John R. Sheppard

Competitive inhibition of the transport of nucleosides, hypoxanthine, choline and deoxyglucose by theophylline, papaverine and prostaglandins

Summary Theophylline, papaverine and prostaglandins E 1 and F 2∝ competitively inhibit the transport of uridine, thymidine, hypoxanthine, choline and deoxyglucose by cultured Novikoff rat hepatoma cells. The transport processes are maximally inhibited immediately upon addition of the drugs and the effects are readily reversed by their removal.

Glial fibrillary acidic protein and Alzheimer's disease

A major protein associated with Alzheimers disease (AD) was detected by an electrophoretic study of temporal cortex obtained at autopsy from patients affected with AD, non-AD dementia, and normal controls matched for age and sex. A markedly increased amount of a 50,000 dalton molecular weight protein, which has been identified as glial fibrillary acidic protein (GFAP), was observed in the crude nuclear fraction of temporal cortex from AD patients. These electrophoretic data may reflect the presence of GFAP immunopositive astrocytic processes that have been shown by immunocytologic methods to infiltrate the neurofibrillary tangles that characterize AD.

β-Adrenergic response in mouse CNS reaggregate cultures

Abstract Primary reaggregate cultures from 17-day embryonic mouse brains were prepared and tested for β-adrenergic response. Isoproterenol stimulated the production of cAMP 3- to 4-fold over basal levels after 10 days in culture. The response to isoproterenol was rapid, reaching a maximum 1–2 min after stimulation, and was blocked by propranolol. The β-adrenergic receptor was characterized in membranes prepared from reaggregates by measurement of specific binding of [125I]iodohydroxybenzylpindolol (IHYP). The Kd for IHYP was 100–300 pM and did not change as a function of time in culture. A maximum receptor density of approximately 80–110 fmol/mg membrane protein was found. Both β-adrenergic receptors and response appeared at times in culture, which were approximately equivalent to birth.

Circadian temperature rhythm and circadian-circaseptan (about 7-day) aspects of murine death from malaria.

Abstract About-7-day (circaseptan) and circadian rhythms were sought and found in host-parasite relations of mice infected with Plasmodium berghei. Five inbred male DBA mice, about 18 weeks of age, were implanted with transsensors for temperature telemetry. Core temperature, monitored every 10 min for 3 days before the intravenous or intraperitoneal inoculation of 105 infected erythrocytes and thereafter until death, was analyzed by cosinor. A statistically highly significant circadian rhythm exhibited similarly synchronized acrophases. Core temperatures on the days immediately after malarial infection were mostly within the range of temperatures observed before injection. A mesor-hypothermic stage preceded death by several days. In a second study, 24 male BALB/c and 42 male DBA mice, 12 weeks of age, housed in three rooms on different regimens of light and darkness (alternating at 12-hr intervals), staggered by 8 hr, were inoculated ip with 104 infected erythrocytes, one-half at noon, the other half at midnight, within 0.5 hr of blood withdrawal. Thus, one endeavored to cover six circadian host stages (02, 06, 10, 14, 18, and 22 hr after light-on). At 54 and 51% overall mortality (irrespective of inoculation time), a circadian rhythm in susceptibility to malaria was demonstrated in these mice by the single cosinor fit of a 24-hr period (P < 0.003 and < 0.020, respectively). The single cosinor fit of a 7-day period further demonstrated a circaseptan rhythm (P = 0.014) in the mortality of both strains following the inoculation of P. berghei. The acrophase (360° = 168 hr) was at −325° from the inoculation time with 95% confidence limits extending from −276 to −378°. Such predictable time relations of P. berghei to its murine host await the exploration of mechanisms underlying the circadian and infradian (7-day) rhythmicities here demonstrated and quantified with their uncertainties.

