Effect of common sedation agents on feline splenic size determined via ultrasonography.

AIMS: To evaluate the effect of IM administration of three sedative drugs, acepromazine, alfaxalone and dexmedetomidine, in combination with morphine, on the size of the feline spleen using ultrasonography. METHODS: Twenty-four client-owned cats undergoing elective de-sexing or minor procedures were recruited for a focused ultrasonographic examination of the spleen prior to and at 10, 20 and 30 minutes following administration of one of three randomly assigned IM sedation protocols: 0.05 mg/kg acepromazine (ACE group), 3 mg/kg alfaxalone (ALF group), or 10 µg/kg dexmedetomidine (DEX group), in combination with 0.5 mg/kg morphine. B-mode images of the spleen were collected and measured following a standardised protocol. Cardiorespiratory parameters and sedation score were also recorded. Mean thickness of the head, body and tail of the spleen for each group at 10, 20 and 30 minutes after drug administration was compared to baseline. RESULTS: Mean splenic thickness increased over time in the ACE group (thickness of body at T0 = 8.9 (SE 2.1) mm and at T30 = 10.5 (SE 2.0) mm; p = 0.001) and the ALF group (thickness of body at T0 = 8.8 (SE 1.0) mm and at T30 = 10.3 (SE 1.7) mm; p = 0.022) but not in the DEX group (thickness of body at T0 = 8.6 mm (1.2) and at T30 = 8.9 mm (0.6); p = 0.67). Mean arterial blood pressure in the DEX group was significantly higher than in the other groups (p = 0.002). Sedation scores in the DEX group were consistently high for the entire period. However, the sedation score in the ACE group increased over 30 minutes (p = 0.007). Sedation score in the ALF group was highest at 10 minutes but gradually decreased over the following 20 minutes (p = 0.003). CONCLUSIONS: Sedation with IM dexmedetomidine and morphine did not change splenic size, whereas acepromazine or alfaxalone and morphine increased it regardless of the degree of sedation. CLINICAL RELEVANCE: Where splenomegaly is identified in a cat sedated with acepromazine or alfaxalone, the effects of the sedation protocol could be considered as a possible cause.

Effects of methionine plus cysteine inclusion on performance and body composition of liquid-fed crossbred calves fed a commercial milk replacer and no starter feed.

This experiment aimed to evaluate the effects of supplying 4 different inclusion levels of Met + Cys to crossbred liquid-fed calves on animal performance and body composition. Thirty-six Holstein-Gyr male calves were separated into 2 age groups: 16 calves, slaughtered at an age of 30 d, representing the physiological phase from 8 to 30 d, and 20 calves, slaughtered at an age of 60 d, representing the physiological phase from 30 to 60 d. At 8 d of age, the animals were randomly distributed among the experimental treatments: 4 Met + Cys inclusion levels (Met + Cys: 8.0, 8.7, 9.4, and 10.2 g/d), provided by an AA supplement added to 1.0 kg (as fed) of commercial milk replacer containing soy protein concentrate and wheat protein isolate reconstituted at 13.8% (dry matter basis). The diet was supplied without allowing leftovers and no starter feed was provided. The experimental diets were supplied without allowing orts, so that the dry matter, crude protein, and ether extract intakes were the same for all animals, independent of Met + Cys level. Total weight gain, average daily gain, gain composition, and body composition were evaluated for both age groups separately. Digestibility of organic matter, crude protein, and ether extract was lower for 8 to 30 d than for 30 to 60 d. The effect of Met + Cys levels on the digestibility of nutrients was not observed; there also was no significant interaction between physiological phase and Met + Cys levels. For the 8 to 30 d group, no responses in performance were observed according to the different Met + Cys levels, which indicates that 8.0 g/d of Met + Cys met the requirements for this physiological phase. The 30 to 60 d group responded positively to higher Met + Cys inclusion in the diet. In conclusion, an optimal Met + Cys dietary level to ensure best performance and protein gain ranges from 8.41 to 9.81 g/d.

Performance and health of Holstein calves fed different levels of milk fortified with symbiotic complex containing pre- and probiotics.

