Rabbit muscle phosphorylase derivatives with oligosaccharides covalently bound to the glycogen storage site

Linear maltooligosaccharides, e.g., maltoheptaose or terminal 4-O-methylmaltoheptaose, activated by cyanogen bromide, react covalently with rabbit muscle phosphorylases b and a (EC 2.4.1.1). Site-specific modification prevents further binding to glycogen and shifts the phosphorylase a tetramer-dimer equilibrium in favor of the dimer. Use was made of these properties to separate by affinity chromatography and gel filtration phosphorylase a dimers with specifically bound oligosaccharide from unspecifically modified products. The phosphorylase a-maltoheptaose derivative carries one oligosaccharide residue per monomer and can be distinguished from the native enzyme by its electrophoretic mobility in polyacrylamide gels or by affinity electrophoresis. Phosphorylase a preparations with covalently bound maltooligosaccharides are enzymatically active in the presence of a primer and alpha-D-glucopyranose 1-phosphate (glucose-1-P). Methylation of the nonreducing chain terminus of the bound oligosaccharide has no effect on glycogen synthesis. These findings exclude the participation of bound oligosaccharides in chain elongation. Purified covalent phosphorylase a-maltoheptaose complexes are stable dimers. They are no longer activated by glycogen. The properties of covalently modified phosphorylase-oligosaccharides are consistent with and provide direct evidence for the existence of a glycogen storage site in rabbit muscle phosphorylases. Covalent occupation of the storage site renders the affinity of glucose-1-P to phosphorylase a independent of modulation by glycogen, supporting the assumption that the glycogen storage site is involved in interactions with the catalytic site.     

An immunocytochemical method for studying the kinetics of osteoclast nuclei on intact mouse parietal bone

Summary An immunocytochemical method using an antibody against 5-bromo-2-deoxyuridine has been applied to the study of the kinetics of osteoclast nuclei on intact mouse parietal bones. Osteoclasts containing tartrate-resistant acid phosphatase show nuclei that are positive for the thymidine analogue within 24 hours of injection into four-day old mice. Labelled osteoclast nuclei decline in number with a half-life of 1.3 days, compatible with a random mechanism of cell death rather than a fixed lifespan. This is shorter than has previously been reported and the possible reasons for this are suggested. The main advantages compared with autoradiography are the shortened processing time and the large number of osteoclasts that can be examined per parietal bone.     

Effects of Frequency and Pattern of Medial Forebrain Bundle Stimulation on Caudate Dialysate Dopamine and Serotonin

In vivo microdialysis was employed to detect changes in extracellular dopamine and serotonin in the rat caudate in response to electrical stimulation of the medial forebrain bundle. Extracellular dopamine concentrations increased linearly as a function of the frequency (4-33 Hz) of evenly spaced stimuli in both the presence and absence of cocaine added to the dialysate. Because dopamine neurons are known to fire in single-spike and burst patterns, stimulation pulses were also delivered in a bursting pattern. The response of extracellular dopamine was augmented in both the presence and absence of cocaine when the same number of stimuli were delivered in bursts as compared to an evenly spaced pattern. Serotonin, which was only assessed in the presence of cocaine, similarly increased linearly with frequency, but, in contrast to the dopamine response, levels of serotonin were not augmented by stimuli presented in bursts. These results suggest that microdialysis can be used to detect physiological changes in synaptic transmitter concentrations.     

DNA insertions distinguish the duplicated renin genes of DBA/2 andM. hortulanus mice

In a survey of inbred and wild mouse DNAs for genetic variation at the duplicate renin loci,Ren-1 andRen-2, a variantNot I hybridization pattern was observed in the wild mouseM. hortulanus. To determine the basis for this variation, the structure of theM. hortulanus renin loci has been examined in detail and compared to that of the inbred strain DBA/2. Overall, the gross features of structure in this chromosomal region are conserved in bothMus species. In particular, the sequence at the recombination site between the linkedRen-1 andRen-2 loci was found to be identical in both DBA/2 andM. hortulanus, indicating that the renin gene duplication occurred prior to the divergence of ancestors of these mice. Renin flanking sequences inM. hortulanus, however, were found to lack four DNA insertions totaling approximately 10.5 kb which reside near the DBA/2 loci. The postduplication evolution of the mouse renin genes in thus characterized by a number of insertion and/or deletion events within nearby flanking sequences. Analysis of renin expression showed little or no difference between these mice in steady state renin RNA levels in most tissues examined, suggesting that these insertions do not influence expression at those sites. A notable exception is the adrenal gland, in which DBA/2 andM. hortulanus mice exhibit different patterns of developmentally regulated renin expression.     

