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PubMed2000

Developmentally regulated and lineage-specific rearrangement of T cell receptor Valpha/delta gene segments.

To quantitate the frequency of Valpha/delta gene utilization by TCRgammadelta T cells we have generated a large panel of gammadelta T cell hybridomas and characterized their productive VDJ rearrangements. Using three novel mAb specific for the Vdelta5 chain and for several members of the Vdelta6 subfamily together with previously described Valpha- and Vdelta-specific mAb we have also quantitated the frequency of gammadelta and alphabeta cells expressing those Valpha/delta gene segments and located in different anatomical sites. We have also characterized the members of the Vdelta7/ADV10 subfamily expressed in C57BL/6 mice and analyzed the representation of individual ADV10 gene segments in alphabeta and gammadelta cells, as well as in precursor cells, in a situation in which TCR-dependent selection is negligible. Our results show that (i) although many Valpha/delta gene segments have the potential to rearrange to either Ddelta and Jdelta segments or to Jalpha segments, only a limited number of Valpha/delta gene segments are expressed by a quantitatively important fraction of gammadelta cells; (ii) such restricted usage of a limited number of Vdelta gene segments by gammadelta cells is mainly established at the level of V(D)J rearrangement, and (iii) there is very little overlap between Valpha/delta gene segments expressed by gammadelta and alphabeta cells.

Animals↗
PubMed1991

Fine structure of the elasmobranch renal tubule: neck and proximal segments of the little skate.

This is the first in a series of studies that examines the renal tubular ultrastructure of elasmobranch fish. Each subdivision of the neck segment and proximal segment of the renal tubule of the little skate (Raja erinacea) has been investigated using electron microscopy of thin sections and freeze-fracture replicas. Flagellar cells, characterized by long, wavy, flagellar ribbons, were observed in both nephron segments. They were found predominantly in the first subdivision of the neck segment, which suggests that propulsion of the glomerular filtrate is a primary function of this part of the renal tubule. In the non-flagellar cells of the neck segment (subdivisions I and II), there were bundles of microfilaments, a few apical cell projections, and, in subdivision II, numerous autophagosomes. In the proximal segment, the non-flagellar cells varied in size, being low in subdivision I, cuboidal in II, tall columnar in III, and again low in IV. Apical cell projections were low and scattered in subdivisions I and IV and were highest in III where the basolateral plasma membrane was extremely amplified by cytoplasmic projections. Furthermore, in these cells the mitochondria were numerous with an extensive matrix and short cristae. A network of tubules of the endoplasmic reticulum characterized the apical region of the non-flagellar cells in subdivisions I, II, and IV. In the late part of subdivision II and the early part of III, the cells were characterized by numerous coated pits and vesicles, large subluminal vacuoles, and basally located dense bodies, all of which are structures involved in receptor-mediated endocytosis. Freeze-fracture replicas revealed gap junctions restricted to the cells of the first three subdivisions of the proximal segment. The zonulae occludentes were not different in the neck and proximal segments, being composed of several strands, suggesting a moderately leaky paracellular pathway.

Animals↗
PubMed2001

Relative variation in human proximal and distal limb segment lengths.

The pattern of variation and covariation of proximal and distal limb segment lengths was examined within and between 20 geographically diverse skeletal samples of modern humans. Analyses of variance-covariance matrices (VCMs) of logarithmically transformed (ln) variates of humerus, radius, femur, and tibia length were performed to test the following hypotheses: first, within populations, the distal and proximal segments will have equal relative (i.e., size-independent) variability. However, between populations, the tibia is predicted to be more variable than the other segments. Tests of fit of computed VCMs to theoretical matrices by an iterative procedure (Anderson [1973] Ann. Stat. 1:135-141) reject the equal variance hypotheses, rather suggesting that the relative variances of the distal limb segments are greater than are those of the proximal. Males and females differ somewhat in that within females, the distal segments of both limbs have equal variance, while within males, the tibia has greater relative variance than the radius. The second hypothesis, regarding between-group variability, is somewhat supported in that between human populations, one cannot reject that the tibia has greater relative variance than the other limb segments. However, neither can one reject an alternative hypothesis that both distal limb segments (tibia and radius) are more variable than the proximal segments. Differential growth allometry is explored, and likely plays a major role in differences seen both within and between human populations.

