Pilot study of propranolol premedication to reduce FDG uptake in brown adipose tissue on PET scans of adolescent and young adult oncology patients.

OBJECTIVE: Physiologic uptake of 18F-fluorodeoxyglucose (FDG) in brown adipose tissue (adipose tissue) of cancer patients may confound interpretation of positron emission tomography (PET) scans. Uptake in adipose tissue occurs in up to half of pediatric oncology patients undergoing PET scans, and is especially common in adolescents. adipose tissue is innervated by the sympathetic nervous system, and beta blockers such as propranolol have shown efficacy in reducing adipose tissue uptake on PET scans done in older adult oncology patients. PARTICIPANTS: Because propranolol may cause hypoglycemia or other side effects in fasting patients, we prospectively assessed the safety of a single dose of 20 mg propranolol in adolescent and young adult oncology patients undergoing FDG-PET imaging. METHODS: Ten patients (median age 18 years, range 14-24) received propranolol premedication prior to FDG-PET. RESULTS: No adverse effects or clinically significant changes in serum glucose, heart rate, or blood pressure were observed. Five of the 10 patients had adipose tissue identified on previous PET scans. However, following propranolol administration only, one patient had persistent uptake in adipose tissue. CONCLUSIONS: Propranolol was convenient and safe in fasting adolescent and young adult oncology patients undergoing PET scans. Larger studies are warranted to better define the effectiveness of this approach.

Effects of polysialic acid on sensory innervation of the cornea.

Sensory trigeminal growth cones innervate the cornea in a coordinated fashion during embryonic development. Polysialic acid (polySia) is known for its important roles during nerve development and regeneration. The purpose of this work is to determine whether polySia, present in developing eyefronts and on the surface of sensory nerves, may provide guidance cues to nerves during corneal innervation. Expression and localization of polySia in embryonic day (E)5-14 chick eyefronts and E9 trigeminal ganglia were identified using Western blotting and immunostaining. Effects of polySia removal on trigeminal nerve growth behavior were determined in vivo, using exogenous endoneuraminidase (endoN) treatments to remove polySia substrates during chick cornea development, and in vitro, using neuronal explant cultures. PolySia substrates, made by the physical adsorption of colominic acid to a surface coated with poly-d-lysine (PDL), were used as a model to investigate functions of the polySia expressed in axonal environments. PolySia was localized within developing eyefronts and on trigeminal sensory nerves. Distributions of PolySia in corneas and pericorneal regions are developmentally regulated. PolySia removal caused defasciculation of the limbal nerve trunk in vivo from E7 to E10. Removal of polySia on trigeminal neurites inhibited neurite outgrowth and caused axon defasciculation, but did not affect Neural Cell Adhesion Molecule (NCAM) expression or Schwann cell migration in vitro. PolySia substrates in vitro inhibited outgrowth of trigeminal neurites and promoted their fasciculation. In conclusion, polySia is localized on corneal nerves and in their targeting environment during early developing stages of chick embryos. PolySias promote fasciculation of trigeminal axons in vivo and in vitro, whereas, in contrast, their removal promotes defasciculation.

A simple method for determining split renal function from dynamic (99m)Tc-MAG3 scintigraphic data.

