Outbreak of tuberculosis in a correctional facility: consequences of missed opportunities.

In May 2006, the Department of Public Health investigated a tuberculosis (TB) outbreak at a correctional facility after two recently discharged inmates were diagnosed with TB. Based on epidemiological and genotyping data, one infectious patient was determined to be the source of infection for the other. Despite prolonged symptoms and abnormal chest radiographs, the index patient was not diagnosed while incarcerated. Among the estimated 910 exposed inmates tested, 53 (5.8%) had newly positive tuberculin skin tests (TSTs). Ten (2.1%) of 485 corrections staff tested converted their TSTs. This investigation highlights the consequences of missed TB diagnoses in prisons.

Green fluorescent protein rendered susceptible to proteolysis: positions for protease-sensitive insertions.

The green fluorescent protein (GFP) is highly resistant to proteolysis and remains uncleaved after prolonged incubation with trypsin or pronase despite several putative tryptic and chymotryptic sites in exposed loops. We have rendered GFP sensitive to proteolysis by inserting five amino acids, IEGRS, in loops at position 157, 172, or 189. Excitation and emission maxima of the three insertion mutants were similar to those of wild type, but quantum yields of mutants Omega172 and Omega189 were lower, indicating increased freedom of the fluorophore. Trypsin cleaved the native (folded) form of each mutant at a unique site defined by the insert. Pronase also yields similar digestion patterns in these variants, but further proteolysis was also observed, suggesting that the primary cleavage relaxes GFP structure and reveals previously inaccessible sites. Fluorescence of Omega189 changed little upon digestion with trypsin but decreased progressively by as much as 40% upon digestion with increasing amounts of pronase. Fluorescence of other variants was not affected significantly by the proteases, further confirming the remarkable stabilities of GFP variants. These constructs define a new conformation-sensitive site around residue 189 of GFP and show that GFP may be useful for design of protease-susceptible molecules for monitoring of specific proteolytic activities in vivo.

Parvalbumin-positive projection neurons characterise the vocal premotor pathway in male, but not female, zebra finches.

Parvalbumin (PV) and calbindin (CB) immunoreactivities were assessed in nucleus robustus archistriatalis (RA) of male and female zebra finches, together with retrograde labelling of RA neurons. The results of double and triple labelling experiments suggested that, in males, moderately and faintly PV-positive neurons were projection neurons, but that all intensely PV-positive cells were not. The latter, which are presumably interneurons, were also intensely CB-positive, and may correspond to the GABAergic inhibitory interneurons identified by others. In addition, the complete RA pathway and its terminal fields in the respiratory-vocal nuclei of the brainstem were strongly PV-positive. In female zebra finches, which do not sing, no evidence was found that PV-positive RA cells were projection neurons, yet the pattern of projections of RA neurons, as determined by anterograde transport of biotinylated dextran amine, was very similar to that of RA in males. Moreover, in females, RA neurons retrogradely labelled from injections of cholera toxin B-chain into the tracheosyringeal nucleus (XIIts) were abundant and included, in the lateral part of the nucleus, a population of cells that were as large as those in the male RA. Parvalbumin immunoreactivity was also present in RA and its projections in males of several other songbird species (northern cardinal, brown headed cowbird, canary) and in the female cardinal, which sings to some extent, but the labelling was not as intense as that in male zebra finches.

Trigeminally innervated iron-containing structures in the beak of homing pigeons, and other birds.

The ophthalmic nerve in the upper beak was labelled with cholera toxin B-chain, and iron was identified using the Prussian Blue reaction. Iron deposits were found in the caudal part of the beak, and some were concentrated in cells that clustered in encapsulated structures densely innervated by ophthalmic nerve fibres. Such structures could form the anatomical basis of a type of mechanoreceptor that transmits magnetic sense information to the brain.

Neural pathways for bilateral vocal control in songbirds.

