Improved two-dimensional electrophoretic mapping of Japanese human hair proteins; application to curved and straight Japanese human hairs; and protein identification by MALDI MS and MS/MS quadrupole time-of-flight mass spectrometry.

OBJECTIVES: To evaluate improved protein extraction and two-dimensional electrophoresis (2DE) separation methods with Japanese reference human hair (JRH); to determine whether fibre curvature is related to protein composition in curly and straight Japanese women's human hair (JHH) samples; and to identify proteins from JRH 2DE maps and expression differences between curly and straight JHH. METHODS: Hair keratin and keratin-associated proteins (KAPs) were extracted intact with dithiothreitol or tris(2-carboxyethyl) phosphine from JRH or from curved or straight JHH. Extracted proteins were isoelectric-focused on first-dimensional pH gradient gel strips, then separated by molecular weight on laboratory-made, second-dimension, large format gels. The software compared protein abundance between duplicate 2DE gels of curved and straight JHH. Thirty-eight proteins from a JRH 2DE gel were enzyme-cleaved for MALDI-TOF-MS analysis to determine peptide composition, and where possible, de novo sequencing gave peptide sequence data. An in-house human hair protein database incorporating ninety-eight annotated protein sequences assisted MS analysis. RESULTS: 2DE gels of tris(2-carboxyethyl) phosphine-extracted JRH improved keratin and KAP resolution and number compared to those of dithiothreitol-extracted JRH and published commercially made second-dimensional gels. Silver-stained 2DE gels of the straight or curved JHH sets were remarkably similar. Over-staining to reveal basic proteins caused poor resolution of the major acidic protein classes. Software comparisons of fifty-nine resolved proteins revealed two were significantly different in abundance between curved and straight hairs but in insufficient amounts for MS analysis. MS identified twelve proteins from a JRH CBBG-stained 2DE gel: six type II keratins, three type I keratins and three high sulphur proteins. A further eight were potential conformational isoforms and isoelectric variants of the identified proteins bringing the total to twenty identified or partially identified proteins. CONCLUSION: Root-end human hair extraction with tris(2-carboxyethyl) phosphine improves protein resolution and visualizes more proteins on large format 2DE gels. The two minor protein differences between duplicate straight or curved JHH 2DE gels were unlikely to change fibre structure from straight to curved hair. MS results confirmed that multiple isoforms exist of various hair proteins. Low sequence coverage prevented distinction between members in rows of homologous protein spots of similar molecular weight.

