Mutations in the human retinal degeneration slow (RDS) gene can cause either retinitis pigmentosa or macular dystrophy.

Mutations in the RDS gene, which encodes the photoreceptor glycoprotein peripherin, have been sought in families with autosomal dominant retinal dystrophies. A cysteine deletion at codon 118/119 is associated with retinitis pigmentosa in one. Three families with similar macular dystrophy have mutations at codon 172, arginine being substituted by tryptophan in two and by glutamine in one. A stop sequence at codon 258 exists in a family with adult vitelliform macular dystrophy. These findings demonstrate that both retinitis pigmentosa and macular dystrophies are caused by mutations in RDS and that the functional significance of certain amino-acids in peripherin-RDS may be different in cones and rods.

A fully capable pianist with a congenital bilateral agenesis of extensor pollicis brevis muscle.

A 28-year-old male musical student has been presented with visible inability of active abduction and extension of the thumbs in both hands beyond the neutral position. The student has not been previously diagnosed and claimed no history of trauma or surgical procedures in the area of hands and no family history of such disabilities. The student remained capable of playing on keyboard instruments on high level due to compensation by hyperextension of the interphalangeal joint of both thumbs and showed no increased frequency of the injuries or playing-related disorders. The ultrasound and magnetic resonance imaging showed complete bilateral agenesis of extensor pollicis brevis muscles and was classified as isolated congenital clasped thumb syndrome. Due to the age of the student and the agenesis of the muscles the conservative treatment was deemed inadequate and due to high functionality of the student as a musician and unforeseeable results it might have on a musician's career, surgical treatment has been disadvised.

Morphology of sesamoid bones in keyboard musicians.

BACKGROUND: The sesamoid bones are small, usually oval bone structures often found in joints and under the tendons. Although their precise function is not fully understood, it is agreed upon that they protect the joints and make movements faster and less energy consuming. Sesamoid bones are found in hands, especially around first, second and fifth metacarpophalangeal joint and the interphalangeal joint of the thumb. MATERIALS AND METHODS: This study compares a group of 32 young musicians to 30 non-musicians of similar age and posture. The hands of the subjects were examined by ultrasound imaging for the presence of sesamoid bones. The results were noted and observed sesamoids were measured. RESULTS: The results seem to prove that although there are no difference in the amount or the location of the sesamoid bones between the musicians and the non-musicians, there is statistically significant tendency for the musicians to have bigger sum of the sesamoid's volume per hand (Fisher's test p-value = 0.034 < 0.05). CONCLUSIONS: There was also observed an unusually shaped "Bactrian" sesamoid bone at the interphalangeal joint of the thumb in 8 cases in the musicians' group and 1 case in the control group. All participants with the aforementioned structure were female.

Glutamate impairs neuronal calcium extrusion while reducing sodium gradient.

The rate of decrease of neuronal [Ca2+]i after an elevation induced by a glutamate pulse is much slower than that after a comparable [Ca2+]i elevation induced by a K+ depolarization. To investigate whether the [Na+]i increase taking place during the glutamate pulse reduces the rate of Ca2+ extrusion, we monitored simultaneously [Na+]i and [Ca2+]i during a K+ depolarization and a glutamate pulse lasting 1 min. The K+ depolarization evoked only a transient increase of [Na+]i from 4 mM to 13 mM, whereas the glutamate pulse increased [Na+]i to 60 mM, and this increase persisted after glutamate removal. An application of bepridil immediately after glutamate pulse when [Na+]i was greatly elevated, but not 14 min after glutamate removal when a basal [Na+]i was restored, evoked a [Ca2+]i increase accompanied by a decrease of [Na+]i, indicating a reverse mode of operation of the Na+/Ca2+ exchanger. These data suggest that the glutamate-evoked increase in [Na+]i may play a role in Ca2+ homeostasis destabilization.

Adenoviral vectors efficiently target cell lines derived from selected lymphocytic malignancies, including anaplastic large cell lymphoma and Hodgkin's disease.

