BOLD-fMRI of PTZ-induced seizures in rats.

PURPOSE: To develop a non-invasive method for exploring seizure initiation and propagation in the brain of intact experimental animals. METHODS: We have developed and applied a model-independent statistical method--Hierarchical Cluster Analysis (HCA)--for analyzing BOLD-fMRI data following administration of pentylenetetrazol (PTZ) to intact rats. HCA clusters voxels into groups that share similar time courses and magnitudes of signal change, without any assumptions about when and/or where the seizure begins. RESULTS: Epileptiform spiking activity was monitored by EEG (outside the magnet) following intravenous PTZ (IV-PTZ; n=4) or intraperitoneal PTZ administration (IP-PTZ; n=5). Onset of cortical spiking first occurred at 29+/-16 s (IV-PTZ) and 147+/-29 s (IP-PTZ) following drug delivery. HCA of fMRI data following IV-PTZ (n=4) demonstrated a single dominant cluster, involving the majority of the brain and first activating at 27+/-23s. In contrast, IP-PTZ produced multiple, relatively small, clusters with heterogeneous time courses that varied markedly across animals (n=5); activation of the first cluster (involving cortex) occurred at 130+/-59 s. With both routes of PTZ administration, the timing of the fMRI signal increase correlated with onset of EEG spiking. CONCLUSIONS: These experiments demonstrate that fMRI activity associated with seizure activity can be analyzed with a model-independent statistical method. HCA indicated that seizure initiation in the IV- and IP-PTZ models involves multiple regions of sensitivity that vary with route of drug administration and that show significant variability across animal subjects. Even given this heterogeneity, fMRI shows clear differences that are not apparent with typical EEG monitoring procedures, in the activation patterns between IV and IP-PTZ models. These results suggest that fMRI can be used to assess different models and patterns of seizure activation.

Mutant beta-spectrin 4 causes auditory and motor neuropathies in quivering mice.

The autosomal recessive mouse mutation quivering (qv), which arose spontaneously in 1953, produces progressive ataxia with hind limb paralysis, deafness and tremor. Six additional spontaneous alleles, qvJ, qv2J, qv3J, qv4J, qvlnd and qvlnd2J, have been identified. Ear twitch responses (Preyer's reflex) to sound are absent in homozygous qv/qv mice, although cochlear morphology seems normal and cochlear potentials recorded at the round window are no different from those of control mice. However, responses from brainstem auditory nuclei show abnormal transmission of auditory information, indicating that, in contrast to the many known mutations causing deafness originating in the cochlea, deafness in qv is central in origin. Here we report that quivering mice carry loss-of-function mutations in the mouse beta-spectrin 4 gene (Spnb4) that cause alterations in ion channel localization in myelinated nerves; this provides a rationale for the auditory and motor neuropathies of these mice.

Differences in electron transport potential, antioxidant defenses, and oxidant generation in young and senescent fetal lung fibroblasts (WI-38).

The activities and mRNA abundances of enzymes that regulate the rate of electron flow through the electron transport chain (ETC), including NADH dehydrogenase, succinate dehydrogenase, and cytochrome c oxidase, were examined in young and senescent fetal lung fibroblasts (WI-38). We also determined the activities and mRNA abundances of antioxidant defenses including superoxide dismutase, catalase, and glutathione peroxidase. We confirmed our previous report of a senescence-related increase in the abundance of ND4, a mitochondrially encoded subunit of NADH dehydrogenase. The activities of cytochrome c oxidase and NADH dehydrogenase were also elevated in senescent cultures. No differences were observed in the mRNA abundances of COX-1, a mitochondrially encoded subunit of cytochrome c oxidase or of nuclearly encoded subunits of various electron transport components (SD, COX-4, and ND 51). Lucigenin-detected chemiluminescence and H2O2 generation were both elevated in senescent cells. Catalase activity was also elevated in senescent fibroblasts. However, no differences in catalase mRNA abundance were observed. A small decrease in GSH peroxidase (GPx) mRNA abundance was observed in senescent cells. No other changes in the activities or mRNA abundances of any of the antioxidant defenses were observed in early and late passage cultures. The relationships between oxidant generation, mitochondrial enzyme activities, and antioxidant defense observed during proliferative senescence are dissimilar to those detected between fetal and postnatal fibroblasts as well as those found between fibroblast lines obtained from young and old individuals. The relevance of the differences between these models is discussed.

