Characterization and Distribution of a Cloned Rat μ-Opioid Receptor

Abstract: We have cloned and expressed a rat brain cDNA, TS11, that encodes a μ-opioid receptor based on pharmacological, physiological, and anatomical criteria. Membranes were prepared from COS-7 cells transiently expressing TS11 bound [³H]diprenorphine with high affinity (K_D = 0.23 ± 0.04 nM). The rank order potency of drugs competing with [³H]diprenorphine was as follows: levorphanol (K_i = 0.6 ± 0.2 nM) ≈β-endorphin (K_i = 0.7 ± 0.5 nM) ≈ morphine (K_i = 0.8 ± 0.5 nM) ≈ [d -Ala², N-Me-Phe⁴,Gly-ol⁵]-enkephalin (DAMGO; K_i = 1.6 ± 0.5 nM) ? U50,488 (K_i = 910 ± 0.78 nM) > [d -Pen^2,5]-enkephalin (K_i = 3,170 ± 98 nM) > dextrorphan (K_i = 4,100 ± 68 nM). The rank order potencies of these ligands, the stereospecificity of levorphanol, and morphine's subnanomolar K_i are consistent with a μ-opioid binding site. Two additional experiments provided evidence that this opioid-binding site is functionally coupled to G proteins: (a) In COS-7 cells 50 µM 5′-guanylylimidodiphosphate shifted a fraction of receptors with high affinity for DAMGO (IC₅₀ = 3.4 ± 0.5 nM) to a lower-affinity state (IC₅₀ = 89.0 ± 19.0 nM), and (b) exposure of Chinese hamster ovary cells stably expressing the cloned μ-opioid receptor to DAMGO resulted in a dose-dependent, naloxone-sensitive inhibition of forskolin-stimulated cyclic AMP production. The distribution of mRNA corresponding to the μ-opioid receptor encoded by TS11 was determined by in situ hybridization to brain sections prepared from adult female rats. The highest levels of μ-receptor mRNA were detected in the thalamus, medial habenula, and the caudate putamen; however, significant hybridization was also observed in many other brain regions, including the hypothalamus.     

Enhanced Glial Fibrillary Acidic Protein RNA Response to Fornix Transection in Aged Mice

Abstract: The effects of age on basal and lesion-induced changes in astrocyte RNA messages reported to respond to neurodegeneration were examined in the mouse brain. The first study found an age-related increase in glial fibrillary acidic protein RNA throughout the brain. Other astrocyte RNAs remained generally stable with age. We hypothesize this increase is due to astrocytes undergoing a mild reaction to the small amount of synaptic degeneration occurring with usual aging. To test this theory, we used an experimental model of modest synaptic loss in the hippocampus by transecting the fimbria/fornix bundle in mice and examined the same series of messages. In situ hybridization revealed the expected increase in glial fibrillary acidic protein RNA after the lesion; however, we unexpectedly found that aged mice showed a greater magnitude of this response, which appeared to develop more slowly. There was no significant change in the hippocampus for any of the other messages, although responses were observed at the site of transection. This study supports the idea that the age-related increase in glial fibrillary acidic protein may be secondary to modest synaptic degeneration. We also demonstrated an exaggerated reactive astrocytic response in aged mice, which may be associated with age-related deficits in reactive synaptogenesis and behavioral recovery in normal aging.     

N-glycans of recombinant human interferon-gamma change during batch culture of chinese hamster ovary cells