Ipsilateral and contralateral projections of nucleus robustus archistriatalis (RA), a telencephalic vocal premotor nucleus, to respiratory-vocal nuclei in the brainstem were defined in adult male Wasserschlager canaries, grey catbirds, and zebra finches, three songbird species that appear to differ in the degree of lateralized syringeal dominance. In all three species, ipsilateral projections of RA to the medulla included the tracheosyringeal part of the hypoglossal nucleus (XIIts), that innervates the syrinx, the bird's vocal organ, the suprahypoglossal area (SH), and two respiratory-related nuclei, retroambigualis (RAm) and parambigualis (PAm; Reinke and Wild [1998] J Comp Neurol 391:147-163). Projections of RA to the contralateral XIIts, SH and RAm, were substantial in canaries, which use the left side of the syrinx predominantly during singing; less pronounced in catbirds, which have no lateral dominance for song control; and least pronounced in zebra finches, in which there is a right-sided dominance for song control. There were no obvious differences in the number of crossed projections in birds injected in the left or right RA. Local sources of inputs to XIIts and RAm were defined anatomically in zebra finches and canaries. RAm, including neurons in close proximity to XIIts, was found to project to XIIts and the suprahypoglossal area bilaterally but predominantly ipsilaterally. RAm also had reciprocal connections with its contralateral homologue. These results suggest a pattern of connections between premotor and motor respiratory-vocal nuclei that may be involved in bilateral control of vocal output at medullary levels, a control that involves a high degree of coordination between vocal and respiratory structures on both sides of the body.

Rostral wulst in passerine birds. I. Origin, course, and terminations of an avian pyramidal tract.

An avian "pyramidal tract" was defined in zebra finches and green finches by making injections of neuronal tracers into the hyperstriatum accessorium (HA) of the rostral Wulst. Extratelencephalic projections of rostral HA traveled in the septomesencephalic tract (TSM) and gave rise to nuclear-specific terminal fields in the precerebellar medial spiriform nucleus of the posterior thalamus, the red nucleus in the mesencephalon, the medial pontine nucleus in the pons, and the subtrigeminal, external cuneate, cuneate, gracile, and inferior olivary nuclei in the medulla. Extensive but more diffuse terminal fields were also present in the stratum cellulare externum of the posterior hypothalamus, the central periaqueductal gray, the prerubral field, and the lateral and ventrolateral tegmentum of the pons and medulla. There was also a sparse projection to the dorsal thalamic nucleus intermedius ventralis anterior, which supplies the somatosensory input to the rostral Wulst, and distinct projections to the intercollicular region surrounding the central nucleus of the inferior colliculus, where they partly overlapped the projections of the dorsal column nuclei. Projections from HA to the cerebellum via the TSM are described separately. In the brainstem the ventral ramus of TSM was situated ventral to the medial lemniscus at the base of the brain, entered the spinal cord in the inner margin of the lateral funiculus, predominantly ipsilaterally, and terminated bilaterally but predominantly contralaterally in the medial part of the base of the dorsal horn of the upper six or seven cervical segments. After injections of tracers into putative targets, numerous retrogradely labeled cells were found in the rostral HA, predominantly ventrally. The results confirm the presence of a major descending fiber system in passerine birds that resembles in its brainstem course and several of its terminations the pyramidal tract of mammals. The reciprocal projections of HA with the hypothalamus suggest that rostral HA may also incorporate neuronal components that in mammals would be considered parts of prefrontal cortex.

A direct cerebrocerebellar projection in adult birds and rats.

The rostral Wulst of birds, like the somatosensory cortex of mammals, receives somatosensory information from the thalamus and projects to the brainstem and spinal cord via a pyramidal-like tract. Using anterograde and retrograde tract-tracers, we show here, in adult zebra finches, that the rostral Wulst also projects directly to the cerebellar cortex and deep nuclei. In the cortex, the cerebrocerebellar fibers resemble neither mossy nor climbing fibers, but more closely resemble the multilayer fibers shown to originate from the hypothalamus in mammals. We also show that a sparse projection to the cerebellum from the mammalian neocortex, originally thought to be lost during early development, is present in the adult rat. Although the functional implications of these results are obscure, they suggest a revision of the concept of the "cerebrocerebellar system", which is generally considered to involve a pontine relay.

Rostral wulst of passerine birds: II. Intratelencephalic projections to nuclei associated with the auditory and song systems.

We have previously shown that the hyperstriatum accessorium (HA) of the rostral wulst in zebra finches and green finches is the origin of a pyramidal-like tract with substantial projections to the brainstem and cervical spinal cord. Here, we show that the HA also is the origin of a set of intratelencephalic projections with terminal fields in the lateral part of the frontal neostriatum, the shell surrounding the lateral magnocellular nucleus of the anterior neostriatum, the lobus parolfactorius surrounding area X, the nucleus interface, auditory fields L1 and L3, the shelf underlying the high vocal center, the dorsolateral caudal neostriatum, the dorsocaudal part of the nucleus robustus archistriatalis, and the ventral archistriatum. The cells of origin of these projections are located predominantly laterally in the HA, close to and sometimes within the intercalated HA, which receives somatosensory projections from the dorsal thalamus. The specific implications of these findings for auditory and vocal function are unclear, but the apparent overlap of auditory and somatosensory inputs in several of these regions suggests the possibility of mechanisms for stimulus enhancement or depression, depending on the congruence of stimuli within a cell's "in-register" multiple receptive fields.