PURPOSE: Commonly used methods for determining split renal function (SRF) from dynamic scintigraphic data require extrarenal background subtraction and additional correction for intrarenal vascular activity. The use of these additional regions of interest (ROIs) can produce inaccurate results and be challenging, e.g. if the heart is out of the camera field of view. The purpose of this study was to evaluate a new method for determining SRF called the blood pool compensation (BPC) technique, which is simple to implement, does not require extrarenal background correction and intrinsically corrects for intrarenal vascular activity. METHODS: In the BPC method SRF is derived from a parametric plot of the curves generated by one blood-pool and two renal ROIs. Data from 107 patients who underwent (99m)Tc-MAG3 scintigraphy were used to determine SRF values. Values calculated using the BPC method were compared to those obtained with the integral (IN) and Patlak-Rutland (PR) techniques using Bland-Altman plotting and Passing-Bablok regression. The interobserver variability of the BPC technique was also assessed for two observers. RESULTS: The SRF values obtained with the BPC method did not differ significantly from those obtained with the PR method and showed no consistent bias, while SRF values obtained with the IN method showed significant differences with some bias in comparison to those obtained with either the PR or BPC method. No significant interobserver variability was found between two observers calculating SRF using the BPC method. CONCLUSION: The BPC method requires only three ROIs to produce reliable estimates of SRF, was simple to implement, and in this study yielded statistically equivalent results to the PR method with appreciable interobserver agreement. As such, it adds a new reliable method for quality control of monitoring relative kidney function.

Nerve repulsion by the lens and cornea during cornea innervation is dependent on Robo-Slit signaling and diminishes with neuron age.

The cornea, the most densely innervated tissue on the surface of the body, becomes innervated in a series of highly coordinated developmental events. During cornea development, chick trigeminal nerve growth cones reach the cornea margin at embryonic day (E)5, where they are initially repelled for days from E5 to E8, instead encircling the corneal periphery in a nerve ring prior to entering on E9. The molecular events coordinating growth cone guidance during cornea development are poorly understood. Here we evaluated a potential role for the Robo-Slit nerve guidance family. We found that Slits 1, 2 and 3 expression in the cornea and lens persisted during all stages of cornea innervation examined. Robo1 expression was developmentally regulated in trigeminal cell bodies, expressed robustly during nerve ring formation (E5-8), then later declining concurrent with projection of growth cones into the cornea. In this study we provide in vivo and in vitro evidence that Robo-Slit signaling guides trigeminal nerves during cornea innervation. Transient, localized inhibition of Robo-Slit signaling, by means of beads loaded with inhibitory Robo-Fc protein implanted into the developing eyefield in vivo, led to disorganized nerve ring formation and premature cornea innervation. Additionally, when trigeminal explants (source of neurons) were oriented adjacent to lens vesicles or corneas (source of repellant molecules) in organotypic tissue culture both lens and cornea tissues strongly repelled E7 trigeminal neurites, except in the presence of inhibitory Robo-Fc protein. In contrast, E10 trigeminal neurites were not as strongly repelled by cornea, and presence of Robo-Slit inhibitory protein had no effect. In full, these findings suggest that nerve repulsion from the lens and cornea during nerve ring formation is mediated by Robo-Slit signaling. Later, a shift in nerve guidance behavior occurs, in part due to molecular changes in trigeminal neurons, including Robo1 downregulation, thus allowing nerves to find the Slit-expressing cornea permissive for growth cones.

Effects of ultraviolet-A and riboflavin on the interaction of collagen and proteoglycans during corneal cross-linking.

Corneal cross-linking using riboflavin and ultraviolet-A (RFUVA) is a clinical treatment targeting the stroma in progressive keratoconus. The stroma contains keratocan, lumican, mimecan, and decorin, core proteins of major proteoglycans (PGs) that bind collagen fibrils, playing important roles in stromal transparency. Here, a model reaction system using purified, non-glycosylated PG core proteins in solution in vitro has been compared with reactions inside an intact cornea, ex vivo, revealing effects of RFUVA on interactions between PGs and collagen cross-linking. Irradiation with UVA and riboflavin cross-links collagen α and ß chains into larger polymers. In addition, RFUVA cross-links PG core proteins, forming higher molecular weight polymers. When collagen type I is mixed with individual purified, non-glycosylated PG core proteins in solution in vitro and subjected to RFUVA, both keratocan and lumican strongly inhibit collagen cross-linking. However, mimecan and decorin do not inhibit but instead form cross-links with collagen, forming new high molecular weight polymers. In contrast, corneal glycosaminoglycans, keratan sulfate and chondroitin sulfate, in isolation from their core proteins, are not cross-linked by RFUVA and do not form cross-links with collagen. Significantly, when RFUVA is conducted on intact corneas ex vivo, both keratocan and lumican, in their natively glycosylated form, do form cross-links with collagen. Thus, RFUVA causes cross-linking of collagen molecules among themselves and PG core proteins among themselves, together with limited linkages between collagen and keratocan, lumican, mimecan, and decorin. RFUVA as a diagnostic tool reveals that keratocan and lumican core proteins interact with collagen very differently than do mimecan and decorin.