Anthropology, Physical↗
PubMed1995

Growth of the myocardial volumes of the individual cardiac segments in the rat embryo.

BACKGROUND: Although the growth of the developing heart in relation to an increase of ventricular systolic pressure and the growth of the entire embryo during development has been described, no data are available on the growth of the individual segments and intersegmental junctions. Because these different portions are known to function differently, the need for data on their individual development is obvious. METHODS: We have measured the volumes of these different compartments by Cavalieri's point counting method in rat embryos from 11 to 17 days. RESULTS: It is shown that sinus venosus and sinu-atrial junction as well as the main compartments atrium, inlet, and proximal outlet segment grow roughly proportional to the total myocardial volume. Atrio-ventricular canal and distal outlet segment show a restricted growth and their proportional volumes decrease in time. The inlet segment is the most important part of the ventricular mass at 11 days of gestation, when it is still larger than the proximal outlet segment and, thus, takes the greater part in systolic action of the ventricular mass. The growth of the primary fold increases from day 13 onwards and can be considered as part of the wall of the inlet segment which gives rise to the main part of the ventricular septum. CONCLUSIONS: The timing of the septal volume increase fits with qualitative descriptions of ventricular septation. The atrio-ventricular canal and distal outlet segment have an important constrictive function in early stages, when valves are not yet present. Slow conduction and contraction patterns have been reported to be a characteristic feature of these portions of the embryonic heart. With development of valves these segments are loosing their mechanical function and, thus, their proportional volume declines.

Animals↗
PubMed1998

Acute anterior wall myocardial infarction entailing ST-segment elevation in lead V1: electrocardiographic and angiographic correlations.

BACKGROUND: The correlation between ST elevation in lead V1 during anterior wall acute myocardial infarction (AMI) and the culprit lesion site in the left anterior descending (LAD) coronary artery is poor. HYPOTHESIS: The study was undertaken to assess the electrocardiographic (ECG) characteristics and angiographic significance of ST-segment elevation in lead V1 during anterior wall acute myocardial infarction (AMI). METHODS: Data from 115 patients with anterior wall AMI, who underwent coronary angiography within 14 days of hospitalization, were studied. The admission 12-lead ECG was examined and the coronary angiogram was evaluated for the nature of the conal branch of the right coronary artery (RCA) and for the culprit lesion site in the left anterior descending (LAD) coronary artery. RESULTS: Mean ST-segment deviation and the frequency of patients with ST-segment elevation > 0.1 mV were significantly lower in lead V1 than in lead V2 (0.136 +/- 0.111 mV vs. 0.421 +/- 0.260 mV, and 37 vs. 96%, for leads V1 and V2, respectively). A small conal branch not reaching the interventricular septum (IVS) was more prevalent among patients with ST-segment elevation > 0.1 mV in lead V1 (67%), whereas a large conal branch was more prevalent in patients with ST-segment deviation (1 mV in that lead (83%, p < 0.001). No relation was found between ST-segment deviation in lead V1 during anterior wall AMI and the culprit lesion site in the LAD. CONCLUSION: ST-segment elevation in lead V1 during first anterior wall AMI was found in one third of the patients, and its magnitude was lower than that in the other precordial leads. ST-segment elevation in lead V1 favors the presence of a small conal branch of the RCA that does not reach the IVS.

Aged↗
PubMed2001

Implications of the absence of ST-segment elevation in lead V4R in patients who have inferior wall acute myocardial infarction with right ventricular involvement.

