RESUMEN
We have described recently an acetylcholinesterase (AChE) knockout mouse. While comparing the tissue distribution of AChE and butyrylcholinesterase (BChE), we found that extraction buffers containing Triton X-100 strongly inhibited mouse BChE activity. In contrast, buffers with Tween 20 caused no inhibition of BChE. Conventional techniques grossly underestimated BChE activity by up to 15-fold. In Tween 20 buffer, the intestine, serum, lung, liver, and heart had higher BChE than AChE activity. Only brain had higher AChE than BChE activity in AChE +/+ mice. These findings contradict the dogma, based mainly on observations in Triton X-100 extracts, that BChE is a minor cholinesterase in animal tissues. AChE +/- mice had 50% of normal AChE activity and AChE -/- mice had none, but all mice had similar levels of BChE activity. BChE was inhibited by Triton X-100 in all species tested, except rat and chicken. Inhibition was reversible and competitive with substrate binding. The active site of rat BChE was unique, having an arginine in place of leucine at position 286 (human BChE numbering) in the acyl-binding pocket of the active site, thus explaining the lack of inhibition of rat BChE by Triton X-100. The generally high levels of BChE activity in tissues, including the motor endplate, and the observation that mice live without AChE, suggest that BChE has an essential function in nullizygous mice and probably in wild-type mice as well.
Asunto(s)
Acetilcolinesterasa/metabolismo , Butirilcolinesterasa/metabolismo , Placa Motora/enzimología , Músculo Esquelético/enzimología , Acetilcolinesterasa/deficiencia , Acetilcolinesterasa/genética , Animales , Inhibidores de la Colinesterasa/farmacología , Humanos , Ratones , Ratones Noqueados , Especificidad de Órganos , Glicoles de Propileno/farmacología , Ratas , Especificidad de la Especie , Tetraisopropilpirofosfamida/farmacologíaRESUMEN
Acetylcholinesterase (AChE; EC 3.1.1.7) is the primary terminator of nerve impulse transmission at cholinergic synapses and is believed to play an important role in neural development. Targeted deletion of four exons of the ACHE gene reduced AChE activity by half in heterozygous mutant mice and totally eliminated AChE activity in nullizygous animals. Butyrylcholinesterase (EC 3.1.1.8) activity was normal in AChE -/- mice. Although nullizygous mice were born alive and lived up to 21 days, physical development was delayed. The neuromuscular junction of 12-day-old nullizygous animals appeared normal in structure. Nullizygous mice were highly sensitive to the toxic effects of the organophosphate diisopropylfluorophosphate and to the butyrylcholinesterase-specific inhibitor bambuterol. These findings indicate that butyrylcholinesterase and possibly other enzymes are capable of compensating for some functions of AChE and that the inhibition of targets other than AChE by organophosphorus agents results in death.
Asunto(s)
Acetilcolinesterasa/fisiología , Crecimiento , Isoflurofato/toxicidad , Acetilcolinesterasa/genética , Animales , Butirilcolinesterasa/metabolismo , Ratones , Ratones Noqueados , Microscopía Electrónica , Unión Neuromuscular/ultraestructuraRESUMEN
BACKGROUND: There is general agreement that lymphocytic and histiocytic (L&H) cells, the variants of Reed-Sternberg cells in nodular lymphocyte-predominant Hodgkin's disease, belong to the B-cell lineage. However, the clonality of L&H cells remains controversial. METHODS: We used complementarity-determining region 3 (CDR3) of the immunoglobulin heavy-chain gene as a clonal marker to study individual L&H cells isolated by micromanipulation from tissue sections of five patients with nodular lymphocyte-predominant Hodgkin's disease. The heavy-chain CDR3 of each cell was amplified by the polymerase chain reaction. The products were analyzed by gel electrophoresis, and representative amplification products from each patient were sequenced. RESULTS: L&H cells whose heavy-chain CDR3 was related, indicating the presence of a clonal population, were detected in all five patients and were the dominant population in three. In four of the five patients, members of the clone were found in different nodules in the tissue section, different tissue blocks from the same tumor, or different lymph nodes from the same patient. The CDR3 sequences in each clone frequently contained nucleotide substitutions indicative of intraclonal mutation. CONCLUSIONS: Clonal populations of L&H cells occur in nodular lymphocyte-predominant Hodgkin's disease. Intraclonal variation in nucleotide sequences suggests that hypermutation of the heavy-chain CDR3 continues to occur among the clonal progeny.
