The role of the MTHFR 677C>T polymorphism in methotrexate-induced liver toxicity: a meta-analysis in patients with cancer.

Methotrexate (MTX), one of the important pillars in the treatment of different forms of cancer, is associated with the development of hepatotoxicity. The 677C>T variant (rs1801133) in the methylenetetrahydrofolate reductase (MTHFR) gene might affect the development of hepatotoxicity. Results in literature are, however, contradictive. The aim of this study was to evaluate the role of the MTHFR 677C>T polymorphism in MTX-induced hepatotoxicity by analyzing a Dutch cohort of pediatric patients treated with high doses of MTX and subsequently performing a meta-analysis. Ninety-eight patients receiving 542 courses of high-dose MTX were genotyped for the MTHFR 677C>T variant. Hepatotoxicity was evaluated retrospectively according to common terminology criteria for adverse events-National Cancer Institute criteria. The influence of MTHFR 677C>T on hepatotoxicity was examined using a generalized estimating equation (GEE) analysis. A fixed-effect meta-analysis based on this and previous studies investigating the association between the MTHFR 677C>T polymorphism and uniformly coded hepatotoxicity was performed. The GEE analysis showed an increased risk of developing hepatotoxicity for T versus C allele (odds ratio (OR) 1.8; 95% confidence interval (CI) 1.0-3.2, P=0.04). This finding was not supported by the meta-analysis including seven studies and 1044 patients; the OR for the 677T versus C allele was 1.1 (95% CI 0.84-1.5, P=0.25). Heterogeneity between studies was observed, possibly related to differences in MTX dose and leucovorin rescue. In conclusion, in patients with cancer, the MTHFR 677T allele has only a minor role in the development of MTX-induced hepatotoxicity. Observed heterogeneity between studies warrants further study into (tailored) leucovorin rescue.

Electromagnetic fields (EMFs) and adenosine receptors modulate prostaglandin E(2) and cytokine release in human osteoarthritic synovial fibroblasts.

Synovial fibroblasts (SFs) contribute to the development of osteoarthritis (OA) by the secretion of a wide range of pro-inflammatory mediators, including cytokines and lipid mediators of inflammation. Previous studies suggest that electromagnetic fields (EMFs) may represent a potential therapeutic approach to limit cartilage degradation and control inflammation associated to OA, and that they may act through the adenosine pathway. Therefore, we investigated whether EMFs might modulate inflammatory activities of human SFs from OA patients (OASFs) treated with interleukin-1ß (IL-1ß), and the possible involvement of adenosine receptors (ARs) in mediating EMF effects. EMF exposure induced a selective increase in A(2A) and A(3) ARs. These increases were associated to changes in cAMP levels, indicating that ARs were functionally active also in EMF-exposed cells. Functional data obtained in the presence of selective A(2A) and A(3) adenosine agonists and antagonists showed that EMFs inhibit the release of prostaglandin E(2) (PGE(2)) and the proinflammatory cytokines interleukin-6 (IL-6) and interleukin-8 (IL-8), while stimulating the release of interleukin-10 (IL-10), an antinflammatory cytokine. These effects seem to be mediated by the EMF-induced upregulation of A(2A) and A(3) ARs. No effects of EMFs or ARs have been observed on matrix degrading enzyme production. In conclusion, this study shows that EMFs display anti-inflammatory effects in human OASFs, and that these EMF-induced effects are in part mediated by the adenosine pathway, specifically by the A(2A) and A(3) AR activation. Taken together, these results open new clinical perspectives to the control of inflammation associated to joint diseases.

Adenosine analogs and electromagnetic fields inhibit prostaglandin E2 release in bovine synovial fibroblasts.

