Comparative evaluation of different DNA extraction procedures from food samples.

Five methodologies for extracting DNA from food samples are described. The food products analyzed are from either soybean or maize. They were selected on the basis of the mechanical, thermal, and chemical treatments that they had been subjected to during industrial processing. DNA preparations were evaluated for purity, yield, and average fragment size. Two endogenous genes, soybean lectin gene and alcohol dehydrogenase gene (adh1), were used to assess the degree of DNA degradation at different stages of the transformation chain. The goal of this study was to determine the role that extraction methods play in DNA amplification in order to select the best protocol for a food sample. This comparative evaluation can be specifically useful for detection of genetically modified ingredients in a variety of food matrices.

RB and RB2/p130 genes demonstrate both specific and overlapping functions during the early steps of in vitro neural differentiation of marrow stromal stem cells.

Marrow stromal stem cells (MSCs) are stem-like cells that are currently being tested for their potential use in cell therapy for a number of human diseases. MSCs can differentiate into both mesenchymal and nonmesenchymal lineages. In fact, in addition to bone, cartilage and fat, it has been demonstrated that MSCs are capable of differentiating into neurons and astrocytes. RB and RB2/p130 genes are involved in the differentiation of several systems. For this reason, we evaluated the role of RB and RB2/p130 in the differentiation and apoptosis of MSCs under experimental conditions that allow for MSC differentiation toward the neuron-like phenotype. To this end, we ectopically expressed either RB or RB2/p130 and monitored proliferation, differentiation and apoptosis in rat primary MSC cultures induced to differentiate toward the neuron-like phenotype. Both RB and RB2/P130 decreased cell proliferation rate. In pRb-overexpressing cells, the arrest of cell growth was also observed in the presence of the HDAC-inhibitor TSA, suggesting that its antiproliferative activity does not rely upon the HDAC pathway, while the addition of TSA to pRb2/p130-overexpressing cells relieved growth inhibition. TUNEL reactions and studies on the expression of genes belonging to the Bcl-2 family showed that while RB protected differentiating MSCs from apoptosis, RB2/p130 induced an increase of apoptosis compared to controls. The effects of both RB and RB2/p130 on programmed cell death appeared to be HDAC- independent. Molecular analysis of neural differentiation markers and immunocytochemistry revealed that RB2/p130 contributes mainly to the induction of generic neural properties and RB triggers cholinergic differentiation. Moreover, the differentiation potentials of RB2/p130 and RB appear to rely, at least in part, on the activity of HDACs.

Amino acids and hepatic encephalopathy.

The consideration of HE and its etiology has undergone a radical turn within the past decade. At present HE is seen in the context of severe metabolic derangements, which failure of the liver, the central biochemical powerhouse of the body, must bring with it. The increased awarenesses on the biochemical mechanisms involved in the pathogenesis of HE have found, step by step, their own place in a complex but consequential mosaic of events, in which amino acid and HE are tightly linked. Clinical and experimental studies are needed to further improve the knowledge in this field, nontheless a certain number of corner-stones can be identified: A profound alteration of the central nervous system neurotransmission is responsible for most, if not all, of the symptoms characterizing HE. The plasma amino acid imbalance observed in cirrhotic patients represents a 'condicio sine qua non' HE may develop. A functional impairment of the amino acid transport systems at the level of the blood-brain barrier seems to play a crucial role in causing deleterious modifications of the synaptic neurotransmission in the central nervous system. The reduction of the brain entry of the "toxic" aromatic amino acids usually obtained by parenteral administration of especially tailored amino acid mixtures is most frequently followed by awakening from HE. In conclusion, most of the results obtained have demonstrated that HE represents a research field in which progresses in the knowledge of some of the pathogenic mechanisms have brought the investigators to new therapeutic approaches which have clearly improved the prognosis of patients suffering from this severe neuropsychiatric syndrome.

Small interfering RNAs and antisense oligonucleotides for treatment of neurological diseases.

