Suppression of proliferation of human coronary artery smooth muscle cells by the nitric oxide donor, S-nitrosoglutathione, is cGMP-independent.

Nitric oxide (NO), delivered by a single addition of S-nitrosoglutathione (GSNO, IC(50) = 60-75 microM), causes the prolonged, multi-day suppression of proliferation of asynchronous, logarithmically growing human (hCASMC, two cell strains), and porcine (porCASMC) coronary artery smooth muscle cells. The inhibition is not cytotoxic, but cytostatic and reversible. Transient exposure (>4-12 h) to GSNO is sufficient to elicit prolonged suppression, but a less than 4 h exposure produces little or no inhibition. Unlike porCASMC and rat and rabbit aortic SMC, hCASMC synthesize little cGMP in response to GSNO stimulation, suggesting loss of NO responsive guanylate cyclase in vitro. The guanylate cyclase inhibitor, ODQ, blocks the slight cGMP synthesis induced by GSNO in hCASMC, but does not prevent GSNO suppression of proliferation. These data support a cGMP independent mechanism for NO induced suppression of hCASMC proliferation which may be significant in the treatment of proliferative coronary artery diseases.

The craniofacial morphology of bruxers versus nonbruxers.

The purpose of this investigation was to test for an association between the craniofacial morphologies of bruxers and nonbruxers. The sample for this retrospective descriptive comparative study consisted of 28 Caucasian dental school subjects. Sixteen were bruxers and 12 were nonbruxers. The determination of bruxism was based on a six-item questionnaire as well as objective measures of the severity of tooth wear as analyzed from dental casts. Craniofacial morphology was determined directly using anthropometric spreading calipers. Craniofacial measurements included glabella-opiscranion, euryon-euryon, nasion-gnathion, zygoma-zygoma, and gonion-gonion. From these measurements, the following indices were calculated: cephalic (Gla-Op/Eu-Eu), facial (Na-Gla/Zy-Zy), gonial (Zy-Zy/Go-Go), and gonial height (Na-Gla/Go-Go). This study found no differences in the craniofacial morphologies of bruxers and nonbruxers, nor was there a difference in overbite. There was, however, a statistically significant difference in the bizygomatic (Zy-Zy) and cranial (Eu-Eu) widths of bruxers compared with nonbruxers.

Nitrosylated bovine serum albumin derivatives as pharmacologically active nitric oxide congeners.

Although nitrosothiols have been suggested to act as regulators of cell (patho)physiology, little is known about the pharmacology of nitrosylated proteins as nitric oxide (NO.) congeners. We describe the molecular consequences of nitrosylating bovine serum albumin (BSA) at multiple specific sites and demonstrate that the product S-nitrosoproteins exert NO.-like activity. The content of nucleophilic nitrosylation sites (i.e., free sulfhydryl groups) in native BSA was increased by either reduction with dithiothreitol or thiolation with N-acetylhomocysteine. Fourteen moles of nitrogen monoxide (NO)/mol BSA equivalent were then selectively positioned on either the endogenous sulfhydryl groups of reduced BSA or the homocysteine moieties of thiolated BSA, respectively. Each resulting S-nitrosoprotein adduct was an oligomeric mixture across the >2000 kDa to approximately 66 kDa molecular mass range. The BSA-derived S-nitrosoproteins were immunoreactive with antibodies against native BSA but evidenced compromised long-chain fatty acid binding. Both types of BSA-derived S-nitrosoproteins suppressed human coronary artery smooth muscle cell proliferation to a similar degree (IC50 approximately 70 microM NO. equivalents) and were significantly more effective antiproliferative agents than a standard NO. donor, DETA NONOate. Antiproliferative bioactivity reflected the NO functionalities carried by each protein, but was independent of molecular mass of the nitrosylated BSA adducts. These data exemplify the rational design and characterization of protein-based S-nitrosothiols as NO. congeners and suggest that such agents could have therapeutic potential as NO delivery systems.

Results of urgent appendectomy for right lower quadrant tenderness.

The outcome was examined for 196 cases of urgent appendectomy in patients with abdominal pain and right lower quadrant tenderness and signs of peritoneal irritation. Appendicitis was found in 94 percent, and there was a 6 percent negative appendectomy rate. There were no complications among the patients with normal appendixes. Complications among cases of appendicitis compared favorably with other published series.

Prognostic factors in head and neck melanoma. Effect of lesion location.

