Anthelmintic efficacy of milbemycin D against Toxocara cati and Ancylostoma tubaeforme in domestic cats.

Anthelmintic efficacy of milbemycin D was evaluated against Toxocara cati and Ancylostoma tubaeforme in domestic cats. Twelve cats naturally infected with each nematode species were allocated among 2 groups of 6 animals each, and milbemycin D was orally administered to the 2 groups of cats in doses of 0.05 mg/kg and 0.1 mg/kg body weight, respectively. In all the cats infected with T. cati, fecal egg counts decreased followed by their disappearance from the feces and 2-35 worms were excreted into the feces after the medication in both doses of 0.05 mg/kg and 0.1 mg/kg. At postmortem of these medicated groups, no worms were detected from 4 cats of each group, but 1 and 2 immature worms were recovered from the other 2 cats respectively. In the cats infected with A. tubaeforme, fecal egg counts decreased followed by the disappearance from the feces and 2-62 worms were excreted into the feces in all the cats of the 2 groups, no nematodes remaining at postmortem. These results indicate that milbemycin D is fully effective against T. cati and A. tubaeforme in cats in a dose of 0.05-0.1 mg/kg.

[Participation of sialic acid residue in the fibrinogen-fibrin conversion by thrombin].

In order to estimate the effects of sialic acid residues in fibrinogen on the fibrinogen-fibrin conversion by bovine thrombin the Michaelis constant (Km) and maximum velocity (Vmax) were determined. The Km value obtained by the use of intact-fibrinogen was smaller than that of asialo-fibrinogen. This fact suggests that the sialic acid residues affected the formation of the enzyme-substrate complex. It was also found that in comparison with the asialo-fibrinogen, the intact-fibrinogen was significantly influenced in the gel formation time by the ionic strength in the reaction solution.

[An arginine esterase in the human sperm].

Human sperm was highly purified by the use of a discontinuous Percoll density gradient placed in an innercolumn of a centrifugation tube. Seminal plasma contamination was only 0.0008 percent in the purified sperm. A new basic arginine ester hydrolyzing enzyme with a weak affinity for lima bean trypsin inhibitor (LBTI) Cellulofine column was found in the purified human sperm, and the characteristics of this enzyme were found to be different from those of human acrosin.

Two forms of acidic arginine amidases in human kidney.

Two forms of acidic arginine amidases were separated from human kidney extract using the techniques of basic ion exchange adsorption and elution as well as lima bean trypsin inhibitor (LBTI) and aprotinin affinity adsorptions and elutions. The enzymes were tentatively named acidic human renal arginine amidase-L (AHRAA-L, with affinity to an LBTI column) and -A (AHRAA-A, with affinity to an aprotinin column). Both enzymes showed a similar molecular mass of approximately 3.0 x 10(4) daltons, differing from that of human renal kallikrein (HRK, molecular mass of 4.8 x 10(4) daltons). The specific activity of AHRAA-L and -A were 106 and 680 nmol/min/A280 of Val-Leu-Arg-pNA amidolysis, respectively, and they were strongly inhibited by LBTI and human urinary trypsin inhibitor (UTI), while ethylenglycol-bis(beta-amino ethylether)-N,N,N',N'-tetraacetic acid (EGTA) showed a weak or no effect on both enzymes.

The effects of pH on the generation of turbidity and elasticity associated with fibrinogen-fibrin conversion by thrombin are remarkably influenced by sialic acid in fibrinogen.

In fibrinogen-fibrin conversion by thrombin, the polymerization of a fibrin monomer is accompanied by gelation and an increase turbidity. Since sialic acids at the terminal of the carbohydrate chains bound to fibrinogen are part of the low affinity calcium binding site necessary for polymerization, they are closely involved in the network structure of fibrin clots. Fibrin clots derived from asialofibrinogen exhibited definite differences in turbidity and elasticity compared with those derived from intact fibrinogen, and were markedly dependent on the pH during the reaction. The turbidity during polymerization of fibrin, evaluated according to the absorbance at 350 nm, was maximum at pH 6.5-7.0, but it decreased in the other pH ranges, with the changes being unremarkable at higher pH levels but remarkable at lower pH ranges. The turbidity of fibrin derived from asialofibrinogen was far higher than that from intact fibrinogen near neutrality, but decreased rapidly and was lower than in intact fibrinogen at higher and lower pH ranges. Concerning the elasticity evaluated by thromboelastography, the coagulation time (k) and the maximum amplitude (ma) were lower in asialofibrinogen, indicating a deterioration of the clotting function of fibrinogen with the loss of sialic acid. These results suggest that sialic acid bound to fibrinogen is closely related to the fibrin network formation in blood coagulation, which is the most important function of fibrinogen, and plays a functional role in the stabilization of fibrin clot formation against environmental changes, including pH.

Evidence of two forms of basic arginine esterases in human male urine.

Two forms of basic arginine ester hydrolyzing enzymes were found in human male urine, and their partial purification was performed using lima bean trypsin inhibitor (LBTI) affinity adsorption and Cellulofine GCL-2000 gel chromatography. The forms tentatively called basic human urinary arginine esterase-1 (BHUAE-1) and -2 (BHUAE-2), had respective estimated molecular weights of about 4.5 x 10(4) and 1.8 x 10(4) daltons, with pH optima observed at 9.5 and 10.0. These two new enzymes in the human male urine were different from human renal urokinases.

Sialic acid in fibrinogen: effects of sialic acid on fibrinogen-fibrin conversion by thrombin and properties of asialofibrin clot.