OBJECTIFS: évaluer l'amélioration de l'extraction de protéines et les méthodes de séparation bidimensionnelle par électrophorèse (2DE) avec des cheveux humains de référence Japonais (JRH), déterminer si la courbure de la fibre est liée à la composition protéique dans les échantillons de cheveux humains des Japonaises (JHH) bouclés et raides et identifier les protéines issues des cartes JRH 2DE et les différences d'expression entre les JHH bouclés et raides. MÉTHODES: la kératine des cheveux et les protéines associées à la kératine (KAP) ont été extraites intactes avec du dithiothréitol ou du tris (2-carboxyéthyl) phosphine des JRH ou des JHH bouclés ou raides. Les protéines extraites ont subi une focalisation isoélectrique sur des bandes de gel à gradient de pH unidimensionnelles, puis ont été séparées par poids moléculaire sur des gels bidimensionnels de grand format, fabriqués en laboratoire. Le logiciel a comparé l'abondance des protéines entre les deux duplicatas de gels 2DE des JHH bouclés et raides. Trente-huit protéines provenant d'un gel 2DE JRH ont été clivés par enzyme pour l'analyse MALDI-TOF-MS afin de déterminer la composition des peptides, et dans la mesure du possible, un séquençage de novo a donné des données de séquence des peptides. Une base de données interne des protéines capillaires humaines incorporant 98 séquences de protéines annotées a aidé l'analyse MS. RÉSULTATS: les gels 2DE de JRH extraits par le tris (2-carboxyéthyl) ont amélioré la résolution et le nombre de la kératine et du KAP par rapport à ceux du JRH extrait par le dithiothréitol et des gels bidimensionnels fabriqués commercialement. Les gels 2DE à coloration argentée des ensembles de JHH raides ou bouclés étaient remarquablement similaires. La sur-coloration pour révéler les protéines de base a provoqué une mauvaise résolution des principales classes de protéines acides. Les comparaisons logicielles des 59 protéines résolues ont révélé que deux présentaient une différence significative d'abondance entre les cheveux bouclés et raides, mais en quantités insuffisantes pour une analyse MS. La MS a identifié douze protéines provenant d'un gel 2DE coloré CBBG JRH : six kératines de type II, trois kératines de type I et trois protéines à forte teneur en soufre. Huit autres étaient des isoformes conformationnels potentiels et des variantes isoélectriques des protéines identifiées, ramenant le total à 20 protéines identifiées ou partiellement identifiées. CONCLUSION: l'extraction des cheveux humains à la racine avec du tris (2-carboxyéthyl) phosphine améliore la résolution des protéines et permet de visualiser plus de protéines sur les gels 2DE grand format. Les deux différences de protéines mineures entre les duplicatas des gels 2DE JHH raides ou bouclés étaient peu susceptibles de changer la structure des fibres de cheveux raides à bouclés. Les résultats de la MS ont confirmé qu'il existe plusieurs isoformes de diverses protéines capillaires. Une faible couverture de séquence a empêché la distinction entre les protéines homologues de poids moléculaire similaire.

Characterisation of white and black merino wools: a proteomics study.

Wool is an important agricultural commodity with merino wool being rated alongside the finest quality fibres, which include the goat fibres Mohair and Cashmere. Although pigmented wool merinos have become extremely rare, the market for this wool is increasing. In Portugal, there are two merino breeds: white and black, descendants of animals originally bred on the Iberian Peninsula. These breeds have the potential to assist in our understanding of how protein expression relates to wool traits of importance to the textile industry. Herein, we study the characteristics and protein expression profiles of wool from ewes of the Portuguese black and white merino (n=15). Both breeds had very similar results for fibre diameter (25 µm) and curvature (105 to 111°/mm). Significant between-breed differences were found in the two types of keratin-associated proteins (KAPs): high-sulphur proteins (HSPs) and high-glycine-tyrosine proteins (HGTPs). The expression of HSPs, KAP2-3 and KAP2-4, decreased expression in the pigmented animals, whereas KAP13-1 was found in higher amounts. Likewise, the expression of the ultra-high-sulphur proteins, KAP4-3 and KAP4-7-like, was reduced in black sheep to half the levels of the white wools, whereas the HGTPs, KAP6, KAP6-1, KAP6-2 and KAP16-2, were more abundant in black sheep. These results suggest structural differences between the black and white merino wool, because of differences among some KAPs. These differences have important implications for the textile industry.

Ovine keratome: identification, localisation and genomic organisation of keratin and keratin-associated proteins.

Wool is composed primarily of proteins belonging to the keratin family. These include the keratins and keratin-associated proteins (KAPs) that are responsible for the structural and mechanical properties of wool fibre. Although all human keratin and KAP genes have been annotated, many of their ovine counterparts remain unknown and even less is known about their genomic organisation. The aim of this study was to use a combinatory approach including comprehensive cDNA and de novo genomic sequencing to identify ovine keratin and KAP genes and their genomic organisation and to validate the keratins and KAPs involved in wool production using ovine expressed sequence tag (EST) libraries and proteomics. The number of genes and their genomic organisation are generally conserved between sheep, cattle and human, despite some unique features in the sheep. Validation by protein mass spectrometry identified multiple keratins (types I and II), epithelial keratins and KAPs. However, 15 EST-derived genes, including one type II keratin and 14 KAPs, were identified in the sheep genome that were not present in the NCBI gene set, providing a significant increase in the number of keratin genes mapped on the sheep genome.