We have hypothesized that adenoviral vectors might mediate gene transfer into cell lines derived from human lymphocytic malignancies, such as lymphoma, lymphocytic leukemia, and myeloma. A panel of 33 cell lines was studied for their ability to be transduced by an adenoviral (AD) vector encoding the Escherichia coli beta-galactosidase gene (AD-betagal). A cytochemical assay and a flow cytometry assay both demonstrated that a subset of lymphocytic cell lines can be efficiently transduced by adenoviral vectors. In particular, three of three anaplastic large cell lymphoma lines, two of two Hodgkin's disease cell lines, two of seven Burkitt's lymphoma cell lines, and three of five myeloma cell lines exhibited efficient gene transfer. The ability of an AD vector expressing the thymidine kinase (tk) gene from herpes simplex virus-1 (AD-tk) followed by ganciclovir (GCV) to kill 11 of these lymphocytic cell lines was studied. In eight of the cell lines tested, more than 68% of the cells were killed by AD-tk/GCV. Similar results were obtained using an adenoviral vector expressing the wild-type p53 tumor suppressor gene (AD-p53). Thus, AD-tk/GCV and AD-p53 both demonstrated efficient killing of these cell lines. These data document that adenoviral vectors are valuable reagents for the introduction of genes into selected lymphocytic cell lines. These data also suggest that adenoviral vectors might be useful for gene therapy of subsets of lymphocytic malignancy.

Expression of the intermediate filament nestin during rodent tooth development.

The developing tooth represents a suitable model for understanding the molecular mechanisms involved in induction, morphogenesis and differentiation of organs. It is conceivable that the developmental changes could be reflected in the distribution of different cytoskeletal components and in this report we analyze the expression of the intermediate filament nestin during rodent tooth development at the protein and mRNA levels (by immuno light and electron microscopy, and by in situ hybridization). Nestin is expressed at all stages of tooth development, but the expression levels increase after birth in both ectodermal and ectomesenchymal derivatives. The shift in nestin distribution, from the proliferating dental lamina to the dental mesenchyme, indicates that nestin may be involved in inductive phenomena. At early stages of mineralization, nestin is seen within the apical parts of the presecretory ameloblasts. Nestin is also expressed in odontoblasts, both during odontogenesis and after tooth eruption. The increase in nestin expression from early to late developmental stages and sustained expression in a differentiated cell type contrasts with previously observed patterns of nestin expression during nerve and muscle development. This suggests that nestin could be used as a specific marker for the odontoblast.

Immunoproteomic analysis of Trichinella spiralis larval crude antigens recognized by sera from patients with trichinellosis after treatment with albendazole.

Antibody responses and antigen recognition were monitored during and after treatment with albendazole (ABZ) in nine patients selected from a trichinellosis outbreak that occurred in north-west Poland in 2007. Seven out of the nine patients yielded positive serum IgG response during treatment. One month after treatment, the IgG response decreased in most patients. Serum levels of ABZ and main metabolites greatly varied among patients without correlation with the IgG response. Two-dimensional electrophoresis and western blot with serum from each patient showed highly immunoreactive spots located between 3- 10 pI and 45-97 kDa in all patients. Matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF) and MALDI-TOF/time-of-flight mass spectrometry (TOF MS) analysis identified actine, enolase, p49 protein, Caenorhabditis elegans-targeted antigen, and serine protease as the most reactive proteins. A minor spot located at 6 pI and 26 kDa identified as annexin I failed recognition in most patients showing decline in IgG response and clinical improvement after treatment. This protein could constitute a sensitive marker for the effectiveness of ABZ against trichinellosis.

Metabotropic glutamate receptor 1 acts as a dependence receptor creating a requirement for glutamate to sustain the viability and growth of human melanomas.

