Efficient random subcloning of DNA sheared in a recirculating point-sink flow system

Based on a high-performance liquid chromatographic pump, we have built a device that allows recirculation of DNA through a 63-microm orifice with ensuing fractionation to a minimum fragment size of approximately 300 base pairs. Residence time of the DNA fragments in the converging flow created by a sudden contraction was found to be sufficiently long to allow extension of the DNA molecules into a highly extended conformation and, hence, breakage to occur at midpoint. In most instances, 30 passages sufficed to obtain a narrow size distribution, with >90% of the fragments lying within a 2-fold size distribution. The shear rate required to achieve breakage was found to be inversely proportional to the 1.0 power of the molecular weight. Compared with a restriction digest, up to 40% of all fragments could be cloned directly, with only marginal improvements in cloning efficiency having been observed upon prior end repair with Klenow, T4 polymerase or T4 polynucleotide kinase. Sequencing revealed a fairly random distribution of the fragments.     

Initial processing of human proenkephalin in bovine chromaffin cells

The opioid peptide precursor preproenkephalin (PPE) contains seven enkephalin sequences and is synthesized by epinephrine-producing adrenal chromaffin cells and various peripheral and central neurons. After removal of its signal peptide, PPE undergoes processing at dibasic amino acid sites to yield its final opioid products-Met-enkephalin, Leu-enkephalin, and various larger, enkephalin-containing peptides. Processing of PPE was examined in bovine chromaffin cells using a plasmid containing the human PPE (hPPE) cDNA under the control of the cytomegalovirus immediate early enhancer/promoter. Following transfection of this hPPE-containing plasmid into bovine chromaffin cells, several proenkephalin-immunoreactive bands were observed on western blots with monoclonal antibodies that recognize human, but not bovine, proenkephalin sequences. The pattern of hPPE-derived peptides observed was similar to that of bovine PPE processing products. A series of recombinant plasmids containing mutations in the hPPE sequence at putative processing sites was then constructed. Conversion of Lys-Lys and Lys-Arg sequences to Lys-Gln and of Arg-Arg to Arg-Gln altered initial hPPE processing at only three of the putative processing sites. When hPPE cDNA containing mutations at all of these initially processed sites was expressed, one or more alternative processing sites were revealed. These data suggest the importance of structural features in addition to the dibasic sequences that limit the processing of proenkephalin.     

A high-resolution map of genes, microsatellite markers, and new dinucleotide repeats from UBE1 to the GATA locus in the region Xp11.23

Several new genes and markers have recently been identified on the proximal short arm of the human X chromosome in the area of Xp11.23. We had previously generated a YAC contig in this region extending from UBE1 to the OATL1 locus. In this report two polymorphic dinucleotide repeats, DXS6949 and DXS6950, were isolated and characterized from the OATL1 locus. A panel of YAC deletion derivatives from the distal portion of the contig was used in conjunction with the rest of the YAC map to position the new microsatellites and order other markers localizing to this interval. The marker order was determined to be DXS1367-ZNF81-DXS6849-ZNF21-DXS6616-DXS 6950-DXS6949. In the proximal region below OATL1, we have isolated a pair of YACs from the GATA locus, B1026 and C01160. Mapping within these YACs indicates the orientation of DXS1126 and DXS1240, while a cosmid near the OATL1 region reveals the overlap between the YAC contigs from the two loci. This cosmid contains the gene responsible for Wiskott-Aldrich syndrome (WAS) and localizes the disease gene between OATL1 and GATA. These data enable the expansion of the present physical map of the X chromosome from UBE1 to the GATA locus, covering a large portion of the Xp11.23 region. Genetic cross-overs in Xp11.23 support the marker orientation and the position of WAS, contrary to previous reports. With the integration of both physical and genetic maps we have predicted the following marker order: Xpter-UBE1-SYN1/ARAF1/ TIMP1-DXS1367-ZNF81-DXS.6849-ZNF21-DXSy6616++ +-(OATL1, DXS6950-DXS6949)- WAS-(GATA, DXS1126)-DXS1240-Xcen.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of various lipid-bile salt mixed micelles on the intestinal absorption of amphotericin-B in rat

We investigated the effect of the somatosensory functions to the outcomes of motor functions in 28 patients with thalamic hemorrhage. The disturbance of the pyramidal tracts was assessed by the destruction of the internal capsule found in computed tomography (CT). The disturbance of the somatosensory function was analyzed by the N20 component of short-latency somatosensory evoked potentials (SSEP). The outcomes of motor function was evaluated after 3 months of ictus. Correlations between the outcomes of motor function, disturbance of the pyramidal tract, and disturbance of the somatosensory function were discussed. The result indicated that functional outcomes statistically correlated with neither disturbance of the internal capsule alone nor disturbance of N20 alone. But, there was statistically significant between functional outcomes and the combination of disturbance of the internal capsule with disturbance of N20 (p < 0.05, Wilcoxon signed-rank). There was not statistical difference in hematoma volume or consciousness. The implications of these results suggest that somatosensory function may affect the recovery of motor functions.     

