Stereospecific assignment of the NH2 resonances from the primary amides of asparagine and glutamine side chains in isotopically labeled proteins

An HMQC-based pulse scheme is presented for the stereospecific assignment of asparagineand glutamine side-chain amide protons. The approach makes use of the recently developedquantitative-J correlation spectroscopy [Bax, A. et al. (1994) Methods Enzymol., 239,79–105] to distinguish the E and Z primary amide protons and, as such, eliminates theneed for assignments derived from more time-consuming and potentially ambiguous NOEmethods. An application of this method to a uniformly 15N,13C-labeled cellulose-bindingdomain is presented. When used in combination with a NOESY-HSQC experiment, thepredominant 2 dihedral angles of two asparagine side chains in this protein can also bedefined.     

Gravitropic reaction of primary seminal roots ofZea mays L. influenced by temperature and soil water potential

The growth of the primary seminal root of maize (Zea mays L.) is characterized by an initial negative gravitropic reaction and a later positive one that attains a plagiotropic liminal angle. The effects of temperature and water potential of the surrounding soil on these gravitropic reactions were studied. Temperatures of 32, 25, and 18C and soil water potentials of −5,−38, and −67 kPa were imposed and the direction of growth was measured for every 1 cm length of the root. The initial negative gravitropic reaction extended to a distance of about 10cm from the graln. Higher temperatures reduced the initial negative gravitropic reaction. Lower soil water potential induced a downward growth at root emergence. A mathematical model, in which it was assumed that the rate of the directional change of root growth was a sum of a time-dependent negative gravitropic reaction and an establishment of the liminal angle, adequately fitted the distance-angle relations. It was suggested that higher temperatures and/or a lower water potential accelerated the diminution of the intitial negative gravitropic reaction.     

Chromosome behavior in the primary nucleus ofAcetabularia calyculus as revealed by epifluorescent microscopy

Summary Chromosome behavior preceding secondary nuclei formation within a giant primary nucleus (50–100 m in diameter) inAcetabularia calyculus was observed by the fluorescence emitted from 4-6-diamidino-2-phenylindole (DAPI)-stained DNA.Throughout the period when the large nucleolus was present in the primary nucleus, thin chromonemata were observed twining around the nucleolus. Nuclear division was initiated by degeneration of the sausage-shaped nucleolus into a number of spherical subunits soon after the initiation of cap formation. On the fourth day of cap development, the chromonemata became thicker and chromomeres appeared. They accumulated adjacent to the single spherical nucleolus. The lump of chromosomes became loosened and thick chromosomes were scattered in the nucleus. The peculiar shapes of chromosomes which suggest the existence of chiasmata were frequently observed until the chromosome segregation started. This sequence of chromosome behavior seems to be the prophase of meiotic division. Chromosome segregation, the first meiotic division, occurred on the seventh day of cap development, probably being accompanied by the second meiotic division. Immediately after nuclear division of the primary nucleus, secondary nuclei were formed and cyst formation started 24 hours after repeated mitoses of the secondary nuclei.     

The effects of induced prenatal hypothyroidism on lamb mandibular third primary molars.

Intrauterine thyroidectomies were performed on nine lambs on or about the ninety-sixth postconception day. Seven other control and shamoperated lambs, and the cretin lambs were sacrificed immediately after birth. The mandibles were removed and sectioned at the midline. The right side molars were removed by dissection and caliper measured. The distal cusps of the third primary molars were sectioned, dehydrated, and embedded in Bioplastic. A slow speed diamond saw was used to section the plastic blocks and the embedded teeth. Subsequent grinding and polishing produced high quality 75 micrometer sections of the lamb molar cusps. No significant differences in tooth size or enamel thickness existed. Microscopic examinations show that parts of the cretin enamel were poorly calcified, an observation that was correlated to the intrauterine thyroidectomies. The data suggest that hypothyroidism alters ameloblastic activity during the secretory phase of enamel formation.     

