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S L Schmidt

Publications and source records attributed to S L Schmidt.

At least 19 recordsLinked to original sources

The effect of corpus callosum agenesis on neocortical thickness and neuronal density of BALB/cCF mice.

We used acallosal and normal adult BALB/cCF mice to test the hypothesis that the development of the corpus callosum is relevant for the establishment of a normal structure of the neocortex. Neuronal density and thickness of individual layers were analyzed in neocortical regions with abundant callosal connections (area 6 and the 17/18a border) and in the relatively acallosal area 17. In area 6, acallosal mice exhibited a total neocortical thickness smaller than that of normal mice, as well as thinner layers II+III and IV. Similar data were obtained at the 17/18a border, where the total thickness of the cortex and of layers II+III was smaller in the acallosal mice than in normal ones. In contrast, no significant thickness differences were documented in area 17 of acallosal versus normal mice. The quantitative data obtained in the analyzed neocortical regions did not show differences in neuronal density between acallosal and normal mice. The reduced cortical thickness, associated with the comparatively normal neuronal density in neocortical regions which normally have abundant callosal connections, provides indirect indication of a reduction in the number of cortical neurons in acallosal mice. This assumption was also supported by the lack of evidence of neocortical alterations in the acallosal area 17. The present findings suggest that during development neocortical neurons destined to receive a massive callosal input may die as a result of lack of afferents. Altogether the present data indicate that the input provided by callosal axons is necessary for a normal development of the neocortex.

Afferent Pathways↗

ATP utilization by yeast replication factor C. IV. RFC ATP-binding mutants show defects in DNA replication, DNA repair, and checkpoint regulation.

Replication factor C is required to load proliferating cell nuclear antigen onto primer-template junctions, using the energy of ATP hydrolysis. Four of the five RFC genes have consensus ATP-binding motifs. To determine the relative importance of these sites for proper DNA metabolism in the cell, the conserved lysine in the Walker A motif of RFC1, RFC2, RFC3, or RFC4 was mutated to either arginine or glutamic acid. Arginine mutations in all RFC genes tested permitted cell growth, although poor growth was observed for rfc2-K71R. A glutamic acid substitution resulted in lethality in RFC2 and RFC3 but not in RFC1 or RFC4. Most double mutants combining mutations in two RFC genes were inviable. Except for the rfc1-K359R and rfc4-K55E mutants, which were phenotypically similar to wild type in every assay, the mutants were sensitive to DNA-damaging agents. The rfc2-K71R and rfc4-K55R mutants show checkpoint defects, most likely in the intra-S phase checkpoint. Regulation of the damage-inducible RNR3 promoter was impaired in these mutants, and phosphorylation of Rad53p in response to DNA damage was specifically defective when cells were in S phase. No dramatic defects in telomere length regulation were detected in the mutants. These data demonstrate that the ATP binding function of RFC2 is important for both DNA replication and checkpoint function and, for the first time, that RFC4 also plays a role in checkpoint regulation.

Adenosine Triphosphate↗

ATP utilization by yeast replication factor C. III. The ATP-binding domains of Rfc2, Rfc3, and Rfc4 are essential for DNA recognition and clamp loading.

The conserved lysine in the Walker A motif of the ATP-binding domain encoded by the yeast RFC1, RFC2, RFC3, and RFC4 genes was mutated to glutamic acid. Complexes of replication factor C with a N-terminal truncation (Delta2-273) of the Rfc1 subunit (RFC) containing a single mutant subunit were overproduced in Escherichia coli for biochemical analysis. All of the mutant RFC complexes were capable of interacting with PCNA. Complexes containing a rfc1-K359E mutation were similar to wild type in replication activity and ATPase activity; however, the mutant complex showed increased susceptibility to proteolysis. In contrast, complexes containing either a rfc2-K71E mutation or a rfc3-K59E mutation were severely impaired in ATPase and clamp loading activity. In addition to their defects in ATP hydrolysis, these complexes were defective for DNA binding. A mutant complex containing the rfc4-K55E mutation performed as well as a wild type complex in clamp loading, but only at very high ATP concentrations. Mutant RFC complexes containing rfc2-K71R or rfc3-K59R, carrying a conservative lysine --> arginine mutation, had much milder clamp loading defects that could be partially (rfc2-K71R) or completely (rfc3-K59R) suppressed at high ATP concentrations.

Adenosine Triphosphate↗

ATP utilization by yeast replication factor C. II. Multiple stepwise ATP binding events are required to load proliferating cell nuclear antigen onto primed DNA.

