Calcium Retrieval from Vacuolar Pools (Characterization of a Vacuolar Calcium Channel)

Voltage patch-clamp experiments at the whole-vacuole and single-channel levels were employed to study the retrieval of Ca2+ from vacuoles into the cytoplasm in sugar beet cell (Beta vulgaris L.) suspension cultures. Channels allowing the movement of Ca2+ out of the vacuole were identified at physiological conditions of pH, vacuolar membrane potential, and vacuole/cytoplasm Ca2+ concentrations. The operation of the channel was voltage dependent and inositol-1,4,5-triphosphate insensitive and displayed high selectivity for Ca2+ ions. These channels bear similarities to the dihydropyridine-sensitive L-type Ca2+ channels from animal cells. Bay K-8644, an agonist, increased the frequency of channel openings, whereas nifedipine, an antagonist, reduced the channel activity. Both effects were elicited only from the vacuolar side of the channel. Channel activities were also inhibited by verapamil, La3+, and cytoplasmic Ca2+ concentrations higher than 1 x 10-6 M. The modulation of the channel currents by cytoplasmic Ca2+ would suggest the role of these channels in triggering the initiation of signal transduction processes in plant cells.     

Cch1 mediates calcium entry in Cryptococcus neoformans and is essential in low-calcium environments

The ability of Cryptococcus neoformans to grow at the mammalian body temperature (37 degrees C to 39 degrees C) is a well-established virulence factor. Growth of C. neoformans at this physiological temperature requires calcineurin, a Ca(2+)/calmodulin-dependent protein phosphatase. When cytosolic calcium concentrations are low ( approximately 50 to 100 nM), calcineurin is inactive and becomes active only when cytosolic calcium concentrations rise ( approximately 1 to 10 microM) through the activation of calcium channels. In this study we analyzed the function of Cch1 in C. neoformans and found that Cch1 is a Ca(2+)-permeable channel that mediates calcium entry in C. neoformans. Analysis of the Cch1 protein sequence revealed differences in the voltage sensor (S4 regions), suggesting that Cch1 may have diminished voltage sensitivity or possibly an alternative gating mechanism. The inability of the cch1 mutant to grow under conditions of limited extracellular calcium concentrations ([Ca(2+)](extracellular), approximately 100 nM) suggested that Cch1 was required for calcium uptake in low-calcium environments. These results are consistent with the role of ScCch1 in mediating high-affinity calcium uptake in Saccharomyces cerevisiae. Although the growth defect of the cch1 mutant under conditions of limited [Ca(2+)](extracellular) ( approximately 100 nM) became more severe with increasing temperature (25 degrees C to 38.5 degrees ), this temperature sensitivity was not observed when the cch1 mutant was grown on rich medium ([Ca(2+)](extracellular), approximately 0.140 mM). Accordingly, the cch1 mutant strain displayed only attenuated virulence when tested in the mouse inhalation model of cryptococcosis, further suggesting that C. neoformans may have a limited requirement for Cch1 and that this requirement appears to include ion stress tolerance.     

Elongation factor 3, EF3, associates with the calcium channel Cch1 and targets Cch1 to the plasma membrane in Cryptococcus neoformans

Ca2+-mediated signaling events in eukaryotic cells are initiated by Ca2+ channels located in the plasma membranes and endomembranes. Cch1, a high-affinity Ca2+ channel in the plasma membranes of Cryptococcus neoformans and other fungi, plays a role in many different cellular processes, but the mechanisms that regulate Cch1 are not well understood. A Ras recruitment two-hybrid screen was used to identify protein partners of Cch1 as a means of identifying possible mechanisms of channel regulation. Here, we show that Cch1 specifically associates with a cytoplasmic protein known as elongation factor 3 (EF3). The robust interaction between the cytosolic C terminus of the Cch1 protein and EF3 shown here was confirmed by demonstrating that Cch1 could coimmunoprecipitate with EF3 in yeast lysates. To examine the effects of EF3 on Cch1 behavior, we altered the EF3 gene function by constructing a C. neoformans antisense EF3 repression strain. Our results show that the repression of EF3 led to the mislocalization of Cch1, suggesting a role for EF3 in targeting Cch1 to the plasma membrane of C. neoformans. Consistent with this notion, the antisense EF3 repression strain displayed a growth defect under conditions of limited extracellular Ca2+. Collectively, these results suggest that EF3 and Cch1 are functionally coupled and that EF3 has a function apart from its role in the protein translation cycle.     

