Testosterone responses to intensive interval versus steady-state endurance exercise.

Free testosterone (FT) hormonal responses were compared between high-intensity interval exercise (IE) and steadystate endurance exercise (SSE) in endurance trained males (no.=15). IE session was repeated periods of 90-sec treadmill running at 100-110% maximal oxygen uptake (VO2max) and 90-sec active recovery at 40% VO2max for 42-47 min. The SSE session consisted of a continuous 45-min run at 60-65% VO2max. Total work output was equal for each exercise session. A 45-min supine rest control session (CON) was also performed. All three sessions were on separate days. Pre-session (PRE), immediate post-session (POST), and 12-h post-session (12POST) blood samples were collected and used to determine FT, SHBG, LH, 3- α-androstanediol glucuronide (3-α Diol G) and cortisol. Analysis of variance compared IE and SSE biomarker responses to the reference CON session. IE and SSE each caused an increase (p<0.01) in FT, but IE more so than SSE (p<0.05). The 5α-reductase marker 3-α Diol G response at 12POST IE was elevated while FT was reduced (p<0.05); no such change occurred following SSE. These findings suggest IE might produce a more pronounced turnover of FT by androgen sensitive tissue than the SSE form of exercise.

Activation of polyubiquitin gene expression during developmentally programmed cell death.

Ubiquitin, a highly conserved 76 amino acid protein, plays a role in targeting intracellular proteins for degradation. Ubiquitin expression was examined during the developmentally programmed atrophy and degeneration of the intersegmental muscles (ISMs) in the hawk-moth, Manduca sexta. A clone containing nine repeats of the ubiquitin coding sequence was isolated from an ISM cDNA library and was used as a probe to examine polyubiquitin expression during development. When the ISMs became committed to degenerate, polyubiquitin gene expression increased dramatically. Injection of 20-hydroxyecdysone, which delays degeneration in this system, prevented the increase in polyubiquitin mRNA. The expression of polyubiquitin occurred without apparent activation of the cell's heat shock response. These data suggest that ubiquitin plays a role in programmed cell death.

Allelic variation of the polyubiquitin gene in the tobacco hawkmoth, Manduca sexta, and its regulation by heat shock and programmed cell death.

The intersegmental muscles (ISMs) of the tobacco hawkmoth, Manduca sexta, undergo programmed cell death (PCD) following adult eclosion in response to a decline in the circulating titer of the hormone 20-hydroxyecdysone. The ability of the ISMs to die requires de novo gene expression and a number of cDNAs representing differentially expressed genes have been isolated from condemned cells. One of the genes that is dramatically up-regulated with ISM death is polyubiquitin, which has been shown in many organisms to function as a heat shock protein and as an essential mediator of proteolysis. Northern blot analysis of ISM RNA samples pooled from multiple individuals demonstrated the presence of several polyubiquitin transcripts. In this study, we sought to determine: 1) if these transcripts were the product of multiple genes or multiple alleles, and 2) if all polyubiquitin genes/alleles in the moth are regulated by both heat shock and the endocrine signals that regulate death. Data from Southern blot analysis suggested that the Manduca genome has a single polyubiquitin gene that is represented by multiple alleles. Transcript analysis supported the hypothesis that all polyubiquitin alleles are regulated by both heat shock and the hormonal cues that regulate muscle death. Polyubiquitin transcripts accumulated to much higher levels and had longer half-lives following hormonal induction relative to that seen in response to heat shock. These data suggest that there are multiple polyubiquitin alleles in the laboratory population of Manduca, all of which share common regulatory sequences that drive expression to meet the needs for proteolysis involved in both heat stress and death.

MyoD cannot compensate for the absence of myogenin during skeletal muscle differentiation in murine embryonic stem cells.

myogenin (-/-) mice display severe skeletal muscle defects despite expressing normal levels of MyoD. The failure of MyoD to compensate for myogenin could be explained by distinctions in protein function or by differences in patterns of gene expression. To distinguish between these two possibilities, we compared the abilities of constitutively expressed myogenin and MyoD to support muscle differentiation in embryoid bodies made from myogenin (-/-) ES cells. Differentiated embryoid bodies from wild-type embryonic stem (ES) cells made extensive skeletal muscle, but embryoid bodies from myogenin (-/-) ES cells had greatly attenuated muscle-forming capacity. The inability of myogenin (-/-) ES cells to generate muscle was independent of endogenous MyoD expression. Skeletal muscle was restored in myogenin (-/-) ES cells by constitutive expression of myogenin. In contrast, constitutive expression of MyoD resulted in only marginal enhancement of skeletal muscle, although myocyte numbers greatly increased. The results indicated that constitutive expression of MyoD led to enhanced myogenic commitment of myogenin (-/-) cells but also indicated that committed cells were impaired in their ability to form muscle sheets without myogenin. Thus, despite their relatedness, myogenin's role in muscle formation is distinct from that of MyoD, and the distinction cannot be explained merely by differences in their expression properties.

Wild-type myoblasts rescue the ability of myogenin-null myoblasts to fuse in vivo.

