A simple and rapid method to assay triacylglycerol in cells and tissues

We have developed a reliable, rapid, and economical assay for the quantification of triacylglycerol (TG) in cells and animal tissues. In a few hours, this assay quantifies microgram amounts of TG from tens or even hundreds of samples. The protocol includes an organic extraction to partition TG away from proteins and other hydrophilic molecules found in cells and tissues that may interfere with the colorimetric enzyme-linked TG detection method. In addition, this assay is economical, as no expensive reagents, supplies, or equipment are needed. Another benefit of this assay is that it does not require environmentally unfriendly halogenated solvents.     

Determination of the calcium antagonist SIM6080 and its N- and O-demethylated metabolites in plasma,urine and tissues by high-performance liquid chromatography

A sensitive and versatile high-performance liquid chromatographic assay for the determination of the calcium antagonist SIM6080 and its four N- and O-demethylated metabolites in plasma, urine and tissues has been developed and validated. A two-step extraction procedure is employed followed by reversed-phase liquid chromatographic analysis using ultraviolet detection. An isomer of SIM6080 was used as the internal standard. The analysis of spiked plasma, urine and tissues demonstrated the accuracy and precision of the assay with quantitation limits of 5 ng/ml (plasma and urine) or 100 ng/g (tissues). This assay has been used for urinary recovery and tissue distribution studies, as well as for toxicokinetic protocols.     

Extraction and isolation of glycoproteins and proteoglycans

A number of diverse approaches have been devised to bring about the efficient extraction and isolation of glycoproteins and proteoglycans from biological tissues. Many of these approachs are general procedures that can also be used for the extraction of nonglycoproteins from cells or tissues. Others, such as lectin affinity chromatography, take advantage of specialized structures found only on discrete sub-classes of glycoproteins. Unfortunately, the development of a protocol suitable for the purification of a given glycoprotein of interest remains largely empirical. This article presents a general overview of some of the potential strategies that can be utilized in the development of new isolation procedures and attempts to point out some of the possible pitfalls that may be encountered.     

Carbonate extraction process for the metabolic, isozymic and proteomic profiling of rose-scented geranium (Pelargonium sp.), a hyper-acidic plant

Rose-scented geranium (Pelargonium sp.) is a valuable monoterpene-yielding plant. It has been well characterised phytochemically through the isolation of >270 secondary metabolites, however, there is hardly any biochemical or metabolic information concerning this plant. Initial attempts to investigate its metabolism failed to produce any enzyme activity in the tissue extracts prepared in routine extraction buffers owing to the intrinsic properties of the tissue matrix. It was recognised that cellular hyper-acidity (cell sap pH approximately 3.0) gave rise to very low protein levels in the extracts, thus prohibiting detection of activities of even primary metabolic enzymes that are usually abundantly present in plants. Tissue extraction in Tris solution without pH adjustment (as used for studies involving citrus and banana) led to little or no improvement. Therefore, a novel approach using sodium carbonate solution as an efficient extraction system for enzymes and proteins from the plant was studied. Functionality of the carbonate extraction has been demonstrated through its effectiveness, a several-fold superior performance, in yielding protein, monitoring primary metabolism and secondary metabolic enzymes, and isozymic and polypeptide profiling. The process may also be helpful in the reliable analysis of other acidic plant tissues.     

Detection of the Grapevine Fanleaf Viruses Away from the Period of Vegetation

Best conditions for the detection of AMV (Arabis Mosaic Virus) and GFV (Grapevine Fanleaf Virus) in grapevine tissues at different seasons in the year have been determined. Leaves, rootlets and wood shavings are good virus sources for ELISA. The commonly used nicotine solution can be replaced by a PBS- or Tris-HCl buffer, as extraction medium, provided its molarity is high and it contains polyvinylpyrrolidone. In our conditions, detection of AMV and GFV was possible in leaves from May to October, There was no clear-cut advantage in using extracts prepared either from leaves obtained from the top, middle or bottom parts of a plant. Away from the period of vegetation, the use of wood shavings allowed virus detection, even when cuttings were harvested a few months ago and kept at 6°C.     

