Laboratory Diagnosis of Canine Pyometra

The principal laboratory test used to confirm the pyometra diagnosis in the bitch has been the determination of the total white blood cell count in venous blood. A marked elevation is known to be a characteristic of the disease. In the present study, the white blood cell count was determined as well as the γ-globulin level. An elevation of the γ-globulin level and the total white blood cell count was very characteristic to the pyometra patients. The increase in the number of white blood cells nor the high γ-globulin level cannot be regarded specific for pyometra, therefore it was regarded important to find out a more specific test for pyometra. When sonicated E. coli bacteria were tested against sera from pyometra patients in electroimmunodiffusion, the precipitation was almost always detected when E. coli had been isolated from the uterus. This technique provides a quick method in detecting the causative E. coli infection. The present study suggests that whenever laboratory tests are used to confirm the pyometra diagnosis by the total white blood cell count, it is advantageous to analyze the total γ-globulin level in the serum as well as specific antibodies against a common E. coli antigen. Because of the reliability of the glutaraldehyde coagulation test and the simple technique, this can be suggested as the method of choice for an average small animal practice.     

A Compact Native 24-Residue Supersecondary Structure Derived from the Villin Headpiece Subdomain

Many small proteins fold highly cooperatively in an all-or-none fashion and thus their native states are well protected from thermal fluctuations by an extensive network of interactions across the folded structure. Because protein structures are stabilized by local and nonlocal interactions among distal residues, dissecting individual substructures from the context of folded proteins results in large destabilization and loss of unique three-dimensional structure. Thus, mini-protein substructures can only rarely be derived from natural templates. Here, we describe a compact native 24-residues-long supersecondary structure derived from the hyperstable villin headpiece subdomain consisting of helices 2 and 3 (HP24). Using a combination of experimental techniques, including NMR and small-angle x-ray scattering, as well as all-atom replica exchange molecular-dynamics simulations, we show that a variant with stabilizing substitutions (HP24stab) forms a densely packed and compact conformation. In HP24stab, interactions between helices 2 and 3 are similar to those observed in native folded HP35, and the two helices cooperatively stabilize each other by completing the hydrophobic core lining the central part of HP35. Interestingly, even though the HP24wt fragment shows a more expanded and less structured conformation, NMR and simulations demonstrate a preference for a native-like topology. Thus, the two stabilizing residues in HP24stab shift the energy balance toward the native state, leading to a minimal folding motif.     

Indigestion in Young Calves. II. The Influence Of Ground Barley,Coarse and Fine Hay

Four experiments comprising 86 calves have been carried out in order to examine the influence of ground barley, coarse and fine hay, when raising young calves on a high lactose milk replacer causing diarrhoea. The frequency of diarrhoea decreased when feeding barley (P < 0.01) or fine hay (P < 0.001), but increased when feeding coarse hay (P<0.01). Barley increased (P < 0.01) live weight gain and carcass weight, reduced (P < 0.001) rumen pH, favoured gram-positive cocci and rods in the rumen, caused hyperkeratosis and gave increased (P < 0.01) empty reticulo-rumen weights, the latter being supposed to be enhanced by hyperkeratosis. The calves ate more (P < 0.001) fine than coarse hay, fine hay being superior (P < 0.01) in promoting growth of the rumen. All hay maintained a predominantly gram-negative rumen flora, but somewhat different from that on the milk replacer, and counteracted (P < 0.01) low pH in the rumen and apparently also hyperkeratosis due to barley. The results with barley indicated no relationship between diarrhoea and hyperkeratosis.     

Comparative Studies of Bacillus Cereus Strains Isolated From Various Foods and Food Poisoning Outbreaks

Certain properties of 22 Bacillus cereus strains isolated from different foods and food poisoning episodes were investigated in order to evaluate possible différences between strains isolated from diarrhoeal and vomiting type food poisoning outbreaks. None of the strains isolated from vomiting type episodes produced acid from salicin and mannose, whereas 80 and 40 % of the strains from diarrhoeal type outbreaks were positive, respectively. No association between the antibiotic sensitivity pattern or the fatty acid composition and the source of a strain could be found, although some strains differed from the general pattern of B. cereus in some instances. No significant differences in the production of the skin factor between strains isolated from the two types of outbreaks were found either. The findings of this study support the observation that the food environment itself essentially affects the enterotoxin formation of B. cereus.     

