Expression of an Nkx3.1-CRE gene using ROSA26 reporter mice.

The genetic locus of Nkx3.1, an early murine marker of sclerotome and prostate development, was disrupted by a knock in of CRE recombinase via homologous recombination in embryonic stem cells. Cell fate mapping revealed previously unidentified cell lineages expanded from Nkx3.1-expressing cell populations and recapitulated reported Nkx3.1 expression patterns. In lineage trace experiments of E18.5 Nkx3.1-CRE; R26R embryos novel staining was observed in areas of the lungs, portions of the duodenum, and vertebral elements of the skeleton. beta-galactosidase activity measured in Nkx3.1-CRE; R26R and Nkx3.2-CRE; R26R embryos was observed in overlapping regions of the sclerotome but no apparent change in Nkx3.1 expression was seen in the Nkx3.2 mutants by in situ hybridization.

S100-mediated signal transduction in the nervous system and neurological diseases.

This article presents new information regarding the complement/level of S100 family members expressed in the brain and reviews the contribution of brain S100 family members to nervous system function and disease. A total of ten S100 family members are reported in the literature to be expressed in brain -S100A1, S100A2, S100A4, S100A5, S100A6, S100A10, S100A11, S100A13, S100B, and S100Z. Quantitative Northern blot analysis detected no S100A3, S100A8, S100A9 or S100A14 mRNA in mouse brain suggesting that these family members are not expressed in the brain. In addition, there was a 100-fold range in the mRNA levels for the six family members that were detected in mouse brain: S100A1/S100B levels were 5-fold higher than S100A6/S100A10 levels and 100-fold higher than S100A4/S100A13 levels. Five of these six family members (S1100A1, S100A6, S100A10, S100A13, and S100B) exhibited age-dependent increases in expression in adult mice that ranged from 5- to 20-fold. Although previous studies on S100 function in the nervous system have focused on S100B, other family members (S100A1, S100A3, S100A4, S100A5) have been implicated in neurological diseases. Like S100B, intra- and inter-cellular forms of these family members have been linked to cell growth, cell differentiation, and apoptotic pathways. Studies presented here demonstrate that ablation of S100A1 expression in PC12 cells results in increased resistance to Abeta peptide induced cell death, stabilization of intracellular [Ca2+] homeostasis, and reduced amyloid precursor protein expression. Altogether, these results confirm that S100-mediated signal transduction pathways play an important role in nervous system function/disease and implicate S100A1 in the neuronal cell dysfunction/death that occurs in Alzheimer's disease.

Elevated ovarian expression and serum concentration of alpha inhibin in the luteal phase during follicular development in the squirrel monkey (Saimiri boliviensis) compared to the human.

The goal of the present investigation was to determine in the squirrel monkey the source and pattern of inhibin, a hormone known to effect reproductive steroid levels via pituitary and ovarian mechanisms. Since this seasonally polyestrous species is known to have elevated serum levels of reproductive steroids compared to other primates, the levels of ovarian alpha subunit mRNA expression and serum total alpha inhibin, estradiol, progesterone, and luteinizing hormone were measured and compared to human levels. Expression of the alpha subunit was robust in monkey luteal tissue compared to expression in human luteal tissue. Squirrel monkey serum inhibin peaked 4 days after the luteinizing hormone surge and correlated with progesterone changes. These luteal serum levels of inhibin were greater than 12 times higher than the human levels yet bio-LH activities were less than in the human during the luteal phase. Inhibin concentrations during the nonbreeding season were generally half the levels measured in the breeding season and undetectable in ovariectomized animals. However, exogenous FSH stimulation induced a marked rise in inhibin, which correlated with an estradiol rise. In conclusion, abundant alpha inhibin subunit expression in the luteal ovary of the squirrel monkey and loss of serum delectability in ovariectomized animals indicates that the principle source of inhibin in the squirrel monkey is the ovary. Elevated serum inhibin levels during the luteal phase concurrent with ovulatory-size follicular development is unique among species studied thus far. Possible simultaneous inhibin production from both follicular and luteal tissue may be responsible for the exceptionally high inhibin levels.

S100A1 utilizes different mechanisms for interacting with calcium-dependent and calcium-independent target proteins.

