Contributions from the left PMd and the SMA during sequence retrieval as determined by depth of training

The dorsal premotor cortex (PMd) and the supplementary motor area (SMA) are critical for the acquisition and expression of sequential behavior, but little is known regarding how these regions are recruited when we must simultaneously acquire multiple sequences under different amounts of training. We hypothesized that these regions contribute to the retrieval of sequences at different familiarity levels, with the left PMd supporting sequences of moderate familiarity and the SMA supporting sequences of greater familiarity. Double-pulse transcranial magnetic stimulation (TMS) was applied during the retrieval of six sequences previously learned under three different amounts of exposure during 30 days of training using a discrete sequence production task. TMS led to a significant interaction of sequence error between depth of training and stimulation location. Stimulation of the left PMd increased error during moderate sequence retrieval, whereas stimulation of the SMA increased error during the retrieval of both moderately and extensively trained sequences. The lack of a double dissociation fails to support a direct correspondence between brain region and putative behavioral learning stage. Instead, the interaction suggests that SMA and PMd support the expression of sequences over different, albeit overlapping, time scales. Separate analysis of sequence initiation time did not demonstrate any significant difference between moderately and extensively trained sequences. Instead, stimulation to either region quickened sequence initiation for these sequences, but not for those sequences with poor retrieval performance. This supports the general role of these premotor regions in the maintenance of specific sequence knowledge prior to movement onset.     

Endothelial cell dynamics under pulsating flows: significance of high versus low shear stress slew rates (d(tau)/dt)

Shear stress modulates endothelial cell (EC) remodeling via realignment and elongation. We provide the first evidence that the upstroke slopes of pulsatile flow, defined as shear stress slew rates (positive / affect significantly the rates at which ECs remodel. We designed a novel flow system to isolate various shear stress slew rates by precisely controlling the frequency, amplitude, and time-averaged shear stress _ave of pulsatile flow. Bovine aortic endothelial cell (BAEC) monolayers were exposed to three conditions: (1) pulsatile flow (1 Hz) at high slew rate (293 dyn/cm² s), (2) pulsatile flow (1 Hz) at low slew rate (71 dyn/cm² s), and (3) steady laminar flow at /t=0. All of the three conditions were operated at _ave=50{dyn/cm}². BAEC elongation and alignment were measured over 17 h. We were able to demonstrate the effects of shear stress slew rates /t on EC remodeling at a fixed spatial shear stress gradient (/x). We found that pulsatile flow significantly increased the rates at which EC elongated and realigned, compared to steady flow at /t=0. Furthermore, EC remodeling was faster in response to high than to low slew rates at a given tau _ave © 2002 Biomedical Engineering Society. PAC2002: 8719Tt, 8719Rr     

A new multiphysics model for the physiological responses of vascular endothelial cells to fluid shear stress

Vascular endothelial cell (VEC) responds to wall shear stress that has not only spatial variation, but also temporal gradient. To simplify the problem, we first studied how the calcium dynamics of VEC responded to the steady wall shear stress of varying magnitude in a stenosed artery. We then studied how the VEC responded to the periodic shear stress that had temporal variation, as in the pulsatile blood flow. To investigate the multiphysics model of VEC in vitro, we used a mathematical model for intracellular calcium dynamics and a computational fluid dynamics (CFD) method for arterial wall shear stress, either steady or periodic. The CFD results showed that for the steady stenotic flow, the wall shear stress in the recirculating flow was lower than the threshold value, 4 dyne/cm(2), at two particular points: flow separation and flow reattachment. For these subthreshold shear stresses, the peak value of the transient calcium response did not hit the normal saturated level, but reached a reduced magnitude. We investigated the effect of severity of stenosis (SOS) of the stenosed artery. For the pulsatile flow, the so-called shear stress slew rate or the temporal gradient of the first upsurge of the periodic flow was an important factor for the VEC calcium dynamics. The calcium response had a finite range of parameter for SOS and shear stress slew rate in which the calcium response was more sensitive than elsewhere, showing a sigmoid pattern.     