OBJECTIVE: To investigate the role of adenosine analogs and electromagnetic field (EMF) stimulation on prostaglandin E(2) (PGE(2)) release and cyclooxygenase-2 (COX-2) expression in bovine synovial fibroblasts (SFs). METHODS: SFs isolated from synovia were cultured in monolayer. Saturation and binding experiments were performed by using typical adenosine agonists: N6-cyclohexyladenosine (CHA, A(1)), 2-[p-(2-carboxyethyl)-phenetyl-amino]-5'-N-ethylcarboxamidoadenosine (CGS 21680, A(2A)), 5'-N-ethylcarboxamidoadenosine (NECA, non-selective), N6-(3-iodobenzyl)2-chloroadenosine-5'-N-methyluronamide (Cl-IB-MECA, A(3)). SFs were treated with TNF-alpha (10 ng/ml) and lipopolysaccharide (LPS) (1 microg/ml) to activate inflammatory response. Adenosine analogs were added to control and TNF-alpha- or LPS-treated cultures both in the absence and in the presence of adenosine deaminase (ADA) which is used to deplete endogenous adenosine. Parallel cultures were exposed to EMFs (75 Hz, 1.5 mT) during the period in culture (24h). PGE(2) release was measured by immunoassay. COX-2 expression was evaluated by RT-PCR. RESULTS: TNF-alpha and LPS stimulated PGE(2) release. All adenosine agonists, except for Cl-IB-MECA, significantly inhibited PGE(2) production. EMFs inhibited PGE(2) production in the absence of adenosine agonists and increased the effects of CHA, CGS 21680 and NECA. In ADA, the inhibition on PGE(2) release induced by CHA, CGS and NECA was stronger than in the absence of ADA and the EMF-inhibitory effect was lost. Changes in PGE(2) levels were associated to modification of COX-2 expression. CONCLUSIONS: This study supports anti-inflammatory activities of A(1) and A(2A) adenosine receptors and EMFs in bovine SFs. EMF activity appears mediated by an EMF-induced up-regulation of A(2A) receptors. Biophysical and/or pharmacological modulation of adenosine pathways may play an important role to control joint inflammation.

Pharmacological characterization of P2X1 and P2X3 purinergic receptors in bovine chondrocytes.

OBJECTIVE: The aim of the present study is that of characterizing, for the first time in a quantitative way, from a biochemical, physico chemical and functional point of view P2X(1) and P2X(3) purinergic receptors in bovine chondrocytes. The affinity and the potency of typical purinergic ligands were studied through competition binding experiments and their role in modulating chondrocyte actvities was investigated by analyzing nitric oxide (NO) and prostaglandin E2 (PGE(2)) release. METHODS: Saturation, competition binding experiments, western blotting and immunohistochemistry assays on the P2X(1) and P2X(3) purinergic receptors in bovine chondrocytes were performed. Thermodynamic analysis of the P2X(1) and P2X(3) purinergic binding was studied to investigate the forces driving drug-receptor coupling. In the functional assays (NO and PGE(2) release) the potency of purinergic agonists and antagonists was evaluated. RESULTS: Bovine chondrocytes expressed P2X(1) and P2X(3) purinergic receptors and thermodynamic parameters indicated that purinergic binding is enthalpy- and entropy-driven for agonists and totally entropy-driven for antagonists. Typical purinergic agonists such as adenosine 5'-triphosphate (ATP) and alpha,beta-methyleneATP were able to increase NO and PGE(2) release. A purinergic antagonist, A317491, was able to block the stimulatory effect on functional experiments mediated by the agonists. CONCLUSIONS: These data demonstrate for the first time the presence of functional P2X(1) and P2X(3) purinergic receptors in bovine chondrocytes. Agonists and antagonists are thermodynamically discriminated and are able to modulate functional responses such as NO and PGE(2) release. These results suggest the potential role of novel purinergic antagonists in the treatment of pathophysiological diseases linked to the inflammation and involved in articular cartilage resorption.

Evaluation of the Electroporation Efficiency of a Grid Electrode for Electrochemotherapy: From Numerical Model to In Vitro Tests.

Electrochemotherapy (ECT) is a local anticancer treatment based on the combination of chemotherapy and short, tumor-permeabilizing, voltage pulses delivered using needle electrodes or plate electrodes. The application of ECT to large skin surface tumors is time consuming due to technical limitations of currently available voltage applicators. The availability of large pulse applicators with few and more spaced needle electrodes could be useful in the clinic, since they could allow managing large and spread tumors while limiting the duration and the invasiveness of the procedure. In this article, a grid electrode with 2-cm spaced needles has been studied by means of numerical models. The electroporation efficiency has been assessed on human osteosarcoma cell line MG63 cultured in monolayer. The computational results show the distribution of the electric field in a model of the treated tissue. These results are helpful to evaluate the effect of the needle distance on the electric field distribution. Furthermore, the in vitro tests showed that the grid electrode proposed is suitable to electropore, by a single application, a cell culture covering an area of 55 cm(2). In conclusion, our data might represent substantial improvement in ECT in order to achieve a more homogeneous and time-saving treatment, with benefits for patients with cancer.