While previous studies have identified target proteins that interact with S100A1 in a calcium-dependent manner as well as target proteins that interact in a calcium-independent manner, the molecular mechanisms of S100A1-target protein interaction have not been elucidated. In this study, point and deletion mutants of S100A1 were used to investigate the contribution of carboxyl terminal amino acids to S100A1 interaction with calcium-dependent and calcium-independent target proteins. First, a recombinant rat S100A1 protein (recS100A1) expressed in bacteria exhibited physical and chemical properties indistinguishable from native S100A1. Next, proteins lacking the carboxyl-terminal nine residues of recS100A1 (Delta85-93), or containing alanine substitutions at Phe 88 (F88A), Phe 89 (F89A), or Trp 90 (W90A), both Phe 88 and Phe 89 (F88/89A), or all three aromatic residues (F88/89A-W90A) were recombinantly expressed. Like recS100A1, F88A, F89A, and W90A proteins interacted with phenyl-Sepharose in a calcium-dependent manner. However, the Delta85-93 protein did not interact with phenyl-Sepharose, indicating that a phenyl-Sepharose-binding region (PSBR) of recS100A1 had been disrupted. The F88/89A and F88/89A-W90A proteins exhibited reduced calcium-dependent interaction with phenyl-Sepharose when compared with recS100A1, demonstrating that the carboxyl-terminal aromatic residues Phe 88, Phe 89, and Trp 90 comprise the PSBR of S100A1. Fluorescence studies showed that the Delta85-93 protein exhibited reduced calcium-dependent interaction with the dodecyl CapZ peptide, TRTK, while W90A bound TRTK with a Kd of 5.55 microM. These results demonstrate that the calcium-dependent target protein-binding site and the PSBR are indistinguishable. In contrast to the calcium-dependent target TRTK, activation of the calcium-independent target protein aldolase A by the point and deletion mutant S100A1s was indistinguishable from native S100A1. These results demonstrate that carboxyl-terminal residues are not required for S100A1 modulation of calcium-independent target protein aldolase A. Alltogether, these results indicate that S100A1 utilizes distinct mechanisms for interaction with calcium-independent and calcium-dependent target proteins.

S100A1 regulates neurite organization, tubulin levels, and proliferation in PC12 cells.

As a first step in determining what cellular processes are regulated by the calcium-modulated protein S100A1 isoform in neurons, the effects of ablated S100A1 expression on neurite organization and microtubule/tubulin levels in PC12 cells were examined. A mammalian expression vector containing the rat S100A1 cDNA in the antisense orientation with respect to a cytomegalovirus promoter was constructed and transfected into PC12 cells. Indirect immunofluorescence microscopy confirmed decreased S100A1 protein levels in all three stable transfectants (pAntisense clones) that expressed exogenous S100A1 antisense mRNA. In response to nerve growth factor, pAntisense clones extended significantly more neurites than control cells (4.01 +/- 0.16 versus 2.93 +/- 0.16 neurites/cell). This increase in neurite number was accompanied by an increase in total alpha-tubulin levels in untreated (4.0 +/- 0.6 versus 1.76 +/- 0.4 ng of alpha-tubulin/mg of total protein) and nerve growth factor-treated pAntisense clones (4.15 +/- 0.4 versus 2. 04 +/- 0.5 ng of alpha-tubulin/mg of total protein) when compared with control cells. At high cell densities, pAntisense clones exhibited a significant decrease in anchorage-dependent growth. In soft agar, pAntisense clones formed significantly more colonies (153 +/- 8%) than control cells (116 +/- 5%). However, the pAntisense soft agar colonies were significantly smaller than those observed in control cells (40.6 +/- 3.0 versus 59.5 +/- 1.2 micron). These data suggest that cell density inhibits both anchorage-independent and -dependent growth of pAntisense clones. In summary, ablation of S100A1 expression in PC12 cells results in increased tubulin levels, altered neurite organization, and decreased cell growth. Thus, S100A1 may directly link the cytoskeleton and calcium signal transduction pathways to cell proliferation.

Differential regulation of A gamma and G gamma fetal hemoglobin mRNA levels by hydroxyurea and butyrate.

