Purification of growth hormone-specific transcription factor GHF-1 containing homeobox.

Pituitary-specific expression of the growth hormone (GH) gene is governed by a transcription factor, GHF-1, that binds to two sites within its promoter. Recently, GHF-1 was shown to be a member of the homeobox family of DNA-binding proteins. An important question is whether GHF-1 controls the expression of other pituitary specific genes, such as prolactin (Prl), expressed in closely related cell types. To this end, GHF-1 was purified from extracts of GH- and Prl-expressing pituitary tumor cells and identified as a 33-kilodalton polypeptide. Although GHF-1 bound to and activated the GH promoter, it did not recognize the Prl promoter. However, at least one other factor in the same extracts, which was easily separated from GHF-1, bound to several sites within the Prl but not the GH promoter. Antibodies to GHF-1 did not react with the Prl binding activity. These results imply that the pituitary-specific expression of GH and Prl is governed by two distinct trans-acting factors.

"The devil's in the detail": Release of an expanded, enhanced and dynamically revised forensic STR Sequence Guide.

The STR sequence template file published in 2016 as part of the considerations from the DNA Commission of the International Society for Forensic Genetics on minimal STR sequence nomenclature requirements, has been comprehensively revised and audited using the latest GRCh38 genome assembly. The list of forensic STRs characterized was expanded by including supplementary autosomal, X- and Y-chromosome microsatellites in less common use for routine DNA profiling, but some likely to be adopted in future massively parallel sequencing (MPS) STR panels. We outline several aspects of sequence alignment and annotation that required care and attention to detail when comparing sequences to GRCh37 and GRCh38 assemblies, as well as the necessary matching of MPS-based allele descriptions to previously established repeat region structures described in initial sequencing studies of the less well known forensic STRs. The revised sequence guide is now available in a dynamically updated FTP format from the STRidER website with a date-stamped change log to allow users to explore their own MPS data with the most up-to-date forensic STR sequence information compiled in a simple guide.

Variability in neuronal activity in primate cortex during working memory tasks.

Persistent elevated neuronal activity has been identified as the neuronal correlate of working memory. It is generally assumed in the literature and in computational and theoretical models of working memory that memory-cell activity is stable and replicable; however, this assumption may be an artifact of the averaging of data collected across trials, and needs experimental verification. In this study, we introduce a classification scheme to characterize the firing frequency trends of cells recorded from the cortex of monkeys during performance of working memory tasks. We examine the frequency statistics and variability of firing during baseline and memory periods. We also study the behavior of cells on individual trials and across trials, and explore the stability of cellular firing during the memory period. We find that cells from different firing-trend classes possess markedly different statistics. We also find that individual cells show substantial variability in their firing behavior across trials, and that firing frequency also varies markedly over the course of a single trial. Finally, the average frequency distribution is wider, the magnitude of the frequency increases from baseline to memory smaller, and the magnitude of frequency decreases larger than is generally assumed. These results may serve as a guide in the evaluation of current theories of the cortical mechanisms of working memory.

Effects of keratinocyte and hepatocyte growth factor in vivo: implications for retrovirus-mediated gene transfer to liver.

We have previously shown that intravenous administration of keratinocyte growth factor (KGF) induces hepatocyte proliferation, allowing for efficient and noninvasive in vivo gene transfer with high-titer retroviral vectors in mice. The distinctive periportal distribution of transduced cells led us to investigate the ability of virus-sized particles to perfuse the liver adequately after growth factor treatment. We found that perfusion was adequate, and that transduction was limited to the periportal region because only those cells were stimulated to divide. Cells in this region also showed increased expression of Ram-1, the receptor for the murine Moloney leukemia virus (MoMLV) amphotropic envelope, after KGF treatment. In further studies we found that recombinant hepatocyte growth factor (HGF) induces a different population of hepatocytes to divide and upregulate Ram-1. The differential pattern of induction suggested that combining KGF and HGF would improve gene transfer efficiency further. Indeed, simultaneous delivery of both growth factors leads to an overall increase in the number of proliferating cells. Importantly, when coupled with MoMLV delivery, efficiency of gene transfer increased. These results confirm the utility of growth factors for noninvasive hepatic gene transfer in mice, and demonstrate how experiments to define the mechanism of transduction can be taken advantage of to develop improved gene transfer protocols.