The perinatal mortality rate as an indicator of quality of care in international comparisons

Priming and recollection are expressions of human memory mediated by different brain events. These brain events were monitored while people discriminated words from nonwords. Mean response latencies were shorter for words that appeared in an earlier study phase than for new words. This priming effect was reduced when the letters of words in study-phase presentations were presented individually in succession as opposed to together as complete words. Based on this outcome, visual word-form priming was linked to a brain potential recorded from the scalp over the occipital lobe about 450 ms after word onset. This potential differed from another potential previously associated with recollection, suggesting that distinct operations associated with these two types of memory can be monitored at the precise time that they occur in the human brain.     

The diastolic debate: is it time to discard Korotkoff phase IV in favour of phase V for blood pressure measurements in pregnancy?

The IgE-mediated allergic response involves a series of physiological pathways that result in the array of symptoms commonly associated with allergy. This article describes the mediators that prompt specific clinical responses and identifies those at risk for latex hypersensitivity.     

Somatic antigens of pseudomonads: structure of the O-specific polysaccharide of Pseudomonas fluorescens biovar B, strain IMV 247

The O-specific polysaccharide of Pseudomonas fluorescens biovar B, strain IMV 247, was studied by acid hydrolysis, GLC-MS and 1H and 13C NMR spectroscopy, including 1D and 2D NOE, 2D hybrid TOCSY and ROESY (TORO), and 2D H-detected heteronuclear multiple-bond correlation (HMBC) experiments. The polysaccharide was found to contain L-rhamnose, 3.6-dideoxy-3-[(S)-3-hydroxybutyramido]-D-glucose (D-Qui3NHb), 2-acetamido- 2,4,6-trideoxy-4-[(S)-3-hydroxybutyramido-D-glucose (D-QuiNAc4NHb) and 2-acetamido-2- deoxy-D-galacturonic acid (D-GalNAcA). Partial acid hydrolysis of the polysaccharide resulted in a non-reducing GalNAcA-->QuiNAc4NHb disaccharide with the 3-hydroxybutyryl group glycosylated intramolecularly by the QuiN4N residue. The following structure of the tetrasaccharide repeating unit of the polysaccharide was established:-->4) -alpha-D-GalpNAcA-(1-->3)- alpha-D-QuipNAc4NHb-(1-->2)-beta-D-Quip3NHb-(1-->2)-alpha-L- Rhap(1-->.     

Abdominal tuberculosis: a presumptive diagnosis

BACKGROUND: Abdominal tuberculosis (TB) is common. But the diagnosis of abdominal TB is fraught with difficulties as it is often not possible to get a microbiological confirmation of the infection. We therefore undertook this study to highlight those pertinent clinical and laboratory features which would enable one to make a provisional diagnosis of abdominal TB early, to pave way for a trial of anti-tuberculosis chemotherapy. METHOD: This is a retrospective study of 12 patients treated for abdominal TB in our department over a period of 2 years. FINDINGS: Seven of the patients suffered from chronic diarrhoea for periods ranging from 4 weeks to 12 months. Four patients had progressive abdominal distension (ascites). The last patient came in with multiple abdominal swellings. Seven patients had clinical and biochemical features of malabsorption. Another 9 patients also had persistent pyrexia. The ascitic fluid was exudative in the 4 patients mentioned earlier. A definitive diagnosis could not be established in any of these patients. The diagnosis of abdominal TB was thus one of exclusion in these patients who showed prompt response to anti-tuberculosis therapy. CONCLUSION: Our study justifies a trial of anti-TB chemotherapy in TB endemic areas in the following clinical situations: (a) patients with chronic diarrhoea of unknown aetiology and (b) patients with exudative ascitic fluid, after all other possible causes, have been excluded. A prompt response to anti-TB therapy should be accepted as sufficient ground for the diagnosis of abdominal TB even when histopathological or microbiological confirmation of the disease is not possible. Our study reflects the experience of other workers from Third World countries.     

Changes in densities of hypothalamic mu opioid receptor during cupric acete-induced preovulatory lh surge in rabbit

Decrease in activity of hypothalamic beta-endorphin (beta-EP) is an important factor for inducing the preovulatory LH surge. To study whether hypothalamic mu opioid receptor is involved in this process, changes in densities of hypothalamic mu opioid receptors were observed in this study by autoradiography and image process during cupric acetate (CuAC)-induced preovulatory LH surge in rabbits. New Zealand female rabbits were injected 1% CuAC 0.9 ml or saline 0.9 ml and sacrificed at different times after the injection. The densities of mu opioid receptor in the medial basal hypothalamus (MBH) and the medial preoptic area (MPO) were measured. A transient increase in densities of MPO mu opioid receptor were observed 1 h after CuAC injection (P < 0.05). The densities of MPO mu opioid receptor decreased significantly before the onset of the LH surge (P < 0.05) and remained at a low level during the surge. The change in densities of mu opioid receptor in the MBH was similar to those in the MPO. No change was observed in the saline control group. There was a negative correlation between the changes in densities of MBH mu opioid receptor and serum LH levels in the process of LH surge. The results suggest that the decrease of hypothalamic mu opioid receptor may be involved in the preovulatory LH surge.