The maltoporin of Salmonella typhimurium: sequence and folding model

The sequence of the lamB gene from Salmonella typhimurium was determined. It encodes the precursor to the LamB protein from S. typhimurium (pre-LamBS.t.; 452 residues) which presents extensive homologies with the pre-LamB protein from Escherichia coli (pre-LamBE.c.; 446 residues). The first third of pre-LamBS.t. is the most conserved, with 4% changes and strict identity between the signal peptides. The last two-third contains five "variable" segments where more than 50% of the residues are changed with respect to LamBE.c.. The three first variable segments are 8 to 14 residues long and contain only substitutions, while the two more distal ones are 24 and 29 residues long and also include insertions and deletions. It is remarkable that the variable segments correspond essentially to regions predicted to be extramembranous loops on our 2D folding model for LamBE.c.; they alternate with conserved predicted transmembranous segments. Four of the variable regions were predicted to be cell-surface-exposed loops on the basis of genetic and immunological data, while one of them (region II) was predicted to be periplasmic on the sole basis of folding rules. The LamB protein from S. typhimurium can substitute for the LamB protein from E. coli for maltodextrins binding and transport, but not for infection by any of the known E. coli phages using LamBE.c. for adsorption. A tetrapeptide, RGDS, assumed to be responsible for mammalian cell aggregation by LamBE.c. is conserved in LamBS.t., suggesting that it could have a functional role. The conservation of the binding and transport activity can be accounted for by the conservation of the regions known to be directly involved, namely the first third of the protein and a region corresponding to 352 to 374 of LamBS.t.. The phage resistance can be attributed to the variability of the four cell-surface-exposed loops previously identified as essential for phage adsorption. These results, together with those obtained with polyclonal and monoclonal antibodies directed against known LamB regions, strongly support the folding model presented for LamBE.c. and the idea that it can essentially be extended to LamBS.t., except perhaps for a region between residues 155 and 245. We propose that the existence of variable regions is due essentially, and perhaps only, to the local lack of structural constraints in the protein. The intergenic region between lamB and the following gene, malM, comprises conserved segments, including one palindromic unit.     

Spatio-temporal alignment of pedobarographic image sequences

This article presents a methodology to align plantar pressure image sequences simultaneously in time and space. The spatial position and orientation of a foot in a sequence are changed to match the foot represented in a second sequence. Simultaneously with the spatial alignment, the temporal scale of the first sequence is transformed with the aim of synchronizing the two input footsteps. Consequently, the spatial correspondence of the foot regions along the sequences as well as the temporal synchronizing is automatically attained, making the study easier and more straightforward. In terms of spatial alignment, the methodology can use one of four possible geometric transformation models: rigid, similarity, affine, or projective. In the temporal alignment, a polynomial transformation up to the 4th degree can be adopted in order to model linear and curved time behaviors. Suitable geometric and temporal transformations are found by minimizing the mean squared error (MSE) between the input sequences. The methodology was tested on a set of real image sequences acquired from a common pedobarographic device. When used in experimental cases generated by applying geometric and temporal control transformations, the methodology revealed high accuracy. In addition, the intra-subject alignment tests from real plantar pressure image sequences showed that the curved temporal models produced better MSE results (P < 0.001) than the linear temporal model. This article represents an important step forward in the alignment of pedobarographic image data, since previous methods can only be applied on static images.     

Unscheduled DNA replication origin activation at inserted HPV 18 sequences in a HPV-18/MYC amplicon

Oncogene amplification is a critical step leading to tumorigenesis, but the underlying mechanisms are still poorly understood. Despite data suggesting that DNA replication is a major source of genomic instability, little is known about replication origin usage and replication fork progression in rearranged regions. Using a single DNA molecule approach, we provide here the first study of replication kinetics on a previously characterized MYC/papillomavirus (HPV18) amplicon in a cervical cancer. Using this amplicon as a model, we investigated the role DNA replication control plays in generating amplifications in human cancers. The data reveal severely perturbed DNA replication kinetics in the amplified region when compared with other regions of the same genome. It was found that DNA replication is initiated from both genomic and viral sequences, resulting in a higher median frequency of origin firings. In addition, it was found that the higher initiation frequency was associated with an equivalent increase in the number of stalled replication forks. These observations raise the intriguing possibility that unscheduled replication origin activation at inserted HPV-18 viral DNA sequences triggers DNA amplification in this cancer cell line and the subsequent overexpression of the MYC oncogene.     