Animal board invited review: advances in proteomics for animal and food sciences.

Animal production and health (APH) is an important sector in the world economy, representing a large proportion of the budget of all member states in the European Union and in other continents. APH is a highly competitive sector with a strong emphasis on innovation and, albeit with country to country variations, on scientific research. Proteomics (the study of all proteins present in a given tissue or fluid - i.e. the proteome) has an enormous potential when applied to APH. Nevertheless, for a variety of reasons and in contrast to disciplines such as plant sciences or human biomedicine, such potential is only now being tapped. To counter such limited usage, 6 years ago we created a consortium dedicated to the applications of Proteomics to APH, specifically in the form of a Cooperation in Science and Technology (COST) Action, termed FA1002--Proteomics in Farm Animals: www.cost-faproteomics.org. In 4 years, the consortium quickly enlarged to a total of 31 countries in Europe, as well as Israel, Argentina, Australia and New Zealand. This article has a triple purpose. First, we aim to provide clear examples on the applications and benefits of the use of proteomics in all aspects related to APH. Second, we provide insights and possibilities on the new trends and objectives for APH proteomics applications and technologies for the years to come. Finally, we provide an overview and balance of the major activities and accomplishments of the COST Action on Farm Animal Proteomics. These include activities such as the organization of seminars, workshops and major scientific conferences, organization of summer schools, financing Short-Term Scientific Missions (STSMs) and the generation of scientific literature. Overall, the Action has attained all of the proposed objectives and has made considerable difference by putting proteomics on the global map for animal and veterinary researchers in general and by contributing significantly to reduce the East-West and North-South gaps existing in the European farm animal research. Future activities of significance in the field of scientific research, involving members of the action, as well as others, will likely be established in the future.

Stability of athlete blood passport parameters during air freight.

INTRODUCTION: Fluctuations in ambient temperature and pressure, as well as physical jostling, may affect the stability of whole blood samples transported by air freight. The aim of this study was to characterize the stability of key blood variables during air freight and to investigate whether vibration or reduced pressure alone affected results. METHODS: Over a 72-h interval, we evaluated the stability of full blood count indices (plus reticulocytes) in tubes that were air-freighted a total of 2, 10 and 28 h. We also examined the impact of 24 h of reduced atmospheric pressure (750 hpa or approximately 2500 m.a.s.l) and vibration (5 Hz). Samples were measured on a Sysmex XT-2000i instrument. RESULTS: The two key variables in the context of antidoping (haemoglobin concentration, reticulocytes) remained stable over a 72-h period regardless of the duration of air freight. Atmospheric pressure and vibration had no discernible effect. CONCLUSION: Whole blood samples stored in NanoCool devices can be relied upon to remain stable for at least 72 h despite interim air freight.

Stability of athlete passport parameters during extended storage.

INTRODUCTION: Extended intervals between sample collection and analyses render athlete's whole-blood specimens collected in the field for antidoping purposes susceptible to storage degradation. The aim of this study was to characterize the stability of key blood variables under different storage durations and temperatures. METHODS: We evaluated stability of full blood count indices (plus reticulocytes) in individual tubes left undisturbed during 36, 48, 72, 96, 120, 144 and 168 h of storage at approximately 4, 6 and 12 °C. Samples were measured on a Sysmex XT-2000i instrument. RESULTS: The two key variables in the context of antidoping (haemoglobin concentration, reticulocytes) were stable for at least 168 h, except under 12 °C (stable 48 h only). Volume-dependent variables changed in a predictable manner that enabled a nomogram to be generated to predict original values provided storage duration and temperature were known. CONCLUSION: Key blood results can be relied upon for at least 7 days if storage temperature is kept at 4-6 °C.

