Defining a Molecular Mechanism for Lead Toxicity via Calcium-Binding Proteins

Essential metals like Ca2+ and Zn2+ play critical roles in biological processes through protein interactions. Conversely, non-essential metals (e.g., Gd3+ and Pb2+) also interact with proteins, often with toxic effects. Molecular metal toxicity is assumed to be due to ionic displacement, and studies have demonstrated that Pb2+ replaces Zn2+, Ca2+ and other essential metals in proteins. The focus of this work was to compare protein Ca2+ and Pb2+ -binding sites and to investigate a mechanism of Pb2+ toxicity in Ca2+-binding proteins, particularly the intracellular trigger protein calmodulin (CaM) which binds four Ca2+ ions and interacts with numerous molecular targets via Ca2+-induced conformational change. A statistical analysis of PDB structural data for Pb2+ and Ca2+-binding (EF-hand and non-EF-hand) proteins revealed fewer binding ligands in Pb2+ sites (4 ± 2), than non-EF-Hand (6 ± 2) and EF-Hand (7 ± 1) Ca2+-binding sites. Pb2+ binds predominantly with sidechain Glu (38.4%), which is less prevalent in both non-EF-Hand (10.4%) and EF-Hand (26.6%) sites. Interestingly, analyses of proteins where Pb2+ replaces Ca2+ (calmodulin) or Zn2+ (5-aminolaevulinic acid dehydratase) revealed structural changes presumably unrelated to ionic displacement. These results suggested that Pb2+ adopts diverse binding geometries and that opportunistic binding outside of known Ca2+-binding sites may play a role in molecular metal toxicity. Ca2+-binding affinities (Kd) using phenylalanine and tyrosine fluorescence were found to be 1.15 ± 0.68 X 10-5 M and 2.04 ± 0.02 X 10-6 M for the N- and C-terminal domains, respectively. The Kd for Pb2+-binding in the N-terminal domain, 1.40 ± 0.30 X 10-6 M, was 8-fold higher than Ca2+. Binding of Pb2+ in the C-terminal domain produced a biphasic response with Kd values 7.34 ± 0.95 X 10-7 M and 1.93 ± 0.32 X 10-6 M, suggesting a single higher affinity Pb2+-binding site in the C-terminal domain with nearly equivalent affinity for the remaining sites. Competitive effects of Pb2+ added to Ca2+-loaded CaM were examined using multiple NMR techniques. Pb2+ was found to displace Ca2+ only in the N-terminal domain, however structural/dynamic changes were observed in the central helix apparently due to Pb2+-binding in secondary sites. These data supported our hypothesis that CaM structure and function is altered by opportunistic Pb2+-binding

Analyses and Applications of Metalloprotein Complexes

The structural characteristics associated with the binding of beneficial metals (i.e. - Mg2+, Zn2+ and Ca2+) to natural proteins has typically received more attention than competitive binding by toxic metals (e.g. – Pb2+, Hg2+, Cd2+, La3+, etc.). In this thesis, a statistical analysis of Pb2+-binding in crystallized protein structures indicates that Pb2+ does not bind preferentially with nitrogen, as generally assumed, but binds predominantly with oxygen, and to a lesser degree, sulfur. A comparison of Ca2+ and Pb2+ indicates that Pb2+ binds with a wider range of coordination numbers, with less formal change, and with less defined structure than Ca2+. The Pb2+ ion also appears to displace Ca2+ with little conformational stress in calcium binding proteins (CaBP’s). Experimental data from the binding of metals with engineered fluorescent proteins indicate that both Pb2+ and Gd3+ will occupy grafted calcium-binding sites with greater affinity than Ca2+, and strong evidence is presented to support the hypothesis that Pb2+ and Gd3+ will bind non-specifically on the protein surface. These results suggest that toxicity is associated with two binding mechanisms: displacement of the metal cofactor which disrupts protein function, and non-specific binding which maintains higher solubility of the metal

Cervical porcupine quill foreign body involving the spinal cord of a dog: a description of various imaging modality findings