BACKGROUND: ST-segment elevation of > or = 1.0 mm in lead V4R has been shown to be a reliable marker of right ventricular involvement (RVI), a strong predictor of a poor outcome in patients with inferior acute myocardial infarction (IMI). However, patients with no ST-segment elevation in lead V4R despite the presence of RVI have received little attention. HYPOTHESIS: The study was undertaken to study the clinical features of patients with no ST-segment elevation in lead V4R despite the presence of RVI, which means false negative, as such patients have received little attention in the past. METHODS: We studied 62 patients with a first IMI, who had total occlusion of the right coronary artery (RCA) proximal to the first right ventricular branch and successful reperfusion within 6 h from symptom onset, to examine the implications of the absence of ST-segment elevation in lead V4R despite the presence of RVI. RESULTS: A standard 12-lead electrocardiogram (ECG) and right precordial ECG (lead V4R) were recorded on admission, and three posterior chest ECGs (leads V7 to V9) were additionally recorded in 34 patients. Patients were classified according to the absence (Group 1, n = 18) or presence (Group 2, n = 44) of ST-segment elevation of > or = 1.0 mm in lead V4R on admission. Patients in Group 1 had a greater ST-segment elevation in leads V7 to V9 (2.9+/-2.4 vs. 1.4+/-3.0 mm. p < 0.05), a higher frequency of a dominant RCA (defined as the distribution score > or = 0.7) (72 vs. 11%, p < 0.001), and a higher peak creatine kinase level (3760+/-1548 vs. 2809+/-1824 mU/ml, p < 0.05) than those in Group 2. CONCLUSIONS: In patients with IMI caused by the occlusion of the RCA proximal to the first right ventricular branch, no ST-segment elevation in lead V4R can occur because of concomitant posterior involvement. In such patients, the incidence of RVI may be underestimated on the basis of ST-segment elevation in lead V4R.

Aged↗
PubMed1985

Heterogeneous properties of segmentally homologous interneurons in the ventral nerve cord of locusts.

The G, B1, and B2 neurons are three prominent interneurons located in adjacent segmental ganglia in the central nervous system of locusts. Previous studies on the adult nervous system have shown that each of these cells has its own distinctive morphology and responsiveness to auditory input. Previous studies on the embryonic nervous system have described the lineage and development of one of these cells, the G neuron, in the mesothoracic (T2) segment. In this paper it is shown that the G, B1, and B2 neurons are segmental homologues in that they arise from equivalent lineages during embryogenesis in the T2, T3, and A1 segments, respectively. Each cell arises (along with its identified sibling neuron) from the division of the second ganglion mother cell of neuroblast 7-4. The segment-specific morphology of the G homologues was determined in the T3 and A1 segments between 60-70% of embryonic development, and their identity was established as the adult B1 and B2 neurons by comparing the distinctive cell-specific features of their morphology between embryo and adult. Although all three neurons display striking morphological differences, they all share certain structural features in common, including the location of their primary axons and neurites in specific tracts in the neuropil. By recording intracellularly from the main neurites of the G, B1, and B2 neurons, clear differences were found in the synaptic inputs each of the neurons receives and the synaptic outputs each makes. For example, G and B2, but not B1, receive direct monosynaptic input from the descending contralateral movement detector (DCMD) interneurons and from auditory afferents; B1, but not B2, connects directly to G; and B2, but not B1 or G, connects directly to flight motoneurons. The main conclusion from these observations is that lineally equivalent neurons in different segments can develop similar primary structures but quite different secondary morphologies and synaptic connections. How these segment-specific differences arise during embryogenesis remains unknown.

Animals↗
PubMed1989

Comparison of met-enkephalin, dynorphin A, and neurotensin immunoreactive neurons in the cat and rat spinal cords: II. Segmental differences in the marginal zone.