The objective of this study was to evaluate the performance and health of Holstein calves fed low or high milk supply (MSP) with or without symbiotic complex (SYM) supplementation, consisting of prebiotics, probiotics, and fibrolytic enzymes. Thirty-two Holstein calves with body weight (BW) of 34 ± 7 kg were distributed in a randomized block design in a 2 × 2 factorial arrangement. Treatments consisted of low and high MSP: 10 % of BW from 1st to 8th weeks after birth (low) and 20 % BW from 1st and 2nd weeks after birth, 15 % BW for the 3rd and 4th weeks after birth, and 10 % BW from 5th and 8th weeks after birth (high). Solid ration was supplied in addition to milk. Intake, ADG, diet digestibility, and fecal consistency index were evaluated. Low and high MSP groups tended (P < 0.10) to differ in calf growth, final BW (69 vs. 73 kg), post-weaning average weight gain (548 vs. 788 g/day), and final average weight gain (549 vs. 646 g/day) in low and high MSP calves, respectively. There was an interaction between MSP level and SYM on the digestibilities of dry matter (DM) and neutral detergent fiber (NDF) (P < 0.10). In the low MSP group, inclusion of SYM increased digestibility of DM (0.720 to 0.736 g/kg) and NDF (0.758 to 0.783 g/kg). The inclusion of SYM improved calf health (P < 0.10) with a fecal score of 0.31 compared to 0.42 without SYM. Milk-feeding level was an important factor in calf performance, while SYM supplementation improved diet digestibility and animal health.

Purification and partial characterization of two phospholipases A2 from Bothrops leucurus (white-tailed-jararaca) snake venom.

Two proteins with phospholipase A(2) (PLA(2)) activity were purified to homogeneity from Bothrops leucurus (white-tailed-jararaca) snake venom through three chromatographic steps: Conventional gel filtration on Sephacryl S-200, ion-exchange on Q-Sepharose and reverse phase on Vydac C4 HPLC column. The molecular mass for both enzymes was estimated to be approximately 14 kDa by SDS-PAGE. The N-terminal sequences (48 residues) show that one enzyme presents lysine at position 48 and the other an aspartic acid in this position, and therefore they were designated blK-PLA(2) and blD-PLA(2) respectively. blK-PLA(2) presented negligible levels of PLA(2) activity as compared to that of blD-PLA(2). The PLA(2) activity of both enzymes is Ca(2+)-dependent. blD-PLA(2) did not have any effect upon platelet aggregation induced by arachidonic acid, ADP or collagen, but strongly inhibits coagulation and is able to stimulate Ehrlich tumor growth but not angiogenesis.

High expression of human carboxypeptidase M in Pichia pastoris: purification and partial characterization.

Carboxypeptidase M (CPM) is an extracellular glycosylphosphatidyl-inositol-anchored membrane glycoprotein, which removes the C-terminal basic residues, lysine and arginine, from peptides and proteins at neutral pH. CPM plays an important role in the control of peptide hormones and growth factor activity on the cell surface. The present study was carried out to clone and express human CPM in the yeast Pichia pastoris in order to evaluate the importance of this enzyme in physiological and pathological processes. The cDNA for the enzyme was amplified from total placental RNA by RT-PCR and cloned in the vector pPIC9, which uses the methanol oxidase promoter and drives the expression of high levels of heterologous proteins in P. pastoris. The cpm gene, after cloning and transfection, was integrated into the yeast genome, which produced the active protein. The recombinant protein was secreted into the medium and the enzymatic activity was measured using the fluorescent substrate dansyl-Ala-Arg. The enzyme was purified by a two-step protocol including gel filtration and ion-exchange chromatography, resulting in a 1753-fold purified active protein (16474 RFU mg protein(-1) min(-1)). This purification protocol permitted us to obtain 410 mg of the purified protein per liter of fermentation medium. SDS-PAGE showed that recombinant CPM migrated as a single band with a molecular mass similar to that of native placental enzyme (62 kDa), suggesting that the expression of a glycosylated protein had occurred. These results demonstrate for the first time the establishment of a method using P. pastoris to express human CPM necessary to the development of specific antibodies and antagonists, and the analysis of the involvement of this peptidase in different physiological and pathological processes.

Pro-rat atrial natriuretic peptide-mimicking peptides as substrates for rat kallikreins rK2 (tonin) and rK9.

