Procedure for whole mount fluorescence in situ hybridization of interphase nuclei on Arabidopsis thaliana

A procedure for whole mount fluorescence in situ hybridization (FISH) on plant tissue is reported. The technique was demonstrated on seedlings and flowers of Arabidopsis thaliana L. with rDNA as a probe, labelled, both for direct and indirect detection. It was found that fixation in 1% formaldehyde yielded the best results with respect to morphology and hybridization efficiency. The combination of whole mount FISH and confocal scanning laser microscopy allowed the nuclear localization of the rDNA loci in all tissues of both seedlings and flowers. Direct labelling yielded the best signal-to-noise ratio, especially in the apical zones of the seedlings. The technique was further illustrated on seedlings of A. thaliana in double labelling experiments with rDNA and a tandemly repeated, 500 bp sequence of A. thaliana. Although nuclei in all tissues in the seedling exhibited both signals, hybridization efficiency for both signals was reduced in the dense, apical zones as compared with single labelling experiments with rDNA.     

[32P]orthophosphate and [35S]methionine label separate pools of neurofilaments with markedly different axonal transport kinetics in mouse retinal ganglion cells in vivo

Newly synthesized neurofilament proteins become highly phosphorylated within axons. Within 2 days after intravitreously injecting normal adult mice with [³²P]orthophosphate, we observed that neurofilaments along the entire length of optic axons were radiolabeled by a soluble³²P-carrier that was axonally transported faster than neurofilaments.³²P-incorporation into neurofilament proteins synthesized at the time of injection was comparatively low and minimally influenced the labeling pattern along axons.³²P-incorporation into axonal neurofilaments was considerably higher in the middle region of the optic axons. This characteristic non-uniform distribution of radiolabel remained nearly unchanged for at least 22 days. During this interval, less than 10% of the total³²P-labeled neurofilaments redistributed from the optic nerve to the optic tract. By contrast, newly synthesized neurofilaments were selectively pulse-labeled in ganglion cell bodies by intravitreous injection of [³⁵S]methionine and about 60% of this pool translocated by slow axoplasmic transport to the optic tract during the same time interval. These findings indicate that the steady-state or resident pool of neurofilaments in axons is not identical to the newly synthesized neurofilament pool, the major portion of which moves at the slowest rate of axoplasmic transport. Taken together with earlier studies, these results support the idea that, depending in part on their phosphorylation state, transported neurofilaments can interact for short or very long periods with a stationary but dynamic neurofilament lattice in axons.Special issue dedicated to Dr. Sidney Ochs.     

Effects of Ca(II) ions on Mn(II) dynamics in chick glia and rat astrocytes: Potential regulation of glutamine synthetase

Previous studies have demonstrated that in glia and astrocytes Mn(II) is distributed with ca. 30–40% in the cytoplasm, 60–70% in mitochondria. Ca(II) ions were observed to alter both the flux rates and distribution of Mn(II) ions in primary cultues of chick glia and rat astrocytes. External (influxing) Ca(II) ions had the greatest effect on Mn(II) uptake and efflux, compared to internal (effluxing) or internal-external equilibrated Ca(II) ions. External (influxing) Ca(II) ions inhibited the net rate and extent of Mn(II) uptake but enhanced Mn(II) efflux from mitochondria. These observations differ from Ca(II)–Mn(II) effects previously reported with brain (neuronal) mitochondria. Overall, increased cytoplasmic Ca(II) acts to block Mn(II) uptake and enhance Mn(II) release by mitochondria, which serve to increase the cytoplasmic concentration of free Mn(II). A hypothesis is presented involving external L-glutamate acting through membrane receptors to mobilize cell Ca(II), which in turn causes mitochondrial Mn(II) to be released. Because the concentration of free cytoplasmic Mn(II) is poised near the K_d for Mn(II) with glutamine synthetase, a slight increase in cytoplasmic Mn(II) will directly enhance the activity of glutamine synthetase, which catalyzes removal of neurotoxic glutamate and ammonia.     