Comparative Endocrinology of Steroid Hormones in Vertebrates

Recent advances in biochemical techniques have resulted in considerableprogress in the comparative qualitative analysis of non-mammalianvertebrate steroids. The literature surveyed herein suggeststhat a number of C21, C19, and C18 steroids occur throughoutthe subphylum. although there may be important qualitative differenceseven among closely related forms. The potential importance,from a phylogenetic or a physiological standpoint, of new steroidsrecently characterized in fishes cannot be evaluated at present. Three widely used techniques—tissue extraction or incubationwithout added precursors, tissue incubation with exogenous steroidsubstrates, and the histochemical visualisation of 3ßhydroxysteroiddehydrogenases—are discussed, and their application tothe study of steroidogenic tissues is illustrated. The adrenocorticalhomologue (interrenal), the ovary, and the testis appear tofunction as sources of steroid hormones in those vertebrateswhich have been examined. However, the existence of additionalsteroidogenic tissues, or of tissues which may modify the steroidcomposition of the body fluids in other ways, is also suggested.     

Determination of cocaine, its metabolites, pyrolysis products, and ethanol adducts in postmortem fluids and tissues using Zymark automated solid-phase extraction and gas chromatography-mass spectrometry

Demonstrating the presence or absence of cocaine (COC) and COC-related molecules in postmortem fluids and/or tissues can have serious legal consequences and may help determine the cause of impairment and/or death. We have developed a simple method for the simultaneous determination of COC and the COC metabolites benzoylecgonine (BE), norbenzoylecgonine (NBE), ecgonine methyl ester (EME), ecgonine (E), and norcocaine (NCOC), as well as anhydroecgonine methyl ester (AEME) (a unique byproduct of COC smoking), cocaethylene (a molecule formed by the concurrent use of COC and ethanol) and their related metabolites, anhydroecgonine (AE), norcocaethylene (NCE), and ecgonine ethyl ester (EEE). This method incorporates a Zymark RapidTrace automated solid-phase extraction (SPE) system, gas chromatography/mass spectrometry (GC/MS) and 2,2,3,3,3-pentafluoro-1-propanol (PFP)/pentafluoropropionic anhydride (PFPA) derivatives. The lower limits of detection ranged from 0.78 to 12.5 ng/mL and the linear dynamic range for most analytes was 0.78-3200 ng/mL. The extraction efficiencies were from 26 to 84% with the exception of anhydroecgonine and ecgonine, which were from 1 to 4%. We applied this method to five aviation fatalities. This method has proven to be simple, robust and accurate for the simultaneous determination of COC and 11 COC metabolites in postmortem fluids and tissues.     

A preparation technique for analysis of explosives in plant tissues

A preparation technique for analysis of explosives compounds in plant tissue has been developed. The technique utilizes acetone extraction followed by ultrafiltration clean up. Ultrafiltration removes the large quantities of chlorophyll and polar organics that interfere with UV/Vis detection used in high‐performance liquid chromatography. The procedure was tested with 14 explosives‐related compounds spiked into reed canary grass extracts and method detection limits (MDLs) were calculated. In addition, the overall extraction efficiency for RDX in parrot feather and reed canary grass tissues was determined using ¹⁴C labeled compound. Scientists may find the technique useful for plant analysis at waste sites and for phytoremediation research.     

Micropreparation of single secretory glands from the carnivorous plant Nepenthes

A rapid mechanical micropreparation technique has been developed to isolate multicellular glands, here from Nepenthes pitchers, based on a microdissection platform. The method is an alternative to laser capture dissection because fresh plant tissue can be used directly without previous fixation. Subsequent experiments, such as polymerase chain reaction (PCR)-based detection of an individual gene encoding a thaumatin-like protein and RNA extraction for gene expression analysis, have been successfully added to prove the quality of the prepared biological material. The procedure described is adaptable to a broad range of plant species and should find wide application in the preparation of multicellular glands or other tissues.     