Cutaneous malignant melanomas of the head and neck are prognostically engimatic. In addition to known prognostic determinants of stage and lesion microstage, lesion location also appears to have prognostic importance. The authors have reviewed a series of 83 microstaged head and neck melanoma patients in order to analyze the relative importance of these factors. There were 36 males and 47 females with a median age of 56 years. Eighty-one percent had pathologic Stage I disease, 7% were Stage II, and 12% were Stage III. The primary location was face in 32 patients, neck in 18, ear in 12, and scalp in 21 patients. The actuarial 5-year survival according to lesion thickness was 86% for melanoma less than 1.0 mm, 56% for 1 to 2 mm thick lesions, 47% for 2.1 to 4 mm thick lesions, and 25% for melanomas greater than 4.0 mm. The 5-year survival according to lesion location was 78% for facial and 58% for neck melanomas; for ear and scalp, the respective survivals were 33% and 37%. Median thickness was 2.0 mm for facial and 1.85 mm for neck lesions. It was 2.7 mm for ear and 2.0 mm for scalp lesions (differences not significant). There were no microstage factors that correlated with the adverse prognosis seen with scalp and ear melanomas. Multivariate analysis in the entire series (all clinical stages) showed the following to be significant: stage, thickness, and location of the primary melanoma (all less than 0.0002). In clinical Stage I melanoma, the significant prognostic factors were location (P = 0.035), thickness (P = 0.008), level (P = 0.024), and ulceration (P = 0.035). The prognosis of head and neck melanoma is uniquely influenced by location of the primary lesions in addition to stage, thickness, level, and ulceration, as observed with other cutaneous melanomas at other sites. Ear and scalp melanomas are high-risk lesions whose poor prognosis is not readily explained by any of the microstage factors reviewed.

Comparison of local, spinal, and general anesthesia for inguinal herniorrhaphy.

Patients who had undergone inguinal herniorrhaphy with local, general, or spinal anesthesia were surveyed for satisfaction with the anesthesia. A review of complications was carried out for these same patients to determine comparative rates in the three anesthesia groups. Patients were stratified by age and concomitant medical illness. Satisfaction ratings were equal among the three groups. Complication rates were highest in the spinal anesthesia group (primarily urinary complications) for patients of all ages. Local anesthesia had the lowest complication rate in those over 65 and those with concomitant illnesses.

Evidence for a transforming growth factor(s) in the serum-free conditioned medium of SV40-transformed 3T3 fibroblasts.

Unlike untransformed 3T3 fibroblasts, Simian Virus 40-transformed 3T3 cells (SV3T3) do not require the presence of exogenous polypeptide mitogens in order to become competent for the initiation of DNA synthesis. As an explanation for this state of perpetual competence, growth activity for 3T3 cells was found in the serum-free conditioned medium of SV3T3 cells. This activity, which is abolished by incubation with pepsin, but withstands to a fair degree denaturants (8 M urea and 4 M guanidinium HCl) and a reducing agent (65 mM dithioerythritol), enhances the growth of Swiss and BALB/c 3T3 cells in 1% (v/v) calf serum medium and ensures the complete survival of Swiss 3T3 cells for several days in the total absence of serum. The growth activity appears to be derived from the SV3T3 cells, since it is not found in the non-conditioned medium (and the insulin and transferrin components of this serum-free medium have only weak growth activity alone) and the chances of it being due to residual serum growth factors are slight because of the method of growing the cells and the collection of the conditioned medium. Moreover, in a separate experiment to test the possible involvement of cell attachment factors, rat fibronectin was found not to affect 3T3 growth in the standard growth assay. The conditioned medium growth activity apparently has the biological capabilities of transforming growth factors. After concentration by ammonium sulfate precipitation, it can stimulate DNA synthesis in confluent, quiescent Swiss 3T3 cells, in both the absence and presence of 10% depleted calf serum. It also permits the growth of large colonies of Swiss 3T3 cells in soft agar. As part of a preliminary characterization of the conditioned medium growth activity, gel filtration in 0.15 M NaCl, 0.001 M HCl resolves the activity into high (mol. wt. greater than or equal to 100,000) and low (mol. wt. 5,000-6,000) molecular weight factors. Since re-chromatography of the high molecular weight factor, which may still be in an aggregated form, does not generate the smaller, the latter is not a dissociation product of the former. The precise relationship of these SV3T3-conditioned medium growth factors to other growth factors, including known transforming growth factors, has not yet been determined.

