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A series of reports in the literature indicated symmetry breaking in assemblies of chiral molecules of opposite handedness. These unexpected observations could be accounted for as being generated by the “parity violation” of the nuclear weak force, combined with an autocatalytic amplification process. However, in many such cases, in particular of chiral fluids, this putative mechanism is far from providing a reasonable explanation for such discrimination. In this article it is suggested that space may have deviated a priori from absolute symmetry, a possibility which complies with observations in atoms and molecules and may even be implicated in the asymmetrical configuration of spiral galaxies. Space asymmetry can be extrapolated to a difference between the relative statistical weights of the “right” versus the “left” directions with respect to Euclidian coordinates or, analogously, to a difference between the clockwise and anticlockwise orientations in polar coordinates. The difference in weights of these directions in space is estimated to be around 1%, based on the differences observed in density values of chiral fluids and chiral crystals of NaClO3. The implied asymmetry of time, as the conjugated fourth dimension, suggests a similar difference in magnitude of the time coordinate in a right-handed versus left-handed space, which is feasible for experimental verification. Chirality 25:308–311, 2013. © 2013 Wiley Periodicals, Inc.

Background: It has been suggested that the etiology of schizophrenia, in a distinct group of patients, originates from an autoimmune reaction against platelets. Previous studies have demonstrated significantly higher blood titers of platelet-associated autoantibodies (PAA) in adult schizophrenia patients as compared to normal healthy subjects. In addition, young adult schizophrenia patients at their early stages of the disorder displayed higher PAA titers than older patients with longer duration of the disorder. Aim: To assess the blood titers of PAA in children with schizophrenia as compared to matched control subjects without psychotic disorders, as a possible diagnostic parameter. Methods: Twenty-nine children with DSM-IV schizophrenia in the active psychotic state, with an age range of 6-12 years (mean ± SD: 9.6 ± 1.5 years), with average Positive and Negative Syndrome Scale scores of 108 ± 19.2, were assessed. The control group consisted of 25 children with DSM-IV conduct disorder in a similar age range of 5-12 years (mean ± SD: 9.5 ± 1.6 years). The blood titers of PAA were evaluated using an optimized ELISA test, expressed by a linear optical density (OD) scale. The blood samples of all participants were tested anonymously and were scored under a code number. A test recording above 1.4 OD units was predefined as positive. Results: The titers of PAA of children with schizophrenia (1.9 ± 0.5 OD units, range: 0.7-2.44 units) were significantly (p < 0.00001) higher than those of the control group (1.0 ± 0.4 OD units, range: 0.45-2.28 units). In 83% of the children with schizophrenia (24 out of the 29 patients) a positive test, i.e. OD >1.4, was detected. In contrast, in the control group, only 12% (3 of the 25 subjects) displayed a positive test, p < 0.00001. Conclusions: High titers of PAA in children with schizophrenia as compared with nonpsychotic controls may indicate an active autoimmune process in the early onset of schizophrenia. The PAA level may therefore provide a supportive diagnostic biomarker for childhood schizophrenia.

Supramolecular chiral assemblies of R(−) and S(+) 2-butanol, in their neat form or when dissolved in their nonchiral isomer isobutanol, were evaluated by isothermal titration calorimetry (ITC) ensuing mixing. Dilution of 0.5 M solution of R(−) 2-butanol in isobutanol into the latter liberated heat of several calories per mole, which was approximately double than that obtained in parallel dilutions of S(+) 2-butanol in isobutanol. The ITC dilution profiles indicated an estimate of about 100 isobutanol solvent molecules surrounding each of the 2-butanol enantiomers, presumably arranged in chiral configurations, with different adopted order between the isomers. Mixings of neat R and S 2-butanol were followed by endothermic ITC profiles, indicating that, in racemic 2-butanol, both the supramolecular order and the intermolecular binding energies are lower than in each of the neat chiral isomers. The diversion from symmetrical ITC patterns in these mixings indicated again a subtle difference in molecular organization between the neat enantiomers. It should be noted that the presence of impurities, α-pinene and teterhydrofuran, at a level totaling 0.5%, did not influence the ITC heat flow profiles. The findings of this study demonstrate for the first time that chiral solutes in organic solvents are expected to acquire asymmetric solvent envelopes that may be different between the enantiomers, thus broadening this phenomenon beyond the previously demonstrated cases in aqueous solutions. Chirality 24:500–505, 2012. © 2012 Wiley Periodicals, Inc.

A homogeneous solution of a chiral substance stores a residual chemical potential, related to its overall anisotropy. Therefore, by mixing solutions of opposite enantiomers, heat release may take place, corresponding to the mutual anisotropy annulment. In the following study we present proofs for this fundamental, yet unexplored, prediction by measuring the heat released upon mixing of aqueous solutions of D-proline with L-proline, as well as D-alanine with L-alanine, using isothermal titration calorimetry. Heat release in the range of 0.6-6 cal/mol was detected in these intermolecular racemizations at 30 degrees C. Its magnitude varied linearly with the apparent optical rotation, which complied with the possibility that the hydration envelope coating the chiral molecule is of a long-range condensed and asymmetrical configuration that can expand by integration with adjacent hydration envelopes. The ordered water in such hydration layers constitutes regions of "negative entropy", a basic medium for information storage. On the basis of our findings, a fundamental expression which combines entropy, information capacity, and thermal energy is proposed.

L-amino acids and D-carbohydrates were incorporated into the first forms of life over 3.5 billion years ago, presumably from racemic mixtures of organic solutes produced by abiotic synthetic pathways. The process by which this choice occurred has not been established, but a consensus view is that it was a chance event, such that life could equally well have used D-amino acids and L sugars. In this review we will explore a second, less plausible alternative that minute differences in the physical properties of certain enantiomers made it more likely that L-amino acids and D-carbohydrates would be incorporated into early life. By all classical criteria, chiral isomers are perfect mirror image structures and, therefore, are expected to be identical in their macroscopic properties. However, scattered reports in the literature suggest that there may be slight differences in the physical properties of L- and D-amino acids and their polymers, which could lead to a preferred incorporation of L-amino acids into primitive forms of life. Here we present a literature survey of this issue and discuss its possible role in the origin of biochirality.