This study examined the number of met-enkephalin, dynorphin A 1-8, and neurotensin immunoreactive (IR) neurons in the marginal zone (lamina I) at one thoracic (T8:cat,T9:rat), one midlumbar (L5:cat,L4:rat), and one lower lumbar or sacral (S1:cat,L6:rat) spinal cord segment in the cat and rat. Marginal zone IR neurons ranged 10-70 microns in diameter in cats and 10-50 microns in rats and were flattened, pyramidal, fusiform, or polygonal in morphology. Immunoreactive neurons for each peptide in both species were found in the marginal zone at all spinal levels, but with a differential segmental distribution. The average number of IR neurons per 50-microns section generally was lowest in thoracic cord and greatest in lower lumbar/sacral cord for all peptides. For enkephalin and dynorphin, the estimated total number of IR neurons per segment and number of IR neurons per volume (mm3) generally were lowest in the midlumbar segments and highest in the thoracic and lower lumbar/sacral cord. For neurotensin, the estimated total number of neurons per segment remained lowest in the thoracic and largest in the lower lumbar/sacral cord. The number of neurotensin IR neurons per volume was equal in the thoracic and midlumbar cord, but remained highest at lower lumbar/sacral levels. The IR neurons quantified in this study may be interneurons or may serve as supraspinal projection neurons. The large number of IR neurons observed in segments receiving a relatively large visceral afferent input suggests that some of these neurons may be involved in visceral sensory processing. In addition, the segmental distribution of the IR neurons indicates that physiological and pharmacological studies on the effects of opioid and/or neurotensin peptides should be interpreted in light of the spinal segment(s) investigated.

Animals↗
PubMed1992

Automated image detection and segmentation in blood smears.

A simple technique which automatically detects and then segments nucleated cells in Wright's giemsa-stained blood smears is presented. Our method differs from others in 1) the simplicity of our algorithms; 2) inclusion of touching (as well as nontouching) cells; and 3) use of these algorithms to segment as well as to detect nucleated cells employing conventionally prepared smears. Our method involves: 1) acquisition of spectral images; 2) preprocessing the acquired images; 3) detection of single and touching cells in the scene; 4) segmentation of the cells into nuclear and cytoplasmic regions; and 5) postprocessing of the segmented regions. The first two steps of this algorithm are employed to obtain high-quality images, to remove random noise, and to correct aberration and shading effects. Spectral information of the image is used in step 3 to segment the nucleated cells from the rest of the scene. Using the initial cell masks, nucleated cells which are just touching are detected and separated. Simple features are then extracted and conditions applied such that single nucleated cells are finally selected. In step 4, the intensity variations of the cells are then used to segment the nucleus from the cytoplasm. The success rate in segmenting the nucleated cells is between 81 and 93%. The major errors in segmentation of the nucleus and the cytoplasm in the recognized nucleated cells are 3.5% and 2.2%, respectively.

Algorithms↗
PubMed1987

Somatic point mutations in unrearranged immunoglobulin gene segments encoding the variable region of lambda light chains.

Somatic point mutations are usually found in the coding and flanking regions of functionally and aberrantly rearranged immunoglobulin variable region gene segments. Mutations in the unrearranged V gene segments of myelomas or hybridomas have not been described so far. We have cloned and sequenced unrearranged V lambda gene segments from several cell lines. There were no nucleotide changes in four unrearranged V lambda segments: one V lambda 1 from a lambda 3-producing hybridoma and one V lambda 2 from a lambda 1-producing myeloma (J558) and two V lambda 2 from a kappa-producing myeloma (P3X63). However, we found somatic mutations in the unrearranged V lambda segments from the lambda 2-producing myeloma MOPC315. The unrearranged V lambda 1 gene segment had two mutations in the coding region and the unrearranged V lambda 2 had one mutation in the 3' flanking region. We also cloned and sequenced the unrearranged J lambda and C lambda gene segments of MOPC315 and found no sequence alterations. This is consistent with the notion that the overall mutation rate is not higher in this cell line. Therefore, we suggest that the somatic hypermutation system can use unrearranged V gene segments as substrates. The extensive sequencing required for this work revealed a number of errors in the reported nucleotide sequences of the Ig lambda locus in BALB/c mice.

Animals↗
PubMed1994

Ischemia-induced delayed-onset paraplegia is accompanied by an unusual form of synaptic degeneration in the lumbosacral segments: an experimental light and electron microscopic study in dogs.

