Chemical and genetic diversity of high-seed-yield sorghum (Sorghum bicolor M.) germplasms.

This study evaluated the chemical and genetic diversity of high-seed-yield sorghum germplasms from Korea, the United States, and South Africa. We identified significant differences in the chemical contents of whole plants at the heading stage in all cultivars, including differences in crude protein, fat, fiber, ash, neutral detergent fiber, acid detergent fiber, mineral, and fatty acid contents. Our results suggest that Banwoldang is the most appropriate cultivar for roughage because of its high protein yield. We identified significant differences in the tannin, flavonoid, amylose, mineral, crude fat, fatty acid, and 3-deoxyanthocyanin contents in the whole grain from all cultivars, but not in the mineral or crude fat contents. Tannin levels were generally low. IS645 contained the highest levels of flavonoids and linolenic acid compounds, and Moktak had the highest amylose and deoxyanthocyanidin content in the grain. To assess genetic diversity, we used 10 simple sequence repeat (SSR) primer sets to identify 38 alleles with 3-8 alleles per locus. Based on phylogenetic analysis of the SSR markers, the sorghum cultivars were divided into three major groups. Comparison of clusters based on chemical compositions with those based on SSRs showed that the groups formed by the three native Korean cultivars clustered similarly in molecular dendrograms. Association analysis was conducted for the 10 SSR marker; 48 chemical and growth traits were present for two marker traits (seed color and whole plant fatty acid content) with significant marker-trait associations. These markers could be used to select sorghum cultivars for breeding programs.

Development of a transposon-based marker system for mutation breeding in sorghum (Sorghum bicolor L.).

Under certain circumstances, transposable elements (TE) can create or reverse mutations and alter the genome size of a cell. Sorghum (Sorghum bicolor L.) is promising for plant transposon tagging due to its small genome size and its low content of repetitive DNA. We developed a marker system based on targeted region amplification polymorphisms (TE-TRAP) that uses the terminal inverted repeats (TIRs) of transposons. A total of 3816 class 2 transposons belonging to the PIF/Harbinger family were identified from the whole sorghum genome that produced five primers, including eight types of TIRs. To define the applicability and utilization of TE-TRAP, we used 21 individuals that had been bred after ɤ-ray irradiation. In total, 31 TE-TRAP, 16 TD, and 21 AFLP primer combinations generated 1133, 223, and 555 amplicons, respectively. The percent polymorphic marker was 62.8, 51.1, and 59.3% for the TE-TRAP, TD, and AFLP markers, respectively. Phylogenetic and principal component analyses revealed that TE-TRAP divided the 21 individuals into three groups. Analysis of molecular variance suggested that TE-TRAP had a higher level of genetic diversity than the other two marker systems. After verifying the efficiency of TE-TRAP, 189 sorghum individuals were used to investigate the associations between the markers and the ɤ-ray doses. Two significant associations were found among the polymorphic markers. This TE-based method provides a useful marker resource for mutation breeding research.

The role of psychological distress in laryngopharyngeal reflux patients: a prospective questionnaire study.

OBJECTIVES: To determine the role of psychological distress in laryngopharyngeal reflux patients and evaluate the correlation between symptoms, laryngeal signs, pH monitoring results and psychological profile. DESIGN: Prospective study. SETTING: Hanyang University Hospital, a university teaching hospital and tertiary referral center. PARTICIPANTS: One hundred and six patients who were diagnosed with laryngopharyngeal reflux by 24-h ambulatory double probe pH monitoring and 119 healthy controls visiting our health promotion center from January 2006 to June 2007. MAIN OUTCOME MEASURES: The psychological profile of laryngopharyngeal reflux patients measured by the Symptom Checklist-90-Revised questionnaire were evaluated and compared with those of healthy controls. The correlation between reflux symptom index, reflux finding score, parameters of pH monitoring and the Symptom Checklist-90-Revised profiles were also evaluated. RESULTS: On the Symptom Checklist-90-Revised questionnaire, the total mean T-scores of the nine symptom dimensions and three global indices of the laryngopharyngeal reflux patients were all below 50. The Global Severity Index, which indicates overall psychological distress, was normal in all of the patients. On comparison with the control group, no statistically significant difference was noted in the psychological profile except on the Somatisation scale where laryngopharyngeal reflux patients showed significantly higher scores. Reflux symptom index showed significant positive correlation with the number of reflux episodes, percentage of time which pH fell below 4 in total positions, and DeMeester score of the upper probe. The nine symptom dimensions and three global indices of Symptom Checklist-90-Revised questionnaire did not show any correlation with reflux symptom index, reflux finding score and the parameters of the 24-h ambulatory double probe pH monitoring. CONCLUSIONS: Laryngopharyngeal reflux patients did not demonstrate any significant level of psychological distress and their symptom severity showed significant positive correlation with reflux severity.

