Calbindin 28 kDa in endocrine cells of known or putative calcium-regulating function

Summary The distribution of calbindin in some endocrine glands (thyroid, parathyroid, ultimobranchial body, pituitary and adrenals) and in the diffuse endocrine cells of the gut and pancreas has been investigated immunohistochemically using an antiserum raised against the 28 kDa calbindin from chicken duodenum. The identity of calbindin-immunoreactive cells in a number of avian and mammalian species was ascertained by comparison with hormone-reactive cells in consecutive sections or by double immunostaining of the same section with both calbindin and hormone antibodies. Calcitonin-producing C cells of the mammalian and avian thyroid, parathyroid or ultimobranchial body, PP, glucagon and insulin cells of the mammalian and avian pancreas, enteroglucagon cells of the avian intestine, secretin cells of the mammalian duodenum, histamine-producing ECL cells of the mammalian stomach, as well as noradrenaline-producing cells of the adrenal medulla and some (TSH?) cells of the adenohypophysis were among the calbindin-immunoreactive cells. Although some species variability has been observed in the intensity and distribution of the immunoreactivity, especially in the pancreas and the gut, a role for calbindin in the mechanisms of calcium-mediated endocrine cell stimulation or of intracellular and extracellular calcium homeostasis is suggested.     

Ornithine decarboxylase activity associated with a particulate fraction of brain

The enzymic decarboxylation of ornithine by adult rat brain largely occurs in the particulate fraction. The activity is primarily due to ornithine decarboxylase (ODC) as evidenced by several criteria: i) the concurrent production of equimolar amounts of CO₂ and putrescine, ii) the sensitivity of the reaction to difluoromethylornithine (DFMO), a specific inhibitor of ODC, iii) the lack of major effect of two inhibitors of ornithine-2-oxo-acid transaminase, upon the DFMO-sensitive component of decarboxylation, iv) the failure to profoundly reduce decarboxylation activity in the presence of a large excess of many aminoacids which could compete for non-specific decarboxylases. The insoluble ODC activity appears largely within synaptosomal and mitochondrial-enriched morphological fractions, yet cannot be attributed to trapped soluble ODC. Particulate ODC has a pH optimum and kinetic parameters that differ from those of soluble cerebral ODC.     

Calbindin D28k is essentially located in the colonic part of the toad intestine

Summary— The distribution of calbindin D28k in the digestive system and the urinary bladder of the toad was investigated using immunohistochemistry and Western blotting. By analogy with mammals and birds, the protein was expected to be located preferentially in the duodenal part of the intestine. Interestingly, absorptive cells of the duodenum were totally devoid of calbindin D28k while the colon contained high amounts of the calcium-binding protein. This reversed polarity of calbindin D28k content in the toad intestine should obviously correspond to a different scheme of calcium absorption regulation between amphibians and higher vertebrates. Calbindin D28k containing neuroendocrine-like cells were found scattered in the proximal parts of the gut with a similar distribution to what has been described in rat and chick intestine. The oesophagus, the stomach, and the intrinsic nervous sytem of the intestine were negative. No significant amounts of the proteins were found in the urinary bladder, which is known to be a site of Ca²⁺ active transport.     

Calbindin D28k is essentially located in the colonic part of the toad intestine

The distribution of calbindin D28k in the digestive system and the urinary bladder of the toad was investigated using immunohistochemistry and Western blotting. By analogy with mammals and birds, the protein was expected to be located preferentially in the duodenal part of the intestine. Interestingly, absorptive cells of the duodenum were totally devoid of calbindin D28k while the colon contained high amounts of the calcium-binding protein. This reversed polarity of calbindin D28k content in the toad intestine should obviously correspond to a different scheme of calcium absorption regulation between amphibians and higher vertebrates. Calbindin D28k containing neuroendocrine-like cells were found scattered in the proximal parts of the gut with a similar distribution to what has been described in rat and chick intestine. The oesophagus, the stomach, and the intrinsic nervous sytem of the intestine were negative. No significant amounts of the proteins were found in the urinary bladder, which is known to be a site of Ca²⁺ active transport.     