Modelling the growth of Clostridium perfringens during the cooling of bulk meat.

A dynamic predictive model was developed to describe the effects of temperature, pH and NaCl concentration on the growth of Clostridium perfringens type A. The model for the specific growth rate was based on 81 growth curves generated in our laboratory or obtained from the publicly available ComBase database. Growth curves obtained during cooling were fitted with the dynamic model of Baranyi and Roberts. This made it possible to determine the parameter value reflecting the physiological state of C. perfringens after heating profiles typically applied to bulk meat. The model with the obtained parameters provided a good description of growth of C. perfringens in 24 heating/cooling curves generated specifically for this work (various non-isothermal treatments with a range of combinations of pH and NaCl concentration), and also for existing literature data. The dynamic model was implemented in Perfringens Predictor, a web-based application that can be accessed free of charge via www.combase.cc. It is anticipated that the use of this model and Perfringens Predictor will contribute to a reduction in the food poisoning incidence associated with C. perfringens.

The microbiological quality of hot water-washed broccoli florets and cut green beans.

AIMS: To determine the effect of hot water washing on the microbiological quality of cut broccoli florets and trimmed green beans. METHODS AND RESULTS: Broccoli florets and trimmed beans were washed for 90 s in tap water at either 20 degrees C or 52 degrees C and stored at 7 and 10 degrees C. The numbers of naturally occurring aerobic mesophilic organisms, Pseudomonas spp., Enterobacteriaceae, yeast and moulds and lactobacilli or lactic acid bacteria were enumerated at intervals for up to 2 weeks. The ability of Listeria monocytogenes, Bacillus cereus and Escherichia coli O157:H7 inoculated onto the tissue post heat treatment to survive or grow was also measured to mimic the effect of postprocess contamination. Using a hot wash treatment improved the initial appearance of the vegetables and resulted in a small, but significant, reduction in populations of all groups of endogenous flora measured. The number of yeast and moulds on the vegetables washed at 52 degrees C remained below the levels observed on the 20 degrees C washed vegetables throughout the observation period, but Pseudomonas spp., lactobacilli and Enterobacteriaceae were better able to grow on the hot-washed vegetables such that the counts at the end of storage were greater on hot-washed than ambient-washed vegetables. All three of the pathogens tested were better able to grow on hot-washed broccoli and beans than on equivalent product washed at 20 degrees C. CONCLUSIONS: Hot water washing can be used to control enzymic browning or yeast and moulds growth but it can also allow more rapid and extensive growth by pathogens and spoilage organisms. SIGNIFICANCE AND IMPACT OF THE STUDY: Reduced protection against growth by pathogens means that the hot wash treatment of vegetables should be used with caution and requires careful assessment of risk.

Use of a novel method to characterize the response of spores of non-proteolytic Clostridium botulinum types B, E and F to a wide range of germinants and conditions.

AIMS: Limited information is available on the germination triggers for spores of non-proteolytic Clostridium botulinum. An automated system was used to study the effect of a large number of potential germinants, of temperature and pH, and aerobic and anaerobic conditions, on germination of spores of non-proteolytic Cl. botulinum types B, E and F. METHODS AND RESULTS: A Bioscreen analyser was used to measure germination by decrease in optical density. Results were confirmed by phase-contrast light microscopy. Spores of strains producing type B, E and F toxin gave similar results. Optimum germination occurred in L-alanine/L-lactate, L-cysteine/L-lactate and L-serine/L-lactate (50 mmol l(-1) of each). A further 12 combinations of factors induced germination. Sodium bicarbonate, sodium thioglycollate and heat shock each enhanced germination, but were not essential. Germination was similar in aerobic and anaerobic conditions. The optimum pH range was 5.5-8.0, germination occurred at 1-40 degrees C, but not at 50 degrees C, and was optimal at 20-25 degrees C. CONCLUSIONS: The automated system enabled a systematic study of germination requirements, and provided an insight into germination in spores of non-proteolytic Cl. botulinum. SIGNIFICANCE AND IMPACT OF THE STUDY: The results extend understanding of germination of non-proteolytic Cl. botulinum spores, and provide a basis for improving detection of viable spores.

