Differentiation of Two Types of Basophils In The Adenohypophysis of The Rat and The Mouse

Pituitaries are fixed for 24 hr. in Bouin's fluid containing 0.5% trichloroacetic acid instead of 5% acetic acid, or in a mixture of 9 parts SUSA and 1 part saturated aqueous solution of picric acid. They are embedded in paraffin and horizontal sections are cut at 3-4 μ. The staining method consists of 3 phases: (a) immersion in aldehyde-fuchsin for the selective demonstration of the beta cell granules, (b) staining of the nuclei with Ehrlich's hematoxylin and (c) a rapid one-step counterstain with light green and orange G dissolved in a phosphotungstic-acetic acid mixture for the differentiation of the acidophilic and the delta cell granules.     

Selective Staining of Mast Cell Granules with Chrysoidin

A simple chrysoidin stain (0.5% aqueous solution, 5-10 min) is selective for mast cell granules; alum-hematoxylin-chrysoidin stains histological features in addition to the granules and periodic acid-Schiff can be used for cytological and histochemical features. Selective chrysoidin staining is probably due to strong basophilia of mast cell granules.     

Azophloxine in Two-Dye and Four-Dye Staining Combination

Tissues are fixed in either 10% formalin or Lavdow-sky's mixture. After the tissues are sectioned and mounted, they are stained in hematoxylin, then counterstained for 2 minutes in 0.1% aqueous azophloxine to which 4 drops of acetic acid have been added to each 100 ml. of stain. Sections are then rinsed in 0.2% acetic acid and dehydrated. Azophloxine GA can be used also in a tetrachrome method. Sections are stained in Harris' hematoxylin, washed, and placed in 0.2% acidified aqueous azophloxine for 2 minutes. They are then rinsed in 0.2% acetic acid, stained 1 minute in an aqueous mixture of 4% phosphotungstic acid and 2% orange G solution and rinsed again in 0.2% acetic acid. Finally, they are stained in 0.2% light green for 2 minutes, and differentiated in 0.2% acetic acid for 5 minutes. The advantage in using azophloxine is that it is clear and delicate and when used in a constant concentration, does not overstain if the recommended procedure is followed.     

Azophloxine in Two-Dye and Four-Dye Staining Combination

Tissues are fixed in either 10% formalin or Lavdow-sky's mixture. After the tissues are sectioned and mounted, they are stained in hematoxylin, then counterstained for 2 minutes in 0.1% aqueous azophloxine to which 4 drops of acetic acid have been added to each 100 ml. of stain. Sections are then rinsed in 0.2% acetic acid and dehydrated. Azophloxine GA can be used also in a tetrachrome method. Sections are stained in Harris' hematoxylin, washed, and placed in 0.2% acidified aqueous azophloxine for 2 minutes. They are then rinsed in 0.2% acetic acid, stained 1 minute in an aqueous mixture of 4% phosphotungstic acid and 2% orange G solution and rinsed again in 0.2% acetic acid. Finally, they are stained in 0.2% light green for 2 minutes, and differentiated in 0.2% acetic acid for 5 minutes. The advantage in using azophloxine is that it is clear and delicate and when used in a constant concentration, does not overstain if the recommended procedure is followed.     

Indirect sulphonation of mucopolysaccharides and glycogen in tissue sections with the use of bisulphite or dithionite after periodic acid oxidation.

Tissue mucopolysaccharides and glycogen can be indirectly sulphated after being oxidized by periodic acid and treated with sodium bisulphite or dithionite in aqueous solution. The sulphated sites are darkly stained by Roluidine Blue and realted dyes at pH less than 1.0. The background is very pale or colourless. The stained sections resist extraction with 1% hydrochloric acid for 48 hr, but can be extracted by 5% ammonium hydroxide in ethanol in 1 hr. Other oxidizing agents cannot be substituted for periodic acid.     

Cell-free in vitro reduction of carboxylates to aldehydes: With crude enzyme preparations to a key pharmaceutical building block

The scarcity of practical methods for aldehyde synthesis in chemistry necessitates the development of mild, selective procedures. Carboxylic acid reductases catalyze aldehyde formation from stable carboxylic acid precursors in an aqueous solution. Carboxylic acid reductases were employed to catalyze aldehyde formation in a cell-free system with activation energy and reducing equivalents provided through auxiliary proteins for ATP and NADPH recycling. In situ product removal was used to suppress over-reduction due to background enzyme activities, and an N-protected 4-formyl-piperidine pharma synthon was prepared in 61% isolated yield. This is the first report of preparative aldehyde synthesis with carboxylic acid reductases employing crude, commercially available enzyme preparations.     