NRE,the major nitrogen regulatory protein of Penicillium chrysogenum,binds specifically to elements in the intergenic promoter regions of nitrate assimilation and penicillin biosynthetic gene clusters

NRE, the nitrogen regulatory protein of Penicillium chrysogenum, contains a single Cys2/Cys2-type zinc-finger motif followed immediately by a highly basic region. The zinc-finger domain was expressed to Escherichia coli as a fusion protein with -galactosidase. In order to test the putative DNA-binding ability of NRE, the intergenic promoter region of the nitrate reductase/nitrite reductase gene cluster (niiA-niaD) of Penicillium was sequenced. Our results show that NRE is a DNA-binding protein and binds to the intergenic promoter regions of the P. chrysogenum niiA-niaD and acvA-pcbC gene cluster, encoding the first two enzymes in penicillin biosynthesis. Three of the four high-affinity NRE-binding sites contained two GATA core elements. In one of the recognition sites for NRE, one GATA motif was replaced by GATT. The two GATA elements showed all possible orientations, head-to-head, head-to-tail and tail-to-tail, and were separated by between 4 and 27 bp. Missing-contact analysis showed that all three purines in both of the GATA core sequences and the single adenine residue in each of the complementary TATC sequences were involved in the binding of NRE. Moreover, loss of purines in the flanking regions of the GATA elements also affect binding of NRE, as their loss causes reduced affinity.     

Behaviour of ring bivalents in holokinetic systems: Alternative sites of spindle attachment in Pachylis argentinus and Nezara viridula (Heteroptera)

Heteropteran chromosomes are holokinetic; during mitosis, sister chromatids segregate parallel to each other but, during meiosis, kinetic activity is restricted to one pair of telomeric regions. This meiotic behaviour has been corroborated for all rod bivalents. For ring bivalents, we have previously proposed that one of the two chiasmata releases first, and a telokinetic activity is also achieved. In the present work we analyse the meiotic behaviour of ring bivalents in Pachylis argentinus (Coreidae) and Nezara viridula (Pentatomidae) and we describe for the first time the chromosome complement and male meiosis of the former (2n = 12 + 2m + X0, pre-reduction of the X). Both species possess a large chromosome pair with a secondary constriction which is a nucleolus organizer region as revealed by in-situ hybridization. Here we propose a new mode of segregation for ring bivalents: when the chromosome pair bears a secondary constriction, it is not essential that one of the chiasmata releases first since these regions or repetitive DNA sequences adjacent to them become functional as alternative sites for microtubule attachment and they undertake chromosome segregation to the poles during anaphase I.     

Comparative analysis of human and mouse 3' Igh regulatory regions identifies distinctive structural features

Immunoglobulin heavy chain (Igh) locus rearrangements are controlled in part by an approximately 30 b complex 3' regulatory region located 3' of C alpha: this region contains several enhancers. We report here the comparison of the genomic sequences of the 3' regulatory region and further downstream sequences from mouse, rat, human and chimpanzee. Only short segments of homology were detected in the 3' regulatory region, and these were located in the vicinity of the known 3' enhancers. The nearest highly conserved segment is the nearest non-Igh gene, hole, which is located approximately 62 kb downstream of mouse C alpha. Analysis of murine 3' Igh sequences by single nucleotide polymorphism (SNP) and restriction fragment length polymorphism (RFLP) detected a transition region (high to low SNP or RFLP density) approximately 120 kb downstream of mouse C alpha. Although there is only limited sequence identity between rodent and primate 3' Igh regulatory regions, all of these regulatory regions contain a palindrome and locally repetitive elements. Locally repetitive elements in primates comprise blocks of "switch-like" sequences that differ from the families of inverted and tandem repeats that are present in rodents. We propose that together with enhancers, these "conserved" structural features are essential for the activity of the 3' Igh regulatory region in vivo.     

