William B. Rhoten

[42]Rat calbindin D28K: Purification, quantitation, immunocytochemical localization, and comparative aspects

Publisher Summary The purification, and biochemical and immunological characterization of vitamin D-dependent rat renal CaBP have only recently been reported. These biochemical and immunological studies have permitted a more detailed analysis of this calcium-binding protein; thus, providing insight into the role of the vitamin D endocrine system in kidney and brain function. The purification method results in the isolation of electrophoretically homogeneous rat renal CaBP. Silver stain after analytical polyacrylamide gel electrophoresis of the purified protein resulted in the migration of CaBP as a single band, suggesting the homogeneity of the preparation. The molecular weight of renal CaBP determined by gel filtration on a calibrated Sephadex G-100 column was found to be 28,000. The calcium-binding properties were determined by equilibrium dialysis performed at 4° for 18 hours on a reciprocating incubation shaker in polystyrene bottles that had been washed five times with deionized water. A number of methods are available for immunolocalization of CaBP at the light microscopic level. The two methods described in this chapter are the peroxidase-antiperoxidase complex technique and the colloidal gold with silver intensification method.

Mouse renal cytochrome p450IIE1: Immunocytochemical localization, sex-related difference and regulation by testosterone☆

Cytochrome P450IIE1 is responsible for the metabolic activation of N-nitrosodimethylamine and a variety of other chemicals. Renal P450IIE1 was shown previously to be regulated by testosterone in C3H/HeJ and BALB/c mice. The present study investigated the distribution of cytochrome P450IIE1 in the kidneys of C3H/HeJ and BALB/c mice. The amount of P450IIE1 was immunotitrated by immunohistochemistry using polyclonal antibodies against rat P450IIE1. Strong immunoreactivity was identified mainly in the cortical tubules, including proximal tubules and some tubules. Weak immunoreactivity was also observed in the outer medulla when higher concentrations of antibodies were used. Much higher immunostaining was observed in male mice than in female mice when identical antibody dilutions were used. The renal P450IIE1 level in females was elevated to the same level as that in males 24 hr after administration of testosterone. The results showed a specific cellular localization of cytochrome P450IIE1 in mouse kidney. The findings may lead to a better understanding of the site-specific renal toxicity and carcinogenesis due to the activation of chemicals by cytochrome P450IIE1.

Vitamin D‐Dependent Calcium‐Binding Protein Is Highly Conserved in the Metanephros

We have previously found a vitamin D-dependent calcium-binding protein (CaBP) with a relative molecular weight ( M , ) of 28,000 in mammalian and avian kidney.’*2 The adult reptile, like other amniotes, has a kidney that arises from metanephrogenic mesenchyme. The present study was initiated to obtain information on the phylesis of this CaBP. The biochemical, immunochemical, and immunocytochemical findings demonstrate that a CaBP with a M, of 28,000 is highly conserved during vertebrate phylogeny.

1 – Calbindin D28K Gene Expression in Neurodegenerative Diseases

Publisher Summary This chapter focuses on calbindin D 28K gene expression in aging and neurodegenerative diseases. The study described in the chapter has permitted an analysis of the role that changes in calbindin D 28K gene expression may play in the etiology and pathogenesis of neurodegeneration in the brain, such as Huntingtons, Parkinsons, and Alzheimers diseases. A method of molecular cloning of calbindin D 28K described in the chapter resulted in the isolation of a cloned calbindin D 28K cDNA via direct immunological screening of a mouse cerebellar λgt11 bacterial expression library. The calbindin D 28K cDNA could then be radiolabeled directly and utilized as a probe for detection of calbindin D 28K mRNA (slot-blot and Northern blot analyses) or converted to radiolabeled cRNA transcripts to detect calbindin D 28K mRNA at the cellular level by in situ hybridization. Slot-blot and Northern blot hybridization analyses revealed that in each disease entity, the brain area that is particularly affected and used as a hallmark for the neuropathological changes exhibits a significant reduction in calbindin D 28K gene expression.

Somatostatin-containing and other endocrine cells in the pancreas of the spectacled caiman.

Light-microscopic immunocytochemistry was used to localize 4 major pancreatic hormones in the pancreas of the spectacled caiman, Caiman fuscus. Somatostatin, insulin, glucagon and pancreatic polypeptide were localized by the peroxidase-antiperoxidase complex technique. A relatively large population of somatostatin-containing D cells was present. The D cells were nearly as numerous as the insulin-containing B cells and glucagon-containing A cells which were the most common cell types. All three cell types were commonly intermingled with one another in endocrine cell areas. Pancreatic polypeptide-reactive F cells were absent from some regions of the pancreas, but where present were related to other endocrine cell types. Functional properties of the pancreatic endocrine cells in this anatomical variant remain to be determined.

LOCALIZATION OF S100 ALPHA IN MAMMALIAN KIDNEY

Publisher Summary This chapter discusses the localization of S100 alpha in mammalian kidney. The alpha subunit of S100, a calcium binding protein, has been found in the mammalian kidney by radioimmunoassay (RIA) and immunocytochemistry (ICC). An antiserum was generated in rabbits against the alpha subunit of S100 protein from bovine brain. For biochemical studies, the kidneys of rats were homogenized, and a supernatant fraction prepared and evaluated for the presence of S100 alpha by RIA. Kidneys for ICC were fixed in Bouins fluid and processed by routine paraffin-methods. S100alpha was localized by the peroxidase-anti-peroxidase technique and the biotin-strepavidin method. Kidneys were positive for S100alpha by RIA and levels of S100alpha were higher in the medulla than in the cortex. Cells reactive for S100alpha by ICC were found in both the medulla and the cortex. Immunoreactivity was localized to the epithelial cells lining the collecting tubules. Positive tubules were especially prominent in the papilla; however, the epithelium covering the papilla was unreactive. Immunoreactivity was eliminated when the antiserum was absorbed with an excess of S100alpha. Limited co-localization suggests the possibility that these two proteins may coordinate signal responses in some cells.

CALBINDIN D28k IS HIGHLY CONSERVED DURING PHYLOGENY

Publisher Summary This chapter discusses the localization and the characterization of calcium binding protein (CaBP) in kidney and brain of diverse species. The highest concentrations of CaBP are present in kidney, pancreas and brain. It is found that although the function of CaBP is unknown, broad distribution and conservation are criteria that should be met by proteins involved in regulating basic intracellular processes. In salietian amphibian, saurian and crocodilian reptiles, chicken, rat, mouse, and baboon, calbindin D 28k are most evident in the distal tubule of the kidney. Reactive tubules become less apparent as one continues beyond the distal tubule, but the baboon has positive cells in collecting tubules all the way to the papilla. The finding of calbindin D D 28k in the anamniotic kidney of the salientian amphibian at all stages of metamorphosis suggests that CaBP has functional significance in the mesonephric and the metanephric kidney. The increase in the number of reactive cells for calbindin D 28k from the premetamorphic period onward suggests that this protein may have a role in the differentiation of kidney function.