Development and mineralization of embryonic avian scleral ossicles.

PURPOSE: To investigate the development and mineralization of avian scleral ossicles using fluorescence microscopy in combination with field emission scanning electron microscopy (FESEM) and energy dispersive spectroscopy (EDS). METHODS: The anterior halves of whole eyeballs from chickens on embryonic (E) days E10 to E21 and Japanese quail on embryonic days E8 to E17 were fixed in 100% methanol for 1 min, stained with Giemsa solution for 5 min, destained with distilled water for 30 min, and then viewed by epifluorescence. Propidium iodide (PI) was used to detect the nuclei of osteocytes in scleral ossicles. FESEM and EDS were then used to show areas of mineralization and to identify differences in the elemental composition of different regions of the ossicles. RESULTS: Using Giemsa as a fluorescence stain, it was possible to observe the detailed morphology and development of both chicken and quail scleral ossicles. In chickens, bone microporosities first became visible at E15. Each microporosity contained a single nucleus, likely that of an osteocyte. The amount of carbon in ossicles steadily decreased during embryogenesis and post-hatching, while the concentration of oxygen showed a distinct increase over this time period. Calcium and phosphate levels in the ossicles increased gradually during embryonic and post-hatching stages. CONCLUSIONS: A novel approach to study the development and mineralization of avian scleral ossicles during embryogenesis is presented. This methodology was validated by studying two different species, both important models for avian developmental research.

Thyroxine increases the rate but does not alter the pattern of innervation during embryonic chick corneal development.

PURPOSE: Embryonic chick corneal nerves reach limbal mesenchyme by embryonic day (E)5, encircle the cornea in several days, then defasciculate into the stroma simultaneously from all sides, while extracellular keratan sulfate proteoglycan (KSPG) accumulates from posterior to anterior stroma. Precocious thyroxine (T4)-induced increases in corneal thinning/transparency are blocked by 2-thiouracil (2-TU) inhibition of T3 synthesis. The hypothesis for this study was that precocious T4 exposure increases corneal innervation similarly. METHODS: E8 embryos received T4, 2-TU, T4+2-TU, or buffer; corneas were harvested on E12. Corneal nerves were stained with neuronal beta-tubulin-specific TuJ1 antibody or chick nerve-specific CN antibody. Corneal thickness was determined from cryostat sections, and mRNA expression was measured by real-time PCR. RESULTS: Nerves avoided the cornea until E9, then entered the anterior stroma, extended toward and reached the cornea center by E14, and never invaded posterior stroma. E7 to E18 corneal expressions of nerve growth factor and neurotrophin-3 genes were unchanged; receptor gene expressions rose. E7 to E12 semaphorin 3A and 3F and ephrin A2 and A5 expressions did not change significantly; semaphorin and ephrin/eph expressions increased from E9 to E18. E8 T4 administration increased nerve extension by E11, but did not alter circumferential penetration, anterior-only penetration, or neurotrophin expressions. 2-TU prevented T4-induced precocious corneal thinning, but augmented T4 nerve stimulation. CONCLUSIONS: No changes in corneal neurotrophin or nerve pathfinding gene expressions accompany corneal transition to nerve growth cone permissiveness. T4 increases corneal nerve penetration rates by a non-T3-dependent mechanism. Results are consistent with possible roles for corneal KSPGs in regulating corneal nerve growth.

On-target derivatization of keratan sulfate oligosaccharides with pyrenebutyric acid hydrazide for MALDI-TOF/TOF-MS.