Polyoma middle T antigen in HL-60 cells accelerates hematopoietic myeloid and monocytic cell differentiation.

Expression of the polyoma virus middle T antigen in HL-60 cells accelerates their differentiation in response to both monocytic and granulocytic differentiation-inducing agents. Middle T-expressing cells treated with the granulocytic inducer retinoic acid or the monocytic inducer 1,25-dihydroxy vitamin D3 differentiated 24 h earlier than parental, mock-electroporated, or vector control cell lines. The rapid onset of differentiation correlated with an increase in the cellular level of the middle T protein as well as two known retinoic-acid-inducible markers in HL-60 cells: the paxillin and transglutaminase gene products. The accelerated functional differentiation response and expression of retinoic-acid-inducible markers indicate that middle T played a causal role in differentiation. Thus, expression of the polyoma middle T antigen in HL-60 cells enhanced a variety of molecular changes associated with cellular differentiation.

An antibody Fab selected from a recombinant phage display library detects deesterified pectic polysaccharide rhamnogalacturonan II in plant cells.

Rhamnogalacturonan II (RG-II) is a structurally complex, low molecular weight pectic polysaccharide that is released from primary cell walls of higher plants by treatment with endopolygalacturonase and is chromatographically purified after alkaline deesterification. A recombinant monovalent antibody fragment (Fab) that specifically recognizes RG-II has been obtained by selection from a phage display library of mouse immunoglobulin genes. By itself, RG-II is not immunogenic. Therefore, mice were immunized with a neoglycoprotein prepared by covalent attachment of RG-II to modified BSA. A cDNA library of the mouse IgG1/kappa antibody repertoire was constructed in the phage display vector pComb3. Selection of antigen-binding phage particles resulted in the isolation of an antibody Fab, CCRC-R1, that binds alkali-treated RG-II with high specificity. CCRC-R1 binds an epitope found primarily at sites proximal to the plasma membrane of suspension-cultured sycamore maple cells. In cells deesterified by alkali, CCRC-R1 labels the entire wall, suggesting that the RG-II epitope recognized by CCRC-R1 is masked by esterification in most of the wall and tha such RG-II esterification is absent near the plasma membrane.

Suppression of the Fix- phenotype of Rhizobium meliloti exoB mutants by lpsZ is correlated to a modified expression of the K polysaccharide.

The rhizobial production of extracellular polysaccharide (EPS) is generally required for the symbiotic infection of host plants that form nodules with an apical meristem (indeterminate nodules). One exception is Rhizobium meliloti AK631, an exoB mutant of Rm41, which is deficient in EPS production yet infects and fixes nitrogen (i.e., is Fix+) on alfalfa, an indeterminate nodule-forming plant. A mutation of lpsZ in AK631 results in a Fix- strain with altered phage sensitivity, suggesting that a cell surface factor may substitute for EPS in the alfalfa-AK631 symbiosis. Biochemical analyses of the cell-associated polysaccharides of AK631 and Rm5830 (AK631 lpsZ) demonstrated that the lpsZ mutation affected the expression of a surface polysaccharide that is analogous to the group II K polysaccharides of Escherichia coli; the polysaccharide contains 3-deoxy-D-manno-2-octulosonic acid or a derivative thereof in each repeating unit. Rm5830 produced a polysaccharide with altered chromatographic and electrophoretic properties, indicating a difference in the molecular weight range. Similar results were obtained in a study of Rm1021, a wild-type isolate that lacks the lpsZ gene: the introduction of lpsZ into Rm1021 exoB (Rm6903) both suppresses the Fix- phenotype and results in a modified expression of the K polysaccharide. Chromatography and electrophoresis analysis showed that the polysaccharide extracted from Rm6903 lpsZ+ differed from that of Rm6903 in molecular weight range. Importantly, the effect of LpsZ is not structurally specific, as the introduction lpsZ+ into Rhizobium fredii USDA257 also resulted in a molecular weight range change in the structurally distinct K polysaccharide produced by that strain. This evidence suggests that LpsZ has a general effect on the size-specific expression of rhizobial K polysaccharides.

The morphology of human neuroblastoma cell grafts in the kainic acid-lesioned basal ganglia of the rat.

