Play and learn team building.

In order to have a team function correctly, power must be distributed equally, with no team member having more perceived power than any other. It is this leveling of the playing field that allows the team to develop and to stimulate the creative juices of its members. This article discusses techniques that can help an organization break down the power barriers and permit its employees to become a cohesive unit--a team.

Anti-inflammatory and wound healing activity of a growth substance in Aloe vera.

Aloe vera improves wound healing and inhibits inflammation. Since mannose-6-phosphate is the major sugar in the Aloe gel, the authors examined the possibility of its being an active growth substance. Mice receiving 300 mg/kg of mannose-6-phosphate had improved wound healing over saline controls. This dose also had anti-inflammatory activity. The function of mannose-6-phosphate in A. vera is discussed.

Regulatory element analysis and structural characterization of the human sarcomeric mitochondrial creatine kinase gene.

Creatine kinase (EC 2.7.3.2) (CK) isoenzymes are crucial to energy metabolism, particularly in tissues with high energy requirements. Nuclear genes encode four known CK subunits: cytoplasmic muscle, cytoplasmic brain, ubiquitous mitochondrial (uMtCK), and sarcomeric mitochondrial (sMtCK). Herein, we report the isolation and complete structural characterization of the human sMtCK gene. It contains 11 exons and encompasses more than 37 kilobase pairs (kb). The sites of exon localization in the sMtCK-coding region and their precise sizes are identical with the human uMtCK gene. The translation start codon is in the third exon and lies 17 kb from the transcription start site. The human sMtCK gene is located on chromosome 5. Sequence analysis of the sMtCK genomic upstream sequences reveals a typical TATAA box within the 80 base pairs (bp) that, by transfection experiments, are sufficient to promote expression of chimeric plasmids with the chloramphenicol acetyltransferase reporter. Cis-acting sequences in a fragment containing 3360 bp of upstream sequence, the first exon, and 750 bp of the first intron are sufficient to mediate tissue-specific expression. However, these sequences only partially regulate induction of sMtCK expression in differentiating mouse myoblasts. MEF1/MYOD and MEF2 sequence motifs present in the sMtCK gene are not sufficient to regulate differentiation-specific expression. The sMtCK gene contains sequences homologous to several motifs that are shared among some nuclear genes encoding mitochondrial proteins and that may be essential for the coordinated activation of these genes during mitochondrial biogenesis.

Structural characterization and tissue-specific expression of the mRNAs encoding isoenzymes from two rat mitochondrial creatine kinase genes.

Creatine kinase (CK; EC 2.7.3.2) isoenzymes play prominent roles in energy transduction. Mitochondrial CK (MtCK) reversibly catalyzes the transfer of high energy phosphate to creatine and exists, in the human, as two isoenzymes encoded by separate genes. We report here the cDNA sequences of the two isoenzymes of MtCK in the rat. Rat sarcomeric MtCK has 87% nucleotide identity in the 1257 bp coding region and 82% in the 154 bp 3' untranslated region as compared with human sarcomeric MtCK. Rat ubiquitous MtCK has 92% nucleotide identity over the 1254 bp coding region with human ubiquitous MtCK and 81% identity of the 148 by 3' untranslated region. Nucleotide identity between the rat sarcomeric and ubiquitous MtCK coding regions is 70%, with no conservation of their 3' untranslated regions. Thus, MtCK sequence is conserved in a tissue-specific, rather than species-specific, manner. Conservation of the 3' untranslated regions is highly unusual and suggests a regulatory function for this region. The NH2-terminal transit peptide sequences share 82% amino acid homology between rat and human sarcomeric MtCKs and 92% homology between rat and human ubiquitous MtCKs, but have only 41% homology to each other. This tissue-specific conservation of the transit peptides suggests receptor specificity in mitochondrial uptake. Rat sarcomeric MtCK mRNA is expressed only in skeletal muscle and heart, but rat ubiquitous MtCK mRNA is expressed in many tissues, with highest levels in brain, gut and kidney. Ubiquitous MtCK mRNA levels are dramatically regulated in uterus and placenta during pregnancy. Coexpression of sarcomeric and ubiquitous MtCK with their cytosolic counterparts, MCK and BCK, respectively, supports the creatine phosphate shuttle hypothesis and suggests that expression of these genes is coordinately regulated.

Regulation of expression of M, B, and mitochondrial creatine kinase mRNAs in the left ventricle after pressure overload in rats.

