The nonuniformity of antibody distribution in the kidney and its influence on dosimetry

The therapeutic efficacy of radiolabeled antibody fragments can be limited by nephrotoxicity, particularly when the kidney is the major route of extraction from the circulation. Conventional dose estimates in kidney assume uniform dose deposition, but we have shown increased antibody localization in the cortex after glomerular filtration. The purpose of this study was to measure the radioactivity in cortex relative to medulla for a range of antibodies and to assess the validity of the assumption of uniformity of dose deposition in the whole kidney and in the cortex for these antibodies with a range of radionuclides. Storage phosphor plate technology (radioluminography) was used to acquire images of the distributions of a range of antibodies of various sizes, labeled with 125I, in kidney sections. This allowed the calculation of the antibody concentration in the cortex relative to the medulla. Beta-particle point dose kernels were then used to generate the dose-rate distributions from 14C, 131I, 186Re, 32P and 90Y. The correlation between the actual dose-rate distribution and the corresponding distribution calculated assuming uniform antibody distribution throughout the kidney was used to test the validity of estimating dose by assuming uniformity in the kidney and in the cortex. There was a strong inverse relationship between the ratio of the radioactivity in the cortex relative to that in the medulla and the antibody size. The nonuniformity of dose deposition was greatest with the smallest antibody fragments but became more uniform as the range of the emissions from the radionuclide increased. Furthermore, there was a strong correlation between the actual dose-rate distribution and the distribution when assuming a uniform source in the kidney for intact antibodies along with medium- to long-range radionuclides, but there was no correlation for small antibody fragments with any radioisotope or for short-range radionuclides with any antibody. However, when the cortex was separated from the whole kidney, the correlation between the actual dose-rate distribution and the assumed dose-rate distribution, if the source was uniform, increased significantly. During radioimmunotherapy, the extent of nonuniformity of dose deposition in the kidney depends on the properties of the antibody and radionuclide. For dosimetry estimates, the cortex should be taken as a separate source region when the radiopharmaceutical is small enough to be filtered by the glomerulus.     

One eye but no vision: cave fish with induced eyes do not respond to light

One of the most intriguing questions in evolutionary biology is the degree to which behavior is a necessary consequence of morphology. We explore this issue by examining phototactic behavior in epigean (eyed surface-dwelling) and troglomorphic (blind cave) forms of the teleost Astyanax fasciatus whose eyes were modified during embryogenesis by removing one or both lens vesicles from the epigean form or by transplanting the lens vesicle from an epigean fish into the optic cup of a blind cave form. Lens removal results in eye degeneration and blindness in adult epigean fish, whereas lens transplantation stimulates growth of the eye, inducing the development of optic tissues in the normally eyeless adult cave fish. Photoresponsiveness was examined by placing fish in an aquarium with one half illuminated and the other half dark and scoring their presence in the illuminated or dark half. Both the eyeless epigean fish and cave fish with induced eyes are indifferent to the illumination whereas the surface forms are scotophilic, suggesting that optic development and phototactic behavior are decoupled.     

Interaction of the bullous pemphigoid antigen 1 (BP230) and desmoplakin with intermediate filaments is mediated by distinct sequences within their COOH terminus

The bullous pemphigoid antigen 1 (BP230) and desmoplakin (DP) are members of the plakin protein family of cytolinkers. Despite their homology, their COOH termini selectively bind distinct intermediate filaments (IFs). We studied sequences within their COOH termini required for their interaction with the epidermal keratins K5/K14, the simple epithelial keratins K8/K18, and type III IF vimentin by yeast three-hybrid, cell transfection, and overlay assays. The results indicate that BP230 interacts with K5/K14 but not with K8/K18 or vimentin via a region encompassing both the B and C subdomains and the COOH extremity, including a COOH-terminal eight-amino-acid stretch. In contrast, the C subdomain with the COOH-terminal extremity of DP interacts with K5/K14 and K8/K18, and its linker region is able to associate with K8/K18 and vimentin. Furthermore, the potential of DP to interact with IF proteins in yeast seems to be regulated by phosphorylation of Ser 2849 within its COOH terminus. Strikingly, BP230 and DP interacted with cytokeratins only when both type I and type II keratins were present. The head and tail domains of K5/K14 keratins were dispensable for their interaction with BP230 or DP. On the basis of our findings, we postulate that (1) the binding specificity of plakins for various IF proteins depends on their linker region between the highly homologous B and C subdomains and their COOH extremity and (2) the association of DP and BP230 with both epidermal and simple keratins is critically affected by the tertiary structure induced by heterodimerization and involves recognition sites located primarily in the rod domain of these keratins.     