High-frequency transitions in cortical spike trains related to short-term memory.

Single-unit spike trains recorded from parietal cortex of monkeys performing a tactile short-term memory task show characteristic fluctuations (transitions) in their firing frequency that are related to memory. Spike trains recorded during the memory period, when the animal must retain information for the short term, show a higher rate of such transitions than spike trains recorded during intertrial baseline periods. In the present study, an analysis of multiple temporal resolutions over which these transitions are observed reveals that the memory-related transitions occur most prominently in the 25-50 Hz range. The results of this study suggest that, in the monkey, high frequency fluctuations of neuronal discharge in the parietal cortex are correlated with haptic short-term memory. The presence of such fluctuations are also consistent with theoretical models of short-term memory.

Auditory memory cells in dorsolateral prefrontal cortex.

The activity of single neurons was recorded extracellularly from dorsolateral prefrontal cortex (DPC) of monkeys during the performance of a cross-modal audio-visual short-term memory task. Cells in DPC show sustained elevated firing levels (higher than spontaneous discharge) during the retention of the auditory stimulus. In some cells this elevated firing was significantly different depending on the particular auditory memorandum of each trial. These results support the notion that DPC participates in auditory short-term memory and the integration of auditory and visual information for prospective action.

Structure and expression of the vasoactive intestinal peptide (VIP) gene in a human tumor.

To identify the VIP biosynthetic pathways, we have isolated the human VIP gene, using synthetic oligodeoxynucleotides. These specific hybridization probes were constructed according to the neuroblastoma VIP-cDNA sequence and contained up to 39 bases. The gene structure was deduced by direct chemical nucleotide sequencing. Six exons were thus far discovered; among them two short exons, one encoding VIP and the second encoding PHM-27 (a peptide having a N-terminal histidine and C-terminal methionine amide, closely related in sequence and activity to VIP). As a model system for VIP gene expression, we used a human buccal tumor producing elevated amounts of VIP. In these cells, a major transcript of the VIP-gene was identified as a long RNA containing intron sequences. The occurrence of elevated quantities of a high molecular weight, intron containing, gene transcript which is not processed directly into mature RNA suggests that VIP gene expression may be regulated at the RNA processing level.

Studies toward the biosynthesis of vasoactive intestinal peptide (VIP).

In view of the potential biological importance of VIP, we have begun to examine the regulation of its biosynthesis. For this purpose we have, as a first step, searched for an enriched source of VIP biosynthesis. By a combination of chromatographic procedures and radioimmunoassays we discovered an as yet unknown source for VIP production, namely a human buccal tumor, containing 0.67 +/- 0.05 ng VIP/micrograms protein which is greater than the richest source in brain (the cerebral cortex). Thus, we decided to use the tumor tissue for VIP-mRNA purification and characterization. To identify VIP-mRNA we are using as hybridization probes, synthetic oligodeoxynucleotides with relatively unambiguous nucleotide sequence complementary to the predicted VIP-mRNA sequence. These probes are synthesized, using the deoxynucleoside phosphoramidite approach, to a length of 17 bases each, and contain all the possible DNA sequences according to the genetic code. These specific probes are then radioactively labelled using the reaction catalyzed by the enzyme polynucleotide kinase and afterwards hybridized to mRNA, which had been resolved on denaturing agarose gels. Employing this approach, we identified a single putative VIP-mRNA band which was then partially purified by sucrose gradient centrifugation. Upon in vitro translation in a rabbit reticulocyte lysate cell free system, this mRNA was found to code for VIP immunoreactive proteins. In conclusion, our studies suggest the existence of high molecular weight precursors to VIP cross-reactive with anti-VIP antibodies, that are coded for by a partially purified mRNA containing VIP sequences.

