Biochemical and histochemical properties of hepatic tumors of rainbow trout, Oncorhynchus mykiss

Biochemical and histochemical studies were conducted in aflatoxinB₁-induced liver tumors in adult, rainbow trout. Specific activitiesof the phase I enzymes, ethoxyresorufin-O-deethylase (EROD),microsomal and cytosolic epoxide hydrolase (mEH and cEH), aldehydedehydrogenase (ALDH) and DT-diaphorase, and the phase II enzymes,-glutamyltransferase (-GT), glutathione transferase (GST) anduridine diphosphoglucuronyl transferase (UDPGT) were measured.Cryostat sections of tumor and surrounding liver from the samecohorts were analyzed immunohistochemically for cytochrome P450IA1and histochemically for ALDH (benzaldehyde and hexanal), DT-diaphorase,-GT and uridine diphosphoglucuronyl dehydrogenase (UDPGdH).In tumor tissues, the largest biochemical changes were foundwith benzaldehyde dehydrogenase, where activity increased fromundetectable levels to 7.4 nmol/min/mg protein, and -GT, whereactivity increased 12-fold over controls. Increases in otherenzymes ranged from 1.26 to 2.84 times that of control liver,except EROD, which decreased, and cEH and mEH, which were unchanged.Histochemical analyses showed mEH, which were unchanged. Hischemicalanalyses showed the induction of ALDH, -GT, DT-diaphorase andUDPGdH, and the depression of cytochrome P450IA1 in hepaticneoplasms. In addition, marker enzyme histochemistry of neoplasmsrevealed heterogeneous populations of hepatocytes and absenceof necrotic areas.     

Direct selection: a method for the isolation of cDNAs encoded by large genomic regions.

We have developed a strategy for the rapid enrichment and identification of cDNAs encoded by large genomic regions. The basis of this "direct selection" scheme is the hybridization of an entire library of cDNAs to an immobilized genomic clone. Nonspecific hybrids are eliminated and selected cDNAs are eluted. These molecules are then amplified and are either cloned or subjected to further selection/amplification cycles. This scheme was tested using a 550-kilobase yeast artificial chromosome clone that contains the EPO gene. Using this clone and a fetal kidney cDNA library, we have achieved a 1000-fold enrichment of EPO cDNAs in one cycle of enrichment. More significantly, we have further investigated one of the "anonymous" cDNAs that was selectively enriched. We confirmed that this cDNA was encoded by the yeast artificial chromosome. Its frequency in the starting library was 1 in 1 x 10(5) cDNAs and after selection comprised 2% of the selected library. DNA sequence analysis of this cDNA and of the yeast artificial chromosome clone revealed that this gene encodes the beta 2 subunit of the human guanine nucleotide-binding regulatory proteins. Restriction mapping and hybridization data position this gene (GNB2) to within 30-70 kilobases of the EPO gene. The selective isolation and mapping of GNB2 confirms the feasibility of this direct selection strategy and suggests that it will be useful for the rapid isolation of cDNAs, including disease-related genes, across extensive portions of the human genome.     

Neighborhood Socioeconomic Status and Cognitive Trajectories in a Diverse Longitudinal Cohort

Objectives: Although individual-level socioeconomic status is associated with poor outcomes, less is known regarding how the social context might affect cognitive outcomes. We examined the effect of neighborhood socioeconomic status (NSES) on baseline cognitive function and trajectories of decline.

Methods: The sample (N = 480) came from a longitudinal cohort recruited to study cognitive function. Mixed effects models examined the influence of NSES on baseline and rate of change in executive function, semantic memory, and episodic memory.

Results: NSES was positively associated with semantic memory scores at baseline, but not with executive function or episodic memory in adjusted models, nor was it associated with cognitive change in longitudinal analyses. In exploratory analyses, for individuals with dementia, those with higher NSES declined faster in executive function and semantic memory than did those with lower NSES.

Conclusions: Results suggest that NSES has limited effects independent of personal characteristics; however, findings showed a complex relation of NSES and decline, with NSES effects observed only for individuals with dementia. Results are discussed in the context of cognitive reserve.

Clinical Implications: Clinical assessments of individuals who present with cognitive impairment might benefit from an understanding of the neighborhood context from which patients come.     

Gamma glutamyl transpeptidase in safrole-induced, presumptive premalignant mouse hepatocytes

A histochemical procedure was used to determine the presenceof -glutamyl transpeptidase (GGT) in the livers of control,regenerating and carcinogen-treated mice. Young Balb/c micewere fed safrole, a naturally occurring hepatocarcinogen (0.4%w/w), for one year. Ten mice from control and ten mice fromthe safrole-treated group were killed at 4,8,16,24,36, and 52weeks of exposure and 9 adult mice were killed after 2/3 hepatectomy.Basophilic and acidophilic foci of altered hepatocytes occurredin safrole-treated mice after 24 weeks. Neoplastic nodules appearedafter 36 weeks. Both foci and nodules displayed elevated GGTactivity as determined by enzyme histochemistry. Variabilityin the pattern of enzyme distribution and staining intensitywas seen between cells of the same focus or nodule, as wellas between different foci and nodules. Hepatocytes from regeneratinglivers of partially hepatectomized mice were negative for GGT.These results demonstrated a sequential development of carcinogen-alteredhepatocyte populations characterized by the appearance of GGTactivity prior to carcinoma formation. The results in the mouseshow marked similarity to those reported in rat liver, whereGGT has been used as a positive marker for premalignant liverlesions induced by a variety of carcinogens.     

