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.     

Real-time ultrasonic imaging of the peripheral arteries: Technique, normal anatomy, and pathology

Real-time ultrasonic echo imaging of peripheral arteries promises to facilitate the management of selected patients with peripheral and extracranial arterial disease. This report outlines the technique of imaging the carotid system and portions of the arteries that supply the lower extremities. It also discusses the normal and pathologic anatomy of these arteries.     

Reduction of REM sleep by a tryptophan-free amino acid diet.

The acute administration of a tryptophan-free amino acid diet to rats has previously been reported to produce a marked reduction in brain serotonin concentrations. The present study examined the effects of such a diet on electroencephalographic sleep measures. There was a decrease in REM sleep and a small increase in nonREM sleep, with no change in total sleep time. In view of these and other observations, the hypothesis that the serotonergic system plays an important role in the maintenance of nonREM sleep should be carefully reevaluated.     

Vimentin is transiently co-localized with and phosphorylated by cyclic GMP-dependent protein kinase in formyl-peptide-stimulated neutrophils.

The effects of cGMP-dependent protein kinase (G-kinase), a major cellular receptor of cGMP, were investigated in activated human neutrophils. Immunocytochemistry demonstrated that G-kinase translocated from a diffuse localization in the cytoplasm to the cytoskeleton and nucleus after stimulation with N-formyl-methionyl-leucyl-phenylalanine (fMLP), and transiently co-localized with the intermediate filament protein, vimentin. During this time period, the most remarkable co-localization of G-kinase and vimentin was observed between 1-2.5 min stimulation with fMLP. At that time co-localization of G-kinase and vimentin was predominantly confined to filaments which extended from regions adjacent to the nucleus into the uropod. Distinctive localization for only G-kinase was observed at the microtubule organizing center and euchromatin of the nucleus. The filamentous staining pattern for G-kinase and vimentin was enhanced in the presence of 8-Br-cGMP. Coincident with co-localization of G-kinase and vimentin in adherent neutrophils was a transient increase in cGMP levels and an increase in the phosphorylation of vimentin in fMLP-stimulated cells. The increase in cGMP levels was dependent upon cell adherence, was enhanced by preincubating neutrophils with L-arginine (the precursor for nitric oxide synthesis), and attenuated with the nitric oxide synthase inhibitor, NG-monomethyl-L-arginine. Phosphorylation of vimentin in the fMLP-stimulated neutrophil was observed in the presence or absence of exogenous cGMP, although in the presence of low concentrations of 8-Br-cGMP a more rapid phosphorylation of vimentin was observed that correlated with the enhanced co-localization of G-kinase and vimentin. Phosphorylation of vimentin was not observed in non-activated cells treated with 8-Br-cGMP, suggesting that phosphorylation only occurs when G-kinase is co-localized with vimentin. The presence of the protein kinase C inhibitors, staurosporine or H-7, did not inhibit vimentin phosphorylation during fMLP stimulation, while 8-Br-cGMP enhanced phosphorylation in fMLP-treated cells. This suggests that neither protein kinase C nor cAMP-dependent protein kinase catalyze the phosphorylation of vimentin in neutrophils activated by fMLP. These results indicate that vimentin and G-kinase are co-localized in neutrophils and that vimentin is phosphorylated by G-kinase in response to the co-localization of the two proteins. A model for the targeting of G-kinase and vimentin is presented which hypothesizes that the transient redistribution of G-kinase may regulate neutrophil activation.     

Perception of the auxin signal at the plasma membrane of tobacco mesophyll protoplasts

Auxin-induced variations of transmembrane potential difference have been shown to be a useful tool for analyzing hormone sensitivity in tobacco protoplasts. Using this technique, we demonstrated that protoplasts derived from wild-type, an auxin-resistant mutant and Agrobacterium-rhizogenes transformed plants differed widely in the sensitivity of their electrical response to naphthalene acetic acid. We have used different antibodies, raised to auxin binding proteins (ABP) from maize coleoptiles, or to the axr1 gene product (ABP1), to test whether changes in auxin sensitivity can be correlated with the presence of tobacco proteins immunologically related to this ABP. Titrations indicated that 0.4 nM anti-ABP IgG inhibited 50% of the auxin-specific response of wild-type protoplasts, whereas 0.04 nM or 4 nM anti-ABP IgG were necessary to inhibit the response of mutant and transformed protoplasts, respectively, to the same extent. On wild-type protoplasts, blocking part of the immunoreactive sites with anti-ABP antibodies resulted in a decrease in auxin sensitivity of the electrical response (0.4 nM anti-ABP IgG inducing a 10–fold decrease), whereas addition of maize ABP increased this auxin sensitivity (1 pM ABP1 raised the sensitivity more than 1000–fold). The results obtained suggest that the auxin sensitivity detected by our assay system correlates with the amount of tobacco proteins immunologically related to the axr¹ gene product from maize. A hypothesis accounting for the presence of these proteins at the external surface of tobacco protoplasts and for the effects of hetero-logous maize ABP on auxin sensitivity is proposed.     

Human herpesvirus 7 is a constitutive inhabitant of adult human saliva.