The reduced sensitivity of the ProC Global test in protein S deficient subjects reflects a reduction in the associated thrombotic risk.

To investigate simultaneously a defect affecting the protein C/protein S (PC/PS) anticoagulant pathway is possible thanks to a methodological approach (ProC(R) Global; Dade Behring) based on the activation of endogenous plasma PC by a snake venom extract. Factor V (FV) Leiden, the most frequent cause of hereditary thrombosis, is well detected by the test with sensitivity of 100% irrespective of the presence/absence of thrombosis in the subjects investigated. The test is also suited to detect PC or PS defect, but in this case the in vitro impairment of the PC/PS pathway is less pronounced particularly for PS defects (sensitivity for PC and PS defect, 85-100 and 30-90%, respectively). In this study, we hypothesized that the lower sensitivity described for PS defect, compared with those of PC and FV Leiden defects, could also be related to the clinical condition of the subject investigated (symptomatic/asymptomatic) rather than solely to the PS plasma activity/level. Therefore, we analyzed 126 subjects with single congenital defects in the PC/PS pathway: 46 subjects with PS deficiency (26 thrombotic cases and 20 asymptomatic relatives), 40 subjects with PC deficiency (25 thrombotic cases and 15 asymptomatic relatives), and 40 heterozygous FV Leiden subjects (25 thrombotic cases and 15 asymptomatic relatives). By a cut-off of normalized Agkistrodon contortix snake venom ratio of 0.84, the sensitivity in the whole group of cases (sensitivity a) was 76.1, 95.0 and 100%, respectively, for PS, PC and FV Leiden defects. The test failed to detect 11 (23.9%) among the 46 PS-deficient subjects, and all these cases except two belonged to the asymptomatic subgroup (9/20; 45%). Excluding the 20 asymptomatic relatives, the new sensitivity (sensitivity b) for the PS defect was 92.3%. The comparison of the sensitivity in the symptomatic PS cases and in the asymptomatic ones was significantly different (P = 0.010). Among the 40 PC-deficient subjects, only two (5.0%) were not detected by the test and they belonged indifferently to the two subgroups. Finally, none of the 40 FV Leiden heterozygotes were misdiagnosed by the test. These results suggest that in symptomatic PS-deficient cases the test could reflect a post-thrombotic effect and/or reveal potential unidentified prothrombotic influences assessing a prothrombotic risk condition.

[The vanishing testis syndrome. Considerations on a case]. / La sindrome del testicolo evanescente. Considerazioni su di un caso

The aetiopathogenesis and clinical and diagnostic features of bilateral absence of the testicles in 46XY subjects are briefly reviewed. A eunuchoid 25-yr-old patient with male sex chromatin, 46 XY genotype, plasma testosterone slightly below normal, normal urinary 17-KS unaltered by gonadotropin stimulation, and bilateral absence of the testicles is presented. Examination of the inguinal canal showed a funiculus and ductus deferens residue terminating in an adipose mass. Section of the fibrous duct showed that this mass was full of capillaries and venules. Lesion between the 14th and 16th week of pregnancy is postulated.

[Synthetic PH-RH as a pituitary function test in anorexia nervosa]. / Il GN-RH sintetico come test di funzionalità ipofisaria nella anoressia nervosa

The hypophyseal gonadotrophin LH reserve was studied in 9 adult women, 5 of whom were suffering from nervous anorexia (N.A.) and 4 normal, by means of subcutaneous injection of 50 mcg of synthetic GN-RH. Basal values of plasmatic LH proved to be significantly lower in N.A. than in the controls where the response to GN-RH was prompt. In patients with N.A., on the other hand, response was practically absent and differences were significant at all stages of the test. The parallel study of basal steroiduria and following methopirone confirmed the torpid hypophyseal response in N.A. These findings confirm previous personal reports and indicate that the GN-RH test is a useful technique for diagnostic and prognostic definition of N.A.

In vivo effect of two different pulsed electromagnetic field frequencies on osteoarthritis.