The uncoupling of macromolecular synthesis from cell division in SV3T3 cells by glucocorticoids: the imposition of a G2 block.

Through a receptor-mediated process glucocorticosteroids block cell division by 20-45 hours in SV40-transformed 3T3 (SV3T3) mouse fibroblasts growing in a low calf serum (0.30% v/v) medium containing biotin. However, the rate of DNA synthesis, determined at various times after dexamethasone addition by the incorporation of radioactive thymidine into acid-insoluble material, is not inhibited by this steroid as late as 66 hours. A modest decrease is observable by 91 hours. There is also no reduction in the uptake of exogenous thymidine into acid-soluble cellular pools. Similarly, RNA synthesis and the uptake of radioactive uridine are not affected by the glucocorticoid up to 69 hours. Measurements of the amounts of cellular DNA (by the fluorescent dye, 4', 6-diamidino-2-phenylindole) and protein revealed that both macromolecules are present in elevated quantities in steroid-treated cells. (The constancy of the protein content in the nonproliferative stage suggests that protein synthesis and degradation are occurring at equal rates.) If the steroid is removed and fresh 10% calf serum medium added, cell division commences (even if nearly 90% of protein synthesis is inhibited by cycloheximide) as early as 45 minutes later such that by 2 hours the viable cell count increases by as much as 70%. Since the growth curve after recovery resembles a step function, it appears that the cells are partially synchronized by the glucocorticoid. These results demonstrate that the glucocorticoid cytostatic effect in SV3T3 cells is the result of a block not in G1, as previously thought, but in G2.

The serum growth and survival requirements of SV40-transformed 3T3 cells.

Simian virus 40-transformed 3T3 cells are dependent on serum for survival and growth. This growth activity can be separated on a pH 2 Sephadex G100 column into two fractions: a high molecular weight activity and a low molecular weight substance that has recently been characterized as containing as its major agent, biotin. To replace the remainder of the serum requirement, hormones and other growth factors were tested. Both insulin at high, nonphysiological concentrations (200 to 500 ng/ml) and transferrin (5 X 10(-8) M) stimulate the growth rate in low serum medium (0.3% v/v bovine calf serum DME) individually and, when added together, are nearly as growth enhancing as 10% serum. The need for the residual serum in this medium can be eliminated by the use of crystalline trypsin during trypsinization. Under these serum-free conditions, biotin and transferrin supplementation provide for moderately good growth (20 to 30 hr population doubling time, 1 X 10(6) cells/3.2-cm dish final cell density). Insulin addition further stimulate the growth rate (16 to 20 hr) and the final density (1.5 X 10(6) cells). Although the protein growth factors, EGF (0.5 to 1.0 ng/ml) and FGF (4 to 10 ng/ml), also appear to enhance growth individually and additively, their effects are slight and very variable. Nevertheless, the complete serum-free medium (DME supplemented with biotin, transferrin, insulin, EGF, and FGF) yields growth comparable but still inferior to 10% serum supplementation (14-versus 12-hr population doubling time, 1 to 2 X 10(6) versus 2 to 3 X 10(6) cells final cell density).

The suppression of cellular proliferation in SV40-transformed 3T3 cells by glucocorticoids.

Glucocorticosteroids, when added two hours after cell plating to SV40-transformed, 3T3 mouse fibroblasts in low serum (0.3% v/v), biotin-supplemented medium, suppress cellular proliferation by 24 hours. While some cell death probably occurs, the growth inhibition is not primarily due to cytotoxicity and cytolysis. This conclusion is supported by the following: 1) both dead and viable cell numbers are suppressed, 2) little cell debris is evident in the medium, and 3) very high concentrations of glucocorticoids do not cause an increase in the dead cell count. Furthermore, this growth suppression, which is specific for glucocorticoids since several non-glucocorticoid steroids have no inhibitory effect, is not permanent nor irreversible. Removal of the glucocorticoid and replacement with 10% serum restore rapid proliferation. Although higher concentrations (1% and 10%) of serum afford some protection against glucocorticoid inhibition, this protection is not simply a consequence of faster growth rates. SV3T3 cells can be grown in serum-free medium supplemented with biotin, transferrin, insulin, and epidermal growth factor (EGF). Under these conditions growth rates are comparable to high serum media, yet glucocorticoids are still powerful inhibitors. However, the omission of insulin from serum-free, glucocorticoid cultures does result in observable cell death and lysis. Flow microfluorometry and autoradiographic studies have determined that glucocorticoid-inhibited cells are partially blocked in G1. The proportions of S phase and G2 + M cells are greatly reduced with an accompanying accumulation of G1 cells. These results suggest that glucocorticoids regulate a biochemical step(s) in G1 which is critical for DNA initiation.