A xylogalacturonan epitope is specifically associated with plant cell detachment

A monoclonal antibody (LM8) was generated with specificity for xyloglacturonan (XGA) isolated from pea (Pisum sativum L.) testae. Characterization of the LM8 epitope indicates that it is a region of XGA that is highly substituted with xylose. Immunocytochemical analysis indicates that this epitope is restricted to loosely attached inner parenchyma cells at the inner face of the pea testa and does not occur in other cells of the testa. Elsewhere in the pea seedling, the LM8 epitope was found only in association with root cap cell development at the root apex. Furthermore, the LM8 epitope is specifically associated with root cap cells in a range of angiosperm species. In embryogenic carrot suspension cell cultures the epitope is abundant at the surface of cell walls of loosely attached cells in both induced and non-induced cultures. The LM8 epitope is the first cell wall epitope to be identified that is specifically associated with a plant cell separation process that results in complete cell detachment.Abbreviations DAA Days after anthesis - 2,4-D 2,4-Dichlorophenoxyacetic acid - ELISA Enzyme-linked immunosorbent assay - GalA Galacturonic acid - HGA Homogalacturonan - HPAEC High-performance anion-exchange chromatography - HPSEC High-performance size-exclusion chromatography - RG-I Rhamnogalacturonan-I - RG-II Rhamnogalacturonan-II - XGA Xylogalacturonan     

Segmental Igfbp5 expression is specifically associated with the bent structure of zigzag hairs

The murine hair coat consists of four different hair types that are characterised by hair length, the number of medulla columns, and the presence and number of bends. The molecular mechanisms underlying the establishment and maintenance of distinct hair follicle fates are unknown. We identify Igfbp5 as the first molecular marker that distinguishes among different hair follicle types. High-resolution expression analysis revealed that its expression in the medulla of hair shafts is associated with the bend-forming zones of zigzag hairs. To directly examine the functional importance of segmental gene expression in the hair follicle, we have generated transgenic mice expressing Igfbp5 in differentiating keratinocytes of the medulla and inner root sheath. Ectopic expression of Igfbp5 resulted in the appearance of remarkable curvatures and thinning of hair shafts, two hallmarks of hair bends. Both effects and the natural bending process are under negative control of IGF signalling. Thus, our data identify Igfbp5 as a central regulator of hair shaft differentiation and hair type determination.     

Interaction of calmodulin with the particulate fraction of cardiac muscle

Tritiated calmodulin (T-CM) was bound to the EGTA-treated particulate fraction of cardiac muscle in a calcium-dependent manner with half-maximal binding occurring between 0.8 to 1.2 microM calcium. Binding exhibited high specificity at an optimum pH of 7.4-7.6. An excess of parvalbumin and other globular proteins did not displace T-CM. The Kd for the interaction was 2.5 +/- 0.83 microM. Binding was trypsin-sensitive, inhibited by high ionic strength and was heat inactivated at a midpoint of 48 - 50 degrees C. Competitive displacement of T-CM occurred with unlabeled troponin C and calmodulin over the same concentration range. The first-order rate constant of T-CM dissociation was 3.27 min-1. Calcium-dependent binding of T-CM was inhibited equally by both mepacrine and trifluoperazine with 50 percent inhibition occurring at 70 microM.     

Calbindin D-27 kDa: preferential localization in non-B islet cells of the rat pancreas

The presence and abundance of calbindin in rat pancreatic islet cells was assessed by immunohistochemistry of either whole islets or purified B and non-B islet cells, as well as by Western blotting of extracts derived from whole islets and purified B and non-B islet cells. Immunohistochemistry of pancreatic sections indicated a higher calbindin content in non-B cells, located at the periphery of the islets, than in the centrally located insulin-producing B cells. Comparable results were obtained in purified islet cells. Likewise, scanning densitometry of the Western blots indicated that, relative to cell volume, the single calbindin band (Mr 27 kDa) was 5-7 times higher in non-B than in B cells. In the splenic lobe of chick pancreas, however, the opposite situation prevailed. Thus, insulin-producing cells clustered in small roundish islets were more intensely labelled after exposure to anti-calbindin serum than non-B islet cells located in large and irregularly shaped islets. Nevertheless, even in the chick pancreas, non-B islet cells contained an appreciable amount of calbindin.     

Underexpression of the 43 kDa inositol polyphosphate 5-phosphatase is associated with cellular transformation.