We previously found autoantibodies against platelets in schizophrenic patients. One of the platelet proteins that bind these antibodies is enolase. Here, we describe the isolation and sequencing of an immunoreactive peptide after enzymatic digestion of enolase. The 3-D structure of enolase indicates that, unexpectedly, this peptide is buried inside the protein. However, 3-D surface analysis leads to the identification of a conformational epitope that resembles the binding peptide and might constitute a specific binder of the autoantibodies. In a screening of antibody binding with the peptide LVVGLCK, we found in 50 serum samples of controls a mean of O.D.=0.46; s= +/- 0.21 relative enzyme immunoassay units, while in sera of 39 schizophrenic patients, we found a mean of O.D.=1.47; s= +/- 0.65; P

We report here an unexpected difference in the solubilities of D- and L-tyrosine in water, which could be discerned by their rate of crystallization and the resulting concentrations of their saturated solutions. A supersaturated solution of 10 mM L-tyrosine at 20 degrees C crystallized much more slowly than that of D-tyrosine under the same conditions, and the saturated solution of L-tyrosine was more concentrated than that of D-tyrosine. Supersaturated solutions of 10 mM DL-tyrosine in water formed precipitates of predominantly D-tyrosine and DL-tyrosine, resulting in an excess of L-tyrosine in the saturated solution. The experimental setups were monitored independently by UV-absorption, radioactivity tracing, optical rotation and X-ray diffraction. The process of nucleation and crystallization of D- and L-tyrosine is characterized by an exceptionally high cooperativity. It is possible that minute energy differences between D- and L-tyrosine, originating from parity violation or other non-conservative chiral discriminatory rules, could account for the observations. The physical process that initiated chiral selection in biological systems remains a challenging problem in understanding the origin of life, and it is possible that chiral compounds were concentrated from supersaturated racemic mixtures by preferential crystallization.

Purpose: The anticancer activity of Ω-3 polyunsaturated fatty acids (Ω-3 PUFA) has been shown in a large number of studies. This study was undertaken to analyze the combined effect of Ω-3 PUFA and antioxidative vitamins on the level of spontaneous metastatic dissemination. The supportive effect of this dietary combination on chemotherapy with cisplatin (CP) was determined in parallel. Methods: C57BL/6J mice bearing the Lewis lung carcinoma 3LL were fed ad libitum one of three isocaloric diets containing 5% soybean oil supplemented with 40 mg/kg α-tocopherol acetate (SO diet), or 4% fish oil plus 1% corn oil, and basal amounts of vitamin E (FO diet) or FO diet supplemented with vitamins E and C (FO+E+C diet). These diets were tested in combination with the conventional cytotoxic agent CP in a series of regimens. Tumor growth, feed consumption, body weight, lung metastasis and lung histology were followed. Results: Both the FO dietary groups showed significantly lower tumor development than the SO group in all examined parameters, indicating that Ω-3 PUFA have anticancer activity. However, the FO diet, in comparison with the FO+E+C diet induced a significantly slower rate of tumor growth, and lower metastatic load, as reflected in lung weight. The decrease in the anticancer activity of FO by the addition of vitamins E and C suggests that in situ oxidation of Ω-3 PUFA underlies their anticancer action. It is thus proposed that oxidized Ω-3 PUFA accumulates in the membranes and the cytosol of tumor cells, reducing their vitality and eventually leading to their death. No signs of anorexia or cachexia were observed in either FO group, in contrast to the SO group. CP treatment with the SO diet had no apparent therapeutic effect, while with the FO diets it reduced the metastatic load. The best regimen of this combined treatment was FO diet followed by CP treatment with FO diet supplemented with vitamins E and C after resection of the primary growth. This regimen could be translated to a combined therapy for human cancer. Conclusions: Diets enriched with Ω-3 PUFA may have beneficial anticancer effects in particular when containing only basal amounts of antioxidants such as vitamin E or C. Furthermore, the addition of drugs which promote oxidation of Ω-3 PUFA, such as ferrous salts (e.g. as prescribed for the treatment of anemia), may further increase these effects. However, the supportive effect of Ω-3 PUFA in chemotherapy (e.g. with CP) increases when vitamins E and C are also included.

Cyclic glycerophosphates can be formed by enzymic degradation of phospholipids. They have only recently attracted attention, and their physiological function is still obscure. In this study, we have searched for signalling functions of the natural 1,3-cyclic and 1,2-cyclic glycerophosphates, their deoxy analogues, and the phenyl esters of the 1,3-cyclic phosphates. Linear sn-glycerol 3-phosphate and glycerol 2-phosphate served as the control compounds. Each of the six-membered ring cyclic phosphates tested induced rapid intracellular tyrosine phosphorylation in CHO and NIH-3T3 cells when applied extracellularly at a concentration of 0.5-4 microM. The phosphorylated intracellular proteins had molecular masses of approximately 35 kDa, approximately 45 kDa, 60-70 kDa and approximately 120 kDa. The five-membered ring cyclic phosphates had a similar effect, but at an external concentration of 2-10 microM, while sn-glycerol 3-phosphate and glycerol 2-phosphate had no effect. The six-membered cyclic phosphates also induced rapid threonine phosphorylation in CHO cells of approximately 18-kDa, approximately 35-kDa, and approximately 38-kDa proteins. Further experiments indicated that the cyclic phosphates partition rapidly into the cell cytosol where they activate kinases, including mitogen-activated protein kinase. When their intracellular level increases, dephosphorylation presumably takes place. This pattern may account for the signalling profile of cyclic phosphates and suggests that they may take part in processes associated with cell differentiation.