Although porcupine quill injuries are common in dogs, the detailed appearance of the quill on diagnostic ultrasound, computed tomography, and magnetic resonance imaging has not been sufficiently described. A 4-year-old, intact, female Jack Russel terrier presented with severe neck pain and ataxia after an altercation with a porcupine 2 weeks earlier. Radiology, diagnostic ultrasound, computed tomography and magnetic resonance imaging were all utilised to identify a quill imbedded in the cervical vertebral canal and cervical musculature and were compared to each other. Surgical removal of the quill, guided by imaging findings, led to the resolution of the clinical signs in the patient. Previous ultrasound imaging reports have just stated that the quill consists of parallel hyperechoic lines, and do not mention the finer hyperechoic lines in between and do not try to provide a reason for the appearance. Previous computed tomography (CT) reports just mention identifying the quill on CT images (whether or not CT could identify the fragments), but do not go into detail about the attenuating appearance of the quill nor try to relate this to the composition of the quill. This is to the authors’ knowledge the first report with detailed imaging descriptions of a case of cranial cervical vertebral canal porcupine quill foreign body in a dog. This is also the first report to allude to a possible difference in imaging findings related to quill structure because of keratin orientation and melanin content. The ideal imaging modality to use remains elusive, but ultrasound, computed tomography and magnetic resonance imaging could all identify the quill

Echocardiographic indices in normal German shepherd dogs

Sixty clinically normal German shepherd dogs, 31 males and 29 females, ranging in age from 1 and 5 years and with a body weight ranging from 22 to 37.2 kg, were examined by the two-dimensional mode, M-mode, and Doppler echocardiography. In Doppler mode, the mitral valve flows were obtained, where the aim was to determine the velocity peaks and ratios of the E and A waves and the mitral E wave deceleration time. The velocity peaks were obtained for the tricuspid, pulmonary and aortic valves. On the left ventricular outflow tract flow, the time velocity integral and aortic cross-sectional area was used to calculate the cardiac output. A statistically significant correlation with the body weight was found for the systolic left atrial and diastolic aortic diameter on two-dimensional mode. On M-mode, there was a significant correlation between the body weight and the systolic left atrium and diastolic aortic dimension, systolic and diastolic left ventricular, septal and posterior wall dimensions. Doppler echocardiography showed that there was no significant correlation between the body weight and the mitral, tricuspid, pulmonary and aortic valves flows. These results demonstrate that it is important to know the normal echocardiographic values for German shepherd dogs because there are some characteristics peculiar to this particular breed. The data obtained is expected to be helpful for studies on small animal cardiology

Diagnostic imaging of migrating kebab (sosatie) sticks - a review of 8 cases

Complications related to extraluminal migration of ingested kebab (sosatie) sticks are infrequently diagnosed in small animals. A total of 8 cases diagnosed with extragastric migration of ingested kebab sticks were retrospectively evaluated. No significant breed or sex predilection was found but there was a tendency for animals to present at a younger age (less than 3 years). Clinical signs (of variable duration) were non-specific and included haemoptysis, abdominal pain, regurgitation, subcutaneous abscessation and chronic draining sinus tracts, making a clinical diagnosis difficult. Ultrasonography proved invaluable in facilitating the diagnosis of kebab stick migration in 6 of the cases and computed tomography unexpectedly identified a kebab stick that had migrated into the thorax in 1 patient. Survey radiography was generally found to be insensitive in identifying the kebab sticks. The aim of this article is to alert veterinarians to a clinical syndrome that may not be considered a differential diagnosis in patients with non-specific inflammatory disease of the thorax, abdomen or pelvic regions and to illustrate the usefulness of the various diagnostic imaging modalities in facilitating a diagnosis of kebab stick ingestion and its possible secondary complications.http://www.journals.co.za/ej/ejour_savet.htm

Osteochondroma and multiple cartilagenous exostosis involving the distal radius and ulna on adjacent cortices : an unusual manifestation of a common condition