Binding of adenosine (3-thiotriphosphate) (ATPgammaS), a nonhydrolyzable analog of ATP, to replication factor C with a N-terminal truncation (Delta2-273) of the Rfc1 subunit (RFC) was studied by filter binding. RFC alone bound 1.8 ATPgammaS molecules. However, when either PCNA or primer-template DNA were also present 2.6 or 2.7 ATPgammaS molecules, respectively, were bound. When both PCNA and DNA were present 3.6 ATPgammaS molecules were bound per RFC. Order of addition experiments using surface plasmon resonance indicate that RFC forms an ATP-mediated binary complex with PCNA prior to formation of a ternary DNA.PCNA.RFC complex. An ATP-mediated complex between RFC and DNA was not competent for binding PCNA, and the RFC.DNA complex dissociated with hydrolysis of ATP. Based on these experiments a model is proposed in which: (i) RFC binds two ATPs (RFC.ATP(2)); (ii) this complex binds PCNA (PCNA.RFC.ATP(2)), which goes through a conformational change to reveal a binding site for one additional ATP (PCNA.RFC.ATP(3)); (iii) this complex can bind DNA to yield DNA.PCNA.RFC.ATP(3); (iv) a conformational change in the latter complex reveals a fourth binding site for ATP; and (v) the DNA.PCNA.RFC.ATP(4) complex is finally competent for completion of PCNA loading and release of RFC upon hydrolysis of ATP.

Adenosine Triphosphate↗

Muf1, a novel Elongin BC-interacting leucine-rich repeat protein that can assemble with Cul5 and Rbx1 to reconstitute a ubiquitin ligase.

The heterodimeric Elongin BC complex has been shown to interact in vitro and in mammalian cells with a conserved BC-box motif found in a growing number of proteins including RNA polymerase II elongation factor Elongin A, SOCS-box proteins, and the von Hippel-Lindau (VHL) tumor suppressor protein. Recently, the VHL-Elongin BC complex was found to interact with a module composed of Cullin family member Cul2 and RING-H2 finger protein Rbx1 to reconstitute a novel E3 ubiquitin ligase that activates ubiquitylation by the E2 ubiquitin-conjugating enzymes Ubc5 and Cdc34. In the context of the VHL ubiquitin ligase, Elongin BC functions as an adaptor that links the VHL protein to the Cul2/Rbx1 module, raising the possibility that the Elongin BC complex could function as an integral component of a larger family of E3 ubiquitin ligases by linking alternative BC-box proteins to Cullin/Rbx1 modules. In this report, we describe identification and purification from rat liver of a novel leucine-rich repeat-containing BC-box protein, MUF1, which we demonstrate is capable of assembling with a Cullin/Rbx1 module containing the Cullin family member Cul5 to reconstitute ubiquitin ligase activity. In addition, we show that the additional BC-box proteins Elongin A, SOCS1, and WSB1 are also capable of assembling with the Cul5/Rbx1 module to reconstitute potential ubiquitin ligases. Taken together, our findings identify MUF1 as a new member of the BC-box family of proteins, and they predict the existence of a larger family of Elongin BC-based E3 ubiquitin ligases.

Amino Acid Sequence↗

Cloning and characterization of ELL-associated proteins EAP45 and EAP20. a role for yeast EAP-like proteins in regulation of gene expression by glucose.

RNA polymerase II elongation factor ELL was recently purified from rat liver as a component of a multiprotein complex containing ELL and three ELL-associated proteins (EAPs) of approximately 45 (EAP45), approximately 30 (EAP30), and approximately 20 (EAP20) kDa (Shilatifard, A. (1998) J. Biol. Chem. 273, 11212-11217). Cloning of cDNA encoding the EAP30 protein revealed that it shares significant sequence similarity with the product of the Saccharomyces cerevisiae SNF8 gene (Schmidt, A. E., Miller, T., Schmidt, S. L., Shiekhattar, R., and Shilatifard, A. (1999) J. Biol. Chem. 274, 21981-21985), which is required for efficient derepression of glucose-repressed genes. Here we report the cloning of cDNAs encoding the EAP45 and EAP20 proteins. In addition, we identify the S. cerevisiae VPS36 and YJR102c genes as potential orthologs of EAP45 and EAP20 and show that they are previously uncharacterized SNF genes with properties very similar to SNF8.