In clinical studies, both hydroxyurea and butyrate increase fetal hemoglobin expression and ameliorate the symptoms of sickle cell anemia. However, comparative studies of the effects of hydroxyurea and butyrate on the expression of the individual fetal hemoglobin genes, A gamma and G gamma, have not been performed. The present study reports the effects of hydroxyurea and butyrate on steady-state A gamma and G gamma mRNA levels in K562 cells. Because the high degree of homology between the A gamma and G gamma cDNA sequences precludes the use of large cDNA probes for detection of individual fetal hemoglobin gene products, we investigated the specificity of two 20-base oligonucleotide probes synthesized from the region of greatest sequence diversity between these genes. Hybridization experiments demonstrated that the A gamma oligonucleotide probe was specific for A gamma DNA and RNA sequences and the G gamma oligonucleotide probe was specific for G gamma DNA and RNA sequences. These oligonucleotide probes detected both A gamma and G gamma mRNAs in K562 cells. In K562 cells treated with 2 mM sodium butyrate for 168 hours, the G gamma mRNA level increased 3.6-fold, whereas the A gamma mRNA level was not significantly different from untreated cells. Similar results were obtained when K562 cells were treated with 80 microM hydroxyurea. The G gamma mRNA level increased 2.3-fold at 168 hours, whereas the A gamma mRNA level did not change. The above results demonstrate that both butyrate and hydroxyurea selectively increase G gamma expression. Selective regulation of individual fetal hemoglobin genes is also seen in human development, where approximately 70% of the total fetal hemoglobin in the fetus is G gamma. Therefore, understanding the mechanisms by which butyrate and hydroxyurea differentially regulate fetal hemoglobin gene expression may provide insights into the developmental regulation of hemoglobin expression as well as the mechanisms of action of pharmacological agents currently being used to treat sickle cell disease.

S100A1 and S100B expression and target proteins in type I diabetes.

Calcium receptor proteins are an essential link between hormones that alter intracellular calcium levels and the generation of cellular responses. However, there is no information available regarding the role of calcium receptor proteins, in particular the S100 family, in insulin action and/or diabetes. This study examines the effects of streptozotocin-induced type I diabetes on the expression of the individual S100A1 and S100B isoforms as well as their binding proteins. Diabetes did not increase (or initiate) S100B expression in any non-S100B-expressing tissue (skeletal muscle, heart, kidney, liver, spleen, and pancreas). In all S100B-expressing tissues examined (brain, white fat, and testes), S100B protein levels increased approximately 2-fold while steady state S100B messenger RNA (mRNA) levels decreased. S100A1-expressing tissues exhibited increased (kidney and lung), decreased (skeletal muscle), and unchanged (brain and heart) S100A1 protein levels. While noncoordinate changes in S100A1 protein and steady state mRNA levels were observed in heart, other S100A1-expressing tissues (brain, slow twitch skeletal muscle, and kidney) exhibited coordinate changes in S100A1 protein and steady state mRNA levels. Altogether, these results suggest that the effects of diabetes on S100 expression are isoform as well as tissue-specific. Gel overlay analysis of the S100-binding protein profile revealed both increases and decreases in binding proteins in all tissues examined. In summary, changes in the expression of S100A1, S100B, and S100-binding proteins occur in type I diabetes and represent important molecular events in the effects of insulin/insulin insufficiency on cell function.

The role of cysteine residues in S100B dimerization and regulation of target protein activity.

Previous studies have demonstrated that the two cysteine residues in the calcium-binding protein S100B are required for its extracellular functions. In the present study, a recombinant S100B protein and mutant S100Bs containing one or no cysteine residue(s) have been used to determine the contribution of cysteine residues to S100B dimerization and interaction with the intracellular target proteins aldolase, phosphoglucomutase, and the microtubule associated tau protein. Mutation of C68 to a valine or C84 to a serine, C68 to valine and C84 to serine, or C68 to valine and C84 to alanine did not significantly alter S100B activation of aldolase. However, mutation of C84 to serine resulted in calcium-independent S100B activation of phosphoglucomutase and a loss of S100B inhibition of tau phosphorylation by Ca2+/calmodulin-dependent protein kinase II. The altered functionality of the C84S mutant with phosphoglucomutase and tau was not due to altered physical properties or dimerization state. All of the mutants exhibited heat stability and calcium dependent conformational changes which were identical to recombinant S100B. In addition, S100B proteins containing two, one or no cysteine residues behaved as dimers in size exclusion chromatography experiments in the presence or absence of calcium as well as in the presence or absence of reducing agent. Dynamic light scattering and analytical ultracentrifugation experiments confirmed that dimerization was not affected by calcium or reducing agent. Altogether these results demonstrate that S100B dimerization is not calcium- or sulfhydryl-dependent. In summary, cysteine residues are not necessary for the noncovalent dimerization of S100B, but are important in certain S100B target protein-interactions.

