.3 microM. A good agreement (R2 = 0.9984) with a commercial lactate testing kit was obtained in beverage sample measurements.     

Kinetic study of phenol hydroxylase and catechol 1,2-dioxygenase biosynthesis by <Emphasis Type="Italic">Candida tropicalis</Emphasis> cells grown on different phenolic substrates

When Candida tropicalis was grown on phenol, catechol or resorcinol, the highest levels of specific activity of phenol hydroxylase (EC. 1.14.13.7) and catechol 1,2-dioxygenase (EC. 1.13.11.1) were attained with phenol. With the three aromatic compounds tested, the yeast cells exhibited sharp peaks of specific activity of both enzymes at particular incubation times. Phenol-induced cells containing high levels of both enzymes were capable of degrading rapidly and without delay 4-chlorophenol and 2,6-dichlorophenol, and to a lesser extend pentachlorophenol. However, the yeast could not grow on chlorophenols as major carbon and energy source.     

Structural and stability effects of phosphorylation: Localized structural changes in phenylalanine hydroxylase

Phosphorylation of phenylalanine hydroxylase (PAH) at Ser16 by cAMP-dependent protein kinase increases the basal activity of the enzyme and its resistance to tryptic proteolysis. The modeled structures of the full-length phosphorylated and unphosphorylated enzyme were subjected to molecular dynamics simulations, and we analyzed the energy of charge-charge interactions for individual ionizable residues in the final structures. These calculations showed that the conformational changes induced by incorporation of phosphate were localized and limited mostly to the region around the phosphoserine (Arg13-Asp17) and a region around the active site in the catalytic domain that includes residues involved in the binding of the iron and the substrate L-Phe (Arg270 and His285). The absence of a generalized conformational change was confirmed by differential scanning calorimetry, thermal-dependent circular dichroism, fluorescence spectroscopy, and limited chymotryptic proteolysis of the phosphorylated and unphosphorylated PAH. Our results explain the effect of phosphorylation of PAH on both the resistance to proteolysis specifically by trypsin-like enzymes and on the increase in catalytic efficiency.     

酪氨酸羟化酶和左旋芳香族氨基酸脱羧酶基因的细胞表达及活性检测

由于在帕金森病中合成多巴胺所需的酪氨酸羟化酶(tyrosine hydroxylase,TH)和左旋芳香族氨基酸脱羧酶(aromatic L-amino acid decarboxylase,AADC)活性明显降低,所以补充多巴胺合成酶成为基因治疗帕金森病研究的主要手段。我们分别构建了重组逆转录病毒载体pLHCX／TH及pLNCX2／AADC,通过脂质体介导将带有目的基因的载体分别转到包装细胞PA317中,经筛选得到产病毒的细胞PA317／TH和PA317／AADC,采用免疫组化、原位杂交方法检测目的基因表达;细胞经裂解后进行的酶促反应产物多巴胺以高压液相电化学方法检测证明所克隆的T‘H及AADC基因具有功能活性;这两种基因工程改造细胞可以完成酶促动力学的功能,使L-dopa及多巴胺产生明显增加。本研究为用TH和AADC双基因对帕金森病进行基因治疗提供了一定的依据。     

Induction of SM-20 in PC12 cells leads to increased cytochrome c levels,accumulation of cytochrome c in the cytosol,and caspase-dependent cell death

Sympathetic neurons deprived of nerve growth factor (NGF) release cytochrome c into the cytosol and undergo caspase-dependent cell death through a process that requires de novo gene expression. Expression of the SM-20 gene increases after NGF withdrawal, and ectopic SM-20 expression induces cell death in NGF-maintained neurons. To further evaluate the mechanism by which SM-20 promotes cell death, we developed a PC12-derived cell line in which SM-20 expression can be induced by addition of doxycycline to the culture medium. Induction of SM-20 in either undifferentiated or NGF-differentiated cells resulted in cell death. Cell death was accompanied by an increase in caspase activity and was inhibited by the caspase inhibitor zVAD-FMK. Analysis of cytochrome c in cytosolic and mitochondria-enriched subcellular fractions revealed that induction of SM-20 led to the accumulation of cytochrome c in the cytosol. Surprisingly, SM-20 expression also resulted in a selective increase in the total amount of cytochrome c protein. Thus, induction of SM-20 expression appears to affect both the amount and subcellular localization of cytochrome c in PC12 cells. These results suggest that SM-20 promotes caspase-dependent cell death through a mechanism involving cytochrome c.     

The pesticide rotenone induces caspase-3-mediated apoptosis in ventral mesencephalic dopaminergic neurons

In vivo, the pesticide rotenone induces degeneration of dopamine neurons and parkinsonian-like pathology in adult rats. In the current study, we utilized primary ventral mesencephalic (VM) cultures from E15 rats as an in vitro model to examine the mechanism underlying rotenone-induced death of dopamine neurons. After 11 h of exposure to 30 nm rotenone, the number of dopamine neurons identified by tyrosine hydroxylase (TH) immunostaining declined rapidly with only 23% of the neurons surviving. By contrast, 73% of total cells survived rotenone treatment, indicating that TH+ neurons are more sensitive to rotenone. Examination of the role of apoptosis in TH+ neuron death, revealed that 10 and 30 nm rotenone significantly increased the number of apoptotic TH+ neurons from 7% under control conditions to 38 and 55%, respectively. The increase in apoptotic TH+ neurons correlated with an increase in immunoreactivity for active caspase-3 in TH+ neurons. The caspase-3 inhibitor, DEVD, rescued a significant number of TH+ neurons from rotenone-induced death. Furthermore, this protective effect lasted for at least 32 h post-rotenone and DEVD exposure, indicating lasting neuroprotection achieved with an intervention prior to the death commitment point. Our results show for the first time in primary dopamine neurons that, at low nanomolar concentrations, rotenone induces caspase-3-mediated apoptosis. Understanding the mechanism of rotenone-induced apoptosis in dopamine neurons may contribute to the development of new neuroprotective strategies against Parkinson's disease.     

BDNF heightens the sensitivity of motor neurons to excitotoxic insults through activation of TrkB

The survival promoting and neuroprotective actions of brain-derived neurotrophic factor (BDNF) are well known but under certain circumstances this growth factor can also exacerbate excitotoxic insults to neurons. Prior exploration of the receptor through which BDNF exerts this action on motor neurons deflects attention away from p75. Here we investigated the possibility that BDNF acts through the receptor tyrosine kinase, TrkB, to confer on motor neurons sensitivity to excitotoxic challenge. We blocked BDNF activation of TrkB using a dominant negative TrkB mutant or a TrkB function blocking antibody, and found that this protected motor neurons against excitotoxic insult in cultures of mixed spinal cord neurons. Addition of a function blocking antibody to BDNF to mixed spinal cord neuron cultures is also neuroprotective indicating that endogenously produced BDNF participates in vulnerability to excitotoxicity. We next examined the intracellular signaling cascades that are engaged upon TrkB activation. Previously we found that inhibition of the phosphatidylinositide-3'-kinase (PI3'K) pathway blocks BDNF-induced excitotoxic sensitivity. Here we show that expression of a constitutively active catalytic subunit of PI3'K, p110, confers excitotoxic sensitivity (ES) upon motor neurons not incubated with BDNF. Parallel studies with purified motor neurons confirm that these events are likely to be occuring specifically within motor neurons. The abrogation of BDNF's capacity to accentuate excitotoxic insults may make it a more attractive neuroprotective agent.