Amino Acid Sequence↗

Sex differences in sensitivity to seizures elicited by pentylenetetrazol in mice.

Sex differences in sensitivity to seizures elicited by intraperitoneally injected pentylenetetrazol (PTZ) were studied in 240 (120 males and 120 females) adult Swiss mice. Animals were separated into four groups according to the dose that was injected: 40, 50, 60 and 70 mg/kg. Seizure severity was expressed by the following scoring scale: (0) no abnormal behavior; (1) myoclonus; (2) running bouncing (RB) clonus; (3) tonic hind limb extension (THE). The analyses of the dose-response curves indicated that females were more susceptible than males when the 50- and 60-mg/kg doses were used. Specifically, females often displayed RB clonus, while males frequently displayed only myoclonus or no abnormal behavior. No significant sex differences were demonstrated when either the 40- or the 70-mg/kg doses were used. These data indicate that, for a specific range of doses, sex differences in seizure susceptibility can be clearly demonstrated with the use of intraperitoneally injected PTZ. In this sense, this method could be used as a tool to investigate the role played by sexual hormones in regulating the sensitivity of the gamma-aminobutiric acid (GABA(A)) receptor complex (GRC).

Animals↗

Depletion of cortical target induced by prenatal ionizing irradiation: effects on the lateral geniculate nucleus and on the retinofugal pathways.

Studies using neonatal surgical lesions to reduce the target area of the retina have supported the idea that developing axons show only a limited specificity in their targeting. This investigation tested whether retinogeniculate axons adjust for partial target depletion by repositioning of axons. We used adult Swiss mice exposed to gamma rays at the time when layer IV cells are generated in the ventricular zone (16 days of gestation). Nissl-stained brain sections were used for histological analyses in thalamus and cortex. Retinal ganglion cells were backfilled from the optic tract with horseradish peroxidase. Intraocular injections of horseradish peroxidase were used to study the retinal projections. In the posterior cortex there was a nearly complete absence of layer IV. The irradiated animals showed a 75% reduction of the dorsal lateral geniculate nucleus. The ventral division, superior colliculus, and other visually related nuclei were not affected. The loss in the ganglion cells (15.7%) was significant but clearly smaller than that observed in the dorsal lateral geniculate nucleus (75%). Therefore, the shrinkage of the dorsal lateral geniculate nucleus led to a reduction in the area available for retinal projections. Despite partial target loss, pattern of retinal projections did not differ from that of the controls. The effect on the dorsal lateral geniculate nucleus is discussed in the light of differences between prenatal and neonatal damage of the presumptive visual cortex. The absence of aberrant retinal projections suggests that repositioning of axons is not the first mechanism employed by retinal axons to match connections in numerically disparate populations.

Animals↗

Effects of prenatal ionizing irradiation on the development of the ganglion cell layer of the mouse retina.

Prenatal exposure to ionizing irradiation has been shown to be an effective method to eliminate selectively certain neuronal population. This investigation studied the effects on the ganglion cell layer of the retinae of adult mice exposed to a gamma source (total dose=3 Gy) at 16 days gestation. There was a significant reduction in the total number of neurons (displaced amacrine+ganglion cells) in the ganglion cell layer (33%) that was mainly caused by a pronounced loss (59%) of displaced amacrine cells. The diameters of the surviving retinal ganglion cells were consistently larger than those of the controls. Prenatal irradiation is the first experimental approach that partially eliminates displaced amacrine cells. It is suggested that the morphogenesis of retinal ganglion cells may be affected by displaced amacrine cells.

Animals↗

Time course of the effects of prenatal gamma irradiation on the dorsal lateral geniculate nucleus of Swiss mice.

A previous study reported that adult mice irradiated at the 16th embryonic day present a severe neuronal number reduction in the dorsal lateral geniculate thalamic nucleus. In the present study, we investigated the time course of the effects of prenatal irradiation on this thalamic nucleus. One day after irradiation, a great number of pyknotic figures were seen mainly in the cerebral proliferative zones. In the geniculate nucleus, only scattered pyknotic figures were identified. On the first week after birth, the geniculate nucleus presented frequent pyknotic figures. From five days after birth onwards, a severe shrinkage of the occipital cortex and a great reduction in the geniculate nucleus neuronal number were found. On the second week after birth this neuronal number reduction reached as high as 75%. At each postnatal analyzed age, severe volumetric geniculate nucleus shrinkage was combined to non-significant neuronal density variations. The presence of few pyknotic figures in the geniculate nucleus one day after irradiation and its delayed neuronal loss indicate an indirect effect of irradiation. We suggest that the effect upon the geniculate nucleus is secondary to the damage of the occipital cortex. A possible interpretation for thalamic neuronal loss is that geniculate neurons fail to establish cortical arbors after major target loss. In this case, the loss of trophic support should also be considered.

