Thermodynamic and structural factors in the removal of bulky DNA adducts by the nucleotide excision repair machinery

The function of the human nucleotide excision repair (NER) apparatus is to remove bulky adducts from damaged DNA. In an effort to gain insights into the molecular mechanisms involved in the recognition and excision of bulky lesions, we investigated a series of site specifically modified oligonucleotides containing single, well-defined polycyclic aromatic hydrocarbon (PAH) diol epoxide-adenine adducts. Covalent adducts derived from the bay region PAH, benzo[a]pyrene, are removed by human NER enzymes in vitro. In contrast, the stereochemically analogous N(6)-dA adducts derived from the topologically different fjord region PAH, benzo[c]phenanthrene, are resistant to repair. The evasion of DNA repair may play a role in the observed higher tumorigenicity of the fjord region PAH diol epoxides. We are elucidating the structural and thermodynamic features of these adducts that may underlie their marked distinction in biologic function, employing high-resolution nuclear magnetic resonance studies, measurements of thermal stabilities of the PAH diol epoxide-modified oligonucleotide duplexes, and molecular dynamics simulations with free energy calculations. Our combined findings suggest that differences in the thermodynamic properties and thermal stabilities are associated with differences in distortions to the DNA induced by the lesions. These structural effects correlate with the differential NER susceptibilities and stem from the intrinsically distinct shapes of the fjord and bay region PAH diol epoxide-N(6)-adenine adducts.     

Evidence of probable scurvy in subadults from archeological sites in North America

The authors surveyed subadult human skeletons from Native American archeological sites in the United States for evidence of skeletal lesions associated with scurvy. Geographic regions surveyed include the Midatlantic area, the Southeast (Florida), the Southwest, and the Plains. The prevalence of probable subadult scurvy ranged from zero in the Plains samples to 38% in a small sample from Florida. These data indicate the likelihood that scurvy was a significant childhood disease in many Native American groups. Reasons for variation in prevalence remain speculative but include regional and seasonal variation in food types and abundance, cultural patterns of storage and utilization, periodic food shortages, and the relative importance of corn in the diet. These factors are part of a nutritional complex that is related to disease prevalence which can be studied through evidence seen in archeological human remains.     

Different long-term effects of bilateral and unilateral nucleus basalis lesions on rat cerebral cortical neurotransmitter content

Young adult rats received either unilateral or bilateral ibotenic acid infusions in their nucleus basalis, destroying most of the cholinesterase-staining neurons in that region. Cerebral cortex levels of choline acetyltransferase, somatostatin, neuropeptide Y, and monoamines were then assayed 2.5 and 10 months after bilateral lesions, or, 2.5, 10, and 14 months after unilateral lesions. Entorhinal and cerebral cortex levels of several amino acid transmitters were also measured. As expected, choline acetyltransferase activity was decreased in the frontal cortex ipsilateral to the ibotenic acid infusion in unilaterally or bilaterally lesioned animals. Parietal cortex concentrations of somatostatin and neuropeptide Y were altered by lesioning in a complicated, time-dependent manner. Thus, while unilateral lesions transiently decreased or had no effect on these neuropeptide levels, bilateral lesions elevated the level of each neuropeptide by over 100% at 10 months. Other cortical transmitter systems investigated appeared to be less affected by nucleus basalis-lesions. Unilateral lesions had no effect on prefrontal cortex norepinephrine, serotonin, or dopamine content at 14 months post-lesioning. These different neurochemical effects of unilateral and bilateral nucleus basalis lesions may be important for developing a model for the trans-synaptic effects of cortical cholinergic deafferentation.     

