Absolute requirement for STAT3 function in small-intestine crypt stem cell survival.

The transcription factor signal transducer and activator of transcription 3 (STAT3) is frequently activated in human cancers. Interestingly, STAT3 also maintains the pluripotency and self-renewal of murine embryonic stem cells, and several tissue stem cell types. To investigate whether STAT3 also maintains the small-intestine crypt stem cell, we conditionally inactivated a Floxed Stat3 allele (Stat3(fl)) in murine small-intestine crypt stem cells. Following Cre recombinase expression, apoptosis increased in Stat3(fl/-) experimental crypts relative to Stat3(wt/-) controls before declining. Control Stat3(wt/-) mice carrying a Flox-STOP LacZ reporter transgene stably expressed LacZ after Cre induction. In contrast, Stat3(fl/-) intestine LacZ expression initially increased modestly, before declining to background levels. Quantitative PCRs revealed a similar transient in recombined Stat3(fl) allele levels. Long-term bromodeoxyuridine labelling directly demonstrated that functional STAT3 is required for +4 to +6 region label-retaining small-intestine stem cell survival. Rapid clearance of recombined Stat3(fl/-) cells involves apoptosis potentially induced by elevated c-Myc in non-recombined cells and involves elevated p53 expression and caspase 3 activation. Intriguingly, Stat3(fl/-) intestine recombination triggered dramatically upregulated polycomb transcriptional repressor Bmi1 - potentially accelerating recombined crypt repopulation. In summary, STAT3 activity is absolutely required for small-intestine crypt stem cell survival at both the +4 to +6 label-retaining and crypt base columnar cell locations.

Early response to light therapy partially predicts long-term antidepressant effects in patients with seasonal affective disorder.

OBJECTIVE: To determine if the antidepressant effect of 1 hour of light therapy is predictive of the response after 1 and 2 weeks of treatment in patients with seasonal affective disorder (SAD). PATIENTS: Twelve patients with SAD. SETTING: National Institutes of Health Clinical Center, Bethesda, Md. INTERVENTIONS: Light therapy for 2 weeks. OUTCOME MEASURES: Scores on the Seasonal Affective Disorder Version of the Hamilton Depression Rating Scale (SIGH-SAD) on 4 occasions (before and after 1 hour of light therapy and after 1 and 2 weeks of therapy) in the winter when the patients were depressed. Change on typical and atypical depressive scores at these time points were compared. RESULTS: Improvement of atypical depressive symptoms after 1 hour of light therapy positively correlated with improvement after 2 weeks of therapy. CONCLUSION: In patients with SAD, the early response to light therapy may predict some aspects of long-term response to light therapy, but these results should be treated with caution until replicated.

Evidence from the waking electroencephalogram that short sleepers live under higher homeostatic sleep pressure than long sleepers.

We used the waking electroencephalogram to study the homeostatic sleep regulatory process in human short sleepers and long sleepers. After sleeping according to their habitual schedule, nine short sleepers (sleep duration < 6 h) and eight long sleepers (> 9 h) were recorded half-hourly during approximately 40 h of wakefulness in a constant routine protocol. Within the frequency range of 0.25-20.0 Hz, spectral power density in the 5.25-9.0 and 17.25-18.0 Hz ranges was higher in short sleepers than in long sleepers. In both groups, increasing time awake was associated with an increase of theta/low-frequency alpha activity (5.25-9.0 Hz), whose kinetics followed a saturating exponential function. The time constant did not differ between groups and was similar to the previously obtained time constant of the wake-dependent increase of slow-wave activity (0.75-4.5 Hz) in the sleep electroencephalogram. In addition, the time constant of the decrease of slow-wave activity during extended recovery sleep following the constant routine did not differ between groups. However, short sleepers showed an abiding enhancement of theta/low-frequency alpha activity during wakefulness after recovery sleep that was independent of the homeostatic process. It is concluded that, while the kinetics of the homeostatic process do not differ between the two groups, short sleepers live under and tolerate higher homeostatic sleep pressure than long sleepers. The homeostat-independent enhancement of theta/low-frequency alpha activity in the waking electroencephalogram in the short sleepers may be genetically determined or be the result of long-term adaptation to chronically short sleep.