Nucleotide homologies in genes encoding members of the S100 protein family.

Members of the S100 protein family exhibit a unique pattern of cell/tissue-specific expression and approx. 50% similarity at the amino-acid level. The cDNAs encoding many of these proteins from a variety of species are now available making a comparison of these family members at the nucleotide level possible. With few exceptions, family members exhibited less nucleotide identity than amino-acid similarity. Furthermore, the pattern of divergence calculated on the basis of nucleotide identity did not always agree with that calculated on the basis of amino-acid similarity. The majority of sequence diversity occurred in the nontranslated regions suggesting that these regions may be involved in directing the expression of particular members of the family to specific cell types. When comparisons of individual family members were made across species, the following order of species diversity was observed: rat/mouse < human/bovine < porcine < rabbit/avian < Xenopus laevis. The structure of the gene loci encoding these proteins was remarkably conserved both within family members of a given species as well as in individual family members from different species. Although there appears to be great diversity in the 5' flanking regions of these genes, members of the family share at least one common potential regulatory element-the S100 protein element. Thus, membership in the S100 family could be ascertained on the basis of gene organization and the presence of an SPE. Although functional data are limited, the available data indicate that the regulation of the expression of S100 family members is complex and involves both positive and negative regulatory elements. Additional nucleic acid sequences and complimentary functional studies will be required to dissect the mechanisms which target the expression of the members of this family to specific cell types during development.

Identification of an S100A1/S100B target protein: phosphoglucomutase.

Phosphoglucomutase was identified as a potential intracellular S100 target protein because it interacted with two members of the S100 family of calcium-modulated proteins, S100A1 and S100B, in gel overlay experiments. These results were confirmed by affinity chromatography experiments demonstrating that S100A1 and S100B bound to phosphoglucomutase-Sepharose in a calcium-dependent manner. In the reverse experiment, phosphoglucomutase bound to S100A1 and S100B-Sepharose in a calcium-dependent manner. S100A1 inhibited phosphoglucomutase activity in a calcium-dependent manner. In contrast, S100B stimulated phosphoglucomutase activity in a calcium-dependent manner. Other calcium-binding proteins (calmodulin, troponin C, parvalbumin, and alpha-lactalbumin) had no effect on phosphoglucomutase. These results suggest that the effects of S100A1 and S100B are not nonspecific effects of low molecular weight, acidic proteins. This is the first report of an S100 target protein whose activity is antagonistically regulated by S100A1 and S100B, suggesting that cellular diversity in intracellular calcium signaling pathways may be due, at least in part, to the complement of S100 proteins expressed in different cell types.

Expression of the rat S100A1 gene in neurons, glia, and skeletal muscle.

Using a rat S100A1 cDNA probe, S100A1 expression has been documented in rat C6 glioma cells, a cell line previously thought to express only the S100B protein. To identify the molecular mechanisms which target S100A1 gene expression to specific cell types, the rat S100A1 gene was cloned, and functional analysis of the 5' flanking region of the gene was performed. The rat S100A1 gene was located in an 8.5 kb BamHI genomic fragment which contained 3 exons plus 1.6 kb of 5'-upstream and 0.37 kb of 3'-downstream flanking sequence. A single transcription initiation start site and a single polyadenylation signal were identified in this gene. A number of potential regulatory consensus sequences were identified in the rat S100A1 gene including general transcription factor binding sequences (TATA box, GC box and CCAAT box), cAMP regulated sequences (CRE), skeletal muscle specific sequences (E-box and M-CAT), an S100 protein element, and a (GCT) trinucleotide repeat. Analysis of an S100A1 promoter-CAT construct by ribonuclease protection assay demonstrated that this gene is functional in three S100A1 expressing cell lines, C6 cells, PC12 cells and L6 cells. CAT constructs containing progressive deletions of the S100A1 promoter region revealed a positive regulatory element in skeletal muscle (L6) cells between -1600/-1081. The fact that these same sequences were negative in glial (C6) cells and neutral in neuronal (PC12) cells suggests that this region plays a major role in targeting S100A1 expression to specific cell types. The -1081/+10 region contained both positive and negative elements, some of which were cell-type specific. Thus, S100A1 expression is under complex transcriptional control which involves positive and negative elements as well as cell type specific elements.

