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Background Chest radiographs are used for diagnosis and severity assessment in tuberculosis (TB). The extent of disease as determined by smear grade and cavitation as a binary measure can predict 2-month smear results, but little has been done to determine whether radiological severity reflects the bacterial burden at diagnosis. Methods Pre-treatment chest x-rays from 1837 participants with smear-positive pulmonary TB enrolled into the REMoxTB trial (Gillespie et al., N Engl J Med 371:1577–87, 2014) were retrospectively reviewed. Two clinicians blinded to clinical details using the Ralph scoring system performed separate readings. An independent reader reviewed discrepant results for quality assessment and cavity presence. Cavitation presence was plotted against time to positivity (TTP) of sputum liquid cultures (MGIT 960). The Wilcoxon rank sum test was performed to calculate the difference in average TTP for these groups. The average lung field affected was compared to log 10 TTP by linear regression. Baseline markers of disease severity and patient characteristics were added in univariable regression analysis against radiological severity and a multivariable regression model was created to explore their relationship. Results For 1354 participants, the median TTP was 117 h (4.88 days), being 26 h longer (95% CI 16–30, p

The gene encoding rat kallikrein-binding protein (RKBP), a serine protease inhibitor, has been isolated and analyzed with the aid of the polymerase chain reaction. The gene is approximately 10 kilobases in length with four introns of approximately 2.2, 1.8, 0.9, and 0.84 kilobases. This gene is composed of five exons and encodes a polypeptide of 416 amino acid residues. The reactive center region of RKBP is encoded by the fifth exon with the putative P1-P1' residues being Lys-Ser. The organization of the RKBP gene is homologous to those of human alpha 1-antitrypsin and alpha 1-antichymotrypsin in size and arrangement of exons and introns, suggesting that they belong to the same subgroup of serpins. In the 5'-flanking region of the RKBP gene, a variant TATA box sequence, ATAAATA, is found 20 base pairs upstream from the transcription initiation site. The 5'-flanking region of the RKBP gene was able to direct transcription of the reporter gene chloramphenicol acetyltransferase when transfected into a rat hepatoma cell line. An internal promoter-like region was found in the first intron of the RKBP gene, downstream from the transcription initiation site and upstream from the translation initiation codon, however, it was unable to direct expression of the chloramphenicol acetyltransferase reporter gene in our experiments. The expression of RKBP in rat liver was induced by sex hormone treatment as indicated by dot-blot analysis. A genomic Southern blot using an RKBP cDNA probe revealed multiple bands suggesting that the RKBP gene belongs to a family of highly conserved genes.

Motivation: Prokaryotic organisms have been identified utilizing the sequence variation of the 16S rRNA gene. Variations steer the design of DNA probes for the detection of taxonomic groups or specific organisms. The long-term goal of our project is to create probe arrays capable of identifying 16S rDNA sequences in unknown samples. This necessitated the authentication, categorization and alignment of the >75 000 publicly available‘ 16S’ sequences. Preferably, the entire process should be computationally administrated so the aligned collection could periodically absorb 16S rDNA sequences from the public records. A complete multiple sequence alignment would provide a foundation for computational probe selection and facilitates microbial taxonomy and phylogeny. Results: Here we report the alignment and similarity clustering of 62 662 16S rDNA sequences and an approach for designing effective probes for each cluster. A novel alignment compression algorithm, NAST (Nearest Alignment Space Termination), was designed to produce the uniform multiple sequence alignment referred to as the prokMSA. From the prokMSA, 9020 Operational Taxonomic Units (OTUs) were found based on transitive sequence similarities. An automated approach to probe design was straightforward using the prokMSA clustered into OTUs. As a test case, multiple probes were computationally picked for each of the 27 OTUs that were identified within the Staphylococcus Group. The probes were incorporated into a customized microarray and were able to correctly categorize Staphylococcus aureus and Bacillus anthracis into their correct OTUs. Although a successful probe picking strategy is outlined, the main focus of creating the prokMSA was to provide a comprehensive, categorized, updateable 16S rDNA collection useful as a foundation for any probe selection algorithm. Availability: http://greengenes.llnl.gov/16S/ Contact: GLAndersen@lbl.gov Supplementary Information: Complete prokMSA in aligned fasta format: http://greengenes.llnl.gov/16S_cgi/download/update_28Dec02/prokMSA_aligned.fasta.Z Sequences within each Operational Taxonomic Unit: http://greengenes.llnl.gov/16S_cgi/download/update_15Mar02/SeqDescByOTU.txt DNA probe sequences: http://greengenes.llnl.gov/16S_cgi/download/update_15Mar02/15Mar02_StaphGrpPrbs.xls * To whom correspondence should be addressed.

We have previously shown that when tested in the morning, older men and women, pretreated with metyrapone to block endogenous cortisol synthesis, exhibit delayed suppression of plasma ACTH in response to cortisol infusion. To confirm this finding and to determine whether aging-related changes in feedback responsiveness are exaggerated near the time of the circadian nadir in adrenocortical secretion, we performed a similar study in the evening. Healthy young (20-35 yr, n = 22) and old (65 yr, n = 21) men and women were administered metyrapone orally (750 mg) at 1600 and 1900 h, followed by a cortisol infusion of 0.06 mg/kg/h for 150 min. Blood samples were taken at 15-min intervals for 4 h following infusion onset for measurement of plasma ACTH, cortisol, 11-deoxycortisol, and corticosteroid binding globulin. When corrections were made for differences in circulating cortisol concentrations achieved among age and gender subgroups, feedback inhibition of ACTH was found to be significantly greater in young than in old subjects of both genders. Our studies support the hypothesis that glucocorticoid responses to stress in aging individuals are likely to be prolonged due to blunted and delayed inhibition of ACTH secretion, thus increasing the total exposure to glucocorticoids.