Multiple splenic infarctions in a dog with immune-mediated hemolytic anemia: therapeutic implications.

BACKGROUND: Splenic infarction (SI) is a rare clinical entity seldom encountered in veterinary medicine. Its most frequent causes include thromboembolic status, splenomegaly, and cardiac disease. Although thrombotic elements from the circulation provide the most common context for thromboembolic SIs, immune-mediated hemolytic anemia (IMHA) has not been reported as an underlying disease in canine SI. CASE DESCRIPTION: A 2-year-old, female spayed Dachshund, was referred with vomiting, hematochezia, and brown colored urine over the preceding 4 days. Physical examination revealed abnormalities including generalized weakness, jaundice, and splenomegaly; blood work showed pancytopenia and hyperbilirubinemia. Erythrocyte agglutination, polychromasia, and spherocytes on a peripheral blood smear were observed and IMHA concurrent with thrombocytopenia was diagnosed. FINDINGS/TREATMENT AND OUTCOME: Although erythrocyte agglutination and leukopenia disappeared after treatment, anemia and thrombocytopenia were unresponsive to oral immunosuppressive drugs and repeated transfusions. Further abdominal ultrasound identified an occlusive splenic vein thrombus. Splenic histopathology found marked multifocal to coalescing necrosis, and hemorrhage consistent with multiple SI. Symptoms resolved following splenectomy combined with 1 month of immunosuppressive medication, and the dog was healthy on follow-up evaluation after 2 years. CONCLUSION: Immune-mediated hemolytic anemia is an incompletely characterized cause of SI. This report establishes a potential and novel causal role for IMHA in canine SI. We believe it to be the first case report of SI in a dog with refractory IMHA and thrombocytopenia, successfully managed by splenectomy combined with short-term immunosuppressive therapy.

Anomalous muscles and nerves in the hand of a 94-year-old cadaver-A case report.

INTRODUCTION: During an anatomical dissection of the distal upper extremities, numerous muscular and nervous anomalies were found in the forearm and hand of a 94-year-old cadaver. These anomalies are clinically relevant with regard to medical or surgical interventions. PRESENTATION OF CASE: The presence of a "flexor digiti minimi longus" muscle was observed passing through Guyon's canal; to our knowledge this passageway has never been previously reported. An aberrant first lumbrical with three origins was noted. Additionally, numerous atypical nerves were found innervating the hand; the dorsal branch of the ulnar nerve contributed to cutaneous innervation of the palm of the hand (Kaplan's anastomosis), the superficial ulnar nerve provided muscular innervation to the flexor digiti minimi brevis muscle, and two connections between the common palmar digital branches of the median and superficial ulnar nerves were observed (Berrettini anastomosis). DISCUSSION: Here, we describe an extranumerary muscle associated with the hypothenar group of muscles. We also describe unusual origins of the first lumbrical muscle, and atypical cutaneous and muscular innervation to the palm of the hand. CONCLUSION: Clinically, understanding the existence of these anatomical variations may influence medical care or surgical procedures.

G protein alpha subunit G alpha z couples neurotransmitter receptors to ion channels in sympathetic neurons.

The functional roles subserved by G(alpha)z, a G protein alpha subunit found predominantly in neuronal tissues, have remained largely undefined. Here, we report that G(alpha)z coupled neurotransmitter receptors to N-type Ca2+ channels when transiently overexpressed in rat sympathetic neurons. The G(alpha)z-mediated inhibition was voltage dependent and PTX insensitive. Recovery from G(alpha)z-mediated inhibition was extremely slow but accelerated by coexpression with RGS proteins. G(alpha)z selectively interacted with a subset of receptors that ordinarily couple to N-type Ca2+ channels via PTX-sensitive Go/i proteins. In addition, G(alpha)z rescued the activation of heterologously expressed GIRK channels in PTX-treated neurons. These results suggest that G(alpha)z is capable of coupling receptors to ion channels and might underlie PTX-insensitive ion channel modulation observed in neurons under physiological and pathological conditions.

