Translation of beta-tubulin mRNA in vitro generates multiple molecular forms

We describe the in vitro expression and characterization of the isolated beta-tubulin subunit in rabbit reticulocyte lysates and compare its assembly and chromatographic properties with that of the isolated alpha-subunit and the tubulin heterodimer. The beta-tubulin polypeptides, derived from a single chicken beta-tubulin cDNA, were found in three distinct molecular forms: a multimeric or lysate-associated form, beta I (Mr approximately 180,000); the free beta-subunit beta II (Mr approximately 55,000); and the hybrid heterodimer alpha(rabbit) beta(chick), beta III (Mr approximately 80,000-100,000). The hybrid heterodimers were 100% assembly competent, whereas beta-tubulin in the "associated" beta I and the monomeric beta II forms displayed only approximately 70 +/- 15 and 25 +/- 10% competence, respectively, in coassembly assays with bovine brain tubulin. This reduced functionality was not a consequence of diminished beta-subunit stability or protein denaturation. By comparing the elution positions of the three beta forms, the monomeric alpha-subunit, and tubulin dimer purified from bovine brain, we demonstrate that anion-exchange columns (Mono-Q) interact preferentially with the alpha-subunit and chromatograph tubulin dimer on the basis of alpha-subunit isotype. The rate of exchange of the free beta-subunit into bovine tubulin dimer was followed chromatographically. The exchange was slow at 4 degrees C and rapid at 37 degrees C where it is essentially complete in 40 min in the presence of 2.5 mg/ml bovine microtubule protein. Exogenous GTP, a potent effector of microtubule assembly, binds exchangeably to beta II and enhances the recovery of this form from the Mono-Q column, suggesting that GTP binding may occur at identical sites in the isolated beta-subunit and in the tubulin heterodimer.     

The early adaptive evolution of calmodulin

Interaction between gene duplication and natural selection in molecular evolution was investigated utilizing a phylogenetic tree constructed by the parsimony procedure from amino acid sequences of 50 calmodulin- family protein members. The 50 sequences, belonging to seven protein lineages related by gene duplication (calmodulin itself, troponin-C, alkali and regulatory light chains of myosin, parvalbumin, intestinal calcium-binding protein, and glial S-100 phenylalanine-rich protein), came from a wide range of eukaryotic taxa and yielded a denser tree (more branch points within each lineage) than in earlier studies. Evidence obtained from the reconstructed pattern of base substitutions and deletions in these ancestral loci suggests that, during the early history of the family, selection acted as a transforming force on expressed genes among the duplicates to encode molecular sites with new or modified functions. In later stages of descent, however, selection was a conserving force that preserved the structures of many coadapted functional sites. Each branch of the family was found to have a unique average tempo of evolutionary change, apparently regulated through functional constraints. Proteins whose functions dictate multiple interaction with several other macromolecules evolved more slowly than those which display fewer protein-protein and protein-ion interactions, e.g., calmodulin and next troponin-C evolved at the slowest average rates, whereas parvalbumin evolved at the fastest. The history of all lineages, however, appears to be characterized by rapid rates of evolutionary change in earlier periods, followed by slower rates in more recent periods. A particularly sharp contrast between such fast and slow rates is found in the evolution of calmodulin, whose rate of change in earlier eukaryotes was manyfold faster than the average rate over the past 1 billion years. In fact, the amino acid replacements in the nascent calmodulin lineage occurred at residue positions that in extant metazoans are largely invariable, lending further support to the Darwinian hypothesis that natural selection is both a creative and a conserving force in molecular evolution.      

The heterochromatin of grasshoppers from the Caledia captiva species complex. I. Sequence evolution and conservation in a highly repeated DNA family