A recombinant Chinese hamster ovary (CHO) cell line making human interfron-gamma (IFN-gamma) was grown in 12-L stirred tank fermentors in three batch fermentations under conditions of constant temperature, pH, and dissolved oxygen tension. In addition to cell growth, metabolite, and productivity data, a detailed analysis of the carbohydrate structures attached to each glycosylation site of IFN-gamma was achieved using matrix-assisted laser desorption mass spectrometry (MALDI-MS) in combination with exoglycosidase array sequencing. Complex biantennary oligosaccharides (particularly Gal(2)GlcNAc(4)Man(3) which was core alephl-6 fucosylated at Asn(25) but not at Asng(97)) were most prevalent at both glycosylation sites. However, considerable microheterogeneity arising from the presence of triantennary and truncated glycan structures was also observed. The proportion of the dominant core glycan structure (Gal(2)GlcNAc(4)Man(3) +/- Fuc(1)) decreased by 15-26% during batch culture, with increases in the proportion of oligomannose and truncated glycans over the same time period. Prolonged culture resulting from an extended lag phase led to further accumulation of oligomannose and truncated structures, reaching up to 52% of total glycans attached to Asng(97) by 240 h of culture. The implications of these glycosylation changes for optimizing the time for harvesting cell cultures, and for the clearance of recombinant therapeutic products in vivo are discussed. (c) 1995 John Wiley & Sons, Inc.     

Taxonomic studies in the Miconieae (Melastomataceae). VII. Miconia howardiana,a new speices from Hispaniola

Miconia howardiana, which is known only from a diverse moist montane forest in the vicinity of Loma Trocha de Pey (or “Monteada Nueva”), the easternmost peak of the Sierra de Baoruco, is described and illustrated. It is compared to phenetically similar (and probably phylogenetically related) species of theMiconia favosa complex, i.e.,M. favosa, M. xenotricha, M. campanensis M. Sintenisii, M. foveolata, andM. pycnoneura. The species of this complex are characterized by bullate leaves with frequently cordate bases and often six secondary veins. Most species also have large berries and flowers, 5-locular ovaries, large inflorescence bracts and bracteoles, and more or less dendritic multicellular hairs.     

Cellular Basis and Developmental Timing in a Size Cline of Drosophila Melanogaster

We examined 20 Drosophila melanogaster populations collected from a 2600-km north-south transect in Australia. In laboratory culture at constant temperature and standard larval density, a genetic cline in thorax length and wing area was found, with both traits increasing with latitude. The cline in wing area was based on clines in both cell size and cell number, but was primarily determined by changes in cell number. Body size and larval development time were not associated among populations. We discuss our results in the context of selection processes operating in natural and experimental populations.     

Recolonization of estuarine organisms: effects of microcosm size and pesticides

Two six-week laboratory experiments were conducted to evaluate effects of pesticides and microcosm size on benthic estuarine macroinvertebrate recolonization. Sediments fortified with the pesticides (fenvalerate: controls, 5 (low) and 50 μg g⁻¹ wet sediment (high); endosulfan: controls, 1 (low) and 10 μg g⁻¹ wet sediment (high)) were fine-grained, organically rich (approximately 3.5% organic carbon and 22% dry weight) material. Relative dominance of the four most abundant taxa in both experiments was consistent among treatments with few exceptions. The amphipod,Corophium acherusicum, dominated abundance in both experiments. In the fenvalerate experiment, large trays (400 cm²) contained significantly (p<0.05) more total number of taxa (TNT) than small microcosms (144 cm²) but tray size was not significantly related to total number of organisms (TNO). When size was adjusted to a common unit area, small trays contained significantly more TNO than large containers. Adjusted abundance of small trays was 2.5 times that of large containers; a ratio close to that of microcosm sizes (i.e., 2.8). This result suggests that larval supply may have been inadequate to ‘aturate’ the available sediment in large containers. Fenvalerate significantly reduced abundance in the high treatment compared to both controls and low treatment but low treatment was not significantly different from controls. The amphipod,Corophium acherusicum, accounted for most of the decrease in abundance in response to fenvalerate. The holothruroid,Leptosynpta sp. and the polychaete,Mediomastus ambiseta, increased in abundance significantly with increased concentration of fenvalerate. Combined effects of actual microcosm size and concentration of endosulfan were not significant for TNO or TNT. As in the fenvalerate experiment, adjusted abundance of small microcosms was 2.6 times that of large trays which approximated the ratio of unit area between microcosm sizes. Abundance of a few taxa responded significantly to adjusted and unadjusted unit area. Abundance of the tunicate,Molgula manhattensis, increased significantly with increased concentration of endosulfan. Abundance was affected by sample location (e.g., interiorvs exterior cores) within microcosms. Abundance adjusted to unit area resulted in significantly greater TNO in externalvs internal cores. This has importance for sequential sub-sampling of microcosms to determine temporal dynamics. Statistically significant effects were measured in benthic community structure associated with microcosm size; however, the magnitude was relatively small. There appears to be no major biological reason to select one microcosm size over the other for screening for contaminant effects. Where feasible, the small trays provide savings in sample preparation and analysis, allow more replicates where laboratory space is limiting and generate less chemical waste. These benefits may be off-set by less ‘artifacts’ associated with edge effects of larger microcosms and the need for a larger mass of sediment to accommodate additional analytical requirements (e.g., thin vertical surficial samples to refine contaminant exposure at the sediment/water interface).     

