Phenylalkyl 1,2-diamines as sigma-receptor ligands: the discovery of novel antidepressant agents.

This article focuses on the study of phenylalkyl 1,2-diamines as sigma-receptor ligands for the discovery of antidepressant agents. We synthesized N-chain substituted phenylalkyl 1,2-diamine derivatives. Phenylalkyl 1,2-diamines are one of the most important class of therapeutical medicines useful in managing central nervous system diseases and have been used mainly to treat obesity, narcolepsy, minimal brain dysfunction, and mild depression. In the present study, we found that phenylalkyl 1,2-diamine and amide compounds were strongly bound to the sigma-receptors and showed a potent activity of mice forced swimming test. Our article, after briefly outlining the background and nature of sigma-receptor ligands, explores the antidepressant activity of phenylalkyl 1,2-diamines.

Origin of lumbar spinal roots and their relationship to intervertebral discs: a cadaver and radiological study.

Previous studies on the anatomy of the lumbar spine have not clarified the precise relationship of the origin of the lumbar roots to their corresponding discs or their angulation to the dural sac. We studied 33 cadavers (25 formalin-preserved and eight fresh-frozen) and their radiographs to determine these details. All cadavers showed a gradual decrease in the angle of the nerve root from L1 to S1. The origin of the root was found to be below the corresponding disc for the L1 to L4 roots. In the formalin-preserved cadavers 8% of the L5 roots originated above, 64% below and 28% at the L4/L5 disc. In the fresh cadavers the values were 12.5%, 62.5% and 25%, respectively. For the S1 root 76% originated above and 24% at the L5-S1 disc in the formalin-preserved cadavers and 75% and 25%, respectively, in the fresh cadavers.A herniated disc usually compresses the root before division of the root sleeve. Thus, compression of the thecal sac before the origin of the root sleeve is common for L1 to L5 whereas compression at the root sleeve is common for S1. Our findings are of value in understanding the pathophysiology of prolapse of the disc and in preventing complications during surgery.

Rapid increase of cytosolic content of acetyl-CoA carboxylase isoforms in H9c2 cells by short-term treatment with insulin and okadaic acid.

Mammalian acetyl-CoA carboxylase (ACC) is present in two isoforms, alpha and beta, both of which catalyze formation of malonyl-CoA by fixing CO2 into acetyl-CoA. ACC-alpha is highly expressed in lipogenic tissues whereas ACC-beta is a predominant form in heart and skeletal muscle tissues. Even though the tissue-specific expression pattern of two ACC isoforms suggests that each form may have a distinct function, existence of two isoforms catalyzing the identical reaction in a same cell has been a puzzling question. As a first step to answer this question and to identify the possible role of ACC isoforms in myogenic differentiation, we have investigated in the present study whether the expression and the subcellular distribution of ACC isoforms in H9c2 cardiac myocyte change so that malonyl-CoA produced by each form may modulate fatty acid oxidation. We have observed that the expression levels of both ACC forms were correlated to the extent of myogenic differentiation and that they were present not only in cytoplasm but also in other subcellular compartment. Among the various tested compounds, short-term treatment of H9c2 myotubes with insulin or okadaic acid rapidly increased the cytosolic content of both ACC isoforms up to 2 folds without affecting the total cellular ACC content. Taken together, these observations suggest that both ACC isoforms may play a pivotal role in muscle differentiation and that they may translocate between cytoplasm and other subcellular compartment to achieve its specific goal under the various physiological conditions.

Primers determine the sensitivity of PCR-mediated hepatitis B virus DNA detection and pretreatment of PCR mixture with 8-methoxypsoralen eliminates false-positive results.

