Toward the Identification of Novel Antimicrobial Agents: One-Pot Synthesis of Lipophilic Conjugates of N-Alkyl d- and l-Iminosugars

In the effort to improve the antimicrobial activity of iminosugars, we report the synthesis of lipophilic iminosugars 10a–b and 11a–b based on the one-pot conjugation of both enantiomeric forms of N-butyldeoxynojirimycin (NBDNJ) and N-nonyloxypentyldeoxynojirimycin (NPDNJ) with cholesterol and a succinic acid model linker. The conjugation reaction was tuned using the established PS-TPP/I₂/ImH activating system, which provided the desired compounds in high yields (94–96%) by a one-pot procedure. The substantial increase in the lipophilicity of 10a–b and 11a–b is supposed to improve internalization within the bacterial cell, thereby potentially leading to enhanced antimicrobial properties. However, assays are currently hampered by solubility problems; therefore, alternative administration strategies will need to be devised.     

rBmTI-6, a Kunitz-BPTI domain protease inhibitor from the tick Boophilus microplus, its cloning, expression and biochemical characterization

Boophilus microplus is a rich source of trypsin inhibitors, numerous Kunitz-BPTI (bovine pancreatic trypsin inhibitor) inhibitors have been described from larvae and eggs, named BmTIs. Among them, were characterized inhibitors for trypsin, human neutrophil elastase, human plasma kallikrein and plasmin. BmTIs elicited a protective immunological response against B. microplus infestation in cattle. However, only a small amount of purified natural BmTIs can be obtained from larvae and eggs by chromatographic methods, thus if BmTIs are to be used as vaccine antigens (immunogens) the production of recombinant BmTIs (rBmTIs) is essential. In this work we describe the cloning, expression, purification and characterization of rBmTI-6. rBmTI-6 is a three-headed Kunitz-BPTI inhibitor, expressed in the Pichia pastoris system. Although rBmTI-6 was processed by proteases and glycosylated during the expression process, these post-translational modifications did not alter the ability of rBmTI-6 to inhibit protease activity. Purified rBmTI-6 inhibited trypsin and plasmin.     

Identification of coagulase-negative staphylococci from bovine mastitis using RFLP-PCR of the groEL gene

Coagulase-negative staphylococci (CNS) have become the predominant pathogens causing bovine mastitis in many countries. CNS infections are associated with damage to milk secretory tissue of the mammary gland by increased connective tissue stroma, moderate increases of somatic cells count in milk and significant production decreases. These consequences impose serious economic losses for the farmers and the dairy industry. Routine veterinary laboratories do not usually identify CNS at the species level. Thereby, the aims of this study were to identify the most common staphylococcal pathogens involved in bovine mastitis using PCR-restriction fragment length polymorphism (RFLP) analysis of a partial groEL gene sequence and to compare our results with the identification carried out by the conventional method. A total of 54 isolates of Staphylococcus, involved in bovine mastitis, were analyzed by this method. The size and number of the fragments obtained by either AluI or HindIII/PvuII digestions made possible to form clear patterns differentiating, among the isolates, 11 of the most common species of animal staphylococcal pathogens. Most of the isolates clustered together with the reference strain of Staphylococcus chromogenes (28) and the type strain of Staphylococcus epidermidis (8). Besides, some isolates clustered together with the type strain of Staphylococcus aureus (5). All patterns were confirmed by the conventional biochemical method, showing concordant results. Thus, the PCR-RFLP of the groEL gene constitutes a reliable and reproducible molecular method for identification of CNS species responsible for bovine mastitis.     