Skeletal muscle is formed via a complex series of events during embryogenesis. These events include commitment of mesodermal precursor cells, cell migration, cell-cell recognition, fusion of myoblasts, activation of structural genes, and maturation. In mice lacking the bHLH transcription factor myogenin, myoblasts are specified and positioned correctly, but few fuse to form multinucleated fibers. This indicates that myogenin is critical for the fusion process and subsequent differentiation events of myogenesis. To further define the nature of the myogenic defects in myogenin-null mice, we investigated whether myogenin-null myoblasts are capable of fusing with wild-type myoblasts in vivo using chimeric mice containing mixtures of myogenin-null and wild-type cells. Chimeric embryos demonstrated that myogenin-null myoblasts readily fused in the presence of wild-type myoblasts. However, chimeric myofibers did not express wild-type levels of muscle-specific gene products, and myofibers with a high percentage of mutant nuclei appeared abnormal, suggesting that the wild-type nuclei could not fully rescue mutant nuclei in the myofibers. These data demonstrate that myoblast fusion can be uncoupled from complete myogenic differentiation and that myogenin regulates a specific subset of genes with diverse function. Thus, myogenin appears to control not only transcription of muscle structural genes but also the extracellular environment in which myoblast fusion takes place. We propose that myogenin regulates the expression of one or more extracellular or cell surface proteins required to initiate the muscle differentiation program.

Cytoplasmic domain affects membrane expression and function of an Ia molecule.

The association of foreign antigen with Ia molecules on the surface of antigen-presenting cells is necessary for the interaction with the clonally distributed antigen receptor on T cells and is therefore critical in the initiation and regulation of immune responses. Ia polypeptides (alpha and beta) are composed of two extracellular domains, a transmembrane domain and a cytoplasmic domain. Although exon-shuffling experiments have demonstrated that antigen associates with the NH2-terminal alpha 1 and beta 1 domains, the roles that the other domains play in Ia function are still poorly understood. The B-hybridoma cell line 2B1 was selected in a series of positive and negative immunoselection steps for a mutation in the Ek alpha polypeptide. It was found to fortuitously contain a mutation in the Ak alpha polypeptide as well. Sequence analysis of the Ak alpha gene showed that a single base transition (C----T) resulted in a stop codon at amino acid residue 222. This caused the loss of 12 amino acids from the cytoplasmic domain of the mature polypeptide. This mutation results in a decreased level of Ak alpha polypeptide expression on the cell surface (50% of wild-type levels), an increased half-life of Ak alpha polypeptide in the cell, and a specific limited defect in antigen presentation.

Antigen presentation abrogated in cells expressing truncated Ia molecules.

Oligonucleotide site-directed mutagenesis was used to introduce a premature stop codon in wildtype A beta k and A alpha k cDNA clones to create truncated A beta k and A alpha k molecules lacking the cytoplasmic domain. Transfected B lymphoma cells expressing an I-Ak molecule with a truncated beta-chain or with truncated alpha- and beta-chains showed profound defects in two Ia-related functions: Ia-restricted Ag presentation and intracytoplasmic signaling. The ability of these transfected cell lines to activate autoreactive T hybrids was markedly impaired whereas loss of Ag presentation to nominal Ag-specific T hybrids was more subtle. Ia-mediated transmembrane signaling as measured by PKC translocation from cytosol to nucleus after stimulation with anti-Ak antibody was greatly affected by truncation of the A beta and A alpha cytoplasmic domains. These results indicate an important role for the highly conserved cytoplasmic domain in Ia-mediated responses.

Differential induction of class II gene expression in murine pre-B-cell lines by B-cell stimulatory factor-1 and by antibodies to B-cell surface antigens.

We have previously reported that BSF-1 and an alloantibody to the B-cell differentiation antigen Lyb2 induce class II gene expression in two Ia negative pre-B-cell lines. Two questions were asked in these studies. The first question is whether the different stimuli which we and others have shown to induce class II expression in B-cells act via the same signal transduction mechanisms. The second question is whether the traditionally accepted pathway of B-cell differentiation, as defined by immunoglobulin (Ig) gene rearrangement, is applicable to other events that occur during B-cell differentiation. In this report, we have therefore examined a large panel of pre-B-cell lines at different stages of Ig gene rearrangement in an attempt to 1) identify the stage in B-cell development where class II gene expression occurs and where it becomes inducible by BSF-1 or anti-Lyb2, and 2) compare the signal transduction mechanisms used by these ligands. The majority of pre-B-cell lines tested did not express BSF-1 receptors and were consequently noninducible for class II by BSF-1; such cell lines were, however, inducible for class II expression by anti-Lyb2 and, in addition, by antibodies to the B220 membrane glycoprotein. The induction of class II molecules by BSF-1 and by anti-Lyb2 and anti-B220 differed in several respects: 1) Induction by anti-Lyb2 and anti-B220 did not require the presence of BSF-1 receptors; 2) BSF-1 selectively induced class II antigen expression while anti-Lyb2 and anti-B220 induced the expression of other surface markers as well; and 3) PGE2 inhibited BSF-1 but not antibody-mediated class II induction. Finally, the presence of receptors for BSF-1 and the baseline expression of cell surface Ia was shown to be unlinked to Ig gene rearrangement and expression in this series of pre-B-cell lines. The independent regulation of Ia and Ig genes observed here may reflect a branching rather than a linear pathway for B-cell differentiation. The differentiation of pre-B-cells to mature Ig-secreting cells should probably not be defined solely by rearrangement of Ig genes, since this is likely to represent an oversimplified view of B-cell differentiation.