A review of the concept of the heart rate deflection point.

The heart rate deflection point (HRDP) is a downward or upward change from the linear HR-work relationship evinced during progressive incremental exercise testing. The HRDP is reported to be coincident with the anaerobic threshold. In 1982, Conconi and colleagues suggested that this phenomenon could be used as a noninvasive method to assess the anaerobic threshold. These researchers developed a field test to assess the HRDP, which has become popularised as the 'Conconi test'. Concepts used to define and assess the anaerobic threshold as well as methodological procedures used to determine the HRDP are diverse in the literature and have contributed to controversy surrounding the HRDP concept. Although the HRDP may be assessed in either field or laboratory settings, the degree of HR deflection is highly dependent upon the type of protocol used. The validity of HRDP to assess the anaerobic threshold is uncertain, although a high degree of relationship exists between HRDP and the second lactate turnpoint. The HRDP appears to be reliable when a positive identification is made; however, not all studies report 100% reproducibility. Although the physiological mechanisms explaining the HRDP are unresolved, a relationship exists between the degree and direction of HRDP and left ventricular function. The HRDP has potential to be used for training regulation purposes. Clinically, it may be incorporated to set exercise intensity parameters for cardiac rehabilitation.

Symmetric temporal patterns in cortical spike trains during performance of a short-term memory task.

The trion model is a highly structured representation of cortical organization, which predicts families of symmetric spatial-temporal firing patterns inherent in cortical activity. The symmetries of these inherent firing patterns are used by the brain in short-term memory to perform higher level computations. In the present study, symmetric temporal patterns were searched for in spike trains recorded from cells in parietal cortex of a monkey performing a short-term memory task. A new method of analysis was used to map neuronal firing into sequences of integers representing relative levels of firing rate about the mean (i.e. -1, 0 and 1). The results of this analysis show families of patterns related by symmetry operations. These operations are: i. the interchanging of all the +1's and -1's in a given pattern sequence (C symmetry), ii. the inverting of the temporal sequence of the mapping (T symmetry), and iii. the combination of the two previous operations (CT symmetry). Patterns of a given family are found across cells, especially in the memory periods of the task; in most cases they reoccur within a given spike train. The pattern families predicted by the model and reported here should be further investigated in multiple microelectrode and EEG recordings.

FMRI study relevant to the Mozart effect: brain areas involved in spatial-temporal reasoning.

Behavioral studies, motivated by columnar cortical model predictions, have given evidence for music causally enhancing spatial-temporal reasoning. A wide range of behavioral experiments showed that listening to a Mozart Sonata (K.448) gave subsequent enhancements. An EEG coherence study gave evidence for a carryover from that Mozart Sonata listening condition to the subsequent spatial-temporal task in specific cortical regions. Here we present fMRI studies comparing cortical blood flow activation by the Mozart Sonata vs. other music. In addition to expected temporal cortex activation, we report dramatic statistically significant differences in activation by the Mozart Sonata (in comparison to Beethoven's Fur Elise and 1930s piano music) in dorsolateral pre-frontal cortex, occipital cortex and cerebellum, all expected to be important for spatial-temporal reasoning. It would be of great interest to explicitly test this expectation. We propose an fMRI study comparing (subject by subject) brain areas activated in music listening conditions and in spatial-temporal tasks.

Effect of supercooling and freezing on photosynthesis in freezing tolerant leaves of Afroalpine 'giant rosette' plants.

