The Combined Perls-Hematoxylin-Eosin Stain

To provide a routine check for the presence of ferric iron in sections, Perls' method was combined with hematoxylin and eosin as follows. Deparaffinized sections of formalin-fixed tissues are stained in Perls' reagent (1:1 2%, w/v, of potassium ferrocyanide in distilled water and 2%, v/v, concentrated HCl in distilled water) for 20 min. After brief rinsing in distilled water stain sections in Mayer's hemalum, wash in tap water for 5 min, counterstain in 0.5% (w/v) eosin B in 50% ethyl alcohol for 15 sec. Rinse in tap water, dehydrate and mount as usual.     

Rapid fluorometric detection for completeness in solid phase coupling reactions

The fluorescamine test for the rapid detection of trace amounts of uncoupled products from solid phase peptide synthesis is reported. This novel procedure can detect much smaller amounts of incomplete coupling with greater simplicity than has previously been possible. Since the test is carried out under mild conditions certain side reactions are circumvented. The fluorophor-resins are easily viewed under long wave ultraviolet light, are stable at room temperature, and may be used for quantitative evaluation.     

Inhibition of prolyl hydroxylase activity by bradykinin analogs containing a prolyl-like residue

A number of substituted bradykinin analogs were prepared in which the proline in position 3 was replaced by analogs of proline. All of the bradykinin analogs, with the exception of l-azetidine-2-carboxyl³-bradykinin showed significant ability to inhibit prolyl hydroxylase activity. Addition of an l-glutamyl residue to the amino terminus of 3,4-dehydro-l-prolyl³-bradykinin and trans-4-hydroxy-l-prolyl³-bradykinin resulted in competitive inhibitors of increased effectiveness with K_i, values approximately 10^?4m. One of the peptides, l-3,4-dehydro-l-prolyl³-bradykinin, appeared to serve as a substrate for prolyl hydroxylase.     

Analysis of ethylene‐induced gene regulation during carposporogenesis in the red seaweed Grateloupia imbricata (Rhodophyta)

Ethylene favors carposporogenesis in the red seaweed Grateloupia imbricata. Analyses of cystocarp development in vitro in thalli treated with ethylene suggest an interconnection between polyamine and ethylene biosynthesis pathways. Yet, little is known about molecular mechanisms underlying carposporogenesis. Here, we used droplet digital PCR to analyze genes encoding enzymes related to polyamine (Spermidine [Spd] synthase) and ethylene (ACC synthase) synthesis; a pivotal compound of both pathways (S‐adenosyl methionine synthase, SAMS); the gene that encodes amine oxidase, which is involved in polyamine degradation, and a candidate gene involved in seaweed reproduction (ornithine decarboxylase, ODC). In addition, we analyzed genes encoding proteins related to stress and reactive oxygen species, ascorbate peroxidase (APX), cytochrome P450 and WD 40. We characterized gene expression in fertilized and fertile thalli from G. imbricata that were exposed to ethylene for 15 min at two time points after treatment (1 and 7 d). The differential gene expression of SAMS, Spd synthase, ACC synthase, and cytochrome P450 was related to disclosure and development of cystocarps in fertilized thalli that transitioned from having no visible cystocarps at 1 d to developing cystocarps at 7 d. Likewise, cytochrome P450 was associated with cystocarp disclosure and maturation. In addition, amine oxidase and APX were involved in fine‐tuning polyamine and reactive oxygen species during carposporogenesis, respectively, whereas WD 40 did so in relation to ethylene signaling. Expression of the candidate gene ODC was increased when cystocarps were not visible (fertilized thalli, 1d), as previously described. This analysis suggests developmental stage‐specific roles for these genes during carposporogenesis.     

Solution structure of contryphan-R, a naturally occurring disulfide-bridged octapeptide containing D-tryptophan: comparison with protein loops.

Contryphan-R is a disulfide-constrained octapeptide containing a D-tryptophan that was isolated recently from venom of the cone shell Conus radiatus. The polypeptide is present in two forms in solution due to cis-trans isomerization at hydroxyproline 3. The solution structure of the major form of this unusual polypeptide, determined from NMR data, consists of a well-defined fold containing a non-hydrogen-bonded chain reversal from Gly1 to Glu5, which includes a cis-hydroxyproline and a D-Trp, and a type I beta-turn from Glu5 to Cys8. The presence of a putative salt bridge between the Glu5 carboxyl group and the N-terminal ammonium group is investigated by using various solvation models during energy minimization and is compared with the results of a pH titration. A comparison of the structure of contryphan-R with other cyclic peptide structures highlights some of the key structural determinants of these peptides and suggests that the contryphan-R fold could be exploited as a scaffold onto which unrelated protein binding surfaces could be grafted. Comparison with small disulfide-bridged loops in larger proteins shows that contryphan-R is similar to a commonly occurring loop structure found in proteins.     

Molecular detection of bacterial indicators in cosmetic/pharmaceuticals and raw materials

PCR assays were compared with standard microbiological methods for rapid detection of the United States Pharmacopoeia (USP) bacterial indicators in artificially contaminated samples of raw materials and cosmetic/pharmaceutical products. DNA primers containing the specific sequences of the uidA gene of the β-glucuronidase enzyme for Escherichia coli, the membrane lipoprotein gene oprL for Pseudomonas aeruginosa, and the 16S ribosomal gene for Staphylococcus aureus were used for detection in the PCR reaction. Contaminated samples were incubated for 24 h at 35°C. After incubation in broth media with and without 4% Tween 20, samples were streaked on selective growth media. After 5–6 days, all microbial indicators were morphologically and biochemically identified using standard methods while detection and identification by the PCR-based assays was completed within 27–30 h. Rapid PCR detection of E. coli, S. aureus, and P. aeruginosa will allow a faster quality evaluation and release of raw materials and cosmetic/pharmaceutical products sensitive to microbial contamination. Received 21 June 1998/ Accepted in revised form 11 January 1999     

Purification and characterization of an acid proteinase from mesophilic Mucor sp. solid-state cultures.

The fourth-day extract of a solid-state culture of the mesophilic Mucor sp. (M-105) strain showed a high milk-clotting activity and a clotting/proteolytic activity ratio similar to that of commercial preparations from microbial origin used in cheese manufacture. After ultrafiltration of the crude extract, the milk-clotting proteinase was purified in two steps: ion-exchange followed by size-exclusion chromatography. Enzyme homogeneity was assessed by HPLC, SDS-PAGE and N-terminal residue determination. A pI value of 4.21 was obtained and a molecular weight of 33 kDa was calculated from size-exclusion chromatography and SDS-PAGE data. The optimum pH for proteolytic activity towards dimethylcasein was in the 3.0-3.5 range. The proteinase retained 26 and 13% of its proteolytic activity after a 30-min incubation period, at pH 5.0 and 50 and 60 degrees C, respectively. This evidenced a lower heat stability than that of the thermophilic enzymes currently used in the cheese industry and also than that of bovine chymosin. The enzyme was fully inhibited by pepstatin A and no effect was observed with PMSF, p-CMPS or EDTA. The N-terminal amino acid sequence: GTGTVPVTDDGNLNEYYXTVTVGXP was compared with those from other fungal enzymes.