Faculty Bibliography

This unit describes methods for preparing zymogen granules from rat pancreas. Zymogen granules are storage organelles in pancreatic acinar cells containing digestive enzymes that are released into the pancreatic duct. The protocols in this unit take advantage of the large size (up to 1 microm diameter) and high density (>1.20 g/cm(3) on sucrose gradients) of the granules as compared to other cellular organelles. They use a combination of differential sedimentation and density gradient separation to accomplish the purification. Similar procedures can be used to isolate zymogen granules from mouse pancreas and canine pancreas. A protocol for preparing zymogen granules from dog pancreas is also included

PURPOSE: We report the first case of subconjunctival mucosa-associated lymphoid tissue (MALT) lymphoma arising in Tenon's capsule (fascia bulbi). METHODS: A 75-year-old woman presented with painless swelling of the superior bulbar conjunctiva in her left eye. During the biopsy of the bulbar lymphoid lesion, it was noticed that the conjunctiva was movable and that the lesion was located in the subconjunctiva. The tissues were studied by conventional light microscopy, immunohistochemistry, flow cytometry, and gene rearrangement analysis. RESULTS: Histopathological examination revealed that a diffuse lymphoid infiltrate consisting of small-sized lymphoid cells was present in Tenon's capsule but not in the substantia propria of the conjunctiva. Immunohistochemical and flow cytometric studies documented tumor cells of B-lymphocyte lineage. Molecular analysis demonstrated positive immunoglobulin heavy chain gene rearrangement. The final diagnosis was subconjunctival MALT lymphoma arising in Tenon's capsule. CONCLUSION: Ophthalmologists and pathologists need to distinguish the subconjunctival lymphoma that arises in Tenon's capsule from the conjunctival lymphoma in the substantia propria during diagnosis of epibulbar lymphoid tumors.

Light intensity and atmospheric CO2 partial pressure are two environmental signals known to regulate stomatal numbers. It has previously been shown that if a mature Arabidopsis leaf is supplied with either elevated CO2 (750 ppm instead of ambient at 370 ppm) or reduced light levels (50 micromol m-2 s-1 instead of 250 micromol m-2 s-1), the young, developing leaves that are not receiving the treatment grow with a stomatal density as if they were exposed to the treatment. But the signal(s) that it is believed is generated in the mature leaves and transmitted to developing leaves are largely unknown. Photosynthetic rates of treated, mature Arabidopsis leaves increased in elevated CO2 and decreased when shaded, as would be expected. Similarly, the levels of sugars (glucose, fructose, and sucrose) in the treated mature leaves increased in elevated CO2 and decreased with shade treatment. The levels of sugar in developing leaves were also measured and it was found that they mirrored this result even though they were not receiving the shade or elevated CO2 treatment. To investigate the effect of these treatments on global gene expression patterns, transcriptomics analysis was carried out using Affymetrix, 22K, and ATH1 arrays. Total RNA was extracted from the developing leaves after the mature leaves had received either the ambient control treatment, the elevated CO2 treatment, or the shade treatment, or both elevated CO2 and shade treatments for 2, 4, 12, 24, 48, or 96 h. The experiment was replicated four times. Two other experiments were also conducted, one to compare and contrast gene expression in response to plants grown at elevated CO2 and the other to look at the effect of these treatments on the mature leaf. The data were analysed and 915 genes from the untreated, signalled leaves were identified as having expression levels affected by the shade treatment. These genes were then compared with those whose transcript abundance was affected by the shade treatment in the mature treated leaves (1181 genes) and with 220 putative 'stomatal signalling' genes previously identified from studies of the yoda mutant. The results of these experiments and how they relate to environmental signalling are discussed, as well as possible mechanisms for systemic signalling.