Chitosan-LiOH-urea aqueous solution—a novel water-based system for chitosan processing

A solution of partially N-deacetylated chitosan in aqueous lithium hydroxide (LiOH)/urea was prepared successfully through a freeze-thawing process and the dissolution behavior was studied. The results indicated that chitosan can directly dissolve in LiOH/urea aqueous solution. LiOH mainly contributed to the breakage of intramolecular and intermolecular hydrogen bonds in chitosan. Urea, LiOH, and chitosan formed inclusion compound (IC) with urea as the IC host, and the LiOH-chitosan complex as the guest. Aqueous 4.8 wt % LiOH/8.0 wt % urea was verified to be the optimal solvent for chitosan. The results of rheology and viscosity characterizations revealed that chitosan/4.8 wt % LiOH/8.0 wt % urea aqueous solution was pseudoplastic fluid, and was more stable than the solution of chitosan in acetic acid at ambient temperature.     

Simultaneous determination of benazepril hydrochloride and benazeprilat in plasma by high-performance liquid chromatography/electrospray-mass spectrometry

An analytical method for simultaneous determination of benazepril and its active metabolite, benazeprilat, in human plasma by high-performance liquid chromatography/electrospray-mass spectrometry was developed and validated. Rutaecarpine was selected as the internal standard. The separation was achieved on a C(18) column with acetonitrile and aqueous solution (0.1% formic acid) as mobile phase with a gradient mode. The quantification of target compounds was using a selective ionization recording at m/z 425.5 for benazepril, m/z 397.5 for benzeprilat and m/z 288.3 for rutaecarpine. The correlation coefficients of the calibration curves were better than 0.992 (n = 6), in the range of 6.67-666.67 ng/ml for benazepril and benazeprilat. The inter- and intra-day accuracy, precision, linear range had been investigated in detail. The method can be used to assess the bioavailability and pharmacokinetics of the drug.     

N-(9-acridinyl)-bromoacetamide--a powerful reagent for phase-transfer-catalyzed fluorescence labeling of carboxylic acids for liquid chromatography

Phase-transfer-catalyzed esterification of various carboxylic acids has been carried out on a microanalytical scale with a new reagent, N-(9-acridinyl)-bromoacetamide, in aqueous/organic two-phase systems. The procedure gives esters showing very high fluorescence intensity in acid solution. The large Stokes' shift (greater than 120 nm) means that a low background can be achieved even when using a large emission bandwidth. These derivatives show very good chromatographic behavior on reversed-phase liquid chromatographic columns with the use of aqueous acetonitrile, containing 0.2% of phosphoric acid, as the mobile phase. Detection limits (S/N = 2) as low as 10 fmol can be obtained under optimal conditions.     

A Nuclear Stain for Escherichia Coli And Related Organisms

The dye base of new fuchsin was precipitated by adding potassium hydroxide to the dye solution. The precipitate was filtered out and washed with water. It was then suspended in water, brought into solution and adjusted to a pH of about 5.0 with nitric acid. The staining solution was prepared by adding 0.3 ml. of a 14% aqueous solution of pyrogallol and 0.1 ml. of a 1% aqueous solution of boric acid to 3.0 ml. of the dye solution. Smears of cells were made in water on a slide and allowed to dry before covering with the staining solution which was also permitted to air dry. The smear was then washed in water and mordanted for 5-20 seconds in a 0.1% aqueous solution of mercuric nitrate. After rinsing in water, the smear was air dried. When dry, the slide was placed on a 50° C. warm plate for a few seconds before covering with a very thin film of a 5% aqueous solution of nigrosin which had a pH of about 5.0.     

A Protargol Method for Staining Nerve Fibers in Frozen or Celloidin Sections

Extensive experimentation with protargol staining of neurons in celloidin and frozen sections of organs has resulted in the following technic: Fix tissue in 10% aqueous formalin. Cut celloidin sections IS to 25 μ, frozen sections 25 to 40 μ. Place sections for 24 hours in 50% alcohol to which 1% by volume of NH₄OH has been added. Transfer the sections directly into a 1% aqueous solution of protargol, containing 0.2 to 0.3 g. of electrolytic copper foil which has been coated with a 0.5% solution of celloidin, and allow to stand for 6 to 8 hours at 37° C. Caution: In this and the succeeding step the sections must not be allowed to come in contact with the copper. From aqueous protargol, place the sections for 24 to 48 hours at 37° C. directly into a pyridinated solution of alcoholic protargol (1.0% aqueous solution protargol, 50 ml.; 95% alcohol, 50 ml.; pyridine, 0.5 to 2.0 ml.), containing 0.2 to 0.3 g. of coated copper. Rinse briefly in 50% alcohol and reduce 10 min. in an alkaline hydroquinone reducer (H₃BO₃, 1.4 g.; Na₂SO₃, anhydrous, 2.0 g.; hydroquinone, 0.3 g.; distilled water, 85 cc; acetone, 15 ml.). Wash thoroly in water and tone for 10 min. in 0.2% aqueous gold chloride, acidified with acetic acid. Wash in distilled water and reduce for 1 to 3 min. in 2% aqueous oxalic acid. Quickly rinse in distilled water and treat the sections 3 to 5 min. with 5% aqueous Na₂S₂O₃+5H₂O. Wash in water and stain overnight in Einarson's gallocyanin. Wash thoroly in water and place in 5% aqueous phosphotungstic acid for 30 min. From phosphotungstic acid transfer directly to a dilution (stock solution, 20 ml.; distilled water, 30 ml.) of the following stock staining solution: anilin blue, 0.01 g.; fast green FCF, 0.5 g.; orange G, 2.0 g.; distilled water, 92.0 ml.; glacial acetic acid, 8 ml.) and stain for 1 hour. Differentiate with 70% and 95% alcohol; pass the sections thru butyl alcohol and cedar oil; mount.     