We studied the effect of high thoracic aorta cross-clamping, complete transverse section of the spinal cord at Th6 level, and combined hemisection at Th6 level followed later by high thoracic aorta cross-clamping upon the morphology and number of identified presynaptic knobs in lumbosacral segments in dogs. In animals surviving 48-72 hours after high thoracic aorta cross-clamping the occurrence of an unusual form of boutons accompanied by periboutonal halo in L3-S1 segments was found. According to the bouton size and light as well as electron microscopic appearance, four types, i.e., light giant (T1), dark enlarged (T2), light giant with periboutonal halo (T3), and giant disintegrating (T4) boutons were detected after 48 and 72 hour reperfusion. The appearance of four boutonal types in the lumbosacral segments is caused by spinal cord ischemia secondary to high thoracic aorta cross-clamping followed by 48 or 72 hour reperfusion. At the end of the sixth reperfusion day no signs of enlarged and giant boutons were detected in L3-S1 segments. A statistically significant increase of enlarged and giant boutons was noted at the end of the third reperfusion day in comparison with 48 hour survival. After spinal cord transection at midthoracic (Th6) level, followed by 72 hour survival, no such unusual synaptic knobs could be found in L3-S1 segments. The laminar distribution pattern of T1-T4 types based on light microscopic analysis and confirmed electron microscopically is characteristic and strictly bound to those spinal cord gray matter layers which serve as main termination sites of the descending cortical, brain stem, as well as long propriospinal projections in the lumbosacral segments (laminae V-VII). A statistically significant increase of enlarged and giant boutons was found in the intermediate zone (lamina VII). Hemisection at midthoracic level (Th6) followed later by 30 minute high thoracic aorta cross-clamping and 48 hour reperfusion caused a marked decrease of enlarged and giant boutons in L3-S1 segments on the hemisectioned side in comparison with the contralateral one. Large amounts of irregularly arranged round vesicles and tubular profiles were disclosed in the boutonal matrix of T1, T3, and T4 types in L3-S1 segments of animals subjected to 30 minute high thoracic aorta cross-clamping followed by 72 hour reperfusion. Accumulation of tubular and membranous materials was invariably seen in the bulbous enlargement of the terminal axonal branch.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1984

Reproductive development of female Schistosoma mansoni (Digenea: Schistosomatidae) following bisexual pairing of worms and worm segments.

Maturation and maintenance of normal reproductive function in female Schistosoma mansoni require a permanent association with the male, but the nature of this relationship is not well understood. The regional localization of a stimulatory factor in the male and its target in the female were investigated. Unisexual female and mature male worms were transected into segments of various lengths. Various combinations of transected male and female segments and intact worms were transferred to the mesenteric veins of recipient hamsters and were also maintained in vitro. In hamsters and in vitro, pairing took place between intact worms of each sex and segments of the other, and between segments of both sexes. The majority of female worms and segments so paired showed some reproductive development, as assessed by vitelline gland differentiation. In intact unisexual females paired with small male segments, vitelline gland development was limited to that portion of the worm that had been held by the male. Worm segments continued to display normal body contractions throughout 24 days of in vitro maintenance and morphological integrity was retained. It is concluded that 1) in the absence of a functioning gut, worm segments can survive for prolonged periods on nutrients absorbed through the tegument; 2) worm pairing, male stimulation, and the female developmental response are independent of central nervous control by the cerebral ganglia; 3) males have no centralized localization for the female-stimulating factor; 4) vitelline gland differentiation in the female requires local stimulation through male contact, and this is not propagated throughout the worm.

Animals↗
PubMed1995

Stability of nuclear segments in human neutrophils and evidence against a role for microfilaments or microtubules in their genesis during differentiation of HL60 myelocytes.

