Microtubule dynamics in axons and dendrites.

We have investigated the stability, alpha-tubulin composition, and polarity orientation of microtubules (MTs) in the axons and dendrites of cultured sympathetic neurons. MT stability was evaluated in terms of sensitivity to nocodazole, a potent anti-MT drug. Nocodazole sensitivity was assayed by quantifying the loss of MT polymer as a function of time in 2 micrograms/ml of the drug.

Spontaneous haemopneumothorax: a rare clinical entity.

A 39 yr old man presented with a spontaneous pneumothorax. On initial pleural drainage 120 ml of haemorrhagic fluid were collected. Twenty four hours, after re-expansion of the lung, shock developed and 1,200 ml of haemorrhagic fluid were spontaneously collected.

Deregulation of alternative processing of Calcitonin/CGRP-I pre-mRNA by a single point mutation.

The Calcitonin/CGRP-I (CALC-I) gene was one of the first examples of a cellular gene exhibiting alternative, tissue-specific processing of its primary transcript. Calcitonin (CT) mRNA is the predominant product in thyroid C-cells, whereas CGRP-I (Calcitonin Gene Related Peptide-I) mRNA is the main product in neurons of the central and peripheral nervous systems. Investigating the molecular mechanism underlying the alternative processing events, we have demonstrated that the CT-specific splice acceptor site is an intrinsical weak site due to usage of a uridine branch acceptor.

Islet amyloid polypeptide: structure and upstream sequences of the IAPP gene in rat and man.

Islet amyloid polypeptide (IAPP) or amylin is a pancreatic islet hormone which was first found in amyloid in insulinomas and in pancreases of patients with type 2 diabetes. In rat a similar polypeptide occurs; however, pancreatic amyloid in this species has not been described. Here we report the structure of the rat and human IAPP gene.

Uridine branch acceptor is a cis-acting element involved in regulation of the alternative processing of calcitonin/CGRP-l pre-mRNA.

The human calcitonin/CGRP-I (CALC-I) gene contains 6 exons and encodes two polypeptide precursors. In thyroid C-cells, calcitonin (CT) mRNA is produced by splicing of exons 1-2-3 to exon 4 (CT-encoding) and polyadenylation at exon 4. CGRP-I mRNA is produced in particular neural cells by splicing of exons 1-2-3 to exon 5 (CGRP-I-encoding) and the polyadenylated exon 6.

Individual microtubules in the axon consist of domains that differ in both composition and stability.

We have explored the composition and stability properties of individual microtubules (MTs) in the axons of cultured sympathetic neurons. Using morphometric means to quantify the MT mass remaining in axons after various times in 2 micrograms/ml nocodazole, we observed that approximately 48% of the MT mass in the axon is labile, depolymerizing with a t1/2 of approximately 5 min, whereas the remaining 52% of the MT mass is stable, depolymerizing with a t1/2 of approximately 240 min. Immunofluorescence analyses show that the labile MTs in the axon are rich in tyrosinated alpha-tubulin, whereas the stable MTs contain little or no tyrosinated alpha-tubulin and are instead rich in posttranslationally detyrosinated and acetylated alpha-tubulin.

Changes in microtubule polarity orientation during the development of hippocampal neurons in culture.

Microtubules in the dendrites of cultured hippocampal neurons are of nonuniform polarity orientation. About half of the microtubules have their plus ends oriented distal to the cell body, and the other half have their minus ends distal; in contrast, microtubules in the axon are of uniform polarity orientation, all having their plus ends distal (Baas, P.W.

In vitro splicing analysis of mini-gene constructs of the alternatively processed human calcitonin/CGRP-I pre-mRNA.

The human calcitonin/CGRP-I (CALC-I) gene can be alternatively expressed into calcitonin mRNA in thyroid C-cells and into CGRP-I mRNA in particular nerve cells. Formation of calcitonin mRNA requires splicing of exons 1, 2, 3 and 4 and addition of poly(A) at exon 4, whereas splicing of exons 1, 2, 3, 5 and 6 and addition of poly(A) at exon 6 yields CGRP-I mRNA. The calcitonin and CGRP-I mRNA-specific splicing reactions were investigated in vitro, in nuclear extracts of HeLa cells, using model precursor RNAs containing the exon 3 to exon 5 region of the gene.

The basis of polarity in neurons.

It has been recognized since the very early studies on the cytology of vertebrate nervous systems that neurons produce two fundamentally different types of neurite, the axon and the dendrite. Contemporary studies using electron microscopy have defined in detail the many structural differences between axons and dendrites. Perhaps the best known of these differences concerns ribosomes and Golgi elements, which are present in dendrites, but are absent from axons.

Isolated regional perfusion in the treatment of subungual melanoma.

Subungual melanoma is rare and represents only 1% to 3% of all diagnosed melanomas in Western countries. The tumor is frequently mistaken for a benign lesion and the delay in diagnosis and final treatment may be responsible for the high local recurrence rate and the low disease-free survival rate. From 1965 to 1982 the combined-modality therapy of amputation and adjuvant isolated regional perfusion with melphalan with or without dactinomycin was used in the treatment of 22 patients with subungual melanoma.