Analysis of IgG kinetic stability by differential scanning calorimetry,probe fluorescence and light scattering

Monoclonal antibodies of the immunoglobulin G (IgG) type have become mainstream therapeutics for the treatment of many life‐threatening diseases. For their successful application in the clinic and a favorable cost‐benefit ratio, the design and formulation of these therapeutic molecules must guarantee long‐term stability for an extended period of time. Accelerated stability studies, e.g., by employing thermal denaturation, have the great potential for enabling high‐throughput screening campaigns to find optimal molecular variants and formulations in a short time. Surprisingly, no validated quantitative analysis of these accelerated studies has been performed yet, which clearly limits their application for predicting IgG stability. Therefore, we have established a quantitative approach for the assessment of the kinetic stability over a broad range of temperatures. To this end, differential scanning calorimetry (DSC) experiments were performed with a model IgG, testing chaotropic formulations and an extended temperature range, and they were subsequently analyzed by our recently developed three‐step sequential model of IgG denaturation, consisting of one reversible and two irreversible steps. A critical comparison of the predictions from this model with data obtained by an orthogonal fluorescence probe method, based on 8‐anilinonaphthalene‐1‐sulfonate binding to partially unfolded states, resulted in very good agreement. In summary, our study highlights the validity of this easy‐to‐perform analysis for reliably assessing the kinetic stability of IgGs, which can support accelerated formulation development of monoclonal antibodies by ranking different formulations as well as by improving colloidal stability models.     

Age-dependent cyclic locomotor activity in the cricket, Gryllus bimaculatus, and the effect of adipokinetic hormone on locomotion and excitability

Excitability and locomotor activity of male and female last instar larvae and adults of the two-spotted cricket are measured under crowded conditions, allowing the animals to interact with conspecifics during observations. Male and female last instar larvae display age-dependent cyclic patterns of activity with maxima during early to mid scotophase and minima during early photophase. A period of low locomotor activity without time of day-dependent cyclic changes starts 1 day before the final moult and lasts until 1 day after the moult. Then, both excitability and locomotor activity increase and become cyclic again within 2 or 3 days. The cyclic changes gradually dampen in adult females older than 6 days and finally cease. When injected into photophase larvae and adults, adipokinetic hormone (AKH) increases excitability and locomotor activity in a dose-dependent manner, whereas it has no such effect when injected into scotophase animals. Other behaviours (jumping, hind wing trembling) that mostly occur in scotophase crickets are also increased by injecting AKH into photophase crickets. We argue that AKH could be responsible for linking the endogenous clock output with the cyclic changes in locomotor activity. Furthermore, AKH may serve to synchronise metabolism and behaviour to optimise larval development and reproduction.     

Elimination of Homologous And Heterologous (Bovine) Serum Albumin from the Blood Circulation in the Dog)

The elimination of intravenously administered I131-labelled bovine serum albumin has been compared to the elimination rate of relabelled homologous serum albumin in normal and bled dogs, which had lost considerable blood volumes. The investigation shows that during the first four to five days after the administration the elimination is similar of heterologous and homologous serum albumin. This proves that bovine serum albumin can be regarded to be an equivalent plasma expander to homologous serum albumin in the dog. Elimination of homologous as well as heterologous serum albumin follows a simple exponential curve during four to five days after administration. The intravascular half-lives for homologous serum albumin were 6.4 ±1.5 days and 6.4 ± 0.6 days respectively in control and bled dogs. Corresponding values for heterologous (bovine) serum albumin were 5.0 ± 0.3 and 4.8 ± 0.4 days respectively. The quote for cencentrations of homologous and heterologous serum albumin in different tissues was found to be relatively constant approximately 1.4. An exception was the stomach wall in bled dogs which had a quote of 1.1 only.     

Conformational stability and dynamics of cytochrome <Emphasis Type="Italic">c</Emphasis> affect its alkaline isomerization

The alkaline isomerization of horse heart ferricytochrome c (cyt c) has been studied by electronic absorption spectroscopy in the presence of the Hofmeister series of anions: chloride, bromide, rhodanide and perchlorate. The anions significantly affect the apparent pK _a value of the transition in a concentration-dependent manner according to their position in the Hofmeister series. The Soret region of the absorption spectra is not affected by the presence of the salts and shows no significant structural perturbation of the heme crevice. In the presence of perchlorate and rhodanide anions, the cyanide exchange rate between the bulk solvent and the binding site is increased. These results imply higher flexibility of the protein structure in the presence of chaotropic salts. The thermal and isothermal denaturations monitored by differential scanning calorimetry and circular dichroism, respectively, showed a decrease in the conformational stability of cyt c in the presence of the chaotropic salts. A positive correlation between the stability, ΔG, of cyt c and the apparent pK _a values that characterize the alkaline transition indicates the presence of a thermodynamic linkage between these conformational transitions. In addition, the rate constant of the cyanide binding and the partial molar entropies of anions negatively correlate with the pK _a values. This indicates the important role of anion-induced solvent reorganization on the structural flexibility of cyt c in the alkaline transitions. Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users.