Development and age-associated differences in electron transport potential and consequences for oxidant generation.

We determined the activities of NADH dehydrogenase (ND), succinate dehydrogenase, and cytochrome c oxidase (COX) in 29 skin fibroblast lines established from donors ranging in age from 12 gestational weeks to 94 years. The results of this study demonstrate that all three of the enzyme activities examined are greater in adult-derived fibroblasts than in the fetal cell lines. The ratio of enzyme activities that control electron entry into and exit from the electron transport chain varied directly with lucigenin-detected chemiluminescence (an indicator of .O2- generation) and inversely with H2O2 generation. These results indicate a clear difference in the predominant oxidant species generated during fetal and adult stages of life. We also examined the mRNA abundances of different components of the electron transport chain complexes. We observed higher abundances of mitochondrial encoded mRNAs (COX 1 and ND 4) in cell lines established from adults than in fetal cells. No differences in the mRNA abundances of the nuclear encoded sequences (COX 4 and ND 51) were observed in fetal and postnatal-derived lines. Succinate dehydrogenase mRNA abundance was greater in cell lines established from postnatal donors than in fetal cell lines. No significant differences between cell lines established from young and old adults were detected in any of the parameters examined.

Expression of hydrogen peroxide and glutathione metabolizing enzymes in human skin fibroblasts derived from donors of different ages.

We have examined the activities and mRNA abundance of two hydrogen peroxide metabolizing enzymes (glutathione peroxidase and catalase), glutathione concentration, and the activities of several enzymes that influence glutathione concentration, including glutathione reductase (GR), glucose-6-phosphate dehydrogenase (G-6-PD), and gamma-glutamylcysteine synthetase (gamma-GCS), in 29 skin fibroblast lines derived from donors ranging in age from 14 gestational weeks to 94 years of age. H2O2 metabolizing enzyme activities and mRNA abundances were greater in skin fibroblast cultures established from postnatal donors than in fetally derived cultures. There were no significant differences in either of these parameters in cell lines established from postnatal donors of different ages. Total glutathione concentration decreased with age, but GR activity appeared to be unaffected by age. In order to estimate the ability of the cultures to produce NADPH (an important component of cellular redox status and a cofactor for GR), we determined glucose-6-phosphate dehydrogenase activity and mRNA abundance. We were unable to directly measure gamma-GCS activity or mRNA abundance in any of the skin lines or in fetal lung fibroblast; however, we were able to indirectly demonstrate the presence of this enzyme by stimulating fetal lung fibroblasts with H2O2 following treatment with L-buthionine-S,R-sulfoximine (BSO), an inhibitor of gamma-GCS activity. These results show that some, but not all, age-associated differences in antioxidant defense levels are maintained in a culture environment and are consistent with the hypothesis that developmental stages of life are associated with lower antioxidant defense levels than are present in postnatal phases of life.

Effects of cellular aging on the induction of c-fos by antioxidant treatments.