Bicortical implant insertion in caudal cervical spondylomyelopathy: a computed tomography simulation in affected Doberman Pinschers

Objective— To evaluate the safety of the angle of insertion of bicortical implants in Doberman Pinschers affected by caudal cervical spondylomyelopathy (CCSM) using simulation on computed tomography (CT) images.
Animals— Doberman Pinschers (n=9) with dynamic spinal compression attributed to CCSM.
Methods— Dogs had CT evaluation of the cervical vertebral column. Digital images were used to evaluate the safety of bicortical implant insertion from the midline at 30, 35, and 40° inclination by drawing a line simulating implant position. Correct surgical positioning of dogs was evaluated by measuring the degree of rotation of the spine along the longitudinal axis.
Results— Insertion of an implant at 30, 35, and 40° inclination was considered safe in 42%, 59%, and 67% of selected sites. Rotation of the spine from 9° counter clockwise to 10° clockwise along the longitudinal axis occurred in 53% of the areas considered.
Conclusions— These results suggest that bicortical implants in the caudal cervical area in Doberman Pinschers affected by CCSM carry a high risk of vertebral canal and intervertebral foramina violation.
Clinical Relevance— Bicortical implants inserted at 30 and 35° in the caudal cervical area in Doberman Pinschers affected by CCSM carry a high risk of vertebral canal and intervertebral foramina violation. CT planning for individual dogs can facilitate selection of the appropriate insertion plane and vertebral column rotation needs to be considered in surgical planning.     

Intranasal Osteosarcoma in a Dog—A Case Report

Osteosarcoma is the most commonly reported bone tumor in dogs, typically affecting the axial and appendicular skeleton. Involvement of the paranasal sinuses and the nasal cavity is extremely rare. This report describes a 6-year-old, female, cross-breed dog presented with signs of 90 days duration and diagnosed with a mass located in the right nasal cavity. Radiography, rhinoscopy, and computed tomography findings suggested the presence of an intranasal tumor. Osteosarcoma in the nasal cavity was diagnosed based on histopathological examination and clinical and imaging findings. Survival time was 12 months from diagnosis.     

An investigation of Leishmania spp. in Didelphis spp. from urban and peri-urban areas in Bauru (São Paulo, Brazil)

Blood and bone marrow samples were taken from 112 Didelphis spp., collected between March 2005 and February 2006, from urban and peri-urban areas of Bauru, São Paulo State, Brazil, to evaluate the hypothesis that these animals might constitute a reservoir of Leishmania spp. Anti-Leishmania ssp. antibodies were screened in the serum samples using an enzyme-linked immuno-sorbent assay (ELISA) and the polymerase chain reaction (PCR). PCR was performed on fragments of DNA samples from Leishmania spp. using primers 13A and 13B, and showed a positive outcome in 91.6% of the 112 samples tested. Of the 107 samples analyzed by ELISA, 71% were positive. Evidence of epidemiological risk factors such as a circulating parasite and freely moving vectors suggests that Didelphis spp. may participate in the transmission cycle of Leishmania spp. in Bauru.     

Agarose gel electrophoresis of cerebrospinal fluid proteins of dogs after sample concentration using a membrane microconcentrator technique

BACKGROUND: Cerebrospinal fluid (CSF) is produced in the cerebral ventricles through ultrafiltration of plasma and active transport mechanisms. Evaluation of proteins in CSF may provide important information about the production of immunoglobulins within the central nervous system as well as possible disturbances in the blood-brain barrier. OBJECTIVE: The objective of this study was to measure the concentration and fractions of protein in CSF samples using a membrane microconcentrator technique followed by electrophoresis, and to compare the protein fractions obtained with those in serum. METHODS: CSF samples from 3 healthy dogs and 3 dogs with canine distemper virus infection were concentrated using a membrane microconcentrator having a 0.5 to 30,000 d nominal molecular weight limit (Ultrafree, Millipore, Billerica, MA, USA). Protein concentration was determined before and after concentration. Agarose gel electrophoresis was done on concentrated CSF samples, serum, and serial dilutions of one of the CSF samples. RESULTS: Electrophoretic bands were clearly identified in densitometer tracings in CSF samples with protein concentrations as low as 1.3 g/dL. The higher CSF protein concentration in dogs with distemper was mainly the result of increased albumin concentration. CONCLUSION: The microconcentrating method used in this study enables characterization of the main protein fractions in CSF by routine electrophoresis and may be useful for interpreting the underlying cause of changes in CSF protein concentrations.