In the present work, a rapid and novel method of on-target plate derivatization of keratan sulfate (KS) oligosaccharides for subsequent analysis by matrix-assisted laser desorption and ionization (MALDI) mass spectrometry is described. MALDI-(time-of-flight)-TOF spectra of labeled KS oligosaccharides revealed that significantly improved ionization can be accomplished through derivatization with pyrenebutyric acid hydrazide (PBH), and the most abundant peak in each spectrum corresponds to the singly charged molecular ion [M - H]- or [M + (n - 1)Na - nH]-, where n = the number of sulfates (n = 1, 2, 3...). The high-energy collision-induced dissociation (heCID) spectra of labeled KS oligosaccharides displayed fragments of compounds similar to those observed with laser-induced dissociation (LID) analysis, suggesting that both heCID and LID fragmentations can be used to analyze KS oligosaccharides. Moreover, fragmentation analysis of all labeled KS oligosaccharides was performed by MALDI-TOF/TOF-MS. With LID mode, sodium adducts showed fragmentation of glycosidic linkages with mainly Y/B/C ions, as well as various cross-ring cleavages providing exact information for the positions of sulfate groups along the KS oligosaccharide chains. This one-step on-target derivatization method makes MALDI-TOF/TOF-MS identification of KS fast, simple and highly throughput for trace amounts of biological samples.

Expression studies of osteoglycin/mimecan (OGN) in the cochlea and auditory phenotype of Ogn-deficient mice.

Genes involved in the hearing process have been identified through both positional cloning efforts following genetic linkage studies of families with heritable deafness and by candidate gene approaches based on known functional properties or inner ear expression. The latter method of gene discovery may employ a tissue- or organ-specific approach. Through characterization of a human fetal cochlear cDNA library, we have identified transcripts that are preferentially and/or highly expressed in the cochlea. High expression in the cochlea may be suggestive of a fundamental role for a transcript in the auditory system. Herein we report the identification and characterization of a transcript from the cochlear cDNA library with abundant cochlear expression and unknown function that was subsequently determined to represent osteoglycin (OGN). Ogn-deficient mice, when analyzed by auditory brainstem response and distortion product otoacoustic emissions, have normal hearing thresholds.

Thyroxine affects expression of KSPG-related genes, the carbonic anhydrase II gene, and KS sulfation in the embryonic chicken cornea.

PURPOSE: Opaque chick corneas become thin and transparent from embryonic day (E)9 to E20 of incubation. Thyroxine (T4) injected in ovo on E9 induces precocious transparency by E12. The present study was conducted to determine whether corneal cells differentially express genes for T4 regulation, keratan sulfate proteoglycan (KSPG) synthesis, crystallins, and endothelial cell ion transporters during transparency development and whether these expressions are altered when E9 embryos are treated with T4. METHODS: E9 eggs received T4 or buffer; corneas were dissected on E12. Corneal transparency was measured digitally and thickness was determined from cryostat cross sections. mRNA expressions were determined by real-time PCR using cDNA synthesized from whole-cell RNA, cells expressing T4 receptor mRNAs assessed by in situ hybridization, and KS disaccharide sulfation measured by electrospray ionization tandem mass spectrometry (ESI-MS/MS). RESULTS: All corneal layers expressed T4 receptor alpha (THRA) mRNA; keratocytes and endothelial cells expressed T4 receptor beta (THRB) mRNA. During normal development, THRB expression increased 20-fold from E12 to E20; THRA expression remained constant. Expressions of most genes involved in KS synthesis increased from E9 to E16, and then decreased from E16 to E20. From E9 to E20, expressions of crystallin genes increased; T4/3-deiodinase DIII (DIO3) increased 10-fold; and sodium-potassium ATPase transporter (ATP1A1), sodium-bicarbonate transporter (NBC), and carbonic anhydrase II (CA2) increased 5- to 10-fold. E9 T4 administration decreased corneal thickness by E12; increased DIO3, THRB, and CA2 expressions 5- to 20-fold; decreased KSPG core protein genes and galactose sulfotransferase CHST1 expressions 2-fold; and reduced KS disulfated/monosulfated disaccharide (DSD/MSD) ratios. CONCLUSIONS: Thyroxine modifies expressions of KSPG synthesis and carbonic anhydrase genes.

