The Alzheimer disease BIN1 locus as a modifier of GBA-associated Parkinson disease.

GBA mutations are among the most common genetic risk factors for Parkinson disease (PD) worldwide. We aimed to identify genetic modifiers of the age at onset (AAO) in GBA-associated PD. The study included a genome-wide discovery phase, including a cohort of 79 patients with the GBA p.N370S mutation, and candidate validation and replication analyses of 8 SNPs in patients with mild (n = 113) and severe (n = 41) GBA mutations. Genotyping was performed using the Affymetrix human SNP 6.0 array and TaqMan assays. In the genome-wide phase, none of the SNPs passed the genome-wide significance threshold. Eight SNPs were selected for further analysis from the top hits. In all GBA-associated PD patients (n = 153), the BIN1 rs13403026 minor allele was associated with an older AAO (12.4 ± 5.9 years later, p = 0.0001), compared to patients homozygous for the major allele. Furthermore, the AAO was 10.7 ± 6.8 years later in patients with mild GBA mutations, (p = 0.005, validation group), and 17.1 ± 2.5 years later in patients with severe GBA mutations (p = 0.01, replication). Our results suggest that alterations in the BIN1 locus, previously associated with Alzheimer disease, may modify the AAO of GBA-associated PD. More studies in other populations are required to examine the role of BIN1-related variants in GBA-associated PD.

A homozygous mutation in SLC1A4 in siblings with severe intellectual disability and microcephaly.

We performed exome analysis in two affected siblings with severe intellectual disability (ID), microcephaly and spasticity from an Ashkenazi Jewish consanguineous family. We identified only one rare variant, a missense in SLC1A4 (c. 766G>A [p. E256K]), that is homozygous in both siblings but not in any of their 11 unaffected siblings or their parents (Logarithm of odds, LOD score: 2.6). This variant is predicted damaging. We genotyped 450 controls of Ashkenazi Jewish ancestry and identified only 5 individuals who are heterozygous for this variant (minor allele frequency: 0.0056). SLC1A4 (ASCT1) encodes a transporter for neutral aminoacids such as alanine, serine, cysteine and threonine. L-Serine is essential for neuronal survival and differentiation. Indeed, L-serine biosynthesis disorders affect brain development and cause severe ID. In the brain, L-serine is synthesized in astrocytes but not in neurons. It has been proposed that ASCT1 mediates the uptake of L-serine into neurons and the release of glia-borne L-serine to neighboring cells. SLC1A4 disruption may thus impair brain development and function by decreasing the levels of L-serine in neurons. The identification of additional families with mutations in SLC1A4 would be necessary to confirm its involvement in ID.

[Focal osteochondral necrosis of the femoral head of an adult after Legg-Calve-Perthes disease in childhood]. / Fokale Osteo-Chondro-Nekrose des Hüftkopfes beim Erwachsenen bei Zustand nach M. Perthes im Kindesalter.

In the treatment of circumscribed osteochondral lesions of the knee and the ankle joint autologous osteochondral transplantation (AOT) has been established as one of the possible operative therapies. However, there is less experience with the use of AOT on other joints (shoulder, elbow). The care of osteochondral defects of the hip joint with autologous osteochondral transplantation can still be regarded as an absolute rarity. Facing a focal osteochondral necrosis in a young female adult after LCP disease in childhood, we report about an autologous osteochondral transplantation at the femoral head using the diamond bone cutting system.

Structure characterization of human serum proteins in solution and dry state.

The present report describes application of advanced analytical methods to establish correlation between changes in human serum proteins of patients with coronary atherosclerosis (protein metabolism) before and after moderate beer consumption. Intrinsic fluorescence, circular dichroism (CD), differential scanning calorimetry and hydrophobicity (So) were used to study human serum proteins. Globulin and albumin from human serum (HSG and HSA, respectively) were denatured with 8 m urea as the maximal concentration. The results obtained provided evidence of differences in their secondary and tertiary structures. The thermal denaturation of HSA and HSG expressed in temperature of denaturation (Td, degrees C), enthalpy (DeltaH, kcal/mol) and entropy (DeltaS kcal/mol K) showed qualitative changes in these protein fractions, which were characterized and compared with fluorescence and CD. Number of hydrogen bonds (n) ruptured during this process was calculated from these thermodynamic parameters and then used for determination of the degree of denaturation (%D). Unfolding of HSA and HSG fractions is a result of promoted interactions between exposed functional groups, which involve conformational changes of alpha-helix, beta-sheet and aperiodic structure. Here evidence is provided that the loosening of the human serum protein structure takes place primarily in various concentrations of urea before and after beer consumption (BC). Differences in the fluorescence behavior of the proteins are attributed to disruption of the structure of proteins by denaturants as well as by the change in their compactability as a result of ethanol consumption. In summary, thermal denaturation parameters, fluorescence, So and the content of secondary structure have shown that HSG is more stable fraction than HSA.