Immunotherapy of cancer has proven to be of limited clinical value thus far, largely because of its failure to overcome the multiple escape strategies deployed by tumor cells. Renewed interest in cancer immunotherapy was stimulated in recent years by the identification of distinct tumor antigens, which are capable of eliciting specific antitumor immune responses. However, the availability of synthetic or purified tumor antigens may not in itself be sufficient to offer an improved therapeutic modality, because their immunogenic hierarchy is largely unknown, and because metastatic tumor cells mutate frequently, thereby altering their overall antigenic character. During the past decade, we have developed a novel method in which tumor cells are exposed to high hydrostatic pressure (P) in the presence of a biologically compatible crosslinker (CL), 2'-3'-adenosine dialdehyde. The newly acquired immunogenic topology of surface proteins created by application of pressure converted the modified cells into potent stimulators of an antitumor response. Recently, we suggested that subsequent reduction of surface protein disulfides with Nacetyl-L-cysteine (NAC) could augment the immunogenic potential of PCL-modified tumor cells. An immunotherapeutic regimen combining PCL+NAC modified carcinoma cells plus intravenous delivery of NAC provoked an effective antitumor response capable of eradicating the metastatic nodules. We propose that this novel two-prong strategy, based on local immunization with irradiated PCL+NAC modified tumor cells and systemic administration of NAC, could provide a practical, effective immunotherapeutic approach for the treatment of human cancer.

Heat shock protein-60 (HSP60) is implicated in several autoimmune diseases as a triggering antigen. Based on the autoimmune hypothesis of schizophrenia, we examined cellular and humoral responses against HSP60 and a series of its peptide fragments with peripheral blood samples of schizophrenic patients and healthy subjects each of group size between 12 to 32 participants. The average stimulation indices of peripheral blood mononuclear cells (PBMC) to HSP60 were 3.17 +/- 0.36 (mean +/- SE) for schizophrenic patients and 2.23 +/- 0.24 (mean +/- SE) for healthy subjects, with a significant difference between the groups (P = 0.0457). In parallel, 38 synthetic peptide fragments of HSP60, each of 18-21 amino acids, were tested for in vitro sensitization of PBMC. With one peptide (p32) the average stimulation index of PBMC from schizophrenic patients was significantly higher than that obtained for PBMC of control subjects (P = 0.0006). Comparing the cellular immune response to p32 between patients who were distinctive responders (n = 10) or non-responders (n = 10) to neuroleptic treatment indicated a similar elevation of cellular response in these groups. Antibodies against HSP60 were screened by dot-blot and ELISA in the sera of the above blood samples. Titers of IgG and IgM against HSP60 were found to be of similar magnitude in schizophrenic patients and in controls. Titers of IgA against HSP60 were somewhat higher in the sera of schizophrenic patients in comparison to sera of control subjects (P = 0.0605).

In this study we examined the influence of different polyunsaturated fatty acid (PUFA) diets on the tumor growth and metastatic dissemination of the well-characterized Lewis Lung Carcinoma (3LL) in C57BL 6J mice. The tumor-bearing mice were fed ad libitum with three different diets of 5% oil; either soybean oil (SO), which is rich in omega-6 (ω-6); perilla oil (PO), which is rich in omega-3 (ω-3) 18:3; and fish oil (FO), which is rich in ω-3, 20:5 and 22:6 PUFA. A significantly slower growth of primary tumor, lower mortality rate, and lower metastatic spread were observed in mice fed FO. This trend was also observed in mice fed first with SO and after tumor inoculation transferred to the FO diet. Indomethacin reduced significantly the metastasis growth in the SO-fed group, and exerted only a small effect in the FO and PO fed counterparts, suggesting that eicosanoid derivatives of ω-6 fatty acids support the process of tumor growth. In addition, the long chain PUFA of fish oil, which are sensitive to oxidation, could act as targets for membrane perforation and eventual elimination of the proliferating tumor cells.

Counteraction of drug resistance is a major challenge in cancer therapy, particularly in adhanced stages. The main mechanism of multidrug resistance is related to an increased drug efflux. In the present study we examined the effect of modifying cell membrane lipid fluidity on uptake of adriamycin (ADR) in cells of AKR lymphoma malignancy variants. Modification of cell membrane fluidity, either by lecithin or by lecithin-cholesterol mixtures, induced in a high proportion of cells of all variants a higher capacity to accumulate ADR. The chemosensitizing effect, for lecithin in particular, was proportional to the vegree of malignancy of the lymphoma variants. The increased ADR uptake was up to 1.4-fold in the variant of lowest malignancy and up to 5-fold in the one of highest aggressiveness. This tenvency correlates with our previous studies and is of particular value since highly-malignant tumors are often drug resistant. The cholesterol-lecithin mixture, induced, however, in part of the variants the appearance of a small subpopulation with very low ADR permeability. Cell membrane rigidification is of value for exposing tumor cell cryptic antigens but may be veleterious when used in conjunction with chemotherapy.

Phospholipase D from Streptomyces chromofuscus hydrolyzes lysophosphatidylcholine or lysophosphatidylethanolamine in aqueous 1% Triton X-100 solution. In situ monitoring of this reaction by 31P NMR revealed the formation of cyclic lysophosphatidic acid (1-acyl 2,3-cyclic glycerophosphate) as an intermediate which was hydrolyzed further by the enzyme at a functionally distinct active site to lysophosphatidic acid (lyso-PA). Synthetic cyclic lyso-PA (1-octanoyl 2,3-cyclic glycerophosphate) was found to be stable in aqueous neutral solutions at room temperature. It was hydrolyzed by the bacterial phospholipase D to lyso-PA at a rate which was approximately 4-fold slower than the rate of formation of cyclic lyso-PA. The addition of 5-10 mM sodium vanadate could partially inhibit the ring opening reaction and thus increase substantially the cyclic lyso-PA accumulation. Cyclic lyso-PA may act as a dormant configuration of the physiologically active lyso-PA or may even possess specific activities which await verification.

The delta 6-desaturase system was isolated from rat liver microsomes by high hydrostatic pressure (1500 bars) and the enzyme components were then separated by size chromatography. The lipids extracted by organic solvents from the pressure shed fractions were phosphatidylcholine (PC) and cholesterol at a mole ratio of 4:1. The acyl chains of the shed PC were 56% saturated and 21% polyunsaturated resulting predominantly from 13% higher and 15% lower contents of palmitic and arachidonic acid, respectively, as compared to those of microsomal PC. The weight ratio of phospholipids to protein in the shed desaturase fraction was 0.2 which corresponds to an average of 31 phospholipid molecules around each desaturase molecule. Differential scanning calorimetry of the lipids associated with the desaturase system showed a phase transition at 41 degrees C. Fluorescence anisotropy studies of the desaturase surrounding lipids indicated the same transition point. We concluded that the delta 6-desaturase has an associated lipid surrounding of PC and cholesterol at an approx. 4:1 mole ratio that constitutes a gel phase at physiological temperature. We suggest that this state is essential for optimal desaturase activity and that the specific acyl chains of the lipid annulus provide a regulatory sensor of the delta 6-desaturase activity.