The release element of the yeast polymerase I transcription terminator can function independently of Reb1p.

The Saccharomyces cerevisiae polymerase I (polI) transcription terminator utilizes a DNA-binding protein (Reb1p) as part of a signal that causes the polymerase to pause prior to release from the template. To study the release element of the terminator, independent of the Reb1p pause signal, we have replaced the Reb1p binding site with the binding site for the lac repressor, which acts as a self-contained heterologous pause signal for polI. Release efficiency is maximal when the lac repressor causes polI to pause in exactly the same position that Reb1p would have caused it to pause, suggesting that polI must be precisely positioned for transcript release to occur. Mutational analysis shows that the release element is a region rich in T residues which codes for the extreme 3' end of the transcript and which has no apparent ability to form hairpins when transcribed into RNA. We discuss possible mechanisms whereby this polI release element might function.

xUBF, an RNA polymerase I transcription factor, binds crossover DNA with low sequence specificity.

Xenopus UBF (xUBF) is a transcription factor for RNA polymerase I which contains multiple DNA-binding motifs. These include a short basic region adjacent to a dimer motif plus five high-mobility-group (HMG) boxes. All of these DNA-binding motifs exhibit low sequence specificity, whether assayed singly or together. In contrast, the HMG boxes recognize DNA structure that is formed when two double helices are crossed over each other. HMG box 1, in particular, requires association of two double helices before it will bind and, either by itself or in the context of the intact protein, will loop DNA and organize it into higher-order structures. We discuss how this mode of binding affects the function of xUBF as a transcription factor.

Effects of age, replicative lifespan and growth rate of human nucleus pulposus cells on selecting age range for cell-based biological therapies for degenerative disc diseases.

Autologous disc cell implantation, growth factors and gene therapy appear to be promising therapies for disc regeneration. Unfortunately, the replicative lifespan and growth kinetics of human nucleus pulposus (NP) cells related to host age are unclear. We investigated the potential relations among age, replicative lifespan and growth rate of NP cells, and determined the age range that is suitable for cell-based biological therapies for degenerative disc diseases. We used NP tissues classified by decade into five age groups: 30s, 40s, 50s, 60s and 70s. The mean cumulative population doubling level (PDL) and population doubling rate (PDR) of NP cells were assessed by decade. We also investigated correlations between cumulative PDL and age, and between PDR and age. The mean cumulative PDL and PDR decreased significantly in patients in their 60s. The mean cumulative PDL and PDR in the younger groups (30s, 40s and 50s) were significantly higher than those in the older groups (60s and 70s). There also were significant negative correlations between cumulative PDL and age, and between PDR and age. We found that the replicative lifespan and growth rate of human NP cells decreased with age. The replicative potential of NP cells decreased significantly in patients 60 years old and older. Young individuals less than 60 years old may be suitable candidates for NP cell-based biological therapies for treating degenerative disc diseases.

Sequestration of G-protein beta gamma subunits by different G-protein alpha subunits blocks voltage-dependent modulation of Ca2+ channels in rat sympathetic neurons.

The membrane-delimited and voltage-dependent inhibition of N-type Ca2+ channels is mediated by Gbeta gamma subunits. Previously, exogenous excess GDP-bound GalphaoA has been shown to dramatically attenuate the norepinephrine (NE)-mediated Ca2+ current inhibition by sequestration of Gbeta gamma subunits in rat superior cervical ganglion (SCG) neurons. In the present study, we determined whether the attenuation of NE-mediated modulation is specific to GalphaoA or shared by a number of closely related (Galphatr, GalphaoB, Galphai1, Galphai2, Galphai3, Galphaz) or unrelated (Galphas, Galphaq, Galpha11, Galpha16, Galpha12, Galpha13) Galpha subunits. Individual Galpha subunits from different subfamilies were transiently overexpressed in SCG neurons by intranuclear injection of mammalian expression vectors encoding the desired protein. Strikingly, all Galpha subunits except Galphaz nearly blocked basal facilitation and NE-mediated modulation. Likewise, VIP-mediated Ca2+ current inhibition, which is mediated by cholera toxin-sensitive G-protein, was also completely suppressed by a number of Galpha subunits overexpressed in neurons. Galphas expression produced either enhancement or attenuation of the VIP-mediated modulation-an effect that seemed to depend on the expression level. The onset of the nonhydrolyzable GTP analog, guanylylimidodiphosphate-mediated facilitation was significantly delayed by overexpression of different GDP-bound Galpha subunits. Taken together, these data suggest that a wide variety of Galpha subunits are capable of forming heterotrimers with endogenous Gbeta gamma subunits mediating voltage-dependent Ca2+ channel inhibition. In conclusion, coupling specificity in signal transduction is unlikely to arise as a result of restricted Galpha/Gbeta gamma interaction.