An ultrastructural study of primary cilia,abnormal cilia and ciliary knobs from the ciliated cells of the guinea-pig trachea

Summary Single primary cilia are found in developing as well as mature ciliated cells of guinea-pig tracheal epithelium. A few biciliated cells were observed, and in a rare case one cell had developed four such processes. Primary cilia are characterized by a 9 + 0 microtubular arrangement near the base, while a transition to an 8 + 1 pattern occurs at a slightly more distal position. Spokes are lacking, and dynein arms are absent or incompletely developed. The function, if any, of primary cilia remains unknown.In the population of the motile 9 + 2 cilia atypical forms are very rare, i.e. <0.1%. Of the various abnormalities cilia with supernumary microtubules are most common. Only one atypical basal body was observed. Although some of the aberrant forms undoubtedly are non-motile, their very low number suggests that they have no practical influence on the muco-ciliary clearance.Local extrusions of the ciliary membrane, here named ciliary knobs, are believed to be fixation artefacts. It is suggested that they represent circumscribed regions of the ciliary membrane which are sensitive to changes in the environmental osmotic pressure.     

Fatty Acid Oxidation and Ketogenesis by Astrocytes in Primary Culture

The oxidation of the fatty acids octanoate and palmitate to CO2 and the ketone bodies acetoacetate and D-(-)-3-hydroxybutyrate was examined in astrocytes that were prepared from cortex of 2-day-old rat brain and grown in primary culture to confluence. Accumulation of acetoacetate (by mass) in the culture medium of astrocytes incubated with octanoate (0.3-0.5 mM) was 50-90 nmol C2 units h-1 mg of protein-1. A similar rate was obtained using radiolabeled tracer methodology with [1-14C]octanoate as labeled substrate. The results from the radiolabeled tracer studies using [1-14C]- and [7-14C]octanoate and [1-14C]-, [13-14C]-, and [15-14C]palmitate indicated that a substantial proportion of the omega-terminal four-carbon unit of these fatty acids bypassed the beta-ketothiolase step of the beta-oxidation pathway and the 3-hydroxy-3-methylglutaryl (HMG)-CoA cycle of the classic ketogenic pathway. The [14C]acetoacetate formed from the 1-14C-labeled fatty acids, obligated to pass through the acetyl-CoA pool, contained 50% of the label at carbon 3 and 50% at carbon 1. By contrast, the [14C]acetoacetate formed from (omega-1)-labeled fatty acids contained 90% of the label at carbon 3 and 10% at carbon 1, whereas that formed from the (omega-3)-labeled fatty acid contained 20% of the label at carbon 3 and 80% at carbon 1. These results indicate that acetoacetate is primarily formed either by the action of 3-oxo-acid-CoA transferase (EC 2.8.3.5) or acetoacetyl-CoA deacylase (EC 3.1.2.11) or both on acetoacetyl-CoA and not by the action of the mitochondrial HMG-CoA cycle involving HMG-CoA lyase (EC 4.1.3.4), which was readily detected, and HMG-CoA synthase (EC 4.1.3.5), which was barely measurable.     

Characterization of [3H]Dopamine Uptake Sites and [3H]Cocaine Recognition Sites in Primary Cultures of Mesencephalic Neurons During In Vitro Development

[3H]Dopamine uptake and [3H]cocaine binding sites were studied in primary cultures of ventral mesencephalon from 14-day-old rat embryos. Specific binding sites for [3H]cocaine and [3H]mazindol were detected only in intact cell cultures of ventral mesencephalon, and were absent in sonicated, washed membranes prepared from these cell cultures. [3H]Cocaine was not taken up by the cells through an active transport process because [3H]cocaine binding occurred also at 4 degrees C. Moreover, the possibility of [3H]cocaine entering the cells by passive diffusion and ion trapping was also excluded because extensive washing failed to remove [3H]cocaine from the cells. [3H]Cocaine binding was reduced to 6% of control when cells were permeabilized with streptolysin O (0.2 U/ml, 5 min). Taken together, these results suggest that in cultured mesencephalic neurons, [3H]cocaine may enter the cell by passive diffusion and then be sequestered by a cytosolic compartment that is lost in the process of permeabilization or sonication and washing of membrane preparations. Permeabilization of cultured neurons failed to alter the storage of [3H]dopamine. When cells were permeabilized with streptolysin O (0.2 U/ml; 5 min) after [3H]dopamine was taken up, [3H]dopamine was retained by the cells and did not leak into the incubation medium, indicating that [3H]dopamine was stored in sites that could not pass through the perforated membranes. In contrast, [3H]dopamine uptake into already permeabilized cells was reduced by 33%, suggesting that a cytosolic protein that had leaked out may play a functional role in the uptake process. In contrast to striatal membrane preparations of adult rats, [3H]cocaine binding in intact mesencephalic cell cultures was Na+ independent.(ABSTRACT TRUNCATED AT 250 WORDS)     