The 43 kDa inositol polyphosphate 5-phosphatase (5-phosphatase) hydrolyses the second messenger molecules inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] and inositol 1,3,4,5-tetrakisphosphate [Ins(1,3,4,5)P4]. We have underexpressed the 43 kDa 5-phosphatase by stably transfecting normal rat kidney cells with the cDNA encoding the enzyme, cloned in the antisense orientation into the tetracycline-inducible expression vector pUHD10-3. Antisense-transfected cells demonstrated a 45% reduction in Ins(1,4,5)P3 5-phosphatase activity in the total cell homogenate upon withdrawal of tetracycline, and an approximately 80% reduction in the detergent-soluble membrane fraction of the cell, as compared with antisense-transfected cells in the presence of tetracycline. Unstimulated antisense-transfected cells showed a concomitant 2-fold increase in Ins(1,4,5)P3 and 4-fold increase in Ins(1,3,4,5)P4 levels. The basal intracellular calcium concentration of antisense-transfected cells (170 +/- 25 nM) was increased 1.9-fold, compared with cells transfected with vector alone (90 +/- 25 nM). Cells underexpressing the 43 kDa 5-phosphatase demonstrated a transformed phenotype. Antisense-transfected cells grew at a 1.7-fold faster rate, reached confluence at higher density and demonstrated increased [3H]thymidine incorporation compared with cells transfected with vector alone. Furthermore, antisense-transfected cells formed colonies in soft agar and tumours in nude mice. These studies support the contention that a decrease in Ins(1,4,5)P3 5-phosphatase activity is associated with cellular transformation.     

Human cathepsin D precursor is associated with a 60 kDa glycosylated polypeptide.

Human cathepsin D is synthesized as a 53 kDa precursor. Most of it is segregated into lysosomal compartments and subjected to a proteolytic fragmentation. Using a cross-linking reagent we show that a large proportion of the precursor is associated with a distinct protein which--under denaturing and reducing conditions--is characterized as a 60 kDa glycopeptide. Studies on cells cultured in the presence of drugs known to affect the intracellular transport (deoxynojirimycin, brefeldin A and NH4Cl) indicated that the association with cathepsin D precursor occurs early after the synthesis and is at least partially maintained after secretion.     

Calbindin D28k-like immunoreactivity in the developing and regenerating circumvallate papilla of the rat

The distribution of calbindin D28k (CB)-like immunoreactivity (-LI) in the circumvallate papilla (CVP) was examined during development and regeneration following bilateral crush injury to the glossopharyngeal nerve in the rat. In the adult CVP, CB-like immunoreactive (-IR) nerve fibers were observed in the subgemmal region and some penetrated into the taste buds. CB-LI was also detected in the cytoplasm of the spindle-shaped gustatory cells in the lower half of the trench epithelium, which contained numerous synaptic vesicles and bundles of intermediate filaments. These CB-IR gustatory cells made synapse-like contacts with CB-IR nerve terminals. Some CB-IR nerve terminals made contacts with the gustatory cells negative for CB-LI. At least three developmental stages were defined with regard to the developmental changes in the distribution of CB-LI: (1) Stage I (embryonic day (E) 18–postnatal day (P)5): CB-IR nerve fibers appeared in the lamina propria just beneath the newly-formed CVP at E18, but the gustatory epithelium of the CVP contained no CB-IR structures. Taste buds with taste pores appeared at P1. (2) Stage II (P5–10): thin CB-IR nerve fibers began entering the trench epithelium, but no CB-IR cells were observed. (3) Stage III (P10–adult): in addition to the intragemmal and perigemmal CB-IR nerve fibers, very few CB-IR cells appeared in the taste buds around P10, and their numbers increased progressively. The changes in the distribution of taste buds and CB-LI following glossopharyngeal nerve injury were similar to those observed during development. On post-operative day (PO) 4, the taste buds and CB-IR cells decreased markedly in number. These CB-IR cells became round in shape, and the number of CB-IR nerve fibers decreased markedly. On PO8, both taste buds and CB-IR cells disappeared completely. The regenerated taste buds were first observed on PO12, increased rapidly in number by PO20, and increased slowly thereafter. CB-IR nerve fibers accumulated at the subgemmal region and began penetrating into the trench wall epithelium around PO16. CB-IR cells appeared between PO20 and PO24, and their numbers increased progressively and reached the normal level on PO40. The topographical localizations of the taste buds and CB-IR cells during development and regeneration were comparable to those of normal animals. The delay of the time courses for appearance of CB-IR nerve fibers and CB-IR cells compared to the appearance of taste buds during development and regeneration suggests that CB in the gustatory epithelium may participate in the survival of the taste bud cells rather than in the induction of the taste buds.     

