The Response to Exercise with Constant Energy Intake in Identical Twins

Seven pairs of young adult male identical twins completed a negative energy balance protocol during which they exercised on cycle ergometers twice a day, 9 out of 10 days, over a period of 93 days while being kept on a constant daily energy and nutrient intake. The total energy deficit caused by exercise above the estimated energy cost of body weight maintenance reached 244 ± 9.8 MJ (Mean ± SEM). Baseline energy intake was estimated over a period of 17 days preceding the negative energy balance protocol. Mean body weight loss was 5.0 kg (SEM = 0.6) (p <0.001) and it was entirely accounted for by the loss of fat mass (p <0.001). Fat-free mass was unchanged. Body energy losses reached 191 MJ (SEM = 24) (p <0.001) which represented about 78% of the estimated energy deficit. Subcutaneous fat loss was slightly more pronounced on the trunk than on the limbs as estimated from skinfolds, circumferences, and computed tomography (CT). The reduction in CT-assessed abdominal visceral fat was quite striking, from 81 cm² (SEM = 5) to 52 cm² (SEM = 6) (p <0.001). At the same submaximal power output level, subjects oxidized more lipids than carbohydrates after the program as indicated by the changes in the respiratory exchange ratio (p <0.05). Intrapair resemblance was observed for the changes in body weight (p <0.05), fat mass (P <0.01), percent fat (p <0.01), body energy content (p <0.01), sum of 10 skinfolds (p <0.01), abdominal visceral fat (p <0.01), fasting plasma triglycerides (p <0.05) and cholesterol (p <0.05), maximal oxygen uptake (p <0.05), and respiratory exchange ratio during submaximal work (p <0.01). We conclude that even though there were large individual differences in response to the negative energy balance and exercise protocol, subjects with the same genotype were more alike in responses than subjects with different genotypes particularly for body fat, body energy, and abdominal visceral fat changes. High lipid oxidizers and low lipid oxidizers during sub-maximal exercise were also seen despite the fact that all subjects had experienced the same exercise and nutritional conditions for about three months.     

Phosphorylation of Rat Brain Choline Acetyltransferase and Its Relationship to Enzyme Activity

Abstract— Choline acetyltransferase catalyzes the formation of acetylcholine from choline and acetyl-CoA in cholin-ergic neurons. The present study examined conditions for modulation of kinase-mediated phosphorylation of this enzyme. By using a monospecific polyclonal rabbit anti-human choline acetyltransferase antibody to immunoprecipi-tate cytosolic and membrane-associated subcellular pools of enzyme from rat hippocampal synaptosomes, we determined that only the cytosolic fraction of the enzyme (67,000 ± 730 daltons) was phosphorylated under basal, unstimulated conditions. The quantity of this endogenous phosphoprotein was dependent, in part, upon the level of intracellular calcium, with ³²P_i incorporation into the enzyme in nerve terminals incubated in nominally calcium-free medium only 43 ± 7% of control. The corresponding enzymatic activity of cytosolic choline acetyltransferase did not appear to be altered by lowered cytosolic calcium, whereas membrane-associated choline acetyltransferase activity was decreased to 58 ± 11 % of control. Depolarization of synaptosomes with 50 μ M veratridine neither altered the extent of phosphorylation or specific activity of cytosolic choline acetyltransferase, nor induced detectable phosphorylation of membrane-associated choline acetyltransferase, although the specific activity of the membrane-associated enzyme was increased to 132 ± 5% of control. In summary, phosphorylation of choline acetyltransferase does not appear to regulate cholinergic neurotransmission by a direct action on catalytic activity of the enzyme.     

Expression of the Saccharomyces cerevisiae Kex2p endoprotease in inset cells. Evidence for a carboxy-terminal autoprocessing event.

The pheromone-processing Kex2p endoprotease of Saccharomyces cerevisiae has been difficult to characterize due to its low level of expression in yeast cells. To overcome this problem, we have overexpressed Kex2p using the baculovirus/insect cell expression system. Spodoptera frugiperda Sf9 insect cells infected with a recombinant baculovirus, containing the complete KEX2 gene which encodes the Kex2p protease (814 amino acids), accumulate an 120-kDa functional form of the enzyme. The inhibition profile of the insect-cell-derived endoprotease is similar to that of the yeast enzyme. The recombinant infected insect cells also secrete into the medium about half of the total Kex2p activity produced. Deleting the carboxyl-terminal tail and the transmembrane domain of Kex2p (Kex2 delta p, 666 amino acids) does not measurably interfere with the enzyme characteristics and results in the secretion of up to 90% of the total enzyme activity. The truncated form, Kex2 delta p, of the endoprotease accumulates in the cell supernatant to 6.7 x 10(5) U/l. The molecular mass of the secreted forms for both the wild-type Kex2p and Kex2 delta p is the same (70 kDa) and is 50-kDa lower than the intracellular form. This result implicates a processing event which gives rise to shorter extracellular forms of both the wild-type Kex2p and Kex2 delta p and which trims their carboxy termini upsteam of amino acid 666. This processing event requires the integrity of the Ser385 of the Kex2p active site.     