Evidence of a role for SHP-1 in platelet activation by the collagen receptor glycoprotein VI.

The Src homology (SH)2 domain-containing protein-tyrosine phosphatase SHP-1 is tyrosine phosphorylated in platelets in response to the glycoprotein VI (GPVI)-selective agonist collagen-related peptide (CRP), collagen, and thrombin. Two major unidentified tyrosine-phosphorylated bands of 28 and 32 kDa and a minor band of 130 kDa coprecipitate with SHP-1 in response to all three agonists. Additionally, tyrosine-phosphorylated proteins of 50-55 and 70 kDa specifically associate with SHP-1 following stimulation by CRP and collagen. The tyrosine kinases Lyn, which exists as a 53 and 56-kDa doublet, and Syk were identified as major components of these bands, respectively. Kinase assays on SHP-1 immunoprecipitates performed in the presence of the Src family kinase inhibitor PP1 confirmed the presence of a Src kinase in CRP- but not thrombin-stimulated cells. Lyn, Syk, and SLP-76, along with tyrosine-phosphorylated 28-, 32-, and 130-kDa proteins, bound selectively to a glutathione S-transferase protein encoding the SH2 domains of SHP-1, suggesting that this is the major site of interaction. Platelets isolated from motheaten viable mice (mev/mev) revealed the presence of a heavily tyrosine-phosphorylated 26-kDa protein that was not found in wild-type platelets. CRP-stimulated mev/mev platelets manifested hypophosphorylation of Syk and Lyn and reduced P-selectin expression relative to controls. These observations provide evidence of a functional role for SHP-1 in platelet activation by GPVI.

Two circadian rhythms in the human electroencephalogram during wakefulness.

The influence of the circadian pacemaker and of the duration of time awake on the electroencephalogram (EEG) was investigated in 19 humans during approximately 40 h of sustained wakefulness. Two circadian rhythms in spectral power density were educed. The first rhythm was centered in the theta band (4.25-8.0 Hz) and exhibited a minimum approximately 1 h after the onset of melatonin secretion. The second rhythm was centered in the high-frequency alpha band (10.25-13.0 Hz) and exhibited a minimum close to the body temperature minimum. The latter rhythm showed a close temporal association with the rhythms in subjective alertness, plasma melatonin, and body temperature. In addition, increasing time awake was associated with an increase of power density in the 0.25- to 9.0-Hz and 13.25- to 20. 0-Hz ranges. It is concluded that the waking EEG undergoes changes that can be attributed to circadian and homeostatic (i.e., sleep-wake dependent) processes. The distinct circadian variations of EEG activity in the theta band and in the high-frequency alpha band may represent electrophysiological correlates of different aspects of the circadian rhythm in arousal.

Gender differences in glycosylated hemoglobin levels in seasonal affective disorder patients and controls.

Seasonal affective disorder (SAD) has been shown to manifest different symptoms in female and male patients. Specifically, women with SAD have been shown to have greater increases in overeating, weight gain, and increased sleep as compared with their male counterparts. Given these dietary changes, we predicted that female SAD patients would exhibit increased glycosylated hemoglobin (HbA1) levels, indicative of chronically elevated glucose levels. Twenty-two patients (15 women and seven men) and matched controls were enrolled during the winter season and tested for HbA1 levels. A three-way analysis of variance (ANOVA; gender x group x season) was insignificant and the result was a negative study. After the initial hypothesis was rejected, we undertook a post-hoc analysis of the data, from which emerged that in winter, women patients had higher HbA1 levels as compared with matched controls. As our original hypothesis was rejected, we cannot accept the results of the post-hoc study. However, numerous other studies have demonstrated that female and male SAD patients differ in their pathophysiology, and are suggestive that in future analyses ought to consider analyzing subjects separately across gender.