Hyperthermic isolated regional perfusion in the treatment of extremity melanoma in children and adolescents.

From 1973 to 1982 six children and eight adolescents with extremity melanomas were treated by local excision and adjuvant hyperthermic isolated regional perfusion with Melphalan (L-phenylalanine mustard, manufactured by Burroughs Wellcome Company, Research Triangle Park, NC). The median Breslow thickness of the melanomas was 2.7 mm (range, 1 to 15 mm).

Compartmentation of alpha-tubulin in neurons: identification of a somatodendritic-specific variant of alpha-tubulin.

The composition of alpha-tubulin in axons was compared with that in cell bodies and dendrites in the rat. Pure axons and highly enriched preparations of cell bodies and dendrites were obtained from explant cultures of sympathetic neurons, dissolved directly into sodium dodecyl sulfate, and analysed by isoelectric focusing gel electrophoresis. Immunoblots using two different monoclonal antibodies against alpha-tubulin and fluorographs of [35S]methionine- and [3H]tyrosine-labeled alpha-tubulin all show several variants common to both compartments, and an additional variant, more basic than the others, prominent in the somatodendritic compartment but absent from the axonal compartment.

Dynamics of alpha-tubulin deacetylation in intact neurons.

The majority of the alpha-tubulin in cultured neurons is acetylated (Black and Keyser, 1987). The present studies examine the relationships of the acetylation and deacetylation reactions to tubulin assembly and disassembly in intact neurons. Extraction assays which separate assembled and unassembled tubulin pools reveal that greater than or equal to 99% of the total acetylated, as well as newly acetylated, tubulin is cytoskeletal associated.

Polarity orientation of microtubules in hippocampal neurons: uniformity in the axon and nonuniformity in the dendrite.

We have analyzed the polarity orientation of microtubules in the axons and dendrites of cultured rat hippocampal neurons. As previously reported of axons from other neurons, microtubules in these axons are uniform with respect to polarity; (+)-ends are directed away from the cell body toward the growth cone. In sharp contrast, microtubules in the mid-region of the dendrite, approximately 75 microns from the cell body, are not of uniform polarity orientation.

Unusual branch point selection involved in splicing of the alternatively processed Calcitonin/CGRP-I pre-mRNA.

To study splice site selection in alternative RNA processing we used the human Calcitonin/CGRP-I (CALC-I) gene. Expression of the CALC-I gene in thyroid C-cells results predominantly in calcitonin (CT) mRNA (containing exons 1 to 4) whereas CGRP-I mRNA (containing exons 1,2,3,5 and 6) is the exclusive product in particular nerve cells. We previously reported that a model precursor RNA containing the exon 3 to exon 5 region is predominantly processed into CGRP-I mRNA in vitro using nuclear extracts of three different cell types.

Model for tissue specific Calcitonin/CGRP-I RNA processing from in vitro experiments.

The Calcitonin/CGRP-I (CALC-I) gene is known to be expressed in a tissue specific fashion resulting in the production of Calcitonin mRNA in thyroid C-cells and CGRP-I mRNA in particular nerve cells. The alternative RNA processing reactions include splicing of exons 1, 2 and 3 to exon 4 and poly (A) addition at exon 4 (Calcitonin mRNA) or splicing of exons 1, 2 and 3 to exons 5 and 6 and poly (A) addition at exon 6 (CGRP-I mRNA). Using a model precursor RNA containing the exon 3 to exon 5 region of the human CALC-I gene we have investigated the Calcitonin- and CGRP-I mRNA-specific processing reactions in vitro, in nuclear extracts of Hela, PC12 and Ewing-1B cells, respectively.

Role of microtubules in the cytoplasmic compartmentation of neurons. II. Endocytosis in the growth cone and neurite shaft.

We investigated the role of microtubules in the compartmentation of motility and endocytosis in the neurite shaft and growth cone of cultured chick sensory neurons. As reported previously by Letourneau and Ressler (J. Cell Biol.

Microtubule stability in severed axons.

We examined severed axons of cat sympathetic nerves and severed neurites of cultured chick sensory neurons for evidence of extensive microtubule depolymerization. Cat sympathetic fibres fixed at various times after severing were cross-sectioned for electron microscopy from both cut ends. The number density of microtubules was determined at various times after severing for matched proximal and distal regions equidistant from cut ends.

Mutational analysis of the bacteriophage phi X174 replication origin.

Bacteriophage phi X174 mutants within the 30 base-pair replication origin were constructed using oligodeoxynucleotide-directed mutagenesis. A total of 18 viable base substitution mutants at 13 different positions within the origin region were obtained. The majority of these ori mutants have a plaque morphology and burst size comparable to that of wild-type phi X174.

Role of microtubules in the cytoplasmic compartmentation of neurons.

We have examined the role of microtubules (MTs) in the development and maintenance of the cytoplasmic compartmentation of ribosomes in cultured embryonic chick sensory neurons. Control neurons show a sharp cytoplasmic demarcation between the ribosome-rich/MT-deficient 'translational' cytoplasm of the perikaryon and the ribosome-deficient/MT-rich 'expressional' cytoplasm of the axon-like neurites. Neurites treated with 1.