Analysis of S100A1 expression during skeletal muscle and neuronal cell differentiation.

To better understand the mechanisms that regulate the function of the calcium-binding proteins S100A1 and S100B in developing systems, we have examined the level of, subcellular distribution of, and target proteins for these proteins in skeletal muscle (L6S4) and neuronal (PC12) cell lines. Both undifferentiated and differentiated L6 and PC12 cells express S100A1 and not S100B. Whereas S100A1 protein levels were higher in differentiated cells than in undifferentiated cells, steady-state mRNA levels did not change in differentiated L6 cells and decreased in differentiated PC12 cells when compared with undifferentiated cells. These results suggest that posttranscriptional rather than transcriptional mechanisms are responsible for increased S100A1 protein expression in myotubes and neurons. The colocalization of S100A1 staining with wheat germ agglutinin staining suggests that S100A1 is associated with the Golgi apparatus and secretory vesicles in PC12 and L6 cells. Using a gel overlay technique, S100A1-binding proteins were detected in undifferentiated and differentiated PC12 and L6 cells and the patterns observed were similar to those observed in brain and skeletal muscle, respectively. Although changes in the intensity of some binding proteins were detected, the overall pattern did not change when differentiated and undifferentiated cells were compared. These results suggest that the complement of S100A1-binding proteins does not change during differentiation, only the levels of some binding proteins. Altogether, our data demonstrate that the L6 and PC12 cell lines are excellent in vitro model systems for studying S100A1 expression and mechanisms that regulate S100A1 expression, subcellular distribution, and interaction with target proteins.

The S100 protein family: history, function, and expression.

The S100 family of calcium binding proteins contains approximately 16 members each of which exhibits a unique pattern of tissue/cell type specific expression. Although the distribution of these proteins is not restricted to the nervous system, the implication of several members of this family in nervous system development, function, and disease has sparked new interest in these proteins. We now know that the original two members of this family, S100A1 and S100B, can regulate a diverse group of cellular functions including cell-cell communication, cell growth, cell structure, energy metabolism, contraction and intracellular signal transduction. Although some members of the family may function extracellularly, most appear to function as intracellular calcium-modulated proteins and couple extracellular stimuli to cellular responses via interaction with other cellular proteins called target proteins. Interaction of these proteins with target proteins appear to involve cysteine residues (one in S100A1 and two in S100B), as well as a stretch of 13 amino acids, in the middle of the molecule called the linker region, which connects the two EF-hand calcium binding domains. In addition to the amino acid sequence and secondary structures of these proteins, the structures of the genes encoding these proteins are highly conserved. Studies on the expression of these proteins have demonstrated that a complex mixture of transcriptional and postranscriptional mechanisms regulate S100 expression. Further analysis of the function and expression of these proteins in both nervous and nonnervous tissues will provide important information regarding the role of altered S100 expression in nervous system development, function and disease.

A role for intronic sequences on expression of thyroid hormone receptor alpha gene.

We have cloned and characterized the organization of the rat thyroid hormone receptor alpha (THR) gene. Multiple transcription start sites were mapped by RNA primer extension analyses. The promoter of the rat THR alpha gene does not contain a TATA or CAAT box. Deletion analyses of the 5' region of THR alpha gene and transfection assays, using NIH3T3 and NG108-15 cells, revealed that the sequences from -137 to +205 (+205 resides in the first intron) are necessary for efficient expression of this gene. This region contains two positively acting elements, the sequence -137 to -60 upstream from the major start of transcription and three copies of an AGG sequence located in the first intron. In contrast, two octamer-binding motifs in the first intron function as the negative regulatory elements. Gel mobility shift assays showed that the purine-rich sequence and the octamer-binding motifs bind to a protein(s) present in NIH3T3 and NG108-15 cells, the recipients in transient transfection assays. Genomic sequence comparison of THR alpha and beta revealed the presence of the purine-rich track in both genes, while the octamer-binding motifs were found only in the alpha gene. These results might explain the differential regulation of THR alpha and beta gene expression previously noted.