Investigation of the substrate specificity of rat tissue kallikreins has shown the importance of an extended site of interaction, and that the proform of rat natriuretic peptides, pro-ANP, could be a substrate for two members of the family, rK2 (tonin) and rK9 (Moreau et al. (1992) J. Biol. Chem. 267, 10045-10051). Synthetic peptide substrates that reproduce the sequence of rat pro-ANP in the region of the activation sites were used to further assess the specificity of these two proteinases. Peptides 95-107 (AGPRSLRRSSCFG) and 91-107 (RALLAGPRSLRRSSCFG) of the rat pro-ANP sequence, which include all the cleavage sites for generating natriuretic peptides (R98, R101, R102), were synthesized and assayed as kallikrein substrates. Despite their homology, the two peptides had different susceptibilities to cleavage by rK2 and rK9. Peptide 91-107 was rapidly and specifically cleaved by both kallikreins, with a single cleavage site at the R98-S99 bond, which is the primary cleavage site in pro-ANP for generating ANP[1-28]. The kcat/Km values were 289,000 M-1 s-1 for rK2 and 39,000 M-1 s-1 for rK9. The N-terminally truncated peptide (95-107) was also cleaved at that bond by both proteinases, but far less rapidly than peptide 91-107, and additional cleavages appeared at secondary sites i.e those generating atriopeptin III (R101) and auriculin (R102) in rat pro-ANP. A commercial fluorogenic tetrapeptide substrate reproducing the sequence of rat pro-ANP was slowly hydrolysed under the same conditions. The kinin-releasing kallikrein rK1 did not cleave synthetic peptides at the R98-S99 bond, further demonstrating the different specificities of tissue kallikreins. The results indicate that residues in positions P5 to P8 with respect to the cleavage site in the substrate, are essential for the substrate binding and specificity of kallikreins rK2 and rK9. They also show that long peptide substrates should be used to identify biological substrates of kallikreins from the investigation of their kinetic properties. The biological significance of pro-ANP processing by these proteinases, remains, however, to be proven.

Hydrolysis by plasma kallikrein of fluorogenic peptides derived from prorenin processing site.

Human plasma kallikrein (HPK) activates plasma prorenin to renin, and the physiological significance of this activation is still unknown. In this paper we investigated the efficiency and the cleavage pattern of the hydrolysis by HPK of the internally quenched fluorescent peptides (qf-peptides) derived from the amino acid sequence of human prorenin cleavage site. The peptide Abz-F-S-Q-P-M-K-R-L-T-L-G-N-T-T-Q-EDDnp (Abz=ortho-aminobenzoic acid, and EDDnp=N-[2,4-dinitrophenyl]-ethylene diamine), that corresponds to the amino acid sequence P(7) to P(7)' of human prorenin cleavage site, is hydrolyzed at the correct processing site (R-L bond) with k(cat)/K(m)=85 mM(-1) s(-1). Alanine was scanned in all positions from P(5) to P(5)' in order to investigate the substrate specificity requirements of HPK. The qf-peptides derived from the equivalent segment of rat prorenin, that has Lys-Lys as basic amino acid pair, and the peptide Abz-NVTSPVQ-EDDnp that contains the proposed cleavage site of rat prorenin have very low susceptibility to hydrolysis by rat plasma kallikrein. These data are according to the previously reported absence of rat plasma prorenin activation by rat plasma kallikrein (RPK), and with the view that prorenin activation in rat requires alternative enzymes and/or mechanism. All the obtained peptides described in this paper were also assayed with bovine trypsin that was taken as a reference protease because it is commonly used to activate prorenin.

Hydrolysis by cathepsin B of fluorescent peptides derived from human prorenin.