Chromatin assembly on plasmid DNA in vitro. Apparent spreading of nucleosome alignment from one region of pBR327 by histone H5.

We have found that histone H5 (or H1) induces physiological nucleosome spacings and extensive ordering on some plasmid constructions, but not on others, in a fully defined in vitro system. Plasmid pBR327 containing DNA insertions with lengths close to 300 base-pairs permitted histone H5 to induce a remarkable degree of nucleosome alignment. Seventeen multiples of a unit 210(+/- 4) base-pair repeat, covering the entire plasmid, were detected. Plasmid pBR327, not containing a DNA insert, permitted continuous alignment of only a few nucleosomes. These observations suggest that a necessary requirement in this system for histone H5 (or H1)-induced nucleosome alignment on small (less than 4 kb; 1 kb = 10(3) bases or base-pairs) circular plasmids may be that the total DNA length must be close to an integer multiple of the nucleosome repeat length generated, a type of boundary effect. Consistent with this hypothesis, five deletion constructs of pBR327 (not containing inserts), that spanned 64% of the plasmid, and possessed DNA lengths close to integer multiples of 210 base-pairs, permitted nucleosome alignment by histone H5. We have also found that plasmid length adjustment is not a sufficient condition for nucleosome alignment. For example, plasmids pBR322 and pUC18 did not permit nucleosome alignment when adjusted to near-integer multiples of 210 base-pairs. Also, for pBR327 that contained a length-adjusted deletion in one particular region, appreciable nucleosome alignment no longer occurred. These data suggest that a contiguous approximately 800 base-pair region of pBR327, interrupted in pBR322 and not present in pUC18, can nucleate histone H5-induced nucleosome alignment, which can then spread to adjacent chromatin. Supporting this idea, a positioned five-nucleosome array appears to originate in the required region. Additionally, on a larger (6.9 kb) plasmid construction, the "chromatin organizing region" of pBR327 and adjacent DNA on one side of it exhibited preferred H5-induced nucleosome alignment.

A systematic survey of the intergenic region between the murine oxytocin- and vasopressin-encoding genes.

The genomic region between the oxytocin (OT) and vasopressin (VP) genes in the two strains of mice was independently sequenced by our two groups. In this report, we present our collated sequence data and analyses. The mouse intergenic region (MUIGR) was aligned to that of the rat, which has been reported to contain 6.4-kb long interspersed nuclear element (LINE). The MUIGR sequences in the two mice strains did not contain any LINE sequences. This suggests that the approximately 3.5-kb sequence that is conserved between the rat and mouse intergenic regions is likely to be involved in the regulation of OT and VP expression. We also observed several conserved putative transcription factor recognition sequences. Analysis of the MUIGR revealed the lack of any significant ORFs, but the presence of several repetitive elements.

Induction of apoptosis in human leukemia cells by 3-deazaadenosine is mediated by caspase-3-like activity.

3-Deazaadenosine (DZA), one of the potent inhibitors of S-adenosylhomocysteine hydrolase, is known to possess several biological properties including an induction of apoptosis. To evaluate a possibility that DZA may be utilized for the treatment of human leukemia, we studied molecular events of cell death induced by DZA in human leukemia HL-60 and U-937 cells. DZA induced a specific cleavage of poly ADP-ribose polymerase (PARP) and an activation of the cysteine protease caspase-3/CPP32 which is known to cleave PARP. DZA-mediated nuclear DNA-fragmentation was completely blocked in the presence of a universal inhibitor of caspases (z-VAD-fmk) or the specific inhibitor of caspase-3 (z-DEVD-fmk) unlike of cycloheximide (CHX). DNA fragmentation was preceded by the lowering of c-myc mRNA in the DZA treated cells. In addition, DZA-induced apoptosis was blocked by pretreatment with adenosine transporter inhibitors such as nitrobenzylthioinosine (NBTI) and dipyridamole (DPD). Taken together, these results demonstrate that DZA-induced apoptosis initiated through an active transport of DZA into human leukemia cells, is dependent on the caspase-3-like activity without de novo synthesis of proteins and possibly involves c-myc down-regulation.