The formation of neuromuscular synapses requires a complex exchange of signals between motor neurons and skeletal muscle fibers. Essential for the formation of neuromuscular junction (NMJ) is the activation of MuSK, a muscle-specific receptor tyrosine kinase (DeChiara et al., 1996). In mice lacking MuSK, motor axons fail to stop and differentiate, acetylcholine receptors (AChRs) fail to cluster, and AChR genes are expressed uniformly in muscle (DeChiara et al., 1996; Gautam et al., 1996). The retrograde signals for presynaptic differentiation are not known. Because synapse-specific transcription, like presynaptic differentiation, is MuSK-dependent, it is possible that retrograde signals for presynaptic differentiation might be encoded by genes that are expressed preferentially by synaptic nuclei. To identify such synapse-specific genes we screened Affymetrix microarrays with RNA from the dissected, synapse-enriched, and extrasynaptic regions of skeletal muscle and further studied those genes that encode for the secreted or cell-surface proteins

The identification of cathepsins in amyloid-beta plaques revealed broad dysfunction of the lysosomal system in Alzheimer's disease (AD). Coinciding with the discovery that proteolysis is required to generate the Abeta-peptide, these findings heralded an era of intense investigation on proteases in neurodegeneration. This review traces lysosomal system pathology from its early characterization to its origins within two pathways leading to the lysosome, the endocytic and autophagic pathways. An understanding has grown about how these two pathways are adversely influenced by normal brain aging and by genetic and environmental risk factors for AD, resulting in increased susceptibility of neurons to injury, amyloidogenesis, and neurodegeneration

The neurotrophins-nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), NT-3 and NT-4-represent a family of proteins essential for neuronal survival and plasticity. Each neurotrophin can signal through two different transmembrane receptors, Trk receptor tyrosine kinases and the p75 neurotrophin receptor, the first member of the TNF receptor superfamily. Neurotrophic factors play an important role in neurodegenerative diseases, as well as neuropsychiatric disorders such as depression, bipolar disease and eating disorders. Indeed, a number of approaches have been taken to use neurotrophins to treat Alzheimer's dementia, amyotrophic lateral sclerosis and peripheral sensory neuropathy. However, many of these clinical trails have failed, due to problems in delivery and unforeseen side effects of neurotrophic factors. An alternative approach is to use ligands in the G protein-coupled receptor (GPCR) family to transactivate trophic activities. We have discovered that treatment with adenosine, a neuromodulator that acts through G protein-coupled receptors, is capable of activating Trk tyrosine kinase receptors. Transactivation of neurotrophic receptors by GPCR ligands raise the possibility that small molecules may be used to elicit neurotrophic effects for the treatment of neurodegenerative diseases. This approach would allow for selective targeting of neurons that express specific G protein-coupled receptors and trophic factor receptors. GPCRs transduce information provided by extracellular signals to modulate synaptic activity and neurotransmission. In addition to the classical G protein signalling, GPCR ligands also activate receptor tyrosine kinases (RTK), including neurotrophin receptors. Activation of Trk neurotrophin receptors can occur by GPCR ligands in the absence of neurotrophins. Adenosine and PACAP (pituitary adenylate cyclase activating polypeptide) induce Trk activation specifically through their respective GPCRs to promote cell survival. Transactivation of Trks by GPCRs has emerged as a new theme in the biology of neurotrophin function. Although the precise role of transactivation is unknown, one possibility is that it adds a safety factor that might protect neurons from death in the absence of neurotrophins. Abnormal activity of the neurotrophin system has been implicated in several psychiatric and neurobiological illnesses. However, the lack of knowledge about the precise site of neurotrophin dysfunction has compromised the ability to improve the efficacy and the safety of drugs used in treatment modalities. If small-molecule GPCR ligands can ameliorate neuronal cell loss through Trk, transactivation may offer a new strategy for promoting trophic effects during neurodegeneration