The complexity of the central nervous system (CNS) exposes it to a number of different diseases, often caused by only small variations in gene sequence or expression level. Antisense oligonucleotides and RNA interference-mediated therapies hold great promise for the treatment of CNS diseases in which neurodegeneration is linked to overproduction of endogenous protein or to synthesis of aberrant proteins coded by dominant mutant alleles. Nevertheless, difficulties related to the crossing of the blood-brain barrier, expression vectors, molecule design and to the choosing of the correct target, should be effectively solved. This review summarizes some of the most recent findings concerning the administration of potential nucleic acid-based therapeutic drugs, as well as the most promising studies performed both in vitro and in animal models of disease. Finally, some current clinical trials involving antisense oligonucleotides or silencing RNA for therapy of neurological disorders are illustrated. Results of current studies and clinical trials are exciting, and further results will be certainly reached with increasing knowledge of blood-brain barrier transporters, of genes involved in neurological disease and in new vectors for efficient delivery to brain.

Methods to improve the yield and quality of DNA from dried and processed figs.

We describe here a molecular method that can be used to detect genome traits of a given horticultural item at each stage from the farm to the market. We developed a procedure to extract and amplify by PCR DNA obtained from complex matrixes, such as dried figs and fig jam. Few fragmented DNA molecules can be recovered from food products. However, we were able to increase the yield of PCR reactions by successfully applying an enzymatic repair protocol to retrieved DNA.

Symmetric transcription of bacteriophage T4 base plate genes.

Dot-blot and Northern-blot experiments, using strand-specific RNA probes, show that part of the bacteriophage T4 DNA that codes for six of the base plate structural genes (gp 51, 27, 28, 29, 48 and 54), is transcribed in vivo from both DNA strands. The r DNA strand transcripts contain sequences which are translated into structural proteins. Antisense l strand RNA is about 100 fold less abundant than RNA molecules transcribed from the r DNA strand.

Eating behavior and adherence to dietary prescriptions in obese adult subjects treated with 5-hydroxytryptophan.

Previous observations have shown that oral administration of 5-hydroxytryptophan (5-HTP) without dietary prescriptions causes anorexia, decreased food intake, and weight loss in obese subjects. To confirm these data over a longer period of observation and to verify whether adherence to dietary restriction could be improved by 5-HTP, 20 obese patients were randomly assigned to receive either 5-HTP (900 mg/d) or a placebo. The study was double-blinded and was for two consecutive 6-wk periods. No diet was prescribed during the first period, a 5040-kJ/d diet was recommended for the second. Significant weight loss was observed in 5-HTP-treated patients during both periods. A reduction in carbohydrate intake and a consistent presence of early satiety were also found. These findings together with the good tolerance observed suggest that 5-HTP may be safely used to treat obesity.

Clinical trials of a new class of therapeutic agents: antisense oligonucleotides.

Antisense oligodeoxynucleotides (ODNs) are short stretches of DNA complementary to a target mRNA. The ODNs selectively hybridise to their complementary RNA by Watson-Crick base pairing rules. In theory, the use of antisense ODNs provides a method to specifically inhibit the intracellular expression of any disorder whose genetic aetiology is well known. For this reason, researchers thought that if antisense drugs proved to be so specific there would be no side effects. However, toxicity-related problems arose in initial animal studies of antisense drugs in the early 1990s and since then companies have been using these compounds cautiously. In order to be useful therapeutically, an ODN must (a) exhibit reasonable stability in the physiological environment, (b) be taken up and retained in adequate quantities by the target cells, (c) specifically bind target mRNA with high affinity, (d) have an acceptable therapeutic ratio, free of unwanted toxic and non-specific side effects and (e) be easily synthesised in sufficient quantities to allow clinical use. Most of these criteria have already been met by ODNs recently used in this way. This review describes certain therapeutic applications of antisense techniques currently under investigation in oncology, haematopathology and inflammatory diseases.

Influence of phenylethanolamine on octopamine plasma determination in hepatic encephalopathy.