Florigenic Acid from fungal culture

The extraction procedures which have been successfully employed in the preparation of a florigenic principle from the tissues of Xanthium, are applicable to the derivation of an entity of similar activity from Calonectria culture. The Xanthium principle is acedic, with pK_a values characteristic of a carboxylic acid (6). Although definitive chemical comparisons have not been completed, the extraction and solvent partition procedures that have been applicable to the extraction of the active entity from higher plant tissues have yielded florigenic preparations from fungal culture. The chemical principle from higher plant tissue may be the same or similar to the florigenic agent of Calonectria.

The many responses of higher plants to growth regulators produced by micro-organisms are well known. The presence of a flower-producing principle from Calonectria (Fusarium) rigidiuscula parallels closely the pattern exhibited by those fungal species capable of the production of auxin and gibberellins.

     

Spectrophotometric assay for diphenylthiohydantoin in biological materials

A new method for the determination in tissues of DPTH, a potent antagonist of thyroxine, has been developed based upon the strong uv absorbance characeristic of the thiohydantoins. Substances present in tissue homogenates which would interfere with the DPTH assay by virtue of their uv absorbance characteristics have been eliminated by means of a simple two-step extraction procedure. The important parameters affecting the specificity and efficiency of the extraction conditions procedure have been identified; considerable latitude in the choice of extraction conditions allows a high degree of adaptability to individual requirements. The assay protocol currently used in our laboratory involves extraction of the DPTH-containing tissue with xylene followed by the extraction of the xylene with 1.0 n NaOH. After removal of traces of dissolved xylene from the DPTH-containing alkali solution by bubbling with N₂, its DPTH concentration is calculated from the absorbance at 264.5 nm. DPTH concentrations in rat tissues as a function of time after a single oral dose have been determined.     

Diaryl dimers of estradiol and of estrone may be formed as major metabolites by mouse mammary glands

The formation of a novel estrogen metabolite by mammary tissues was investigated. Polar and nonpolar metabolites of endogenous estrogens are formed in liver and other tissues. Polar products such as the catechol estrogens are implicated in tumorigenesis in breast tissue, whereas a nonpolar metabolite, 2-methoxyestradiol, may be protective. Diaryl ether dimers, as a novel form, have been reported as nonpolar products from liver microsomes. We have noted major amounts of nonpolar metabolites in other tissues that were neither 2-methoxyestrogens nor estrogen fatty acid esters. The possible formation of such novel metabolites by breast tissues from adult nulliparous mice with [³H]-labeled estrogens as substrates was considered. Steroids were recovered from media by solid-phase extraction and profiles were obtained from HPLC (acetonitrile:water). Saponification was done with an internal standard of estradiol stearate. Major amounts of nonpolar metabolites were formed in all instances, with one or two principal peaks. Alkaline hydrolysis had no effect on the nonpolar product(s) but released estradiol from its stearate. Strong acid treatment also had no effect as shown by HPLC. Thus, it is suggested that diaryl dimers of estrogens may be formed as major metabolites by mouse mammary glands.     

High-performance liquid chromatographic quantitation of desmosine plus isodesmosine in elastin and whole tissue hydrolysates

Quantitation of desmosine and isodesmosine, the major crosslinks in elastin, has been of interest because of their uniqueness and use as markers of that protein. Accurate measurement of these crosslinks may allow determination of elastin degradation in vivo and elastin content in tissues, obviating lengthy extraction procedures. We have developed a method of quantitating desmosine plus isodesmosine in hydrolysates of tissue and insoluble elastin using high-performance liquid chromatographic separation and absorbance detection that is rapid (21-35 min) and sensitive (accurate linearity from 100 pmol to 5 nmol). This method has been used to quantitate desmosines in elastin from bovine nuchal ligament and lung and in whole aorta from hamster. The ability to completely separate [3H]lysine from desmosine plus isodesmosine allows the method to be used to study incorporation of lysine into crosslinks in elastin.     