Activation of caspase-8 in 3-deazaadenosine-induced apoptosis of U-937 cells occurs downstream of caspase-3 and caspase-9 without Fas receptor-ligand interaction.

3-Deazaadenosine (DZA), a cellular methylation blocker was reported to induce the caspase-3-like activities-dependent apoptosis in U-937 cells. In this study, we analyzed the activation pathway of the caspase cascade involved in the DZA-induced apoptosis using specific inhibitors of caspases. In the U-937 cells treated with DZA, cytochrome c release from mitochondria and subsequent activation of caspase-9, -8 and -3 were observed before the induction of apoptosis. zDEVD-Fmk, a specific inhibitor of caspase-3, and zLEHD-Fmk, a specific inhibitor of caspase-9, prevented the activation of caspase-8 but neither caspase-3 nor caspase-9, indicating that caspase-8 is downstream of both caspase-3 and caspase-9, which are activated by independent pathways. zVAD-Fmk, a universal inhibitor of caspases, kept the caspase-3 from being activated but not caspase-9. Moreover, ZB4, an antagonistic Fas-antibody, exerted no effect on the activation of caspase-8 and induction of apoptosis by DZA. In addition, zVAD-Fmk and mitochondrial permeability transition pore (MPTP) inhibitors such as cyclosporin A (CsA) and bongkrekic acid (BA) did not block the release of cytochrome c from mitochondria. Taken together, these results suggest that in the DZA-induced apoptosis, caspase-8 may serve as an executioner caspase and be activated downstream of both caspase-3 and caspase-9, independently of Fas receptor-ligand interaction. And caspase-3 seems to be activated by other caspses including IETDase-like enzyme and caspse-9 seems to be activated by cytochrome c released from mitochondria without the involvement of caspases and CsA- and BA- inhibitory MPTP.

Functional and genetic assessment of IFN-gamma receptor in patients with clinical tuberculosis.

OBJECTIVE: The molecular basis of the genetic vulnerability underlying the most common form of clinical tuberculosis (TB) remains largely unknown. We speculated that mild genetic defects in the interferon-gamma (IFN-gamma) signalling pathway caused a subtle functional impairment of IFN-gamma which would explain susceptibility to Mycobacterium tuberculosis in clinical TB. DESIGN: A case-control study. RESULTS: We evaluated functional responsiveness to IFN-gamma in monocytes from patients with clinical TB (n = 10), and analysed the genetic sequences of the IFN-gamma receptor 1 (IFN-gammaR1) and STAT1 genes in patients with disseminated TB (n = 18). IFN-gamma stimulated an increase in the expression of HLA-DR and CD64 on monocytes of both controls and patients; the rate of increase in expression was the same in both groups. Treatment with IFN-gamma before lipopolysaccharide (LPS) stimulation further increased tumour necrosis factor-alpha (TNF-alpha) production as compared to TNF-alpha production with LPS stimulation alone; the rate of increase in TNF-alpha production was the same in both groups. The known mutations in the coding sequences of the IFN-gammaR1 and STAT1 genes were not found in the patients with disseminated tuberculosis. CONCLUSION: These results suggest that impairment of the IFN-gamma signalling pathway did not account for cases of clinical TB in this study.

Heterologous expression of a green fluorescent protein-pertussis toxin S1 subunit fusion construct disrupts calcium channel modulation in rat superior cervical ganglion neurons.