Octopamine and phenylethanolamine levels were measured by a radioenzymatic procedure in 30 cirrhotic patients with and without hepatic coma and in 15 normal controls. Octopamine data were obtained either by direct extraction with 40% isoamyl alcohol in toluene according to Molinoff et al. (Molinoff, P.B., Landsberg, L. and Axelrod, J. (1969) J. Pharm. Exp. Ther. 170, 253), or after pre-extraction of phenylethanolamine with 3% isoamyl alcohol in toluene. Phenylethanolamine was statistically correlated with the grade of hepatic encephalopathy. Octopamine levels also appeared to parallel the grade of coma, although the values obtained after pre-extraction were lower and less significant than those obtained with 40% isoamyl alcohol in toluene extraction. The higher values of directly extracted octopamine are due to contamination of other beta-hydroxylated phenylethylamines, among which is phenylethanolamine.

Octopamine and ammonia plasma levels in hepatic encephalopathy.

It has been recently proposed that hepatic encephalopathy could be due to the accumulation of octopamine acting as a false neurotransmitter, and the increase of ammonia might reflect this accumulation. The simultaneous determination of octopamine and ammonia was performed in 88 cases with or without encephalopathy. The correlation between the two substances appeared to be good (P less than 0.01; r = 0.5), except in shunted patients. All the cases with low octopamine and high ammonia were patients who had been submitted to surgical portal-systemic anastomosis. This finding does not seem to be coincidental; in this type of patients, the mechanism of hepatic encephalopathy could involve other beta-hydroxyphenylethanolamines in addition to octopamine. The presence of the inhibition of the reaction of transmethylation constantly observed during octopamine plasma assay is in favour of this hypothesis.

Octopamine plasma levels and hepatic encephalopathy: a re-appraisal of the problem.

An investigation on the blood levels of octopamine was carried out on 70 adult individuals. There was a statistically significant correlation between the levels of octopamine and hepatic encephalopathy. Normal subjects had values below 1 ng/ml, while patients with grade 3 or grade 4 encephalopathy constantly showed values above 3.2 ng/ml. In these two groups the distribution was fairly homogeneous. Through the differences between cirrhotics without neurologic involvement and those with grade 1 or 2 hepatic encephalopathy displayed statistical significance, distribution of values in these groups was rather non-homogeneous. Octopamine levels paralleled variations in mental state in 3 out 4 cases. No difference was found between venous and arterial values. The reaction of transmethylation used in the assay of octopamine was constantly found to be inhibited by the presence of plasma. This inhibition is probably due to the presence of one or more beta-hydroxyphenylethanolamines other than octopamine.

Effect of energy substrate manipulation on tumour cell proliferation in parenterally fed cancer patients.

The effects of isocaloric carbohydrate-based vs. fat-based total parenteral nutrition (TPN) regimens on cancer cell proliferation and host nutritional status were evaluated in 27 patients with tumours of the gastro intestinal tract consecutively assigned to receive for 14 days: a glucose-based (A) or a lipid-based (B) TPN formula, or an oral diet (C) isocaloric and isonitrogenous to A and B. Cancer cell replication rate was evaluated by thymidine labelling index (LI) on tumour samples before and at the end of each nutritional regimen. The number of replicating cells increased by 32.2% in patients receiving regimen A. LI decreased by 24.3% in patients given regimen B. LI values were slightly increased (+15%) in patients maintained on regimen C. Nutritional status remained within normal limits. None of the LI changes observed between and within the three arms of the trial were found to be statistically significant. Thus we failed to prove that glucose consistently stimulates or lipids inhibit tumour proliferation despite a trend in this sense.

In vivo effects of partial phosphorothioated AT1 receptor antisense oligonucleotides in spontaneously hypertensive and normotensive rats.