Establishing physiological blood parameters in the loggerhead sea turtle (Caretta caretta)

The loggerhead sea turtle (Caretta caretta), one of the three sea turtle species inhabiting the Mediterranean Sea, is an endangered species. However, although frequently treated in marine animal rescue centers, and the subject of several studies in literature, as yet, few studies have been conducted on large numbers of loggerhead sea turtles in order to establish physiological reference ranges that enable the identification of pathological values. The lack of studies on reference parameters probably depends on the fact that marine turtles treated in wildlife rescue centers are usually in a critical conditions, thus precluding the collection of data on healthy animals and compromising the reliability of any data obtained from them. The present biological study was therefore conducted in order to obtain a database from healthy animals in natural conditions. Serum biochemistry and serum protein electrophoresis were performed on blood samples obtained from 65 healthy adult loggerhead sea turtles captured and delivered to the Sea Turtle Rescue Center of Linosa (Italy); the blood samples were collected during the clinical examination of rescued animals. Laboratory analyses of serum samples were made in order to establish reference parameters, commonly required for laboratory diagnoses in mammals and diseased animals.

The euchromatic 9p+ polymorphism is a locus-specific amplification caused by repeated copies of a small DNA segment mapping within 9p12

A large duplication involving the proximal euchromatic region of chromosome 9p was detected by conventional cytogenetics in a healthy 33-year-old woman and in two unrelated foetuses; both of them received the rearrangement from their healthy father. The duplicated segment was R(RBG) and C(CBG)-negative and G(GTG)-positive and was also positive for a 9-specific painting probe. It was preliminarily interpreted as a pathological quantitative change of the genome in the foetuses. FISH analyses allowed us to characterise the chromosome boundaries of this polymorphism, being identified by the RP11-15E1 BAC clone, proximally, and by the RP11-402N8 clone, distally, both probes falling within the 9p12 region. The contiguous, distally, RP11-916H19 probe was not included in the amplification, and may represent the discriminating genetic locus between chromosome polymorphism and chromosome mutation. The 9p12 amplification was approximately 12, 7 and 8 Mb in the three different families and was stable through generations. Our observations confirm the already provided evidence that proximal 9p duplications represent a benign euchromatic polymorphism. However, we demonstrated that these variants are not a simple duplication of the region 9p11.2-p13.1, as already suggested, but that they result from a many-fold amplification of a segment mapping within 9p12. These results provide important insights both in the genetic counselling and in the prenatal diagnosis of rare euchromatic chromosome variants and in understanding the architecture of the human genome.     

Activity of the Calcium Channel Pore Cch1 Is Dependent on a Modulatory Region of the Subunit Mid1 in Cryptococcus neoformans

Calcium (Ca²⁺)-mediated signaling events in fungal pathogens such as Cryptococcus neoformans are central to physiological processes, including those that mediate stress responses and promote virulence. The Cch1-Mid1 channel (CMC) represents the only high-affinity Ca²⁺ channel in the plasma membrane of fungal cells; consequently, cryptococci cannot survive in low-Ca²⁺ environments in the absence of CMC. Previous electrophysiological characterization revealed that Cch1, the predicted channel pore, and Mid1, a binding partner of Cch1, function as a store-operated Ca²⁺-selective channel gated by depletion of endoplasmic reticulum (ER) Ca²⁺ stores. Cryptococci lacking CMC did not survive ER stress, indicating its critical role in restoring Ca²⁺ homeostasis. Despite the requirement for Mid1 in promoting Ca²⁺ influx via Cch1, identification of the role of Mid1 remains elusive. Here we show that the C-terminal tail of Mid1 is a modulatory region that impinges on Cch1 channel activity directly and mediates the trafficking of Mid1 to the plasma membrane. This region consists of the last 24 residues of Mid1, and the functional expression of Mid1 in a human embryonic cell line (HEK293) and in C. neoformans is dependent on this domain. Substitutions of arginine (R619A) or cysteine (C621A) in the modulatory region failed to target Mid1 to the plasma membrane and prevented CMC activity. Interestingly, loss of a predicted protein kinase C (PKC)-phosphorylated serine residue (S605A) had no effect on Mid1 trafficking but did alter the kinetics of Cch1 channel activity. Thus, establishment of Ca²⁺ homeostasis in C. neoformans is dependent on a modulatory domain of Mid1.     

Intermediate-sized filament proteins (keratin, vimentin, desmin) in metaplastic carcinomas, carcinosarcomas and stromal sarcomas of the breast

The distribution of intermediate-filament (IF) proteins of the keratin, vimentin and desmin type in breast stromal sarcomas, carcinosarcomas, metaplastic carcinomas and phyllodes tumors has been compared using the avidin-biotin complex immunoperoxidase technique. Keratin reactivity was found in carcinomatous and pseudosarcomatous areas of all metaplastic carcinomas, in the cuboidal epithelial cells of carcinosarcomas and in the epithelial component of phyllodes tumors. Vimentin and desmin were detected in the sarcomatous portion of carcinosarcoma, focally in the stromal component of phyllodes tumors and not always in the stromal sarcomas. These data confirm that combined analysis of IF expression is a reliable and convincing way to differentiate stromal sarcomas, metaplastic carcinomas and carcinosarcomas in breast pathology.