Repetitive sequences in the ITS1 region of ribosomal DNA in congeneric microphallid species (Trematoda: Digenea)

In searching for species-specific DNA sequences of microphallid species (Digenea, Trematoda) we examined the ribosomal internal transcribed spacer regions (ITS) of three closely related species (Levinseniella group) hosted by mud snails (first intermediate host) and marine crustaceans (second intermediate host). In the ITS1 region we found consistent patterns of repeating sequences of 130 bp. Within each main repeat there was a varying number of subrepeats specific for each of the species. All repeats including subrepeats were identified by a similar starting sequence: 5-CCTGTGG-3. As this sequence has close resemblance to the chi sequence 5-GCTGGTGG-3 found in phage we speculate if it serves the same function as a recombination hotspot. Alternatively but less likely, it could be an inactive, mutational relic of a sequence that once served this purpose.     

Spatially-localized NMR spectroscopy employing an inhomogeneous surface-spoiling magnetic field gradient. 2. Surface coil experiments with multicompartment phantom and rat in vivo

The use of inhomogeneous surface-spoiling magnetic field gradients for elimination of signal from surface lying regions of a sample was theoretically examined in the companion article (W. Chen and J.J.H. Ackerman, NMR Biomed. 3, 147-157 (1990)). Using the spoiling gradient coil design described therein, this article presents experimental verification of the feasibility of such an approach to enhanced spatial localization. Single coil mode 31P NMR surface coil interrogation of both a two compartment phantom and rat in vivo are shown to provide excellent suppression of surface lying regions with minimal degradation of signal from the deep lying region of interest. Both pulse-and-collect and spin echo sequences were highly efficient in concert with spoiling gradient periods of 0.5-2 ms and driving currents of 0.5-2 A. The use of a current-generated surface spoiling gradient offers a robust means to remove surface tissue signal contributions and can be implemented with a wide range of localizing pulse sequences and imaging protocols.     

A complex genetic rearrangement in a t(10;14)(q24;q11) associated with T-cell acute lymphoblastic leukemia.

The t(10;14)(q24;q11) is observed in the leukemia cells of 5-10% of cases of T-cell acute lymphoblastic leukemia (T-ALL). Recently, molecular analyses of a number of these translocations revealed simple reciprocal translocations between the T-cell receptor delta chain gene (TCRD) and a region of 10q24. We have characterized, at the molecular level, a t(10;14)(q24;q11) in a patient with T-ALL. The translocation in this case, in contrast to the previous cases, is part of a complex genetic rearrangement. In addition to a reciprocal translocation between the D delta 3 gene segment of TCRD and a region of 10q24, a local inversion occurred within TCRD, involving the D delta 2 and V delta 2 gene segments. As a consequence, the entire joining and constant regions and most of the diversity regions of TCRD are located on the derivative 14 chromosome, whereas the joining and constant regions of TCRA are positioned on the derivative 10 chromosome. The chromosome 10 breakpoint in our patient, as in other t(10;14), clusters within a 9 kb breakpoint region. The occurrence of seven breakpoints within a localized region of chromosome 10 implies the existence of a nearby gene whose activation may have conferred a selective advantage on the leukemia cells. Moreover, as in the previous cases, the translocation in the present study exhibits recombination signal sequences or signal-like sequences adjacent to the breakpoint junction. The presence of such motifs suggests the involvement of the recombinase enzyme system in the generation of this genetic alteration.     

Structural correlates to the rabbit immunoglobulin heavy chain a100 allotype

Three a100/a100 homozygous rabbits immunized with Micrococcus lysodeikticus produced large amounts of anti-polysaccharide antibodies of restricted heterogeneity. These antibodies were purified by either immunoabsorption or ion exchange chromatography. The almost complete sequence of one heavy chain spanning residues 1-123, with the exception of 10 residues (66-67 and 79-86), was determined. Partial sequence data were also obtained for the two other heavy chains. The identity of these three sequences in the first framework region unraveled a prototype sequence of the a100 allotype that differs from homologous sequence of VH regions that determine other allotypic specificities. The gradient of sequence conservation was found to be a100 greater than a3 greater than a1 greater than a- greater than a2. Homologies in sequence paralleled previously described serological cross-reactions observed between a100, a3 and a1 specificities. This remarkable conservation of framework residues suggests that the VH regions of the rabbit immunoglobulins represent a paucigene system, in which each basic allotypic specificity might be encoded a discrete subgroup of genes.     