Practice guidelines and tort reform: the legal system confronts the technocratic wish.

Recent scholarly writing has argued that the advent of managed care within the health policy arena can be seen as a contemporary manifestation of a broader set of concerns focusing on how objective decision procedures become politically legitimated--what one recent commentator has characterized as a faith in the technocratic wish. In the 1990s, this faith in objective decision procedures has manifested itself through the emergence of outcomes assessment and the development of practice guidelines. Notably, a few states have sought to couple the practice guidelines movement with tort reform by enacting demonstration projects permitting physicians to introduce evidence that they followed practice guidelines as an affirmative defense. In this article, I argue that even though the introduction of practice guidelines may promote the policy objective of cost-effectiveness in the delivery of health care services, their use to establish culpability in actual cases may be more difficult because the structure of legal reasoning focuses on the particular facts in the case at hand rather than appealing to abstract decision procedures. By highlighting the potential difficulties of invoking practice guidelines in the adjudication of actual malpractice cases, I demonstrate how a process of ongoing political negotiation will be necessary if the technocratic faith in practice guidelines is to become justified in reality.

Monorhinal odor identification and depression scores in patients with seasonal affective disorder.

BACKGROUND: Visual and olfactory pathways are interconnected. Olfactory deafferentation unmasks photoperiodic responsiveness in some nonphotoperiodic animals such as laboratory rats. By analogy, we hypothesized that olfactory deficits may unmask seasonal rhythms in certain individuals, namely those with seasonal affective disorder (SAD). Since previous studies suggest lateralized hemispheric dysfunction in SAD, and since olfactory neurons' primary projections are largely ipsilateral, we assessed olfactory identification performance on both the right and left side of the nose. METHODS: Twenty-four patients with SAD and 24 matched controls were studied using a phenyl ethyl alcohol detection threshold test bilaterally and the University of Pennsylvania Smell Identification Test unilaterally. Subjects rated their mood using the Self Assessment Mood Scale for SAD. Patients' testing was done in both 'depressed' and 'improved on light' states. RESULTS: No difference in olfactory performance was found between patients and controls or between patients before and after light treatment. However, right-side identification scores were negatively correlated with 'typical' depression scores (r = -0.56, P = 0.006), while left-side olfactory scores were not. Atypical depression scores were unrelated to olfactory performance. Similar correlations emerged between the olfactory identification laterality quotient (Right - Left)/(Right + Left) and typical depressive scores (r = - 0.64, P < 0.001) and total depression scores (r = - 0.59, P < 0.004). LIMITATIONS: We studied a demographically heterogeneous sample and did not control for menstrual factors. DISCUSSION: Our results add to previous evidence of lateralized hemispheric involvement in SAD and suggest that olfaction may be related to seasonal emotional rhythms in humans.

Greater improvement in summer than with light treatment in winter in patients with seasonal affective disorder.

OBJECTIVE: The authors sought to compare the degree of mood improvement after light treatment with mood improvement in the subsequent summer in patients with seasonal affective disorder. METHOD: By using the Seasonal Affective Disorder Version of the Hamilton Depression Rating Scale, the authors rated 15 patients with seasonal affective disorder on three occasions: during winter when the patients were depressed, during winter following 2 weeks of light therapy, and during the following summer. They compared the three conditions by using Friedman's analysis of variance and the Wilcoxon signed ranks test. RESULTS: The patients' scores on the depression scale were significantly higher after 2 weeks of light therapy in winter than during the following summer. CONCLUSIONS: Light treatment for 2 weeks in winter is only partially effective when compared to summer. Further studies will be necessary to assess if summer's light or other factors are the main contributors to this difference.

Effects of tryptophan depletion vs catecholamine depletion in patients with seasonal affective disorder in remission with light therapy.