Partial phosphorothioate (PS) antisense oligodeoxynucleotides (ODNs) targeted against rat AT1 receptor mRNA have been used to control blood pressure in normotensive (WKY) and spontaneously hypertensive (SHR) rats. Molecules were injected intracerebroventricularly (i.c.v., right lateral ventricle) in freely moving animals. The antisense ODN lowered the mean arterial pressure (MAP) 24 hours (-43 mmHg+/-10) and 48 hours (-30 mmHg+/-13) after injection, while the control ODN molecule had no significant effects. The observed decrease of blood pressure was due to a specific inhibition of AT1 receptor gene expression, since the level of its mRNA, monitored by reverse transcription (RT)- polymerase chain reaction (PCR), was significantly reduced by antisense molecule (-40%), compared to sense one. In normotensive rats no effect on MAP have been observed, while AT1 receptor gene expression is reduced (-40%) by antisense treatment. It is known that SHRs have an enhanced basal activity of the central renin-angiotensin system that induces an increase in central sympathetic outflow. Instead in WKY rats the central sympathetic outflow is not conditioned by the enhanced activity of brain renin-angiotensin system. Therefore in normotensive rats although partial PS ODN reduces the AT1 mRNA level this will not result in a modification of the sympathetic outflow and no change in MAP level would be observed.

Plasma amino acid imbalance in patients with lung and breast cancer.

In order to evaluate whether different solid tumors may specifically influence plasma free amino acid (PFAA) profile, PFAA were analysed in seventy-four patients with lung (41 patients) and breast cancer (33 patients) and 28 healthy subjects. In lung cancer patients a significant reduction of gluconeogenic amino acids, threonine, serine, glycine and a significant increase of free tryptophan and glutamic acid was found. In breast cancer patients a significant increase of ornithine, glutamic acid and free tryptophan was found. The comparison of PFAA profiles between lung and breast cancer suggests that different tumors have a different influence on the host's PFAA pattern.

Cytokines, tryptophan and anorexia in cancer patients before and after surgical tumor ablation.

Serotoninergic system activity and cytokine production have been both implicated in the pathogenesis of cancer anorexia. To verify the existence of relationships between tryptophan, cytokines and anorexia, twenty cancer and six non-neoplastic patients were studied. Plasma amino acid concentration, including tryptophan, and spontaneous and LPS stimulated tumor necrosis factor and interleukin-1 release from peripheral blood mononuclear cells were determined before and after surgery in both groups of patients. A close relationship between plasma free tryptophan concentration and anorexia was observed, whereas no relationship between cytokine production and either anorexia or plasma tryptophan was found in cancer patients.

Increased plasma free tryptophan levels in human cancer: a tumor related effect?

High free tryptophan (F-TRP) plasma levels are found in cancer patients (CP). F-TRP plasma concentrations are affected by the levels of its carrier, albumin (ALB), and free fatty acids (FFA) competing with TRP for ALB binding sites. The lack of correlation between F-TRP, ALB and FFA in CP suggests a tumor-dependent effect on the rise in F-TRP. To verify this hypothesis, F-TRP, ALB and FFA levels were assayed in 12 lung and 16 breast CP susceptible to radical surgery, before and 15 days after surgical removal of the tumor. F-TRP levels significantly decreased after tumor ablation. Since no correlation was found between F-TRP, ALB and FFA variations, it is conceivable that the tumor itself may be responsible for the high F-TRP levels in CP.

Tumor-induced changes in host metabolism: a possible marker of neoplastic disease.

A large number of "biologic markers" for cancer have been described, including tumor-associated antigens, ectopic hormones, enzymes, and effects of tumor on the host's metabolism. Although tumors may metabolically differ from each other, they may induce similar derangements in glucose, lipid, and protein metabolism in the host. In particular, changes in carbohydrate metabolism may induce glucose intolerance that may be early and easily detected using an oral glucose tolerance test. Hypertriglyceridemia and reduced exogenous lipid clearance may represent an early marker of deranged lipid metabolism. Changes in protein metabolism, as reflected by plasma amino acid profile, may also represent a new diagnostic tool for cancer. Among other amino acids, free tryptophan seems to be the best candidate as a new useful marker for monitoring neoplastic disease. It is conceivable that, based on the understanding of the differences in plasma amino acid profiles, more specific and rational antineoplastic strategies may arise.

Cancer anorexia: new pathogenic and therapeutic insights.