Most methods for the diagnosis of hepatitis B virus (HBV) infection largely depend on viral DNA detection by polymerase chain reaction (PCR) or radioimmunological assay of viral antigens or antibodies. The quality assurance program recently established in Europe reported that PCR-mediated HBV DNA detection methods used in many laboratories produced a high rate of false-positive and false-negative results. Thus, we attempted to improve the conditions of current PCR methods for detection of HBV DNA. In the present study, we applied a recently developed method of releasing HBV DNA from virion by NaOH treatment of patient serum. Using four different primer sets specific to the HBV core region, we found that the sensitivity of first-round PCR can be improved by more than two orders of magnitude depending on the primers. The second round of PCR using nested primers was sensitive enough to detect up to 10(-6) pg of the HBV DNA, which is equivalent to approximately 3 copies of the HBV genome. Among the approximately 800 HBV-infected patient sera investigated in our laboratory, more than 60% of the tested samples gave positive results in the first-round PCR. The rate of positive results obtained using our experimental conditions is very high in comparison with other reports. The reamplification of the first-round PCR reaction mixture with the nested primers produced practically 100% positive results. For diagnosis of HBV infection, we routinely used 1 microliter of patient serum, which was found to be optimum in our laboratory. Surprisingly, from 20% of our positive results, even serum diluted to 1/100 (0.01 microliter) produced a stronger signal than 1 microliter. This observation suggests that direct PCR amplification of HBV DNA released from serum by NaOH treatment has to be compensated by other DNA detection methods for correct quantitation. In order to eliminate the false positive signal resulting from the carry-over due to massive screening of a large number of samples, PCR reaction mixture containing 8-methoxypsoralen was exposed to ultraviolet light prior to thermal cycle amplification. This exercise did not decrease the sensitivity of the detection method, but almost completely removed the false positive results caused by contaminated templates. We are in the process of improving PCR-mediated HBV DNA detection methods to attain more reliable and easily applicable methods.

Effect of urea treatment on recovery of staphylococcal enterotoxin A from heat-processed foods.

The effects of urea treatment on the potential reactivation of heat-damaged antigenic components of staphylococcal enterotoxin A (SEA) were examined with cooked foods, including mushrooms, ham, bologna, salami, and turkey. The thermal stability of purified SEA spiked into foods and native SEA produced by Staphylococcus aureus in foods was also examined. Food samples containing either spiked or native SEA were thermally processed by autoclaving or retorting. This was followed sequentially by toxin extraction, urea treatment, dialysis, reconstitution, and SE assays with the reversed passive latex agglutination and/or enzyme immunoassay kit. The results indicate that (i) urea treatment did not result in any reactivation of heat-inactivated antigenic components of SEA in any of the foods tested, (ii) the serological components of purified SEA were destroyed (> or = 96%) by autoclaving at 121.1 degrees C for 5 to 15 min or by retorting at an F0 of 4 to 18, and (iii) the immunological property of the native SEA was approximately threefold-more heat resistant than that of the purified SEA. The study suggested that the current urea method is not suitable for the detection of heat-denatured SEA in the thermally processed foods.

A collaborative study on the detection of staphylococcal enterotoxins in foods by an enzyme immunoassay kit (RIDASCREEN).

The RIDASCREEN SET kit (R-Biopharm GmbH, Darmstadt, Germany), a commercial staphylococcal enterotoxin (SE) immunoassay kit, utilizes monovalent capture antibodies against SE types A to E (SEA to SEE); therefore, it simultaneously detects and identifies the enterotoxin types. A collaborative study was conducted to ascertain whether the specificity, sensitivity, repeatability and reproducibility of the kits would meet food safety criteria. Twelve Canadian laboratories participated in this study to analyze various foods to which 1.0 to 2.0 ng of SE/g had been added and negative control samples. The results indicate that the sensitivity and specificity of the kit were excellent; all collaborators were able to detect the minimum toxin levels of 1.0 ng SEA/g in ham and cheese, 1.0 ng SEB/g in salami and turkey, and 2.0 ng SED/g in other samples without any false-negative results. With regard to negative control samples, all analysts obtained correct results except for one analyst who recorded weak false-positive results with several foods detecting SEC or SEA. The overall rate of false-positive results was 0.7% for 600 triplicate assays. In addition, it was confirmed that the RIDASCREEN kit did not yield false-positive results with mussels in contrast to some other EIA kits. Overall repeatability and reproducibility of the kit were in the range of 11.69-42.57% and 17.25-68.05%, respectively.

A Collaborative Study on the Detection of Staphylococcal Enterotoxins in Foods with an Enzyme Immunoassay Kit (TECRA).

One of the commercially available enzyme immunoassay kits for the detection of staphylococcal enterotoxins (SEs) in foods, the TECRA screening kit (Bioenterprises Pty. Ltd., Roseville, New South Wales, Australia), has microtiter plates coated with a mixture of antibodies to all of the SEs. A collaborative study was conducted to ascertain whether specificity, sensitivity, repeatability, and reproducibility of the results obtained using this kit would meet food-safety criteria. Thirteen Canadian collaborators participated in this study to analyze both various foods to which 1.0 to 3.0 ng of SE/g of food had been added and negative control samples. In addition, the effect of animal serum in these analyses was examined. The results indicate that all collaborators (100%) were able to detect the minimum toxin levels of 1.0 ng of SEA/g of ham and 1.0 ng of SEB/g of salami and SE or SEs in other samples (chicken, turkey, and cheese) containing 2.0 to 3.0 ng/g, without any false-negative results. With regard to negative control samples, all collaborators obtained correct results except when analyzing two types of food: two collaborators (15%) showed weak false-positive results with salami and all analysts found strong false-positive results with mussels. The problem regarding specificity could be largely corrected by treating the sample with rabbit serum (0.1 volume in 1.0 volume food extract). The repeatability and reproducibility of results from the kit were acceptable.

