Spectrum of spontaneous missense mutations causing cyclic AMP-resistance phenotypes in cultured S49 mouse lymphoma cells differs markedly from those of mutations induced by alkylating mutagens

Mutants of S49 mouse lymphoma cells resistant to cytolysis by analogs of cyclic AMP (cAMP) generally have missense mutations in the gene encoding the regulatory subunit of cAMP-dependent protein kinase. We have compared the mutations in 95 spontaneous isolates with those in 60 mutagen-induced isolates by sequence analysis of amplified cDNAs. Twenty-nine single basepair substitutions in 19 codons produced selectable phenotypes. The spontaneous mutant spectrum was dominated by a CpG transition hotspot in the codon for Arg334. This and other nearby CpG sites were found to be methylated in genomic S49 cell DNA by restriction enzyme analyses. Most of the remaining spontaneous mutants had either G-CC-G or T-AG-C transversions, which have been associated with damage caused by oxygen radicals. In contrast, the majority of mutants induced with the alkylating mutagens ethyl methanesulfonate (EMS) and N-methyl-N-nitro-N-nitrosoguanidine had G-CA-T mutations at non-CpG sites; in addition, EMS induced several A-TG-C, A-TT-A, and G-CT-A substitutions. A single ICR191-induced mutant analyzed had a unique A-TG-C lesion. A number of spontaneous and mutagen-induced isolates had closely linked double or triple substitutions, and two isolates had tandem triple substitutions.     

Prognostic factors determining response to antidepressant drugs in psychiatric out-patients and general practice

The outcome of antidepressant drug treatment was measured in 200 patients, 145 seen in psychiatric out-patient clinics and 55 in general practice, after 4 weeks of therapy. The results of the 200 patients taken together suggested that prognosis was largely determined by factors dependent on the natural history of the disorder and that clinical symptoms were unimportant, but when the results for patients in each drug group were analysed separately symptoms were more important than natural history factors. We conclude that clinical symptoms are only important predictors of response to antidepressant drugs when the patients studied are homogeneous with regard to natural history factors, particularly duration of illness.     

Neuroendocrine differentiation in endocrine and nonendocrine lung carcinomas

Bronchial carcinoids and small cell lung cancer (SCLC) are currently recognized as neuroendocrine (NE) neoplasms. However, non-SCLC (NSCLC) may also express NE properties. Paraffin-embedded sections of a comprehensive panel of 113 lung carcinomas were analyzed for the expression of three general markers common to all NE cells, namely, chromogranin A, Leu-7 and neuron-specific enolase (NSE), five specific NE secretory products, and four other tumor markers by immunohistochemistry using the sensitive avidin-biotinylated peroxidase technique. The authors were able to demonstrate the following: (1) most, but not all carcinoids and SCLCs expressed multiple NE markers in a high percentage of tumor cells; (2) up to a half of NSCLC cases contained small subpopulations of cells expressing NE in a high percentage of tumor cells; (2) up to half of NSCLC cases contained small subpopulations of cells expressing NE markers; and (3) occasional NSCLCs showed staining patterns indistinguishable from SCLC. Specifically, 7 of 77 NSCLCs expressed four or more NE markers. NE markers in NSCLCs were more commonly expressed in adenocarcinomas and large cell carcinomas and rarely in squamous cell carcinomas. For comparison, the mean number of NE markers expressed by all cases of NSCLC was 1.5, carcinoids 6.0, and SCLCs 3.8. Individual "marker counts" were not useful in categorizing lung tumors as carcinoids and SCLC versus NSCLC. Instead, 95% of the tumors were correctly classified, applying a statistical model created from staining indices of the three general NE markers (chromogranin A, Leu-7, NSE) and three other tumor markers (carcinoembryonic antigen, keratin, vimentin). Because NSCLCs with NE features might have different clinical characteristics than other NSCLCs, immunohistochemistry provides an effective manner to identify this biologically interesting subset of NSCLCs in routine paraffin sections.     

Alkaline phosphatase isoenzymes and osteocalcin in serum of normal subjects

Clinical laboratory tests are increasingly being used to evaluate individuals for osteoporosis and other metabolic bone diseases. Serum bone alkaline phosphatase (AP) [EC 3.1.3.1, orthophosphoric-monoester phosphohydrolase (alkaline optimum)] and osteocalcin are used to assess osteoblastic activity. Although methods for assessing relative amounts of AP isoenzymes continuously appear in the literature, no single method is satisfactory for quantification. Polyacrylamide gel electrophoresis with densitometric scanning combined with two-point heat inactivation was used to obtain quantitative values for AP isoenzymes. Serum bone AP concentrations correlated positively and significantly with serum osteocalcin concentrations obtained by radioimmunoassay for women. Men had significantly higher total alkaline phosphatase and bone AP than women, whereas liver AP concentrations did not differ between the two groups. Bone AP correlated negatively and significantly with age in men, but not women. Osteocalcin concentrations tended to be higher in men, but not significantly.     

Study of lymphocyte subpopulations in normal humans and patients with systemic lupus erythematosus by fractionation of peripheral blood lymphocytes on a discontinuous Ficoll gradient.