Steady-state mRNA levels of the sarcolemmal Na(+)-Ca2+ exchanger peak near birth in developing rabbit and rat hearts.

To functionally compensate for an underdeveloped sarcoplasmic reticulum in immature cardiomyocytes, it has been proposed that the sarcolemmal Na(+)-Ca2+ exchanger may assume a more predominant role for regulating cytosolic Ca2+. Previous studies using sarcolemma prepared from developing rabbit hearts demonstrated that Na(+)-dependent Ca2+ uptake and exchanger protein content were highest at birth and declined postnatally. To further investigate the significance of the Na(+)-Ca2+ exchanger during normal myocardial development, steady-state mRNA levels of the cardiac Na(+)-Ca2+ exchanger were quantitated by Northern blot and slot-blot analyses using poly(A+) RNA isolated from rabbit and rat ventricles at various fetal and postnatal ages. Northern analyses were performed with a 1.35-kb guinea pig cardiac Na(+)-Ca2+ exchanger cDNA probe. Exchanger mRNA levels were quantitated by densitometric scans of the slot blots, and results were normalized by reprobing the same blots with 32P 5'-end-labeled oligo(dT). In both species, exchanger mRNA levels peaked near birth and declined postnatally. Maximal levels were approximately sixfold greater in the late fetal rabbit (gestational day 29) and eightfold greater in the early newborn rat (postnatal day 1) compared with adults of the respective species. The parallel changes in exchanger mRNA and protein levels suggest that developmental regulation of cardiac Na(+)-Ca2+ exchanger expression involves pretranslational control mechanisms. These results support the concept that during normal cardiac development, Na(+)-Ca2+ exchanger expression is maximal near the time of birth and then declines postnatally as Ca2+ regulation by the sarcoplasmic reticulum reaches functional maturity.

Identification of an S100 target protein: glycogen phosphorylase.

An S100 binding protein from skeletal muscle, R95 000, has been purified, identified as glycogen phosphorylase, and shown to be regulated in vitro by the S100 alpha isoform. When a soluble skeletal muscle fraction was subjected to a standard purification procedure for glycogen phosphorylase, R95 000 copurified with the 95 000 molecular weight glycogen phosphorylase protein standard on SDS-polyacrylamide gels, as well as having glycogen phosphorylase activity. In addition, purified glycogen phosphorylase a and b interacted with both S100 isoforms, S100 alpha and S100 beta, by gel overlay and affinity chromatography. While S100 beta had no effect on the enzymatic activity of glycogen phosphorylase a, S100 alpha inhibited the enzymatic activity of glycogen phosphorylase a in a calcium-independent manner. Altogether, these data suggest that glycogen phosphorylase may be an intracellular S100 alpha target in skeletal muscle fibers. Furthermore, these results suggest that the inhibition of glycogen phosphorylase a activity may be responsible for the lack of fatigability of slow-twitch fibers, which express S100 alpha, when compared to fast-twitch fibers, which do not express S100 proteins.

Isolation of a rat S100 alpha cDNA and distribution of its mRNA in rat tissues.

In order to clarify the reported discrepancies in S100 alpha protein and mRNA distribution in rat tissues, a rat S100 alpha cDNA has been isolated and this species homologous probe along with a rat S100 beta cDNA probe has been used to examine S100 mRNA expression in rat tissues. Although the rat S100 alpha cDNA was missing approximately 30 nucleotides of coding sequence, only 4 conservative changes in amino acid sequence were observed when the deduced amino acid sequence was compared to the bovine S100 alpha amino acid sequence. Thus, S100 alpha proteins, like S100 beta proteins, are highly conserved among species. All nineteen of the tissues examined (including cerebrum and cerebellum) contained S100 alpha mRNA. In addition, S100 beta mRNA was detected in thirteen of the nineteen tissues examined. These results are in agreement with previous protein distribution studies and further demonstrate that S100 proteins are not brain-specific and are expressed in a large number of tissues. Although S100 alpha and S100 beta mRNAs were detected in rat tissues which had previously been reported to contain S100 alpha and S100 beta protein, a direct correlation between the protein and mRNA levels were not observed, suggesting that different mechanisms regulate S100 expression in various tissues. S100 alpha exhibited a single similar size mRNA species (0.5 Kb) in all tissues examined, as did S100 beta (1.5 Kb), suggesting that the individual S100 proteins are expressed as single mRNA and protein products in rat tissues.

