Production of the 19-kDa antigen of Mycobacterium tuberculosis in Escherichia coli and its purification

The 19-kDa antigen (19Ag) of Mycobacterium tuberculosis (Mt) is a lipoprotein which is released from the organism during growth. In order to study the possible involvement of this antigen in the host protective response against Mt infection, it would be helpful to obtain high-level production of 19Ag from a recombinant organism. We have found that overexpression of the native 19Ag gene in Escherichia coli or yeast leads to products which are aggregated and insoluble. By site-directed mutagenesis of the 19Ag lipoprotein leader sequence, we have generated a mutant gene which directs the production of 19Ag into the periplasmic space of E. coli, from where it can be easily purified in high yield. 19Ag obtained from this mutant construct lacks the lipid-modified N-terminal Cys residue found in the native 19Ag, and is not glycosylated, but is otherwise indistinguishable from 19Ag isolated from Mt culture supernatant.     

An integrated energy planning model for Illinois

An integrated energy supply and demand model for the state of Illinois is described. This equilibrium model is an important tool for integrating information from a variety of planning studies and data bases. A wide range of energy issues affecting diverse areas of the energy system, such as Illinois coal production under alternate oil price assumptions, renewable energy resource potential, and conservation options, can be analyzed by using the model.     

Quantity,controls and functions of large woody debris in Midwestern USA streams

Large woody debris (LWD) can increase stream habitat heterogeneity by providing structure, altering flow patterns, enhancing sediment deposition, forming pools and retaining organic matter. In North America, the role of LWD has been studied extensively in streams of mature forests (e.g. Pacific Northwest), but few studies have assessed LWD in streams of younger forests (e.g. Midwestern USA). Our objectives were to: (1) quantify the volume and abundance of LWD in a set of Midwestern streams; (2) evaluate possible factors influencing LWD quantity; (3) identify the functional roles of LWD; and (4) compare LWD levels in the upper Midwest to those elsewhere in North America. In 2002 and 2003, we measured LWD and geomorphological variables in 15 low‐gradient streams draining previously logged watersheds in the Upper Peninsula of Michigan. Mean (±SE) LWD volume (0.77 ± 0.12 m³ 100 m⁻²) and abundance (33 ± 3 pieces 100 m⁻¹) were 71% and 10% lesser, respectively, than in streams of similar gradient elsewhere in North America. Channel shape (width:depth ratio) explained 30% of the variation in LWD volume (multiple stepwise regression, P = 0.015) while LWD length and length:channel width combined, explained 72% of the variation in LWD density (multiple stepwise regression, P < 0.0001). About 50% of the LWD either stored sediment or stabilized banks and 14% of the LWD formed pools, although pool density was not significantly related to LWD volume or density. LWD levels, overall, were low in upper Midwestern streams, but the relative importance of that LWD to ecosystem function may be magnified in these wood‐poor systems. Copyright © 2006 John Wiley & Sons, Ltd.     

Testing an implantable intraspinal drug delivery device in the ewe

OBJECTIVE: The authors report the use of multiple implanted intraspinal port and catheter systems per test animal to study the in vivo functional characteristics and reliability of a new implantable spinal drug delivery port system. METHODS: Four ewes were each implanted with two epidural and one subarachnoid silicone elastomer catheters at the lumbar level. Each catheter was connected in series to one of three Therex filtered spinal delivery ports implanted subcutaneously in a similar grid pattern in each ewe to facilitate percutaneous identification. Saline (2 ml) was injected 3 times weekly in each port. The ease of injection and behavioral responses were recorded for 207-213 days of implantation until sacrifice/necropsy. RESULTS: All ports functioned reliably during the study. However, injection through two of the four subarachnoid catheters resulted in behavioral withdrawal responses intermittently. This behavioral pattern was much less common after epidural port injections. All four subarachnoid and four of eight epidural port and catheter systems were tested with local anesthetic just before sacrifice. Motor block was observed in three of four subarachnoid and three of four epidural port and catheter systems tested. Integrity of the other four epidural ports was tested by injection of methylene blue at sacrifice. This dye did not distribute in the epidural space in one of the latter four epidural ports (not local anesthetic tested) because of a concentric fibrotic reaction about the catheter. Similar fibrotic reactions surrounded the catheters that failed a functional test with local anesthetic. CONCLUSIONS: The implantable intraspinal port system tested functions reliably under repetitive percutaneous access. However, filtering such ports, though desirable to prevent entry of debris into the spinal canal, did not eliminate pericatheter chronic subarachnoid and epidural reaction. The number of test animals required to test 12 ports chronically was reduced by two-thirds without undue trauma to the individual test subject. Chronic percutaneous injection of an implanted subarachnoid system is feasible but may be associated with behavioral effects similar to that seen with chronic epidural systems. Fibrosis around chronic silicone catheters limited functional utility in one-fourth of the implanted test systems. Further study of the potential reactivity of chronic epidural and subarachnoid catheters is indicated.     

Damage to cochlear efferents following AF64A intoxication

Damage to cochlear efferents in chinchillas was assessed using transmission electron microscopy following unilateral treatment with the cholinotoxin ethylcholine mustard aziridinium ion (AF64A). AF64A was diluted in artificial perilymph to concentrations ranging from 0.5 to 100 microM. Survival times ranged from 1 to 12 weeks. At concentrations above 10 microM, widespread damage was noted to efferent fibers within the inner spiral bundle (ISB), tunnel spiral bundle (TSB), tunnel radial fibers (TRF) and efferent terminals at the base of OHCs. This damage included degeneration of fibers and terminals, delamination of mitochondria, vacuolization, and loss of cell membrane. However, at high concentrations, non-specific damage was also noted as thinnings or discontinuities of the membrane of OHCs and afferent fibers. At concentrations between 3 and 10 microM, selective damage was observed to efferent fibers within the ISB, TSB, TRF, and to terminals at the base of the OHCs, with all other structures appearing normal. At concentrations of 0.5 and 1 microM, damage was limited to efferent fibers within the TSB and ISB below the inner hair cells. In general, insult was greatest to middle- and basal-turn efferents, and longer survival times did not produce greater damage to, or loss of, efferents. These data suggest that at low concentrations, AF64A produces a partial yet selective degeneration of cochlear efferents within both the medial and lateral tracts, and that at the lowest concentrations used in these studies, AF64A produces a preferential insult on lateral olivocochlear efferents.