Aging↗

Contralateral rotatory bias in the free-swimming test after unilateral hemispherectomy in adult Swiss mice.

In the free-swimming rotatory test mice spend most of the time swimming close to the wall of the container attempting to escape from an aversive test situation. The attraction to the wall may suggest that turning behavior in the free-swimming test reflects the existence of intrinsic sensory asymmetries, which determine preferential attention adhesion to one side. In order to test this hypothesis, we investigated the rotatory swimming behavior of mice submitted to a unilateral hemispherectomy at adulthood, a condition of extreme sensory asymmetry. Fifteen days after surgery procedures, each mouse was tested for 5 min on 3 different days. We found that the hemispherectomized mice had a significant strong bias to turn in the direction contralateral to their lesion. These data could be explained considering that, in attempting to escape from the test situation, animals bring the recipient wall into their intact sensory field and, as a consequence, set the direction of locomotion. Thus, the free-swimming test may be useful to investigate sensory asymmetries during an aversive test situation.

Animals↗

Influences of handedness and gender on the grooved pegboard test.

We studied performance on the Grooved Pegboard Test upon repeated trials and transfer of training between the hands in the first trial. The classification of handedness was based on the writing hand. We employed three trials for each hand and two different protocols for the order in which the hands started the test. For the three trials combined, women were faster than men. From the first to the second trial, there was an improvement in performance for both sexes. Within the first trial, sex differences reached significance and the protocol interacted with handedness. In this trial, only left-handed men were found to benefit from previous opposite-hand performance. It is speculated that a larger corpus callosum in left-handed men allows for the greater transfer of training between the hands.

Adult↗

The effects of hand preference and gender on finger tapping performance asymmetry by the use of an infra-red light measurement device.

We used an infra-red device to study the effects of gender and handwriting preference on manual asymmetry in tapping rate and intertap variability. Our sample (n=102) consisted of approximately equal number of subjects with respect to gender (52 women and 50 men) and handedness (52 right-handers and 50 left-handers). Data on overall performance indicated that men performed more quickly and regularly than women. The index used for measuring manual asymmetry was the difference between the hands as a proportion of the total. Therefore, the asymmetry index was adjusted to remove the influence of overall performance. The analyses based on asymmetry scores indicated a significant handedness effect: right-handers showed greater manual asymmetries than left-handers for both tapping rate and intertap variability. In addition, right handers exhibited a significant greater asymmetry for intertap variability than tapping rate. Taken together, these data may reflect greater hemispheric differences in right-handers, specially for intertap variability.

Adolescent↗

Empirical analysis of the selective attention and associated behavior checklists of the Aggregate neurobehavioral student health and educational review.

For 111 referred children, the Behavior Observation (BO) and the Selective Attention (SA) components of the Aggregate Neurobehavioral Student Health and Educational Review (ANSER) were completed by mothers and teachers and the Pediatric Examination of Educational Readiness at Middle Childhood was completed by the pediatrician. Smaller samples were used to study concurrent validity. The questionnaires presented high internal reliability. Factor analyses of the BO questionnaires resulted in a common primary factor of "Aggressiveness" for ratings by mothers and teachers. For the SA, "Hyperactivity" (mothers) and "Inattention" (teachers) accounted for the majority of variance. There were modest correlations between teachers and mothers. The correlation between the pediatrician and teachers reached the highest value. The BO-Teachers form was significantly related to the Teacher Report Form and the Conners Teacher Rating Scale. The authors concluded that the ANSER presents concurrent validity for teacher ratings, items predictive of atypical behavior, and a unique pool for defining a subgroup of children with attentional disorders. The limited relationship between mothers and teachers may be ascribed to different interpretations of the same items.

Attention Deficit Disorder with Hyperactivity↗

Cloning and characterization of the EAP30 subunit of the ELL complex that confers derepression of transcription by RNA polymerase II.