Modulation of cisPlatin cytotoxicity by interleukin-1α and resident tumor macrophages

The modulation of cisPlatin cytotoxicity by interleukin-1 (IL-1α) was studied in cultures of SCC-7 tumor cells with and without tumor macrophages to examine potential mechanisms for the synergistic antitumor activity of cisPlatin and IL-1α in SCC-7 solid tumors. Neither IL-1α nor tumor macrophages affected the survival of clonogenic tumor cells and IL-1α had no direct effect on tumor cell growthin vitro. Macrophages had no direct effect on cisPlatin sensitivity (IC₉₀=6.0 μM), but, the addition of IL-1α (500–2000U/ml) to co-cultures of cisPlatin pretreated tumor cells and resident tumor macrophages increased cell killing (IC₉₀=3.1 μM). Similar responses were seen in primary cultures treated with cisPlatin before IL-1α. The modulation of cisPlatin cytotoxicity by IL-1α exhibited a biphasic dose response that paralleled the IL-1α dose dependent release of H₂O₂by resident tumor macrophages. Further, IL-1α modification of cisPlatin cytotoxicity was prompt and inhibited by catalase. CisPlatin and exogenous H₂O₂ (50 μM) produced more than additive SCC-7 clonogenic cell kill and hydroxyl radicals played an important role in the response. Interleukin-1 modulation of cisPlatin cytotoxicity was schedule dependent. IL-1α treatment for 24 hrs, before cisPlatin, produced drug resistance (IC₉₀=11.1 μM). Our study shows that IL-1α can stimulate tumor macrophages to release pro-oxidants that modify cellular chemosensitivity in a schedule and dose dependent fashion. Our findings may also provide a mechanistic explanation for the synergistic antitumor activity of cisPlatin and IL-1αin vivo.     

Relictual vegetative anatomical characters in Cactaceae: the genusPereskia

The genusPereskia, which contains numerous morphological features considered relictual in the Cactaceae, has numerous anatomical features that we consider to be relictual also. These were studied to establish a basis for determining the ways that morphogenic mechanisms and anatomical characters diversified as the family evolved. ThesePereskia features may be relictual in the family: epidermis predominantly unistratose and lacking crystals; hypodermis absent or of about three layers of weakly collenchymatous cells with druses; cortex thin and predominantly parenchyma with druses and mucilage cells but lacking cortical bundles; secondary phloem without early differentiation of sclerenchyma but with secondary sclereids developing later, either idioblastically or in clusters; ergastic substances lacking from old secondary phloem; wood with a matrix of libriform fibers (mostly septate and nucleate), scanty paratracheal parenchyma, vessels solitary or in small clusters, perforations simple, pitting circular, oval or very broad; wide-band tracheids absent; ray cells slightly thick-walled, lignified, upright, isodiametric or procumbent; all primary rays narrow; pith without medullary bundles; leaves lacking hypodermis, with only weak development of palisade mesophyll; veins of four orders, strongly distinct in size, none with fibers; vessels in leaves narrower than those in stems.