In vitro enzyme activation with calbindin-D28k, the vitamin D-dependent 28 kDa calcium binding protein.

Purified porcine erythrocyte membrane Ca(2+)-ATPase and 3':5'-cyclic nucleotide phosphodiesterase were stimulated in a dose-dependent, saturable manner with the vitamin D-dependent calcium binding protein from rat kidney, calbindin-D28k (CaBP-D28k). The concentration of CaBP-D28k required for half-maximal activation (K0.5 act.) of the Ca(2+)-ATPase was 28 nM compared to 2.2 nM for calmodulin (CaM), with maximal activation equivalent upon addition of either excess CaM or CaBP-D28k. 3':5'-Cyclic nucleotide phosphodiesterase (PDE) also showed equivalent maximum saturable activation by calbindin (K0.5 act. = 90 nM) or calmodulin (K0.5 act. = 1.2 nM). CaBP-D28k was shown to effectively compete with CaM-Sepharose for PDE binding. Immunoprecipitation with CaBP-D28k antiserum completely inhibited calbindin-mediated activation of PDE but had no effect on calmodulin's ability to activate PDE. While the physiological significance of these results remains to be established, they do suggest that CaBP-D28k can activate enzymes and may be a regulator of yet to be identified target enzymes in certain tissues.     

The cytotoxin of Actinobacillus pleuropneumoniae (pleurotoxin) is distinct from the haemolysin and is associated with a 120 kDa polypeptide

Mutants of Actinobacillus pleuropneumoniae strain HK 361 (serotype 2) were isolated which were deficient in type II (Ca2(+)-dependent) haemolysin activity (Hly-). Some of the Hly- mutants secreted a potent, heat-labile extracellular cytotoxic activity against porcine alveolar macrophages. Comparison of cell-free culture supernatant from the parent strain and some Hly- mutants by SDS-PAGE and immunoblotting revealed the loss of a major extracellular polypeptide of 109 kDa. Two Hly- mutants which in addition failed to secrete a 120 kDa polypeptide produced no extracellular cytotoxic activity, suggesting that the 120 kDa protein was the cytotoxin. Antiserum raised to the culture supernatant from a Hly- mutant lacking the 109 kDa polypeptide recognized the 120 kDa band, but not the 109 kDa band, in immunoblots and neutralized the cytotoxic activity, but not the haemolytic activity, of A. pleuropneumoniae. The 120 kDa polypeptide and extracellular cytotoxic activity were widespread among A. pleuropneumoniae strains, but absent from related bacterial pathogens of the pig: Actinobacillus suis, Haemophilus parasuis and Pasteurella multocida. A clear correlation was found between the presence of the 120 kDa polypeptide and cytotoxic activity in culture supernatants. The cytotoxic activity of all the strains tested was neutralized by antibody to the Hly- extracellular material and by convalescent pig serum. It is proposed that the 120 kDa polypeptide represents the cytotoxin of A. pleuropneumoniae, that it is distinct from the haemolysin, and that it be termed pleurotoxin.     

Proteins specifically associated with the microtubules of the mammalian mitotic spindle

The molecular species that determine the unique structure and functions of the microtubules in the mitotic spindle are not known. We describe the results of two new approaches to the molecular structure of the spindle. Both approaches rely on detergent-extracted preparations of synchronized populations of cells metabolically labeled with 35S-methionine or 32P-phosphate. In these preparations, the original cellular microtubules are preserved. The microtubule components can be released from the detergent-extracted preparations by selective depolymerization with calcium ions. Alternatively, the microtubules can be stabilized by taxol, freed of chromatin by digestion with DNAase and freed of the surrounding cage of intermediate filaments by further extraction at low ionic strength. Gel electrophoresis of each of these preparations of mitotic microtubules demonstrates that they contain microtubule-associated proteins that we have previously shown to be present in interphase microtubules. They also contain a protein of 150,000 daltons, which is the first mitosis-specific microtubule-associated protein identified in mammalian cells.