New views on RPE65 deficiency: the rod system is the source of vision in a mouse model of Leber congenital amaurosis.

Leber congenital amaurosis (LCA) is the most serious form of the autosomal recessive childhood-onset retinal dystrophies. Mutations in the gene encoding RPE65, a protein vital for regeneration of the visual pigment rhodopsin in the retinal pigment epithelium, account for 10-15% of LCA cases. Whereas previous studies of RPE65 deficiency in both animal models and patients attributed remaining visual function to cones, we show here that light-evoked retinal responses in fact originate from rods. For this purpose, we selectively impaired either rod or cone function in Rpe65-/- mice by generating double- mutant mice with models of pure cone function (rhodopsin-deficient mice; Rho-/-) and pure rod function (cyclic nucleotide-gated channel alpha3-deficient mice; Cnga3-/-). The electroretinograms (ERGs) of Rpe65-/- and Rpe65-/-Cnga3-/- mice were almost identical, whereas there was no assessable response in Rpe65-/-Rho-/- mice. Thus, we conclude that the rod system is the source of vision in RPE65 deficiency. Furthermore, we found that lack of RPE65 enables rods to mimic cone function by responding under normally cone-isolating lighting conditions. We propose as a mechanism decreased rod sensitivity due to a reduction in rhodopsin content to less than 1%. In general, the dissection of pathophysiological processes in animal models through the introduction of additional, selective mutations is a promising concept in functional genetics.

Lack of an endothelial store-operated Ca2+ current impairs agonist-dependent vasorelaxation in TRP4-/- mice.

Agonist-induced Ca2+ entry into cells by both store-operated channels and channels activated independently of Ca2+-store depletion has been described in various cell types. The molecular structures of these channels are unknown as is, in most cases, their impact on various cellular functions. Here we describe a store-operated Ca2+ current in vascular endothelium and show that endothelial cells of mice deficient in TRP4 (also known as CCE1) lack this current. As a consequence, agonist-induced Ca2+ entry and vasorelaxation is reduced markedly, showing that TRP4 is an indispensable component of store-operated channels in native endothelial cells and that these channels directly provide an Ca2+-entry pathway essentially contributing to the regulation of blood vessel tone.

p53 activates expression of HIC-1, a new candidate tumour suppressor gene on 17p13.3.

For several human tumour types, allelic loss data suggest that one or more tumour suppressor genes reside telomeric to the p53 gene at chromosome 17p13.1. In the present study we have used a new strategy, involving molecular analysis of a DNA site hypermethylated in tumour DNA, to identify a candidate gene in this region (17p13.3). Our approach has led to identification of HIC-1 (hypermethylated in cancer), a new zinc-finger transcription factor gene which is ubiquitously expressed in normal tissues, but underexpressed in different tumour cells where it is hypermethylated. Multiple characteristics of this gene, including the presence of a p53 binding site in the 5' flanking region, activation of the gene by expression of a wild-type p53 gene and suppression of G418 selectability of cultured brain, breast and colon cancer cells following insertion of the gene, make HIC-1 gene a strong candidate for a tumour suppressor gene in region 17p13.3.

Induction of STAT3-related genes in fast degenerating cone photoreceptors of cpfl1 mice.

Cone dystrophies are genetic diseases characterized by loss of cone photoreceptor function and severe impairment of daylight vision. Loss of function is accompanied by a progressive degeneration of cones limiting potential therapeutic interventions. In this study we combined microarray-based gene-expression analysis with electroretinography and immunohistochemistry to characterize the pathological processes in the cone photoreceptor function loss 1 (cpfl1) mouse model. The cpfl1-mouse is a naturally arising mouse mutant with a loss-of-function mutation in the cone-specific Pde6c gene. Cpfl1-mice displayed normal rod-specific light responses while cone-specific responses were strongly diminished. Despite the lack of a general retinal degeneration, the cone-specific functional defect resulted in a marked activation of GFAP, a hallmark of Müller-cell gliosis. Microarray-based network-analysis confirmed activation of Müller-glia-specific transcripts. Unexpectedly, we found up-regulation of the cytokine LIF and the anti-apoptotic transcription factor STAT3 in cpfl1 cone photoreceptors. We postulate that STAT3-related pathways are induced in cpfl1 cone photoreceptors to counteract degeneration.