Osteoarthritis (OA) is a joint pathology characterized by fibrillation, reduced cartilage thickness and subchondral bone sclerosis. There is evidence that pulsed electromagnetic fields (PEMFs) counteract OA progression, but the effect of two different PEMF frequencies has not yet been shown. The aim of this study was to test the effectiveness of PEMFs at two different frequencies (37 and 75 Hz) in a late OA stage in 21-month-old Guinea pigs. After 3 months of 6 h/day PEMF stimulation, histological and histomorphometric analyses of the knees were performed. At both frequencies, PEMFs significantly reduced histological cartilage score, fibrillation index (FI), subchondral bone thickness (SBT) and trabecular number (Tb.N) and increased trabecular thickness (Tb.Th) and separation (Tb.Sp) in comparison to the not treated SHAM group. However, PEMFs at 75 Hz produced significantly more beneficial effects on the histological score and FI than 37 Hz PEMFs. At 75 Hz, PEMFs counteracted cartilage thinning as demonstrated by a significantly higher cartilage thickness values than either those of the SHAM or 37 Hz PEMF-treated groups. Although in severe OA both PEMF frequencies were able to limit its progression, 75 Hz PEMF stimulation achieved the better results.

Identification of in vitro electropermeabilization equivalent pulse protocols.

Exposure of cells to an external sufficiently strong electric field results in the formation of pores across the membrane. This phenomenon, termed electropermeabilization, permits the transport of poorly permeant molecules into cytosol. In clinical practice, cell membrane permeabilization for drug electrotransfer is achieved using the ESOPE pulse protocol (1000 V/cm, 8 pulses, 100 µs, 5 kHz). The aim of this study was to investigate several combinations of electric field amplitude and pulse number able to induce electropermeabilization as the one observed when the ESOPE protocol was applied. Decreasing electric field amplitudes (1000 to 300 V/cm) in combination with increasing number of pulses (8 to 320) were applied to in vitro MG63 cells. Propidium iodide and Calcein blue AM uptake were used to evaluate cell electropermeabilization and viability. Results showed that the threshold of local electric field needed to obtain electropermeabilization decreased exponentially with increasing the number of pulses delivered (r2 5 0.92, p<0.0001). The absorbed dose threshold was dependent on the number of pulses for each voltage applied (r2 5 0.96, p<0.0001). In conclusion, the possibility of applying an increased number of pulses rather than increasing the electric field amplitude to perform electropermeabilization, may become an important tool for electropermeabilization-related clinical applications.

Expression and functional role of adenosine receptors in regulating inflammatory responses in human synoviocytes.

BACKGROUND AND PURPOSE: Adenosine is an endogenous modulator, interacting with four G-protein coupled receptors (A(1), A(2A), A(2B) and A(3)) and acts as a potent inhibitor of inflammatory processes in several tissues. So far, the functional effects modulated by adenosine receptors on human synoviocytes have not been investigated in detail. We evaluated mRNA, the protein levels, the functional role of adenosine receptors and their pharmacological modulation in human synoviocytes. EXPERIMENTAL APPROACH: mRNA, Western blotting, saturation and competition binding experiments, cyclic AMP, p38 mitogen-activated protein kinases (MAPKs) and nuclear factor (NF)-kappaB activation, tumour necrosis factor alpha (TNF-alpha) and interleukin-8 (IL-8) release were assessed in human synoviocytes isolated from patients with osteoarthritis. KEY RESULTS: mRNA and protein for A(1), A(2A), A(2B) and A(3) adenosine receptors are expressed in human synoviocytes. Standard adenosine agonists and antagonists showed affinity values in the nanomolar range and were coupled to stimulation or inhibition of adenylyl cyclase. Activation of A(2A) and A(3) adenosine receptors inhibited p38 MAPK and NF-kappaB pathways, an effect abolished by selective adenosine antagonists. A(2A) and A(3) receptor agonists decreased TNF-alpha and IL-8 production. The phosphoinositide 3-kinase or G(s) pathways were involved in the functional responses of A(3) or A(2A) adenosine receptors. Synoviocyte A(1) and A(2B) adenosine receptors were not implicated in the inflammatory process whereas stimulation of A(2A) and A(3) adenosine receptors was closely associated with a down-regulation of the inflammatory status. CONCLUSIONS AND IMPLICATIONS: These results indicate that A(2A) and A(3) adenosine receptors may represent a potential target in therapeutic modulation of joint inflammation.