Evaluation of a commercial enzyme immunoassay kit (RIDASCREEN) for detection of staphylococcal enterotoxins A, B, C, D, and E in foods.

The RIDASCREEN SET kit (R-Biopharm GmbH, Darmstadt, Germany), a commercial staphylococcal enterotoxin (SE) visual immunoassay kit, was evaluated for its efficacy. The kit utilizes monovalent capture antibodies against SE types A to E (SEA to SEE); therefore, it simultaneously detects and identifies the enterotoxin types. The major advantages of the kit are (i) a high degree of specificity (except for naturally occurring peroxidases, food compositions or ingredients and microbiological products due to growth of nonstaphylococcal microorganisms did not cause false-positive results; additionally, no cross-reactions among reagents of the kits were observed), (ii) excellent sensitivity (minimum detectable limits were 0.20 to 0.30 ng of SEs per ml of extracts of ham, salami, and mushroom and 0.30 to 0.35 ng of SEs per ml of cheese extracts, or 0.50 to 0.75 ng of SEs per g of foods such as noodles, ham, salami, cheese, and turkey), (iii) simplicity (the kit enabled direct assay of SEs in food extracts without the need for lengthy extraction or concentration procedures), (iv) rapidity (it took less than 3 h to complete the analysis of individual enterotoxin types SEA to SEE), and (v) its semiquantitative results (optical density values could be read against a standard curve to estimate the amount of SE in the extract). The RIDASCREEN kit is a convenient, rapid, and reliable tool for the detection and identification of SEs in foods.

Simple solutions to false-positive staphylococcal enterotoxin assays with seafood tested with an enzyme-linked immunosorbent assay kit (TECRA).

The TECRA kit, a commercial staphylococcal enterotoxin visual immunoassay kit, is an enzyme-linked immunosorbent assay system which utilizes polyvalent antisera against staphylococcal enterotoxin types A to E. The test is simple and rapid to perform (4 h) and has therefore been widely used for screening purposes. In this study, the TECRA kit produced a number of false-positive reactions with seafood; 25% of 218 samples of seven types of seafood gave false-positive results, particularly shellfish such as mussels (85%), clams (32%), oysters (23%), winkles (20%), and squid (13%). Some nonshellfish samples also gave false-positive results with the TECRA kit (smelt [20%] and trout [10%]). The substance contributing to the false-positive results differed from true staphylococcal enterotoxins in that it was: (i) heat labile, being completely inactivated by heating for 3 min at 70 degrees C, compared with 5% inactivation of true staphylococcal enterotoxins by the same heat treatment, (ii) in a selective reaction with normal rabbit or calf serum (nonspecific reactions were completely abolished by these sera, whereas staphylococcal enterotoxins were not affected), and (iii) incapable of binding to a copper-chelate Sepharose gel (all of the substance remained in the unbound wash fraction, whereas staphylococcal enterotoxins were quantitatively bound to the gel). The false-positive reactions occurring with seafood were not associated with substances produced by microorganisms, since the bacterial isolates from the samples did not give positive results with the TECRA kit.(ABSTRACT TRUNCATED AT 250 WORDS)

Nonspecific reactions of a commercial enzyme-linked immunosorbent assay kit (TECRA) for detection of staphylococcal enterotoxins in foods.

A staphylococcal enterotoxin visual immunoassay kit (TECRA) has recently become commercially available. Since the kit is an enzyme-linked immunosorbent assay system equipped with polyvalent antisera against staphylococcal enterotoxin types A to E (SEA to SEE) and the test is simple and rapid to perform (4 h), it has been widely used for screening purposes. In this study, the sensitivity of the kit for detection of SEA, SEB, and SEC in ham, cheese, and mushrooms was similar to those of kits based on an enzyme immunoassay and reversed passive latex agglutination: 0.75 to 1.0 ng of SEA per ml, 0.5 to 0.75 ng of SEB per ml, and 1.0 to 1.25 ng of SEC per ml. However, the TECRA kit showed nonspecific reactions with food samples contaminated by microorganisms other than Staphylococcus aureus, such as Enterobacter agglomerans, Enterobacter cloacae, Proteus mirabilis, Pseudomonas aeruginosa, and Serratia marcescens. The substance contributing to the false-positive results differed from true staphylococcal enterotoxins in that it was (i) heat labile (completely inactivated by heating for 2 min at 100 degrees C, whereas true staphylococcal enterotoxins were inactivated by about 10% with this treatment), (ii) lower in molecular weight than staphylococcal enterotoxins, and (iii) not bound to a copper chelate Sepharose gel (all of the substance remained in the unbound wash fraction, whereas staphylococcal enterotoxins were quantitatively bound to the gel). The problem of false-positive results with the TECRA kit could be resolved by heat treatment (2 min at 100 degrees C) or by cleanup procedures involving metal chelate affinity chromatography with copper chelate Sepharose for 4 h before use of the TECRA kit.