During tumor growth, anorexia and reduced food intake markedly contribute to the development of malnutrition, thus worsening overall patients' survival. A better understanding of the pathophysiology of eating behavior may lead to new and more effective therapies, aiming at counteracting the detrimental effects of anorexia and reduced food intake on nutritional status and survival in cancer patients. Brain tryptophan and serotonin concentrations seem to play a pivotal role in the regulation of eating behavior. Increased brain serotonin activity is indeed associated with a reduction of food intake. It has been recently hypothesized that increased availability of tryptophan to the brain and the consequent increased serotonin activity may represent the pathogenic mechanism for cancer-associated anorexia. According to this hypothesis, the modulation of brain serotonin activity may result in an improvement of anorexia. Reducing brain tryptophan availability represents a possible mechanism to restore brain serotonin activity to normal. There is evidence that the oral administration of neutral amino acids competing with tryptophan for brain entry results in a significant improvement of cancer anorexia. The same treatment may also be effective in improving secondary anorexia, which is associated with other chronic illnesses, including renal and liver failure, sepsis, and so forth, sharing a similar pathogenic mechanism.

Plasma amino acid concentrations in patients with acute myelogenous leukemia.

Changes in plasma-free amino acid (PFAA) concentrations in the presence of solid tumors have been widely described. Conversely, the PFAA profile in patients with acute leukemias is less well defined. The aim of the present study was to clarify whether the PFAA profile is altered in patients with acute myeloid leukemia (AML), whether the profile differs from the PFAA profile of solid tumors, and whether it may predict outcome of AML. Fasting PFAA were measured in 40 untreated, normally nourished patients with AML (17 males, 23 females), ages 22-78 y, with white blood cell (WBC) counts ranging from 1.08 to 276.5 x 10(3)/cm2, and in 24 healthy volunteers. Plasma concentrations (mu mol/L, mean +/- SE) of glutamic acid (GLU), free tryptophan (FTRP), ornithine (ORN), and glycine (GLY) were significantly higher in AML (GLU: 90.2 +/- 6.1 versus 37 +/- 8; FTRP: 7.0 +/- 0.6 versus 4.8 +/- 0.3, P < 0.005; ORN: 108.7 +/- 5.8 versus 78 +/- 6, P < 0.001; GLY: 295.0 +/- 14.8 versus 239 +/- 9, P < 0.01), whereas serine (SER), methionine (MET), and taurine (TAU) were significantly lower in AML than in controls (SER: 109.0 +/- 5.8 versus 130 +/- 4, P < 0.03; MET: 25.5 +/- 1.3 versus 33 +/- 3, P < 0.03; TAU: 46.5 +/- 3.5 versus 81 +/- 2, P < 0.001), and tended to be even lower in patients who had not responded to chemotherapy or had relapsed within 18 mo of enrollment. Such changes were unrelated to age, sex, and WBC count. Changes in PFAA that occur in AML are only in part similar to those observed in solid tumors. The reduction of TAU appears to be a typical feature of AML and might be secondary to the deficiency of its precursors SER and MET. Further studies are under way aimed at clarifying whether PFAA might predict prognosis in AML, whether PFAA is normalized by remission induction, and if its correction may be of any benefit for patients with hematologic malignancies.

Plasma clearance of exogenous lipids in patients with malignant disease.

Abnormalities in lipid metabolism have been widely described in cancer-bearing patients and animals. In particular, the presence of the tumor seems to profoundly affect triglyceride (TG) utilization by interfering with lipoprotein lipase (LPL) activity. Exogenous TG plasma clearance was evaluated in 10 nonmalnourished patients with cancers of various origin and 10 normolipidemic volunteers by means of a three-stage intravenous lipid clearance test. This technique allows precise determination of both the fractional removal rate (K2) and the maximal clearing capacity (K1) for exogenous lipids. Mean K2 values (min-1) were found to be significantly reduced in cancer patients compared with control subjects (0.07 +/- 0.006 vs 0.13 +/- 0.013 SEM, p less than 0.005). K1 values (mumol/L/min) were also found to be significantly lower in cancer patients than in the control group (109.43 +/- 6.3 vs 143 +/- 6.3 SEM, p less than 0.01). The data obtained indicate that the capacity to eliminate exogenous lipids from the bloodstream is reduced in cancer-bearing patients. This may be the consequence of a tumor-related impairment of LPL system activity.