The effect of supercooling and freezing on the photosynthetic capability of representatives of the permanent frost hardy giant rosette plants Dendrosenecio keniodendron, D. brassica and Lobelia telekii, of the tropical alpine regions was investigated with the non-invasive chlorophyll a fluorescence technique. While supercooling, normal chlorophyll a fluorescence kinetics exhibiting the sequence 0, I, (D), P, S, M, were recorded, however with some retardation of both, the fast and the slow characteristics as compared to those obtained at day-time temperature. As long as the leaves remained unfrozen, the rise of the variable fluorescence F ν from the level 0 to P was inversely related to a drop of the temperature from about 0°C to-8°C. The increase of F ν with lower temperature is understood to result from a decrease of the velocity of the quenching reactions while photoreduction of the primary electron acceptor appeared to be unimpeded. The second fluorescence maximum (M), usually interpreted to indicate the commencement of the biochemical reactions of photosynthesis was consistenly to be observed during supercooling. Fluoescence induction kinetics of frozen leaves showed only fast rise to presumably F max which was not followed by a significant decay for as long as 4 min. The lack of substantial quenching indicates that in the freeze-dehydrated state neither reoxidation of the primary acceptor nor energetization of the thylakoid membrane was accomplished. This effect however was immediately and fully reserved upon thawing of the leaves when the usual fluorescence induction kinetics as well as normal rates of CO2-uptake were observed. Thus the permanent frost-hardy afroalpine plants do not exhibit any even short-term memory effect of the nocturnal frost on such a delicate process as is photosynthesis.

Music enhances spatial-temporal reasoning: towards a neurophysiological basis using EEG.

Motivated by predictions from the structured trion model of the cortex, based on Mountcastle's columnar organizational principle, behavioral experiments have demonstrated a causal short-term enhancement of spatial-temporal reasoning in college students following listening to a Mozart Sonata (K.448) but not in control conditions. An EEG coherence study reported presence of right frontal and left temporoparietal activity induced by listening to the Mozart Sonata, which carried over into the spatial-temporal tasks in three of the seven subjects. In this paper, we present further predictions from the trion model and discuss how the new SYMMETRIC analysis method can be used in EEG recordings to help determine the neurophysiological basis of specific music enhancing spatial-temporal reasoning. We conclude with potential clinical applications of major significance.

Detecting symmetric patterns in EEG data: a new method of analysis.

Theoretical models of higher cognitive function predict that cortical activity will exhibit families of spatial-temporal patterns of activity whose individual members are related to each other by specific symmetry transformations. In the trion model, it is suggested that these inherent symmetries play a vital role in how we think and reason. We have developed a method of analysis (SYMMETRIC analysis), which detects families of patterns in EEG data, and characterizes the symmetry relationships between members of those pattern families. Using this analysis, significant symmetry families have been found in EEG and single unit spike train data. If symmetry is a crucial aspect of brain function, it is possible that different pathologies are associated with specific types of symmetry relationships in brain activity that could be detected in EEG data by a SYMMETRIC analysis.

Blood transfusion and blood conservation: cost and utilization issues.

Approximately 12 million red blood cell units are transfused to nearly 4 million patients annually in the United States (1). The conservation of blood has historically arisen from awareness that the inventory of this resource is limited (2), as well as the knowledge that blood transfusion carries a risk (3). Estimates of current blood transfusion risks (4-12), and the costs of transfusion complications (13-17), are summarized in Table 1. In addition, emphasis on the costs of health care has raised issues related to the costs of blood transfusion (18, 19). Finally, recent guidelines have emphasized that in the elective transfusion setting, no blood transfusion is a desirable outcome (20, 21). Furthermore, these guidelines along with consensus conference recommendations (22) have emphasized that if blood is to be transfused, autologous (the patient's own) blood is preferable to allogeneic (from an anonymous, volunteer donor) blood. Thus, the costs of blood conservation, for which an increasing array of technologic procedures and products have become available (Table 2), have also become an issue (23). The purpose of this review is to provide an overview of emerging data on the cost-effectiveness of blood and blood conservation interventions in order to help identify areas important for future investigation.

Patterned firing of parietal cells in a haptic working memory task.