The proto-oncogene c-fos (the cellular homolog of v-fos, Finkel-Biskis-Jenkins (FBJ) murine osteogenic sarcoma virus) encodes a major component of the activator protein-1 (AP-1) transcription factor. Serum stimulation as well as oxidizing treatments induce transitory increases in c-fos mRNA abundance. The induction of c-fos by serum stimulation is also known to decline during proliferative senesence. In this study, we examined the effects of two classes of antioxidants on the induction of c-fos in early and late passage human fetal lung fibroblasts (WI-38). N-acetyl cysteine (NAC) induces c-fos transcription in both early and late passage cells, while nordihydroguaiaretic acid (NGA) induced c-fos transcription in early passage cells but fails to stimulate it in late passage cells. Since we had previously observed an age-related decline in protein kinase C (PKC) translocation from the cytosol to the membrane, following its activation, and because PKC activation appears to be involved in the NGA induction of c-fos we examined the relative protein abundances of several PKC isoforms in early and late passage cells. Additionally, we examined the protein abundance of several members of the MAP kinase pathway which could play a role in c-fos induction by the PKC-dependent pathway. We were unable to detect PKC-beta or theta in early or late passage cells. Late passage cells contained a slightly greater abundance of PKC alpha, gamma and epsilon than cells at an early passage. No other differences in PKC isoforms or in members of the MAP kinase family were observed in early or late passage cells. These results clearly demonstrate that at least some pathways leading to c-fos induction remain intact in late passage cells. While we were unable to detect any decreases in PKC isoforms or MAP kinase proteins we cannot exclude the possibility that functional decrements accumulate in these proteins during senesence.

Expression and regulation of superoxide dismutase activity in human skin fibroblasts from donors of different ages.

We have determined the activities, protein, and mRNA abundances as well as the level of transcriptional activation of two intracellular forms of the free radical metabolizing enzyme superoxide dismutase in 29 human skin fibroblast lines established from donors of different ages. SOD-1 (a copper and zinc containing form of SOD) and SOD-2 (a manganese containing form of the enzyme) activities were both observed to be significantly lower in cell lines derived from fetal skin than in lines established from postnatal skin (ages 17-94 years). The percent of total activity contributed by SOD-1 decreased in an age-associated manner from approximately 50% in the fetal lines to less than 20% in lines established from old tissue donors. All of the cell lines were screened to exclude the possibility that they contained a polymorphism known to influence SOD-2 activity. Northern blot analysis revealed three SOD-1 mRNA transcripts that were 0.5, 0.7, and 1.9 kb in length. Although SOD-1 protein abundance was lower in fetal lines than in lines derived from postnatal donors, SOD-1 mRNA abundance did not differ between fetal cells and cell lines derived from young donors. SOD-2 protein abundance and mRNA abundance were both significantly lower in fetal lines than in postnatal lines. No postnatal age-dependent differences were observed in any of the SOD-2 parameters examined. Nuclear run-on analysis revealed that fetal cell lines exhibited a lower level of transcriptional initiation for SOD-1 than postnatal lines. The transcription of SOD-2 was readily detected in postnatal lines, but undetectable in fetal lines. These results are consistent with multiple levels of control for SOD-1 expression and with a strong transcriptional influence on SOD-2 expression.

Adsorption of Bacteriophage eb7 on Streptococcus cremoris EB7.

When M17 broth was used as growth medium before preparation of cell walls, adsorption of phage eb7 on Streptococcus cremoris EB7 at pH 4.0 was stimulated. When preparation included treatment with trypsin, absorption of phage was 65%. Without trypsin treatment, absorption was 81%. Only 10 to 20% of the adsorption was irreversible. Treatment with pepsin or commercial rennet but not pure chymosin prevented adsorption on non-trypsin-treated cell walls. d-Galactosamine treatment of phage eb7 had an inactivating effect which was enhanced by l-rhamnose.

Possible role of coryneform bacteria in age gelation of ultrahigh-temperature-processed milk.

Two strains of psychrotrophic, gram-positive, proteolytic bacteria isolated from raw milk were identified as Arthrobacter or closely related coryneform species. We inoculated raw-milk samples with the strains (one strain per sample) and compared rates of gelation after ultrahigh-temperature processing with that of ultrahigh-temperature-processed controls. The trial indicated that either organism could play a role in age gelation of ultrahigh-temperature-processed milk.

Aberrant Forms of Streptococcus cremoris HP.