Vitamin E succinate inhibits proliferation and migration of retinal pigment epithelial cells in vitro: therapeutic implication for proliferative vitreoretinopathy

Background Retinal pigment epithelial (RPE) cells play an important role in proliferative vitreoretinopathy (PVR). Vitamin E succinate is an ester form of a potent biological antioxidant, vitamin E, and has unique effects on various cells. We examined the effect of vitamin E succinate on proliferation and migration of cultured bovine RPE cells, since these are critical steps in the development of PVR. Methods Bovine RPE cells were cultured in minimal essential medium (MEM) containing 10% fetal calf serum (MEM-10). Cells were incubated with MEM-10 containing 25 M vitamin E, vitamin E succinate, butylated hydroxytoluene BHT) or d-mannitol. Cell proliferation was assessed by counting cell numbers on days 2, 4 and 6. ³H-Thymidine uptake was also examined in RPE cells incubated with various forms of vitamin E — vitamin E, vitamin E succinate, Trolox, -tocopherol, vitamin E acetate, vitamin E phosphate, vitamin E nicotinate — or antioxidants — BHT or d-mannitol (25 M each). RPE cell migration was studied as follows: small area (5×15 mm) of confluent cultured RPE cells was denuded using a straight razor blade and incubation was continued for 20 h with MEM-10 containing vitamin E, vitamin E succinate, -tocopherol or BHT. The number of cells that migrated into the denuded area from the wound edge in each microscopic field (×20) was counted and expressed as a percentage of control (MEM-10 alone). Results The antioxidants, vitamin E and BHT, stimulated RPE cell proliferation and ³H-thymidine incorporation compared with the control, while vitamin E succinate significantly inhibited both proliferation and ³H-thymidine uptake (IC₅₀, 23 M Other forms of vitamin E or d-mannitol had no effect. Neither vitamin E nor BHT had a significant effect on RPE cell migration (108.2% and 112.6% of control, respectively), but vitamin E succinate inhibited migration (58.3%). Cell viability, assessed by the trypan blue dye exclusion test, was not impaired by a 3-day incubation with 50 M of vitamin E succinate. Conclusions An ester form of a physiological antioxidant, vitamin E succinate, inhibits RPE cell proliferation and migration without causing cellular toxicity. These findings suggest its therapeutic potential for the pharmacological treatment of PVR.     

Involvement of the medial prefrontal cortex in mediating behavioural responses to odour cues rather than olfactory recognition memory

Sheep form an olfactory recognition memory for their lambs within 2 h of parturition and will subsequently reject the approaches of any strange lamb and protest vocally. In this study we report that following olfactory memory formation, ewes exposed to either their own or a strange lamb show c-fos mRNA expression in the medial frontal cortex, although levels of expression in the pyramidal output cell layer V were significantly higher in ewes that rejected strange lambs. Reversibly inactivating this region by the retrodialysis of the anaesthetic tetracaine before birth reduced aggressive motor responses towards lambs but not protest vocalisations. Similar treatment during the critical period for olfactory memory formation and lamb recognition (0-4 h post-partum) had no effect on ewes maternal behaviour towards their own lambs. It did, however, prevent the normal selective expression of aggressive rejection, and reduced protest vocalisation behaviours directed towards strange lambs. These rejection behaviours did appear 1 h after the termination of tetracaine infusions despite the ewes not being given the opportunity to interact with their own lambs during this time. Therefore, tetracaine blockade of the medial frontal cortex prevents animals from responding with motor aggression, but not vocal aggression, to odour cues from strange lambs, but has no effect on the formation of an olfactory recognition memory for their own lambs. Both pre- and post-partum aggressive rejection of strange lambs was associated with increased concentrations of dopamine, serotonin, glutamate and GABA. When these behaviours were inhibited by the tetracaine infusions, extracellular concentrations of these neurotransmitters were all increased by the anaesthetic but did not change in response to lambs. These findings suggest that a functional medial frontal cortex is not required for the formation of an olfactory recognition memory or for mediating pro-active maternal behaviours. It is however required for the mediation of motor but not vocal aspects of aggressive rejection responses directed towards aversive odour cues from strange lambs.     

Sequential infection of glial cells by the murine hepatitis virus JHM strain (MHV-4) leads to a characteristic distribution of demyelination.