Abstract Cells in the somatosensory cortex of the monkey are known to exhibit sustained elevations of firing frequency during the short-term mnemonic retention of tactile information in a haptic delay task. In this study, we examine the possibility that those firing elevations are accompanied by changes in firing pattern. Patterns are identified by the application of a pattern-searching algorithm to the interspike intervals of spike trains. By sequential use of sets of pattern templates with a range of temporal resolutions, we find patterned activity in the majority of the cells investigated. In general, the degree of patterning significantly increases during active memory. Surrogate analysis suggests that the observed patterns may not be simple linear stochastic functions of instantaneous or average firing frequency. Therefore, during the active retention of a memorandum, the activity of a 'memory cell' may be characterized not only by changes in frequency but also by changes in pattern.

A pituitary-specific trans-acting factor can stimulate transcription from the growth hormone promoter in extracts of nonexpressing cells.

The growth hormone (GH) gene is specifically expressed in the anterior pituitary. The first 289 bp of the human (h)GH 5' region contains a promoter that is active only in pituitary-derived cell lines. These cell lines also contain a specific factor, GHF-1, that binds to two sites upstream to the hGH TATA box. Using in vitro transcription systems we demonstrate greater hGH promoter activity in extracts of GH-expressing GC cells than in extracts of nonexpressing HeLa cells. The two GHF-1 binding sites are essential promoter elements in GC extracts. Addition of GHF-1-containing fractions to HeLa extracts stimulates hGH promoter activity. Thus, GHF-1 appears to be a positively acting, cell-type- and promoter-specific transcription factor. Furthermore, the tissue specificity of the hGH promoter appears to be positively controlled by the abundance or activity of GHF-1.

Antiemetic efficacy of ondansetron after outpatient laparoscopy.

The safety and efficacy of ondansetron were evaluated for the treatment of postoperative nausea and vomiting after laparoscopic surgical procedures. Seventy-one healthy, consenting outpatients were randomly assigned to one of two treatment groups according to a double-blind, placebo-controlled protocol. A standardized anesthetic technique consisting of alfentanil-thiopental-succinylcholine for induction and alfentanil-nitrous oxide-succinylcholine for maintenance of anesthesia was used. Patients in whom postoperative nausea and/or vomiting developed and persisted for greater than or equal to 10 min received equivolemic intravenous injections of either ondansetron (8 mg) or saline (placebo) over a 2-5 min period. Ondansetron significantly decreased the posttreatment nausea scores (vs placebo) without increasing sedation or producing changes in cardiorespiratory parameters. In the placebo-treated group, 92% of the patients experienced subsequent episodes of vomiting in the postanesthesia care unit compared with 51% of the patients in the ondansetron group. Finally, only 43% of the ondansetron-treated patients required a "rescue" antiemetic compared with 86% in the placebo group. Thus, ondansetron (8 mg IV) was associated with a decreased incidence of nausea and vomiting after outpatient laparoscopic procedures.

Coding sequences for vasoactive intestinal peptide and PHM-27 peptide are located on two adjacent exons in the human genome.

The human precursor gene for vasoactive intestinal peptide (VIP) and PHM-27, a peptide that has an NH2-terminal histidine and COOH-terminal methionine amide and is closely related in sequence and activity to VIP, was detected with synthetic oligodeoxynucleotide probes. These specific hybridization segments were constructed according to the neuroblastoma VIP cDNA sequence and contained up to 39 bases. The gene structure was partly deduced by hybridization to synthetic oligodeoxynucleotide probes and partly by direct chemical nucleotide sequencing. Four exons were discovered thus far; among them are two short exons separated by a 0.75-kilobase DNA stretch, one encoding PHM-27 and the second encoding VIP (exons 1 and 2). Each of these two exons encodes both the hormone amino acid residues as well as the post-translational processing signal sequences. The 3' splice sites of the two exons contain an identical stretch of nine nucleotides. At the cDNA level, the 3' splice sites contain the same stretch of six nucleotides, which are identically spliced. The occurrence of VIP and PHM-27 coding sequences on two separate exons of the human genome and the homology of their 3' splice site may allow alternative RNA processing as discussed below.