Occurrence of thermotolerant campylobacters in fresh vegetables sold at farmers' outdoor markets and supermarkets.

A total of 1564 fresh samples of 10 vegetable types from two different retail levels (533 samples from farmers' outdoor markets and 1031 samples from supermarkets) were surveyed for the occurrence of thermotolerant campylobacters. In samples from the outdoor markets, campylobacters were detected on six types of vegetables; the detection rates were spinach, 3.3; lettuce, 3.1; radish, 2.7; green onions, 2.5; parsley, 2.4; and potatoes, 1.6%. Campylobacter jejuni was the predominant species (88%), with the remainder being C. lari (8%) and C. coli (4%). When the outdoor market samples were thoroughly washed with chlorinated water, all were negative for campylobacters. Of the samples from supermarkets, all were negative for campylobacters whether purchased in summer or winter. These results suggest that vegetables sold at farmers' outdoor markets are produced and (or) stored under less sanitary conditions than those sold at supermarkets, and they could constitute health hazards. Therefore, vegetables (e.g., potatoes and spinach) from farmers' markets must be decontaminated by washing with chlorinated water or cooked thoroughly before consumption.

Thermal inactivation of Campylobacter species, Yersinia enterocolitica, and hemorrhagic Escherichia coli O157:H7 in fluid milk.

Heat treatment of raw milk in an HTST pasteurizer operated at 60.0 to 72.0 degrees C for a minimum holding time of 16.2 s rapidly inactivated mixtures of hemorrhagic Escherichia coli O157:H7, Yersinia enterocolitica and Campylobacter spp. (C. fetus, C. coli, and C. jejuni). Each of the three genera in the mixture was inoculated at a level of approximately 1.0 x 10(5) cfu/ml. At 60.0 degrees C, hemorrhagic E. coli showed a maximum 2 log10 reduction in counts and no viability at greater than or equal to 64.5 degrees C. Yersinia enterocolitica and Campylobacter spp. showed greater heat sensitivity with a 4 log10 reduction in counts at 60.0 degrees C and absence of viable cells at greater than or equal to 63.0 degrees C. These findings reiterate the need for stringent control of thermal processes in the manufacture of dairy products from raw or heat-treated (non-pasteurized) milk.

Evaluation of the reversed passive latex agglutination (RPLA) test kits for detection of staphylococcal enterotoxins A, B, C, and D in foods.

Four lots of the SET-RPLA kit (Denka Seiken Co. Ltd., Tokyo), a commercial reverse passive latex agglutination test kit for the detection of staphylococcal enterotoxins A, B, C, and D in foods, have been evaluated for their efficacy. The kits showed high specificity and sensitivity with a detection limit of 0.75 ng enterotoxin/g of food. The test is simple, is completed within 24 h, and does not require complicated extraction or concentration procedures nor expensive equipment. In addition, the assay is semiquantitative. However, as in any other immunological system, routine verification of the specificity of the latex reagents against standard enterotoxins and toxin-free food extracts is necessary.

Effect of temperature, duration of incubation, and pH of enrichment culture on the recovery of Campylobacter jejuni from eviscerated market chickens.

To establish an enrichment system of high efficiency for recovery of Campylobacter jejuni from market chickens, the effects of the temperature, duration of incubation, and pH of the enrichment culture on the isolation of the bacterium were evaluated. Whole chickens or chicken parts in plastic bags were individually rinsed, and the washings filtered through cheesecloth. The cells were separated from the washings by centrifugation, and the pellet was inoculated into 100 mL of enrichment broth. Isolation of C. jejuni from poultry samples was significantly increased by incubating these samples in an enrichment medium at 42 degrees C as opposed to 35 degrees C; for 48 h as opposed to 24 h or 72 h; and at pH 7.0 as opposed to pH 6.0, 6.5, 7.5, or 8.0.