Characterization of adenosine receptors in bovine chondrocytes and fibroblast-like synoviocytes exposed to low frequency low energy pulsed electromagnetic fields.

OBJECTIVE: The present study describes the presence and binding parameters of the A1, A2A, A2B and A3 adenosine receptors in bovine chondrocytes and fibroblast-like synoviocytes. The effect of low frequency low energy pulsed electromagnetic fields (PEMFs) on the adenosine receptor affinity and density was studied. METHODS: Saturation, competition binding experiments and Western blotting assays in the absence and in the presence of PEMFs on the adenosine receptors in bovine chondrocytes or fibroblast-like synoviocytes were performed. Thermodynamic analysis of the A2A or A3 binding was studied to investigate the forces driving drug-receptor coupling. In the adenylyl cyclase and proliferation assays the potency of typical high-affinity A2A or A3 agonists in the absence and in the presence of PEMFs was evaluated. RESULTS: Bovine chondrocytes and fibroblast-like synoviocytes expressed all adenosine receptors. PEMFs evoked an up-regulation of A2A and A3 receptors and thermodynamic parameters indicate that adenosine binding is enthalpy and entropy driven. In PEMF-treated cells the potency of typical A2A or A3 agonists on cyclic AMP assays was significantly increased when compared with the untreated cells. PEMFs potentiated the effect of A2A or A3 agonists on cell proliferation in both cell types. CONCLUSIONS: PEMFs mediate an up-regulation of A2A and A3 receptors related to an increase of their functional activities in bovine chondrocytes and fibroblast-like synoviocytes. No differences are present in adenosine affinity and in the drug-receptor interactions. Our data could be used as a trigger to future studies addressed to PEMFs and adenosine therapeutic intervention in inflammatory joint diseases.

Proteoglycan synthesis in bovine articular cartilage explants exposed to different low-frequency low-energy pulsed electromagnetic fields.

OBJECTIVE: To investigate the role of pulsed electromagnetic field (PEMF) exposure parameters (exposure length, magnetic field peak amplitude, pulse frequency) in the regulation of proteoglycan (PG) synthesis of bovine articular cartilage explants. METHODS: Bovine articular cartilage explants were exposed to a PEMF (75 Hz; 2 mT) for different time periods: 1, 4, 9, 24 h. Then, cartilage explants were exposed for 24 h to PEMFs of different magnetic field peak amplitudes (0.5, 1, 1.5, 2 mT) and different frequencies (2, 37, 75, 110 Hz). PG synthesis of control and exposed explants was determined by Na2-35SO4 incorporation. RESULTS: PEMF exposure significantly increased PG synthesis ranging from 12% at 4 h to 17% at 24 h of exposure. At all the magnetic field peak amplitude values, a significant PG synthesis increase was measured in PEMF-exposed explants compared to controls, with maximal effect at 1.5 mT. No effect of pulse frequency was observed on PG synthesis stimulation. CONCLUSIONS: The results of this study show the range of exposure length, PEMF amplitude, pulse frequency which can stimulate cartilage PG synthesis, and suggest optimal exposure parameters which may be useful for cartilage repair in in vivo experiments and clinical application.

[Controlled application of cynarin in the treatment of hyperlipemic syndrome. Observations in 60 cases]. / Kontrollierte Anwendung von Cynarin in der Behandlung hyperlipämischer Syndrome. Beobachtungen von 60 Fällen

In a controlled trial 2 groups of 30 patients presenting various dislipidemic pictures were treated for 50 days with either 3,5-dihydroxy-1,4-bis-(3,4-dihydroxy-cinnamoyloxy)-cyclohexan-carbonic acid-(1) (cynarin, Listrocol) or placebo. Cynarin provied to be able to induce a significant reduction of the hypercholesteremia, the level of pre-beta-lipoproteins, the beta/alpha-lipoprotein ratio and patients' body weight. The administration of cynaria is, therefore, deemed useful in the prophylaxis and therapy of dislipidemic pictures of the arteriosclerotic disease.

Coexistence of factor V G1691A and factor II G20210A gene mutations in a thrombotic family is associated with recurrence and early onset of venous thrombosis.