Characterization of soluble amaranth and soybean proteins based on fluorescence, hydrophobicity, electrophoresis, amino Acid analysis, circular dichroism, and differential scanning calorimetry measurements.

Intrinsic fluorescence (IF), surface hydrophobicity (S(o)), electrophoresis, amino acid analysis, circular dichroism (CD), and differential scanning calorimetry (DSC) were used to study folded and unfolded soluble proteins from Amaranthus hypochondriacus (A. h.) and soybean (S). Globulin (Glo) and albumin subfractions (Alb-1 and Alb-2) were extracted from A. h. and S and denatured with urea. Electrophoretic and functional properties indicated a significant correlation between soluble protein fractions from soybean and amaranth. The protein fractions shared some common electrophoretic bands as well as a similar amino acid composition. The larger percent of denaturation in protein fractions, which is associated with enthalpy and the number of ruptured hydrogen bonds, corresponds to disappearance of alpha-helix. The obtained results provided evidence of differences in their secondary and tertiary structures. The most stable was Glo followed by the Alb-2 fraction. Predicted functional changes in model protein systems such as pseudocereals and legumes in response to processing conditions may be encountered in pharmaceutical and food industries. These plants can be a substitute for some cereals.

Beer consumption and changes in stability of human serum proteins.

The aim of this study was to evaluate the influence of beer consumption (BC) on the functional and structural properties of human serum proteins (HSP). Thirty-eight volunteers (after coronary bypass) were divided into two groups: experimental (EG) and control (CG). Nineteen volunteers of the EG consumed 330 mL per day of beer (about 20 g of alcohol) for 30 consecutive days. The CG volunteers consumed mineral water instead of beer. Blood samples were collected from EG and CG patients before and after the experiment. Albumin (Alb), globulin (Glo), and methanol-precipitable proteins (MPP) from human serum were denatured with 8 M urea. Fluorescence and electrophoresis were employed in order to elucidate urea-induced conformational changes and structural behavior of proteins. The measured fluorescence emission spectra were used to estimate the stability of native and denatured protein fractions before and after BC. It was found that before BC the fractions most stable to urea denaturation were Glo, Alb, and MPP fractions. After BC in most of the beer-consuming patients (EG) some changes in native and denatured protein fractions were detected: a tendency to lower stability and minor structural deviations. These qualitative changes were more profound in MPP than in Alb and Glo. Thus, Glo is more resistible to alcohol influence than Alb, which in turn is more resistible than MPP. No serum protein changes were detected in patients of CG.

Monitoring of the natural environment by chemical speciation of elements in aerosol and sediment samples.

The development of a monitoring network for chemical speciation of elements of aerosol and sediment samples collected at Lake Balaton has been carried out. Sequential leaching procedures for the determination of the distribution of elements in aerosols (3 steps) and sediments (4 steps) were used. These methods were recently successfully applied to describe environmentally mobile and stable fractions of toxic metals. In aerosol matrices the partition of elements was accomplished by particle size and chemical bonding. In sediments the distribution was performed by chemical bonding. The processes are called fractionation of elements. Particular attention was paid to distinguishing between environmentally mobile and environmentally immobile fractions because these represent the two extreme modes by which the metals are bound to solid matrices. The monitoring objectives were to assess pollution effects on man and his environment and to identify any possible cause and effect relationship between pollutant concentrations and health effects. The results of dry and wet deposition rates showed that most of the toxic metals were dissolved in an aqueous phase and the wet deposition played an important role. It has been found that, while the concentration of Cd and Pb in aerosols is low (0.7 and 29 ng m(-3), respectively), environmentally mobile fractions are considerable. Based upon the data it can be concluded that the effect of the anthropogenic sources on the quality of the lake is minor. This has been the first attempt to correlate speciation results between aerosols and sediments.

Comparative contents of dietary fiber, total phenolics, and minerals in persimmons and apples.

