Cathepsin K expression in basal cell carcinoma.

BACKGROUND: Cathepsin K is a cysteine protease with strong collagenolytic and elastolytic properties. Recently, cathepsin K expression in tumour cells of malignant melanoma and in the stromal cells of squamous cell carcinoma of the skin has been reported to play an important role in tumour progression. However, its expression profile in basal cell carcinoma (BCC) has not yet been clarified. OBJECTIVE: The aim of this study is to examine the expression profile of cathepsin K in both the tumour cells and the peritumoural stromal cells of BCC in comparison with its expression in normal skin. METHODS: Fifty consecutive operative cases of BCC, 10 cases of actinic keratosis, 10 cases of Bowen's disease and five normal skin tissues were assessed for cathepsin K expression by immunohistochemical methods. RESULTS: In normal skin, cathepsin K expression was observed in the stratum corneum, mature sebaceous cells and outer root sheath of the hair follicles. Cathepsin K was expressed in the tumour cells of all BCC cases, in which 90% showed diffuse expression (>51% of tumour cells), as well as in the peritumoural stromal cells in all BCC cases. Focal cathepsin K expression was observed in the tumour cells of Bowen's disease (2/10 cases), but not in any of actinic keratosis (0/10 cases). CONCLUSION: Cathepsin K expression may contribute to tumour invasion and peculiar histopathological features, such as fibromucinous stroma around the tumour nests by mediating extracellular matrix degradation in BCC.

Fluid overload deteriorate chylothorax: evaluation in a canine model.

No conservative treatments for chylothorax have yet been established, and surgical ligation of the thoracic duct is required in many cases. In the present study, we investigated the management of body fluid in a canine chylothorax model. Twelve beagle dogs were divided evenly into three groups: A, B, and C. Under general anesthesia, the thoracic duct was cut and opened, and the amount of lymph fluid leakage was measured. Intravenous extracellular fluid infusion was started at 5mL/kg/h for the first 2h, and then between 2 and 4h, the infusion rate was increased to 10 mL/kg/h in group A and to 20mL/kg/h in group B. During the first 2h after cutting the thoracic duct, the mean lymph fluid leakage rates in groups A, B, and C were 0.466, 0.635, and 0.575 mL/kg/h, respectively. The rates of leakage did not differ significantly among the groups. Between 2 and 4h, the mean rates of leakage were 0.750, 1.43, and 0.544mL/kg/h, respectively, being significantly higher in groups A and B than in group C. The amount of lymph fluid ascending the thoracic duct correlates with the amount of intravenous fluid infusion. For the management of chylothorax, it is important to avoid fluid overload.

Various enzyme activities in muscle and other organs of dystrophic mice.

To elucidate the metabolic abnormality of musclar dystrophy, 27 kinds of enzyme activity in various organs of control and dystrophic mice were examined. The organs examined included muscle, bone, heart, testis, uterus, spleen, thymus, submaxillary gland, stomach, pancreas, liver, kidney, brain, and lung. The activities of 14 different aminopeptidases, 5 endopeptidases, 4 glycosidases, phosphatase, esterase, and ribonuclease were measured. Most of the enzyme activities were significantly elevated in muscles and bones of dystrophic mice. These organs were similar in their patterns of enzyme abnormality. Among the 14 kinds of aminopeptidase activity studied, the degree of increased activity was greater for the aminopeptidases (AP):Ala-AP, Leu-AP, Met-AP, Phe-AP, Trp-AP, Gly-Pro-Leu-AP. In addition to aminopeptidases, there were significant increases in activities of chymotrypsinlike enzyme, cathepsin C, cathepsin D, several glycosidases and neutral ribonuclease in the muscles of dystrophic mice. Similarly increased enzyme activity was also observed in organs other than muscle and bone. Furthermore, protein content in most organs was higher in dystrophic mice than in those of control mice. These abnormalities were seen in both males and females. The present results suggest that there are extensive abnormalities in the protein metabolism in dystrophic mice. It seems therefore that the therapeutic approach to muscular dystrophy should be studies not only from the well-known abnormality of intramuscular endopeptidases, but from other aspects as well.

Purification by affinity chromatography using amastatin and properties of aminopeptidase A from pig kidney.

