LEAF DEVELOPMENT OF RUMEX PATIENTIA L. (POLYGONACEAE) EXPOSED TO UV IRRADIATION (280-320 NM)

Two factors which affect leaf ontogeny and ultimate leaf size: (1) the rate and duration of cell expansion, and (2) the rate and duration of cell division, were examined for their role in the slower early leaf growth rate and the smaller size of fully expanded leaves of plants exposed to ultraviolet-it (UV-B 280-320 nm) radiation. Rumex patientia L. was grown in controlled environment chambers under enhanced UV-B radiation (equivalent to daily solar UV-B irradiation at 40°N latitude in mid-May with an atmospheric ozone concentration of 0.20 atm-cm) and control treatments. The pattern of growth as expressed in changes of mean cell size in two distinct cell types, tissue cell density, and length of the entire leaf blades are consistent with the hypothesis that the radiation primarily affects cell division rather than cell expansion. Furthermore, it appears that the radiation probably alters the rate rather than the duration of the cell division phase. An understanding of the mechanism of radiation damage should facilitate prediction of how this stress may interact with other stresses to which plants are normally subjected. Species with normally prolonged periods of cell division during leaf expansion may be particularly impacted if solar UV radiation were intensified as a result of atmospheric ozone reduction.     

Reduction of rapid eye movement (REM) sleep by glucose alone or glucose and insulin in rats

Administration of a high dose of glucose (2.5 g/kg, i.p.) that is known to produce severe hyperglycemia in euglycemic rats suppressed rapid eye movement (REM) sleep time significantly during the first three hours of 8 hr total electroencephalogram (EEG) recording period. Co-administration of glucose (2.5 g/kg, i.p.) and a non-convulsive dose of insulin (1.0 I.U./kg, i.p.) produced a significant reduction in REM sleep time during 1st through 5th hour and an increase in slow-wave sleep (NREM) time in the 3rd and 4th hour of 8 hr total EEG recording period. However, awake, NREM and REM sleep time in the 8 hr total EEG recording period were unaffected by either glucose alone or glucose plus insulin treatments. These results strongly suggest that the insulin's effects on the sleep-awake cycle i.e. reduction in REM and a slight increase in NREM sleep times of rats is not due to indirect effects of insulin on the central nervous system via hypoglycemia as reported by us previously, but could possibly be due to its direct effects on brain chemistry of neurotransmitters such as serotonin, catecholamines and acetylcholine which are believed to modulate the sleep-awake cycle pattern in rats.     

Functionally distinct roles for TET-oxidized 5-methylcytosine bases in somatic reprogramming to pluripotency

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Optical Spectral Surveillance of Breast Tissue Landscapes for Detection of Residual Disease in Breast Tumor Margins

We demonstrate a strategy to “sense” the micro-morphology of a breast tumor margin over a wide field of view by creating quantitative hyperspectral maps of the tissue optical properties (absorption and scattering), where each voxel can be deconstructed to provide information on the underlying histology. Information about the underlying tissue histology is encoded in the quantitative spectral information (in the visible wavelength range), and residual carcinoma is detected as a shift in the histological landscape to one with less fat and higher glandular content. To demonstrate this strategy, fully intact, fresh lumpectomy specimens (n = 88) from 70 patients were imaged intra-operatively. The ability of spectral imaging to sense changes in histology over large imaging areas was determined using inter-patient mammographic breast density (MBD) variation in cancer-free tissues as a model system. We discovered that increased MBD was associated with higher baseline β-carotene concentrations (p = 0.066) and higher scattering coefficients (p = 0.007) as measured by spectral imaging, and a trend toward decreased adipocyte size and increased adipocyte density as measured by histological examination in BMI-matched patients. The ability of spectral imaging to detect cancer intra-operatively was demonstrated when MBD-specific breast characteristics were considered. Specifically, the ratio of β-carotene concentration to the light scattering coefficient can report on the relative amount of fat to glandular density at the tissue surface to determine positive margin status, when baseline differences in these parameters between patients with low and high MBD are taken into account by the appropriate selection of threshold values. When MBD was included as a variable a priori, the device was estimated to have a sensitivity of 74% and a specificity of 86% in detecting close or positive margins, regardless of tumor type. Superior performance was demonstrated in high MBD tissue, a population that typically has a higher percentage of involved margins.     

Design, expression, and characterization of a synthetic human cannabinoid receptor and cannabinoid receptor/ G-protein fusion protein.

We report here the synthesis and characterization of two gene constructs designed to facilitate structure/function studies of the human neuronal cannabinoid receptor, CB1. The first gene, which we call shCB1, is a synthetic gene containing unique restriction sites spaced roughly 50-100 bases apart to facilitate rapid mutagenesis and cloning. A nine amino acid epitope tag (from the rhodopsin C-terminus) is also present in the shCB1 C-terminal tail to enable detection and purification using the monoclonal antibody 1D4. We find that that the shCB1 gene can be transiently expressed in COS cells with yield of approximately 10-15 micro g receptor per 15 cm plate and is wild type like in its ability to bind cannabinoid ligands. Our confocal microscopy studies indicate shCB1 targets to the membrane of HEK293 cells and is internalized in response to agonist. To facilitate functional studies, we also made a chimera in which the C-terminus of shCB1 was fused with the N-terminus of a G-protein alpha subunit, Galphai. The shCB1/Galphai chimera shows agonist stimulated GTPgammaS binding, and thus provides a simplified way to measure agonist induced CB1 activation. Taken together, the shCB1 and shCB1/Galphai gene constructs provide useful tools for biochemical and biophysical examinations of CB1 structure, activation and attenuation.     

Subcellular localization of uric acid storage in the fat body of Manduca sexta during the larval-pupal transformation

The fat body of the tobacco hornworm, Manduca sexta, serves as the major site for uric acid storage during metamorphosis. Light and electron microscopic examinations of fat body stained with reduced silver to show the location of stored uric acid have revealed that most, if not all, fat body cells store uric acid. The extent of specific staining is proportional to the increase in uric acid concentration in fat body during the initial stages of metamorphosis. Storage is associated with discrete membrane-bound structures, designated as uric acid storage vacuoles. In larval fat body, the structures are round or elliptical-shaped vacuoles with electron-dense fibrous interiors and are about the size of observed mitocondria (0.5–1.0 μm). During the larval-pupal transformation, the storage vacuoles double in size and appear as fibrous cores with spaces between the cores and the surrounding membranes. Before pupal ecdysis, the storage vacuoles are concentrated around the nucleus of each cell but after that event they are more uniformly distributed within fat body cells.