Comparison of phylogenetic relationships based on phospholipid fatty acid profiles and ribosomal RNA sequence similarities among dissimilatory sulfate-reducing bacteria

Abstract Twenty-five isolates of dissimilatory sulfate-reducing bacteria were clustered based on similarity analysis of their phospholipid ester-linked fatty acids (PLFA). Of these, 22 showed that phylogenetic relationships based on the sequence similarity of their 16S rRNA directly paralleled the PLFA relationships. Desulfobacter latus and Desulfobacter curvatus grouped with the other Desulfobacter spp. by 16S rRNA comparison but not with the PLFA analysis as they contained significantly more monoenoic PLFA than the others. Similarly, Desulfovibrio africanus clustered with the Desulfovibrio spp. by 16S rRNA but not with them when analyzed by PLFA patterns because of higher monoenoic PLFA content. Otherwise, clustering obtained with either analysis was essentially congruent. The relationships defined by PLFA patterns appeared robust to shifts in nutrients and terminal electron acceptors. Additional analyses utilizing the lipopolysaccharide-lipid A hydroxy fatty acid patterns appeared not to shift the relationships based on PLFA significantly except when completely absent, as in Gram-positive bacteria. Phylogenetic relationships between isolates defined by 16S rRNA sequence divergence represent a selection clearly different from the multi-enzyme activities responsible for the PLFA patterns. Determination of bacterial relationships based on different selective pressures for various cellular components provides more clues to evolutionary history leading to a more rational nomenclature.     

Influence of endotoxin on the stereoselective pharmacokinetics of oxprenolol,propranolol, and verapamil in the rat

The influence of endotoxin-induced inflammation on the enantioselective pharmacokinetics of propranolol, oxprenolol, and verapamil, which bind to α₁-acid glycoprotein, was studied in the rat. The racemic mixtures were given orally. In the control animals, for propranolol and oxprenolol, the plasma concentrations of the (R)-enantiomer were higher than those of the (S)-enantiomer, while for verapamil the reverse was true. Protein binding and intrinsic clearance are the main factors responsible for this enantioselectivity. After endotoxin treatment, for the three drugs tested the plasma concentrations and the plasma binding of both enantiomers were significantly increased. This effect was more pronounced for (R)-propranolol, (R)-oxprenolol, and (S)-verapamil than for their respective antipodes. The enantioselective effect of endotoxin on the plasma concentrations of the drugs studied seems mainly due to the enantioselective increase in binding to α₁-acid glycoprotein. © 1994 Wiley-Liss, Inc.     

Production of a membrane-bound proteins,the human gamma-glutamyl transferase,by CHO cells cultivated on microcarriers,in aggregates and in suspension

Recombinant Chinese Hamster Ovary (CHO) cells, engineered for the production of human gamma-glutamyl transferase (GGT), have been grown on Cytodex 1 microcarriers, as aggregates, or as single cells in suspension after adaptation. GGT is a membrane bound enzyme which was not secreted during the culture period. The maximal enzyme activity was found to be directly related to the achieved maximal cell density. Culture of CHO on microcarriers yielded the fastest growth, with a specific growth rate of 0.04 h^–1, the highest cell density (near 1.3×10⁶ cells ml^–1), and the highest enzyme activity around 300 mU ml^–1, which corresponded to a specific cellular level of 20 mU 10^–5 cells. GGT could also be produced by growing CHO cells in suspension as single cells or as aggregates. Under these conditions, however, the specific CHO growth rate was significantly slower and the GGT level per cell was divided by a factor 6. Growing CHO cells without microcarriers also resulted in differences in cell metabolism, with a higher conversion yield of glutamine into ammonia, and a higher cell lysis. The catalytic kinetic constants of the enzyme were found identical for the three culture systems.     

The effect of protein synthesis inhibitors on the glycosylation site occupancy of recombinant human prolactin

The relationship between synthesis and N-liked glycosylation site occupancy of recombinant human prolactin produced from C127 cells was studied with the aid of a battery of protein synthesis inhibitors. Non-lethal concentrations of sodium fluoride, gougerotin, puromycin, anisomycin, and emetine did not alter site occupancy, but low concentrations (<10g ml^–1) of cycloheximide increased the fraction of secreted prolactin bearing oligosaccharide from 20% to 80% of the total. Cycloheximide is an inhibitor of the elongation step of protein synthesis. The observed increase in glycosylation site occupancy upon addition of cycloheximide is consistent with the current opinion that the initial glycosylation event occurs cotranslationally during a limited time period. Cycloheximide may extend this time period by reducing elongation rate. However, the absence of any effect from treatment with other inhibitors of elongation suggests that cycloheximide is unique in its behavior on this system.Abbreviations clp-PRL clipped form of prolactin - DMEM/F12 11 Dulbecco's Modified Eagle's Medium/Ham's nutrient mixture F12 - G-PRL glycosylated (N-linked) fraction of prolaction - NG-PRL prolactin fraction without N-linked glycosylation - PMSF phenylmethylsulfonylfluoride     