The fusion construct pEGFP-PTXS1 was assembled by ligating cDNA encoding the S1 subunit of Bordetella pertussis toxin (PTX) into the plasmid pEGFP-C1 (which codes for enhanced green fluorescent protein). Microinjection of pEGFP-PTXS1 (1-100 ng/microl) into the nucleus of dissociated rat sympathetic ganglion neurons resulted in functional expression as determined from the diffuse green fluorescence and disruption of norepinephrine-mediated N-type Ca2+ channel modulation. The heterologously expressed toxin retained specificity for G alpha(i/o)-dependent pathways as VIP-mediated modulation of N-type Ca2+ channels and muscarine-mediated inhibition of M-type K+ channels persisted in pEGFP-PTXS1 expressing neurons. These data demonstrate that the S1 subunit of PTX is readily expressed in mammalian neurons and remains functional following fusion to the C-terminus of another protein.

Focal lesion in the splenium of the corpus callosum in epileptic patients: antiepileptic drug toxicity?

BACKGROUND AND PURPOSE: Discrete focal lesions in the splenium of the corpus callosum on MR images in epileptic patients have received little attention in the literature. Our purpose was to describe these lesions, which may be related to the toxicity of antiepileptic drugs (AEDs), and to discuss the possible mechanisms of their development. METHODS: We examined six patients with epilepsy whose brain MR imaging findings showed a discrete focal nonhemorrhagic lesion in the splenium of the corpus callosum. The medical records and MR images were reviewed retrospectively with respect to the patients' clinical history, medication, and laboratory findings to determine the etiology of the lesion. RESULTS: In all six patients MR imaging showed a focal lesion in the splenium of the corpus callosum, which was ovoid in shape and 15 to 19 mm in size. In the three patients who received contrast material, there was no enhancement of the lesion. Four of six patients had a history of medication with dilantin, in two of whom the level of serum dilantin was found to be elevated (22.3 micrograms/mL and 70.4 micrograms/mL, respectively). Vigabatrin was administered in three patients, one of whom took dilantin together with vigabatrin. In two patients, the focal lesion in the corpus callosum disappeared on follow-up MR images after withdrawal of dilantin and/or vigabatrin. CONCLUSION: A discrete, focal, ovoid, nonhemorrhagic lesion in the splenium of the corpus callosum may be seen on brain MR images of patients with epilepsy. The lesion is considered to be reversible demyelination related to AEDs toxicity.

Evidence that ginsenosides prevent the development of opioid tolerance at the central nervous system.

The analgesic effect of ginsenosides or morphine was first determined following intrathecal (i.t.) administration in rat tail-flick test. The effect of chronic i.t. co-administration of ginsenosides with morphine on the development of opioid tolerance were also examined using rat tail-flick test. Administration of ginsenosides (i.t.) produced a weak antinociception in a dose-dependent manner. Administration of morphine (i.t.) also produced antinociception in a dose-dependent manner. The ED50 was 1.20 microg (1.14-1.29 microg). However, acute i.t. co-administration of ginsenosides with morphine was not additive in antinociception. Repeated i.t. co-administration of 200 microg ginsenosides with 10 microg morphine inhibited the development of tolerance induced by 10 microg morphine in rat tail-flick test, although i.t. co-administration of 50 or 100 microg ginsenosides with morphine was without effect. In conclusion, these results indicate that i.t. administered ginsenosides produce an antinociception in rat tail-flick test and also prevent opioid tolerance caused by chronic treatment with morphine at the spinal sites.

Bronchial hyperreactivity in patients with endobronchial tuberculosis.

Some patients with endobronchial tuberculosis (EBTB) have wheeze on physical examination and normal chest PA, which mimic bronchial asthma. Non-specific bronchial challenge tests have been used to confirm the presence of bronchial hyperreactivity, which is a hallmark of bronchial asthma. To evaluate the effect of endobronchial tuberculous inflammation on bronchial responsiveness to histamine, the provocation concentrations of histamine required to reduce FEV1 by 20% of the pre-challenge baseline (PC20) were compared between patients with EBTB, patients with symptomatic bronchial asthma and normal, healthy controls. PC20 in EBTB patients (17.2 +/- 2.3 mg ml-1) and normal controls (19.5 +/- 1.4 mg ml-1) were significantly higher than in bronchial asthma patients (0.99 +/- 0.15 mg ml-1). PC20 in EBTB patients was not affected by disease location in the bronchial tree was not correlated with FVC or FEV1. In conclusion, one should consider the possibility of EBTB for differential diagnosis from bronchial asthma, if airway responsiveness appears normal in wheezy patients.