The findings of two canine patients presenting with distal antebrachial multiple cartilaginous exostoses (MCE) highlights the difference in presentation and clinical significance of MCE impinging on the adjacent radius and ulna. We suspect that a lesion in this region, affecting the adjacent bones, may be missed as a cause of lameness. This may be due to a lack of knowledge of the normal radiological anatomy of this region on a mediolateral view, and because of the close anatomical association between the distal ulna and radius and thus superimposition on radiographs. Additionally, skeletal immaturity may mask the exostosis as the thicker cartilage cap is not visible radiologically. CT allows a much better understanding of the pathology involved due to its cross-sectional imaging.http://vetrecordcasereports.bmj.comhb201

Low-field magnetic resonance imaging and multislice computed tomography for the detection of cervical syringomyelia in dogs

Background: Syringomyelia (SM) is defined as the presence of fluid- containing cavities within the parenchyma of the spinal cord. Sagittal magnetic resonance (MR) images have been described as the preferred technique for visualizing SMin dogs and humans. Objective: To investigate whether computed tomography (CT) can be used to diagnose SM. Animals: Thirty- two client- owned dogs referred for investigation of the cervical spine on magnetic resonance imaging (MRI) and CT. Methods: Two reviewers retrospectively analyzed sagittal and transverse T1- weighted spin echo (T1WSE) MR images and CT images from each dog for the presence of SM and, if SM was present, the width (mm, syrinx width [ SW]) was measured. The results were analyzed statistically. Results: For the presence of SM there was a moderate interobserver agreement for MR (81%, j = 0.54) and almost perfect agreement for CT (94%, j = 0.87). There was a moderate intramodality agreement for both observers (observer 1 81%, j = 0.59; observer 2 81%, j = 0.57). For measurement of SW the repeatability was the best on the midsagittal T1WSE images (95% repeatability coefficient < 0.52 mm) and the reproducibility was the best on midsagittal images in both modalities (95% limits of agreement 0.55- 0.45; P = 0.002). Conclusion and Clinical Importance: Both techniques can be used to detect SM. Midsagittal MR and CT images are best used for measuring SW. Computed tomography can be used as a diagnostic tool for SM when MRI is not available, but CT cannot replace MRI as the standard screening technique for the detection of SM in Cavalier King Charles Spaniel for breeding purposes

Hepatocutaneous syndrome in a Maltese, diagnosis, treatment and the value of CT in the diagnosis

A 10-year-old Maltese neutered female was presented for lip dermatitis and mild generalised pruritus. On examination, there were multiple lesions affecting the peri-oral and peri-nasal mucocutaneous junctions as well as distal limbs. Serum chemistry and a bile acids test were indicative of hepatocellular injury and hepatic dysfunction. Plasma amino acids were markedly depleted. Ultrasonography of the liver revealed a honeycombed appearance. A CT scan was performed to rule out a porto-vascular abnormality, hepatic or pancreatic neoplasia. Abnormalities in hepatic architecture are described in this confirmed case of hepatocutaneous syndrome (HCS) for the first time on CT. This is the first reported case of HCS in Hong Kong. The dog was treated with a high-quality protein diet and amino acids transfusions. Eleven months after presentation the dog was still alive with good control of clinical signs.http://vetrecordcasereports.bmj.comhj2020Companion Animal Clinical Studie

Computed tomographic, ultrasonographic, MRI and radiographic findings of a venous malformation with phleboliths in the thoracic limb of a dog

An 11-month-old entire male Rottweiler was referred for further investigation of raised, soft, compressible, non-pulsatile, subcutaneous tubular structures along the right antebrachium that had been diagnosed to be a vascular malformation on histopathology of a subcutaneous biopsy. Computed tomographic (CT) with angiography did not reveal an arteriovenous malformation or fistula. Ultrasonography of the limb demonstrated a venous malformation with thrombi and phleboliths. An MRI study of the limb was consistent with a venous malformation with small rounded luminal signal voids that were confirmed to be phleboliths. Radiography at follow-up demonstrated phleboliths. This is the first report describing the combined CT, ultrasonographic, MRI and radiographic characteristics of venous malformation with phleboliths in a limb of a dog.https://wileyonlinelibrary.com/journal/vrc2Companion Animal Clinical Studie