BACKGROUND: Although hypotheses about the therapeutic mechanism of action of light therapy have focused on serotonergic mechanisms, the potential role, if any, of catecholaminergic pathways has not been fully explored. METHODS: Sixteen patients with seasonal affective disorder who had responded to a standard regimen of daily 10000-lux light therapy were enrolled in a double-blind, placebo-controlled, randomized crossover study. We compared the effects of tryptophan depletion with catecholamine depletion and sham depletion. Ingestion of a tryptophan-free amino acid beverage plus amino acid capsules was used to deplete tryptophan. Administration of the tyrosine hydroxylase inhibitor alpha-methyl-paratyrosine was used to deplete catecholamines. Diphenhydramine hydrochloride was used as an active placebo during sham depletion. The effects of these interventions were evaluated with measures of depression, plasma tryptophan levels, and plasma catecholamine metabolites. RESULTS: Tryptophan depletion significantly decreased plasma total and free tryptophan levels. Catecholamine depletion significantly decreased plasma 3-methoxy-4-hydroxyphenylethyleneglycol and homovanillic acid levels. Both tryptophan depletion and catecholamine depletion, compared with sham depletion, induced a robust increase (P<.001, repeated-measures analysis of variance) in depressive symptoms as measured with the Hamilton Depression Rating Scale, Seasonal Affective Disorder Version. CONCLUSIONS: The beneficial effects of light therapy in the treatment of seasonal affective disorder are reversed by both tryptophan depletion and catecholamine depletion. These findings confirm previous work showing that serotonin plays an important role in the mechanism of action of light therapy and provide new evidence that brain catecholaminergic systems may also be involved.

Hb Auckland [alpha 87(F8) His-->Asn]: a new mutation of the proximal histidine identified by electrospray mass spectrometry.

Hb Auckland is a newly described unstable hemoglobin with a mutation of alpha 97(F8)His-->Asn. This substitution, involving the proximal histidine, does not lead to methemoglobinemia, but to instability and accelerated heme loss. The clinical picture is of a mild compensated hemolytic anemia. The presence of an abnormal hemoglobin was first demonstrated by the isopropanol stability test and confirmed by electrospray ionization mass spectrometry of total lysate. This showed that 14% of the alpha chains had a mass of 15,103.4 Da, i.e. 23 Da less than normal. Examination of tryptic digests showed an identical decrease in mass for peptide alpha T-9 (from 2,997.4 to 2,974.5 Da). Subdigestion with endoproteinase Asp-N located the 23 Da loss to residues alpha 85-90, and further digestion with thermolysin identified the mutation as His-->Asn at position 87 of the alpha chain. This was confirmed by sequence analysis of the peptide alpha 85-90.

Insects as teaching tools in primary and secondary education.

Insects offer a vast array of teaching opportunities for precollege students. Here we address the basics teachers need in order to use insects successfully in their curricula. We identify exemplary resources in the printed North American literature and point out potentially productive places for teachers and students to search for ideas and materials. We review the roles of entomology in the educational framework, highlight favorite classroom arthropods and less well-known examples, and guide readers to entomological resources. Tips to help teachers identify, rear, and maintain classroom insects and find equipment and supplies are included. The review concludes with a plea for greater classroom and curricular involvement by those in the entomological profession.

The Stroke Prevention Policy Model: linking evidence and clinical decisions.

Simulation models that support decision and cost-effectiveness analysis can further the goals of evidence-based medicine by facilitating the synthesis of information from several sources into a single comprehensive structure. The Stroke Prevention Policy Model (SPPM) performs this function for the clinical and policy questions that surround stroke prevention. This paper first describes the basic structure and functions of the SPPM, concentrating on the role of large databases (broadly defined as any database that contains many patients, regardless of study design) in providing the SPPM inputs. Next, recognizing that the use of modeling continues to be a source of some controversy in the medical community, it discusses the philosophical underpinnings of the SPPM. Finally, it discusses conclusions in the context of both stroke prevention and other complex medical decisions. We conclude that despite the difficulties in developing comprehensive models (for example, the length and complexity of model development and validation processes, the proprietary nature of data sources, and the necessity for developing new software), the benefits of such models exceed the costs of continuing to rely on more conventional methods. Although they should not replace the clinician in decision making, comprehensive models based on the best available evidence from large databases can support decision making in medicine.