Asunto(s)
Genes de Inmunoglobulinas , Enfermedad de Hodgkin/patología , Cadenas Pesadas de Inmunoglobulina/genética , Linfocitos B , Secuencia de Bases , Células Clonales , Enfermedad de Hodgkin/inmunología , Humanos , Región Variable de Inmunoglobulina/genética , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Células de Reed-SternbergRESUMEN
Molecular analysis of isolated single cells is a powerful tool for analyzing heterogeneity within a population of cells and for clarifying issues of cell origin and clonality. Current techniques are limited by the availability of suitable fresh tissue. To broaden the applicability of molecular techniques at single-cell level, we have developed an approach that uses routinely processed archival tissue. Immunoglobulin heavy chain (IgH) gene rearrangement was analyzed in large tumor cells from four cases of diffuse large cell B-non-Hodgkin's lymphoma and in small reactive T and B lymphocytes from three cases of lymphocytic predominance Hodgkin's disease. One case of Epstein-Barr virus (EBV)-encoded RNA (EBER)-positive angiocentric pulmonary T-cell lymphoma was assayed for the presence of the BamHI-W multiple-copy fragment of the EBV genome. T- and B-lymphoid cells were immunostained with anti-CD3 and CD20, respectively. The tissue sections from the EBER-positive T-cell lymphoma were stained by nonisotopic in situ hybridization. Single cells were mobilized after proteolytic treatment under an inverted microscope using a hydraulic micromanipulator at a magnification of 400 x. Isolated cells were aspirated into a micropipette fixed to a second micromanipulator and transferred into a PCR tube. The IgH complementarity determining region (CDR)3 was successfully amplified in 17 of 52 (33%) small B-lymphocytes from lymphocytic predominance Hodgkin's disease using a previously reported semi-nested PCR method, and the products from each case differed in size as expected of a polyclonal population. None of the 49 small T lymphocytes demonstrated any amplifiable IgH CDR3 products, indicating no significant cellular contamination. The IgH CDR3 sequence analysis of the PCR products indicated a clonal relationship among harvested cells. In the T-cell lymphoma case, the harvested EBER-positive cells were amplifiable for the multiple-copy fragment BamHI-W of the EBV genome. Our study indicates that single-cell analysis can be performed on paraffin-embedded archival tissue after being subjected to immunoperoxidase and in situ hybridization procedures.
Asunto(s)
Inmunohistoquímica , Hibridación in Situ , Micromanipulación/métodos , Adhesión en Parafina , Reacción en Cadena de la Polimerasa/métodos , Antígenos CD20 , Linfocitos B/citología , Secuencia de Bases , Complejo CD3/análisis , Herpesvirus Humano 4/genética , Enfermedad de Hodgkin/genética , Humanos , Cadenas Pesadas de Inmunoglobulina/genética , Linfoma/genética , Datos de Secuencia Molecular , Receptores de Antígenos de Linfocitos T alfa-beta/análisis , Linfocitos T/citologíaRESUMEN
Long-term venous access for leukapheresis, repeated blood sampling, and administration of drugs and fluids can be accomplished nonsurgically in Yucatan miniature swine. The catheter is placed under fluoroscopic guidance into the inferior vena cava using a needle and guidewire. This procedure has the advantage that it avoids a surgical incision, allows high flow rates, exists conveniently on the lower back, and can be replaced easily in the event of mechanical failure or thrombosis. Actuarial analysis of the duration of patency disclosed that of 41 catheters placed in 30 animals, the probability of function at 28, 42, and 54 days was 75%, 50%, and 25%, respectively. Eleven nonfunctioning catheters were replaced and nine of these continued to function until the completion of the experiment. No catheters were removed due to infection. Chronic catheterization of the inferior vena cava is a convenient method for long-term venous access in swine.