We report the frequent isolation of human herpesvirus 7 from the saliva of healthy adults. Virus isolates recovered from different individuals exhibited minimal restriction enzyme polymorphism, which was mostly confined to heterogeneous (het) sequences in the genome. DNAs of isolates recovered from the same individual over a period of several months showed the same characteristic het fragments, indicating the stability of the het sequences upon virus replication and shedding in vivo. In contrast to the results of previous reports, human herpesvirus 6, the causative agent of roseola infantum, could not be isolated from the saliva specimens, raising questions regarding oral transmission of human herpesvirus 6 and human herpesvirus 7 to young children.     

Both ankyrin and band 4.1 are required to restrict the rotational mobility of band 3 in the human erythrocyte membrane.

A population of band 3 proteins in the human erythrocyte membrane is known to have restricted rotational mobility due to interaction with cytoskeletal proteins. We have further investigated the cause of this restriction by measuring the effects on band 3 rotational mobility of rebinding ankyrin and band 4.1 to ghosts stripped of these proteins as well as spectrin and actin. Rebinding either ankyrin or 4.1 alone has no detectable effect on band 3 mobility. Rebinding both these proteins together does, however, reimpose a restriction on band 3 rotation. The effect on band 3 rotational mobility of rebinding ankyrin and 4.1 are similar irrespective of whether or not band 4.2 is removed from the membrane. We suggest that ankyrin and 4.1 together promote the formation of slowly rotating clusters of band 3.     

Characterization of murine cell lines from Diethylstilbestrol-Induced uterine endometrial Adenocarcinomas

Neonatal treatment with estrogens is associated with development of uterine adenocarcinomas in CD-1 mice. Treatment with the synthetic estrogen diethylstilbestrol (DES) on Days 1 to 5 after birth results in 90% incidence of these hormonedependent lesions in 18-mo.-old mice. Three cell lines were established from these DES-associated tumors. Each of these cell lines exhibited morphologic and ultrastructural characteristics of transformed epithelial cells, including an increased nuclear:ytoplasmic ratio, enlarged and irregular nuclei with multiple nucleoli and areas of chromatin condensation, positive staining for cytokeratin, desmosomes, and microvilli. After subcutaneous injection into nude mice, all three cell lines formed solid tumors within 4 wk. Although the primary uterine tumors and tumor transplants in nude mice had been shown to be estrogen-dependent and estrogen-receptor positive, neither the monolayer growth nor the tumorigenicity of any of the three cell lines in this study was enhanced by or dependent on estrogen. Estrogen receptor levels were low in early and intermediate passage cells. Allele-specific oligonucleotide hybridization analysis of PCR-amplified cell line DNA revealed no point mutations in the 12th, 13th, or 61st codons of the K-ras or H-ras protooncogenes. Southern analysis revealed no changes in genomic organization of the putative tumor suppressor gene DCC, but demonstrated a three-to four-fold amplification of the c-myc gene in one cell line. Expression of c-myc RNA was concomitantly increased in the same cell line. These three transformed cell lines represent the end point in the process of hormone-associated tumorigenesis and as such should prove useful in investigating the molecular changes and the mechanisms involved in hormonal carcinogenesis.     

EVIDENCE FOR POLLINATION ECOTYPES IN THE YELLOW-FRINGED ORCHID,PLATANTHERA CILIARIS

Platanthera ciliaris is a butterfly-pollinated, terrestrial orchid with a loose terminal raceme of 10–50 orange flowers, characterized by a long nectariferous spur. In the southeastern United States, P. ciliaris occurs in the Appalachian mountains and coastal-plain physiographic provinces, but it is found rarely in the intervening Piedmont. In 1983 and 1984, detailed observations of two populations within these disjunct areas revealed that the butterfly species that serve as the primary pollinators differ sharply. In the mountains, Papilio troilus (spicebush swallowtail) was the most frequent and effective visitor, whereas in the coastal plain, P. palamedes (palamedes swallowtail) was the predominant pollinator. Proboscis lengths of P. troilus (mean = 23.3 mm) were significantly shorter than those of P. palamedes (mean = 28.7 mm). Floral characters, most notably spur length, also differed significantly between mountain (mean = 23.8 mm) and coastal-plain (mean = 25.6 mm) plants. In both 1983 and 1984, levels of pollinator service, as assessed by rates of removal and insertion of pollinia, were higher in the mountains (0.81 and 0.86) than in the coastal plain (0.63 and 0.67). In addition, fruit-set was significantly greater in mountain (83.9% in 1983, 86.5% in 1984) than in coastal-plain (63.8% in 1983, 65.5% in 1984) populations. We hypothesize that selection pressure exerted through pollinator proboscis lengths has resulted in pollination ecotypes of P. ciliaris. The short-spurred mountain plants appear to be in equilibrium with their short-tongued butterfly pollinators, receiving high levels of effective pollination and achieving high levels of fruit-set. Coastal-plain plants produce flowers with longer spurs which, nevertheless, are shorter than optimal for insuring that very long-tongued butterflies make contact with their pollinia. Thus, effective pollinator service and fruit-set are reduced. Correlation analyses in 1984 showed a positive relationship between spur length and fruit-set only in the coastal plain. The presence of long-tongued nectar thieves in the coastal plain may also contribute to lower effective pollinator service and fruit-set. Results of reciprocal-transplant studies attempting to determine the genetic basis of floral characters, including spur length, were inconclusive but suggest that differences between mountain and coastal-plain plants are not due solely to phenotypic plasticity.