Examination of the calcium-modulated protein S100 alpha and its target proteins in adult and developing skeletal muscle.

In this study radioimmunoassay, immunohistochemistry, Northern blot analysis, and a gel overlay technique have been used to examine the level, subcellular distribution, and potential target proteins of the S100 family of calcium-modulated proteins in adult and developing rat skeletal muscles. Adult rat muscles contained high levels of S100 proteins but the particular form present was dependent on the muscle type: cardiac muscle contained exclusively S100 alpha, slow-twitch skeletal muscle fibers contained predominantly S100 alpha, vascular smooth muscle contained both S100 alpha and S100 beta, and fast-twitch skeletal muscle fibers contained low but detectable levels of S100 alpha and S100 beta. While the distribution of S100 mRNAs paralled the protein distribution in all muscles there was no direct correlation between the mRNA and protein levels in different muscle types, suggesting that S100 protein expression is differentially regulated in different muscle types. Immunohistochemical analysis of the cellular distribution of S100 proteins in adult skeletal muscles revealed that S100 alpha staining was associated with muscle cells, while S100 beta staining was associated with nonmuscle cells. Radioimmunoassays of developing rat skeletal muscles demonstrated that all developing muscles contained low levels of S100 alpha at postnatal day 1 and that as development proceeded the S100 alpha levels increased. In contrast to adult muscle S100 alpha expression was confined to fast-twitch fibers in developing skeletal muscle until postnatal day 21. At postnatal day 1, developing contractile elements were S100 alpha positive, but no staining periodicity was detectable. At postnatal day 21, S100 alpha exhibited the same subcellular localization as seen in the adult: colocalization with the A-band and/or longitudinal sarcoplasmic reticulum. Comparison of the S100 alpha-binding protein profiles in fast- and slow-twitch fibers of various species revealed few, if any, species- or fiber type-specific S100 binding proteins. Isolated sarcoplasmic reticulum fractions and myofibrils contained multiple S100 alpha-binding proteins. The colocalization of S100 alpha and S100 alpha-binding proteins with the contractile apparatus and sarcoplasmic reticulum suggest that S100 alpha may regulate excitation and/or contraction in slow-twitch fibers.

Immunohistochemical localization of phosphoenolpyruvate carboxykinase in adult and developing mouse tissues.

We used immunohistochemical techniques to analyze the cell distribution of phosphoenolpyruvate carboxykinase (PEPCK) in adult and developing mouse tissues. PEPCK immunoreactivity was detected in many tissues, including some that had not been previously reported to contain PEPCK enzyme activity (bladder, stomach, ovary, vagina, parotid gland, submaxillary gland, and eye). In some multicellular tissues, PEPCK immunoreactivity was observed in multiple cell types. Several tissues (spleen, thyroid, and submaxillary gland) contained no detectable PEPCK immunoreactivity. During development, PEPCK immunoreactivity was associated with the developing nervous system and somites in 15-day embryos. At prenatal day 18, PEPCK immunoreactivity was detected only in the nervous system. At prenatal day 20, PEPCK immunoreactivity was observed in many of the tissues that contain PEPCK in the adult, with the exception of liver, lung, and stomach. PEPCK immunoreactivity was detected in liver at postnatal day 1, lung at postnatal day 7, and stomach after postnatal day 21. The only tissue in which PEPCK immunoreactivity decreased during development was the pancreas, where PEPCK immunoreactivity was detected at prenatal day 20 and was present until postnatal day 21. These results suggest that PEPCK expression is cell-type specific, more widespread than previously thought, and differentially expressed during development.