Metabotropic glutamate 1 (mGlu) receptor has been proposed as a target for the treatment of metastatic melanoma. Studies have demonstrated that inhibiting the release of glutamate (the natural ligand of mGlu1 receptors), results in a decrease of melanoma tumor growth in mGlu1 receptor-expressing melanomas. Here we demonstrate that mGlu1 receptors, which have been previously characterized as oncogenes, also behave like dependence receptors by creating a dependence on glutamate for sustained cell viability. In the mGlu1 receptor-expressing melanoma cell lines SK-MEL-2 (SK2) and SK-MEL-5 (SK5), we show that glutamate is both necessary and sufficient to maintain cell viability, regardless of underlying genetic mutations. Addition of glutamate increased DNA synthesis, whereas removal of glutamate not only suppressed DNA synthesis but also promoted cell death in SK2 and SK5 melanoma cells. Using genetic and pharmacological inhibitors, we established that this effect of glutamate is mediated by the activation of mGlu1 receptors. The stimulatory potential of mGlu1 receptors was further confirmed in vivo in a melanoma cell xenograft model. In this model, subcutaneous injection of SK5 cells with short hairpin RNA-targeted downregulation of mGlu1 receptors resulted in a decrease in the rate of tumor growth relative to control. We also demonstrate for the first time that a selective mGlu1 receptor antagonist JNJ16259685 ((3,4-Dihydro-2H-pyrano[2,3-b]quinolin-7-yl)-(cis-4-methoxycyclohexyl)-methanone) slows SK2 and SK5 melanoma tumor growth in vivo. Taken together, these data suggest that pharmacological inhibition of mGlu1 receptors may be a novel approach for the treatment of metastatic melanoma.

Inhibitory effects of basic fibroblast growth factor on chondrocyte differentiation.

The role of basic fibroblast growth factor (bFGF) and insulin-like growth factor I (IGF-I) in cartilage growth was studied in primary cultures of rat rib growth plate chondrocytes. Growth factors effects on expression of the proto-oncogene c-fos, DNA synthesis, differentiation, and morphological changes were analyzed by in situ hybridization, 3H-thymidine incorporation, and light and fluorescence microscopy. In serum-deprived cells, bFGF induced a transient expression of c-fos with a maximal effect 15-30 minutes after stimulation. After 24 h of culture it had a slightly lower stimulatory effect on DNA synthesis than IGF-I, but became a significantly more potent mitogen than IGF-I after 48 and 72 h. The stimulatory effect of bFGF on DNA synthesis coincided with a decrease in collagen type II and IGF-II expression. In contrast, IGF-I alone stimulated expression of these genes. In bFGF-treated cultures, cell morphology and the appearance of actin filaments was changed. Polygonal chondrocytes became elongated, fibroblast-like, and the smooth actin filaments were brush-like and disrupted. Addition of IGF-I reduced these changes without affecting c-fos expression induced by bFGF. Our results suggest that bFGF stimulates cell proliferation by preventing terminal differentiation of chondrocytes. This effect is mediated by induction of c-fos expression and a decrease in the steady-state levels of transcripts for collagen II and IGF-II.

Demonstration of estrogen receptor-beta immunoreactivity in human growth plate cartilage.

Estrogens affect longitudinal bone growth through their action on endochondral bone formation. Two estrogen receptors are known, the classical estrogen receptor-alpha (ER alpha), newly demonstrated in human growth plate cartilage, and a recently cloned estrogen receptor-beta (ER beta). The present study aimed to localize a possible expression of ER beta protein in human growth plates. Tissue samples were obtained from tibial and femoral growth plates in four female pubertal patients undergoing epiphyseal surgery. Immunohistochemistry, using two different ER beta-specific antibodies, demonstrated positive staining for ER beta in hypertrophic epiphyseal chondrocytes from all patients. No staining was noted in resting or proliferative chondrocytes. These data suggest that in addition to ER alpha, human epiphyseal chondrocytes also express ER beta. The physiological role of ER beta in the regulation of longitudinal bone growth in humans remains to be elucidated.

Differential effects of oral losartan and enalapril on local venous and systemic pressor responses to angiotensin I and II in healthy men.

This double-blind, placebo-controlled crossover study was designed to differentiate the pharmacodynamic effects of the angiotensin II receptor antagonist losartan from the angiotensin converting enzyme inhibitor enalapril. Effects of placebo, enalapril (10 mg), and losartan (20 and 100 mg) on local venous and systemic pressor responses to angiotensin I and II were compared in eight healthy male subjects. Treatments were administered orally approximately 4 hours before agonist infusions into a dorsal hand vein. Local changes in hand vein diameter and systemic blood pressure were monitored during the infusions. The 100 mg dose of losartan attenuated local venoconstrictor and systemic pressor responses to angiotensin I and II. In contrast, enalapril blocked only responses to angiotensin I. Both losartan and enalapril increased plasma renin concentration compared with placebo. These results are consistent with direct antagonism of angiotensin II receptors by losartan and with indirect effects of enalapril through inhibition of angiotensin converting enzyme.

