Ras p21 protein promotes survival and differentiation of human embryonic neural crest-derived cells

We have previously shown that the oncogene product p21 Ras is essential for the survival and neurite outgrowth-promoting activity of nerve growth factor on cultured chick embryonic sensory, but not sympathetic neurons. In order to extend our observations to the human system and to non-neuronal cells, we introduced the oncogenic form of p21 Ras into the cytoplasm of three different types of cultured human embryonic neural crest derivatives (8th-11th gestational week): dorsal root ganglion neurons, sympathetic neurons, and adrenal chromaffin cells. These cells are dependent on nerve growth factor for survival and/or fibre outgrowth in vitro. In dorsal root ganglion neurons, p21 Ras promoted survival and fibre outgrowth which was quantitatively and qualitatively comparable to the nerve growth factor effect (84% vs. 95%, control 18%). Sympathetic neurons showed a similar effect, albeit with a higher background survival (91% vs. 93%, control 58%). On chromaffin cells, which respond to nerve growth factor with pronounced fibre outgrowth in culture, the effect of p21 Ras was again comparable to that of nerve growth factor (35% vs. 30%, control 5%). The survival and fibre outgrowth-promoting effects of p21 Ras on human embryonic dorsal root ganglion neurons, sympathetic neurons and chromaffin cells suggest an involvement of p21 Ras in the intracellular signal transduction of nerve growth factor in human neural crest-derived cell populations.     

The affinities of procolipase and colipase for interfaces are regulated by lipids

It has been suggested that at physiological pH, the trypsin-catalyzed activation of the lipase cofactor, procolipase, to colipase has no consequence for intestinal lipolysis and serves primarily to release the N-terminal pentapeptide, enterostatin, a satiety factor (Larsson, A., and C. Erlanson-Albertsson 1991. The effect of pancreatic procolipase and colipase on pancreatic lipase activation. Biochim. Biophys. Acta 1083:283-288). This hypothesis was tested by measuring the adsorption of [14C]colipase to monolayers of 1-stearoyl-2-oleoyl-sn-3-glycerophosphocholine and 13, 16-cis, cis-docosadienoic acid in the presence and absence of procolipase. With saturating [14C]colipase in the subphase, the surface excess of [14C]colipase is 29% higher than that of procolipase, indicating that colipase packs more tightly in the interface. With [14C]colipase-procolipase mixtures, the proteins compete equally for occupancy of the argon-buffer interface. However, if a monolayer of either or both lipids is present, [14C]colipase dominates the adsorption process, even if bile salt is present in the subphase. If [14C]colipase and procolipase are premixed for > 12 h at pH approximately 8, this dominance is partial. If they are not premixed, procolipase is essentially excluded from the interface, even if procolipase is added before [14C]colipase. These results suggest that the tryptic cleavage of the N-terminal pentapeptide of procolipase may be of physiological consequence in the intestine.     

Mechanisms for the maintenance and eventual degradation of neurofilament proteins in the distal segments of severed goldfish mauthner axons

Cellular mechanisms that might affect the degradation of neurofilament proteins (NFPs) were examined in the distal segments of severed goldfish Mauthner axons (M-axons), which do not degenerate for more than 2 months after severance. Calpain levels, as determined by reactivity to a polyclonal antibody, remained constant for 80 d postseverance in distal segments of M-axons and then declined from 80 to 85 d postseverance. Calpain activity in rat brain, as determined by a spectrophotometric assay, was much higher than calpain activity in control and severed goldfish brain, spinal cord, muscle, or M-axons. Calpain activity was extremely low in M-axons compared with that in all other tissues and remained low for up to 80 d postseverance in distal segments of M-axons. Phosphorylated NFPs, as determined by Stains-All treatment of SDS gels, were maintained for up to 72 d postseverance and then decreased noticeably at 75 d postseverance when NFP breakdown products appeared on silver-stained gels. By 85 d post-severance, phosphorylated NFPs no longer were detected, and NFP breakdown products were the most prominent bands on silver-stained gels. These results suggest that the distal segments of M-axons survive for months after severance, because NFPs are maintained in a phosphorylated state that stabilizes and protects NFPs from degradation by low levels of calpain activity in the M-axon; the distal segments of severed M-axons degenerate eventually when NFPs no longer are maintained in a phosphorylated state and become susceptible to degradation, possibly by low levels of calpain activity in the M-axon.     