The systematic utility of floral and vegetative fragrance in two genera of nyctaginaceae

We examined relationships between fragrance and phylogeny using a number of approaches to coding fragrance data and comparing the hierarchical information in fragrance data with the phylogenetic signal in a DNA sequence data set. We first used distance analyses to determine which coding method(s) best distinguishes species while grouping conspecifics. Results suggest that interspecific differences in fragrance composition were maximized by coding as presence/absence of fragrance compounds and biosynthetic pathways rather than when quantitative information was also included. Useful systematic information came from both compounds and pathways and from fragrance emitted by both floral and vegetative tissues. The coding methods that emerged from the distance analyses as best distinguishing species were then adapted for use in phylogenetic analysis. Although hierarchical signal among fragrance data sets was congruent, this signal was highly incongruent with the phylogenetic signal in the DNA sequence data. Notably, topologies inferred from fragrance data sets were congruent with the DNA topology only in the most distal portions (e.g., sister group pairs or closely related species that had similar fragrance profiles were often recovered by analyses of fragrance). Examination of consistency and retention indices for individual fragrance compounds and pathways as optimized onto one of the most-parsimonious trees inferred from DNA data revealed that although most compounds were homoplastic, some compounds were perfectly congruent with the DNA phylogeny. In particular, compounds and pathways found in a few taxa were less homoplastic than those found in many taxa. Pathways that synthesize few volatiles also seem to have lower homoplasy than those that produce many. Although fragrance data as a whole may not be useful in phylogeny reconstruction, these data can provide additional support for clades reconstructed with other types of characters. Factors other than phylogeny, including pollinator interactions, also likely influence fragrance composition.     

Renal C-type natriuretic peptide and natriuretic peptide receptor B mRNA expression are affected by water deprivation in the Spinifex Hopping mouse

This study investigated the effect of water deprivation on the expression of C-type natriuretic peptide (CNP) and natriuretic peptide receptor B (NPR-B) mRNA, and the ability of NPR-B to generate cGMP in the Spinifex Hopping mouse, Notomys alexis. This rodent is a native of central and western Australia that is well adapted to survive in arid environments. Initially, CNP and NPR-B cDNAs (partial for NPR-B) were cloned and sequenced, and were shown to have high homology with those of rat and mouse. RT-PCR analysis showed CNP mRNA expression in the kidney, proximal and distal colon and small intestine, whilst NPR-B mRNA expression was found in the kidney, proximal and distal colon and the atria. Using a semi-quantitative multiplex PCR technique, the expression of renal CNP and NPR-B mRNA was determined in 7- and 14-day water-deprived hopping mice, in parallel with control hopping mice (access to water). Water deprivation significantly decreased the relative levels of CNP and NPR-B mRNA expression in both the 7- and 14-day water-deprived hopping mice, when compared to control hopping mice. In contrast, the ability of CNP to stimulate cGMP production was significantly increased after 14 days of water deprivation. This study shows that alterations in the renal CNP/NPR-B system may be an important physiological adjustment when water is scarce.     

Husbandry, handling, and nutrition for marmosets

Marmosets, especially Callithrix jacchus, have become an established part of the laboratory animal community. Information on marmoset life history, behavior, and diet acquired from experience with natural and captive habitats has increased, but the early information from workers with colonies, principally those of tamarins, has led to some common perceptions about how to house, handle, and especially, feed callitrichids that may not apply to marmoset requirements. The availability of commercially produced, almost-complete base diet components and a wider variety of cage construction materials, combined with the recent emphasis on the integration of engineering and performance standards for housing, have made captive life and the implementation of research requirements better for the animals and the people that work with them. We will review some of the routine aspects of husbandry, handling, and nutrition for marmoset monkeys maintained in a research setting.     

CCK-58 is the only detectable endocrine form of cholecystokinin in rat

CCK-58 differs from CCK-8 in patterns of expression of pancreatic secretion of fluid and amylase and gallbladder contraction. These differences have physiological relevance only if CCK-58 release is stimulated by nutrients entering the intestine and if CCK-58 circulates in sizeable amounts. In this study, we report that when radiolabeled CCK-58 is added to rat blood and plasma is formed, there is extensive loss and degradation of the radioactive peptide. Therefore, a new method was developed to minimize loss and degradation of this label. This method recovered >85% of the label with no detectable degradation. Furthermore, the optimized method recovered all unlabeled exogenous cholecystokinin molecular forms in >80% yields. Blood from fasted rats and rats in which cholecystokinin release was stimulated by the trypsin inhibitor camostat contained only CCK-58 (3.5 +/- 0.5 and 17 +/- 1.5 fmol/ml, respectively). Because CCK-58 predominates in the blood, this molecular form should be used in studies on the physiology and pathophysiology of cholecystokinin.     

Mass distributions of a macromolecular assembly based on electrospray ionization mass spectrometric masses of the constituent subunits

Macromolecular assemblies containing multiple protein subunits and having masses in the megadalton (MDa) range are involved in most of the functions of a living cell. Because of variation in the number and masses of subunits, macromolecular assemblies do not have a unique mass, but rather a mass distribution. The giant extracelular erythrocruorins (Ers), ∼ 3.5 MDa, comprized of at least 180 polypeptide chains, are one of the best characterized assemblies. Three-dimensional reconstructions from cryoelectron microscopic images show them to be hexagonal bilayer complexes of 12 subassemblies, each comprised of 12 globin chains, anchored to a subassembly of 36 nonglobin linker chains. We have calculated the most probable mass distributions forLumbricus andRiftia assemblies and their globin and linker subassemblies, based on theLumbricus Er stoichiometry and using accurate subunit masses obtained by electrospray ionization mass spectrometry. The expected masses ofLumbricus andRiftia Ers are 3.517 MDa and 3.284 MDa, respectively, with a possible variation of ∼ 9% due to the breadth of the mass distributions. TheLumbricus Er mass is in astonishingly good agreement with the mean of 23 known masses, 3.524 ± 0.481 MDa.