1. Amastatin, a specific inhibitor of aminopeptidase A (L-alpha-aspartyl(L-alpha-glutamyl)-peptide hydrolase, EC 3.4.11.7), was linked to an agarose matrix and by this affinity chromatography aminopeptidase A of pig kidneys was purified as a single protein shown by acrylamide gel electrophoresis. 2. Aminopeptidase A which was purified 710-fold, hydrolyzed only acidic amino acid beta-naphthylamide. The optimum pH and the optimum temperature was 7.5 and 45-50 degrees C, respectively. 3. The molecular weight was approx. 300 000 as determined by Sephadex G-200 gel filtration. 4. The activity of aminopeptidase A was not affected by sulfhydryl agents, S-S dissociating agents and serine proteinase inhibitor, but was inhibited strongly by metal chelating agents, and enhanced by alkaline earth metals. 5. Amastatin inhibited aminopeptidase A in a competitive manner with L-glutamic acid beta-naphthylamide, and the Ki value was calculated to be 2.5 x 10(-7) M. The inhibitory effect of amastatin on aminopeptidase A was not reversed by addition of Ca2+.

Effects of prepartum lipid supplementation on FSH superstimulation and transferable embryo recovery in multiparous beef cows.

The objective of this experiment was to determine the effect of prepartum lipid supplementation on the number and quality of embryos recovered following ovarian super-ovulation in postpartum suckled beef cows. Mature cows (n = 40) were assigned to one of two treatments (lipid versus. no lipid) and supplemented for approximately 40 days prior to calving. Supplements provided to cows were isocaloric and isonitrogenous. The treatment group was fed 1.6 kg hd(-1) per day of whole soybeans (WSB; 19.8% ether extract, and 41.8% crude protein) and the control group received a supplement consisting of 1.8 kg hd(-1) day of a soybean meal and soy-hull combination (SBS; 2.15% EE and 36.81% CP). Cows were synchronized using a GnRH [Cystorelin((R)) 100 microg im]-GnRH-PGF(2alpha) [Lutalyse 25 mg im] protocol. Cows were administered two injections of GnRH seven days apart and PG seven days after the second GnRH injection. Twenty-eight cows (WSB, n = 15; SBS, n = 13) responded to estrus synchronization and were superstimulated. Super-ovulation was initiated on day 8-10 of the synchronized cycle by twice-daily injections of pFSH (Pluset) over four days in decreasing doses using a total of 608.4 IU per cow. Prostaglandin F(2alpha) was administered 96 and 108 h after super-stimulation was initiated with FSH. Days postpartum (WSB = 59 days; SBS = 57 days) at initiation of FSH treatments were similar (P > 0.10) for both treatments. Cows were monitored for estrus activity by the HeatWatch Estrus Detection System. Twenty-seven cows (WSB, n = 15; SBS, n = 12) exhibited estrus after FSH and inseminated at 0, 12, and 24 h after the onset of estrus with 1, 2, and 1 units of semen, respectively. Embryos were recovered and evaluated 7-8 days later. Only cows that responded to FSH and that were inseminated were used for statistical analysis. Data were analyzed using the General Linear Models Procedure of SAS. Body condition scores did not differ (P > 0.10) between treatments when cows were evaluated at the initiation of the experiment, two weeks prior to calving, and at initiation of superovulation with FSH. Estrous cyclicity prior to the initiation of estrus synchronization did not differ (P > 0.10) between treatments. There was no difference (P > 0.10) between treatments in recovery of total embryos (WSB, 14.7 +/- 3.5; SBS, 17.5 +/- 3.0), transferable embryos (WSB, 10.3 +/- 2.5; SBS, 13.6 +/- 2.6), degenerate embryos (WSB, 3.3 +/- 1.1; SBS, 1.6 +/- 1.7) or unfertilized ova (WSB, 1.1 +/- 0.5; SBS, 2.3 +/- 1.2). Cows that were supplemented with whole soybeans prior to parturition failed to produce an increased total number of ova or transferable embryos following super-ovulation.

Recent advances in bovine reproductive endocrinology and physiology and their impact on drug delivery system design for the control of the estrous cycle in cattle.

When methods of drug intervention are being developed to control estrous cycles, a thorough understanding of the endocrine and functional changes together with the reproductive behavior of the animals are essential. This review presents our current knowledge on reproductive endocrinology, physiology and behavior, and the methods of drug intervention to control estrous cycles. It also describes current efforts to develop advanced drug delivery systems that meet the animal scientist's demands to control the estrous cycle in cattle.

