Enzyme profiles of single muscle fibers never exposed to normal neuromuscular activity.

Do muscle fiber properties commonly associated with fiber types in adult animals and the population distribution of these properties require normal activation patterns to develop? To address this issue, the activity of an oxidative [succinic dehydrogenase (SDH)] and a glycolytic [alpha-glycerophosphate dehydrogenase (GPD)] marker enzyme, the characteristics of myosin adenosinetriphosphatase (myosin ATPase, alkaline preincubation), and the cross-sectional area of single fibers were studied. The soleus and medial gastrocnemius of normal adult cats were compared with cats that 6 mo earlier had been spinally transected at T12-T13 at 2 wk of age. In control cats, SDH activity was higher in dark than light ATPase fibers in the soleus and higher in light than dark ATPase fibers in the medial gastrocnemius. After transection, SDH activity was similar to control in both muscles. GPD activity appeared to be elevated in some fibers in each fiber type in both muscles after transection. The cross-sectional areas most affected by spinal transection were light ATPase fibers of the soleus and dark ATPase fibers of the medial gastrocnemius, the predominant fiber type in each muscle. These data demonstrate that although the muscle fibers of cats spinalized at 2 wk of age presumably were never exposed to normal levels of activation, the activity of an oxidative marker enzyme was maintained or elevated 6 mo after spinal transection. Furthermore, although the absolute enzyme activities in some fibers were elevated by transection, three functional protein systems commonly associated with fiber types, i.e., hydrolysis of ATP by myosin ATPase and glycolytic (GPD) and oxidative (SHD) metabolism, developed in a coordinated manner typical of normal adult muscles.     

Polypeptide folding with off-lattice Monte Carlo dynamics: the method

The MC dynamics of an off-lattice all-atom protein backbone model with rigid amide planes are studied. The only degrees of freedom are the dihedral angle pairs of the C-atoms. Conformational changes are generated by Monte Carlo (MC) moves. The MC moves considered are single rotations (simple moves, SM's) giving rise to global conformational changes or, alternatively, cooperative rotations in a window of amide planes (window moves, WM's) generating local conformational changes in the window. Outside the window the protein conformation is kept invariant by constraints. These constraints produce a bias in the distribution of dihedral angles. The WM's are corrected for this bias by suitable Jacobians. The energy function used is derived from the CHARMM force field. In a first application to polyalanine it is demonstrated that WM's sample the conformational space more efficiently than SM's.Abbreviations CPU Central Processing Unit - MC Monte Carlo - MCD Monte Carlo Dynamics - MD Molecular Dynamics - RMS Root-Mean-Square - RMSD Root-Mean-Square-Deviation - SM Simple Move - WM Window Move     

Biology ofTrichogramma ostriniae (Hym.: trichogrammatidae) reared onOstrinia nubilalis (Lep.: pyralidae) and survey for additional hosts

Trichogramma ostriniae has been identified as a candidate for biological control ofOstrinia nubilalis. As little was known about the biology of this parasitoid when reared onO. nubilalis, we undertook experiments to quantify biological parameters important to mass-rearing and ase ofT. ostriniae. When reared continuously onO. nubilalis, femaleT. ostriniae on average lived 2.7 days and produced 24 progeny. Continuous access to honey resulted in a four-fold increase in longevity and fecundity and a significant increase in the percentage of females parasitizing eggs. Rates of fecundity and parasitism decreased with age of female. Likewise, emergence rates and percentage of female progeny decreased with age of parental female.T. ostriniae successfully parasitizedO. nubilalis eggs until the blackhead stage. Most parasitism of eggs and eclosion of adults occurred during the first half of photophase. Eggs of 13 Lepidopterans were parasitized byT. ostriniae. Eggs of the Noctuidae, Pyralidae, and Plutellidae experienced higher levels of parasitism than others tested.T. ostriniae appears to be similar to other species ofTrichogramma in several respects and does not possess any characteristics that limit its potential for mass rearing and use for augmentative biological control ofO. nubilalis.     

Inactivation of a Synechocystis sp strain PCC 6803 gene with homology to conserved chloroplast open reading frame 184 increases the photosystem II-to-photosystem I ratio.

