Modulation of protein splicing of the Saccharomyces cerevisiae vacuolar membrane ATPase intein

Protein splicing of the Saccharomyces cerevisiae vacuolar membrane ATPase intein involves four highly coordinated reactions that result in precise cleavage and formation of peptide bonds. In this study, we investigated the roles of the last N-extein residue (-1 residue) and the intein penultimate residue in modulating splicing reactions. Most of the 20 amino acid substitutions at the -1 position had no effect on overall protein splicing but could lead to significant accumulation of thioester intermediates when splicing was blocked by mutation. A subset of -1 substitutions attenuated the initiation of protein splicing and enabled us to demonstrate in vitro splicing of a mesophilic intein containing all wild-type catalytic residues. Substitutions involving the intein penultimate residue allowed modulation of the branch resolution and C-terminal cleavage reaction. Our data suggest that the N-S acyl rearrangement, which initiates splicing, may also serve as the rate-limiting step. Through appropriate amino acid substitutions, we were able to modulate splicing reactions in vitro by change in pH or temperature or addition of thiol reagents. Both insertion and deletion were tolerated in the central region of the intein although splicing or structure of the intein may have been affected.     

Gamma irradiation and ozone treatment for inactivation of Escherichia coli O157:H7 in culture media

A study was conducted to investigate the reduction and elimination of Escherichia coli O157:H7 by the effects of gamma irradiation and ozone treatment. Log phase cells were found to be more sensitive to gamma irradiation than stationary phase cells. E. coli O157:H7 was found to be considerably more resistant to irradiation at -18 degrees C than at 20 degrees C. The D values for this organism for treatment with ozone in tryptic soy agar were higher than those for treatment with ozone in phosphate buffer. Gamma irradiation at a dose of 1.5 kGy or ozone treatment at a concentration of 3 to 18 ppm for 20 to 50 min was required to assure the elimination of E. coli O157:H7.     

The astrocytic response to afferent activity blockade in chick nucleus magnocellularis is independent of synaptic activation, age, and neuronal survival

Astrocytes in nucleus magnocellularis (NM) of the chick respond to afferent activity blockade with increased immunoreactivity for glial fibrillary acidic protein (GFAP). NM neurons respond to the same manipulations with reduced protein synthesis, ribosomal dissociation, and subsequent death of a subset of these neurons. In the present study, we sought to evaluate the relationship between these neuronal and glial responses and to determine if similar activity-dependent mechanisms mediate them. We first examined the anatomical relationship between NM neurons and astrocytic processes by electron microscopy and GFAP immunostaining. Both methods showed that NM neurons deprived of activity for 6 hr were apposed by more glial processes than active NM neurons. However, we found no preferential positioning of GFAP-immunoreactive processes near neurons of the dying or surviving populations, and there were no differences in glial process apposition to dying versus surviving neurons at the EM level. To determine whether the astrocytic response is similar to the neuronal response in age dependence, GFAP immunoreactivity was analyzed in adult chickens following unilateral afferent activity blockade. Unlike the neuronal response to activity blockade, the astrocytic response is equally strong in adult animals. These results imply an independence of the neuronal and astrocytic responses to activity blockade, raising the possibility that these two cell types may be responding to different activity-related signals. This possibility was tested using an in vitro slice preparation. Unilateral stimulation of NM was provided in three ways: orthodromically, antidromically, and orthodromically in a low-calcium medium. The regulation of astrocytic GFAP immunoreactivity by these manipulations of activity was then analyzed. The results of these experiments show that, unlike neuronal protein synthesis, astrocytic GFAP immunoreactivity can be suppressed by either presynaptic or postsynaptic neuronal activity. Therefore, the astrocytes and neurons are regulated by different activity-dependent signals and, by the present measures, their responses to activity blockade appear independent of one another.     

The comparative anatomical study of the parietal region of the skull of the Korean native goat (Capra hircus)

In the skull of the Korean native goat, the parietal region was classified into four types by the degree of the fusion of the bones, the os interparietale, the os parietale and the squama occipitalis of the os occipitale, and the structural variations of these fusions. The fusion appeared first in the sutura interparietoparietalis and that of the sutura sagittalis of both ossa parietalia was followed. There was no fusion between the os parietale and the squama occipitalis of the os occipitale. These results suggest that the os interparietale developed independently but fused to the os parietale after birth, and the os parietale were developed as paired bones in prenatal life and then fused together according to age.     

Applying the realized probability of dying to cancer survival

Age comparisons of survival in cancer cohorts generally utilize relative survival rates, which are based on indicators of the probability of survival for a given number of years after diagnosis. Cancer relative survival rates for the same number of years tend to decline as age at diagnosis increases. However, the same number of years of survival reflects higher relative longevity at older ages than at younger ages. The realized probability of dying (RPD) is a survival measure that expresses individual survival time after diagnosis relative to the survival distribution of an age-, race-, and sex-specific reference population, in effect weighing individual survival time more heavily as age at diagnosis increases. The purpose of this study was to apply the RPD as a survival measure in cancer epidemiology. Two cohorts of cancer patients, white males with prostate cancer and white females with breast cancer, aged 55 years and over at diagnosis, were followed for 15 years. Although older subjects survived less time after diagnosis than younger subjects, they achieved more favorable RPD values. We present survival analysis methods for analyzing the RPD in this population, an approach not previously used with this measure. The implications for use of the RPD in cancer epidemiology are discussed.     