Application of proteomics for determining protein markers for wool quality traits.

The technique of two-dimensional electrophoresis (2-DE) has been under investigation for its usefulness in identifying protein markers for wool quality traits in sheep. However, before this could be achieved, unique problems relating to the detection and quantitation of wool proteins needed to be overcome so that 2-DE protein maps could be examined using computational programs like Melanie II. Four protein staining regimes were examined. Colloidal Coomassie Blue G-250 was found to be superior to Coomassie Blue R-250 and gave satisfactory staining of all protein classes. Silver staining detects minor strings of keratinous proteins, but unfortunately it negatively stains intermediate filament proteins, the major high sulphur proteins (HSPs) and the high glycine tyrosine proteins and the latter two classes can only be seen by overstaining the background of the gel. In contrast, labeling reduced keratins with [14C]iodoacetamide, followed by autoradiography detection, results in a protein map with low background and all protein spots stained positively. 2-DE has been used to obtain wool protein maps of Lincoln/Merino chimeric sheep to examine wool originating from two genotypes grown with different crimp frequencies within the same fleece. Between fleece, variations have also been examined. Work to date suggests that several major HSPs may be associated with the fibre curvature trait known as crimp frequency. From matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectral mapping, one of these proteins has been identified as being from the B2A family from the HSP class.

Structural features of a peptide corresponding to human kappa-casein residues 84-101 by 1H-nuclear magnetic resonance spectroscopy.

The peptide Val-Arg-Arg-Pro-Asn-Leu-His-Pro-Ser-Phe-Ile-Ala-Ile-Pro-Pro- Lys-Lys-Ile, which corresponds to residues 84-101 of human kappa-casein, has been synthesized and its conformation preferences determined by 1H-nuclear magnetic resonance spectroscopy in dimethyl sulphoxide. The peptide adopted a largely extended chain conformation in solution and there was evidence for the presence of a beta-turn involving residues Pro87-His90 of human kappa-casein. The presence of a turn in this position would make the physiologically significant Arg85 residue of human kappa-casein (which is equivalent to Arg97 in bovine kappa-casein) unavailable for interaction with Asp249 of bovine chymosin, and may partly explain why human kappa-casein is hydrolysed more slowly than its bovine counterpart by bovine chymosin.

Micelle stability: kappa-casein structure and function.

The stability of the casein micelle is dependent on the presence of kappa-casein (CN) on the surface of the micelle where it functions as an interface between the hydrophobic caseins of the micelle interior and the aqueous environment. kappa-Casein is also involved in thiol-catalyzed disulfide interchange reactions with the whey proteins during heat treatments and, after rennet cleavage, in the facilitation of micelle coagulation. These functions of kappa-CN are regulated by the three-dimensional structure of the protein on the micelle surface. The usual means of determining structure are not available for kappa-CN because this protein is strongly self-associating and has never been crystallized. Instead, algorithms were used to predict selected secondary structures and circular dichroism spectroscopy on kappa-CN and the macropeptide released by chymosin. Three peptides were synthesized to cover the chymosin-sensitive site (His98-Lys111), the region in the macropeptide that could be helical (Pro130-Ile153), and the region between. Nuclear magnetic resonance spectroscopy showed that the peptide His98-Lys111 was probably a beta-strand with tight turns at each end. This hypothesis was confirmed by a study of the molecular dynamics showing that the C variant of kappa-CN interacted less strongly with chymosin; consequently, the slow renneting time of milk that contains this protein was explainable. Both circular dichroism and nuclear magnetic resonance indicated that the peptide Pro130-Ile153 was probably helical under normal physiological conditions. A preliminary study using nuclear magnetic resonance showed that the intervening peptide had no discernible secondary structure. Consequently, most of the beta-sheet structure of kappa-CN is likely in the para-kappa-CN region.