Cathepsin B is a lysosomal thiolprotease that, because of its colocalization with renin and its ability to activate prorenin, has been proposed as a prorenin processing enzyme. To characterize the biochemical aspect of this potential cathepsin B activity in more detail, we synthesized and assayed with human cathepsin B the internally quenched fluorescent peptide Abz-FSQPMKRLTLGNTTQ-EDDnp (Abz, ortho-aminobenzoic acid fluorescent group and EDDnp, N-¿2, 4-dinitrophenyl-ethylenediamine quencher group) that contains 7 amino acids for each side of the R-L bond that is the processing site of human prorenin. Human cathepsin B hydrolyzed this peptide at the correct site (R-L bond), with k(cat)/K(m)=75 mmol/L(-1) s(-1). Analogues of this peptide obtained by Ala scanning at positions P(5) to P(5)' were also synthesized and assayed as substrates for human cathepsin B. The obtained specificity constant (k(cat)/K(m)) values have a significant parallel with the previous data of prorenin activation by AtT-20 cells and in vitro by cathepsin B. In addition, we demonstrated the presence of cathepsin B-like activity in rat mesangial cells and the ability of its whole soluble fraction lysates, as well as that of purified cloned rat cathepsin B, to hydrolyze Abz-IKKSSF-EDDnp at the K-S bond, which contains 6 amino acids of rat prorenin processing site. The specificity data of cathepsin B toward peptides derived from prorenin processing site support the view that human or rodent cathepsin B could be involved in the intracellular processing of prorenin that is locally synthesized or taken up from the extracellular compartment.

Inhibition of cathepsin B by its propeptide: use of overlapping peptides to identify a critical segment.

Ten overlapping 15-mer peptides (peptidyl amides) spanning the proregion of rat cathepsin B (residues 1p-60p) were constructed to identify minimal segments having inhibitory activity towards the mature enzyme, that could be used to develop a new generation of peptide-derived inhibitors specifically targeting the active site of the corresponding proteinase. Three synthetic peptides, containing the pentapeptide Leu-Cys-Gly-Thr-Val (residues 41p-45p) in their sequence, inhibited cathepsin B with Ki values in the micromolar range. Alkylation of the thiol group of Cys-42p of peptide PB8 (36p-50p) resulted in its rapid proteolytic degradation, suggesting that this residue is essential for inhibition. The inhibition constant was slightly improved (Ki = 2 microM) using a longer peptide (26p-50p) which was completely resistant to cleavage even after a prolonged incubation. Alkylation of its cysteinyl residue also resulted in rapid cleavage of the peptide chain. Peptides derived from the rat cathepsin B prosequence also inhibited human cathepsin B with similar Ki values. Unlike rat cathepsin B, which cleaves peptide PB8 at the G47p-G48p bond after prolonged incubation, the human enzyme cleaved both PB8 and PB11 at the Lys-40p-Leu-41p bond, in agreement with the different kinetic properties of these two proteinases. New probes with improved specificity for cysteine proteinases may therefore be designed based on the sequences of their propeptides.

A comparison of the enzymatic properties of the major cysteine proteinases from Trypanosoma congolense and Trypanosoma cruzi.

Congopain and cruzipain, the major cysteine proteinases from Trypanosoma congolense and Trypanosoma cruzi, were compared for their activities towards a series of new, sensitive fluorogenic substrates of the papain family of cysteine proteinases and for their sensitivity to inhibition by cystatins and related biotinylated peptidyl diazomethanes. Low Ki values, in the 10 pM range, were found for the interaction of both proteinases with natural cystatin inhibitors. The kinetic constants for the hydrolysis of cystatin-derived substrates, and the inhibition by related diazomethanes were essentially identical. Unlike cathepsins B and L, the related mammal papain family proteinases, congopain and cruzipain accomodate a prolyl residue in P2'. Substrates having the sequence VGGP from P2 to P2' were hydrolysed by both congopain and cruzipain with a k(cat)/Km greater than 4.10(3) mM(-1) s(-1). Irreversible diazomethane inhibitors, deduced from the unprime sequence of cystatin-derived substrates, inhibited the two parasite proteinases. N-terminal labelling of diazomethanes with a biotin group did not alter the rate of inhibition significantly, which provides a useful tool for examining the distribution of these enzymes in the parasite and in the host. Despite their similar activities on cystatin-derived substrates, congopain and cruzipain had significantly different pH-activity profiles when assayed with a cystatin-derived substrate. They were correlated with structural differences, especially at the presumed S2 subsites.

Identification of Random Amplified Polymorphic DNA Markers Linked to the Co-4 Resistance Gene to Colletotrichum lindemuthianum in Common Bean.