Selective elimination (purging) of contaminating malignant cells from hematopoietic stem cell autografts using recombinant adenovirus.

In this work we have explored the use of adenoviral vectors for the purging of cancer cells from hematopoietic stem cell (HSC) autografts. We showed that a recombinant adenovirus expressing the herpes simplex-1 thymidine kinase gene (AD-tk) plus ganciclovir (GCV) killed HELA cells more effectively than did GCV alone. HELA cells were then mixed with human HSCs and exposed to AD-tk/GCV. AD-tk/GCV reduced the number of HELA colonies to 4% of control values, with no detectable reduction in the hematopoietic progenitor, colony forming unit-granulocyte/monocyte (CFU-GM). Similar studies of the JB6 non-Hodgkins lymphoma cell line showed a reduction to 5% of controls; studies of MCF-7, a breast carcinoma cell line, showed a reduction to 30% of controls, with no CFU-GM toxicity. Thus, AD-tk mediated selective killing of contaminating tumor cells. We also evaluated a recombinant adenovirus encoding the tumor suppressor gene p53 (AD-p53). AD-p53 was able to selectively kill all three cell lines (reducing tumor colonies approximately 100-fold) without any toxicity to CFU-GM. Although both AD-tk/GCV and AD-p53 were effective in these experiments, AD-p53 seemed to be more potent. Adenoviral vectors show promise for selectively targeting cancer cells that contaminate HSC autografts.

Learning impairment in rats by N-methyl-D-aspartate receptor antagonists.

2-Amino-5-phosphonovalerate (APV, icv) phencyclidine (PCP, ip) and scopolamine (sc) dose-dependently disrupted short term working memory in radial maze. These drugs injected before, but not after training attenuated retention of long term memory in passive avoidance task. A relation of PCP action to its antagonism at NMDA receptors may be suggested.

Profile of phosphatidylinositol metabolism stimulated by carbachol and glutamate in primary cultures of rat cerebellar neurons.

The formation of inositol phosphates, after stimulation of primary cultures of cerebellar neurons of the neonatal rat, in the presence of lithium chloride, by glutamate, carbachol, norepinephrine, histamine and Mg2+-free conditions, was measured by anion exchange high-pressure liquid chromatography (HPLC) with on-line radioactivity detection. All of the above agents caused a persistent, dose-dependent and calcium-sensitive preferential accumulation of inositol-4-phosphate, while the levels of inositol-1-phosphate were virtually unaffected. Agonist stimulation produced also a transient increase of a second peak which co-eluted with the standard for inositol 1,4-bisphosphate. However, no significant accumulation of inositol-1,4,5-trisphosphate and inositol-1,3,4,5-tetrakisphosphate was detected, possibly due to the fast kinetics of the metabolism of inositol phosphate. The results indicate that receptor-stimulated metabolism of inositol phosphate, in cultures of cerebellar granule cells, is due to a preferential hydrolysis of polyphosphoinositides and leads to the formation of inositol-4-phosphate through several calcium- and lithium-sensitive enzymatic steps.

Different coupling of excitatory amino acid receptors with Ca2+ channels in primary cultures of cerebellar granule cells.

Cerebellar granule cells in primary culture express receptors for excitatory amino acids. The activation of these receptors results in an increased uptake of Ca2+, however, the effects are different depending on the agonists used. Aspartate, NMDA and ibotenate are active only in depolarized conditions, whereas kainate and glutamate activate Ca2+ uptake independently from depolarization. The results indicate the presence of two receptor types: kainate recognition site coupled with voltage-independent Ca2+ channels and NMDA recognition site coupled with voltage-dependent Ca2+ channels.

Pharmacological modulation of GABAergic transmission in cultured cerebellar neurons.

GABA activates specific ion channels in post-natal cerebellar neurons in primary culture generating Cl- currents that can be recorded with the whole-cell patch-clamp technique. Evoked and spontaneous GABA-mediated synaptic activity can be recorded from cells kept in culture for a few days. Benzodiazepine and beta-carboline derivatives which bind with high affinity to the domains for allosteric regulation of GABA receptors facilitated and inhibited directly applied GABA responses and synaptically evoked Cl- currents recorded under voltage-clamp.