Nucleotide-amino acid interactions and their relation to the genetic code

Summary The apparent dissociation constants of the complexes of AMP with the methyl esters of amino acids in aqueous solution exhibit good correlations with features of the genetic code and with the frequencies of occurrence of amino acid residues in proteins. Thus it is likely that chemically selective nucleotide-amino acid interactions were involved in the processes of chemical evolution that have led to the emergence of the genetic code. Based on these correlations a storage device for the information regarding nucleotide-amino acid interactions is proposed. It involves processes of simultaneous polymerization to polynucleotides and polypeptides.     

A Self-Limiting, Highly Selective Chromium-Hematoxylin Stain

A compound, which is probably a cationic chelate, can be isolated as a dry powder from a hematoxylin-chrome alum lake. In aqueous acid solution this compound is an excellent nuclear stain which is extremely selective, very resistant to acids and alcohols, and self-limiting. Staining time may vary from 20 min to 16 hr without causing significant differences in staining intensity. To prepare the dry stain, dissolve 10 gm of hematoxylin, 10 gm of NaOH and 70 gm of chrome alum in 600 ml of distilled water, boil 20 min, cool and filter, allowing the filtrate to drop into 3.5 liters of absolute alcohol. Filter off the precipitate formed in the alcohol, and air dry it at room temperature. The staining solution is prepared by dissolving 3 gm of the dried precipitate in 100 ml of 3% HCl.     

A Controllable Differential Stain for the Hypophysis by Adaptation of the Mallory Triple Stain

A selective and controllable staining method for the hypophysis has been developed with rat material, using Mallory's triple stain as a basis.

Fix in Zenker neutral formol for 6 hours. Longer fixation is undesirable. Transfer to 30% alcohol plus a few drops of a saturated solution of I₂ in aqueous KI over night. Gradually complete dehydration and clear in cedar oil. Infiltrate with a paraffin mixture (paraffin, rubber-paraffin, bayberry wax and beeswax). Section 3-Sμ. Hydrate to distilled water, placing a few drops of a KI-I₂ solution in the 50% alcohol. Stain in 1% acid fuchsia for 30 minutes. Rinse, and differentiate in a weak NH₄OH solution (one drop 28% NH₄OH to 200 cc. HOH). When differentiation is complete, transfer to a 0.5% phosphomolybdic acid solution for 3 minutes, after first stopping the differentiation with a 0.1% HC1 solution and then rinsing with distilled water. Stain for one hour in a solution of: 1% anilin blue, water soluble, 2% orange 6, and 1% phosphomolybdic acid. Rinse in distilled water plus a few cubic centimeters of the stain. Differentiate in 95% alcohol, transfer to absolute alcohol and clear in a mixture of 30% oil of cedar, 40% oil of thyme, 15% absolute alcohol and 15% xylene. Finally, transfer to xylene and mount.     

Release of bovine serum albumin from a hydrogel-cored biodegradable polymer fiber

We have developed a novel biodegradable, polymeric fiber construct that is coextruded using a wet-spinning process into a core-sheath format with a polysaccharide pre-hydrogel solution as the core fluid and poly(L-lactic acid) (PLLA) as the sheath. The biodegradable, biocompatible fibers were extruded from polymeric emulsions comprised of solutions of various molecular weights of PLLA dissolved in chloroform and containing dispersed, protein-free aqueous phases comprising up to 10% of the emulsion volume. Biologically sensitive agents can be loaded via a dispersed aqueous phase in the polymer, and/or directly into the polysaccharide. We show that this core-sheath fiber format will load a model protein that can be delivered for extended periods in vitro. Bovine serum albumin (BSA) was loaded into the fiber core as a model protein. We have shown that the greater the volume of the protein-free aqueous phase dispersed into the polymeric continuous-phase emulsion, the greater the total release of BSA encapsulated by a core gel comprised of 1% sodium alginate solution. We conclude this fiber format provides a promising vehicle for in vivo delivery of biological molecules. Its biocompatibility and biodegradability also allow for its use as a possible substrate for tissue engineering applications.     