Chloroplast biogenesis 88. Protochlorophyllide b occurs in green but not in etiolated plants

It has recently been reported that protochlorophyllide (Pchlide) b is an abundant pigment in barley etioplasts but is rather unstable, as it is rapidly converted to Pchlide a by 7-formyl reductase during pigment extraction with conventional 80% acetone (Reinbothe, S., Pollmann, S., and Reinbothe, C. (2003) J. Biol. Chem. 278, 800-806). It has also been claimed that extraction of barley etioplasts with 100% acetone containing 0.1% diethyl pyrocarbonate prevents the conversion of Pchlide b to Pchlide a and leads to the detection of large amounts of Pchlide b in the isolated etioplasts. In this work the extraction protocol of Reinbothe et al. is compared with the more conventional 80% aqueous acetone extraction method. No Pchlide b was detected either in etiolated barley leaves or isolated barley etioplasts irrespective of the extraction protocol. On the other hands, small amounts of Pchlide b were detected in green barley leaves and isolated chloroplasts, extracted either with 80% acetone or 100% acetone containing 0.1% diethyl pyrocarbonate. It is concluded that the proposed occurrence of a light-harvesting POR-Pchlide-a,b complex in etiolated plant tissues is untenable, and its ensuing consequences and implications, for the greening process, are irrelevant.     

A simple, rapid and quantitative method for preparing Arabidopsis protein extracts for immunoblot analysis

Although Arabidopsis has numerous well documented advantages for genetic and molecular analyses, its small size can be a limitation for biochemical and immunochemical assays requiring protein extraction. We have developed a rapid method to extract total protein from small amounts of Arabidopsis tissue that can be used for quantitative immunoblot analysis. The procedure involves direct extraction of tissue into SDS-containing buffer under conditions permitting immediate protein quantification in the extract, using commercially available kits without prior fractionation. This approach provides maximal extraction and quantitative recovery of total cellular protein, together with accurate evaluation of target protein levels as a proportion of the total. We have examined the utility and sensitivity of the procedure using monoclonal antibodies to phytochromes A and C (phyA and phyC), which are high- and low-abundance members, respectively, of the phytochrome family in Arabidopsis. Both phytochromes could be rapidly and readily quantified in the tissues examined, with phyC being detectable in extracts representing as few as five dark-grown seedlings, two light-grown seedlings, or half a single leaf from 3-week-old adult plants. The data indicate that the procedure may have broad utility for the detection and quantitative analysis of many proteins, including those of low abundance, in a variety of applications in Arabidopsis. In one such application, we used transgenic Arabidopsis phyC-overexpressor seedlings to demonstrate that the procedure can be used to detect transgene-encoded protein early at the segregating T2 generation, thereby offering the capacity for accelerated screening and selection of lines engineered to overexpress target proteins.     

Analysis of sorbitol, galactitol, and myo-inositol in lens and sciatic nerve by high-performance liquid chromatography

Accumulation of sorbitol or galactitol and depletion of myo-inositol in hyperglycemic conditions such as diabetes and galactosemia involve the activity of aldose reductase and are implicated in hyperglycemia-induced complications such as cataract and neuropathy. A high-performance liquid chromatographic method has been developed for the measurement of polyols in the lens and sciatic nerve of rats. This method comprises polyol extraction from tissues, lyophilization of extracts, derivatization of polyols by the reaction with phenylisocyanate, and HPLC of derivatives with detection at 240 nm. The time needed for each run is less than 25 min, which allows the testing of a large number of samples per day. Sensitivity is very high: as low as 0.5 nmol each of sorbitol, galactitol, and myo-inositol in lyophilized extracts of tissues can be determined. The present method offers a reliable tool to evaluate the in vivo activities of aldose reductase and its inhibitors.     