Keratan sulfate and chondroitin/dermatan sulfate in maximally recovered hypocellular stromal interface scars of postmortem human LASIK corneas.

PURPOSE: To analyze the amounts and distributions of nonsulfated and sulfated keratan sulfate (KS) and chondroitin/dermatan sulfate (CS/DS) disaccharides in the interface wound of human postmortem LASIK corneas in comparison with normal control corneas. METHODS: Corneal stromal tissue samples from central and paracentral hypocellular primitive stromal interface scars of human LASIK corneas and from similar regions of normal control corneas were collected by laser capture microdissection (LCM) and subsequently were digested with specific glycosidase enzymes. Digests were directly analyzed by electrospray ionization tandem mass spectrometry (ESI-MS/MS). RESULTS: Concentrations of both monosulfated GlcNAc(6S)-beta-1,3-Gal (MSD2) and disulfated Gal (6S)-beta-1,4-GlcNAc(6S) (DSD) KS disaccharides from the LASIK interface scars were significantly lower than in normal control corneal stromas. No significant difference was found for the concentration of nonsulfated (NSD) KS disaccharides in LASIK interface scars compared with normal controls. The concentration of DeltaUA-beta-1,3-GalNAc(6S) (Deltadi-6S) CS/DS disaccharides from the LASIK interface scar was significantly higher than normal corneal stroma, whereas concentrations of DeltaUA-beta-1,3-GalNAc(4S) (Deltadi-4S) and nonsulfated Deltadi-0S CS/DS disaccharides demonstrated no significant differences from normal corneas. CONCLUSIONS: The profiles of KS and CS/DS disaccharides in LASIK interface scars are significantly different from those in normal cornea stromal tissue, as revealed by LCM and ESI-MS/MS.

Differentiation of diiodothyronines using electrospray ionization tandem mass spectrometry.

Diiodothyronines 3,5-diiodothyronine (3,5-T2), 3',5'-diiodothyronine (3',5'-T2), and 3,3'-diiodothyronine (3,3'-T2) are important metabolites of 3,5,3'-triiodothyronine (T3) and 3,3',5'-triiodothyronine (rT3; reverse T3). In this paper, a novel and rapid method for identifying and quantifying 3,5-T2, 3',5'-T2 and 3,3'-T2 has been introduced using electrospray ionization tandem mass spectrometry (ESI-MS/MS). Fragmentation patterns were proposed on the basis of our data obtained by ESI-MS/MS. MS2 spectra in either negative ionization mode or positive ionization mode can be used to differentiate 3,5-T2, 3',5'-T2 and 3,3'-T2. On the basis of the relative abundance of fragment ions in MS2 spectra under the positive ionization mode, quantification of the 3,5-T2, 3',5'-T2 and 3,3'-T2 isomers in mixtures is also achieved without prior separation.

An x-ray diffraction study of corneal structure in mimecan-deficient mice.