Peripheral blood lymphocytes of thirty normal volunteers and fifty-two patients with systemic lupus erythematosus were fractionated using a discontinuous (5-30 percent) Ficoll gradient. Such fractionation permitted the isolation, identification of study of null cells, T cells and B cells. Patients with inactive SLE were found to have a cell distribution and responsiveness to PHA, Con A and Pokeweed mitogen (PWM) similar to controls. In contrast, patients with active SLE showed a significant decrease in T-cell fractions as well as a relative increase in null cells, a normal distribution of B cells, a marked reduction in responsiveness to Con A, a lesser reduction to PHA and only a minor reduction to PWM. With increasing disease activity, the number of null cells increased despite lymphopenia. Spontaneous lymphocyte transformation was observed in patients with SLE. This occurred predominantly in the fractions enriched in B cells and was observed both early (0-16 hr) and late (68-72 hr) in the lymphocyte cultures. The method of discontinuous Ficoll gradients is both versatile and reproducible with good correlations between isolated lymphoid subpopulations and disease activity.     

Electrical stimulation in the treatment of osteonecrosis of the femoral head--a 1-year follow-up

To date, there is no completely satisfactory method for the treatment of osteonecrosis of the femoral head. "Conservative" management leads to a high failure rate, and surgical results have been inconsistent and disappointing. The study described in this article seeks to evaluate the role of electrical stimulation in conjunction with decompression and bone grafting. A total of 82 hips have been included to date, of which 42 with a minimum follow-up of 1 year have been evaluated. Pain relief and improved function have been noted in the majority of hips operated on, both with and without electrical stimulation. Careful radiologic assessment using a comprehensive new method of evaluation has shown some degree of progression in the majority of cases, however. No effects of electrical stimulation per se have been demonstrated to date. It must be emphasized that this is a preliminary report with a minimum follow-up of 1 year. Final conclusions await the completion of the study, when all patients have been followed for a minimum of 3 years.     

Isolation of the non-glycosylated proteins of desmosomes and immunolocalization of a third plaque protein: desmoplakin III.

The cytoplasmic plaque of the spot desmosome or macula adhaerens mediates the attachment of bundles of intermediate filaments to the plasma membrane. We have isolated from a bovine epidermal desmosome preparation a fraction that is highly enriched in the non-glycosylated desmosomal proteins. Plastic-embedded and thin-sectioned high-speed pellets of this fraction reveal closely packed filaments that resemble plaque regions of the low pH whole desmosome preparation from which they are derived. NaDodSO4/polyacrylamide gel electrophoresis reveals four major, non-glycosylated proteins of 240, 210, 81, and 77 kDa. In agreement with a previous study, we find the 240- and 210-kDa proteins (desmoplakins I and II) to be closely related, whereas the 81- and 77-kDa proteins are unique. This is shown both immunologically and by one-dimensional proteolytic peptide mapping. Monospecific, polyclonal rabbit antibodies were prepared against the 81-kDa protein and used, in conjunction with protein A-complexed colloidal gold particles (PAG), to immunolocalize this antigen on ultrathin sections of bovine muzzle epidermis. On antibody-labeled sections, PAG particles were associated principally with the desmosomal cytoplasmic plaque. Sections exposed to preimmune serum showed little or no labeling. We conclude that the 81-kDa protein, like the 240/210-kDa protein family, is one of the major components of the desmosomal plaque. We designate it as "desmoplakin III." The location of the 77-kDa protein remains to be definitively established.     

EFFECT OF NITROGLYCERNE-INDUCED HYPOTENSION ON CANINE SPINAL CORD BLOOD FLOW

Twenty-four mongrel dogs were anaesthetized with pentobarbitoneand morphine sulphate. Neuromuscular blockade was achieved usingpancuronium. Spinal cord blood flow was measured using the radionuclidemicrosphere and hydrogen washout methods before, during, andfollowing nitroglycerine-induced hypotension. Heart rate, meanarterial pressure, cardiac output, pulmonary capillary wedgepressure, and acid-base balance were determined with each measurement.Mean arterial pressure was reduced by 50%. Spinal cord bloodflow, as measured by the microsphere method, increased duringthe period of hypotension, whereas values obtained using thehydrogen washout method were not significantly different fromthose at normotension. No significant change in spinal cordblood flow was detected by either method after the applicationof spinal distraction. Nitroglycerine acts predominantly onvenous capacitance vessels and it is postulated that perfusionpressure, and therefore flow, is maintained despite a reductionin arterial pressure. Presented in part at the Anual Meeting of the American Societyof Anesthesiologists, October 1985, San Francisco, California. ^*Shackleton Department of Anaesthetics, Southampton GeneralHospital, Shirley, Southampton, Hants SO9 4XY. Section of Orthopedic Surgery, Madison, Wisconsin.     

The Effects of Magnetic Resonance Imaging on Implantable Pulse Generators

The effects of magnetic resonance imaging were assessed on four dual chamber and two single chamber pulse generators. The tests were performed with a resistive, water-cooled magnet operating at 0.15 T. The 6.4-MHz radiofrequency (RF) field was operated at a maximum power of 1,000 watts with a period adjusted from 130 to 500 ms. Reed switch closure occurred in all six pulse generators tested when placed near the entrance of the magnetic resonance imaging scanner, and the generators reverted to asynchronous operation unless programmed to the "magnet off" mode. None of the pulse generators exhibited any alterations in programmed parameters or in the ability to be reprogrammed after RF pulsing. When the RF field was turned on, there was no change in the asynchronous paced cycle length in four pulse generators; however, during RF scanning there was rapid cardiac stimulation at the RF pulse period in one single chamber and one dual chamber pulse generator.