Cells from a human neuroblastoma cell line (SH-SY5Y) have been used to examine their potential suitability as donor cells for neural transplantation. Grafts of SH-SY5Y cells were placed in the basal ganglia of the rat brain 7 days after kainic acid lesions of the striatum. The animals were killed 4 or 8 weeks following grafting, and light and electron microscopic studies showed that the graft formed a well-vascularized compact mass of cells in the host brain. At both time points grafted cells showed evidence of cellular differentiation with process formation, especially at the graft-host interface where there was intermingling of graft and host neuronal process. Electron microscopic studies showed that graft cell processes containing irregularly-shaped, clear vesicles or membrane-bound dense core vesicles, established regions of specialized contact with other graft cells and formed close associations with host neuronal processes. There was little difference between the grafts of different ages, except that in the older grafts there were early signs of neurodegeneration. Since the SH-SY5Y cells used in these grafts express the enzyme tyrosine hydroxylase and synthesize dopamine in vitro, these cells were used in the hope that they may potentially be useful for repairing lesions in the dopamine pathway, such as that seen in Parkinson's disease. Our behavioural studies show that grafting SH-SY5Y cells into the striatum of rats with 6-hydroxydopamine lesions of the median forebrain bundle result in a reduction of amphetamine-induced rotation. However, this was unlikely to be due to dopamine release since there was no tyrosine hydroxylase immunoreactivity seen in the region of the grafts. Thus grafted human neuroblastoma cells survive, establish specialized morphological associations with graft and host processes and improve behavioural deficits resulting from 6-hydroxydopamine lesions. We suggest that grafted differentiated human neuroblastoma cells can interact with cells in the host brain with beneficial effects, and that in the medium-term, neuroblastoma grafts will make useful models for examining graft-host interactions. However, the presence of early degenerative changes in the older grafts suggests that neuroblastoma cells may not be suitable for long-term neural transplantation therapy for neurodegenerative diseases.

Differential foci and synaptic organization of the principal and spinal trigeminal projections to the thalamus in the rat.

The thalamus is known to receive single-whisker 'lemniscal' inputs from the trigeminal nucleus principalis (PrV) and multiwhisker 'paralemniscal' inputs from the spinal trigeminal nucleus (SpV), yet the responses of cells in the thalamic ventroposteromedial nucleus (VPM) are most similar to and contingent upon inputs from PrV. This may reflect a differential termination pattern, density and/or synaptic organization of PrV and SpV projections. This hypothesis was tested in adult rats using anterograde double-labelling with fluorescent dextrans, horseradish peroxidase (HRP) and choleragenoid, referenced against parvalbumin and calbindin immunoreactivity. The results indicated that PrV's most robust thalamic projection is to the whisker-related barreloids of VPM. The SpV had robust projections to non-barreloid thalamic regions, including the VPM 'shell' encapsulating the barreloid area, a caudal and ventral region of VPM that lacks barreloids and PrV inputs, the posterior thalamic nucleus, nucleus submedius and zona incerta. Within the barreloid portion of VPM, SpV projections were sparse relative to those from PrV, and most terminal labelling occurred in the peripheral fringes of whisker-related patches and in interbarreloid septae. Thus, PrV and SpV have largely complementary projection foci in the thalamus. Intra-axonal staining of a small sample of trigeminothalamic axons with whisker or guard hair receptive fields revealed highly localized and somatotopic terminal aggregates in VPM that spanned areas no larger than that of a single barreloid. In the electron microscopic component of this study, HRP transport to the barreloid region of VPM from left SpV and right PrV in the same cases revealed PrV terminals contacting dendrites with a broad range of minor axis diameters (mean +/- SD: 1.51 +/- 0.10 microns). SpV terminals were indistinguishable from those of PrV, but they had a disproportionate number of contacts on narrow dendrites (1.27 +/- 0.07 microns, P < 0.01). PrV endings were also more likely to contact VPM somata (11.0 +/- 4.2% of all labelled terminals) than those from SpV (3.0 +/- 1.0%, P < 0.01). Insofar as primary dendrites are thicker than distal dendrites in VPM, these data suggest a differential distribution of PrV and SpV inputs onto VPM cells that may account for their relative efficacies in dictating the responses of VPM cells to whisker stimulation. Multiwhisker receptive fields in VPM may also reflect direct transmission of convergent inputs from PrV.

Elevated expression of jun and fos-related proteins in transplanted striatal neurons.