Dietary fibers, major phenolics, main minerals, and trace elements in persimmons and apples were analyzed and compared in order to choose a preferable fruit for an antiatherosclerotic diet. Fluorometry and atomic absorption spectrometry following microwave digestion were optimized for the determination of major phenolics and minerals. Total, soluble, and insoluble dietary fibers, total phenols, epicatechin, gallic and p-coumaric acids, and concentrations of Na, K, Mg, Ca, Fe, and Mn in whole persimmons, their pulps, and peels were significantly higher than in whole apples, pulps, and peels (P < 0.01-0.0025). Conversely, the contents of Cu and Zn were higher in apples than in persimmons. In persimmons and apples all of the above components were higher in their peels than in whole fruits and pulps. The relatively high contents of dietary fibers, total and major phenolics, main minerals, and trace elements make persimmon preferable for an antiatherosclerotic diet.

The 50-millilitre syringe as an inexpensive training aid in the application of cricoid pressure.

A recent study in our department demonstrated that depressing the plunger of a 50-mL syringe was reliably and linearly related to the force applied between 20 N and 50 N. Using a 50-mL syringe we constructed a simple device to help train anaesthetic assistants to apply cricoid pressure correctly. We then tested anaesthetists, operating department practitioners (non-physicians) and nurses in our hospital to see if they could correctly apply forces of 20 and 40 N. All subjects were then trained using this apparatus and once confident were retested immediately afterwards, and again 1 week and 1 month later. The results show a wide variation in the force applied with only 30% of subjects applying appropriate force at 20 N, and 40% at 40 N. Training leads to a significant improvement in performance (P < 0.005 at 20 N and P < 0.001 at 40 N) which is maintained for 1 week for both 20 N (P < 0.05) and 40 N (P < 0.05) but not for 1 month. Therefore training should be practised on a weekly basis. This is an inexpensive and simple device that we believe to be useful in helping anaesthetic assistants to apply effective cricoid pressure.

Entonox equipment as a potential source of cross-infection.

A survey of the hospitals with obstetric units within the Anglia and Oxford Region was performed to assess current practices regarding the cleaning of, and use of filters with, Entonox apparatus. The survey revealed that there was no consensus regarding the cleaning of the equipment and, in contrast to anaesthetic machines in which microbiological filters are recommended and in widespread use, only 10% of the hospitals surveyed were using such filters with the Entonox apparatus in their units. Cleaning procedures were changed in 75% of hospitals when dealing with known 'high-risk' patients, the remaining hospitals treating all patients as 'high-risk' or denied caring for such patients. All patients should be protected from potential cross-infection, and the recommendation that a microbiological filter should be placed between patients and the breathing system should be extended to Entonox equipment.

Intrinsic tryptophan fluorescence of human serum proteins and related conformational changes.

The unfolding of human serum proteins (HSP) was studied by measuring the intrinsic fluorescence intensity at a wavelength of excitation corresponding to tryptophan's or typosine's fluorescence and surface hydrophobicity. The maxima emission wavelengths (lambdamax) of human serum albumin (HSA) and human serum globulin (HSG) before beer consumption (BC) were 336.0 and 337.0 nm and after BC shifted to 335.0 and 334.0 nm, respectively. The surface hydrophobicity slightly increased after BC. In a solution of 8 M urea the lambdamax of BSA shifted to 346.4 and that of BSG to 342.5 nm. In contrast, in the same solution but after BC the lambdamax positions of HSA and HSG shifted to 355.9 and 357.7 nm, respectively. A decrease in fluorescence intensity, a shift in the maximum of emission, and an increase in surface hydrophobicity which reflected unfolding of proteins were observed. Here we provide evidence that the loosening of the HSP structure takes place primarily in various concentrations of urea before and after beer consumption. Differences in the fluorescence behavior of the proteins are attributed to disruption of the structure of proteins by denaturants as well as by the change in their compactability as a result of ethanol consumption.

Novel Ras antagonist blocks human melanoma growth.