Effects of caffeine withdrawal on isoproterenol-stimuiated cyclic adenosine monophosphate

The effect of short‐term caffeine withdrawal on lymphocyte beta‐adrenoceptor sensitivity was studied in seven men who had been drinking coffee daily for at least 3 mo before the study. The ratio of cyclic adenosine monophosphate production after in vitro incubation with isoproterenol and a blank was used to assess receptor sensitivity. There was a reduction in sensitivity to isoproterenol but not to prostaglandin E1 3 days after caffeine withdrawal. The mechanism of this effect is not clear, but may represent a biochemical correlate of caffeine‐withdrawal syndrome

Reconstitution of the Gs protein from B16 melanoma clones of high and low experimental metastatic potential into S49 cyc-membranes.

The ability of a series of B16 melanoma clones to form experimental lung metastases in syngeneic mice has been shown to correlate positively with adenylate cyclase activity. (Sheppard et al, Int. J. Cancer 37 (1986) 713-722). To begin to identify the components of the adenylate cyclase complex that account for enhanced enzyme activity in highly metastatic tumor populations, cholate extracts containing the GTP-binding protein GS from B16 melanoma clones of different metastatic capacities were reconstituted with membranes prepared from S49 cyc-, a variant lymphoma cell line that lacks GS function. The results revealed that extracts from a highly metastatic B16 clone (F10-C23) reconstituted significantly greater adenylate cyclase activities in S49 cyc- membranes than parallel preparations from a B16 clone (F1-C29) of low metastatic capacity. The data suggest that aberrations in GS function may contribute to the heightened responsiveness of adenylate cyclase observed in B16 melanoma clones of increased metastatic potential.

Elution of the regulatory subunit of cAMP-dependent protein kinase Type I isozyme derived from epididymal fat within the Type II isozyme chromatographic peak

No cAMP-dependent protein kinase activity is found upon DEAE-cellulose chromatography of mouse fat extracts at the low salt concentration characteristic of the Type I isozyme. The RI detected in fat extracts by photoincorporation of the analog, 8-N3 [32P]cAMP, elutes within the high salt Type II isozyme peak. The multiple charge variants of this photolabeled RI which can be resolved by two-dimensional gel electrophoresis are similar to those of the histoptypically-related cultured cells, SV3T3 and 3T6, which do contain Type I kinase isozyme activity peaks. This high salt-eluting RI may be part of a Type I holoenzyme whose elution properties are altered by interactions with other substances present in the extract.

The induction and detection of diploid spermatozoa in Gallus domesticus.

Colcemid at the dose level of 0.37 mg/kg/day was injected intraperitoneally to 3 sexually active chicken males for 3 consecutive days. 10-12 days after the first colcemid injection, 14-25% of the sperm population in the semen samples from the treated males was found to be diploid in DNA content by flow microfluorometric analysis. Cytogeneic and developmental analyses on early embryos indicate that, during the process of spermatogenesis, the male germ cells are most susceptible to colcemid treatment 10-12 days prior to the maturation of the spermatozoa which is equivalent to the primary through secondary spermatocyte stages in chicken males. By the application of an extremely unequal chromosomal translocation as a cytological marker of parentage, it is confirmed that the diploid sperm induced are capable of uniting with a normal haploid or diploid egg to produce a triploid or tetraploid zygote.

Expression of a specific protein in spontaneously metastatic fibrosarcoma cell lines and its enhanced synthesis by growth on laminin or fibronectin

A panel of tumor cell lines and clones was generated by selecting for different metastatic capacity in 3AM fibrosarcoma cells. These cell lines were exposed to substrata of various purified extracellular matrix (ECM) proteins and labeled in culture with [35S]methionine. Following analysis by two-dimensional polyacrylamide gel electrophoresis the profiles of total cellular proteins produced by the cell lines displaying different phenotypes were examined. The presence of a specific protein, p54, was related to the cellular metastatic potential, and the synthesis of p54 was influenced by growth on extracellular matrix components. The amount of p54 was minimal and non-inducible in tumor cell lines exhibiting two different phenotypes: (1) experimentally metastatic (EM) and (2) transformed, non-tumorigenic (NTT) cell types. In contrast, all of the five different cell lines capable of both spontaneous and experimental metastasis (SEM), produced p54 either constitutively or through induction by growth on ECM protein substrata of either laminin of fibronectin, but not collagen type IV. These data suggest that the p54 protein may be a unique biochemical marker associated with spontaneous metastatic cell types.