Calbindin-D(28k) controls [Ca(2+)](i) and insulin release. Evidence obtained from calbindin-d(28k) knockout mice and beta cell lines

The role of the calcium-binding protein, calbindin-D(28k) in potassium/depolarization-stimulated increases in the cytosolic free Ca(2+) concentration ([Ca(2+)](i)) and insulin release was investigated in pancreatic islets from calbindin-D(28k) nullmutant mice (knockouts; KO) or wild type mice and beta cell lines stably transfected and overexpressing calbindin. Using single islets from KO mice and stimulation with 45 mM KCl, the peak of [Ca(2+)](i) was 3.5-fold greater in islets from KO mice compared with wild type islets (p < 0.01) and [Ca(2+)](i) remained higher during the plateau phase. In addition to the increase in [Ca(2+)](i) in response to KCl there was also a significant increase in insulin release in islets isolated from KO mice. Evidence for modulation by calbindin of [Ca(2+)](i) and insulin release was also noted using beta cell lines. Rat calbindin was stably expressed in betaTC-3 and betaHC-13 cells. In response to depolarizing concentrations of K(+), insulin release was decreased by 45-47% in calbindin expressing betaTC cells and was decreased by 70-80% in calbindin expressing betaHC cells compared with insulin release from vector transfected betaTC or betaHC cells (p < 0.01). In addition, the K(+)-stimulated intracellular calcium peak was markedly inhibited in calbindin expressing betaHC cells compared with vector transfected cells (225 nM versus 1,100 nM, respectively). Buffering of the depolarization-induced rise in [Ca(2+)](i) was also observed in calbindin expressing betaTC cells. In summary, our findings, using both isolated islets from calbindin-D(28k) KO mice and beta cell lines, establish a role for calbindin in the modulation of depolarization-stimulated insulin release and suggest that calbindin can control the rate of insulin release via regulation of [Ca(2+)](i).     

Reduced transforming growth factor-beta signaling in cartilage of old mice: role in impaired repair capacity

Osteoarthritis (OA) is a common joint disease, mainly effecting the elderly population. The cause of OA seems to be an imbalance in catabolic and anabolic factors that develops with age. IL-1 is a catabolic factor known to induce cartilage damage, and transforming growth factor (TGF)-beta is an anabolic factor that can counteract many IL-1-induced effects. In old mice, we observed reduced responsiveness to TGF-beta-induced IL-1 counteraction. We investigated whether expression of TGF-beta and its signaling molecules altered with age. To mimic the TGF-beta deprived conditions in aged mice, we assessed the functional consequence of TGF-beta blocking. We isolated knee joints of mice aged 5 months or 2 years, half of which were exposed to IL-1 by intra-articular injection 24 h prior to knee joint isolation. Immunohistochemistry was performed, staining for TGF-beta1, -2 or -3, TGF-betaRI or -RII, Smad2, -3, -4, -6 and -7 and Smad-2P. The percentage of cells staining positive was determined in tibial cartilage. To mimic the lack of TGF-beta signaling in old mice, young mice were injected with IL-1 and after 2 days Ad-LAP (TGF-beta inhibitor) or a control virus were injected. Proteoglycan (PG) synthesis (³⁵S-sulfate incorporation) and PG content of the cartilage were determined. Our experiments revealed that TGF-beta2 and -3 expression decreased with age, as did the TGF-beta receptors. Although the number of cells positive for the Smad proteins was not altered, the number of cells expressing Smad2P strongly dropped in old mice. IL-1 did not alter the expression patterns. We mimicked the lack of TGF-beta signaling in old mice by TGF-beta inhibition with LAP. This resulted in a reduced level of PG synthesis and aggravation of PG depletion. The limited response of old mice to TGF-beta induced-IL-1 counteraction is not due to a diminished level of intracellular signaling molecules or an upregulation of intracellular inhibitors, but is likely due to an intrinsic absence of sufficient TGF-beta receptor expression. Blocking TGF-beta distorted the natural repair response after IL-1 injection. In conclusion, TGF-beta appears to play an important role in repair of cartilage and a lack of TGF-beta responsiveness in old mice might be at the root of OA development.     

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.     

Calmodulin and rat vitamin D-dependent calcium-binding proteins: Biochemical and immunochemical comparison

Purified vitamin D-dependent rat intestinal (M_r 10,000) and rat renal (M_r 28,000) calcium-binding proteins (CaBPs) have been compared to vertebrate calmodulin, and the vitamin D-dependent CaBPs have been found to be distinct from calmodulin by biochemical and immunochemical criteria. Rat renal and rat intestinal CaBPs do not stimulate 3′,5′-cyclic nucleotide phosphodiesterase, do not compete with iodinated calmodulin for binding to phenothiazine-Sepharose conjugates, do not cross-react immunochemically, and do not contain N^?-trimethyllysine. In addition, although calmodulin exhibits a characteristic calcium-dependent mobility shift on polyacrylamide gels in the presence of sodium dodecyl sulfate, a similar mobility shift is not observed for the vitamin D-dependent CaBPs. Immunocytochemically, calmodulin has a widespread localization in the kidney, whereas CaBP is present specifically in the distal tubules of the kidney. These localizations suggest a specialized role for CaBP in the kidney. Thus, although the vitamin D-dependent CaBPs and calmodulin are similar in that they are small, acidic, calcium-binding proteins, these two classes of proteins are biochemically and immunochemically distinct.     