Water Relation Alterations Observed during Hypersensitive Reaction Induced by Bacteria

Upon exposure to pathogenic bacteria, resistant and nonhost plants undergo a hypersensitive reaction (HR) that is expressed as rapid plant cell death. If sufficient concentrations of these bacteria are inoculated to such plant tissue, then that portion of the tissue rapidly collapses and becomes necrotic. As the tissue collapses the water relations of inoculated tissues become markedly disturbed. We measured a decline in the relative water content (RWC) in the leaf-like cotyledons of cotton (Gossypium hirsutum cv Immune 216) within the first 4 h (cut at 1 h) after inoculation with Pseudomonas syringae pv tabaci. However, the decrease in RWC was not caused by a decrease in initial fresh weight but by increased water uptake during incubation in water. By 8 h after inoculation, cotyledons still on the plant had lost turgidity, and their area decreased. K+ efflux was also observed concurrently with the decrease in RWC, providing a reason for the loss of turgidity in the tissue. These observations suggest that cells lose turgor and change shape from cylinders with large intercellular spaces to those of a more tabular shape. During this change cell walls come closer together, providing an avenue for increased water uptake through capillary action. The stomatal diffusive resistance of intact cotyledons increased; hence, water loss through stomata is not the cause of the observed wilting and RWC decline. An increase in K+ per dry weight suggests that phloem loading or movement may also be impaired during bacterially induced HR.     

Clutch size, offspring performance, and intergenerational fitness

It is now generally recognized that clutch size affects morethan offspring number. In particular, clutch size affects asuite of traits associated with offspring reproductive performance.Optimal clutch size is therefore determined not by the numericallymost productive clutch but by the clutch that maximizes collectiveoffspring reproductive success. Calculation of optimal clutchsize thus requires a consideration of ecological factors operatingduring an intergenerational time frame, spanning the lifetimeof the egglaying adult and the lifetimes of her offspring. Theoptimal clutch cannot define reproductive values in advance,but instead requires that the strategy chosen is the best responseto the set of reproductive values that it itself generates.In this article, we introduce methods for solving this problem,based on an iterative solution of the equation characterizingexpected lifetime reproductive success. We begin by consideringa semelparous organism, in which case lifetime reproductivesuccess is a function only of the state of the organism. Foran iteroparous organism, lifetime reproductive success dependsupon both state and time, so that our methods extend the usualstochastic dynamic programming approach to the evaluation oflifetime reproductive success. The methods are intuitive andeasily used. We consider both semelparous and iteroparous organisms,stable and varying environments, and describe how our methodscan be employed empirically.     

Detection of Proteins in Normal and Alzheimer's Disease Cerebrospinal Fluid with a Sensitive Sandwich Enzyme-Linked Immunosorbent Assay

Abstract— Alzheimer's disease is a progressive degenerative dementia characterized by the abundant presence of neurofibrillary tangles in neurons. This study was designed to test whether the microtubule-associated protein, a major component of neurofibrillary tangles, could be detected in CSF. Additionally, we investigated whether CSF levels were abnormal in Alzheimer's disease as compared with a large group of control patients. We developed a sensitive sandwich enzyme-linked immunosorbent assay using AT120, a monoclonal antibody directed to human, as a capturing antibody. With this technique, the detection limit for was less than 5 pg/ml of CSF. Using ATS, which recognizes abnormally phosphorylated ser-ines 199–202 in, the detection limit was below 20 pg/ml of CSF. However, with AT8, we found no immunoreactiv-ity in CSF, suggesting that only a small fraction of CSF contains the abnormally phosphorylated AT8 epitope. Our results indicate that CSF levels are significantly increased in Alzheimer's disease. Also, CSF levels in a large group of patients with a diversity of neurological diseases showed overlap with CSF levels in Alzheimer's disease.