Solution conformation of a peptide corresponding to bovine kappa-casein B residues 130-153 by circular dichroism spectroscopy and 1H-nuclear magnetic resonance spectroscopy.

The peptide Pro130-Thr-Ser-Thr-Pro-Thr-Ile-Glu-Ala-Val-Glu140- Ser-Thr-Val-Ala-Thr-Leu-GLu-Ala-Ser-Pro150-Glu-Val-Ile, which corresponds to residues 130-150 of kappa-casein B, was synthesized and the conformation of the peptide in solution investigated by circular dichroism (CD) spectroscopy, structure prediction algorithms and 1H-nuclear magnetic resonance spectroscopy. In a solution containing the structure-enhancing solvent trifluoroethanol the CD spectrum was typical of a peptide in the alpha-helical conformation and nuclear magnetic resonance showed that the amino acids between Ile136 and Ser149 (kappa-casein numbering) were predominantly in the alpha-helical conformation but that Pro130 to Thr135 and Pro150 to Ile153 were not. In addition, Thr133-Pro134 and Ser-149-Pro150 were primarily in the trans conformation, the residues from Thr131 to Thr135 were in unordered structures and the residues from Glu151 to Ile153 were in an extended conformation. Residues Glu137 to Glu140 and Thr145 to Ala148 also displayed some 3(10)-helix character. When the peptide was dissolved in 10 mM-cetyltrimethylammonium chloride solution at pH 6, the CD spectra indicated that the proportion of helical structure was comparable to that of the peptide in trifluoroethanol solution (400 ml/l), whereas when the peptide was dissolved in buffer alone in 10 mM-SDS solution, the CD spectra were consistent with a low helical content. Acidification of these solutions to pH 2.85 resulted in a slight increase in the helical content of the peptide in buffer and more markedly in buffer containing SDS. When the peptide was in 5 mM-CaCl2 solution at neutral pH, the CD spectrum indicated that some ordered structure was present. Taken together these results indicate that the ionizable residues Glu137, Glu140, Glu147 and Glu151 could be important in determining the stability of the putative helix. The structure predictions found that the sequence from Glu137 to Pro150 would be more likely to be in a helical than any other conformation in the intact bovine protein, but that pig, sheep and goat kappa-caseins did not give a prediction of a strongly helical region in this part of the molecule.

Preclinical antitumor activity of bizelesin in mice.

Bizelesin (U-77779, NSC 615291), a synthetic analogue of the cytotoxic antibiotic CC-1065, is a bifunctional alkylating agent that produces DNA interstrand cross-links. Bizelesin was evaluated for antitumor activity against a broad spectrum of syngeneic murine tumors and human tumor xenografts in mice. Systemic drug administration produced >6.7 log10 cell kill against i.p. implanted P388 and L1210 leukemias and 80% tumor-free survivors against s.c. implanted L1210. Against i.p. implanted B16 melanoma, i.p. drug administration produced a 158%; increase in life span with 25% tumor-free survivors, whereas i.v. drug administration produced only a 67% increase in life span with no tumor-free survivors. More than 1.0 log10 cell kill was observed at low microgram/kg doses in several human tumor models representing diverse histiotypes (CAKI-1 renal, LX-1 lung, HT-29 colon, LOX IMVI and UACC-62 melanomas, and MX-1 mammary). Less than 1.0 log10 cell kill was exhibited in other tumor models (Lewis lung, colon 38, pancreatic 02, MCF7 mammary, and SK-MEL-3 melanoma). Bizelesin was optimally active when administered i.v. Although antitumor activity was independent of the schedule of administration, greater total doses were tolerated on the more prolonged schedules in any given experiment. Therapeutic doses of bizelesin did not produce delayed deaths, which had previously been observed for the parent compound CC-1065. However, recovery of lost weight was not attained until 16-30 days posttherapy. Bizelesin was as active against murine leukemia sublines resistant to cisplatin, melphalan, and 1,3-bis-(2-chloroethyl)-1-nitrosourea as against the parental line but was totally inactive against a doxorubicin-resistant subline. The complete cross-resistance of the doxorubicin-resistant subline to bizelesin suggests that bizelesin may be a substrate for the efflux pump that causes multidrug resistance. Due to its breadth of antitumor activity, potency, unique mechanism of action, and lack of cross-resistance with other alkylating agents, bizelesin was selected for development in clinical trials by the National Cancer Institute and the Upjohn Company. Toxicological studies and pharmaceutical development have been completed, and clinical trials are planned to start in the summer of 1996.