The product of the human oncogene ELL encodes an RNA polymerase II transcription factor that undergoes frequent translocation in acute myeloid leukemia (AML). In addition to its elongation activity, ELL contains a novel type of RNA polymerase II interaction domain that is capable of repressing polymerase activity in promoter-specific transcription. Remarkably, the ELL translocation that is found in patients with AML results in the deletion of exactly this functional domain. Here we report that the EAP30 subunit of the ELL complex has sequence homology to the Saccharomyces cerevisiae SNF8, whose genetic analysis suggests its involvement in the derepression of gene expression. Remarkably, EAP30 can interact with ELL and derepress ELL's inhibitory activity in vitro. This finding may reveal a key role for EAP30 in the pathogenesis of human leukemia.

Amino Acid Sequence↗

Morphological brain asymmetries in male mice with callosal defects due to prenatal gamma irradiation.

We have previously suggested a relationship between the development of the corpus callosum and the direction of cerebral asymmetries (Schmidt and Caparelli-Daquer, 1989; Schmidt et al., 1991). Here we report a study on morphological brain asymmetries using a distinct experimental approach. At embryonic day 16, mice were exposed to a gamma source, receiving a total dose of 2 Gy. At adulthood 32 irradiated and 28 normal male Swiss mice were evaluated for individual and populational interhemispheric asymmetries of weight, dorsal area, and neocortical volume. All irradiated mice showed callosal defects ranging from total absence to a partial reduction of the midsagittal callosal area. The normal and irradiated groups displayed a pronounced individual asymmetry in all measurements. In contrast, populational asymmetry could be identified only in the normal group. These results are consistent with our previous data in acallosal mutant mice and support the hypothesis that the corpus callosum may play a role in directing interhemispheric morphological brain asymmetries.

Agenesis of Corpus Callosum↗

Effects of prenatal gamma irradiation on the development of the corpus callosum of Swiss mice.

The temporal sequence of events related to the effects of prenatal gamma irradiation on the development of the corpus callosum and cerebral cortex was studied in Swiss mice. Pregnant females on gestational day 16 were exposed to a 60Co source receiving total doses of 2 or 3 Gy. The offspring were analyzed at both prenatal and postnatal days. One day after irradiation, a great number of pyknotic figures was seen along the whole extension of the cerebral wall, especially in the proliferative zones. At perinatal ages, the thickness of the proliferative zones was reduced and the glial sling was never identified. From 5 days after birth onwards, we observed a severe shrinkage of layers II + III and IV. The majority of the irradiated mice were totally acallosal (particularly when the 3 Gy dose was used), but some animals presented callosal remnants. These remnants were identified above the ventral hippocampal commissure, except for two animals in which a larger callosal remnant extended from the columns of the fornix to the dorsal hippocampal commissure. The presence of callosal remnants in animals irradiated with 3 Gy was dependent on the age at which the animals were analyzed since remnants were observed in some animals analyzed at perinatal ages, but never in older animals. Callosal defects can be explained at least by three factors: (1) Death of a great part of callosal neurons located at layer III. (2) Postnatal axonal elimination. (3) Absence of the glial sling. The callosal agenesis in the absence of the glial sling indicates that this structure may play a crucial role in guiding callosal axons. However, the presence of callosal remnants indicates that surviving callosal axons can use structures other than the sling to cross the midplane. Our data indicate that axons of the middle portion of the callosum can cross the midplane using the ventral hippocampal commissure as a guide. Additionally, the dorsal hippocampal commissure may play a role in directing axons of the posterior part of the corpus callosum.

Aging↗

Evaluation of Candida acidothermophilum in ethanol production from lignocellulosic biomass.

A Saccharomyces-cerevisiae-based simultaneous saccharification and fermentation (SSF) of lignocellulosic biomass is limited to an operating temperature of about 37 degrees C, and even a small increase in temperature can have a deleterious effect. This points to a need for a more thermotolerant yeast. To this end, S. cerevisiae D5A and a thermotolerant yeast, Candida acidothermophilum, were tested at 37 degrees C, 40 degrees C, and 42 degrees C using dilute-acid-pretreated poplar as substrate. At 40 degrees C, C. acidothermophilum produced 80% of the theoretical ethanol yield, which was higher than the yield from S. cerevisiae D5A at either 37 degrees C or 40 degrees C. At 42 degrees C, C. acidothermophilum showed a slight drop in performance. On the basis of preliminary estimates, SSF with C. acidothermophilum at 40 degrees C can reduce cellulase costs by about 16%. Proportionately greater savings can be realized at higher temperatures if such a high-temperature SSF is feasible. This demonstrates the advantage of using thermophilic or thermotolerant yeasts.

Biomass↗