The nucleus of the mature human neutrophil is segmented into three to five interconnected lobes. The physiological purpose of this segmentation is unknown, as is the mechanism by which the lobes are formed during differentiation. Using video observation of migrating human neutrophils simultaneously illuminated for fluorescence and phase-contrast microscopy, we analyzed nuclear movements with respect to cell shape changes. The number of nuclear lobes and their relative size remained constant during observation (up to 1 h). The thin connecting segments between the lobes elongated and attenuated extensively but never separated. Electron microscopic analysis of neutrophil nuclei revealed no specialized nuclear or cytoplasmic structures in the vicinity of connecting segments. With fluorescence in situ hybridization of whole chromosome probes, we determined that chromosomes are randomly distributed among neutrophil nuclear lobes. HL60 cells are a human myelocytic line that, with retinoic acid treatment, segment their nuclei and differentiate into neutrophil-like cells over several days. Using a rapidly responding variant line termed HL60/S4 (Cancer Res. 52, 949-954), we found that segmentation could be induced within 24 h. We tested the role of cytoskeletal elements in the process of nuclear segmentation. Neither the microtubule inhibitor nocodazole nor the microfilament inhibitor cytochalasin D prevented nuclear segmentation. Together, our studies suggest that nuclear lobes in neutrophils are relatively stable structures that are not generated by microtubule- or microfilament-dependent forces.

Actin Cytoskeleton↗
PubMedDate not supplied

In vivo validation of tissue segmentation based on a 3D feature map using both a hamster brain tumor model and stereotactically guided biopsy of brain tumors in man.

The purpose of this study was to validate our MR tissue segmentation technique using a hamster brain tumor model and malignant brain tumors in man. We used a multispectral tissue segmentation analysis. Three sets of MRI data were included: proton density, T2-weighted fast spin echo, and T1-weighted spin echo, as inputs. Three image preprocessing steps included correcting image nonuniformity, application of an anisotropic diffusion type filter, and data point selection by a qualified observer. We used the k-Nearest Neighbor segmentation algorithm, which does not require prior knowledge of the sample distribution. This choice allowed us to optimize the different tissue clusters present in three-dimensional (3D) feature space. In vivo validation of the technique was performed in hamsters harboring tumors induced with JC virus-transformed HJC-15 cells, as compared to three control animals. Human brain tumors obtained by stereotactically guided biopsy in six patients were also included in the study. Finally, brain tumors were removed from two patients who underwent conventional craniotomy using segmentation-derived images as a guide. In the hamsters, 10 tissues were correctly identified by segmentation and were confirmed histologically (P < .02). In the patients, there was also a strong correlation between our segmentation results and the tissue obtained by stereotactic biopsy (P < .01). In one of the two patients who underwent open craniotomy, segmentation images were useful in revealing tumor spread into vital areas of the brain (motor area). In conclusion, the results of segmentation correlate well with the tissues in vivo and thus warrant further clinical utilization and evaluation.

Adult↗
PubMed2005

Dependence of axon initial segment formation on Na+ channel expression.

Spinal motor neurons were isolated from embryonic rats, and grown in culture. By 2 days in vitro, the axon initial segment was characterized by colocalization and clustering of Na+ channels and ankyrinG. By 5 days, NrCAM, and neurofascin could also be detected at most initial segments. We sought to determine, as one important aim, whether Na+ channels themselves played an essential role in establishing this specialized axonal region. Small hairpin RNAs (shRNAs) were used to target multiple subtypes of Na+ channels for reduced expression by RNA interference. Transfection resulted in substantial knockdown of these channels within the cell body and also as clusters at initial segments. Furthermore, Na+ currents originating at the initial segment, and recorded under patch clamp, were strongly reduced by shRNA. Control shRNA against a nonmammalian protein was without effect. Most interestingly, targeting Na+ channels also blocked clustering of ankyrinG, NrCAM, and neurofascin at the initial segment, although these proteins were seen in the soma. Thus, both Na+ channels and ankyrinG are required for formation of this essential axonal domain. Knockdown of Na+ channels was somewhat less effective when introduced after the initial segments had formed. Disruption of actin polymerization by cytochalasin D resulted in multiple initial segments, each with clusters of both Na+ channels and ankyrinG. The results indicate that initial segment formation occurs as Na+ channels are transported into the nascent axon membrane, diffuse distally, and link to the cytoskeleton by ankyrinG. Subsequently, other components are added, and stability is increased. A computational model closely reproduced the experimental results.

Animals↗
PubMed1997

In vivo forces during remodeling of a two-segment anterior cruciate ligament graft in a goat model.