Dynamics of the human EEG during prolonged wakefulness: evidence for frequency-specific circadian and homeostatic influences.

The electroencephalogram (EEG) of nine healthy individuals was recorded at half-hourly intervals during approximately 40 h of sustained wakefulness in a constant routine protocol. EEG power density in the 0.75-9.0 Hz range exhibited a global increasing trend, and a local trough in the evening, centered approximately 6 h prior to the temperature minimum. The former could be attributed to a wake-dependent influence, and the latter to a circadian influence. Power density in the 9.25-12.0 Hz band showed a circadian modulation, the trough coinciding with the minimum of the endogenous rhythm of body temperature, whereas a wake-dependent influence was not evident. Power density in the 12.25-25.0 Hz range exhibited a wake-dependent increase, whereas a circadian modulation was absent. It is concluded that the circadian pacemaker and the wake-dependent (i.e. homeostatic) process affect the waking EEG in a frequency-specific manner.

I(kappa)B(gamma) inhibits DNA binding of NF-kappaB p50 homodimers by interacting with residues that contact DNA.

NF-(kappa)B is an inducible transcription factor that activates many cellular genes involved in stress and immune response and whose DNA binding activity and cellular distribution are regulated by I(kappa)B inhibitor proteins. The interaction between NF-(kappa)B p50 and DNA was investigated by protein footprinting using chemical modification and partial proteolysis. Both methods confirmed lysine-DNA contacts already found in the crystal structure (K-147, K-149, K-244, K-275, and K-278) but also revealed an additional contact in the lysine cluster K-77-K-78-K-80 which was made on an extended DNA. Molecular modelling of such a DNA-protein complex revealed that lysine 80 is ideally placed to make phosphate backbone contacts in the extended DNA. Thus, it seems likely that the entire AB loop, containing lysines 77, 78, and 80, forms a C-shaped clamp that closes around the DNA recognition site. The same protein footprinting approaches were used to probe the interaction of p50 with the ankyrin repeat containing proteins I(kappa)B(gamma) and I(kappa)B(alpha). Lysine residues in p50 that were protected from modification by DNA were also protected from modification by I(kappa)B(gamma) but not I(kappa)B(alpha). Similarly, proteolytic cleavage at p50 residues which contact DNA was inhibited by bound I(kappa)B(gamma) but was enhanced by the presence of I(kappa)B(alpha). Thus, I(kappa)B(gamma) inhibits the DNA binding activity of p50 by direct interactions with residues contacting DNA, whereas the same residues remain exposed in the presence of I(kappa)B(alpha), which binds to p50 but does not block DNA binding.

Increased regional cerebral glucose metabolism and semantic memory performance in Alzheimer's disease: a pilot double blind transdermal nicotine positron emission tomography study.

Nicotinic receptor dysfunction and impaired semantic memory occur early in Alzheimer's disease patients (AD). Previous research implied that nicotine's ability to enhance alertness, arousal, and cognition in a number of nonclinical populations was a function of its ability to stimulate CNS nicotinic cholinergic receptors. In this study it was hypothesized that transdermal administration of nicotine would increase both regional cerebral glucose metabolism (rCMRglc) and semantic memory (as assessed by verbal fluency). Two mild AD and two elderly controls underwent positron emission tomography scanning during a double blind nicotinic agonist verbal fluency challenge procedure. rCMRglc increases occurred in both AD patients, but not controls. In the two AD patients, verbal fluency scores increased by an average of 17%. One elderly control's verbal fluency increased, and the other decreased. These findings suggest that nicotine's effect on metabolism and verbal fluency is due to its ability to stimulate the cholinergic system.

Inhibition of NF-kappaB DNA binding by nitric oxide.

