Polyglucosan body density in the aged mouse hippocampus is controlled by a novel modifier locus on chromosome 1.

Aging can be associated with the accumulation of hypobranched glycogen molecules (polyglucosan bodies, PGBs), particularly in astrocytes of the hippocampus. While PGBs have a detrimental effect on cognition in diseases such as adult polyglucosan body disease and Lafora disease, the underlying mechanism and clinical relevance of age-related PGB accumulation remains unknown. Here, we have investigated the genetic basis and functional impact of age-related PGB accumulation in 32 fully sequenced BXD-type strains of mice which exhibit a 400-fold variation in PGB burden in 16-18 month old females. We mapped a major locus controlling PGB density in the hippocampus to chromosome 1 at 72-75 Mb (linkage of 4.9 -logP), which we defined as the Pgb1 locus. To identify potentially causal gene variants within Pgb1, we generated extensive hippocampal transcriptome datasets and identified two strong candidate genes for which mRNA correlates with PGB density-Smarcal1 and Usp37. In addition, both Smarcal1 and Usp37 contain non-synonymous allele variations likely to impact protein function. A phenome-wide association analysis highlighted a trans-regulatory effect of the Pgb1 locus on expression of Hp1bp3, a gene known to play a role in age-related changes in learning and memory. To investigate the potential impact of PGBs on cognition, we performed conditioned fear memory testing on strains displaying varying degrees of PGB burden, and a phenome-wide association scan of ~12,000 traits. Importantly, we did not find any evidence suggesting a negative impact of PGB burden on cognitive capacity. Taken together, we have identified a major modifier locus controlling PGB burden in the hippocampus and shed light on the genetic architecture and clinical relevance of this strikingly heterogeneous hippocampal phenotype.

Monodrug efficacies of sulfonamides in prophylaxis for Pneumocystis carinii pneumonia.

A remarkably high rate of adverse events is associated with the use of trimethoprim-sulfamethoxazole in patients with human immunodeficiency virus type 1 infection. We examined the efficacies of sulfonamides alone in the prevention of Pneumocystis carinii pneumonitis, with the assumption that at least some of the adverse events with the drug combination might be due to trimethoprim. With the immunosuppressed rat model, eight sulfonamides were studied at 100, 10, and 1.0 mg/kg/day (10 rats per dosage and drug). P. carinii infection was prevented in all animals (100%) receiving dosages of as little as 1.0 mg of sulfamethoxazole, sulfamethoxypyridazine, and sulfadimethoxine per kg per day, as little as 10 mg of sulfameter, sulfachlorpyridazine, and sulfaquinoxaline per kg per day; and 100 mg of sulfaguanidine and sulfanilamide per kg per day. These studies suggest that a sulfonamide, such as sulfamethoxazole, might provide effective prophylaxis for P. carinii pneumonitis without trimethoprim.

Relative potency of 10 drugs with anti-Pneumocystis carinii activity in an animal model.

Several drugs have been shown to have anti-Pneumocystis carinii activity in clinical trials. Because of the large number of patients required, no more than 3 drugs can be compared for efficacy in human studies. However, the experimental animal model for P. carinii pneumonitis is remarkably similar to the human disease and was used to compare 10 drugs for the relative potency against this infection. All drugs were compared at doses known to prevent the pneumonitis in > 80% of animals and at one-tenth of this dose. Drugs effective at the lowest dose were further tested at one-hundredth the original doses, and drugs ineffective were retested at 10 and 100 times the original dose. Trimethoprim-sulfamethoxazole was the most effective drug, with azithromycin-sulfamethoxazole and clarithromycin-sulfamethoxazole next most effective. Intravenous pentamidine and clindamycin-primaquine were the least effective. Atovaquone, sulfadoxine-pyrimethamine, erythromycin-sulfisoxazole, PS-15, and dapsone-trimethoprim had intermediate activity.

Anti-Pneumocystis carinii activity of PS-15, a new biguanide folate antagonist.