Pressure overload of the left ventricle induces synthesis of creatine kinase isoenzymes. To determine whether this response is associated with an altered pattern of creatine kinase gene expression, we induced arterial hypertension in rats by suprarenal aortic banding. After 4 days, left ventricular myocardium from hypertensive (n = 7) and normotensive, sham-operated (n = 5) rats was analyzed for isoenzyme activities by chromatography; M and B creatine kinase subunit protein by Western blot; and M, B, and mitochondrial creatine kinase mRNA by Northern blot. Although total creatine kinase activity increased in hypertensive (1,096 +/- 214 IU/g left ventricle) compared with normotensive rats (648 +/- 81 IU/g left ventricle, p less than 0.01), the relative proportions of the cytoplasmic and mitochondrial isoenzymes did not change. The mass of M and B subunits increased 1.9- and 2.7-fold, respectively, in hypertensive compared with control rats. Similarly, the mRNA for M and B subunits as well as mitochondrial creatine kinase increased 2.6-, 1.6-, and 1.8-fold, respectively, in hypertensive rats compared with control rats. Thus, increased energy requirements in acute pressure overload are met by generalized induction of creatine kinase mRNA and subunit protein and not by an isoenzyme switch.

Separate nuclear genes encode sarcomere-specific and ubiquitous human mitochondrial creatine kinase isoenzymes.

Creatine kinase (EC 2.7.3.2) isoenzymes play a central role in energy transduction. Nuclear genes encode creatine kinase subunits from muscle, brain, and mitochondria (MtCK). We have recently isolated a cDNA clone encoding MtCK from a human placental library which is expressed in many human tissues (Haas, R. C., Korenfeld, C., Zhang, Z., Perryman, B., Roman, D., and Strauss, A. W. (1989) J. Biol. Chem. 264, 2890-2897). With nontranslated and coding region probes, we demonstrated by RNA blot analysis that the MtCK mRNA in sarcomeric muscle is distinct from this placenta-derived, ubiquitous MtCK cDNA. To compare these different mRNAs, a MtCK cDNA clone was isolated from a human heart library and characterized by complete nucleotide sequence analysis. The chemically determined NH2-terminal 26 residues of purified human heart MtCK protein are identical to those predicted from this sarcomeric MtCK cDNA. The human sarcomeric and ubiquitous cDNAs share 73% nucleotide and 80% predicted amino acid sequence identities, but have less than 66% identity with the cytosolic creatine kinases. The sarcomeric MtCK cDNA encodes a 419-amino acid protein which contains a 39-residue transit peptide essential for mitochondrial import. Primer extension analysis predicts a 348-base pair 5'-nontranslated region. RNA blot analysis demonstrates that heart-derived MtCK is sarcomere-specific, but the ubiquitous MtCK mRNA is expressed in most tissues. Thus, separate nuclear genes encode two closely related, tissue-specific isoenzymes of MtCK. Our finding that multiple genes encode different mitochondrial protein isoenzymes is rare.

Isolation and characterization of the gene and cDNA encoding human mitochondrial creatine kinase.

Creatine kinase (CK; EC 2.7.3.2) isoenzymes play prominent roles in energy metabolism. Nuclear genes encode three known CK subunits: cytoplasmic muscle (MCK), cytoplasmic brain (BCK), and mitochondrial (MtCK). We have isolated the gene and cDNA encoding human placental MtCK. By using a dog heart MCK cDNA-derived probe, the 7.0-kb EcoRI fragment from one cross-hybridizing genomic clone was isolated and its complete nucleotide sequence determined. A region of this clone encoded predicted amino acid sequence identical to residues 15-26 of the human heart MtCK NH2-terminal protein sequence. The human placental MtCK cDNA was isolated by hybridization to a genomic fragment encoding this region. The human placental MtCK gene contains 9 exons encoding 416 amino acids, including a 38-amino acid transit peptide, presumably essential for mitochondrial import. Residues 1-14 of human placental MtCK cDNA-derived NH2-terminal sequence differ from the human heart MtCK protein sequence, suggesting that tissue-specific MtCK mRNAs are derived from multiple MtCK genes. RNA blot analysis demonstrated abundant MtCK mRNA in adult human ventricle and skeletal muscle, low amounts in placenta and small intestine, and a dramatic increase during in vitro differentiation induced by serum-deprivation in the non-fusing mouse smooth muscle cell line, BC3H1. These findings demonstrate coordinate regulation of MtCK and cytosolic CK gene expression and support the phosphocreatine shuttle hypothesis.