Melanopsin and rod-cone photoreceptive systems account for all major accessory visual functions in mice.

In the mammalian retina, besides the conventional rod-cone system, a melanopsin-associated photoreceptive system exists that conveys photic information for accessory visual functions such as pupillary light reflex and circadian photo-entrainment. On ablation of the melanopsin gene, retinal ganglion cells that normally express melanopsin are no longer intrinsically photosensitive. Furthermore, pupil reflex, light-induced phase delays of the circadian clock and period lengthening of the circadian rhythm in constant light are all partially impaired. Here, we investigated whether additional photoreceptive systems participate in these responses. Using mice lacking rods and cones, we measured the action spectrum for phase-shifting the circadian rhythm of locomotor behaviour. This spectrum matches that for the pupillary light reflex in mice of the same genotype, and that for the intrinsic photosensitivity of the melanopsin-expressing retinal ganglion cells. We have also generated mice lacking melanopsin coupled with disabled rod and cone phototransduction mechanisms. These animals have an intact retina but fail to show any significant pupil reflex, to entrain to light/dark cycles, and to show any masking response to light. Thus, the rod-cone and melanopsin systems together seem to provide all of the photic input for these accessory visual functions.

HCN channels: structure, cellular regulation and physiological function.

Hyperpolarization-activated and cyclic nucleotide-gated (HCN) channels belong to the superfamily of voltage-gated pore loop channels. HCN channels are unique among vertebrate voltage-gated ion channels, in that they have a reverse voltage-dependence that leads to activation upon hyperpolarization. In addition, voltage-dependent opening of these channels is directly regulated by the binding of cAMP. HCN channels are encoded by four genes (HCN1-4) and are widely expressed throughout the heart and the central nervous system. The current flowing through HCN channels, designated I(h) or I(f), plays a key role in the control of cardiac and neuronal rhythmicity ("pacemaker current"). In addition, I(h) contributes to several other neuronal processes, including determination of resting membrane potential, dendritic integration and synaptic transmission. In this review we give an overview on structure, function and regulation of HCN channels. Particular emphasis will be laid on the complex roles of these channels for neuronal function and cardiac rhythmicity.

The roles of the subunits in the function of the calcium channel.

Dihydropyridine-sensitive voltage-dependent L-type calcium channels are critical to excitation-secretion and excitation-contraction coupling. The channel molecule is a complex of the main, pore-forming subunit alpha 1 and four additional subunits: alpha 2, delta, beta, and gamma (alpha 2 and delta are encoded by a single messenger RNA). The alpha 1 subunit messenger RNA alone directs expression of functional calcium channels in Xenopus oocytes, and coexpression of the alpha 2/delta and beta subunits enhances the amplitude of the current. The alpha 2, delta, and gamma subunits also have pronounced effects on its macroscopic characteristics, such as kinetics, voltage dependence of activation and inactivation, and enhancement by a dihydropyridine agonist. In some cases, specific modulatory functions can be assigned to individual subunits, whereas in other cases the different subunits appear to act in concert to modulate the properties of the channel.

Primary structure of the beta subunit of the DHP-sensitive calcium channel from skeletal muscle.

Complementary DNAs for the beta subunit of the dihydropyridine-sensitive calcium channel of rabbit skeletal muscle were isolated on the basis of peptide sequences derived from the purified protein. The deduced primary structure is without homology to other known protein sequences and is consistent with the beta subunit being a peripheral membrane protein associated with the cytoplasmic aspect of the sarcolemma. The protein contains sites that might be expected to be preferentially phosphorylated by protein kinase C and guanosine 3',5'-monophosphate-dependent protein kinase. A messenger RNA for this protein appears to be expressed in brain.

HIC1 hypermethylation is a late event in hematopoietic neoplasms.

HIC1, a candidate tumor suppressor gene on 17p13.3, is hypermethylated and silenced in a large number of solid tumors. To determine its potential role in leukemias, we studied its methylation status in normal and neoplastic hematopoietic cells. We found HIC1 to be unmethylated in peripheral blood cells, bone marrow cells, and CD34+ cells. HIC1 was rarely methylated in newly diagnosed acute myelogenous leukemias (10%) but was relatively frequently methylated in newly diagnosed non-Hodgkin's lymphoma (25%), acute lymphocytic leukemia (ALL; 53%), and chronic-phase chronic myelogenous leukemia (50%). By contrast, HIC1 was hypermethylated in 100% of recurrent ALL and 100% of blast crisis chronic myelogenous leukemia. In two patients with ALL for whom paired diagnosis/relapse samples were available, HIC1 was unmethylated at diagnosis but was highly methylated at relapse after a chemotherapy-induced complete remission. HIC1 methylation, therefore, seems to be a progression event in hematopoietic neoplasms.