The isolation of a low molecular weight serum factor which enhances the viability of SV3T3 cells.

A serum factor which enhances the growth/viability of SV40 transformed 3T3 mouse fibroblasts, but not untransformed 3T3 cells in tissue culture has been partially purified from calf serum. The purification, which involves acidification to pH 2, chromatography on Sephadex G-100 at pH 2 followed by either Sephadex G-25 or BioGel P-2, results in material exhibiting two ninhydrin positive spots on thin layer chromatography and five to six dansylatable bands on SDS gels. This substance, termed Peak III or Serum Factor III, has been found in every serum examined thus far (including calf, rat, mouse, goat, lamb, rabbit, pig, horse, and chicken) and appears to be a low molecular weight, heat-insensitive molecule. Attempts to characterize it by chemical analyses and specific enzymatic inactivation have been either negative or inconclusive. Its biological mode of action is presently obscure. While it does not effect DNA synthesis, it does greatly increase the viability of SV3T3 cultures growing in low serum (0.15-0.30% calf serum) medium and, when added to "stationary phase" cells, restores healthy proliferation. This and evidence reported by others suggest that Peak III may affect SV3T3 cells in either overcoming a potentially lethal block in G1 or in passage through G2/M.

The effects of biotin and fatty acids on SV3T3 cell growth in the presence of normal calf serum.

The growth of SV3T3 cells in medium containing a low concentration (0.20% v/v) of normal calf serum is enhanced by the addition of biotin or certain unsaturated fatty acids. The biotin effect on the final viable cell density is 5- to 10-fold over the control and is extremely potent, exerting a saturating response at a a concentration of approximately 200 pg/ml. The optimal growth response observed with fatty acids in 5-fold over the control and requires the combination of nervonic acid, palmitoleic acid, and arachidonic acid. The fatty acids are probably not replacing the function of biotin since these two substances are additive in their growth effects.

The partial replacement of the serum growth factor requirement of SV3T3 cells by lactalbumin hydrolyzate.

Lactalbumin hydrolyzate (LH), a commercially prepared, enzymatic digest of a milk protein fraction, can partially replace the serum requirement of SV3T3 cells. In a low, but obligatory, background of calf serum (0.15% v/v), LH carses a large increase in the final cell density (5-10 x over the control) while modestly steimulating the actual growth rate. Lactalbumin hydrolyzate does not contain survival activity for SV3T3 cells and does not affect the growth of 3T3 cells. Since even prolonged exposure to pH 4 or 2 results in complete abolishment of the growth-rate stimulatory capability without affecting the capacity to increase the final cell density, it is possible that the LH growth activity may consist of two dissociable components. All of LH growth activity is water soluble, autoclavable, and resists proteolytic treatment. On Sephadex G15 growth activity appears as a single peak at the void volume but on G25 it is retained beyond the void volume as a broad, skewed peak. The relevant molecular weight range lies between 1,500 and 4,000. The putative low pH resistant material is strongly adsorbed to Dowex 1 x 2 and can be displaced from the column by a reduction in the pH. A comparison of the properties of the LH factors with those of known growth promoting agents isolated from serum and various enzymatic digests indicates that these new factors do not correspond to any of these agents.

Growth of ribonucleic acid bacteriophage f2 in a conditional putrescine auxotroph of Escherichia coli: evidence for a polyamine role in translation.

The ribonucleic acid (RNA) bacteriophage, f2, grows poorly in a conditional putrescine auxotroph during polyamine starvation. The addition of putrescine simultaneously with f2 enhances phage growth, shortens the latent period, and increases the burst size. The stimulation of f2 growth is reflected in higher rates of phage RNA and protein syntheses as measured by radioactive labeling of infected cells in the presence of rifampin. Putrescine does not affect f2 adsorption or the penetration of its RNA. Rather, in vitro assays demonstrate that in putrescine-supplemented cells more molecules of f2 replicase are made per incoming parental RNA than in polyamine-starved cultures. The ability of polyamines to stimulate the translation of a preformed messenger suggests a physiological role for these organic cations in normal protein synthesis.