An existing goat model was used to measure in vivo graft forces during walking, to determine if the forces set at surgery change over time under the same external load and if the forces in the graft during in vivo function can be dictated by the forces set at surgery. The anterior cruciate ligament was reconstructed in 12 goats with use of a composite graft consisting of a bone-patellar tendon-bone autograft and a synthetic augmentation segment. The forces in the graft segments were established intraoperatively by a force-setting technique. In five animals, the tendon segment was set to carry 90% of the total graft force, and in the seven other animals, the augmentation segment was set to carry 90% of the total force. The total graft force was the same in all animals. Graft forces due to anterior tibial loads of 67 N were measured before and after fixation and 6 weeks after surgery with the use of buckle transducers mounted extra-articularly over the anterior tibia. They were also measured during straight, level walking at 6 weeks. The forces changed significantly from just after surgery to 6 weeks later, such that the initially set load-sharing was eliminated by 6 weeks. At 6 weeks, a relatively smooth gait had been achieved, and the maximum total graft force in each animal during walking averaged 35 N and was of similar magnitude to forces generated by the anterior tibial loads of 67 N with the animal anesthetized. After fixation, forces in the tendon graft segments were significantly different between the group with high set forces and that with low set forces. At 6 weeks, when functional joint loads were approaching normal levels, the graft segment forces for the two groups were not significantly different. Load-sharing between tendon and augmentation segment and load in the tendon segment at 6 weeks could not be dictated at surgery.

Animals↗
PubMed1999

Microassay of 5'-nucleotidase and adenosine deaminase activity in microdissected nephron segments.

The present study describes a new method for microassay of the activity of 5'-nucleotidase (5'-ND) and adenosine deaminase (ADA) in the microdissected nephron segments. The nephron segments including glomeruli, proximal convoluted and straight tubules (PCT and PST), cortical and medullary thick ascending limbs, and cortical and medullary collecting ducts were microdissected. 5'-ND and ADA in the nondenatured lysate of 20-mm microdissected tubules and 20 glomeruli were separated by agarose gel electrophoresis and by isoelectric focusing, respectively. The gels were incubated with specific substrates and staining dyes to exhibit the dephosphorylation by 5'-ND or deamination by ADA. The enzyme activity was estimated by measuring the intensity of the reaction bands on the gels. The 5'-ND activity was detected in all microdissected tubular segments and glomeruli. Among these nephron segments, PCT and PST exhibited the greatest enzyme activity, averaging 1142 and 939 mU/mg tissue protein, respectively. The activity of ADA was also detected in all tubular segments and glomeruli. However, the greatest activity of this enzyme was found in the glomeruli (649.8 mU/mg protein). Using reverse transcriptase-polymerase chain reaction technique, we verified the presence of mRNA of 5'-ND and ADA in all microdissected tubular segments and glomeruli. Based on these results, we conclude that 5'-ND and ADA are present in all nephron segments studied, but the activity of these enzymes is nonuniformly expressed along the nephron. This microassay is a highly specific, sensitive, and reliable method for the segmental analysis of adenosine metabolism in the kidney.

5'-Nucleotidase↗
PubMed1994

NGF-induced stabilization of GAP-43 mRNA is mediated by both 3' untranslated region and a segment encoding the carboxy-terminus peptide.

NGF stabilizes cytoplasmic mRNA for GAP-43, a neuronal growth cone protein. A 292 b segment of rat GAP-43 mRNA was determined to mediate this stabilizing effect. Insertion of the 292 b segment into heterologous mRNA was also shown to constitutively destabilize the mRNA in the absence of NGF. This 292 b segment contains the sequences encoding the carboxy-terminus peptide and part of the 3' untranslated region. Neither one of these sequences can fully mediate the NGF-dependent stability control independently from each other. Premature termination of translation before the 292 b segment reduces both the constitutive destabilization by the 292 b segment and the NGF-induced stabilization. These findings indicate that the constitutive destabilizing function of the 292 b segment is dependent on the direct translation of this segment and that NGF suppresses the destabilizing function of this segment.

Animals↗