Antitumor 2,3-dihydro-2-(aryl)-4(1H)-quinazolinone derivatives. Interactions with tubulin.

A series of derivatives of 2,3-dihydro-2-(aryl)-4(1H)-quinazolinone (DHQZ) with known antitumor activity was re-evaluated in the National Cancer Institute cancer cell line screen. Analysis by the COMPARE algorithm suggested that their cytotoxicity derived from interactions with tubulin. Significant inhibition of tubulin assembly and of the binding of radiolabeled colchicine to tubulin was demonstrated with several of the compounds, particularly NSC 145669, 175635, and 175636. The DHQZ derivatives are structurally analogous to a number of antimitotic agents, flavonols and derivatives of 2-styrylquinazolin-4(3H)-one and of 2-phenyl-4-quinolone. Structure-activity analogies between these agents, the combretastatins, and the colchicinoids were analyzed and summarized.

Antiproliferative effect in vitro and antitumor activity in vivo of brefeldin A.

PURPOSE: An empiric in vitro screen of human tumor cell lines found brefeldin A inhibited the growth of immortalized human cell lines, with particular sensitivity to brefeldin in a series of immortalized melanoma cell lines and nonimmortalized prostate carcinoma explants. Brefeldin A alters the morphology and function of the Golgi apparatus, endosomal, and trans-Golgi compartments in different cell types. The studies presented here sought to obtain evidence of in vivo antitumor activity by brefeldin A. METHODS: Antiproliferative activity was studied in prostate carcinoma cells in vitro using cell counts, protein, and viable stains. Activity was also studied in vivo against subcutaneous and subrenal capsule melanoma models. RESULTS: Protracted exposures in vitro (between 24 and 72 hours) are necessary to cause persistent growth inhibition of immortalized PC3 prostate carcinoma cells. In human melanoma athymic mouse xenografts, brefeldin A showed antitumor activity in vivo when given 16 to 64 mg/kg/injection intraperitoneally q 7 h x 2, daily for 5 days. Activity was also observed in the intraperitoneal LOX IMVI (65%-100% increase in life span, with 17%-50% day 60 survivors); early-stage subcutaneous LOX IMVI and SK-MEL-5 (86%-100% growth inhibition), and subrenal capsule SK-MEL-5 and M19-MEL models. CONCLUSIONS: Brefeldin A possesses noteworthy antitumor activity in vivo and antiproliferative effects in vitro in certain cell types. Strategies to allow protracted exposure of tumor cells to brefeldin A while preserving a therapeutic index are needed to assess the clinical potential of brefeldin A.

Synthesis, antitumor activity, and chemical properties of silaplatin and related platinum (II) and platinum (IV) complexes derived from beta-silyl amines.