ABSTRACT New cultivars of the common bean (Phaseolus vulgaris) with durable resistance to anthracnose can be developed by pyramiding major resistance genes using marker-assisted selection. To this end, it is necessary to identify sources of resistance and molecular markers tightly linked to the resistance genes. The objectives of this work were to study the inheritance of resistance to anthracnose in the cultivar TO (carrying the Co-4 gene), to identify random amplified polymorphic DNA (RAPD) markers linked to Co-4, and to introgress this gene in the cultivar Rudá. Populations F(1), F(2), F(2:3), BC(1)s, and BC(1)r from the cross Rudá x TO were inoculated with race 65 of Colletotrichum lindemuthianum, causal agent of bean anthracnose. The phenotypic ratios (resistant/susceptible) were 3:1 in the F(2) population, 1:1 in the BC(1)s, and 1:0 in the BC(1)r, confirming that resistance to anthracnose in the cultivar TO was monogenic and dominant. Six RAPD markers linked to the Co-4 gene were identified, four in the coupling phase: OPY20(830C) (0.0 centimorgan [cM]), OPC08(900C) (9.7 cM), OPI16(850C) (14.3 cM), and OPJ01(1,380C) (18.1 cM); and two in the repulsion phase: OPB03(1,800T) (3.7 cM) and OPA18(830T) (17.4 cM). OPY20(830C) and OPB03(1,800T), used in association as a codominant pair, allowed the identification of the three genotypic classes with a high degree of confidence. Marker OPY20(830C), which is tightly linked to Co-4, is being used to assist in breeding for resistance to anthracnose.

Proteinase activity regulation by glycosaminoglycans.

There are few reports concerning the biological role and the mechanisms of interaction between proteinases and carbohydrates other than those involved in clotting. It has been shown that the interplay of enzymes and glycosaminoglycans is able to modulate the activity of different proteases and also to affect their structures. From the large number of proteases belonging to the well-known protease families and also the variety of carbohydrates described as widely distributed, only few events have been analyzed more deeply. The term "family" is used to describe a group of proteases in which every member shows an evolutionary relationship to at least one other protease. This relationship may be evident throughout the entire sequence, or at least in that part of the sequence responsible for catalytic activity. The majority of proteases belong to the serine, cysteine, aspartic or metalloprotease families. By considering the existing limited proteolysis process, in addition to the initial idea that the proteinases participate only in digestive processes, it is possible to conclude that the function of the enzymes is strictly limited to the cleavage of intended substrates since the destruction of functional proteins would result in normal tissue damage. In addition, the location as well as the eventual regulation of protease activity promoted by glycosaminoglycans can play an essential role in the development of several physiopathological conditions.

The value of therapeutic planning and the degree of experience of the surgical team on the results of cancer treatment of the larynx and hypopharynx.

This is a prospective study supported by 170 cases of epidermoid carcinoma of the larynx or hypopharynx, treated during the period from January of 1981 to January of 1988, at the Head and Neck Surgery Service of the Heliópolis Hospital Complex, São Paulo. The objective of this project was to evaluate the importance of surgeon experience with regard to the rates of post-operative complications and the rates of relapse and survival. The results of the 8 surgical specialists who integrate the permanent staff at the institute and who different varying degrees of experience with regard to time spent exercising their specialties were compared. The results obtained did not show a significant difference among the various surgeons, and this uniformity is explained by the fact that all the therapeutic planning was elaborated through consensus of the whole group, and this could have minimized the effect of experience of a surgeon responsible for the operation. The authors emphasize the importance of pre-operative evaluation for good results and propose that it is in the direction of complete mastery of preliminary work in the area that programs for the formation of new specialists should be directed.

Flape cutâneo em padrão axial auricular caudal para correção de defeito extenso após extirpação de fibrossarcoma facial em felino / Use of a caudal auricular axial pattern flap for correction of large defect after facial fribrosarcoma extirpation in a cat

Um felino que apresentava massa de grandes dimensões, localizada no lábio superior direito, foi submetido à cirurgia para exérese do aumento de volume mantendo-se margem de tecido sadio. O defeito facial foi reconstituído com um flape cutâneo em padrão axial auricular caudal. A ferida apresentou cicatrização em primeira intenção em sua maior extensão, com adequados resultados cosmético e funcional. A avaliação histopatológica revelou se tratar de fibrossarcoma, o qual não apresentou recidivas locais pelo período mínimo de 21 meses de pós-operatório.