Role of individual phosphorylation sites in inactivation of pyruvate dehydrogenase complex in rat heart mitochondria

1. A method is described using trypsin/formic acid cleavage for unambiguously measuring occupancies of phosphorylation sites in rat heart pyruvate dehydrogenase [³²P]phosphate complexes. 2. In mitochondria oxidizing 2-oxoglutarate+l-malate relative initial rates of phosphorylation were site 1>site 2>site 3. 3. Dephosphorylation and reactivation of fully phosphorylated complex was initiated in mitochondria by inhibiting the kinase reaction. Using dichloroacetate relative rates of dephosphorylation were site 2>(1=3). Using sodium dithionite or sodium pyruvate or uncouplers+sodium arsenite or steady state turnover (³¹P replacing ³²P in inactive complex) relative rates were site 2>site 1>site 3. With dithionite reactivation was faster than site 3 dephosphorylation, i.e. site 3 is apparently not inactivating. 4. The steady state proportion of inactive complex was varied (92–48%) in mitochondria oxidizing 2-oxoglutarate/l-malate by increasing extramitochondrial Ca²⁺ (0–2.6μm). This action of Ca²⁺ induced dephosphorylation (site 3>site 2>site 1). These experiments enable prediction of site occupancies in vivo for given steady state proportions of inactive complexes. 5. The proportion of inactive complex was related linearly to occupancy of site 1. 6. Sodium dithionite (10mm) and Ca²⁺ (0.5μm) together resulted in faster dephosphorylations of each site than either agent alone; relative rates were site 2>(1=3). 7. Dephosphorylation and possibly phosphorylation of sites 1 and 2 was not purely sequential as shown by detection of complexes phosphorylated in site 2 but not in site 1. Estimates of the contribution of site 2 phosphorylation to inactivation ranged from 0.7 to 6.4%. 8. It is concluded that the primary function of site 1 phosphorylation is inactivation, phosphorylation of site 2 is not primarily concerned with inactivation and that phosphorylation of site 3 is non-inactivating.     

Cranial morphology and adaptations in Eocene Adapidae. II. The Cambridge skull of Adapis parisiensis

One of the most complete skulls of the early primate Adapis parisiensis is in the collection of the Department of Zoology, Cambridge University. This exceptionally well-preserved male skull, from Quercy in southern France, is important in showing relatively small orbits that are highly convergent, a distinct ethmoid component in the medial orbital wall, very small infraorbital foramina, a well-preserved auditory region with the stapedial canal about twice the diameter of the canal for the promontory artery, and a well-preserved braincase 8.8 cm³ in endocranial volume. The frontal lobe of the brain in the Cambridge skull described here is less expanded than that reported previously in a British Museum skull. The average body weight of Adapis parisiensis is estimated to have been about 2.0 kg, and that of Adapis magnus is estimated to have been about 8.4 to 9.0 kg. The encephalization quotient (EQ) of Adapis parisiensis is estimated to have been 0.45, which is well below the range found in modern prosimians. There is some indication that the size of the foramen magnum has increased with increasing brain size during primate evolution. Adapis parisiensis appears to have been a medium-sized, visually oriented, diurnal, sexually dimorphic arboreal folivore.     

Interactions among coexisting larval Odonata: an in situ experiment using small enclosures

Field experiments using small replicated enclosures focused on interactions between larval populations of Epitheca cynosura and Ladona deplanata (Odonata: Anisoptera) — two species that emerge in early spring. The presence of Epitheca reduced the total biomass of Ladona, but Ladona had no significant effect on Epitheca. These early-emerging species reduced the biomass of small instars of late-emerging Anisoptera which colonized enclosures during the experiments; and the late-emerging Anisoptera seem to have inhibited colonization by Zygoptera larvae. Results are consistent with the importance of predatory (cannibalism or mutual predation) interactions in this community.     

Isolation of selective 3H-chlornaltrexamine-bound complexes,possible opioid receptor components in brains of mice

The nonequilibrium narcotic antagonist, chlornaltrexamine (CNA) was used to bind selectively and covalently pioid specific sites on brain membrane preparations. Selective binding of [³H]CNA occured with a saturation maximum of 185 fmol/mg protein. Bound [³H]CNA was extracted with Triton X-100, dialyzed against Brij 36T, precipitated with trichloroacetic acid and chromatographed on an ultrogel AcA 22 column. The elution profile suggests that this extract contains a minimum of four selective [³H]CNA complexes. At least two of these complexes migrate in a single large peak. Column calibration showed that this peak eluted at 590,000 daltons. One of these specific [³H]CNA complexes elutes at the elution volume of the column and is dialyzable. Finally, putative aggregate of these complexes elutes with the void volume.