The restriction enzyme TaqI digests 0.2% of the genomic DNA from the grasshopper Caledia captiva to a family of sequences 168 bp in length (length of consensus sequence). The sequence variation of this "Taq family" of repeat units was examined among four races from C. captiva to assay the pattern of evolution within this highly repeated DNA. The Taq-family repeats are located in C-banded heterochromatin on at least one member of each homologous pair of chromosomes; the locations range from centromeric to telomeric. Thirty-nine cloned repeats isolated from two population 1A individuals along with 11 clones from seven populations taken from three of the races demonstrated sequence variation at 72 positions. Pairwise comparisons of the cloned repeats, both within an individual and between different races, indicate that levels of intraspecific divergence, as measured by reproductive incompatibility, do not correlate with sequence divergence among the 168-bp repeats. A number of subsequences within the repeat remain unchanged among all 50 clones; the longest of these is 18 bp. That the same 18-bp subsequence is present in all clones examined is a finding that departs significantly (P less than 0.01) from what would be expected to occur at random. Two other cloned repeats, from a reproductively isolated race of C. captiva, have sequences that show 56% identity with this 18-bp conserved region. An analysis showed that the frequency of occurrence of an RsaI recognition site within the 168- bp repeat in the entire Taq family agreed with that found in the cloned sequences. These data, along with a partial sequence for the entire Taq family obtained by sequencing uncloned repeats, suggest that the consensus sequence from the cloned copies is representative of this highly repeated family and is not a biased sample resulting from the cloning procedure. The 18-bp conserved sequence is part of a 42-bp sequence that possesses dyad symmetry typical of protein-binding sites. We speculate that this may be significant in the evolution of the Taq family of sequences.      

The alloantibody response in the allogeneically pregnant rat. II. Primary pregnancy-induced anti-RT1Aa alloantibodies are not as cross-reactive as secondary pregnancy-induced or conventionally raised alloantibodies

We compared the cross-reactive pattern of alloantibodies from conventional anti-DA alloimmunizations with anti-RT1Aa alloantibodies brought about by secondary and primary pregnancy-induced alloimmunizations caused by pregnancies by DA males. As expected, conventional alloimmunization produces alloantibodies that are cross-reactive; secondary pregnancy-induced anti-RT1Aa alloantibodies induced by pregnancies by DA males in females previously immunized against DA were as cross-reactive as conventionally raised antibodies. But primary pregnancy-induced anti-RI1Aa alloantibodies brought about by DA pregnancies in unimmunized females were not as cross-reactive as alloantibodies from conventional or secondary pregnancy-induced alloimmunizations. The cross-reactive pattern of the alloantibodies from primary pregnancy-induced alloimmunizations was distinctive for each of the three responding strains, but the cross-reactive pattern observed for a particular strain was not uniformly seen in all the responding females of that strain. One such pattern between two congenic strains defines an Ir gene between two RT1 phenotypes that are known to be high responders to RT1Aa.     

Electromyographic analysis of abdominal muscle activity using portable abdominal exercise devices and a traditional crunch

The purpose of this study was to compare the abdominal muscle activity elicited while using 4 portable abdominal training devices vs. a traditional crunch. Thirty-three adults participated in this study. The exercise devices tested included the Ab Roller Plus, Torso Track 2, AB-DOer Pro, and the Perfect Abs. All subjects were tested on the Perfect Abs in both a seated and supine position using low-, medium-, and high-resistance bands. The Torso Track 2 was also tested at low- and high-resistance settings. Surface electromyography (EMG) was recorded from the upper and lower portions of the rectus abdominis, external oblique, and the rectus femoris during each repetition. Statistical analyses were performed on the mean EMG values using a repeated analysis of variance (ANOVA) procedure. There was no significant difference in abdominal muscle activity between the Ab Roller Plus, the Torso Track 2 (high resistance), and a traditional crunch. The mean abdominal muscle activity was significantly lower than a normal crunch, however, when using the AB-DOer, Torso Track (low resistance), and the Perfect Abs seated with the low-resistance band. In contrast, the Perfect Abs, when used in the supine position with the medium- and high-resistance bands, elicited significantly greater mean abdominal muscle activity than a crunch. Of the 4 devices tested, only the Perfect Abs when used in the supine position with the medium- and high-resistance bands, elicited more abdominal activity than a crunch. The results suggest that portable abdominal devices are most effective if they not only mimic the mechanics of a traditional crunch, but also provide external resistance to increase the involvement of the abdominal musculature.     