Persistent resetting of the cerebral oxygen/glucose uptake ratio by brain activation: evidence obtained with the Kety-Schmidt technique

Global cerebral blood flow (CBF), global cerebral metabolic rates for oxygen (CMRO2), and for glucose (CMRglc), and lactate efflux were measured during rest and during cerebral activation induced by the Wisconsin card sorting test. Measurements were performed in healthy volunteers using the Kety-Schmidt technique. Global CMRO2 was unchanged during cerebral activation, whereas global CBF and global CMRglc both increased by 12%, reducing the molar ratio of oxygen to glucose consumption from 6.0 during baseline conditions to 5.4 during activation. Data obtained in the period following cerebral activation showed that the activation-induced resetting of the relation between CMRglc and CMRO2 persisted virtually unaltered for > or = 40 min after the mental activation task was terminated. The activation-induced increase in cerebral lactate efflux measured over the same time period accounted for only a small fraction of the activation-induced excess glucose uptake. These data confirm earlier reports that brain activation can induce resetting of the cerebral oxygen/glucose consumption ratio, and indicate that the resetting persists for a long period after cerebral activation has been terminated and physiologic stress indicators returned to baseline values. Activation-induced resetting of the cerebral oxygen/glucose uptake ratio is not necessarily accounted for by increased lactate production from nonoxidative glucose metabolism.     

Determination of free acetaldehyde in total blood for investigating the effect of aspartate on metabolism of alcohol in mice

The cerebral cortex of anaesthetised 2- to 12-day-old rats was superfused with artificial cerebrospinal fluid containing 100 mM acetate substituted for chloride to condition the brain for spreading depression (SD). After such superfusion, the earliest SD-like events were found at day 9 and full blown SD at day 10, whereas in the unconditioned brain the first SD occurred between days 12 and 15. Acetate conditioning of the cerebral cortex may be used to unmask neuronal and glial properties that are hidden in early stages of development.     

Differences in receptor binding of LDL subfractions

Differences in low density lipoprotein (LDL) receptor-binding affinity among LDL particles of different size were examined in competitive binding assays in human skin fibroblasts and LDL (d = 1.020 to 1.050 g/mL) from subjects with a predominance of large (> or = 272 A), medium (259 to 271 A), and small (< or = 257 A) LDL. Among 57 normolipidemic subjects with LDL cholesterol (-C) levels < 160 mg/dL, binding affinity was reduced by 16% in those with predominantly large LDL and by 14% in those with small LDL compared with most subjects who had a predominance of medium-size LDL and in all LDL size subgroups in 66 subjects with LDL-C > or = 160 mg/dL. Differences in LDL receptor-binding affinity were further investigated by using LDL density subfractions (I, d = 1.026 to 1.032 g/mL; II, d = 1.032 to 1.038 g/mL; and III, d = 1.038 to 1.050 g/mL) from three subjects with predominantly large (pattern A) and small (pattern B) LDL particles. The binding affinity (Kd) of LDL-II was similar for patterns A and B (9.2 +/- 1.4 and 9.4 +/- 0.7, respectively) and 30% lower in LDL-III from both groups (P < .05). The binding affinity of LDL-I in pattern A (12.6 +/- 1.5 micrograms/mg) was lower (P < .05) than that in LDL-II and LDL-I from pattern B (8.0 +/- 2.4 micrograms/mg). After incubation with a monoclonal antibody that specifically blocked the LDL receptor-binding domain of apoE, LDL-I from two pattern B subjects showed substantially lower binding affinity (Kd = 20.0 and 19.2 micrograms/mg) than in pattern A (Kd = 13.2 and 14.2 micrograms/mg), a result consistent with our finding of a higher apoE content in pattern B LDL-I (P < .001). Thus, factors associated with variations in particle size and apoE content in LDL subclasses in normolipidemic subjects contribute to the differences in LDL receptor binding that may result in differing metabolic behavior in vivo.     

Identification of a BRCA1-associated kinase with potential biological relevance

A biochemical approach was used to identify proteins which interact with human BRCA1. Through this work, a kinase activity which co-purifies with BRCA1 has been identified. This kinase activity, which phosphorylates BRCA1 in vitro, was originally identified in Sf9 insect cells but is also present in cells of human origin including breast and ovarian carcinoma cell lines. The BRCA1 kinase activity in vitro is associated with a fragment of BRCA1 encompassing amino acids 329-435. This peptide is also phosphorylated in various human cell lines. A computer-assisted sequence analysis revealed that this peptide was a potential substrate for phosphorylation by PKA, PKC, or CKII. However, phosphorylation by these kinases could not be demonstrated in vitro indicating the presence of another kinase activity. Phosphorylation in vitro requires a minimal domain of BRCA1 encompassing amino acids 379-408. Notably, deletion of this minimal domain abolishes growth suppression by BRCA1 indicating that this domain, as well as phosphorylation within this domain, may be important for BRCA1 function.