mu-Opioid receptors are the pharmacological targets of endogenous opioid peptides and morphine-like alkaloid drugs. Previous studies of transfected cells and peripheral neurons indicate that opioid receptors are rapidly internalized after activation by the alkaloid agonist etorphine but not after activation by morphine. To determine whether opioid receptors in the central nervous system are regulated by a similar process of agonist-selective internalization, mu-opioid receptors were examined in rat brain neurons after treatment of animals with opioid drugs. Internalized mu receptors were observed within 30 min after intraperitoneal injection of the alkaloid agonist etorphine, and this process was blocked by the antagonist naloxone. Colocalization of internalized opioid receptors with transferrin receptors in confocal optical sections indicated that receptor internalization observed in vivo is mediated by a membrane trafficking pathway similar to that observed previously in vitro using transfected human embryonic kidney 293 cells. Morphine failed to induce detectable rapid internalization of receptors, even when administered to animals at doses far in excess of those required to induce analgesia. To quantify these agonist-selective differences and to analyze an array of opioid ligands for their ability to trigger internalization, we used flow cytometry on stably transfected 293 cells. These studies indicated that the different effects of individual agonists are not correlated with their potencies for receptor activation and that a variety of clinically important agonists differ significantly in their relative abilities to stimulate the rapid internalization of opioid receptors.

1. We have cloned and characterized a new species of kallikrein cDNA from a human colon cDNA library. The new kallikrein cDNA clone contains a part of intron 2 of the tissue kallikrein gene which is spliced to the remaining exon sequences. It does not contain exons 1 and 2. 2. An in-frame open reading frame is present in the new kallikrein cDNA allowing translation of a 216-amino acid product. The intron-containing kallikrein transcript was detected in salivary glands, pancreas, kidney, colon, prostate gland, testis, spleen, and lung by reverse-transcription/polymerase chain reaction followed by Southern blot analysis using an intron-containing kallikrein-specific oligonucleotide probe. 3. The results indicate that the new species of kallikrein may be processed by alternative splicing or arises from a different transcription initiation site.

All kallikrein-like genes that have been studied to date are composed of 5 exons and the tertiary structures of the encoded enzymes are remarkably similar. In the mouse and rat, these genes are highly conserved, tightly linked and tandemly arranged. In other species, such as the human, the family is less well defined and seems to be much smaller than that of the mouse and rat. Although extensively studied, the exact physiologic significance is not known for many kallikrein gene family members, however, they are thought to play important roles in processing biologically important peptide precursors. Given the potential importance of these mammalian enzymes as a group of highly selective peptide processing enzymes, it would be helpful to know more about the ways in which this family varies from species to species, especially with respect to the size of the family in each species. The evolutionary mechanisms which have shaped this family of genes are largely unknown, however, enough data has been generated to begin understanding the pathway by which this gene family has evolved.

The gene encoding rat kallikrein-binding protein (RKBP), a serine protease inhibitor, has been isolated and analyzed with the aid of the polymerase chain reaction. The gene is approximately 10 kilobases in length with four introns of approximately 2.2, 1.8, 0.9, and 0.84 kilobases. This gene is composed of five exons and encodes a polypeptide of 416 amino acid residues. The reactive center region of RKBP is encoded by the fifth exon with the putative P1-P1' residues being Lys-Ser. The organization of the RKBP gene is homologous to those of human alpha 1-antitrypsin and alpha 1-antichymotrypsin in size and arrangement of exons and introns, suggesting that they belong to the same subgroup of serpins. In the 5'-flanking region of the RKBP gene, a variant TATA box sequence, ATAAATA, is found 20 base pairs upstream from the transcription initiation site. The 5'-flanking region of the RKBP gene was able to direct transcription of the reporter gene chloramphenicol acetyltransferase when transfected into a rat hepatoma cell line. An internal promoter-like region was found in the first intron of the RKBP gene, downstream from the transcription initiation site and upstream from the translation initiation codon, however, it was unable to direct expression of the chloramphenicol acetyltransferase reporter gene in our experiments. The expression of RKBP in rat liver was induced by sex hormone treatment as indicated by dot-blot analysis. A genomic Southern blot using an RKBP cDNA probe revealed multiple bands suggesting that the RKBP gene belongs to a family of highly conserved genes.

Tissue kallikreins are a group of closely related serine proteinases that are represented by multigene families in mice and rats. The existence of similar, large, kallikrein-like gene families in other mammalian species is currently a matter of dispute. We have surveyed a number of vertebrate species using genomic DNA Southern blotting and screened a human genomic library with a monkey kallikrein cDNA probe. The hybridization patterns of the genomic Southern blots and the characterization of 19 independent human clones using restriction analysis and Southern blotting indicate that other mammalian species may have multiple kallikrein-like genes as well. The regulatory mechanisms that govern the expression, activity, and bioavailability of tissue kallikreins are likewise complex. At the level of transcription, hormones, dietary factors, and tissue-specific factors are known to affect the expression of tissue kallikrein genes. At the posttranslational level, kallikrein activity and bioavailability are regulated by enzymatic activation, circulating autoantibodies, and binding proteins. We have demonstrated the presence of kallikrein-binding proteins in humans and rats, and, furthermore, we have shown reduced levels of this binding protein in a hypertensive rat model.