Pertussis toxin inhibits signal transduction at a specific metabolotropic glutamate receptor in primary cultures of cerebellar granule cells.

In primary cultures of cerebellar granule cells, glutamate receptors have been classified into metabolotropic (GP1 and GP2) and ionotropic (GC1 and GC2). The GP1 and GC1 receptors are negatively modulated by magnesium and noncompetitively inhibited by phencyclidine; GP2 and GC2 receptors are insensitive to inhibition by magnesium and phencyclidine (Costa, Fadda, Kozikowski, Nicoletti and Wroblewski, 1988). Exposure of cultured cerebellar granule cells to pertussis toxin (PTX, 1 microgram/ml for 14-16 hr) reduced the stimulation of the hydrolysis of inositol phospholipids (PI) by the GP2 receptor agonists, glutamate and quisqualate in the presence of magnesium, but did not inhibit the stimulation of the hydrolysis of PI by GP1 receptor agonists. The stimulation of the hydrolysis of PI by the muscarinic cholinergic receptor agonist, carbamylcholine, remained unchanged after pretreatment with pertussis toxin. In membranes prepared from cerebellar granule cells in primary culture, the addition of guanosine 5'-0-(3-thiotriphosphate) (GTP-gamma-s), a nonhydrolyzable analogue of GTP, enhanced the hydrolysis of PI and reduced the Bmax of quisqualate-displaceable binding of [3H]glutamate. These results indicate that, in primary cultures of cerebellar granule cells, a specific class of metabolotropic glutamate receptors (the GP2 receptor) is coupled with the hydrolysis of PI through a pertussis toxin-sensitive GTP-binding protein.

Activation of N-methyl-D-aspartate-sensitive glutamate receptors stimulates arachidonic acid release in primary cultures of cerebellar granule cells.

In cultured granule cells prelabeled with [3H]arachidonate the activation of excitatory amino acid receptors by various agonists results in a dose-dependent stimulation of [3H]arachidonic acid release. Glutamate and aspartate were the most potent agonists, whereas N-methyl-D-aspartate, kainate and quisqualate were less potent. Other neurotransmitter receptor agonists--GABA, baclofen and norepinephrine--were inactive, while carbachol induced only a slight effect. Since the transmitter-mediated release of [3H]arachidonate was blocked by phencyclidine, a selective inhibitor of NMDA-sensitive glutamate receptors, it can be inferred that the effects of all other receptor agonists were indirectly mediated via the release of glutamate from granule cells. Aspartate-evoked release was Ca2+-dependent and was abolished by the glutamate receptor inhibitors: Mg2+ ions and 2-amino-5-phosphonovalerate. The inhibitors of phospholipase A2, quinacrine and p-bromophenacyl bromide, decreased the release of [3H]arachidonate in a dose-related manner.

Glycine and D-serine increase the affinity of N-methyl-D-aspartate sensitive glutamate binding sites in rat brain synaptic membranes.

In previously frozen and extensively washed brain membranes [3H]glutamate binds to a single population of sites characteristic of the NMDA-sensitive glutamate receptor subtype. This binding cannot be displaced by glycine and D-serine, but actually is enhanced by these amino acids in a dose-dependent manner. Glycine and D-serine increase the affinity of glutamate binding without changing the density of binding sites. These results delineate glycine as an allosteric modulator of the recognition site for the NMDA-sensitive glutamate receptor.

Serine-O-phosphate, an endogenous metabolite, inhibits the stimulation of inositol phospholipid hydrolysis elicited by ibotenic acid in rat hippocampal slices.

Serine-O-phosphate (PS) inhibits the accumulation of 3H-inositolmonophosphate elicited by ibotenic acid in rat hippocampal slices incubated in the presence of 7 mM Li+. This inhibition is concentration- dependent and stereoselective, L-PS being 5 fold more potent than D-PS. Among different structural analogues of PS, only L-serine weakly antagonizes the action of ibotenic acid, whereas phosphorylcholine, phosphorylethanolamine and phosphothreonine are inactive even at high concentrations. These results are consistent with the hypothesis that L-PS may act as an endogenous regulator of excitatory amino acid receptor function.