Evidence of thermonuclease production by Bacillus spp. and enterococci in naturally contaminated cheese.

Of 105 thermonuclease-positive (TNase-positive) cheese samples comprising 13 types, 92 (87.6%) contained coagulase-positive staphylococci, whereas 9 (8.6%) contained microorganisms other than staphylococci as the major contaminants. Of the latter group, six samples contained Bacillus spp. comprising three species (B. cereus, B. licheniformis, and B. subtilis), and three contained mainly enterococci (Streptococcus faecalis), which were proven to be TNase producers. The organisms responsible for TNase production in the other four samples (3.8%) are not known, because isolates from these samples failed to produce the enzyme. Unlike staphylococcal TNase, a greater part of nonstaphylococcal TNase remains in the cheese homogenate after extraction of the enzyme at pH 3.8 instead of pH 4.5.

A survey of microorganisms for thermonuclease production.

A total of 1204 cultures comprising 16 genera were surveyed for production of thermonuclease (TNase) in milk. Cultures other than Staphylococcus capable of TNase production were restricted to two genera, Streptococcus and Bacillus. Nineteen percent of 338 group D streptococci comprising four species (85% of which were Streptococcus faecalis) and 17% of 60 streptococci belonging to other groups produced TNase. Nine percent of 130 Bacillus cultures comprising six species produced the enzyme. On the other hand, 99% of coagulase-positive staphylococci produced TNase and only 18% of the coagulase-negative staphylococci produced the enzyme. The amount of TNase produced by streptococci and bacilli was significantly lower than that produced by coagulase-positive staphylococci. The pH profile of the streptococci and Bacillus TNases was similar to that of the staphylococcal TNase; each enzyme exhibited a minor peak at pH 7.0 and a broad major peak ranging from pH 8.5 to 10. The nuclease produced by coagulase-positive Staphylococcus was more heat stable than the nucleases produced by Streptococcus and Bacillus; there was little loss in activity of the staphylococcal enzyme after 60 min at 100 degrees C, whereas 50% of the activity of the streptococcal and Bacillus nucleases was destroyed in 40-60 min and 60-80 min, respectively.

A modified pork plasma agar for the enumeration of Staphylococcus aureus in foods.

The coagulase reaction of Staphylococcus aureus on the PPSA (pork plasma for S. aureus) agar of Devoyod et al. was found to be fibrinogen-deficient. By including bovine fibrinogen (BFG) in the medium, the fibrin halos around S. aureus colonies became more distinct, preparations of pork plasma previously unacceptable for inclusion in the original PPSA agar were performing well, and the amount of pork plasma required in PPSA agar could be reduced by nearly 90%. In the modified medium, designated PPF (pork plasma fibrinogen) agar, the agar base (Baird-Parker agar without egg yolk) was unchanged. After surface plating, the base was covered with 8 mL of a modified overpour agar: 2.5% pork plasma, 0.38% BFG, and 0.0015% soy trypsin inhibitor in 0.7% Bacto agar. Most S. aureus strains could be enumerated after 24 h of incubation at 35 degrees C; the others required 44 h. Without soy trypsin inhibitor, a number of strains showed considerable fibrinolysis between 24 and 44 h of growth; this activity was neutralized by the inhibitor. The S. aureus counts of 27 food samples on PPF agar were essentially the same as the confirmed S. aureus counts obtained by the Baird-Parker method.

Inhibitory effect of cocoa powder on the growth of a variety of bacteria in different media.

The inhibitory effect of cocoa powder on 102 organisms belonging to 13 genera was determined. All organisms tested were inhibited by 5% cocoa. Shigella, Staphylococcus, Micrococcus, and Bacillus were the most sensitive. The degree of inhibition depended on the organism, temperature of incubation, and the medium in which the cocoa powder was suspended. Of six media tested, lactose broth and nutrient broth were the most inhibitory, while non-fat dry milk was the least inhibitory. Supplementing NB with tryptone or casein reduced the toxicity of cocoa.

Effect of non-fat dry milk on recovery of staphylococcal thermonuclease from foods.

Modification of the method of Tatini et al. (1976) by addition of non-fat dry milk (NFDM) to food samples and subsequent acid precipitation at pH 3.8 enhanced the recovery of staphylococcal thermonuclease (TNase) from most of 37 foods tested. The modified TNase assay method allowed detection of 10 ng (0.002 units) of the enzyme per gram of each of the following foods: ground beef, boiled egg products, whey powder, fruit-containing yogurt, dressings and spreads, potato and egg salads, and pastas, all of which gave false-negative results without NFDM.