Asunto(s)
Cateterismo Venoso Central/veterinaria , Porcinos Enanos , Vena Cava Inferior , Animales , Cateterismo Venoso Central/efectos adversos , Cateterismo Venoso Central/métodos , PorcinosRESUMEN
A large animal model is needed to evaluate new apheresis technologies. These technologies include novel methods of harvesting the blood mononuclear cell population which contains the hematopoietic stem cells needed to restore hematopoiesis in recipients of hematopoietically lethal therapy and the use of cytokines to provide a safe and predictable method of manipulating these circulating hematopoietic stem cells. We describe the methods used to collect mononuclear cells by leukapheresis from Yucatan miniature swine. These animals are of sufficient size to tolerate the procedures and have many physiologic and hematologic similarities to man. They are of good temperament and are easily trained. Long-term venous access was obtained using single lumen silicone rubber catheters placed in the inferior vena cava. The animals were apheresed while fully awake using a Haemonetics Model V50 machine and a modified lymphocyte collection protocol. The procedure was highly efficient for the collection of mononuclear cells and a 10 pass procedure yielded a product which contained 19.7 x 10(9) mononuclear cells, 10.7 x 10(9) granulocytes, and 17 ml of erythrocytes in a volume of approximately 100 ml. This product can be cryopreserved and used for subsequent transplantation. The content of four apheresis procedures provides hematopoietic reconstitution of lethally irradiated swine on a time scale equivalent to transplantation of optimal numbers of bone marrow cells.
Asunto(s)
Leucaféresis , Leucocitos Mononucleares/trasplante , Modelos Biológicos , Animales , Células de la Médula Ósea , Cateterismo Venoso Central , Monitoreo Fisiológico , Manejo de Especímenes/métodos , Porcinos , Porcinos Enanos , Vena Cava Inferior , Irradiación Corporal TotalRESUMEN
Sociosexual behavior was monitored on a daily basis for 3 months in 5 pairs of golden lion tamarins (Leontopithecus rosalia). Urine samples were collected daily from each female and urinary estrogen cycles were determined by radioimmunoassay. Mounts and copulations were observed during all phases of the estrogen cycle. Peaks or regular cycles in sexual behavior were not documented. There were no significant changes in affiliative behavior by females or males that were associated with changes in urinary estrogen values. A negative relationship between pair bond duration and frequency of sexual interactions was observed: newly established pairs exhibited 2-6 times more frequent sexual behavior than a long-established pair. The lack of a conspicuous sexual signal in female golden lion tamarins may be related to a pattern of continuous sexual receptivity. Both reproductive patterns, concealed estrus and continuous receptivity, are explicable in relation to either monogamous or polyandrous mating systems.
Asunto(s)
Callitrichinae/fisiología , Estro/fisiología , Conducta Sexual Animal , Animales , Callitrichinae/orina , Estrógenos/orina , Femenino , Masculino , RadioinmunoensayoRESUMEN
Daily urine samples were collected from 5 female golden lion tamarins (Leontopithecus rosalia) over a period of 3 or more months, and urinary oestrogen concentrations were determined by radioimmunoassay. Four females exhibited regular patterns of oestrogen excretion, with a peak-to-peak periodicity of 19.6 +/- 1.4 days. Levels of oestrogen excretion tended to vary between, but not within, individual females. Post-partum oestrogen patterns included periods of clear oestrogen cyclicity before conception, with dramatic elevations in oestrogen excretion following conception. Oestrone was the predominant urinary oestrogen excreted by female lion tamarins. Enzyme hydrolysis with Helix pomatia beta-glucuronidase/sulphatase was an efficient method of liberating conjugated oestrogens in tamarin urine. Urinary oestrogen determinations can provide useful information about reproductive status in female lion tamarins.