Migrations and swimming capabilities of endangered pallid sturgeon (Scaphirhynchus albus) to guide passage designs in the fragmented Yellowstone River

Fragmentation of the Yellowstone River is hypothesized to preclude recruitment of endangered Scaphirhynchus albus (pallid sturgeon) by impeding upstream spawning migrations and access to upstream spawning areas, thereby limiting the length of free‐flowing river required for survival of early life stages. Building on this hypothesis, the reach of the Yellowstone River affected by Intake Diversion Dam (IDD) is targeted for modification. Structures including a rock ramp and by‐pass channel have been proposed as restoration alternatives to facilitate passage. Limited information on migrations and swimming capabilities of pallid sturgeon is available to guide engineering design specifications for the proposed structures. Migration behavior, pathways (channel routes used during migrations), and swimming capabilities of free‐ranging wild adult pallid sturgeon were examined using radiotelemetry, and complemented with hydraulic data obtained along the migration pathways. Migrations of 12–26% of the telemetered pallid sturgeon population persisted to IDD, but upstream passage over the dam was not detected. Observed migration pathways occurred primarily through main channel habitats; however, migrations through side channels up to 3.9 km in length were documented. The majority of pallid sturgeon used depths of 2.2–3.4 m and mean water velocities of 0.89–1.83 m/s while migrating. Results provide inferences on depths, velocities, and habitat heterogeneity of reaches successfully negotiated by pallid sturgeon that may be used to guide designs for structures facilitating passage at IDD. Passage will provide connectivity to potential upstream spawning areas on the Yellowstone River, thereby increasing the likelihood of recruitment for this endangered species.     

Use of chromomycin A3 staining in bovine sperm cells for detection of protamine deficiency

Sperm chromatin integrity is essential for accurate transmission of male genetic information, and normal sperm chromatin structure is important for fertilization. Protamine is a nuclear protein that plays a key role in sperm DNA integrity, because it is responsible for sperm DNA stability and packing until the paternal genome is delivered into the oocyte during fertilization. Our aim was to investigate protamine deficiency in sperm cells of Bos indicus bulls (Nelore) using chromomycin A₃ (CMA₃) staining. Frozen semen from 14 bulls were thawed, then fixed in Carnoy's solution. Smears were prepared and analyzed by microscopy. As a positive control of CMA₃ staining, sperm from one bull was subjected to deprotamination of nuclei. The percentage of CMA₃-positive bovine sperm did not vary among batches. Only two bulls showed a higher percentage of CMA₃-positive sperm cells compared to the others. CMA₃ is a simple and useful tool for detecting sperm protamine deficiency in bulls.     

The genome of Rhizobium leguminosarum has recognizable core and accessory components

Background

Rhizobium leguminosarum is an α-proteobacterial N₂-fixing symbiont of legumes that has been the subject of more than a thousand publications. Genes for the symbiotic interaction with plants are well studied, but the adaptations that allow survival and growth in the soil environment are poorly understood. We have sequenced the genome of R. leguminosarum biovar viciae strain 3841.

Results

The 7.75 Mb genome comprises a circular chromosome and six circular plasmids, with 61% G+C overall. All three rRNA operons and 52 tRNA genes are on the chromosome; essential protein-encoding genes are largely chromosomal, but most functional classes occur on plasmids as well. Of the 7,263 protein-encoding genes, 2,056 had orthologs in each of three related genomes (Agrobacterium tumefaciens, Sinorhizobium meliloti, and Mesorhizobium loti), and these genes were over-represented in the chromosome and had above average G+C. Most supported the rRNA-based phylogeny, confirming A. tumefaciens to be the closest among these relatives, but 347 genes were incompatible with this phylogeny; these were scattered throughout the genome but were over-represented on the plasmids. An unexpectedly large number of genes were shared by all three rhizobia but were missing from A. tumefaciens.

Conclusion

Overall, the genome can be considered to have two main components: a 'core', which is higher in G+C, is mostly chromosomal, is shared with related organisms, and has a consistent phylogeny; and an 'accessory' component, which is sporadic in distribution, lower in G+C, and located on the plasmids and chromosomal islands. The accessory genome has a different nucleotide composition from the core despite a long history of coexistence.