Ultrastructural changes in Blastomyces dermatitidis after in vitro exposure to lidocaine

Electron microscopic examination of yeasts of Blastomyces dermatitidis, exposed in vitro to concentrations of lidocaine that occur when the drug is used for topical anesthesia, showed that lidocaine rapidly damaged intracellular structures. The extent of damage was dependent on the concentration of drug and length of exposure. The observed ultrastructural changes were very similar to those reported for other drugs that directly damage membranes. This relationship suggests that the antifungal effect of lidocaine is the result of direct membrane damage.     

Nitrogen fixation efficiency,natural15N abundance,and morphology of mesquite (Prosopis glandulosa) root nodules

Summary The relative nitrogen fixation efficiencies (RE 1-[H₂ evolved÷C₂H₂ reduced]·100) of four mesquite (Prosopis glandulosa var.torreyana) rhizobia (Strains WR 1001, WR 1002, L5, L9) and a cowpea rhizobia (Strain 176A32) on mesquite were evaluated in a glasshouse experiment. Plant yield, shoot N accumulation, and the natural¹⁵N abundance (¹⁵N) of nodule tissue were determined. Strain WR 1002 failed to nodulate mesquite and strain L5 produced ineffective nodules. Among the three effective strains (WR 1001, L9, 176A32) the cowpea strain (176A32) and strain L9 had significantly higher RE than strain WR 1001. Differences in RE, however, were not accompanied by significantly higher plant yield and shoot N accumulation. The difference in¹⁵N abundance between foliar tissue and nodules (nodules minus leaves) was 0.47 ¹⁵N for the ineffective L5 nodules, while for the effective WR 1001, L9, and 176A32 nodules, respectively, this difference was 8.35, 7.81, and 8.35 ¹⁵N. This indicates a similar relationship between N₂-fixing effectiveness and natural¹⁵N enrichment of nodules that was previously observed in soybeans (Glycine max, L. Merr.). Strains WR 1001 and L9 produced elongate, indeterminate nodules typical for mesquite. The ineffective L5 nodules had few infected cells and an abundance of cortical amyloplasts. Mesquite nodules produced by the cowpea strain were spherical and were somewhat more similar in internal morphology to determinate nodules typical of cowpea than indeterminate nodules normally associated with mesquite.     

Stereoselective action of acylated crude papain toward mandelic and atrolactic hydrazides

Acylated crude papain has been shown to exert stereoselective behavior toward racemic hydrazides devoid of an amino acid residue, namely, (RS)-mandelic and (RS)-atrolactic hydrazides. These hydrazides functioned as nucleophiles to yield N¹,N²-diacylhydrazines. Several achiral acylating agents for the enzyme were chosen, including Z-glycine, BOC-glycine, AOC-glycine, and hippuric acid. With the exception of hippuric acid as the acylating agent, the reaction product, in every instance for these achiral hydrazides, consisted of an excess of the (+)-N¹,N²-diacylhydrazine. The relative rates of product formation for the mandelic hydrazides were considerably greater than for corresponding reactions with racemic atrolactic hydrazide. When chiral Z-l-alanine was employed to acylate crude papain, the stereoselective action was most pronounced, with the formation of a mixture of diastereoisomers consisting of 73% N¹-(Z-l-alanyl)-N²-[(R)-mandelyl]hydrazine. The relative reactivities for the electrophiles was Z-l-alanine ? Z-glycine ? hippuric acid ? AOC-glycine > BOC-glycine. The hydrazides of (R)-, (S)-mandelic, and (RS)-atrolactic acids were prepared by conversion of the corresponding acids to their esters by means of a catalytic dehydrating agent and subsequent treatment with a methanolic solution of hydrazine.     

Hydrazides of N-blocked amino acids as sole substrates with unique difunctional behavior under papain catalysis

Hydrazides of five N-acylamino acids have been used alone as substrates for papain catalysis to yield N¹,N²-diacylhydrazines. With the exception of N-(benzyloxycarbonyl)(Z)- -alanine hydrazide, they were very effective as both acylating agents of the enzyme and nucleophiles in attacking the enzyme-substrate intermediate. Although Z- -alanine hydrazide was a minimal acylating agent, it was a satisfactory nucleophile. The most favorable reaction involved Z- -alanine hydrazide in producing N¹,N²-bis(Z- -alanyl)hydrazine. When Z- -alanine hydrazide was the substrate, this same chiral diacylhydrazine was formed along with meso N¹-(Z- -alanyl)-N²-(Z- -alanyl)hydrazine. For the acylation step, the enzyme displayed powerful, essentially stereospecific, bias toward the enantiomer. Once the thioester intermediate was formed, little preference was detected for attack by the enantiomers as nucleophiles. The most direct procedure for synthesis of substrates was conversion of Z-amino acids to their esters by means of dry HCl in an absolute alcohol. Treatment with hydrazine produced the hydrazides in excellent yield.     

Liquid preservation of highly purified human granulocytes

Highly purified human granulocytes isolated from continuous flow centrifugation leukapheresis concentrates by counterflow centrifugation-elutriation were stored at 4 °C in concentrations of 6 × 10⁶ to 1 × 10⁷ granulocytes per milliliter for up to 14 days. The in vitro physiological function assays of phagocytosis, oxygen consumption associated with phagocytosis, bacterial growth inhibition, chemotaxis, and five enzyme analyses indicated good storage survival for up to 4 days. Stored granulocytes separated from other blood cells have greater storage stability than granulocytes stored as leukapheresis concentrates. After 14 days of storage a small percentage of granulocytes still maintained all physiological functions, with the exception of chemotaxis. Of the five enzymes assayed, only the enzyme activity of leucine aminopeptidase decreased significantly by the 14th day of storage. The storage stability of each physiological function assayed decreased as follows: bacterial growth inhibition (most stable), phagocytosis, oxygen consumption, and chemotaxis (least stable).     