A series of reports in the literature indicated symmetry breaking in assemblies of chiral molecules of opposite handedness. These unexpected observations could be accounted for as being generated by the "parity violation" of the nuclear weak force, combined with an autocatalytic amplification process. However, in many such cases, in particular of chiral fluids, this putative mechanism is far from providing a reasonable explanation for such discrimination. In this article it is suggested that space may have deviated a priori from absolute symmetry, a possibility which complies with observations in atoms and molecules and may even be implicated in the asymmetrical configuration of spiral galaxies. Space asymmetry can be extrapolated to a difference between the relative statistical weights of the "right" versus the "left" directions with respect to Euclidian coordinates or, analogously, to a difference between the clockwise and anticlockwise orientations in polar coordinates. The difference in weights of these directions in space is estimated to be around 1%, based on the differences observed in density values of chiral fluids and chiral crystals of NaClO3. The implied asymmetry of time, as the conjugated fourth dimension, suggests a similar difference in magnitude of the time coordinate in a right-handed versus left-handed space, which is feasible for experimental verification.

A procedure using high hydrostatic pressure without detergent has been applied in this study to subfractionate the components of the Δ 6 -desaturase system from the rat iiver endoplasmic reticulum. Microsomes were suspended in a buffer containing liposomes made of phosphatidylcholine. The mixture was placed in a sealed pressure bomb and subjected to hydrostatic pressure of up to 1500 bars. Under these conditions the total desaturase activity was found in the liposomal fraction thus indicating that the three components of the desaturase system, namely NADH-cytochrome b 5 reductase, cytochrome b 5 and the Δ 6 -desaturase co-extracted. Size chromatography and FPLC of the released proteins followed by SDS-PAGE confirmed the independent release of the three components corresponding to the Δ 6 -desaturase system. Δ 6 -Desaturase activity could be full regenerated by mixing the aqueous dispersions of the three components without further purification. Our results indicate that these components are physically facing a similar lipid environment in the microsotnal membrane.

Supramolecular chiral assemblies of R(-) and S(+) 2-butanol, in their neat form or when dissolved in their nonchiral isomer isobutanol, were evaluated by isothermal titration calorimetry (ITC) ensuing mixing. Dilution of 0.5 M solution of R(-) 2-butanol in isobutanol into the latter liberated heat of several calories per mole, which was approximately double than that obtained in parallel dilutions of S(+) 2-butanol in isobutanol. The ITC dilution profiles indicated an estimate of about 100 isobutanol solvent molecules surrounding each of the 2-butanol enantiomers, presumably arranged in chiral configurations, with different adopted order between the isomers. Mixings of neat R and S 2-butanol were followed by endothermic ITC profiles, indicating that, in racemic 2-butanol, both the supramolecular order and the intermolecular binding energies are lower than in each of the neat chiral isomers. The diversion from symmetrical ITC patterns in these mixings indicated again a subtle difference in molecular organization between the neat enantiomers. It should be noted that the presence of impurities, α-pinene and teterhydrofuran, at a level totaling 0.5%, did not influence the ITC heat flow profiles. The findings of this study demonstrate for the first time that chiral solutes in organic solvents are expected to acquire asymmetric solvent envelopes that may be different between the enantiomers, thus broadening this phenomenon beyond the previously demonstrated cases in aqueous solutions.

Platelet-associated antibodies (PAA) from schizophrenic patients and healthy individuals were determined by conventional enzyme immunoassay. The platelets of the schizophrenic patients were found to carry notably higher levels of PAA than were those of the healthy controls. PAA from the schizophrenics inhibited specifically the uptake of dopamine (DA) by platelets. The DA uptake by normal platelets could be reduced to a level similar to that observed in platelets of schizophrenics upon binding of PAA from schizophrenics. Serotonin uptake was virtually unaffected by these antibodies. The Vmax value of dopamine uptake by platelets of schizophrenics was similar to that for normal platelets treated with PAA of schizophrenics, as well as that for normal untreated platelets. However, the Km values of the uptake were higher in the platelets from schizophrenics and normals treated with PAA from schizophrenics than in the untreated platelets of normals. PAA from schizophrenics were found to bind to rat brain P2m membranes and to interfere with the binding of the dopamine receptor ligands [3H]-dopamine and [3H]-spiperone. These results suggest that blood platelets may function as a peripheral epitope for the formation of PAA, which, when reaching the brain, may react with the dopamine receptor and elicit mental disorder.

In the preceding paper we have demonstrated an increase in presentation of both major histocompatibility complex antigens (MHC) and a tumor-associated antigen of the weakly immunogenic B16 melanoma by a straight-forward technique. The method consists in modulating the tumor cell membrane by hydrostatic pressure and simultaneous chemical crosslinking of the cell-surface proteins. In B16-BL6 melanoma, the induced antigenic modulation was found to persist for over 48 h, which permitted the evaluation of the ability of modified B16-BL6 cells to induce immunity against unmodified B16-BL6 cells. In the present study, we have shown that a significant systemic immunity was induced only in mice that were immunized with modified B16-BL6 melanoma cells, whereas immunization with unmodified B16-BL6 cells had only a marginal effect when compared to the results in control sham-immunized mice. The induced immunity was specific since a single immunization affected the growth of B16-BL6 tumors but had no effect on MCA 106, an antigenically unrelated tumor. The addition of interleukin-2 to the immunization regimen had no effect on the antitumor responses induced by the modified B16-BL6 cells. The cell-mediated immunity conferred by immunization with treated B16-BL6 cells was confirmed in experiments in vitro where splenocytes from immunized mice could be sensitized to proliferate by the presence of B16-BL6 cells. In addition, the altered antigenicity of these melanoma cells appeared to correlate with their increased susceptibility to specific effectors. Thus, 51Cr-labeled B16-BL6 target cells, modified by pressure and crosslinking, in comparison to control labeled target cells, were lysed in much greater numbers by effectors such as lymphokine-activated killer cells and allogeneic cytotoxic lymphocytes (anti-H-2b), while such cells remained resistant to lysis by natural killer cells. Our findings indicate that the physical and chemical modifications of the tumor cells that are described here may be considered as a simple yet effective method for the preparation of tumor vaccines, which could be applied in tumor-bearing hosts.