It has been suggested that the NF-kappaB transcription factor family may mediate expression of the gene encoding the cytokine-inducible form of nitric oxide synthase (iNOS). To establish if nitric oxide (NO) could in turn affect activity of NF-kappaB, the ability of NO-donor compounds to influence NF-kappaB DNA binding activity in vitro was investigated. NO-donor compounds sodium nitroprusside (SNP) and S-nitroso-N-acetylpenicillamine (SNAP) both inhibited the DNA binding activity of recombinant NF-kappaB p50 and p65 homodimers and of p50-p65 heterodimers. Inhibition of NF-kappaB p50 DNA binding by NO-donor compounds involved modification of the conserved redox-sensitive C62 residue, as a C62S p50 mutant was significantly more resistant to SNP-mediated inactivation. Non-reducing SDS-polyacrylamide gel electrophoresis demonstrated that SNP could inhibit p50 DNA binding by mechanisms other than the formation of intersubunit disulphide bonds involving p50 residue C62. Electrospray ionization mass spectrometry of a synthetic NF-kappaB p5O peptide containing the C62 residue suggested that NO gas can modify C62 by S-nitrosylation. This study indicates that NO-donors can directly inhibit the DNA binding activity of NF-kappaB family proteins, suggesting that cellular NO provides another control mechanism for modulating the expression of NF-kappaB-responsive genes.

Regulation of the DNA binding activity of NF-kappa B.

The DNA binding activity of the dimeric sequence-specific transcription factor NF-kappa B can be controlled by a variety of post-translational mechanisms, including interactions with inhibitor proteins and by its redox state. The NF-kappa B family of transcription factors bind to kappa B motif sequences found in promoter and enhancer regions of a wide range of cellular and viral genes. Normally NF-kappa B family proteins are held in the cytoplasm in an inactive, non-DNA binding form by labile I kappa B inhibitor proteins. When the cell is activated by one of a wide range of stimuli, typically those associated with the cellular response to pathogens or stress, proteolytic degradation of I kappa B inhibitor proteins allows active NF-kappa B to translocate to the nucleus where it activates transcription of responsive genes. The initial trigger for I kappa B degradation is a signal-induced site-specific phosphorylation by an as yet unidentified kinase, which appears to target I kappa B for the covalent addition of multiple copies of the ubiquitin polypeptide. This modification subsequently allows the proteolytic degradation of the ubiquitinated I kappa B by the cellular 26S multicatalytic proteinase (proteasome) complex. It was recently shown that increased I kappa B-alpha expression in the cytoplasm leads to I kappa B-alpha accumulating in the nuclear compartment, removing template-bound NF-kappa B, and reducing NF-kappa B-dependent transcription. These NF-kappa B-I kappa B-alpha complexes could then be actively re-exported to the cytoplasm, allowing the cell to respond to further stimuli.

Conformational changes induced by DNA binding of NF-kappa B.

The transcription factor NF-kappa B makes extensive contacts with its recognition site over one complete turn of the double helix. Structural transitions, in both protein and DNA, that accompany formation of the DNA-protein complex were analysed by proteinase sensitivity and circular dichroism (CD) spectroscopy. In the absence of DNA chymotrypsin cleaved p50 after residues Y60 and N78, while proteinase K cleaved p50 after residues S74 and Q180. Previous experiments had indicated that trypsin cleaved p50 after K77. Cleavages after Y60, S74, K77 and N78 were blocked in the presence of bound DNA, whereas cleavage after Q180 was enhanced. Y60, S74, K77 and N78 are all located in the p50 N-terminal domain AB loop, whereas Q180 is located in the mainly alpha-helical region between p50 N-terminal domain beta-strands G' and H. As only Y60 makes direct contact with the DNA it is likely that the AB loop is highly unstructured in the absence of DNA, but is held in a rigid, proteinase-resistant structure by bound DNA. These conclusions were supported by CD spectroscopic studies of recombinant p50 and p65 homodimers, which indicated that both species changed conformation when binding DNA. Examination of the near UV CD spectra revealed that with some DNA sequences the bound and free forms of the DNA assumed different conformations. While this was evident for a fully symmetrical, high affinity recognition site DNA, it was not apparent with less tightly bound DNA.