Restoring Light Sensitivity in Blind Retinae Using a Photochromic AMPA Receptor Agonist.

Retinal degenerative diseases can have many possible causes and are currently difficult to treat. As an alternative to therapies that require genetic manipulation or the implantation of electronic devices, photopharmacology has emerged as a viable approach to restore visual responses. Here, we present a new photopharmacological strategy that relies on a photoswitchable excitatory amino acid, ATA. This freely diffusible molecule selectively activates AMPA receptors in a light-dependent fashion. It primarily acts on amacrine and retinal ganglion cells, although a minor effect on bipolar cells has been observed. As such, it complements previous pharmacological approaches based on photochromic channel blockers and increases the potential of photopharmacology in vision restoration.

Methylation of the HIC-1 candidate tumor suppressor gene in human breast cancer.

HIC-1 (hypermethylated in cancer) is a candidate tumor suppressor gene which is located at 17p13.3, a region which frequently undergoes allelic loss in breast and other human cancers. HIC-1 is proposed to be commonly inactivated in human cancers by hypermethylation of a normally unmethylated dense CpG island which encompasses the entire gene. To study whether HIC-1 inactivation may be important to the development of breast cancer, we first measured methylation of the HIC-1 gene in normal breast ductal tissues from microdissected frozen breast tissues and from epithelial cells purified from mammoplasty specimens. Surprisingly, in all normal breast ductal tissues we found approximately equal amounts of densely methylated HIC-1 and completely unmethylated HIC-1. This is in contrast to most normal tissues, in which all copies of HIC-1 are completely unmethylated. We then evaluated 39 primary breast cancer tissues and found virtually complete methylation of the HIC-1 gene in 26 (67%) of the cases. We also found loss of heterozygosity at the telomeric portion of chromosomal arm 17p in 22 of the 26 cases with strongly methylated HIC-1, suggesting that loss of an unmethylated HIC-1 allele may contribute to the inactivation of HIC-1 in cells with a pre-existing methylated allele. Finally, by RNase protection analysis, HIC-1 was found to be expressed in microdissected normal breast ductal tissues and unmethylated tumors but not in tumors with hypermethylation of the HIC-1 gene. These results indicate that hypermethylation of HIC-1 and associated loss of HIC-1 expression is common in primary breast cancer. Furthermore, the HIC-1 gene is densely methylated in approximately one-half of the alleles in normal breast epithelium, which may predispose this tissue to inactivation of this gene by loss of heterozygosity.

Cyclic nucleotide-gated cation channels molecular diversity, structure, and cellular functions.

Cyclic nucleotide-gated (CNG) channels comprise an extended family of cation channels that are directly activated by the binding of cGMP or cAMP. These channels are abundantly expressed in visual and olfactory neurons, where they play important roles in sensory signal transduction. Recent studies have shown that CNG channels are also present in many nonsensory cell types. The major function of CNG channels probably is to provide a cGMP/cAMP-mediated pathway for influx of calcium. This review describes recent progress in our understanding of the genetic diversity, the molecular determinants of channel function, and the physiological roles of CNG channels. ©1996, Elsevier Science Inc. (Trends Cardiovasc Med 1996;6:274-280).

Tissue-specific expression of calcium channels.

The high-voltage-activated calcium channel is a multimeric protein complex containing α(1), α(2)/δ, ß, and γ subunits. The α(1) subunit is the ion conduction channel and contains the binding sites for calcium channel blockers and toxins. Three genes code for distinct L-type, dihydropyridine-sensitive α(1) subunits; one gene codes for the neuronal P-type (Purkinje) α(1) subunit; and one gene codes for the neuronal N-type α(1) subunit. The smooth and cardiac muscle L-type calcium channel α(1) subunits are splice variants of the same gene. The α(1) subunits are coexpressed with a common α(2)/δ subunit and tissue-specific ß subunits (at least three genes). The γ subunit apparently is expressed only in skeletal muscle. The properties of these cloned and expressed calcium channels are discussed here.