A newly synthesized biguanide inhibitor of dihydrofolate reductase in Plasmodium species was evaluated for its anti-Pneumocystis carinii activity. The compound N-3-(2,4,5-trichlorophenoxypropyloxy)-N'-(1-methylethyl)imidoca rbonimidic diamide hydrochloride, designated PS-15, was administered prophylactically and therapeutically to immunosuppressed rats latently infected with P. carinii. Doses of 5 and 25 mg of PS-15 per kg of body weight per day given orally during 7 weeks of dexamethasone immunosuppression prevented P. carinii infection in all (100%) 19 rats given the drug, while 6 of 9 (67%) untreated control rats developed P. carinii pneumonitis. A single weekly dose of 50 mg of PS-15 per kg also prevented the infection in all 10 rats. P. carinii pneumonitis was established after 4 weeks of immunosuppression and was then treated orally for 3 weeks with 25, 5, and 1 mg of PS-15 per kg/day. Complete resolution of the infection occurred in all (100%) 10 rats given 25 mg of PS-15, 6 of 9 (67%) rats given 5 mg of PS-15, and 6 of 8 (75%) rats given 1.0 mg of PS-15 per kg per day and in all (100%) 9 rats treated with trimethoprim-sulfamethoxazole. PS-15 was well tolerated at all doses. Because drug studies in the P. carinii rat model have been highly predictable of the effects of drugs on the disease in humans, these experiments suggest that PS-15 may have promise as a drug for the treatment of P. carinii pneumonitis in humans.

Synergistic anti-Pneumocystis carinii effects of erythromycin and sulfisoxazole.

Pneumocystis carinii pneumonitis was effectively prevented in 90% of immunosuppressed rats by the administration of 100 mg of erythromycin and 300 mg/kg/day of sulfisoxazole. All of the untreated control and erythromycin-treated animals developed the infection and 80% of rats given sulfisoxazole alone had the pneumonitis. A similar pattern of response occurred when the drugs were used therapeutically for rats with established P. carinii pneumonitis. The erythromycin and sulfisoxazole ratio of 1:3 was the most effective of several dose combinations tested. The established safety record from three decades of clinical use of this drug combination plus the broad spectrum of coverage for other causes of diffuse pneumonitis such as Chlamydia, Mycoplasma, and Legionella warrant further study of erythromycin-sulfisoxazole in AIDS patients.

Characterization of beta-endorphin in human pituitary by fast atom bombardment mass spectrometry of trypsin-generated fragments.

A novel mass spectrometric method possessing a high level of structural specificity is described for characterization in biological fluids and tissues of endogenous beta-endorphin of the human amino acid sequence (beta h-EP). The method is based upon purification of tissue extracts by an RP-HPLC gradient, followed by trypsinolysis of that particular HPLC fraction corresponding to the elution time of synthetic beta h-EP. The tryptic digest of that endogenous beta h-EP fraction was purified further by a second RP-HPLC gradient. A unique tryptic fragment selected from the second gradient was analyzed by fast atom bombardment mass spectrometry and B/E linked-field scan MS/MS techniques to provide molecular weight and amino acid sequence-determining fragment ion information, respectively, of that fragment. Collectively, these independent analytical methodologies provided unequivocal structure evidence for the presence of endogenous beta h-EP in human pituitary. The method was established first by utilizing synthetic beta h-EP to optimize experimental parameters, and then applied to the analysis of beta h-EP in post-mortem human pituitary extracts. The suitability of the present method for semi-quantitation of tissue extracts is also demonstrated. The corresponding detection limit of the synthetic beta h-EP was 90 fmol, and human pituitary contained 1.5 pmol of beta h-EP mg-1 protein. The method can be extended readily to the analysis of beta-endorphin derived from other species and tissues.

Effects of orthodontic force on methionine enkephalin and substance P concentrations in human pulpal tissue.

Orthodontic treatment typically involves intermittent periods of patient discomfort caused by forces on the teeth and adjacent tissues. This sensation of discomfort presumably is caused by the action of neuropeptides in the peripheral and central nervous systems. The effects of orthodontic force on the concentrations of two endogenous neuropeptides, methionine enkephalin (ME) and substance P (SP), measured as immunoreactive-methionine enkephalin (ir-ME) and immunoreactive-substance P (ir-SP), in human tooth pulp were evaluated in 20 patients from whom premolars were extracted before orthodontic treatment. The teeth from nine controls were not subjected to a force, whereas the 11 experimental patients had force applied to their maxillary premolars either by a transpalatal spring ligature or, in one case, by a headgear. The ligature applied a force within the range of 120 to 245 gm; the headgear applied 600 gm. Reversed-phase high-performance liquid chromatography (RP-HPLC) was used to purify the neuropeptides in the pulp homogenate, and radioimmunoassay (RIA) was used to quantify ir-ME and ir-SP in their appropriate HPLC fractions. (1) Females subjected to orthodontic force had significantly greater ir-ME concentrations than males. (2) The ir-SP concentration decreased significantly from the first to the third tooth extracted, then increased from the third to the fourth tooth. (3) Ir-SP and ir-ME concentrations are positively intercorrelated. The association was highest in the first tooth extracted from controls; surgical extraction decreased the correlation, although it continued to be positive. (4) The concentrations of ir-ME and ir-SP each correlated negatively with the magnitude of the orthodontic force and that correlation was enhanced when the value of the force was log-transformed.