A gene of the unicellular cyanobacterium Synechocystis sp strain PCC 6803 that is homologous to the conserved chloroplast open reading frame orf184 has been cloned and sequenced. The nucleotide sequence of the gene predicts a protein of 184 amino acids with a calculated molecular mass of 21.5 kD and two membrane-spanning regions. Amino acid sequence analysis showed 46 to 37% homology of the cyanobacterial orf184 with tobacco orf184, rice orf185, liverwort orf184, and Euglena gracilis orf206 sequences. Two orf184-specific mutants of Synechocystis sp PCC 6803 were constructed by insertion mutagenesis. Cells of mutants showed growth characteristics similar to those of the wild type. Their pigment composition was distinctly different from the wild type, as indicated by an increase in the phycocyanin-to-chlorophyll ratio. In addition, mutants also had a two- to threefold increase in photosynthetic electron transfer rates as well as in photosystem II-to-photosystem I ratio-a phenomenon hitherto not reported for mutants with altered photosynthetic characteristics. The observed alterations in the orf184-specific mutants provide strong evidence for a functional role of the orf184 gene product in photosynthetic processes.     

The Na-K-2Cl cotransporter is in a permanently activated state in cytoplasts from Ehrlich ascites tumor cells

Brief incubation of Ehrlich ascites tumor cells with cytochalasin B causes the formation of blebs in the surface membrane. Gentle homogenization removes the blebs as intact cytoplasts which contain neither mitochondrian or nucleus, nor other cytoplasmic membranous organelles. The Na-K-2Cl cotransporter is present in the cytoplasts in a permanently activated state, whereas the Na-K-2Cl transport system in unperturbed intact cells is silent. Pretreatment of intact cells with cytochalasin B for l min stimulates the bumetanide-inhibitable K⁺ influx fivefold. The influx into purified cytoplasts when expressed per g protein is three- to fourfold higher than the influx into cytochalasin B-treated intact cells. Thus, the membrane vesicles are enriched with the cotransporter, and the cotransporter is present in an activated state. The K influx into cytoplasts is inhibited about 40% by Na-free, Cl-free or bumetanide-containing media and to a similar extent by Fab fragments prepared from antiserum against purified proteins of the cotransporter. The K _I for bumetanide was 0.19±0.06 m for the cytoplasts as compared to 0.67±0.11 m for the intact cells. SDS gel electrophoresis of membrane proteins from the cytoplast membranes compared to the membranes of intact cells shows a reduced number of bands and a majority of bands showing reduced staining, whereas a few bands are stained more intensely. Particularly notable is a band at 80 kD, which is similar to the molecular weight previously reported for the main membrane protein isolated from intact cells using a bumetanide-Sepharose affinity column. An immunoblot of the cytoplast preparation using antibodies against the purified bumetanide binding proteins showed strong immunodetection of the 80 kD protein.We are grateful to Marianne Schiødt, Birgit Blytmann Jørgensen, Thomas Krarup and Beverley Dyer for expert assistance. This work was supported by grants from the Danish Natural Science Research Council (11-6835 to E.K.H.) and the National Institutes of Health (DK 33640 to P.B.D.) and by a Carlsberg Foundation research fellowship (to F.J.).     

The effects of acclimation and rearing conditions on the response of tropical and temperate populations ofDrosophila melanogaster andD. simulans to a temperature gradient (Diptera: Drosophilidae)

The effects of rearing and acclimation on the response of adultDrosophila to temperature were investigated in a gradient.D. melanogaster flies preferred a higher mean temperature and were distributed over a wider range of temperatures thanD. simulans flies. Acclimating adults at different temperatures for a week did not influence the response of either species. Adults reared at 28°C as immatures had a lower mean preference than those reared at cooler temperatures, suggesting that flies compensated for the effects of rearing conditions. Adults from tropical and temperate populations ofD. melanogaster andD. simulans did not differ in the mean temperature they preferred in a gradient, suggesting little genetic divergence for this trait within species. The species differences and environmental responses may be related to changes in optimal physiological conditions for the flies.     

Fusion protein mediated prodrug activation (FMPA) in vivo

A two component system, consisting of a fusion protein and an appropriate prodrug, suited to perform selective tumor therapy in vivo, is presented. The fusion protein, owing to its humanized carcinoembryonic antigen (CEA)-specific variable region, specifically binds to CEA-expressing tumors and has an enzymatic activity comparable to human β-glucuronidase. The prodrug is a nontoxic glucuronide-spacer-derivative of doxorubicin decomposing to doxorubicin by enzymatic deglucuronidation. In vivo studies in nude mice bearing human CEA-expressing tumor xenografts revealed that 7 d after injection of 20 mg/kg fusion protein, a high specificity ratio (>100:1) was obtained between tumor and plasma. Injection of 250 mg/kg of prodrug at d 7 resulted in tumor therapeutic effects superior to conventional chemotherapy without any detectable toxicity. These superior therapeutic effects that were observed using established human tumor xenografts can be explained by the approx 10-fold higher drug concentrations found in tumors of mice treated with fusion protein and prodrug than in those treated with the maximal tolerable dose of drug alone.