A cat presented for clinical evaluation with a large mass on the superior lip was submitted to surgery to remove the tumor with the conservation of healthy tissue around it. The facial defect was reconstructed with a cutaneous flap in the caudal auricular axial pattern. The wound presented first intention healing on the major extension, with adequate functional and cosmetic results. According to the histopathological findings, it was a fibrosarcoma, which did not show any sign of relapse in the next 21 months after the surgical intervention.

Intramolecularly quenched fluorogenic tetrapeptide substrates for tissue and plasma kallikreins.

Five intramolecularly quenched fluorogenic substrates for arginyl hydrolases with the sequence Abz-Phe-Arg-X-Y-EDDnp (X = Arg or Ser; Y = Val, Pro, or Arg) were synthesized by classical solution methods. Kinetics of their hydrolysis by tissue and plasma kallikreins, trypsin, and thrombin characterized Abz-Phe-Arg-Ser-Arg-EDDnp as a specific and sensitive substrate for the continuous assay of tissue kallikreins while Abz-Phe-Arg-Arg-Pro-EDDnp was the best substrate for human plasma kallikrein. The five peptides were poor substrates for trypsin and resistant to thrombin.

The anti-leishmanial activity of dipeptide esters on Leishmania amazonensis amastigotes.

L-Amino acid esters, such as L-Leu-OMe, kill Leishmania amazonensis amastigotes by a mechanism which appears to involve ester hydrolysis by cysteine proteinases located in the parasite megasomes. We have examined the killing of isolated amastigotes by L-dipeptide esters and derived some structure-activity correlations. Toxicity of the compounds for the parasites was measured by a tetrazolium (MTT) reduction assay. The results show that active dipeptide esters contained at least 1 hydrophobic amino acid (Leu, Ile, Val, Phe or Trp). The activity of homodipeptide methyl esters depended on the nature of the amino acid, as indicated by the following series: Phe-Phe-OMe greater than Val-Val-OMe greater than Leu-Leu-OMe greater than Trp-Trp-OMe greater than Ile-Ile-OMe. The nature of the amino acids in Leu-X-OMe and X-Leu-OMe was relatively unimportant when X was Phe, Trp or Val. However, when X was Ala or Gly, Leu-X-OMe was several-fold more active than X-Leu-OMe. A similar preference for the more hydrophobic residue in the amino terminal position was also found in esters containing a single phenylalanine or valine. Protection of the amino group by benzyloxycarbonyl (Z) or t-butyloxycarbonyl (BOC) substituents markedly enhanced the activity of the esters. An-mPhe-Gly-OEt, a retro-inverso analogue of Bz-Phe-Gly-OEt, was several-fold more active than the parent compound. Selected esters were assayed on infected macrophages and concentrations that induced minimal toxicity to the host cells were estimated. The ED50s for intracellular parasites were 1.5 to 5-fold higher than those for isolated amastigotes.(ABSTRACT TRUNCATED AT 250 WORDS)

Photographic defocusing: a means to render recognizable line traced models of negatively stained biological specimens.

A simple method of obtaining analogue images from traced models of biological specimens is presented. It consists of the photographic defocusing of traced models and it is illustrated with negatively stained cylindrical forms of the ASFV; the black lines of the trace in the model correspond to the negative stain surrounding the viral morphological subunits as seen in the electron micrograph. The photographic defocusing is the means by which the traced model is filtered and is used to introduce grey levels on an otherwise black and white image. The right amount of defocusing is attained when the width of the trace of the model equals the width of the rim of the negative stain appearing between the morphological subunits in the electron micrograph.

Determinants of tissue kallikrein cleavage specificity in the limited proteolysis of kininogens.

Further evidence for interactions at tissue kallikrein extended binding sites, as determinants of the kininogen cleavage specificities is presented. Differences in the cleavage sites in kininogen hydrolysis by rat and other tissue kallikreins is related to subsite S1' specificity, while the low susceptibility of rat kininogen to horse tissue kallikrein is explained by the difference in their subsite S3'.