The cDNA and deduced amino acid sequence of the gamma subunit of the L-type calcium channel from rabbit skeletal muscle.

Complementary DNAs for the gamma subunit of the calcium channel of rabbit skeletal muscle were isolated on the basis of peptide sequences derived from the purified protein. The deduced primary structure is without homology to other known protein sequences and is consistent with the gamma subunit being an integral membrane protein.

Primary structure and functional expression of a high voltage activated calcium channel from rabbit lung.

The complete amino acid sequence of the receptor for organic calcium channel blockers (CaCB) from rabbit lung has been deduced by cloning and sequence analysis of the cDNA. Synthetic RNA derived from this cDNA induces the formation of a functional CaCB-sensitive high voltage activated calcium channel in Xenopus oocytes.

Mutations in the S4 domain of a pacemaker channel alter its voltage dependence.

In an attempt to study the functional role of the positively charged amino acids present in the S4 segment of hyperpolarization-activated cyclic nucleotide-gated cation (HCN) channels, we have introduced single and sequential amino acid replacements throughout this domain in the mouse type 2 HCN channel (mHCN2). Sequential neutralization of the first three positively charged amino acids resulted in cumulative shifts of the midpoint voltage activation constant towards more hyperpolarizing potentials. The contribution of each amino acid substitution was approximately -20 mV. Amino acid replacements to neutralize either the first (K291Q) or fourth (R300Q) positively charged amino acid resulted in the same shift (about 20 mV) towards more hyperpolarized potentials. Replacing the first positively charged amino acid with the negatively charged glutamic acid (K291E) produced a shift of approximately -50 mV in the same direction. None of the above amino acid substitutions had any measurable effect on the time course of channel activation. This suggests that the S4 domain of HCN channels critically controls the voltage dependence of channel opening but is not involved in regulating activation kinetics. No channel activity was detected in mutants with neutralization of the last six positively charged amino acids from the S4 domain, suggesting that these amino acids cannot be altered without impairing channel function.

Modulation of cardiac Ca2+ channels in Xenopus oocytes by protein kinase C.

L-Type calcium channel was expressed in Xenopus laevis oocytes injected with RNAs coding for different cardiac Ca2+ channel subunits, or with total heart RNA. The effects of activation of protein kinase C (PKC) by the phorbol ester PMA (4 beta-phorbol 12-myristate 13-acetate) were studied. Currents through channels composed of the main (alpha 1) subunit alone were initially increased and then decreased by PMA. A similar biphasic modulation was observed when the alpha 1 subunit was expressed in combination with alpha 2/delta, beta and/or gamma subunits, and when the channels were expressed following injection of total rat heart RNA. No effects on the voltage dependence of activation were observed. The effects of PMA were blocked by staurosporine, a protein kinase inhibitor. beta subunit moderate the enhancement caused by PMA. We conclude that both enhancement and inhibition of cardiac L-type Ca2+ currents by PKC are mediated via an effect on the alpha 1 subunit, while the beta subunit may play a mild modulatory role.

Primary structure and functional expression of a cyclic nucleotide-gated channel from rabbit aorta.

Sequences specific for cyclic nucleotide-gated channels (CNG channels) have been amplified by PCR from cDNA of heart, aorta, sino-atrial node, cerebellum, C-cells and kidney. The complete amino acid sequence of a CNG channel from rabbit aorta has been deduced by cloning and sequence analysis of the cDNA. Synthetic RNA derived from this cDNA induces the formation of a functional CNG channel in Xenopus oocytes.

Neurologists' knowledge of medication costs.

Using mailed questionnaires, neurologists were asked for (1) estimates of retail prices for 39 common drugs, (2) attitudes about drug costs, and (3) implications for clinical practice. Among these practitioners, (1) more product prices were overestimated than underestimated, (2) old products were as unfamiliar as new products, and (3) community practitioners were more aware of prices than academic neurologists and trainees, but still made errors. Future studies should also consider physician prescribing behavior in terms of adherence to recommended laboratory tests and patient inconvenience factors. Neurologists should be aware of alternative prescription outlets for patients.