PURPOSE: Keratan sulfate proteoglycans (KSPGs) in the corneal stroma are believed to influence collagen fibrillar arrangement. This study was performed to investigate the fibrillar architecture of the corneal stroma in mice homozygous for a null mutation in the corneal KSPG, mimecan. METHODS: Wild-type (n = 9) and mimecan-deficient (n = 10) mouse corneas were investigated by low-angle synchrotron x-ray diffraction to establish the average collagen fibrillar spacing, average collagen fibril diameter, and level of fibrillar organization in the stromal array. RESULTS: The mean collagen fibril diameter in the corneas of mimecan-null mice, as an average throughout the whole thickness of the tissue, was not appreciably different from normal (35.6 +/- 1.1 nm vs. 35.9 +/- 1.0 nm). Average center-to-center collagen fibrillar spacing in the mutant corneas measured 52.6 +/- 2.6 nm, similar to the 53.3 +/- 4.0 nm found in wild-type mice. The degree of local order in the collagen fibrillar array, as indicated by the height-width (H:W) ratio of the background-subtracted interfibrillar x-ray reflection, was also not significantly changed in mimecan-null corneas (23.4 +/- 5.6), when compared with the corneas of wild-types (28.2 +/- 4.8). CONCLUSIONS: On average, throughout the whole depth of the corneal stroma, collagen fibrils in mimecan-null mice, unlike collagen fibrils in lumican-null mice and keratocan-null mice, are of a normal diameter and are normally spaced and arranged. This indicates that, compared with lumican and keratocan, mimecan has a lesser role in the control of stromal architecture in mouse cornea.

Detection and quantification of sulfated disaccharides from keratan sulfate and chondroitin/dermatan sulfate during chick corneal development by ESI-MS/MS.

PURPOSE: To identify and quantify changes in keratan sulfate (KS) and chondroitin/dermatan sulfate (CS/DS) sulfated disaccharides in the developing chick cornea using electrospray ionization tandem mass spectrometry (ESI-MS/MS). METHODS: Cryostat sections of fresh nonfixed corneas were obtained from White Leghorn embryonic day (E)8 to E20 chicks, and from 4- and 70-week-old chickens. Tissue sections on glass slides were incubated with selected glycosidase enzymes. Digest solutions were analyzed directly by ESI-MS/MS. RESULTS: The concentration of KS monosulfated disaccharide (MSD) Gal-beta-1,4-GlcNAc(6S) in E8 cornea equaled that at E20, declined to its lowest level by E10, increased to a second peak by E14, decreased to a second low by E18, peaked again by E20, and remained high in adult corneas. A similar concentration profile was observed for KS disulfated disaccharide (DSD) Gal(6S)-beta-1,4-GlcNAc(6S), and thus also for total sulfated KS disaccharides. The molar percent of DSD was higher than that of MSD from E8 to E18, equivalent at E20, and less than that of MSD in adult corneas. In contrast, total concentration of CS/DS Deltadi-4S plus Deltadi-6S decreases as development progresses and is lowest in adult corneas. Concentration and molar percent of Deltadi-6S is highest at E8, then decreases through development as the concentration and molar percent of Deltadi-4S increases from E8 and exceeds that of Deltadi-6S after E14. CONCLUSIONS: New, rapid, direct chemical analysis of extracellular matrix components obtained from sections from embryonic and adult chick corneas reveals heretofore undetected changes in sulfation characteristics of KS and CS/DS disaccharides during corneal development.

Expression and localization of leucine-rich B7 protein in human ocular tissues.

PURPOSE: Using a web-based tool to screen the human proteome for potential mimecan/osteoglycin interacting proteins, we found that the leucine-rich B7 protein may have functional associations with several small leucine-rich proteoglycans (SLRP), including mimecan. The purpose of this study was to determine the expression of leucine-rich B7 protein in human eye tissues and its subcellular localization in MG-63 cells. METHODS: Primers were synthesized to amplify the two known differentially spliced B7 mRNA transcripts. Reverse transcription-polymerase chain reaction (RT-PCR) amplification was used to determine the expression of B7 mRNAs in human ocular and nonocular tissues. A rabbit anti-human B7 antibody was generated that specifically immunostained B7 proteins. The expression of B7 proteins in the human eye was determined by immunohistochemistry (IHC). Intracellular localization of leucine-rich B7 and mimecan proteins were determined using transient co-transfections, cell immunostaining, and laser scanning confocal microscopy. RESULTS: RT-PCR analysis showed moderate expression of B7, transcript variant 2, in human cornea, iris, sclera, and retina. In contrast, B7, transcript variant 1, was strongly expressed only in the cornea. The two B7 mRNAs were highly expressed in human brain, blood, peripheral mononuclear cells, and other human tissues. By IHC, immunostaining for leucine-rich B7 protein was found in epithelial and endothelial layers of the cornea, epithelial and fiber cells of the lens, in sclera, and in the rod and cone layer of the retina of adult human eye. Leucine-rich B7 protein was found to localize to both nucleus and cytoplasm of MG-63 cells, whereas mimecan was found only in the cytoplasm of these cells. Merged images obtained by confocal microscopy revealed certain cytoplasmic regions in MG-63 cells where B7 and mimecan proteins appeared to co-localize. CONCLUSIONS: The present work is the first to demonstrate the expression and localization of leucine-rich B7 protein in human eye and other human tissues. The results reported here are an essential prerequisite for future studies aimed at understanding the biological roles of leucine-rich B7 proteins in health and disease.