The size and configuration of the hydration layer of solutes play a major role in their thermodynamic features. With respect to amino acids in water, a series of indirect evidence strongly suggest that their hydration layer acquires a chiral configuration induced by their chiral centers. Such a chiral hydration may act as a recognition factor in the various biochemical interactions, but information on it remains rather scarce. In this study, we determined by dilution microcalorimetry the fraction of the hydration energy invested in the chiral distortion of the hydration layer surrounding D- and I-alanine in water. The results indicate that in dilute solutions, a multilayered chiral hydration surrounds each of these solutes and amounts to over 100 water molecules. In concentrated solutions, the immediate chiral hydration layer decreases to ∼30 water molecules. The energy invested in the induction of the chiral twist in the hydration layer is predominantly attributed to TΔS, the energy associated with “configurational entropy,” which amounts to only several cal/mol, about a thousandth of the total energy of the hydration shell. Chirality 2010. © 2009 Wiley-Liss, Inc.

Background: In our preceding study, we assayed in a blind fashion the blood sera of young normal subjects and schizophrenic patients for levels of platelet autoantibodies (PAA). The recorded PAA titers of the schizophrenic patients were significantly higher than those of the normal subjects. This observation has lent support to this test being used as an objective evaluation of schizophrenia in young subjects in the future. In addition, this finding strongly suggested that the etiology of a distinct group of sufferers of this disorder could have originated from an autoimmune reaction against platelets which can, under certain conditions, cross-react with brain tissue. Aims: In the present study, PAA titers in the sera of adult schizophrenic patients and matched normal subjects were determined analogously to the preceding study. The effect of hospitalization and drug treatments on the apparent blood test scoring in adult subjects could thus be evaluated. Methods: A total of 46 schizophrenia patients (30 men and 16 women) aged 19–45 years (mean ± SD: 31.7 ± 8.0 years) with a minimum score of 60 on the Positive and Negative Symptom Scale and 43 healthy control subjects (22 men and 21 women) aged 21–44 years (mean ± SD: 31.9 ± 6.9 years) participated in the study. The blood titers of PAA were evaluated in a single-blind fashion using an optimized ELISA test scored by optical density (OD) units. A positive test was defined as a value above 1.3 OD units. Results: The titers of PAA in the group of schizophrenic patients (1.1 ± 0.55 OD units, range: 0.360–2.285 OD units) were significantly higher in comparison to those of the healthy control subjects (0.81 ± 0.37 OD units, range: 0.360–1.704 OD units; p = 0.004, two-tailed unpaired t-test). Significantly more schizophrenic patients showed a positive test (15 patients out of 46) than the control subjects (5 out of 43). However, significantly higher OD values of 1.55 ± 0.5 were recorded in the group of patients with less than 3 years of registered disease (n = 16, age 19–30 years), while in the group with 4–20 years of hospitalization (n = 30, age 24–45 years) the recorded OD values (0.85 ± 0.4 OD units) were practically indistinguishable from those of the control group. Conclusions: In the adult schizophrenic patients, the PAA blood test remains valid for patients who were hospitalized for less than 3 years. Drug treatment, length of disease and age can be assumed to reduce the PAA level considerably.

Background: It has been hypothesized that the etiology of schizophrenia, in a distinct group of patients, originates from an autoimmune reaction against platelets. Previous open screenings have recorded significantly higher blood titers of platelet-associated autoantibodies (PAA) in schizophrenic patients as compared to normal healthy subjects. In addition, young schizophrenic patients at the early stages of their disorder displayed higher PAA titers than older patients with a longer duration of the disorder. A blood test based on these observations was proposed. Aim: To verify by a blind test a significant difference in PAA between young schizophrenic patients and matched healthy control subjects, for the validation of a blood test for schizophrenia. Methods: A total of 36 young schizophrenic patients in an active psychotic state, aged 13–20 years (mean ± SD: 16.2 ± 2.1 years) with an average PANSS score of 115.6 ± 14.5 and illness duration of 9.5 ± 9.4 months, were examined. The control group consisted of 49 healthy young subjects between the ages of 13 and 21 years (16.2 ± 2.2 years). The blood titers of PAA were evaluated blindly using an optimized ELISA test, expressed by a linear optical density (OD) scale. The blood samples of all participants were tested anonymously, and were scored under a code number. A test recording above 1.3 OD units was defined as positive. Results: The PAA titers of schizophrenia patients (1.6 ± 0.4 OD units, range: 0.7–2.3 OD units) were significantly higher than those of the control group (1.0 ± 0.4 OD units, range: 0.4–1.8 OD units; p < 0.0001). In 61% of the young schizophrenic patients (22 out of the 36 patients), a positive result (OD >1.3 units) was recorded. In the control group, only 12.2% (6 of the 49 subjects) displayed a positive result (p < 0.0001). Conclusions: These findings support further assessment of PAA titers as a potential biomarker for patients with clinical signs and symptoms of schizophrenia.

Current procedures for solubilization of membrane proteins involve the use of detergents. A procedure using high hydrostatic pressures without detergent has been applied in this study to subfractionate membrane proteins and their endogenously associated lipids. Rat brain membrane preparations were suspended in hypotonic buffer containing the membrane fluidizer benzyl alcohol in a sealed pressure cell and subjected to hydrostatic pressures of up to 1500 atmospheres (approx 22,000 psi) in a French press. Under these conditions, specific membrane proteins including protein kinase C, phospholipase A2, calmodulin-binding proteins, G-proteins, and microtubule-associated proteins all coextracted and were associated to lipid particles, suggesting inherent physical contact. Two populations of membrane-associated protein kinase C were identified according to molecular weight estimations. The first coeluted with the lipid particles composed predominantly of phospholipids, while the second contained much less lipid and was similar to the soluble monomer, i.e., cytosolic protein kinase C. This procedure provides an important technique for selective subfractionation of membrane proteins in their native lipid environment which could be used for structure-function studies.