Electromyographic comparison of a stability ball crunch with a traditional crunch

The purpose of this study was to compare abdominal muscle activity while performing a crunch on a stability ball with a traditional crunch. Forty-one healthy adults (23 men and 18 women) participated in the study. The subjects performed the crunch with the ball in 2 positions, 1 with the ball at the level of the inferior angles of the scapula (SB-high) and 1 with the ball at the level of the lower lumbar region of the back (SB-low). Surface electromyography was recorded from the upper and lower portions of the rectus abdominis and the external oblique during each repetition. Electromyography values were analyzed using repeated measures analyses of variance and pair-wise comparisons. Muscle activity for the upper and lower portions of the rectus abdominis and external oblique for a traditional crunch was significantly lower than for the crunch performed in the SB-low position but significantly greater than the SB-high position. Our data also showed that, on average, the abdominal muscle activity doubled when the stability ball was moved from the upper to the lower back position. These results support previous findings that a stability ball is not only effective for training the abdominal musculature, but, with the correct placement, it can also significantly increase muscle activity when compared with a traditional crunch. In addition, our results suggest that ball placement is critical for matching the appropriate overload to the condition level of the user.     

Two soluble glycosyltransferases glycosylate less efficiently in vivo than their membrane bound counterparts

Many Golgi glycosyltransferases are type II membrane proteins which are cleaved to produce soluble forms that are released from cells. Cho and Cummings recently reported that a soluble form of alpha1, 3- galactosyltransferase was comparable to its membrane bound counterpart in its ability to galactosylate newly synthesized glycoproteins (Cho,S.K. and Cummings,R.D. (1997) J. Biol. Chem., 272, 13622-13628). To test the generality of their findings, we compared the activities of the full length and soluble forms of two such glycosyltransferases, ss1,4 N-Acetylgalactosaminyltransferase (GM2/GD2/ GA2 synthase; GalNAcT) and beta galactoside alpha2,6 sialyltransferase (alpha2,6-ST; ST6Gal I), for production of their glycoconjugate products in vivo . Unlike the full length form of GalNAcT which produced ganglioside GM2 in transfected cells, soluble GalNAcT did not produce detectable GM2 in vivo even though it possessed in vitro GalNAcT activity comparable to that of full length GalNAcT. When compared with cells expressing full length alpha2,6-ST, cells expressing a soluble form of alpha2,6-ST contained 3-fold higher alpha2,6-ST mRNA levels and secreted 7-fold greater alpha2,6-ST activity as measured in vitro , but in striking contrast contained 2- to 4-fold less of the alpha2,6-linked sialic acid moiety in cellular glycoproteins in vivo . In summary these results suggest that unlike alpha1,3-galactosyltransferase the soluble forms of these two glycosyltransferases are less efficient at glycosylation of membrane proteins and lipids in vivo than their membrane bound counterparts.      

Site-specific inductive and inhibitory activities of MMP-2 and MMP-3 orchestrate mammary gland branching morphogenesis

During puberty, mouse mammary epithelial ducts invade the stromal mammary fat pad in a wave of branching morphogenesis to form a complex ductal tree. Using pharmacologic and genetic approaches, we find that mammary gland branching morphogenesis requires transient matrix metalloproteinase (MMP) activity for invasion and branch point selection. MMP-2, but not MMP-9, facilitates terminal end bud invasion by inhibiting epithelial cell apoptosis at the start of puberty. Unexpectedly, MMP-2 also represses precocious lateral branching during mid-puberty. In contrast, MMP-3 induces secondary and tertiary lateral branching of ducts during mid-puberty and early pregnancy. Nevertheless, the mammary gland is able to develop lactational competence in MMP mutant mice. Thus, specific MMPs refine the mammary branching pattern by distinct mechanisms during mammary gland branching morphogenesis.     

The stromal proteinase MMP3/stromelysin-1 promotes mammary carcinogenesis.

Matrix metalloproteinases (MMPs) are invariably upregulated in the stromal compartment of epithelial cancers and appear to promote invasion and metastasis. Here we report that phenotypically normal mammary epithelial cells with tetracycline-regulated expression of MMP3/stromelysin-1 (Str1) form epithelial glandular structures in vivo without Str1 but form invasive mesenchymal-like tumors with Str1. Once initiated, the tumors become independent of continued Str1 expression. Str1 also promotes spontaneous premalignant changes and malignant conversion in mammary glands of transgenic mice. These changes are blocked by coexpression of a TIMP1 transgene. The premalignant and malignant lesions have stereotyped genomic changes unlike those seen in other murine mammary cancer models. These data indicate that Str1 influences tumor initiation and alters neoplastic risk.