Relative deficiency of serine proteinase activities in spleens of aged mice.

We examined the relation of hydrolytic enzymes in spleen to the aging process in mice over a period of 30 months. When the enzymatic activities were expressed as activities per milligrams protein, those of serine proteinases and dipeptidyl peptidase IV (Gly-Pro-AP) significantly decreased with age, whereas that of L-leucine aminopeptidase (Leu-AP) increased significantly. However, when expressed as total activities, the enzymatic activities in spleen generally tended to increase with age, except in the case of serine proteinases, because of the age-related increase in spleen weight. The results were taken to indicate that the activities of serine proteinases become relatively more deficient in the spleen as age increases. The results of a multivariate study maintained this peculiarity of serine proteinases in comparison with other enzymes. The relative deficiency of serine proteinases in spleen may be somehow related to immunodeficiency in aged animals, as judged from similar findings in animal models of systemic erythematodes.

Characterization of filamentous phages of Vibrio cholerae O139 and O1.

We have analyzed our collection of Vibrio cholerae O139 strains to determine whether filamentous phages are produced in their culture supernatants, and whether any replicative form of DNA is detectable in cell lysates. Two types of filamentous phage, designated fs1 (6.4 kb) and fs2 (8.5 kb), were found in strains of Vibrio cholerae O139, fs1 was commonly produced from clinical isolates of Vibrio cholerae O1. Infectious particles (filamentous phages) were inducible by subculture, mitomycin C, and cultivation in a ligated ileal loop of a rabbit. Type 4 fimbriae of Vibrio cholerae O1 sensitive to D-glucose and D-mannose were suggested to be receptors for fs1 and fs2. The genome of fs1 was revealed to encode a potential new enterotoxin homologous to zonula occludens toxin. Clarification of the relation of type 4 fimbriae and these filamentous phages will provide a new understanding of the colonization of Vibrio-cholerae O1 and O139. Thus the presence of a new enterotoxin encoded by the genome of filamentous phage like fs1 may clarify the pathogenesis of cholera toxin negative clinical isolates of Vibrio cholerae O1 and non-O1. Our findings combined with the earlier report by Ehara et al. [Microbio. Immunol. 37 (1993) 679-688] suggest that type 4 fimbriae of Vibrio cholerae O1 are important for the development of an effective vaccine against cholera.

Two different modes of enzymatic changes in serum with progression of Duchenne muscular dystrophy.

Nineteen serum enzymes from patients with Duchenne muscular dystrophy and asthma, and normal subjects were studied. These enzymes include aminopeptidases, cathepsin C, angiotensin-converting enzyme, serine proteinase, sulphatase, phosphatase, esterases and ribonuclease. The enzymatic changes in dystrophic patients were related to two parameters: severity of the disease as judged from symptomatology, and duration of the disease. Most of the enzyme levels tested were increased in milder cases, but they tended to decrease with severity of the disease. On the other hand, there was a group of enzymes showing just opposite tendencies: serine proteinase, cathepsin C and ribonuclease. Even when viewed from the relationship to duration of the disease, the above mentioned grouping of enzymes was generally valid. Most of the enzyme levels, including those routinely applied as clinical parameters, tended to decrease, logarithmically, with an increase in duration of the disease. On the contrary, some others, including serine proteinase, cathepsin C and ribonuclease, tended to increase toward their control levels. Such tendencies were not found in the patients with asthma. The discrepancy between the above two groups of enzymes may have some implications for the process of protein degradation in dystrophic patients.

Poststatin, a new inhibitor of prolyl endopeptidase, produced by Streptomyces viridochromogenes MH534-30F3. I. Taxonomy, production, isolation, physico-chemical properties and biological activities.

Poststatin, a new inhibitor of prolyl endopeptidase (PEP) was discovered in the fermentation broth of Streptomyces viridochromogenes MH534-30F3. It was purified by Diaion HP-20, Sephadex LH-20 and YMC-gel (ODS-A) column chromatography and then isolated as a colorless powder. Poststatin has the molecular formula C26H47N5O7. The IC50 value of poststatin against the PEP of partially purified porcine kidney was 0.03 microgram/ml. It has low acute toxicity. No deaths occured after iv injection of 250 mg/kg of this agent to mice.

Synthesis of histargin and related compounds and their inhibition of enzymes.