An antigenic variant of the neurotropic murine coronavirus JHMV, designated 2.2-V-1, causes marked demyelination in the relative absence of encephalitis. It is thus useful for the study of the pathogenesis of demyelinating lesions. To better understand the sequential events leading to demyelination, we have examined murine brain and spinal cord tissue at daily intervals after intracerebral inoculation, evaluating them for the distribution of viral antigen, leukocyte infiltration, and demyelination. Immunohistochemical staining indicated that virus established primary infection in the ependymal cells in both brain and spinal cord before spreading into nearby structures and along white matter tracts by cell-to-cell contact. Spread from brain to spinal cord appeared to occur via cerebrospinal fluid. Viral replication was focally cytocidal for ependymal cells, and essentially noncytocidal for other neural cells including glia. In brain, viral antigen and inflammation reached a peak at day 5 postinfection, and rapidly subsided by day 10 postinfection. In spinal cord, viral antigen was less abundant than in brain and was maximal between days 7 and 9 postinfection. The inflammatory response and demyelination, however, were more severe persisting from day 7 through day 19. In the spinal cord, demyelinating lesions developed initially in areas closer to the central canal and were detected most prominently in the anterior funiculi. This finding suggests that the permissiveness of the ependymal cell is crucial to viral entry and that sequential infection of glial cells leads to the characteristic distribution of demyelination.     

Study of 250 children with idiopathic mental retardation reveals nine cryptic and diverse subtelomeric chromosome anomalies

Cryptic subtelomeric chromosome anomalies have been recognized as a significant cause of dysmorphology and mental retardation. To determine whether the clinical cytogenetics laboratory should screen routinely for these aberrations, we have tested 250 patients with idiopathic mental retardation/developmental delay, either isolated (53) or associated with dysmorphic features and/or malformations in the absence of a recognizable syndrome (197). All had normal karyotypes at the 550-850 band level. Subtelomeric anomalies were found in 1/53 of the first group (1.9%) and 8/197 of the second group (4.1%). In one patient, two separate anomalies were present: a deletion (not inherited) and a duplication (inherited). It is possible that one of these 10 observed aberrations might represent a rare and previously unreported polymorphism and one a rare cross-hybridization. Our study supports the proposition that cryptic subtelomeric rearrangements are a significant cause of idiopathic mental retardation/developmental delay, but both the diversity of the phenotypes of the positive cases and the wide diversity of their associated chromosome abnormalities emphasize the central problem for the clinical cytogenetics laboratory-that of choosing the most productive patient base for this useful diagnostic test.     

Dilated ascending aorta in a child with ring chromosome 21 syndrome

Ring chromosome 21 syndrome is a rare condition with a well-characterized phenotype. Affected individuals have recognizable dysmorphic features, developmental delays, growth retardation, and a predisposition for congenital malformations involving the neurologic, craniofacial, digestive, genitourinary, skeletal, and hematologic systems. Structural cardiac anomalies have also been described, but dilated ascending aorta has not been previously reported in association with ring 21 (r(21)). Although rarely seen in this syndrome, the presence of ectopia lentis, abdominal herniae, and dilated ascending aorta suggest an underlying connective tissue disorder. A possible explanation is haploinsufficiency of the COL6A1, COL6A2, and/or COL18A genes located on the distal portion of chromosome 21q, which are lost when the ring chromosome is formed. This article contains supplementary material, which may be viewed at the American Journal of Medical Genetics website at http://www.interscience.wiley.com/jpages/0148-7299/suppmat/index.html.     

Morphometric changes of the lung induced by inhaled bacterial endotoxin

Due to the ubiquitous nature of airborne endotoxin, an understanding of pulmonary alterations which follow inhalation of environmentally realistic concentrations of purified bacteria derived lipopolysaccharide (LPS) is important. Using LPS derived from Enterobacter agglomerans, a bacterium found in cotton and cotton mill dust, aqueous aerosols (effective LPS concentration 4 micrograms/m3) were generated and used to expose either normal hamsters (N = 6) or those rendered endotoxin tolerant by pre-ip injection of 0.1 LD50 LPS. Control groups (normal--N = 6; tolerant--N = 6) received saline aerosol only. At 6 hr after 5-hr aerosol exposure, lungs of all animals were fixed, processed for light and transmission electron microscopy, and subject to qualitative and to multitiered morphometric analysis using standard point counting techniques. Qualitative evaluation of TEM micrographs from LPS aerosolized-nontolerant hamsters showed endothelial alteration (focal disruption, subendothelial space formation, and cytoplasmic blebbing) but volume and number of endothelial cells were not changed indicating only slight, focal endothelial damage. Quantitatively, septal capillary blood space in nontolerant, LPS aerosolized hamsters showed increased Vv of PMNs and platelets. These changes were not seen in tolerant induced-LPS aerosolized hamsters. Independent of tolerization treatment, LPS inhalation led to a decrease in fixed lung volume and an increase in numerical density of endothelial pinocytotic vesicles. It is concluded that the inhalation of realistic, environmental levels of bacterial endotoxin may induce significant changes in distal lung and may be important in the pathogenesis of byssinosis and adult respiratory distress syndrome.