Development and progression of malignancy in human colon tissues are correlated with expression of specific Ca(2+)-binding S100 proteins

The expression levels of seven different S100 proteins (S100A1, S100A2, S100A3, S100A4, S100A5, S100A6, and S100B) were characterized by immunohistochemistry in the epithelial versus connective tissues of a series of 35 colon specimens, including 6 normal samples, 5 adenomas with low-grade dysplasia, 5 adenomas with high-grade dysplasia, and 19 cancers. The results showed that S100A2, S100A3, and S100B proteins could not (or only marginally) be detected in colon tissues. On the other hand, the expression of S100A6 increased in epithelial tissues directly proportional to the increase of malignancy. The percentage of epithelial (or connective tissue) cells expressing S100A4 significantly decreased as the malignancy grade increased. The expression level of S100A1 proteins was somewhat higher in the connective tissues of normal cases and adenomas with low-grade dysplasia than in adenomas with high-grade dysplasia and cancers. This pattern of expression was not observed in epithelial tissues. While the node-positive cancers did not express S100A1, about half of the node-negative specimens did. The expression levels of S100A5 were similar in different epithelial tissues. However, in the connective tissues the expression levels decreased inversely proportional to the increase in pathological grading of the specimens. Therefore, the present study implicates several S100 proteins as useful tools for histochemical typing of colon cancer malignancy development.     

The oncoprotein kinase chaperone CDC37 functions as an oncogene in mice and collaborates with both c-myc and cyclin D1 in transformation of multiple tissues

CDC37 encodes a 50-kDa protein that targets intrinsically unstable oncoprotein kinases including Cdk4, Raf-1, and v-src to the molecular chaperone Hsp90, an interaction that is thought to be important for the establishment of signaling pathways. CDC37 is required for proliferation in budding yeast and is coexpressed with cyclin D1 in proliferative zones during mouse development, a finding consistent with a positive role in cell proliferation. CDC37 expression may not only be required to support proliferation in cells that are developmentally programmed to proliferate but may also be required in cells that are inappropriately induced to initiate proliferation by oncogenes. Here we report that mouse mammary tumor virus (MMTV)-CDC37 transgenic mice develop mammary gland tumors at a rate comparable to that observed previously in MMTV-cyclin D1 mice. Moreover, CDC37 was found to collaborate with MMTV-c-myc in the transformation of multiple tissues, including mammary and salivary glands in females and testis in males, and also collaborates with cyclin D1 to transform the female mammary gland. These data indicate that CDC37 can function as an oncogene in mice and suggests that the establishment of protein kinase pathways mediated by Cdc37-Hsp90 can be a rate-limiting event in epithelial cell transformation.     

Autoinhibition of a calmodulin-dependent calcium pump involves a structure in the stalk that connects the transmembrane domain to the ATPase catalytic domain

The regulation of Ca(2+)-pumps is important for controlling [Ca(2+)] in the cytosol and organelles of all eukaryotes. Here, we report a genetic strategy to identify residues that function in autoinhibition of a novel calmodulin-activated Ca(2+)-pump with an N-terminal regulatory domain (isoform ACA2 from Arabidopsis). Mutant pumps with constitutive activity were identified by complementation of a yeast (K616) deficient in two Ca(2+)-pumps. Fifteen mutations were found that disrupted a segment of the N-terminal autoinhibitor located between Lys(23) and Arg(54). Three mutations (E167K, D219N, and E341K) were found associated with the stalk that connects the ATPase catalytic domain (head) and with the transmembrane domain. Enzyme assays indicated that the stalk mutations resulted in calmodulin-independent activity, with V(max), K(mATP), and K(mCa(2+)) similar to that of a pump in which the N-terminal autoinhibitor had been deleted. A highly conservative substitution at Asp(219) (D219E) still produced a deregulated pump, indicating that the autoinhibitory structure in the stalk is highly sensitive to perturbation. In plasma membrane H(+)-ATPases from yeast and plants, similarly positioned mutations resulted in hyperactive pumps. Together, these results suggest that a structural feature of the stalk is of general importance in regulating diverse P-type ATPases.     

Fibrils formed in vitro from alpha-synuclein and two mutant forms linked to Parkinson's disease are typical amyloid

Two missense mutations in the gene encoding alpha-synuclein have been linked to rare, early-onset forms of Parkinson's disease (PD). These forms of PD, as well as the common idiopathic form, are characterized by the presence of cytoplasmic neuronal deposits, called Lewy bodies, in the affected region of the brain. Lewy bodies contain alpha-synuclein in a form that resembles fibrillar Abeta derived from Alzheimer's disease (AD) amyloid plaques. One of the mutant forms of alpha-synuclein (A53T) fibrillizes more rapidly in vitro than does the wild-type protein, suggesting that a correlation may exist between the rate of in vitro fibrillization and/or oligomerization and the progression of PD, analogous to the relationship between Abeta fibrillization in vitro and familial AD. In this paper, fibrils generated in vitro from alpha-synuclein, wild-type and both mutant forms, are shown to possess very similar features that are characteristic of amyloid fibrils, including a wound and predominantly unbranched morphology (demonstrated by atomic force and electron microscopies), distinctive dye-binding properties (Congo red and thioflavin T), and antiparallel beta-sheet structure (Fourier transform infrared spectroscopy and circular dichroism spectroscopy). alpha-Synuclein fibrils are relatively resistant to proteolysis, a property shared by fibrillar Abeta and the disease-associated fibrillar form of the prion protein. These data suggest that PD, like AD, is a brain amyloid disease that, unlike AD, is characterized by cytoplasmic amyloid (Lewy bodies). In addition to amyloid fibrils, a small oligomeric form of alpha-synuclein, which may be analogous to the Abeta protofibril, was observed prior to the appearance of fibrils. This species or a related one, rather than the fibril itself, may be responsible for neuronal death.     