Platinum (II) and platinum (IV) coordination complexes derived from beta-silyl-substituted amines were prepared. The solubility of selected complexes in water and physiological saline was measured, and the effect of the beta-silicon on the reactivity of the complex in aqueous solution was determined by HPLC. The stabilities of selected silyl complexes were compared to the carbon analogues. The cyclic complexes 2a ("silaplatin") and its Pt(IV) analogue, 2b, were very active against L1210 leukemia in vivo. Both the platinum (II) complex 2a and the platinum (IV) complex 2b produced a significant number of cures over the dose range 10-40 mg/kg. The platinum (II) complex 2a, silaplatin, was very active in vivo against an L1210 leukemia subline that was resistant to cisplatin; 2a was also active, when given ip, against ic implanted L1210. The cyclobutanedicarboxylic acid complex 3c was synthesized; this complex was active against both cisplatin sensitive and resistant L1210 leukemia but was less potent than the analogous dichloro compound 2a. The acyclic platinum (II) and platinum (IV) complexes 1a,b were synthesized and unexpectedly found to be inactive in vivo against L1210 leukemia. More lipophilic silaplatin analogues were prepared--Pt(II) complex 2c and Pt(IV) complex 2d have one additional methylene carbon compared to 2a,b, whereas Pt(II) complex 2e and Pt(IV) complex 2f have two additional methylene carbons. Cyclization of the alkyl groups attached to the silicon gave the spiro bicyclic Pt(II) complexes 10a and 11a and the Pt(IV) complexes 10b and 11b.

Restrained molecular dynamics study of the interaction between bovine kappa-casein peptide 98-111 and bovine chymosin and porcine pepsin.

The cleavage of bovine kappa-casein at the Phe105-Met106 bond by chymosin or pepsin is the first stage in casein micelle coagulation and casein digestion. The nature of the interaction of the peptide His98-Pro-His-Pro-His-Leu-Ser-Phe105-Met-Ala-Ile-Pro-Pro- Lys111 with chymosin and porcine pepsin was investigated using molecular modelling and energy minimization techniques. This study verified and extended a proposed model that electrostatic binding (involving His98, His100, His102 and Lys111 or Lys112) at either end of the active site cleft of chymosin is important for the positioning of residues 103-108 in the cleft. The peptide conformation remained unchanged in going from solution to binding into the active site cleft, with the exception that optimum binding of substrate to chymosin required the isomerization of the His98-Pro99 peptide bond from the trans to the cis conformation. The study also identified an acidic region in porcine pepsin that is in a position to form strong electrostatic interactions with the histidines at the N-terminus of the peptide.

Efficacy of the quinocarmycins KW2152 and DX-52-1 against human melanoma lines growing in culture kand in mice.

Quinocarmycin monicitrate (KW2152) and its analogue, DX-52-1, demonstrated specificity for melanomas in the National Cancer Institute in vitro human tumor cell line drug screen. In contrast to most cell lines, a 50% reduction in tumor cell burden (as measured protein) at the end of a 48-h drug incubation was produced in five of eight melanoma lines by KW2152 concentrations (LC50s) ranging from 0.49 to 10.93 microM and by DX-52-1 concentrations ranging from 0.71 to 7.33 microM. Using the COMPARE algorithm, the patterns of differential cytotoxicity for both agents at the LC50 level of effect most closely resembled those for actinomycin D, mithramycin, and Adriamycin. In in vivo studies, both KW2152 (40 mg/kg/day) and DX-52-1 (90 mg/kg/day) caused partial and complete regressions of staged s.c.-implanted LOX IMVI melanoma xenografts following i.p. administration on days 5, 9, and 13 and produced tumor growth delays of 231 and 181%, respectively (P < 0.001). Activity was augmented by more prolonged therapy. Statistically significant growth inhibition of SK-MEL-2, UACC-62, UACC-257, and M14, but not SK-MEL-5 and MALME-3M, melanoma xenografts also was observed following every fourth or seventh day i.p. treatments. Based on these findings, DX-52-1 has been selected by the National Cancer Institute for development to clinical trial especially against melanomas. This agent represents one of the first to be selected for preclinical development based on disease-panel specificity discovered in the National Cancer Institute cancer drug screen.