Two G-to-A mutations at positions 1691 of the factor V (FV) gene and 20210 of the prothrombin (FII) gene have been associated with an increased risk of venous thromboembolism. We report a thrombosis-prone family in which one subject--the propositus who exhibited combined heterozygous FV G1691A and FII G20210A mutations--showed spontaneous and early clinical onset (at 23 years), recurrences of deep-vein thrombosis and pulmonary embolism. His asymptomatic father carried the FII G20210A substitution and his mother, characterized by an isolated thrombotic episode on occasion of surgery (at 48 years), carried the FV G1691A substitution. In the maternal lineage, one of the propositus' uncles had thrombosis on occasion of a bone fracture (at 65 years) despite the absence of known prothrombotic defects. A sister of the propositus carried the FII G20210A and the brother the FV G1691A mutation. They have been asymptomatic until now. The propositus' two children, 20 and 16 years old, both carry the FV G1691A substitution and have been asymptomatic until now. The plasma levels of FII were higher in carriers of the FII G20210A allele if compared with noncarriers, and the activated protein C resistance phenotype, associated with the FV Leiden mutation, showed a complete correlation with the FV G1691A mutation. Despite the very limited number of thrombotic cases involved in this survey, which does not allow statistically sound conclusions, the data obtained from this family suggest that the synergy of inherited factors and transient risk conditions could play a key role in the occurrence of thrombotic accidents.

Chemico-physical and functional properties of inorganic sunscreens in cosmetic products.

Synopsis This paper reports preliminary results of a study carried out on liquid crystal emulsions added to three different inorganic sunscreens: ultrafine zinc oxide, ultrafine titanium dioxide (inorganic-treated) and ultrafine titanium dioxide (organic-treated hydrophobically). The aim of the work was to investigate the influence of chemico-physical properties of inorganic sunscreens on the microstructure of cosmetic emulsions. The study was carried out using three different techniques: rheological measurements performed in dynamic conditions, to study the homogeneity of samples and their structural features; dispersion of powders in emulsions by optical microscopy and SEM/EDX analysis; and functionality of emulsions by UV spectroscopy, with adhesive tape as substrate. Results show that the different chemico-physical properties of the micropigments lead to different interactions with emulsion components; these interactions may affect the functionality and microstructure of the whole system, with loss of stability.

A common mutation in the gene for coagulation factor XIII-A (VAL34Leu): a risk factor for primary intracerebral hemorrhage is protective against atherothrombotic diseases.

The role of a common polymorphism in the factor XIII A-subunit gene (FXIII Val34Leu) has been recently investigated as a protective genetic factor against arterial and venous thrombosis. In addition, the less frequent Leu34 allele has been described as a risk factor for intracerebral hemorrhage. We evaluated the prevalence of this polymorphism by PCR in three case-control studies of patients diagnosed as having primary intracerebral hemorrhage (PCH, n = 130), coronary heart diseases (CHD, n = 240; myocardial infarction/no myocardial infarction, 120/120), and cerebrovascular diseases (CVD, n = 240; cerebral infarction/transient ischaemic attack, 120/120). The matched control groups consisted of patients admitted to the hospital without history of vascular disease. In addition, 200 healthy subjects were investigated. The frequency of the mutated allele (Leu34) was higher in patients with PCH than in controls (33.8% vs. 23.1%, P = 0.009) and lower in CHD and CVD patients compared to controls (18.1% vs. 25.2%, P = 0.010 and 17.3% vs. 24.2%, P = 0.011, respectively). Moreover, among the patients with CHD, the Leu34 allele was underrepresented in cases with myocardial infarction than without (12.9% vs. 23.3%, P = 0.004) and than in controls (12.9% vs. 25.2%, P < 0.001). Similar findings were obtained in patients with CVD comparing the cases with cerebral infarction versus cases with transient ischaemic attack (12.5% vs. 22.1%, P = 0.008) and versus controls (12.5% vs. 24.2%, P < 0.001). Finally, considering altogether the groups of ischaemic patients (CHD and CVD, n = 480), it was noted a trend towards a higher mean age of the clinical onset in homozygotes for the Leu allele than in the wild types (P = 0.078). This study indicates that in our population possession of the FXIII Val34Leu mutation predisposes to the occurrence of primary intracerebral hemorrhage and protects against cerebral and myocardial infarction. A wider modulatory role in the progression and onset of atherothrombotic diseases could be ascribed to FXIII Val34Leu.