Ixodes dammini: Induced skin lesions in guinea pigs and rabbits compared to erythema chronicum migrans in patients with lyme arthritis

Erythematous skin lesions occurred in rabbits 2 days after being fed upon by larvae or nymphs of the tick, Ixodes dammini. Similar lesions occurred in guinea pigs 7 days after a primary infestation with either larvae or nymphs. Host resistance to secondary feeding by larvae was demonstrated in guinea pigs and rabbits. Host resistance to secondary feeding by nymphs was seen in guinea pigs, but not in rabbits. Guinea pigs developed resistance to nymphs after being previously fed upon twice by larvae. All skin lesions in resistant guinea pigs contained large accumulations of basophils (49–76% of cells) with smaller (20–33%), but significant, numbers of eosinophils. These responses were characteristic of strong cutaneous basophil hypersensitivity reactions. Primary and secondary lesions in rabbits fed upon by larvae contained mostly mononuclear cells (46–52%) and moderate numbers (16–30%) of basophils and eosinophils. Primary and secondary lesions in rabbits fed upon by nymphs had few (3–11%) basophils and eosinophils and were dominated by mononuclear cells (73–86%). Thus, acquired resistance in guinea pigs and rabbits was associated with cutaneous basophil and eosinophil responses and the lack of resistance of rabbits to nymphs was associated with erythematous lesions dominated by mononuclear cells. The mononuclear nature of rabbit lesions induced by nymphal feeding was similar to that seen in erythema chronicum migrans in Lyme arthritis patients who are thought to have been fed upon by I. dammini nymphs. This study confirms the cutaneous basophil hypersensitivity characteristics of lesions in guinea pigs resistant to ticks and demonstrates a relationship between the mononuclear cell response of rabbits to nymphal I. dammini and the cellular response seen in patients with erythema chronicum migrans and Lyme arthritis.     

Sodium-Dependent Proline Uptake in the Rat Hippocampal Formation: Association with Ipsilateral-Commissural Projections of CA3 Pyramidal

Na+-dependent uptake of L-[3H]proline was measured in a crude synaptosomal preparation from the entire rat hippocampal formation or from isolated hippocampal regions. Among hippocampal regions, Na+-dependent proline uptake was significantly greater in areas CA1 and CA2-CA3-CA4 than in the fascia dentata, whereas there was no marked regional difference in the distribution of Na+-dependent gamma-[14C]aminobutyric acid ([14C]GABA) uptake. A bilateral kainic acid lesion, which destroyed most of the CA3 hippocampal pyramidal cells, reduced Na+-dependent proline uptake by an average of 41% in area CA1 and 52% in area CA2-CA3-CA4, without affecting the Na+-dependent uptake of GABA. In the fascia dentata, neither proline nor GABA uptake was significantly altered. Kinetic studies suggested that hippocampal synaptosomes take up proline by both a high-affinity (KT = 6.7 microM) and a low-affinity (KT = 290 microM) Na+-dependent process, whereas L-[14C]glutamate is taken up predominantly by a high-affinity (KT = 6.1 microM) process. A bilateral kainic acid lesion reduced the Vmax of high-affinity proline uptake by an average of 72%, the Vmax of low-affinity proline uptake by 44%, and the Vmax of high affinity glutamate uptake by 43%, without significantly changing the affinity of the transport carriers for substrate. Ipsilateral-commissural projections of CA3 hippocampal pyramidal cells appear to possess nearly as great a capacity for taking up proline as for taking up glutamate, a probable transmitter of these pathways. Therefore proline may play an important role in transmission at synapses made by the CA3-derived Schaffer collateral, commissural, and ipsilateral associational fibers.     