We earlier reported that we had observed quantifiable differences in crystallization rates of d and l tyrosine. It has been suggested that these results were due to the presence of impurities. Here we argue that it is premature to conclude that impurities entirely explain the results. More generally, there is an accumulating weight of evidence that d and l enantiomers display unexpected differences in their physical properties and behavior. These should be taken into account as we attempt to understand the origin of biochirality.

The two hydrogen atoms attached to the carbon in the methylene group are of two different spin configurations, similar to those in the case of water: ortho, where the two proton spins are parallel to each other, and para, where they are antiparallel. The ortho configuration has three degenerate states, while the para configuration is singular, leading to a statistical ratio of these isomers 3:1 ortho/para. Such spin isomers are present in glycine and most chiral amino acids where they may induce broadening of structural zones, a possibility which remains to be assessed. The implications of this neglected possibility could be far-reaching, in particular with respect to protein structure and the origins of biochirality.

Mirror-image asymmetric molecules, i.e., chiral isomers or enantiomers, are classically considered as chemically identical. Recent studies, however, have indicated that parity violation by the nuclear weak force induces a tiny energy difference between chiral isomers. Upon combination with a massive amplification process, expansion of this difference to a detectable macroscopic level may be achieved. Yet, experimental tests of this possibility, where one enantiomer is compared to the other in solution, are hampered by the possible presence of undetectable impurities. In this study we have overcome this problem by comparing structural and dynamic features of synthetic D- and L-polyglutamic acid and polylysine molecules each of 24 identical residues. In these water-soluble polypeptides helix formation is an intramolecular autocatalytic process amplified by each turn, which is actually unaffected by low level of putative impurities in the solvent. The helix and random coil configurations and their transition were determined in this study by circular dichroism (CD) and isothermal titration calorimetry (ITC) in water and deuterium oxide. Distinct differences in structure and transition energies between the enantiomeric polypeptides were detected by both CD and ITC when dissolved in water. Intriguingly, these differences were by and large abolished in deuterium oxide. Our findings suggest that deviation from physical invariance between the D- and L-polyamino acids is induced in part by different hydration in water which is eliminated in deuterium oxide. Based on the recent findings by Tikhonov and Volkov (V. I. Tikhonov and A. A. Volkov, Science 2002, 296, 2363) we suggest that ortho-H(2)O, which constitutes 75% of bulk H(2)O, has a preferential affinity to L-enantiomers. Differential hydration of enantiomers may have played a role in the selection of L-amino acids by early forms of life.

Cyclic glycerophosphates and their deoxy analogs were previously found to induce intracellular tyrosine and threonine phosphorylation in Chinese hamster ovary (CHO) cells. Further studies have indicated that these compounds induce neuronal outgrowth in PC-12 cells, as well as elevation of the state of cellular differentiation in human breast cancer cell lines. The mechanism by which these cyclic phosphates operate is not yet fully delineated. Using an affinity labeling approach we probed for possible cyclic phosphate target proteins in CHO cells. A 170 kDa protein that was labeled by an affinity cyclic phosphate reagent was identified by mass spectrometry as the largest subunit of the eukaryotic initiation factor 3 (eIF3). Using In-Gel kinase assays allowed the detection of a approximately 70 kDa target kinase directly activated by cyclic phosphates. Identification of these proteins may provide a basis for deciphering the mechanisms, by which cyclic phosphates exert their effects.

Breast cancer cells in their virulent undifferentiated state are characterized by lack of functional estrogen receptors (ER) and/or progesterone receptors (PR) as well as relatively low levels of other normal differentiation markers such as milk proteins and lipid droplets. To date, no method for in situ elevation of the state of differentiation of breast cancer cells has yet been proven effective in patients. We have recently shown that 1,3 cyclic propanediol phosphate (1,3 cPP), an analog of 1,3 cyclic glycerophosphate (1,3 cGP), can promote morphological, neuronal-like differentiation in pheochromocytoma-12 cells in vitro. In view of this observation, we tested the potential of 1,3 cPP to elevate the state of cellular differentiation of the human breast cancer cell lines MCF-7 (ER(+)) and HCC1954 (ER(-)), as characterized by the expression of steroid receptors, casein kinase, lipid droplet histology and signal-transduction gene profiles. In the range of 5-100 microM 1,3 cPP the in vitro expression of ER-alpha, PR and casein kinase increased by approximately 2-fold while the mRNA transcription increased by 2-6-fold. Moreover, following 9-12 days of incubation with 1,3 cPP, HCC1954 cells exhibited a significant increase in the production of lipid droplets as observed by Oil Red O staining. The in vivo effect of 1,3 PP on MCF-7 xenografted into nude mice was also determined. After 4 biweekly i.p. injections of 0.5 mg 1,3 cPP per mouse, tumors in the 1,3 cPP treated virtually did not grow at all while the tumors in the control group grew rapidly. Based on these findings, we propose that this novel differentiating compound has the potential to transform the malignant tumor phenotype into a near-normal phenotype, as well as to sensitize the tumor cells to anti-estrogen therapy via upgrading the status of steroid hormone receptors.

Stress and depression are characterized by elevation of circulating cortisol, as well as by changes in physiological functions. In this study, we addressed the possibility that elevated cortisol is also associated with the origin and development of depression. We report here that cortisol at the nM-microM concentration range induces a substantial increase in serotonin uptake both in vitro, by human peripheral blood lymphocytes (PBLs) and cortical neuronal cells, and in vivo, by rabbit PBLs, owing to promotion of synthesis of the serotonin transporter. These findings offer a novel molecular mechanism for depression associated with stress. Accordingly, the elevated cortisol induced by stress increases serotonin uptake, under both rest and nerve stimulation, which is overtly expressed in symptoms of depression.