The final stage in a series of blood coagulating reactions is fibrinogen-fibrin conversion by thrombin. This reaction consists of fibrinopeptide A and fibrinopeptide B release, polymerization of fibrin monomer, and stabilized fibrin formation by factor XIII. The latter two reactions require calcium. In the present study there was no difference in the rate of thrombin-induced fibrinopeptide release between fibrinogen and asialofibrinogen where sialic acid in the terminal end of carbohydrate moiety of fibrinogen was removed by neuraminidase, but turbidity associated with asialofibrin clot formation was increased more rapidly. In asialo-derivatives, the dissolution time of the clots in high concentrated urea solution tended to be shortened and rigidity as a gel tended to be decreased. In measurement by thromboelastography there was no difference in the reaction time (r) between fibrinogen and asialofibrinogen, but the maximum amplitude (ma) was obviously decreased in asialofibrinogen. Furthermore, when the rate of cross-link formation between gamma chains by F-XIII was compared, the production of gamma-dimer in the same reaction time was found to be lower and formation of stabilized fibrin tended to be retarded in asialofibrinogen. Sialic acid in fibrinogen thus may clearly influence the polymerization of fibrin-monomer and the formation of cross-linked fibrin in a series of reactions for fibrinogen-fibrin conversion. This may be consistent with the theory that fibrinogen sialic acid residues are low affinity calcium-binding sites and influence fibrin assembly.

Detection and separation of some arginine amidases including tissue kallikrein from human seminal plasma.

A plasminogen/plasmin like substance (AHSAA-1), with affinity to lysine column was separated from DEAE-cellulose adsorbed human seminal plasma. Two forms of acidic arginine amidase with different affinities to LBTI (AHSAA-2) and aprotinin columns (AHSAA-3) were separated from the DEAE-cellulose adsorbed preparation and AHSAA-3 was identified as tissue kallikrein. Two basic arginine amidase preparations having affinity to LBTI (BHSAA-1) and aprotinin column were also separated from the CM-cellulose adsorbed human seminal plasma. Three basic arginine amidases with different molecular mass (BHSAA-2 to 4) were separated by Cellulofine GCL-2000 gel filtration from aprotinin adsorbed material and some of their properties were examined.

Selective isolation of acrosome-reacted human spermatozoa with progressive motility by using cell affinity chromatography on concanavalin A Sepharose.

In order to ensure fertility, mammalian spermatozoa have to undergo acrosome reaction, the most obvious morphological change during this being the exposure of the inner acrosomal membrane. In the present study, the acrosome-reacted human spermatozoa were successfully separated without loss of viability by using cell affinity chromatography on Concanavalin A (Con A) Sepharose. Con A demonstrated affinity for both the intact and the acrosome-reacted spermatozoa regardless of their viability; the latter, however, gave higher affinity than the former against Con A. Prior to the column chromatography, the immotile spermatozoa and the seminal plasma were excluded by means of a modified swim-down procedure and the resulting spermatozoa were subsequently immobilized by slow rate cooling in ice-cold water. Cell affinity chromatography was performed at 4 degrees C. To prevent mechanical trapping of the spermatozoa among the packed gel beads, the column was interconnected with a reservoir, the vertical drive of which was allowed to lose the gel bed and thereby release the trapped spermatozoa. Stepwise competitive elution with 5.0 microM mannose and 25% heat-inactivated human serum was capable of separating the intact spermatozoa and the acrosome-reacted spermatozoa from each other. The acrosome reaction rate of sperm fraction which was adsorbed to Con A Sepharose and eluted with 25% serum was found to be 83 +/- 2.3%, and motility and viability of these fractions were measured to be 80 +/- 6.3% and 83 +/- 7.6%, respectively (n = 8, mean +/- SD). The status of the acrosome in a final preparation (motility 92%, acrosome reaction rate 88%) was observed by scanning electron microscopy, and 81% spermatozoa lost their acrosome cap.

Clinical application of basic arginine amidase in human male urine.

Basic human urinary arginine amidase (or esterase, called BHUAE) which is only found in male urine, was measured from normal volunteers between the age of 4 and 70 years using D-valyl-L-leucyl-L-arginine-p-nitroanilide as a substrate. BHUAE increases during early adolescence, between 8 to 17 years of age. Then, BHUAE decreases in the twenties and takes a certain range of value in the mature age group, between the late thirties and fifties. In patients with prostate cancer, a significant increase in BHUAE was demonstrated in comparison with the healthy male group (control) over 55 years old. On the other hand, patients with benign prostatic hypertrophy showed no significant elevation of this enzyme activity. It would appear that the measurement of BHUAE in urine can be used as a marker of prostate cancer in an advanced age group.

Urinary kallikrein and non-kallikrein arginine esterase activities in beagle dogs.

The activity of urinary kallikrein and non-kallikrein arginine esterase was measured in the 16hrs urine of 16 beagle dogs. Enzyme activity was assayed using D-valyl-L-leucyl-L-arginine-p-nitroanilide as a substrate at 30 degrees C, pH 8.0, and the unit of measurement was defined as the activity which catalyzes the hydrolysis of 1 mumol of the substrate per minute. Kallikrein activity ranged from 3.87 to 48.92 (28.48 average) mU/ml of urine, or from 1.05 to 12.51 (4.56 average) U/16 hrs; whereas that of non-kallikrein arginine esterase ranged from 0.06 to 24.28 (7.11 average) mU/ml, or from 0.01 to 9.78 (1.43 average) U/16 hrs. The ratio between the activity of these two enzymes was 1: 0.002-1: 2.98 (1: 0.42 average). Male dogs had a tendency to show a higher enzyme activity than females for urinary kallikrein and non-kallikrein arginine esterase.