Ghosting of pulmonary nodules with respiratory motion: comparison of helical and conventional CT using an in vitro pediatric model

OBJECTIVE: The study was designed to compare helical CT with varying pitch and reconstruction intervals and conventional CT for revealing pulmonary nodules in a model that simulates respiratory motion in children. MATERIALS AND METHODS: CT scans were obtained in an experimental model with one nodule (3 or 10 mm) in each scan. One-second scans were obtained at rates of 10, 20, and 30 respirations per minute using conventional CT with 4-mm collimation and table incrementation and helical CT with 4-mm collimation and either 4-mm/sec (pitch, 1:1) or 8-mm/sec (pitch, 2:1) table speed. Reconstructions were at 1-, 2-, and 4-mm intervals for scans obtained using 4-mm/sec table speed and at 1- and 4-mm intervals for scans obtained using 8-mm/sec table speed. Images were independently reviewed by three radiologists who estimated the number of nodules on each image. RESULTS: Ghosting (depiction of more than one nodule in a study) was seen in 79%, 80%, and 75% of helical CT scans obtained with a 1:1 pitch using 1-, 2-, and 4-mm reconstruction intervals, respectively. By comparison, ghosting was seen in only 54% and 58% of helical CT scans with a 2:1 pitch using 1-mm reconstruction intervals and 4-mm reconstruction intervals, respectively, and in 56% of conventional CT scans (p < .0001). A single nodule was detected on all other scans, and at least one nodule was seen on all scans. CONCLUSION: Ghosting of nodules is common in this model. Ghosting was seen less often on conventional scans and helical scans with 2:1 pitch than it was on helical scans with 1:1 pitch. Nonetheless, ghosting was seen on more than 50% of all scans with each technique.     

Expression of an olfactory receptor in Escherichia coli: purification, reconstitution, and ligand binding

An olfactory receptor has been expressed in bacterial cells as a fusion protein with glutathione S-transferase (GST). Overexpression of receptor protein yielding about 10% of the cell protein was achieved with mutants lacking the N-terminus and the first transmembrane region or with mutants carrying three positively charged residues in the first intracellular loop. The overexpressed fusion protein accumulated in inclusion bodies and could be solubilized in detergent. It was purified by metal chelation chromatography based on a C-terminal 6-histidine tag, and the GST portion was removed after proteolytic cleavage. The purified receptor was reconstituted into lipid vesicles and specific binding of odor ligands was shown by photoaffinity labeling and tryptophan fluorescence measurements. Thus, for the first time, an odorant receptor/ligand pair becomes available in large amounts for biophysical and screening studies.     

Allelic imbalance at NME1 in microdissected primary and metastatic human colorectal carcinomas is frequent but not associated with metastasis to lymph nodes or liver

Allelic imbalance at the NME locus on chromosome 17q21 was analyzed in colorectal cancer patients using a highly polymorphic microsatellite repeat sequence within NME1 itself. Duplicate samples of carcinoma and adjacent normal tissue was obtained by microdissection from 6 to 7-microns paraffin sections of 94 primary carcinomas (treatment years 1979-1993) and available lymph node and liver secondaries. In 55 patients informative (heterozygous) at this locus, allelic imbalance was examined in primary and secondary carcinomas. Microsatellite instability prevented assessment of allelic balance in two cases, and there was no evidence of homozygous loss at NME1 in any case analyzed. Allelic imbalance at the NME locus in carcinomas was frequent (27/53; 51%), and concordant results were obtained between primary carcinoma and secondary deposits in 30 of 33 (91%) cases. Three discordant cases showed allelic imbalance in secondary deposits but not the primary lesion. Although frequent, allelic imbalance at NME1 had no relationship to Dukes' stage at presentation or with subsequent hepatic metastasis, nor with the primary carcinoma site (proximal versus distal), tumor size, or mitotic or apoptotic index. Moreover, neither disease-free nor overall survival differed between patients with carcinomas showing NME1 allelic imbalance and patients with carcinomas that did not. Our results show that although allelic imbalance is frequent at the NME locus in primary and secondary colorectal carcinomas, there is no evidence to link this with clinical or pathological features or with metastatic potential. Microsatellite PCR and microdissection of enriched populations of carcinoma cells allowed uniformly successful analysis of samples from archival formalin-fixed paraffin-embedded tissue up to 15 years old and clear assessment of allelic imbalance in tumor specimens. Target sequences (e.g., microsatellites and minisatellites) up to approximately 200-250 bp may be reliably analyzed for allelic balance, suggesting that this method is of general utility in the genetic analysis of primary and metastatic neoplasia.     

Haida perspectives on living with non-insulin-dependent diabetes

OBJECTIVE: To understand the experience of Haida people living with non-insulin-dependent diabetes mellitus (NIDDM), in order to provide a basis for a culturally sensitive community-based approach to managing NIDDM. DESIGN: Qualitative study using grounded theory. SETTING: The villages of Skidegate and Old Massett in Haida Gwaii (Queen Charlotte Islands), British Columbia. PARTICIPANTS: Nine focus groups met at the beginning and six at the end of the project. The focus groups had 8 to 12 members each and roughly the same number of men and women overall. The groups included people with diabetes, family members of people with diabetes, community leaders and elders. FINDINGS: Conceptual findings related to the participants' views on the impact of NIDDM on their lives, their views on what life was like before the effects of NIDDM were felt and their beliefs about the prevention and treatment of NIDDM. Six themes recurred in the discussions: fear; grief and loss; the loss of and desire to regain control; food and eating; physical and personal strength; and traditional ways. CONCLUSIONS: Insights into the illness experience of different cultural groups can inform program development and the creation of culturally sensitive health care interventions.     