During past decades, knowledge of melanoma biology has increased considerably. Numerous therapeutic modalities based on this knowledge are currently under investigation. Advanced melanoma, nevertheless, remains a prime example of poor treatment response that may, in part, be the consequence of activated N-Ras oncoproteins. Besides oncogenic Ras, wild-type Ras gene products also play a key role in receptor tyrosine kinase growth factor signaling, known to be of importance in oncogenesis and tumor progression of a variety of human neoplasms, including malignant melanoma; therefore, it is reasonable to speculate that a pharmacological approach that curtails Ras activity may represent a sensible approach to inhibit melanoma growth. To test this concept, the antitumor activity of S-trans, trans-farnesylthiosalicylic acid (FTS), a recently discovered Ras antagonist that dislodges Ras from its membrane-anchoring sites, was evaluated. The antitumor activity of FTS was assessed both in vitro and in vivo in two independent SCID mouse xenotransplantation models of human melanoma expressing either wild-type Ras (cell line 518A2) or activated Ras (cell line 607B). We show that FTS (5-50 microM) reduces the amounts of activated N-Ras and wild-type Ras isoforms both in human melanoma cells and Rat-1 fibroblasts, interrupts the Ras-dependent extracellular signal-regulated kinase in melanoma cells, inhibits the growth of N-Ras-transformed fibroblasts and human melanoma cells in vitro and reverses their transformed phenotype. FTS also causes a profound and statistically significant inhibition of 518A2 (82%) and 607B (90%) human melanoma growth in SCID mice without evidence of drug-related toxicity. Our findings stress the notion that FTS may qualify as a novel and rational treatment approach for human melanoma and possibly other tumors that either carry activated ras genes or rely on Ras signal transduction more heavily than nonmalignant cells.

A new functional Ras antagonist inhibits human pancreatic tumor growth in nude mice.

Constitutively active Ras proteins, their regulatory components, and overexpressed tyrosine kinase receptors that activate Ras, are frequently associated with cell transformation in human tumors. This suggests that functional Ras antagonists may have anti-tumor activity. Studies in rodent fibroblasts have shown that S-trans, transfarnesylthiosalicylic acid (FTS) acts as a rather specific nontoxic Ras antagonist, dislodging Ras from its membrane anchorage domains and accelerating its degradation. FTS is not a farnesyltransferase inhibitor, and does not affect Ras maturation. Here we demonstrate that FTS also acts as a functional Ras antagonist in human pancreatic cell lines that express activated K-Ras (Panc-1 and MiaPaCa-2). In Panc-1 cells, FTS at a concentration of 25-100 microM reduced the amount of Ras in a dose-dependent manner and interfered with serum-dependent and epidermal growth factor-stimulated ERK activation, thus inhibiting both anchorage-dependent and anchorage-independent growth of Panc-1 cells in vitro. FTS also inhibited tumor growth in Panc-1 xenografted nude mice, apparently without systemic toxicity. Daily FTS treatment (5 mg/kg intraperitoneally) in mice with tumors (mean volume 0.07 cm3) markedly decreased tumor growth (after treatment for 18 days, tumor volume had increased by only 23+/-30-fold in the FTS-treated group and by 127+/-66-fold in controls). These findings suggest that FTS represents a new class of functional Ras antagonists with potential therapeutic value.

Growth inhibition of ras-dependent tumors in nude mice by a potent ras-dislodging antagonist.

A lipophilic farnesyl moiety attached to the carboxyl terminal cysteine of ras proteins structurally supports their membrane anchorage, required for ras-dependent growth-factor signaling and for transforming activity of ras oncoproteins. It has been shown that inhibition of ras farnesylation can block tumor growth in nude mice but that some ras-dependent tumors escape such blockage as a result of prenylation of ras. S-trans-transfarnesylthiosalicylic acid (FTS) is a potent ras-dislodging antagonist that does not affect ras prenylation but rather acts on the mature, membrane-bound ras and facilitates its degradation. Here we demonstrate that FTS induces reappearance of stress fibers in H-ras-transformed rat-1 cells (EJ cells) in vitro, inhibits their anchorage-independent growth in vitro, and blocks EJ-tumor growth in nude mice. The anchorage-independent growth of cells expressing ErbB2 (B104), but not that of v-raf-transformed cells, is also inhibited by FTS, suggesting specificity towards activated ras. FTS treatment (5 mg/kg i.p. daily) caused inhibition (75-80%) of tumor growth in nude mice implanted with EJ, but not in mice implanted with v-raf-transformed cells, with no evidence of systemic toxicity. Moreover, FTS treatment increased the survival rate of EJ-tumor-bearing mice from 48 to 68 days. Here we demonstrate anti-tumor potency in a synthetic, non-toxic, ras-dislodging antagonist acting independently of farnesyltransferases.

Stringent structural requirements for anti-Ras activity of S-prenyl analogues.