Summary: Purpose: We examined whether a dietary supplement containing omega-3 polyunsaturated fatty acids (n-3 PUFAs) can alleviate and/or reduce the frequency of epileptic seizures in patients with central nervous system (CNS) diseases treated with anticonvulsive drugs (ACDs). Methods: A special spread containing 65% n-3 PUFAs was added to the daily diet. The patients consumed 5 g of this spread at every breakfast for 6 months. Results: Five patients completed the study. In all of them, a marked reduction in both frequency and strength of the epileptic seizures was recorded. Conclusions: Incorporation of the dietary supplement containing n-3 PUFAs may be beneficial in suppression of some cases of epileptic seizures.

A series of cyclic glycerophosphates and their deoxy analogues were tested for induction of neuronal outgrowth in PC12 cells. Under chronic presence of a cyclic phosphate PC12 cells developed distinct isles of neuronal networks which covered up to 20% of the culture area, while alpha and beta glycerophosphates (the negative control compounds) did not induce any neuronal outgrowth. Distinct isles of neuronal networks were also observed upon short term application (i.e. 2 pulses of 3 hours each at day 1 and day 4) of the tested cyclic phosphates in contrast to an analogous short term exposure to NGF which was abortive. Analysis of tyrosine phosphorylation indicated a battery of phosphorylated proteins after several minutes of application of the cyclic phosphates, among which was an ERK protein of approximately 63 kD (possibly ERK7). Nerve rescue experiments were carried out with NGF differentiated PC12 cells where NGF was replaced with either 1,2 or 1,3 cyclic propanediolphosphate (1,2 cPP and 1,3 cPP) for 7 days. A distinct dose dependent preservation of neuronal network by these compounds was observed. In the control cultures NGF deprivation resulted in massive neuronal retraction and cell death. Preliminary experiments indicated that the nerve rescue by the cyclic phosphates involves the increase in the level of CASPase 6. The above findings suggest that cyclic glycerophosphates and their analogues may bear important physiological and pharmacological implications which are currently under investigation.

Peripheral blood lymphocytes (PBL) isolated from 17 healthy subjects were engrafted i.p. each to an individual SCID mouse. After 48 hours the mice were tested for skin response to the common recall antigen PPD at various sites on the body periphery. In 8 of them PPD elicited a distinct positive skin reaction only at the abdomen. This procedure could provide a basis for detection of specific cellular immune response such as in autoimmune diseases.

Hydrostatic pressure (P) combined with membrane protein crosslinking (CL) by adenosine dialdehyde (AdA) can render tumor cells immunogenic. We have recently shown that PCL treatment of murine tumor cells augmented the presentation of MHC-restricted tumor-associated antigens and enhanced cell-mediated immunity. In cancer patients inoculated with autologous PCL-modified tumor cells, a significant delayed-type hypersensitivity response was elicited. Since the balance between cell-mediated immunity and humoral immunity is reciprocally controlled by immunoregulatory cytokines, we have examined the proliferative response and cytokine secretion pattern in cultures of human peripheral blood mononuclear cells (PBMC) stimulated by autologous PCL-modified and unmodified tumor cells. These tumor cells were obtained from freshly resected tumor tissue of 16 patients with colon (8), lung (4) and renal (4) carcinomas. The results demonstrated that PCL-modified tumor cells promoted an increase in PBMC proliferation in 5 out of 8 (63%), 1 out of 4 (25%) and 4 out of 4 (100%) colon, lung and renal cell carcinomas. Fourteen of the above cultures were also analyzed for the secretion of interleukin-10 and interferon-gamma. Overall, a substantial decrease in IL-10 secretion was detected in 9 out of 14 (64%) cultures while a reciprocal increase in interferon-gamma secretion was noted in 8 out of 14 (57%) cultures. Our results confirmed that PCL-modified human tumor cells of different etiologies can modulate the pattern of cytokines released from stimulated autologous lymphocytes. Such a procedure could prove valuable in the production of autologous tumor vaccines.

In line with the autoimmune hypothesis of schizophrenia we have tested in this study whether the commonly used neuroleptics, clozapine and haloperidol can also act as systemic immunosuppressants. Twenty one hospitalized chronic schizophrenic patients participated in the study. Five were free of neuroleptic treatment while the other 16 were under chronic treatment with either clozapine (n = 8), or haloperidol (n = 8). Fourteen age matched normal subjects served as the control group. Conventional in vitro mitogenic stimulation of peripheral blood lymphocytes with phytohaemagglutinin (PHA) indicated a clear suppression of responsiveness of approximately 50% in all treated patients. The PHA response of the untreated patients was virtually identical to that of the control group. The in vitro effect of haloperidol and clozapine on PHA stimulation of lymphocytes from normal subjects was determined by 3H-thymidine uptake and secretion of interleukin-2, interleukin-4 and interferon-gamma. Both clozapine and haloperidol suppressed thymidine incorporation and cytokine secretion at a drug concentration of above 1 microM, reaching full suppression at 50 microM. Similar suppressive effects of clozapine and haloperidol were also observed in mixed lymphocyte reaction of mouse lymphocytes. Assays with radioactive ligands indicated that clozapine is not incorporated into the lymphocytes but presumably exerts its action by binding to specific surface sites. The long term immune suppression induced by neuroleptic treatment may inhibit putative autoimmune responses against neurological sites and could thus act synergistically with the direct antagonistic action on brain receptors for the overt amelioration of psychotic behaviour.

Various findings suggest auto-immune changes in schizophrenia. We have recently demonstrated that platelets from schizophrenic patients bear autoantibodies (PAA) which cross-react with brain antigens. Accordingly, treatment of schizophrenia with an immunosuppressant might be of potential benefit. In a recent case study, a chronic schizophrenic patient treated with azathioprine has demonstrated a clear psychiatric improvement preceded by a decrease in PAA level. A phase I study designed for assessing side-effects of short-term azathioprine treatment in a group of schizophrenic patients is described here. From a group of 40 chronic non-responsive patients, 14 patients demonstrating high PAA level have entered the study and 11 have complied all along. Two groups were tested in parallel. In the first (6 patients) 150 mg/day was given for 7 weeks while in the second (5 patients) the same regimen was given for two periods of 7 weeks with an interval of 6 weeks. Blood biochemistry and cell count, as well as determination of PAA were carried out weekly, starting 3 weeks before the trial and continuing up to 7 weeks after the treatment. Two out of 11 patients developed leucopenia in week 4. No other side-effects were recorded in any of the patients. A substantial reduction in PAA was observed in 3 out of 6 patients in group I and 4 out of 5 in group II. Two patients showed improvement of psychiatric symptomatology. Our results demonstrate that short-term azathioprine treatment induces transient leucopenia in 18% of the patients receiving the drug, much alike the percentage reported for other patient populations.