Dysphonia in the elderly: diagnosis and management of age-related voice changes

In our laryngology practice, we have noted an increasing number of elderly patients referred to us for problematic dysphonia. We present our findings of the most common disorder affecting this age group. A sample of 47 consecutive patients over age 60 with dysphonia revealed presbylaryngis, ie, age-related anatomic and physiologic changes, as the most common etiology found in this tertiary referral practice, accounting for 30% (14 patients) of new diagnoses. None of the patients with presbylaryngis received this diagnosis from the referral source. Understanding the anatomic and physiologic changes of the aging vocal tract, along with the clinical correlation of each change, is crucial in evaluating this group of patients. Managing this disorder includes specific goal-oriented speech therapy, with surgery as an adjunct should conservative therapy prove unsuccessful. Earlier recognition of this disorder and prompt intervention are key factors in reversing vocal decompensation, with a primary effect of improving the quality of life for the patient with age-related dysphonia.     

Nitric oxide reduces tumor cell adhesion to isolated rat postcapillary venules

Adhesion of circulating tumor cells to microvascular endothelium plays an important role in tumor metastasis to distant organs. The purpose of this study was to determine whether nitric oxide (NO) would attenuate tumor cell adhesion (TCA) to naive or lipopolysaccharide (LPS)-treated postcapillary venules. A melanoma cell line, RPMI 1846, was shown to be much more adhesive to postcapillary venules isolated from rat mesentery than to corresponding precapillary arterioles. Although venules exposed to LPS for 4 h demonstrated an increased adhesivity for the melanoma cells, TCA to LPS-treated arterioles was not altered. Isolated venules exposed to DETA/NO (1 mM), an NO donor, for 30 min prior to tumor cell perfusion prevented the increment in adhesion induced by LPS and attenuated TCA to naive postcapillary venules. While L-arginine (100 microM), an NO precursor, failed to decrease TCA to naive postcapillary venules, this treatment abolished LPS-stimulated TCA to postcapillary venules. The effect of L-arginine was reversed by administration of N(omega)-nitro-L-arginine methyl ester (L-NAME, 100 microM), an NO synthase (NOS) inhibitor. These observations indicate that both exogenous and endogenous NO modulate TCA to postcapillary venules. To assess the role of NO-induced activation of cGMP in the reduction in TCA produced by DETA/NO, two additional series of experiments were conducted. In the first series, LY-83583 (10 microM), a guanylyl cyclase inhibitor, was shown to completely reverse the effect of DETA/NO on TCA to both naive and LPS-activated postcapillary venules. On the other hand, administration of 8-bromoguanosine 3',5'-cyclic monophosphate (8-B-cGMP) (1 mM), a cell permeant cGMP analog, mimicked the effect of DETA/NO and reduced TCA to LPS-stimulated postcapillary venules. These data suggest that (a) tumor cells are more likely to adhere to postcapillary venules than to corresponding precapillary arterioles, (b) LPS enhances TCA to postcapillary venules, (c) both exogenously applied (DETA/NO) and endogenously generated (L-arginine) NO attenuate the enhanced adhesion induced by LPS, but only DETA/NO reduced TCA to naive postcapillary venules, and (d) the NO-induced reduction in TCA to LPS-activated postcapillary venules occurs by a cGMP-dependent mechanism.     

An analysis of the effects of retinoic acid and other retinoids on the development of adrenergic cells from the avian neural crest

In the present work, we have investigated the role of all-trans-retinoic acid (all-trans RA), and several other natural and synthetic retinoids, in the development of adrenergic cells in quail neural crest cultures. Dose response studies using all-trans RA and 13-cis RA revealed a dose-dependent increase in the number of adrenergic cells in neural crest cultures. Similar dose response studies using RA isomers and other natural retinoids did not result in the same increases. In order to determine the receptor mediating the effects of all-trans RA in the neural crest, we tested several synthetic analogs which specifically bind to a particular RA receptor (RAR) subtype. We found that the compound AM 580, which activates the RAR-alpha, produced an increase in adrenergic cells similar to that seen with all-trans RA. The compound TTNPB, which activates all RAR subtypes, also resulted in an increase in adrenergic cells. We conclude that the increase in adrenergic cells seen with all-trans RA is mediated by RAR-alpha and possibly RAR-beta. To further define the actions of all-trans RA on the neural crest we incubated cultures with 5-bromo-2'-deoxyuridine (BrdU) to determine whether all-trans RA could affect the rate of proliferation. The results show that while all-trans RA did not increase the fraction of cells incorporating BrdU into their nuclei at early time points (24 h), it did increase BrdU incorporation by tyrosine hydroxylase (TH) positive cells at 5 days in culture. These findings demonstrate that the increase in adrenergic cells seen with all-trans RA in neural crest cultures is likely due to an increase in the proliferation of cells already expressing TH.