The carboxy terminal S-farnesylcysteine of Ras oncoproteins is required for their membrane anchorage and transforming activities. We showed previously that S-farnesylthiosalicylic acid (FTS) affects the membrane anchorage of activated H-Ras in EJ cells and inhibits their growth. We report here on structural elements in S-prenyl derivatives that specifically inhibit the growth of EJ cells, but not of untransformed Rat-1 cells. Inhibition of the Ras-dependent extracellular signal-regulated protein kinase (ERK), of DNA synthesis and of EJ cell growth were apparent after treatment with FTS or its 5-fluoro, 5-chloro and 4-fluoro derivatives or with the C20 S-geranylgeranyl derivative of thiosalicylic acid. The 4-Cl-FTS analogue was a weak inhibitor of EJ cell growth. The 3-Cl-FTS analogue and the FTS carboxyl methyl ester were inactive, as were the C10 S-geranyl derivative of thiosalicylic acid, farnesoic acid, N-acetyl-S-farnesyl-L-cysteine and S-farne-sylthiopropionic acid. The structural requirements for anti-Ras activity of S-prenyl analogues thus appear to be rather stringent. With regard to chain length, the C15 farnesyl group linked to a rigid backbone seems to be necessary and sufficient. A free carboxyl group in an appropriately rigid orientation, as in thiosalicylic acid, is also required. Halogenic substitutents on the benzene ring of the thiosalicylic acid are tolerated only at position 5 or 4. This information may facilitate the design of potent Ras antagonists and deepen our understanding of the mode of association of Ras with the plasma membrane.

Dislodgment and accelerated degradation of Ras.

Membrane anchorage of Ras oncoproteins, required for transforming activity, depends on their carboxy-terminal farnesylcysteine. We previously showed that S-trans,trans-farnesylthiosalicylic acid (FTS), a synthetic farnesylcysteine mimetic, inhibits growth of ErbB2- and Ras-transformed cells, but not of v-Raf-transformed cells, suggesting that FTS interferes specifically with Ras functions. Here we demonstrate that FTS dislodges Ras from membranes of H-Ras-transformed (EJ) cells, facilitating its degradation and decreasing total cellular Ras. The dislodged Ras that was transiently present in the cytosol was degraded relatively rapidly, causing a decrease of up to 80% in total cellular Ras. The half-life of Ras was 10 +/- 4 h in FTS-treated EJ cells and 27 +/- 4 h in controls. The dislodgment of membrane Ras and decrease in total cellular Ras were dose-dependent: 50% of the effects occurred at 10-15 microM, comparable to concentrations (7-10 microM) required for 50% growth inhibition in EJ cells. Higher concentrations of FTS (25-50 microM) were required to dislodge Ras from Rat-1 cell membranes expressing normal Ras, suggesting some selectivity of FTS toward oncogenic Ras. Membrane localization of the prenylated G beta gamma of heterotrimeric G proteins was not affected by FTS in EJ cells. An FTS-related compound, N-acetyl-S-farnesyl-L-cysteine, which does not inhibit EJ cell growth, did not affect Ras. FTS did not inhibit growth of Rat-1 cells transformed by N-myristylated H-Ras and did not reduce the total amount of this Ras isoform. The results suggest that FTS affects docking of Ras in the cell membrane in a rather specific manner, rendering the protein susceptible to proteolytic degradation.

The Ras antagonist S-farnesylthiosalicylic acid induces inhibition of MAPK activation.

Inhibition of Ras-dependent signaling and of oncogenic Ras function by farnesyl transferase inhibitors that block Ras membrane anchorage is limited due to alternative prenylation of Ras. Here we demonstrate that inhibition of the Ras-dependent Raf-1-MAPK (mitogen activated protein kinase) cascade is achieved by S-farnesylthiosalicylic acid (FTS) which affects Ras membrane association but not Ras farnesylation. FTS interferes with the activation of Raf-1 and MAPK and inhibits DNA synthesis in Ras-transformed EJ cells at concentrations similar to those at which it inhibits EJ cell growth (5-25 microM). FTS also inhibits MAPK activity and DNA synthesis stimulated by serum, EGF or thrombin in serum-starved untransformed Rat-1 cells, demonstrating the generality of its effects on Ras-dependent signaling. The effects of FTS on MAPK activity developed relatively rapidly (within 2-6 h) consistent with its rapid effect on Ras membrane anchorage. FTS represents a new class of Ras antagonists that may be useful for the inhibition of various types of oncogenic Ras isoforms independently of their prenylation.

Treatment of acute migraine with sumatriptan--response in 40 consecutive patients.

Forty consecutive headache patients self-administered sumatriptan for migraine, diagnosed by the criteria of the International Headache Society (IHS). Eighty percent reported excellent response. Thirty-three percent had recurrence of headache within four to twelve hours, while 67% had no recurrence. Fifty-five percent of the patients reported mild side effects, but only 8% stopped therapy because of adverse reactions. No serious cardiovascular events occurred. Recommendations for safe use of sumatriptan are suggested.