Augmentation of surface presentation of the major histocompatibility complex (MHC) is a leading trend for preparation of tumor vaccines. Exposure of weakly immunogenic tumor cells, such as murine B16 melanoma, to hydrostatic pressure (P) in the presence of the membrane-impermeable protein crosslinker (CL) 2',3'-adenosine dialdehyde, was previously shown to induce a substantial increase in surface presentation of MHC molecules. When B-16 melanoma cells, used here as a model, were first treated for 72 h with interferon-gamma or tumor necrosis factor-alpha at concentrations of 10 and 100 units/ml, respectively, followed by application of pressure and cross-linking (PCL), the surface presentation of H2b molecules increased by 40% compared to treatment with cytokines alone, and by up to 1,700% when compared to treatment with PCL alone. Neither P nor CL alone enhanced the MHC presentation when cells were pretreated with these cytokines. The changes in MHC observed after the cytokine treatment were transient and decayed within several hours. However, the changes induced by the sequential treatment with cytokines and PCL were sustained for at least 96 h post-PCL which is of prime importance for immunogenic expression. A series of analogous experiments in the presence of cycloheximide indicated that approximately 50% of the observed PCL-induced increase in MHC projection originates from protein synthesis while the other 50% corresponds to passive translocation of MHC compartments. B16 melanoma cells, modified by the sequential treatment of cytokines and PCL, proved to be substantially more immunogenic by an in vitro sensitization assay than cells treated by either one of these treatments alone. These results may provide a guideline for the preparation of tumor vaccines which could be applied in immunotherapy treatment of cancer.

In the present study we observed that the number of dense granules per platelet increases with age, attaining a maximum level above the age of about 40 years. Platelets of newborns apparently contain only a small number of dense granules per platelet. The numbers of platelet dense granules and platelet cell size in schizophrenic patients increase compared to age-matched healthy controls. In contrast, in Alzheimer-type dementia the number of platelet dense granules tends to decrease compared to healthy persons.

The action of phospholipase C (PLC) from Bacillus cereus on phosphatidylglycerol (PG), derived from egg yolk phosphatidylcholine (PC), was examined in an ether-water mixture. The PLC cleavage of PG and PC followed a Michaelis-Menten kinetics with apparent Vmax values per 1 microgram enzyme of 0.26 and 0.91 mumol.min-1 and Km values of 10 and 12 mM, respectively. When the same enzymic reaction was carried out in minimally buffered aqueous solution of 1% Triton X-100, the decrease in pH with respect to phospholipid cleavage was as expected with PC but much less pronounced with PG. This could be accounted for by the formation of a cyclic glycerophosphate, rather than alpha-glycerophosphate, in the PLC hydrolysis of PG. Examination of the chemical nature of the water-soluble product of PG by phosphorus nuclear magnetic resonance (31P NMR) revealed a single band at 2.31 ppm, while the bands of alpha-glycerophosphate and beta-glycerophosphate appeared at 5.12 and 4.57 ppm, respectively. Basic hydrolysis of the phospholipase cleavage product of PG (0.1 M NaOH for 1 min at 80 degrees C) followed by neutralization shifted its 31P NMR band to 5.18 ppm, which practically coincided with that of alpha-glycerophosphate. Analogous experiments were carried out with PG labeled with 3H at the carbon 2 of the glycerol headgroup ([3H]PG). Autoradiography of thin layer chromatography (TLC) of the [3H]PG enzymic hydrolyzate displayed a single 3H-labeled compound, which could be converted to alpha-glycerophosphate by basic hydrolysis. These results strongly suggest that the phosphate headgroup of PG is cleaved off by PLC as 1,3-cyclic glycerophosphate. A series of PLC experiments with phosphatidyl dihydroxyacetone and phosphatidyl 1,3-propanediol as model substrates supported this assignment. Two-dimensional homonuclear 1H NMR correlated spectra as well as infrared spectra carried out on the isolated sodium salt of this product could further confirm such a structure. The unique structure and chemical nature of 1,3-cyclic glycerophosphate may bear a distinct physiological function.

The B16-BL6 melanoma, like most spontaneously arising tumors, is poorly immunogenic and expresses low levels of major histocompatibility complex (MHC) antigens. Treatment of cells of this tumor in vitro by hydrostatic pressure in the presence of adenosine 2′,3′-dialdehyde (oxAdo), a membrane-impermeant crosslinker, caused elevated projection of MHC and a specific tumor antigen as demonstrated by flow-cytometric analysis. Maximum projection of both the MHC and the tumor antigens could be reached by application of 1200 atm for 15 min in the presence of 20 mM oxAdo. It is not yet clear whether this passive increase in availability of antigens on the cell surface originated from a dormant pool of antigens in the plasma membrane or from pressure-induced fusion of antigen-rich intracellular organelles (e.g. the endoplasmic reticulum). The immunogenic properties of the antigen-enriched B16-BL6 cells are described in the following paper.

The immunological approach to cancer treatment and prevention is based on the notion that tumor cells bear antigens that can evoke the production of specific antibodies or more likely cytotoxic T-cells. Numerous studies have indeed implicated such immune reactions, though the number of cases where specific tumor antigens were explicitly demonstrated are much fewer and still controversial. There is, however, an agreement that most of these tumor-associated antigens (TAA) reside in normal cells as well, though, at a concentration and distribution which are below die threshold for immune recognition. Operationally, the long and frustrating debate on whether at all immune approaches for therapy and prevention of cancer can be constituted, is actually behind us.