Detection and quantification of 3,5,3'-triiodothyronine and 3,3',5'-triiodothyronine by electrospray ionization tandem mass spectrometry.

A novel and rapid method for identifying and quantifying 3,5,3'-triiodothyronine (T3) and 3,3',5'-triiodothyronine (rT3; reverse T3) has been introduced using electrospray ionization tandem mass spectrometry (ESI-MS/MS). MS(2) spectra in either negative ionization mode or positive ionization mode can be used to differentiate T3 and rT3. Quantification of the T3 and rT3 isomers under the negative ionization mode is also achieved without prior separation by HPLC.

Visualization of bullet track and bullet by radionuclide brain scintigraphy.

Radionuclide brain scintigraphy is a commonly performed examination for the confirmation of brain death. Although the absence of scintigraphically detectable flow of lipophilic tracers to the brain combined with the lack of uptake in the brain is considered consistent with brain death in the appropriate clinical scenario, the cause of death itself is usually not apparent on the scan. A case of bullet track and bullet visualization during a radionuclide brain death study with Tc-99m hexamethylpropyleneamine oxime (HMPAO) is described.

The keratocan gene is expressed in both ocular and non-ocular tissues during early chick development.

Extracellular matrix (ECM) keratan sulfate proteoglycans (KSPGs) are core proteins with sulfated polylactosamine side chains (KS). The KSPG core protein keratocan gene (Kera) is expressed almost exclusively in adult vertebrate cornea, but its embryonic expression is little known. Embryonic chick in situ hybridization reveals Kera mRNA expression in corneal endothelium from embryonic day (E) 4.5, Hamburger-Hamilton (HH) 25, in stromal keratocytes from E6.5, HH30, and in iris distal surface cells from E8, HH34. As highly sulfated, antibody I22-positive KS increases extracellularly from posterior to anterior across the stroma, nerves enter and populate only anterior stroma and epithelium. RT-PCR and in situ hybridization demonstrate that developmentally regulated Kera mRNA expression initiates in midbrain and dorsolateral mesenchyme at E1, HH7, then spreads caudally in hindbrain and cranial and trunk mesenchyme flanking the neural tube through E2, HH20. Cranial expression extends ventrally through the developing head, and concentrates in mesenchyme surrounding eye anterior regions and cranial ganglia, and in subepidermal pharyngeal arch mesenchyme by E3.5, HH22. Kera expression in the trunk at E3.5, HH22 and E4.5, HH25, is strong in dorsolateral subepidermal, sclerotomal and nephrogenic mesenchymes, but absent in neural tube, dorsal root ganglia, nerve outgrowths, notochord, heart and gut. Early limb buds express Kera mRNA throughout their mesenchyme, then in restricted proximal and distal mesenchymes. I22-positive KS appears only in notochord in E3.5, HH22 and E4.5, HH25, embryos. Results suggest the hypothesis that keratocan, or keratocan with minimally sulfated KS chains, may play a role in structuring ECM for early embryonic cell and neuronal migrations.

Scintigraphy as a confirmatory test of brain death.