Sleep states alter ventral medullary surface responses to blood pressure challenges

Ventral medullary surface (VMS) activity declines during rapid eye movement (REM) sleep, suggesting a potential for reduced VMS responsiveness to blood pressure challenges during that state. We measured VMS neural activity, assessed as changes in reflected 660-nm wavelength light, during pressor and depressor challenges within sleep/waking states in five adult, unrestrained, unanesthetized cats and in two control cats. Phenylephrine elevated blood pressure and elicited an initial VMS activity decline and a subsequent rise in VMS activity in all states, although the initial decline during quiet sleep occurred only in rostral placements. Phasic REM periods elicited a momentary recovery from the evoked activity rise, and arousals diminished the overall elevation in activity. A sodium nitroprusside depressor challenge increased VMS activity more in REM sleep than in quiet sleep, with the increase being even less in waking. Enhanced responses to depressor challenges during REM sleep suggest a loss of dampening of evoked activity during that state; state-related differential baroreflex sensitivity may result from sleep-waking changes in VMS responses to blood pressure challenges.     

Calmodulin activation of an endoplasmic reticulum-located calcium pump involves an interaction with the N-terminal autoinhibitory domain

To investigate how calmodulin regulates a unique subfamily of Ca(2+) pumps found in plants, we examined the kinetic properties of isoform ACA2 identified in Arabidopsis. A recombinant ACA2 was expressed in a yeast K616 mutant deficient in two endogenous Ca(2+) pumps. Orthovanadate-sensitive (45)Ca(2+) transport into vesicles isolated from transformants demonstrated that ACA2 is a Ca(2+) pump. Ca(2+) pumping by the full-length protein (ACA2-1) was 4- to 10-fold lower than that of the N-terminal truncated ACA2-2 (Delta2-80), indicating that the N-terminal domain normally acts to inhibit the pump. An inhibitory sequence (IC(50) = 4 microM) was localized to a region within valine-20 to leucine-44, because a peptide corresponding to this sequence lowered the V(max) and increased the K(m) for Ca(2+) of the constitutively active ACA2-2 to values comparable to the full-length pump. The peptide also blocked the activity (IC(50) = 7 microM) of a Ca(2+) pump (AtECA1) belonging to a second family of Ca(2+) pumps. This inhibitory sequence appears to overlap with a calmodulin-binding site in ACA2, previously mapped between aspartate-19 and arginine-36 (J.F. Harper, B. Hong, I. Hwang, H.Q. Guo, R. Stoddard, J.F. Huang, M.G. Palmgren, H. Sze ?1998 J Biol Chem 273: 1099-1106). These results support a model in which the pump is kept "unactivated" by an intramolecular interaction between an autoinhibitory sequence located between residues 20 and 44 and a site in the Ca(2+) pump core that is highly conserved between different Ca(2+) pump families. Results further support a model in which activation occurs as a result of Ca(2+)-induced binding of calmodulin to a site overlapping or immediately adjacent to the autoinhibitory sequence.     

Genetic diversity and gene flow in the endangered dwarf bear poppy, Arctomecon humilis (Papaveraceae)

Arctomecon humilis is a critically endangered species endemic to the Moenkopi shale of Washington County, Utah. Recovery plans for the species would be improved by an understanding of genetic diversity and gene flow among its remaining populations. Ten variable isozyme loci were used to calculate genetic diversity statistics for study populations. Westerly populations possessed higher levels of genetic variability than other populations at the same isozyme loci. Three of the populations exhibited significant deviations from Hardy-Weinberg expectations. No correlation existed between genetic distance and geographic distance. Most of the genetic diversity was distributed among populations with little gene flow between populations, suggesting that observed genetic differences may arise from genetic drift. For the westerly populations, similar genotypes were observed in the seedling and old age classes, while intermediate age classes typically possessed an alternate set of genotypes at Pgi-2. Mean heterozygosity increased with age class across populations. Westerly populations of A. humilis shared more alleles with the nearest geographic population of A. californica than other populations. Since the westerly populations contained more genetic variability and more alleles in common with a near relative, they may be relictual. Other populations may contain less genetic diversity due to founder effects and/or genetic drift.     

14-3-3 proteins activate a plant calcium-dependent protein kinase (CDPK)

Plants and protozoa contain a unique family of calcium-dependent protein kinases (CDPKs) which are defined by the presence of a carboxyl-terminal calmodulin-like regulatory domain. We present biochemical evidence indicating that at least one member of this kinase family can be stimulated by 14-3-3 proteins. Isoform CPK-1 from the model plant Arabidopsis thaliana was expressed as a fusion protein in E. coli and purified. The calcium-dependent activity of this recombinant CPK-1 was shown to be stimulated almost twofold by three different 14-3-3 isoforms with 50% activation around 200 nM. 14-3-3 proteins bound to the purified CPK-1, as shown by binding assays in which either the 14-3-3 or CPK-1 were immobilized on a matrix. Both the 14-3-3 binding and activation of CPK-1 were specifically disrupted by a known 14-3-3 binding peptide LSQRQRSTpSTPNVHMV (IC₅₀=30 μM). These results raise the question of whether 14-3-3 can modulate the activity of CDPK signal transduction pathways in plants.