We report a long-term survival case of Arima syndrome requiring hemodialysis. The patient, now 25 years of age, was hypotonic at birth. She was diagnosed with Dandy-Walker syndrome at an early month of age when she underwent posterior cranial fossa cystectomy and vermian agenesis was confirmed. With some delay in psychomotor development, she showed the development of language comprehension and meaningful speech and started to walk without aid at the age of 7 years. Polycystic kidneys were found at 11 years, and Arima syndrome was diagnosed at 16 years when she presented herself to our hospital with rupture of esophageal varices. With progressive deterioration of renal function, she was placed on chronic hemodialysis at 23 years. She presented short stature, right blepharoptosis and telecanthus on physical examination; pancytopenia, liver dysfunction and renal failure on laboratory studies agenesis of cerebellar vermis on magnetic resonance imaging reduced amplitude of electroretinographic response, and retinal pigmentary changes under funduscopy. Hemodialysis was initiated uneventfully except that nafamostat mesilate was used as anticoagulant because of her bleeding tendency. Arima syndrome, also known as cerebro-oculo-hepato-renal syndrome, is a disorder characterized by cerebellar vermis aplasia and other clinical features such as profound psychomotor retardation, severe visual impairment, characteristic facial appearance with blepharoptosis, hepatic fibrosis and progressive renal insufficiency. The clinical findings of our patient were consistent with Arima syndrome though her psychomotor retardation and visual impairment were relatively moderate as compared with those previously reported. As most patients with Arima syndrome may die of uremia in their early teens, dialysis therapy should be considered to improve the patient's survival and quality of life depending on the severity of psychomotor retardation and other systemic disorders.

Current treatments for damaged articular cartilage (i.e., shaving the articular surface, perforation or abrasion of the subchondral bone, and resurfacing with periosteal and perichondrial resurfacing) often produce fibrocartilage, or hyaline-appearing repair that is not sustained over time (Henche 1967, Ligament and Articular Cartilage Injuries. Springer-Verlag, New York, NY, pp. 157-164; Insall 1974, Clin. Orthop. 101: 61-67; Mitchell and Shepard 1976, J. Bone Joint Surg. [Am.] 58: 230-233; O'Driscoll et al. 1986, J. Bone Joint Surg. [Am.] 68: 1017-1035; 1989, Trans. Orthop. Res. Soc. 14: 145; Kim et al. 1991, J. Bone Joint Surg. [Am.] 73: 1301-1315). Autologous chondrocyte transplantation, although promising, requires two surgeries, has site-dependent and patient age limitations, and has unknown long-term donor site morbidity (Brittberg et al. 1994, N Engl. J. Med. 331: 889-895; Minas 2003, Orthopedics 26: 945-947; Peterson et al. 2003, J. Bone Joint Surg. Am. 85-A(Suppl. 2): S17-S24). Osteochondral allografts remain a widely used method of articular resurfacing to delay arthritic progression. The present study compared the histological response to four types of osteochondral implants in a rabbit model: autograft, frozen, freeze-dried, and fresh implants. Specimens implanted in the femoral groove were harvested at 6 and 12 weeks. Results showed similar restoration of the joint surface regardless of implant type, with a trend toward better repair at the later timepoint. As has been observed in other studies (Frenkel et al. 1997, J. Bone Joint Surg. 79B: 281-286; Toolan et al. 1998, J. Biomed. Mater. Res. 41: 244-250), each group in this study had at least one specimen in which a healthy-appearing surface on the implant was not well-integrated with host tissues. Although the differences were not statistically significant, freeze-dried implants at both timepoints had the best histological scores. The osteochondral grafts tested successfully restored the gross joint surface and congruity. At 12 weeks, no significant differences were observed between the various allografts and autologous osteochondral grafts

Following absorption, lead can concentrate in bodily compartments where it disrupts cellular processes and can result in detrimental health consequences. The concentration and impact of lead within follicular fluid has not been characterized and we used inductively coupled plasma mass spectroscopy (ICP-MS) to determine lead levels in blood and follicular fluid from nine patients undergoing in vitro fertilization (IVF) treatment. Lead levels within follicular fluid were found to be significantly higher in non-pregnant patients compared to pregnant patients suggesting that elevated concentrations of the environmental toxicant lead adversely affect female reproduction