Survival and transfer in the human gut of poorly mobilizable (pBR322) and of transferable plasmids from the same carrier E. coli

We examined the survival of a host Escherichia coli K-12 bacterium containing two transferable plasmids (pLM2, pSL222-4) and one poorly mobilizable plasmid (pBR322), and the transfer of these three plasmids to endogenous bacteria in the human intestinal tract. The survival of this plasmid-carrying host organism in four human volunteers was 3.5 to 6 days at recovery rates of 10^?1 to 10^?4. This finding was similar to our previous survival data on the same organism bearing a single plasmid. The K-12 strain appeared to be under a strong selective disadvantage in the human gut, since, even when bearing a tetracycline-resistant plasmid, its titer did not increase despite the administration of tetracycline. Studies of transferability showed that, while the transfer-depressed incFII plasmid pSL222-4 transferred at a frequency of 10^?1 in culture, its transfer in the human gut was much less frequent. The number of new recipients per donor cell ingested was about 10^?5, which included new recipients arising by multiplication. The recovery of pSL222-4 transcipients was enhanced by the administration of tetracycline on day 6. Neither the transfer-repressed, broad host range incP plasmid pLM2, nor the plasmid pBR322, could be detected in any endogenous host bacteria. Using the transfer and mobilization frequencies obtained in culture and the number of new recipients of pSL222-4 in the intestinal tract, we estimated that any in vivo mobilization of pBR322 to a new recipient could not occur at a frequency higher than 10^?12.     

The structure of immunoglobulin variable regions in the horned shark,Heterodontus francisci

The heavy and light chains of pooled antibodies of the hybodont shark,Heterodontus francisci (horned shark), were subjected to amino acid sequence analysis. Yield determinations showed that more than 90% of the available polypeptides in the respective pools were sequenced. The heavy chains were homogeneous in the initial framework segment and showed a sequence homology of approximately 70% with the corresponding region of the more recently evolved nurse shark and a 45% homology with a human myeloma heavy chain. The light chains were less homogeneous and not identifiable as either kappa or lambda chains as known in higher species. The first half-cystine characteristics of the variable domain intrachain disulfide bridge of immunoglobulins was present in the same position (22 for heavy chains; 23 for light chains) in the horned shark as in mammalian species. The sequence analysis also suggested the presence of a hypervariable region in the horned shark light chains. The combined data imply that the antigen-binding function of immunoglobulins is mediated in much the same manner in this primitive shark as in more recently evolved species, including mammals.     

Enucleation eliminates a differentiation-specific surface marker from normal and leukemic murine erythroid cells

Mammalian erythropoiesis includes a step in which the nucleus is extruded through the cell membrane. We have investigated the relationship between concanavalinA (conA) plasma membrane receptors, which are known to leave the incipient reticulocyte during enucleation, and regions of the plasma membrane which bind merocyanine 540, a differentiation-specific marker of hematopoietic cells. The distribution of these two fluorescent probes was examined on living cells from the spleens of neonatal mice and on erythroleukemia cells induced to enucleate in culture. In both cases, the region of the membrane extruded with the nucleus preferentially binds conA and merocyanine 540, whereas the plasma membrane which is left behind retains the capacity to bind another lectin, wheat germ agglutinin (WGA). The implications of these findings are discussed with respect to the mechanism by which markers are eliminated from the erythrocyte cell surface.     

CD2 can mediate TCR/CD3-independent T cell activation.

T lymphocytes can be activated clonotypically through TCR/CD3 complex or polyclonally via the CD2 molecule. Whether CD2-mediated activation is dependent on TCR/CD3 expression or signaling is controversial. We have re-explored this issue by using a series of CD2-transfected, TCR/CD3 surface membrane-negative human and mouse T cells. Our results clearly show that such T cells can be triggered for IL-2 secretion and increases in intracellular Ca2+ through the CD2 molecule in the absence of surface expression of TCR/CD3 complexes. These responses are only observed when cells express high levels of CD2 and there is a critical threshold of CD2 expression necessary for such activation in the absence of CD3. Concomitant expression of TCR/CD3 complex markedly lowers the level of CD2 required for activation via the latter pathway. These results provide a clear resolution of the controversy concerning the requirement for surface CD3 expression in T cell activation through CD2 and further suggest a possible role for CD2 in activation of TCR/CD3-negative cells.