A novel method for the synthesis of histargin and its analogs is described. It includes two kinds of N-alkylation reactions that prevent the formation of side products. The inhibition of enzymes by these compounds was also measured. Some of the compounds strongly inhibited carboxypeptidase B, carboxypeptidase A, carboxypeptidase N (kininase I), and angiotensin converting enzyme.

Bequinostatins A and B new inhibitors of glutathione S-transferase, produced by Streptomyces sp. MI384-DF12. Production, isolation, structure determination and biological activities.

New benzo[a]naphthacenequinone metabolites, designated bequinostatins A and B, have been isolated from the culture broth of the benastatin-producing strain Streptomyces sp. MI384-DF12. The structures of bequinostatins A and B were determined by spectral analyses to be 5,6,8,13-tetrahydro-1,6,7,9,11-pentahydroxy-8,13-dioxo-3- pentylbenzo[a]naphthacene-2-carboxylic acid and 2-decarboxybequinostatin A, respectively. Bequinostatin A showed considerable inhibitory activity against human pi class glutathione S-transferase (GST pi).

Benastatins A and B, new inhibitors of glutathione S-transferase, produced by Streptomyces sp. MI384-DF12. I. Taxonomy, production, isolation, physico-chemical properties and biological activities.

Benastatins have been isolated as part of a program designed to find microorganism-produced inhibitors of glutathione S-transferase from Streptomyces sp. MI384-DF12. They were purified by chromatography of reversed-phase silica gel, silica gel and Capcell Pak C18 (HPLC) followed by solvent extraction and then isolated as yellow powders. Benastatins A and B have the molecular formulae, C30H28O7 and C30H30O7, respectively. They were competitive with 3,4-dichloronitrobenzene as the substrate, and the inhibition constants (Ki) of benastatins A and B were 5.0 x 10(-6) and 3.7 x 10(-6), respectively.

Nagstatin, a new inhibitor of N-acetyl-beta-D-glucosaminidase, produced by Streptomyces amakusaensis MG846-fF3. Taxonomy, production, isolation, physico-chemical properties and biological activities.

Nagstatin, a new inhibitor of N-acetyl-beta-D-glucosaminidase (NAG-ase) was discovered in the fermentation broth of Streptomyces amakusaensis MG846-fF3. It was purified by chromatography on Dowex 50W, Avicel and Sephadex LH-20 followed by the treatment of active carbon and then isolated as colorless powder. Nagstatin has the molecular formula of C12H17N3O6. It is competitive with the substrate, and the inhibition constant (Ki) was 1.7 x 10(-8) M.

Cyclooctatin, a new inhibitor of lysophospholipase, produced by Streptomyces melanosporofaciens MI614-43F2. Taxonomy, production, isolation, physico-chemical properties and biological activities.

Cyclooctatin has been isolated from Streptomyces melanosporofaciens MI614-43F2 as part of a program designed to find microorganism-produced inhibitors of lysophospholipase. It was purified by chromatography on silica gel, Capcell Pak C18 (HPLC) and Sephadex LH-20 followed by solvent extraction and then isolated as a colorless powder. Cyclooctatin has the molecular formula of C20H34O3. It is competitive with the substrate, and the inhibition constant (Ki) was 4.8 x 10(-6) M.

Benarthin: a new inhibitor of pyroglutamyl peptidase. I. Taxonomy, fermentation, isolation and biological activities.

We found benarthin, a new inhibitor of pyroglutamyl peptidase, in the fermentation broth of Streptomyces xanthophaeus MJ244-SF1. It was purified by column chromatography and centrifugal partition chromatography (CPC) and then was isolated as a colorless powder. The binding of benarthin was competitive with substrate and its inhibition constant (Ki) was 1.2 x 10(-6) M.

Epostatin, new inhibitor of dipeptidyl peptidase II, produced by Streptomyces sp. MJ995-OF5. I. Taxonomy of producing strain, fermentation, isolation, physico-chemical properties and biological properties.

A new inhibitor of dipeptidyl peptidase II (DPP-II, EC 3.4.14.2), designated as epostatin, was discovered in the fermentation broth of a strain isolated in our institute. The strain has been identified as Streptomyces sp. MJ995-OF5 on the basis of taxonomic studies. Epostatin was obtained as a yellow powder after seqential purification by chromatography on Diaion HP-20, n-butanol extraction, Sephadex LH-20 column chromatography and centrifugal partition chromatography (CPC). Epostatin inhibited DPP-II competitively in a dose dependent manner. The compound was slightly inhibitory against other dipeptidyl peptidases.