The concept of "brain death" was introduced to medicine in the second half of the 20th century, when technological advancements began to allow sustaining cardiorespiratory functioning of the body in the absence of brain function. Although physicians generally agree that a patient can be declared brain dead when the loss of brain function is total and irreversible, different approaches have been taken to define what constitutes brain death. A thorough clinical examination is essential to the diagnosis. The role of confirmatory tests differ among countries in the world but generally are indicated when a specific part of the clinical examination cannot be performed or is deemed unreliable. Under certain circumstances, confirmatory tests can be used to shorten the clinical observation. Of the confirmatory tests recommended by the American Academy of Neurology and the American Academy of Pediatrics, cerebral scintigraphy is a safe, reliable, and widely available alternative. Once the radiopharmaceutical is properly compounded, cerebral scintigraphy can be performed rapidly and can be interpreted in a straightforward manner. It is tolerant of metabolic aberrations and pharmacologic intoxicants. It is not affected by electrical interference, and the presence of skull defects or scalp trauma do not preclude its performance. The radiopharmaceuticals used in scintigraphy have no deleterious effects on potential donor organs. Cerebral radionuclide angiography has been highly sensitive. Either cerebral planar scintigraphy or cerebral scintitomography with Tc-99m hexamethylpropyleneamineoxime also are highly sensitive, but, in addition, appear to be 100% specific.

The UV responsive elements in the human mimecan promoter: a functional characterization.

PURPOSE: A major environmental stress encountered by humans is solar UV light, which can cause a spectrum of eye diseases, such as photokeratitis, cataract, pterygia, and ocular neoplasms. Mammalian defense mechanisms in response to adverse effects of UV light result in induction of a number of genes. Studies on the transcriptional regulation of genes that are expressed in the eye will increase understanding of both the physiological functions of these genes in the mammalian UV response, and the molecular bases for abnormalities associated with the above diseases. Mimecan is an extracellular matrix proteoglycan that is abundantly expressed in the cornea. The purpose of this study was to determine and characterize the UV responsive regulatory elements of the human mimecan promoter. METHODS: Transcriptional activity of the promoter was evaluated, before and after UV irradiation, using transient transfection of human mimecan promoter/luciferase reporter constructs into corneal keratocytes and non-corneal cells. Site directed mutagenesis and corresponding functional assays were used to determine the contribution of UV responsive regions to human mimecan transcription. Co-transfection experiments were used to investigate the role of transcription factors that bind these elements in the promoter and mediate the UV response. mRNA expression was analyzed by reverse transcription-polymerase chain reaction (RT-PCR). RESULTS: The shortest promoter construct that was strongly activated following UV irradiation contained three initiator elements, an E-box element that is conserved between species, and the entire first intron of the human mimecan gene. Deletion of the intronic p53 binding site from this construct considerably diminished transcription and the UV response of the promoter. Surprisingly, deletion of the E-box sequence from this construct completely abolished both transcription and UV response of the promoter. These results demonstrated that the E-box is essential to transcription of the human mimecan gene and also is required for activation by p53. The role of the E-box, and the E-box binding protein, USF-1, in transcription and UV responses of the human mimecan promoter were confirmed by co-transfection experiments using dominant negative transcription factor, A-USF. In addition to these positive regulators, we demonstrate that the region between nucleotides -1314 and -1907 contains a transcriptional repressor site that is active in a time dependent manner following UV irradiation. Finally, we show that UV irradiation results in changes in mimecan mRNA levels in bovine corneal keratocytes in a time-dependent manner. CONCLUSIONS: The human mimecan promoter contains several UV responsive regulatory elements that are conserved between human and bovine species and include the intronic p53 DNA binding site, the E-box in the proximal promoter, and the region between nucleotides -1314 and -1907. The E-box plays an important role in transcription and UV response of the human mimecan promoter. UV irradiation modulates expression of mimecan mRNA in bovine corneal keratocytes and non-corneal cells.