Enxymological characterization of a putative canine analogue of primary hyperoxaluria type 1

This paper concerns an enzymological investigation into a putative canine canalogue of the human autosomal recesive disease primary hyperoxaluria type 1 (alanine:glyoxylate / serine:pyruvate aminotransferase deficiency). The liver and kidney activities of alanine:glyoxylate aminotransferase and seribe:pyruvate aminotransferase in two Tibetan Spaniel pups with familial oxalate nephripathy were markedly reduced when compared with a variety of controls. There were no obvious deficiencies in a number of other enzymes including d-glycerate dehydrogenese / glyoxylate reductase which have been shown previously to be deficient in primary hyperoxaluria type 2. Immunoblotting of liver and kidney homogenates from oxalotic dogs also demonstrated a severe deficiency of immunoreactive alanine:glyoxylate aminotransferase. The developmental expression of alanine:glyoxylate / serine:pyruvate aminotransferase was studied in the livers and kidneys of control dogs. In the liver, enzyme activity and immunoreactive protein were virtually undetectable at 1 day old, but then increased to reach a plateau between 4 and 12 weeks. During this period the activity was similar to that found in normal humanb liver. The enzyme activities and the levels of immunoreactive protein in the kidneys were more erratic, but they appeared to increase up to 8 weeks and then decrease, so that by 36 weeks the levels were similar to those found at 1 day. The data presented in this paper suggest that these oxalotic dogs have a genetic condition that is anlogous, at least enzymologically, to the human disease primary hyperoxaluria type 1.     

ESR and ENDOR of primary reactants in photosynthesis

The primary reactants in photosynthesis are defined as the chemical entities on which charges are generated and stabilized after capture of a photon by the photochemical trap: PIX ^hv P ^* IX P ⁺ I ^– X P ⁺ IX ^–, where P stands for the primary electron donor, P ^* for its excited singlet state, I for the first (ESR-detectable) electron acceptor and X for the secondary acceptor complex. The ESR and ENDOR experiments which have played a rÔle in the identification and characterization of P, I, and X in the bacterial and plant photosystems are comprehensively reviewed. The structural and kinetic information obtained with magnetic resonance techniques are integrated with results obtained with optical spectroscopy to give a unified picture of the pathway of primary photochemistry in photosynthesis. Nomenclature of Primary Reactants In the interest of uniformity this review introduces a nomenclature of the primary reactants that deviates in some respects from the commonly used labels. The nametags used here and listed below are abbreviations of the molecules that are identified as primary reactants, with the exception of the donors, for which I have retained the commonly accepted designation. Photosystems: PS 1, photosystem 1 of plants; PS 2, photosystem 2 of plants; pBPS, the photosystem of purple bacteria; gBPS, ditto of green bacteria. P: Primary donors: P700 (PS 1), P680 (PS 2), P860 (generic label for BChl a containing purple bacteria), P960 (generic label for BChl b containing purple bacteria), P840 (generic name for green bacteria). I: First acceptors: Chl a (PS 1), Ph a (PS 2), BPh a,b (pBPS). X: Secondary acceptors: F _x (PS 1), pQ ₁ (PS 2), uQ ₁ or mQ ₁ (pBPS), B (gBPS). Tertiary acceptors: F _A,B (PS 1), pQ ₂ (PS 2), uQ ₂ (pBPS), F ₁ (gBPS).This paper is based on a lecture given at the Joint Meeting of the Belgium, German (FRG), and Netherlands Societies for Biophysics, Aachen 1980     

Studies on aneuploids of Petunia

Summary The seven possible primary trisomics of Petunia (2 n= 14) located in the progenies of triploid, hypertriploid and hypotriploid plants were distinguished from one another and from diploid on the basis of cytological and morphological criteria. They were provisionally named as Oval, Semi, Slender, Pseudonormal, Arrow, Narrow and Giant. In three of the trisomics, the extra chromosome was identified for the first time at pachytene stage. Postpachytene studies revealed no precise relationship between the length of extra chromosome and the frequency of multiple association.