The basal expression of the immediate-early gene protein products fos, fos-related antigens (FRA's), jun and krox-24 was examined using immunocytochemical methods in intrastriatal grafts derived from fetal striatal primordia. Whereas very few, if any, normal adult striatal neurons expressed jun, many neurons in the striatal graft expressed jun at high levels. FRA's, but not fos, were also occasionally induced in some grafted striata. krox-24 was expressed in normal adult striatal neurons, and to a lesser extent in transplanted striatal neurons. These results show that neurons of intrastriatal grafts express jun at substantially higher levels than host striatal neurons, and this may be related to the previously reported increased transcription of neuropeptides in striatal grafts, and/or to the possible failure of transplanted neurons to fully establish normal connections with the host tissue.

Differential sensitivity of calbindin and parvalbumin immunoreactive cells in the striatum to excitotoxins.

The neurotoxic effects of ibotenic acid, quinolinic acid and kainic acid on cells in the rat striatum were investigated using immunocytochemistry with antibodies to the calcium binding proteins, calbindin and parvalbumin. The results showed that both ibotenic acid and quinolinic acid affected calbindin and parvalbumin cells to the same extent. However, parvalbumin immunopositive neurons were more sensitive than calbindin immunopositive neurons to the neurotoxic effects of kainic acid. Although the reason for this increased sensitivity of parvalbumin striatal neurons to kainic acid is unclear, these results suggest that the neurotoxicity produced by kainic acid is different to that occurring with quinolinic acid and ibotenic acid.

Restriction fragment length polymorphism analysis of polymerase chain reaction products amplified from mapped loci of rice (Oryza sativa L.) genomic DNA.

Thirty mapped Indica rice genomic (RG) clones were partially sequenced from each end. From such sequence data, pairs of oligonucleotides were synthesized to act as primers for polymerase chain reaction (PCR) amplification of the corresponding loci in crude total DNA preparations. The PCR products from DNA of Indica varieties were of the sizes expected from the sizes of the corresponding RG clones. However, size polymorphisms were seen between PCR products from Indica and Japonica varieties, and among wildOryza species. Restriction fragment length polymorphism (RFLP) was observed between PCR products of Indica varieties simply by electrophoretic analysis of restricted products, without the need for Southern hybridization or radiolabelling. The RFLPs noted between varieties ARC6650 and Phalguna were inherited in recombinant inbred lines derived from a cross between them. The RFLPs were detectable in PCR products amplified from DNA extracted by a simple procedure from single seedlings or leaves, and revealed genetic heterogeneity in cultivated lines. An approach is described that is relevant to the acceleration of classical plant breeding through molecular techniques.

Rhizobium meliloti chromosomal loci required for suppression of exopolysaccharide mutations by lipopolysaccharide.

Mutants of alfalfa symbiont Rhizobium meliloti SU47 that fail to make extracellular polysaccharide (exo mutants) induce the formation of nodules that are devoid of bacteria and consequently do not fix nitrogen. This Fix- phenotype can be suppressed by an R. meliloti Rm41 gene that affects lipopolysaccharide structure. Here we describe mutations preventing suppression that map at two new chromosomal loci, lpsY and lpsX, present in both strains. Two other lps mutations isolated previously from SU47 also prevented suppression.

The symbiotic defect of Rhizobium meliloti exopolysaccharide mutants is suppressed by lpsZ+, a gene involved in lipopolysaccharide biosynthesis.

exo mutants of Rhizobium meliloti SU47, which fail to secrete acidic extracellular polysaccharide (EPS), induce Fix- nodules on alfalfa. However, mutants of R. meliloti Rm41 carrying the same exo lesions induce normal Fix+ nodules. We show that such induction is due to a gene from strain Rm41, which we call lpsZ+, that is missing in strain SU47. lpsZ+ does not restore EPS production but instead alters the composition and structure of lipopolysaccharide. In both SU47 and Rm41, either lpsZ+ or exo+ is sufficient for normal nodulation. This suggests that in R. meliloti EPS and lipopolysaccharide can perform the same function in nodule development.

Metabolism of Tryptophan and Tryptophan Analogs by Rhizobium meliloti.

The alfalfa symbiont Rhizobium meliloti Rm1021 produces indole-3-acetic acid in a regulated manner when supplied with exogenous tryptophan. Mutants with altered response to tryptophan analogs still produce indole-3-acetic acid, but are Fix(-) because bacteria do not fully differentiate into the nitrogen-fixing bacteriod form. These mutations are in apparently essential genes tightly linked to a dominant streptomycin resistance locus.

Host Restriction and Transduction in Rhizobium meliloti.

A host restriction difference exists between Rhizobium meliloti Rm41 and SU47 exists as indicated by the reduce plating efficiency of transducing phage PhiM12h1. Restriction can be attenuated by incubating cells at 42 degrees C for 3 h; this procedure overcomes a block to transduction from SU47 to Rm41.