A case of kerion celsi due to Arthroderma benhamiae identified by DNA sequences of nuclear ribosomal internal transcribed spacer 1 regions.

We describe a case of a 4-year-old boy with a 1-month history of a purulent lesion on his scalp. His hair samples revealed fungal organisms and Trichophyton mentagrophytes was cultured from the sample. We analysed the DNA sequences of the nuclear ribosomal internal transcribed spacer 1 (ITS1) region of the isolated fungus. These sequences were in accordance with T. mentagrophytes animal 4 type. In mating experiments, our strain only responded to the Arthroderma benhamiae Americano-European race (+) mating type tester. We speculate that the patient was infected from contact with his pet guinea pig. This is the first case of a clinical isolate of A. benhamiae being identified by DNA sequences of nuclear ribosomal ITS1 regions.     

Parallel Cascade Recognition of Exon and Intron DNA Sequences

Many of the current procedures for detecting coding regions on human DNA sequences combine a number of individual techniques such as discriminant analysis and neural net methods. Recent papers have used techniques from nonlinear systems identification, in particular, parallel cascade identification (PCI), as one means for classifying protein sequences into their structure/function groups. In the present paper, PCI is used in a pilot study to distinguish exon (coding) from intron (noncoding; interspersed within genes) human DNA sequences. Only the first exon and first intron sequences with known boundaries in genomic DNA from the T-cell receptor locus were used for training. Then, the parallel cascade classifiers were able to achieve classification rates of about 89% on novel sequences in a test set, and averaged about 82% when results of a blind test were included. In testing over a much wider range of human nucleotide sequences, PCI classifiers averaged 83.6% correct classifications. These results indicate that parallel cascade classifiers may be useful components in future coding region detection programs. © 2002 Biomedical Engineering Society. PAC2002: 8715Cc, 8714Gg, 8715Aa     

Nonrandom distribution of interspersed repeat elements in the BCR and ABL1 genes and its relation to breakpoint cluster regions

The Philadelphia translocation, t(9;22)(q34;q11), is the microscopically visible product of recombination between two genes, ABL1 on chromosome 9 and BCR on chromosome 22, and gives rise to a functional hybrid BCR-ABL1 gene with demonstrated leukemogenic properties. Breakpoints in BCR occur mostly within one of two regions: a 5 kb major breakpoint cluster region (M-Bcr) and a larger 35 kb minor breakpoint cluster region (m-Bcr) towards the 3' end of the first BCR intron. By contrast, breakpoints in ABL1 are reported to occur more widely across a >200 kb region which spans the large first and second introns. The mechanisms that determine preferential breakage sites in BCR, and which cause recombination between BCR and ABL1, are presently unknown. In some cases, Alu repeats have been identified at or near sequenced breakpoint sites in both genes, providing indications, albeit controversial, that they may be relevant. For the present study, we carried out a detailed analysis of genomic BCR and ABL1 sequences to identify, classify, and locate interspersed repeat sequences and to relate their distribution to precisely mapped BCR-ABL1 recombination sites. Our findings confirm that Alu are the most abundant class of repeat in both genes, but that they occupy fewer sites than previously estimated and that they are distributed nonrandomly. r-Scan statistics were applied to provide a measure of repeat distribution and to evaluate extremes in repeat spacing. A significant lack of Alu elements was observed across the major and minor breakpoint cluster regions of BCR and across a 25-kb region showing a high frequency of breakage in ABL1. These findings counter the suggestion that occurrence of Alu at BCR-ABL1 recombination sites is likely by chance because of the high density of Alu in these two genes. Instead, as yet unidentified DNA conformation or nucleotide characteristics peculiar to the preferentially recombining regions, including those Alu elements present within them, more likely influence their fragility.     