Cuticular lesions induced in grass shrimp exposed to hexavalent chromium

Adult grass shrimp were exposed to four concentrations (0.5, 1.0, 2.0 and 4.0 ppm) of hexavalent chromium for 28 days. At the end of the exposure period, over 50% of the surviving shrimp possessed cuticular lesions that had many of the gross characteristics of “shell disease.” These lesions were usually associated with articulations of the appendages and abdomen. Furthermore, it was found that at increasing levels of chromium exposure, there was a proportionate increase in the loss of limbs such that nearly 50% of the limbs were lost in grass shrimp exposed to the highest test concentration of chromium. Histological and ultrastructural examination of numerous lesions demonstrated a range of degenerative features within the subcuticular epithelium that included cytoplasmic vacuolization, mitochondrial swelling, chromatin emargination, and the presence of unusual nuclear inclusions that appear to indicate direct chromium toxicity. Additionally, a marked retardation in new epicuticle and exocuticle formation was observed in viable tissues associated with lesions in late premolt shrimp. It is proposed that chromium interferes with the normal functions of subcuticular epithelium, particularly cuticle formation, and subsequently causes structural weaknesses or perforations to develop in the cuticle of newly moted shrimp. Because of these chromium-induced exoskeletal deficiencies, a viaduct for pathogenic organisms (e.g., bacteria) and direct chromium influx is formed that perpetuates lesion development.     

Nucleus tractus solitarius lesions block the behavioral actions of cholecystokinin

Cholecystokinin (CCK) has been implicated as a signal for the syndrome of satiety in a variety of species. Several lines of evidence point to a peripheral site of action for the behavioral effects of CCK. Peripheral CCK receptors appear to activate a gut-brain pathway involving the sensory fibers of the vagus nerve. To investigate the central anatomical substrate of this visceral-behavioral control system, the terminal regions of the sensory tract of the vagus were lesioned. Radiofrequency lesions of the nucleus tractus solitarius abolished the effects of acute doses of CCK on exploratory behaviors. Sham lesions had no effect on baseline exploratory behaviors and did not influence the ability of CCK to decrease spontaneous exploratory behaviors. These findings delineate the first central site along the ascending sensory pathway which appears to mediate the satiety-related behavioral effects of CCK.     

Anatomical substrate for neurotensin-acetylcholine interactions in the rat basal forebrain

We have previously shown by combined radioautography and acetylcholinesterase histochemistry that the distribution of ¹²⁵I-neurotensin (NT) binding sites was in register with that of cholinergic neurons in the rat nucleus basalis magnocellularis (NBM). The present study utilized three experimental approaches to elaborate on the type and cellular localization of NT binding sites in the NBM. Competition studies using levocabastine, a selective blocker of the low affinity NT binding component, revealed that most of the ¹²⁵I-NT binding sites labeled in the NBM are of the levocabastine-insensitive high affinity type, known to correspond to the physiologically active receptor. Ibotenic acid-induced lesions of the NBM produced a marked reduction in both cholinesterase reactivity and cellular ¹²⁵I-NT binding suggesting that most of the labeled sites are associated with the cholinergic neurons themselves rather than with an afferent input to those cells. Finally, examination of the high resolution radioautographic distribution of ¹²⁵I-NT binding sites in semithin sections revealed that a proportion of ¹²⁵I-NT-labeled receptors is associated with the plasma membrane of magnocellular perikarya and proximal processes, thereby providing an anatomical substrate for a local action of NT in the NBM.     

Desipramine Binding: Relationship to Central and Sympathetic Noradrenergic Activity

We examined the effects of treatments affecting norepinephrine release on the number of norepinephrine reuptake recognition sites as reflected by desipramine binding. To do this, we used manipulations having similar presynaptic but contrasting postsynaptic effects. Presynaptic inhibition by 6-hydroxydopamine lesion or by clonidine, and postsynaptic receptor stimulation by isoproterenol, reduced desipramine binding. Presynaptic stimulation by d-amphetamine and postsynaptic receptor blockade by prazosin increased desipramine binding. Similar effects and binding properties were seen in cerebral cortex, heart, and soleus muscle. After unilateral noradrenergic lesions, reduction in desipramine binding correlated with reduction in norepinephrine uptake. These results show that norepinephrine reuptake appears to be regulated by transmitter release regardless of effects on postsynaptic transmission, and that this regulation is analogous in the central and sympathetic nervous systems.