Dimedone as an Aldehyde Blocking Reagent to Facilitate the Histochemical Demonstration of Glycogen

The demonstration of glycogen by the periodic acid-Schiff technique can be clarified by the interposition of a short dimedone blockade after the periodic acid oxidation. This blocks the PAS reaction of the vast majority of materials, but a very much longer blockade is required to abolish the reaction of glycogen. The dimedone-PAS method is valuable in situations where the demonstration of glycogen is otherwise difficult because of the proximity of diastase-fast PAS positive materials. For this purpose dimedone is best used in alcoholic solution (5% in absolute alcohol for about 3 hr at 60°C), since an aqueous solution permits diffusion of the aldehydes produced from the oxidation of glycogen. A saturated aqueous solution, or a 5% solution in 80% alcohol, is much more rapid in its blocking action, however, and may be more satisfactory when dimedone is used simply as an aldehyde blocking reagent.     

Ion-pair extraction of bile acids with Lipidex gel

A new method for the extraction of bile acids from aqueous solutions, urine, plasma, and bile is described. A buffered solution of decyltrimethylammonium bromide is added to the sample to give a 0.03 m concentration of the counter-ion. The mixture is passed through a bed of Lipidex 1000, which is then washed with the buffered solution of counter-ion followed by water. The decyltrimethylammonium salts of bile acids are sorbed by the Lipidex and are eluted with methanol. Recoveries of unconjugated, taurine- and glycine-conjugated, sulfated, and glucuronidated bile acids are close to 100%. Unconjugated bile acids can also be quantitatively extracted from aqueous solutions and urine after acidification with acetic acid.     

Synthesis and bio-imaging application of highly luminescent mercaptosuccinic acid-coated CdTe nanocrystals

Here we present a facile one-pot method to prepare high-quality CdTe nanocrystals in aqueous phase. In contrast to the use of oxygen-sensitive NaHTe or H(2)Te as Te source in the current synthetic methods, we employ more stable sodium tellurite as the Te source for preparing highly luminescent CdTe nanocrystals in aqueous solution. By selecting mercaptosuccinic acid (MSA) as capping agent and providing the borate-citrate acid buffering solution, CdTe nanocrystals with high quantum yield (QY >70% at pH range 5.0-8.0) can be conveniently prepared by this method. The influence of parameters such as the pH value of the precursor solution and the molar ratio of Cd(2+) to Na(2)TeO(3) on the QY of CdTe nanocrystals was systematically investigated in our experiments. Under optimal conditions, the QY of CdTe nanocrystals is even high up to 83%. The biological application of luminescent MSA-CdTe to HEK 293 cell imaging was also illustrated.     

Spontaneous Onset of Homochirality in Oligopeptide Chains Generated in the Polymerization of N-Carboxyanhydride Amino Acids in Water

This article is concerned with the spontaneous onset of homochiral oligopeptide sequences. We will show that the polymerization of hydrophobic NCA (N-carboxyanhydride = cyclic anhydride)-amino acid racemates (i.e. tryptophane, leucine and isoleucine) in aqueous solution yields oligopeptides that are characterized by a high degree of homochiral sequences. Furthermore we will show that quartz enhances efficiently the mole fraction of oligopeptides with homochiral sequence by selectively adsorbing the more stereoregular oligopeptides from an aqueous solution of oligo-D,L-leucine. We find in particular that the mole fraction of the adsorbed homochiral 7mers is 17 times larger than the mole fraction calculated for a theoretical, random process. Experimentally the stereoisomer distribution for each oligomer length can be determined by the use of enantio-labeling and LC-MS (Liquid Chromatography-Mass Spectrometry). Furthermore, if we start the polymerization with an enantiomeric excess (e.e.) of 20% of L-leucine (L-amino acid: D-amino acid = 6:4, molar ratio) we observe a chiral amplification in the enantiomeric homochiral oligopeptides. We think that such processes are relevant to the chemical evolution of single handedness.     

Characterization and application of a new aqueous two phase system based on gum acacia (ex.Acacia senegal), a naturally occuring polysaccharide,and polyethylene glycol

Summary Gum acacia has been used with polyethylene glycol to form a two phase aqueous polymeric system. The system followed Newtonian fluid behaviour and the acacia rich phase supported a high concentration of protein (>1.3 mg/ml) in solution. The data on partitioning of various proteins in this system indicates that it can be used for selective separation of a particular protein from a protein mixture.