Benastatins C and D, new inhibitors of glutathione S-transferase, produced by Streptomyces sp. MI384-DF12. Production, isolation, structure determination and biological activities.

Benastatin C, a new member of the benastatins, has been isolated from the culture broth of Streptomyces sp. MI384-DF12. The structure of benastatin C was elucidated as 2-decarboxy-benastatin A by NMR studies. Benastatin D, 2-decarboxybenastatin B, was derived from benastatin B. Benastatins C and D showed inhibitory activities against human pi class glutathione S-transferase (GST pi) and excellent stimulatory activities on the murine lymphocyte blastogenesis in vitro.

Fluostatins A and B, new inhibitors of dipeptidyl peptidase III, produced by Streptomyces sp. TA-3391. I. Taxonomy of producing strain, production, isolation, physico-chemical properties and biological properties.

New inhibitors of dipeptidyl peptidaseIII (EC 3.4.14.4) from human placenta, designated as fluostatins A and B, were discovered in the fermentation broth of a strain isolated in our institute. The strain has been identified as Streptomyces sp. TA-3391 on the basis of taxonomic studies. Fluostatins A and B were purified by Diaion HP-20 chromatography, ethyl acetate extraction, silica gel chromatography and reverse phase preparative HPLC. With the synthetic substrate, arginyl-arginine-2-naphthylamide, the IC50 values of fluostatins A and B were 0.44 and 24.0 micrograms/ml, respectively. Fluostatins A and B were slightly inhibitory against other dipeptidyl peptidases. Fluostatin A showed mixed-type (competitive and noncompeptitive) inhibition with human leucine-enkephalin as a substrate, and the inhibition constant (Ki) was 14.2 microM.

Synchronisation of oestrus in dairy cows using prostaglandin F2alpha, gonadotrophin-releasing hormone, and oestradiol cypionate.

An oestrous synchronisation protocol was developed for use in lactating dairy cows using PGF(2alpha), GnRH, and oestradiol cypionate (ECP). In experiment 1, lactating dairy cows received two injections of PGF(2alpha) (on days 0 and 11) (PP; n=10) or two injections of PGF(2alpha) (days 0 and 11) and 100 microg of GnRH on day 3 (PGP; n=10). In experiment 2, cows were treated with PGP (n=7), or PGP and 1 mg of ECP at the same time (PGPE(0); n=7) or 1 day after the second PGF(2alpha) injection (PGPE(1); n=7). In experiment 3, 101 lactating dairy cows in a commercial herd were assigned to one of three treatments; PP, PGP, or PGPE(1). Follicular growth was measured by ultrasound in experiments 1 and 2. Every cow (experiments 1, 2, and 3) was blood sampled at selected intervals for progesterone and oestradiol assays and inseminated at oestrus. In experiment 1, a higher percentage of GnRH-treated cows ovulated after the first PGF(2alpha) injection (90% versus 50%; P<0.05). The GnRH-treated cows tended to have a larger dominant follicle present at the time of the second PGF(2alpha) injection (16.5+/-0.5 mm versus 15.0+/-0.7 mm; P<0.10). The percentage of cows that ovulated after the second PGF(2alpha) injection was similar (60%). In experiment 2, cows treated with ECP had higher peak preovulatory concentrations of oestradiol in plasma (6.99+/-0.63 versus 3.63+/-0.63; P<0.01) following the second PGF(2alpha) injection and a higher percentage ovulated (86% versus 43%; P<0.05). A higher percentage of PGPE(1)-treated cows in experiment 3 were observed in standing oestrus and ovulated after the second PGF(2alpha) injection (standing oestrus, 26.4, 34.3, and 62.6%, P<0.01; ovulated, 56, 63, and 78%, P<0.05; PP, PGP, and PGPE(1), respectively). In conclusion, the PGP protocol increased the number of cows that ovulated after the first PGF(2alpha) injection and produced a more mature dominant follicle at the time of the second PGF(2alpha) injection. Adding ECP to PGP (PGPE(1)) enhanced the expression of oestrus and increased ovulation percentage. The combination of PGP and ECP is potentially a new method to routinely synchronise oestrus and ovulation in dairy cows.