Immunoreactive evidence of beta-endorphin and methionine-enkephalin-Arg-Gly-Leu in human tooth pulp.

For the first time, beta-endorphin-like immunoreactivity (BE-LI) has been measured in human tooth pulp. Separation of peptides from pulp tissue was achieved by acid extraction followed by chromatographic separation through a Sep-Pak disposable cartridge. Reversed phase high performance liquid chromatographic (RP-HPLC) was performed on the peptide-rich fractions for further peptide separation. Radioreceptor assay (RRA) data of the HPLC fractions was used to construct a profile of opioid-receptor active peptides. Radioimmunoassay (RIA) data provided further information. Following acute mechanical stress, a monotonic decrease in BE-LI concentrations was evident according to a four bicuspid extraction order.

Purification, characterization, and localization of neuropeptides in the cornea.

The immunologically detected neuropeptides methionine enkephalin (ME), substance P (SP), beta-endorphin (beta-End), and alpha-melanocyte stimulating hormone (alpha-MSH) were purified from bovine corneal extracts by gradient, followed by isocratic, reversed phase-high performance liquid chromatography (RP-HPLC) and characterized, after both chromatographic steps, by radioimmunoassay (RIA). Immunologically detected ME and SP were purified from canine corneal extracts by gradient RP-HPLC and characterized by RIA. An anatomical study of the bovine cornea separated the cornea into an epithelium-enriched and a stroma-enriched portion. After gradient RP-HPLC, RIA demonstrated that all the ME-like immunoreactivity was located in the corneal epithelium, whereas the SP-like immunoreactivity was distributed between the stroma and epithelium in an approximate two-to-one ratio.

Methionine-enkephalin peptides in human teeth.

The presence of the free opioid pentapeptide methionine enkephalin (ME) and of ME-containing peptide(s) was established firmly in decalcified, depulped human teeth by using a combination of methods including RP-HPLC, radioimmunoassay, radioreceptorassay, trypsin, carboxypeptidase B, fast atom bombardment mass spectrometry, and MS/MS methodology. Positive structural identification of ME was made with mass spectrometry. Those data demonstrate the presence of the preproenkephalinergic A system in the human trigeminal sensory termini.

Opioid peptide profile in human pituitary.

The comprehensive metabolic profile of endogenous opioid peptides is established here for human pituitary for the first time. Sixteen human pituitaries, obtained postmortem, were analyzed individually by gradient reversed-phase high-performance liquid chromatography together with a radio-receptor assay with [3H]etorphine as ligand. This combination was used to detect opioid receptor activity. The 16 assay profiles were sufficiently consistent for a composite of them to serve as a comparative basis for other studies on the pathophysiology of the human pituitary. To demonstrate one selected comparison, we present data on a distinctively different profile of opioid receptor activity in the pituitary of one patient who died from a drug overdose.

Estradiol is responsible for reduced renal prostaglandin dehydrogenase activity in female rats.

The contribution of sex steroids to sex-related differences in renal prostaglandin dehydrogenase activity and urinary prostaglandin excretion was examined in 7-8-week-old male and female rats subjected to sham-operation or gonadectomy at 3 weeks of age. Rats were injected subcutaneously twice over a 6-day interval with vehicle (peanut oil, 0.5 mg/kg) or with depot forms of testosterone (10 mg/kg), estradiol (0.1 mg/kg), progesterone (5 mg/kg), or with estradiol and progesterone combined (0.1 and 5 mg/kg). After the second injection, 24-h urine samples were collected for prostaglandin measurement by radioimmunoassay; the rats were killed, and renal and pulmonary prostaglandin dehydrogenase activities were determined by radiochemical assay. Renal prostaglandin dehydrogenase activity was 10-times higher in intact male rats than in intact females. Gonadectomy increased renal prostaglandin dehydrogenase activity 4-fold in females, but had no effect in males; estradiol, alone or combined with progesterone, markedly suppressed renal prostaglandin dehydrogenase activity in both sexes, while testosterone or progesterone alone had no effect. Pulmonary prostaglandin dehydrogenase did not differ between the sexes and was unaffected by gonadectomy or sex-steroid treatment. Intact female sham-operated rats excreted 70-100% more prostaglandin E2, prostaglandin F2 alpha, and 6-keto-prostaglandin F1 alpha in urine than did males; gonadectomy abolished the difference in urinary prostaglandin E2 excretion. Estradiol decreased urinary prostaglandin E2 in females but not in males; treatment with other sex steroids did not alter urinary prostaglandin excretion.