Efficient extraction of proteins from formalin-fixed paraffin-embedded tissues requires higher concentration of tris(hydroxymethyl)aminomethane

Background

Numerous formaldehyde-fixed and paraffin-embedded clinical tissues have been created in the past decades and stored in pathological depositories at hospitals as well as in clinical laboratories worldwide. In addition to the archived tissues, formaldehyde-fixation is also mandatory for preparing proteomics samples from diseased patients or animal models in order to inactivate contagious agents. Protein extraction from formaldehyde-fixed tissues is hampered by the Schiff base formation between the amino groups of proteins and formaldehyde. Although achievement of the highest extraction efficiency of proteins from the formaldehyde-fixed tissues is essential for obtaining maximum proteomics information, no attention has been paid to the concentration dependence of tris(hydroxymethyl)aminomethane on the extraction efficacy. We suspected that the concentration of tris(hydroxymethyl)aminomethane affects the protein extraction efficiency because of its property as a primary amine that reverses the Schiff base formation between the primary amines of proteins and formaldehyde. Thus we pursued optimization of the component and protocol of protein extraction buffer to achieve better extraction efficiency of proteins from formaldehyde-fixed and paraffin-embedded tissues.

Results

In order to simulate protein extraction from diseased tissues we made formaldehyde-fixed and paraffin-embedded samples from mouse liver slices and investigated the protein extraction efficiency and speed by changing the concentration of the protein extraction buffer component tris(hydroxymethyl)aminomethane under various extraction conditions. We find, as expected, that tris(hydroxymethyl)aminomethane significantly affects the performance of protein extraction from the formaldehyde-fixed and paraffin-embedded samples both in the extraction yield and in the extraction speed.

Conclusions

We recommend the concentration of tris(hydroxymethyl)aminomethane in protein extraction buffer to be higher than 300 mM when extraction is conducted for 90 min at 90°C to achieve the most efficient protein extraction in a shorter time. The information will be essential for performing the most efficient protein extraction from formaldehyde-fixed and paraffin-embedded tissue samples for proteomics analysis.     

Complexities and pitfalls in analyzing and interpreting mitochondrial DNA content in human cancer

Mutations in the human mitochondrial genome have been observed in all types of human cancer, indicating that mutations might contribute to tumorigenesis, metastasis, recurrence, or drug response. This possibility is appealing because of the known shift from oxidative metabolism to glycolysis, known as the Warburg effect, that occurs in malignancy. Mitochondrial DNA (mtDNA) mutations could either be maternally inherited and predispose to cancer (germ line mutations) or occur sporadically in the mtDNA of specific tissues (tissue- or tumor-specific somatic mutations) and contribute to the tumor initiation and progression process. High-throughput sequencing technologies now enable comprehensive detection of mtDNA variation in tissues and bodily fluids, with the potential to be used as an early detection tool that may impact the treatment of cancer. Here, we discuss insights into the roles of mtDNA mutations in carcinogenesis, highlighting the complexities involved in the analysis and interpretation of mitochondrial genomic content, technical challenges in studying their contribution to pathogenesis, and the value of mtDNA mutations in developing early detection, diagnosis, prognosis, and therapeutic strategies for cancer.     

Differentiation of neuronal from non-neuronal Substance P

Substance P (SP) originally found as a neuropeptide in capsaicin-sensitive sensory neurons, had more recently been identified in non-neuronal cells, especially under pathological conditions. Neuronal and non-neuronal SP may perform distinct functions. A simple technique to differentiate different SP sources is currently unavailable. Herein, we describe a two-step sequential acetic acid extraction to differentiate SP source. The efficiency of this two-step extraction in differentiating SP in capsaicin-sensitive neurons was verified by using capsaicin as a tool to deplete SP in sensory neurons. Specifically, Balb-c mice were treated with high dose capsaicin (200 mg/kg). Skin was removed two weeks after treatment. In a separate experiment, lung and skin tissues from control animals (untreated) were incubated in-vitro with capsaicin, and sequential acetic acid extraction was performed. Following capsaicin treatment, both in-vivo and in-vitro, SP recovered in first extraction decreased significantly in lung and skin. Lastly, presence of capsaicin solvent (10% methanol and 10% Tween 80) or protease inhibitor cocktail in solution altered SP EIA test, yielding false positive results. These results demonstrated that SP in capsaicin sensitive sensory neurons was extracted in initial extraction of 15 min while non-neuronal SP was present in second extraction. Because SP in non-neuronal tissues may possibly be more important in pathological conditions, this technique could be useful in determining effects of various treatments on neuronal and non-neuronal SP levels and their consequences.