Genetic variation among isolates of <Emphasis Type="Italic">White spot syndrome virus</Emphasis>

Summary. White spot syndrome virus (WSSV), member of a new virus family called Nimaviridae, is a major scourge in worldwide shrimp cultivation. Geographical isolates of WSSV identified so far are very similar in morphology and proteome, and show little difference in restriction fragment length polymorphism (RFLP) pattern. We have mapped the genomic differences between three completely sequenced WSSV isolates, originating from Thailand (WSSV-TH), China (WSSV-CN) and Taiwan (WSSV-TW). Alignment of the genomic sequences of these geographical isolates revealed an overall nucleotide identity of 99.32%. The major difference among the three isolates is a deletion of approximately 13kb (WSSV-TH) and 1kb (WSSV-CN), present in the same genomic region, relative to WSSV-TW. A second difference involves a genetically variable region of about 750bp. All other variations >2bp between the three isolates are located in repeat regions along the genome. Except for the homologous regions (hr1, hr3, hr8 and hr9), these variable repeat regions are almost exclusively located in ORFs, of which the genomic repeat regions in ORF75, ORF94 and ORF125 can be used for PCR based classification of WSSV isolates in epidemiological studies. Furthermore, the comparison identified highly invariable genomic loci, which may be used for reliable monitoring of WSSV infections and for shrimp health certification.     

Loss of heterozygosity on the X chromosome in human breast cancer

The analysis of loss of heterozygosity (LOH) in tumours can be a powerful tool for mapping the sites of tumour suppressor genes in the human genome. A panel of breast cancer patients was assembled as pairs of tumour and lymphocyte DNA samples and LOH studies carried out by Southern hybridisation with polymorphic loci mapping to the X chromosome with appropriate controls. Deletion mapping revealed a high frequency of small regionalised deletions, defining at least three independent regions, one of which is particularly well mapped to a 500 kb stretch of DNA in the distal portion of the pseudoautosomal region of Xp. A second region has been identified within the pseudoautosomal region close to the pseudoautosomal boundary, and there is a third discrete site of loss on distal Xq. Perturbations of sequences at these regions represent independent events in a number of patients. This study represents the first detailed analysis of LOH on the X chromosome in human breast tumours, the results of which indicate that at least three regions of this chromosome are involved in the disease. © 1995 Wiley-Liss, Inc.     

Analysis of the cellular proto-oncogene mht/raf: relationship to the 5' sequences of v-mht in avian carcinoma virus MH2 and v-raf in murine sarcoma virus 3611

The avian carcinoma virus MH2 contains a hybrid gene delta gag-mht with a contiguous open reading frame of 2682 base pairs as well as v-myc and avian helper virus-related sequences. delta gag is a partial retroviral core protein gene while v-mht and v-myc are cell-drived sequences. The v-mht sequence can be divided into two regions: the v-raf-related region at its 3' end contains 969 nucleotides which are 94% related as amino acid sequence to the onc-specific v-raf sequence of murine sarcoma virus 3611 (MSV 3611), and the v-mht-specific region at its 5' end contains 173 nucleotides which are unrelated to either MSV 3611 or avian helper virus sequences. To study the origin of the v-mht-specific sequences, the 5' region of the proto-mht/raf gene was molecularly cloned from a phage lambda library containing genomic chicken sequences. Nucleic acid hybridization, heteroduplex and DNA sequence analyses indicate that the v-mht-specific sequences are encoded in three exons. The first and second exons are separated by a 3.4-kb intron while the second and third exons are separated by a 90-bp intron. The last 14 bp of the third exon are shared with v-raf and thus represent the start of v-raf-related sequences. The junction between v-mht-unrelated and related cellular sequences occurs within the first exon. There is no homology between the v-mht-unrelated sequences and the retroviral helper sequences indicating that the viral transduction of the proto-mht/raf sequences occurred through illegitimate recombination. The predominant v-mht-related messenger RNA (4.0 kb) hybridizes to several noncontiguous regions on the molecularly cloned cellular proto-mht/raf DNA indicating that the proto-mht/raf gene is distributed over at least 10 kb of DNA in the chicken genome. Thus the v-mht oncogene is a subset of its normal cellular homolog in that it lacks intervening sequences and possibly lacks 5'-coding sequences.