The cheese starter strain, Streptococcus cremoris HP, produced variant colonies when streaked on the surface of solid media and incubated at 30 or 37 degrees C or in the presence of penicillin. Serial plating and incubation at 37 degrees C or in the presence of penicillin resulted in the production of variants. Subculture followed by incubation at 25 degrees C or in the absence of penicillin resulted in the reversion or partial reversion to the parent form. Colony morphology and cell morphology exhibited the characteristics of the L-phase. Evidence suggested that the aberrant forms of S. cremoris at 30 degrees C were transitional phase variants but at 37 degrees C and in the presence of penicillin they were L-phase variants. Electron micrographs showed that the cell walls of the variant cells were defective and that there were differences in the density and the organization of the cytoplasmic constituents compared with the parent cell.

Appraisal of media and methods for assay of bacteriophages of lactic streptococci.

To assess the relative merits of tryptone yeast extract agar, the same medium unbuffered, and medium M17 for the assay of nine bacteriophages of lactic streptococci, comparative plaque counts were made with an overlay of 3 or 9 ml. Four of the phages exhibited no significant difference in plating efficiency between media. The effect of overlay volume varied from strain to strain and was different for different media. The 3-ml overlay created suboptimal atmospheric conditions for those strains which had a special requirement for CO(2). The use of a 9-ml overlay obviated the need to incubate plates under CO(2) and overcame the problems related to special calcium requirements when tryptone yeast extract agar was used. The organic buffer (disodium beta-glycerophosphate) was inhibitory to Streptococcus cremoris ML1 and showed no advantage over the inorganic phosphate buffer (K(2)HPO(4)) for most other strains.

Morphology of the bacteriophages of lactic streptococci.

Electron microscope studies have been made of a number of phages of lactic streptococci, seven of which were phages of Streptococcus lactis C10. Two of the phages are thought to be identical; five have been classified by the method of Tikhonenko as belonging to group IV (phages with noncontractile tails) with type III tail plates; one belongs to group V (phages with tails possessing a contractile sheath). Both prolate polyhedral heads and isometric polyhedral heads are represented among the group IV phages. The phage drc3 of S. diacetilactis DRC3 has been shown to have similar structure to the group IV phages of S. lactis C10 with prolate polyhedral heads. The phages ml1, hp, c11, and z8 of the S. cremoris strains ML1, HP, C11, and Z8, respectively, were shown to belong to the group IV phages with type III tail plates by the method of Tikhonenko. All had octahedral heads and tended to be larger than most of the other phages studied.

Survival and activity of frozen starter cultures for cheese manufacture.

A study has been conducted on the effect of freezing and storage in liquid nitrogen on 13 strains of lactic streptococci. Cultures were frozen in droplet form and collected in mesh bags. After rapid thawing, the activity of the frozen cultures was compared with a culture of the same organism of the age usually used in cheese-making. The activities of the test and control cultures were traced simultaneously by continuous recording of the pH changes in inoculated milks. Viable counts were performed before and after freezing in liquid nitrogen and after storage in liquid nitrogen. There was no decrease in viable count or loss in activity of the cultures due to freezing and storage. Frozen cultures of some strains showed a shorter lag period after inoculation of milk than control cultures. Frozen concentrated cheese-starter cultures behaved normally in the manufacture of Cheddar cheese.

Long-term storage of bacteriophages of lactic streptococci.

Four phage strains representing phages of Streptococcus lactis, S. cremoris, and S. diacetilactis were selected for the observation of the effect of cold storage on their viability. Phages were stored at 4 C and at -18 C, or were frozen at approximately -70 C and stored at -18 C. They were found to display a high degree of stability with these storage methods. The same phage strains showed good stability to storage at room temperature for 3 weeks after thawing and also to alternate freezing and thawing eight times. Three series consisting of from 23 to 31 lactic streptococcal phage preparations were observed over periods extending up to 6 years, and with only a few exceptions were found to store satisfactorily at -18 C after quick freezing. Although the same phage preparations stored at 4 C were generally somewhat less stable, many were stable when stored by both methods.