The genome of Zea mays,its organization and homology to related grasses

The pattern of genome organization of Zea mays has been analyzed, and the relationship of maize to possible progenitor species assessed by DNADNA hybridization. Reassociation of 470 and 1,350 bp fragments of maize DNA to various C₀t values demonstrates that the genome is composed of 3 major kinetic classes: highly repetitive, mid-repetitive, and unique. Mini-C₀t curves of the repetitive sequences at short fragment length indicate that the highly repetitive sequence class is 20% of the genome and is present at an average reiteration frequency of 800,000 copies; the mid-repetitive sequence class is 40% of the genome and is present at an average reiteration frequency of 1,000 copies. Thermal denaturation studies show that the highly repetitive sequences are 12% divergent and mid-repetitive sequences are 6% divergent. Most of the genome is organized in two interspersion patterns. One, approximately one-third of the genome, is composed of unique sequences of average length 2,100 bp interspersed with mid-repetitive sequences; the other, also one-third of the genome, is mid-repetitive sequences interspersed with highly repetitive sequences. The repetitive sequences are 500 to 1,000 bp by electron microscopic measurement. The remaining third of the genome is unique sequences farther than 5,000 bp from a palindromic or repetitive sequence. Hybridization of maize DNA from Midwestern Dent to popcorn and related grasses indicates that both the unique and repetitive sequence elements have diverged. Teosinte and popcorn are approximately equally divergent from Midwestern Dent whereas Tripsacum is much more divergent. The divergence times calculated from the depression of T_m in heterologous duplexes indicate that the divergence within Zea mays and between maize and near relatives is at least an order of magnitude greater than expected. This high degree of divergence may reflect the pressures of domestication of maize.     

Non-random X-chromosome expression early in mouse development

We have used a sensitive electrophoretic technique for estimating the activity, or ratio, of two allozymes of the X-chromosome-linked enzyme phosphoglycerate kinase (PGK-1), in order to investigate the randomness of X-chromosome expression in the derivatives of the three primary cell lineages of the early mouse conceptus. The maternally derived Pgk-1 allele is preferentially expressed in the derivatives of the primitive endoderm and trophectoderm lineages at 6 1/2 days post coitum in Pgk-1^a/Pgk-1^b heterozygous conceptuses, and in the one informative 5 1/2-day heterozygous conceptus analysed. This evidence for preferential expression of the maternally derived X chromosome (X^m), so soon after the time of X-chromosome inactivation, favors the possibility that the preferential expression of X^m is a consequence of primary non-random X-chromosome inactivation, rather than a secondary selection phenomenon. The majority of embryos analysed at 4 1/2 and 5 1/2 days pc produced only a single PGK-1 band, corresponding to the allozyme produced by the Pgk-1 allele on X^m, although 50% of these embryos should have been heterozygous females. Possible explanations are discussed.     

Adenine deaminase of a eukaryotic animal cell,Crithidia fasciculata

Adenine deaminase (adenine aminohydrolase, EC 3.5.4.2) has been found to occur in Crithidia fasciculata with a specific activity higher than that of the same enzymes of bacteria and yeasts. It is remarkable for its stability to heat, exhibiting no appreciable loss of activity after 60 min at 55 °C. It occurs in the soluble portion of cell extracts but can be released into the suspending medium by osmotic and/or cold shock.     

Selective solubilization and purification of cell-surface concanavalin A-binding glycoproteins from Novikoff hepatocellular carcinoma cells

Novikoff hepatocellular carcinoma cells possess cell-surface glycoproteins that bind the lectin, concanavalin A. A subset of Con A-binding plasma membrane glycoproteins was solubilized by addition of n-butanol to a suspension of Novikoff cells. Glycoproteins solubilized into the n-butanol-saturated aqueous phase of the two-phase mixture were purified by sequential chromatography on DEAE-cellulose and Sepharose-conjugated concanavalin A. Glycoproteins specifically bound to the Sepharose-conjugated Con A exhibited apparent M_r = 72,000 to 125,000. The plasma membrane localization of these components was inferred by their isolation from cells surface labeled with NaIO₄/ NaB³H₄. A xenoantiserum, raised against glycoproteins specifically bound to Sepharose-conjugated concanavalin A was employed to identify reactive components in nonionic detergent extracts of Novikoff tumor cells or rat hepatocytes surface labeled using lactoperoxidase-catalyzed iodination (¹²⁵I). Major reactive peptides in extracts of Novikoff cells exhibited apparent M_r = 74,000, 82, 000, 110,000, and 135,000, while those in extracts of hepatocytes possessed apparent M_r = 98,000 and 105,000. The reactivity of the antiserum with extracts of ¹²⁵I-labeled Novikoff cells was abolished by absorption of the antiserum with hepatocytes, indicating that the qualitative differences observed may result from structural modification of one or more cell-surface glycoproteins, rather than the expression of new or inappropriate glycoproteins. This antiserum will provide a useful probe to investigate alterations in the expression or structure of glycoproteins that occur as a consequence of malignant transformation or adaptation of malignant cells to growth in the ascitic form.