Cloning, characterization and construction of htrA and htrA-like mutants of Brucella abortus and their survival in BALB/c mice

A genomic library of Brucella abortus S2308 was screened for expression of recombinant proteins recognized by sera from mice and from cattle infected with B. abortus. A positive clone, BA1, expressing a 50 kDa peptide was recognized by both sera. Plasmid pBA1, isolated from BA1, was shown by restriction enzyme digestion to possess a 1.9 kb insert. The nucleotide sequence of the pBA1 insert revealed an open reading frame with of 1539 bases with a coding capacity of 513 amino acids and a predicted molecular weight of 50,992. The predicted amino acid sequence showed 37% identity to E. coli HtrA, a temperature inducible serine protease. A second B. abortus htrA gene, designated htrA-like, was identified on a different cloned fragment that also encoded B. abortus recA. The nucleotide sequence of the htrA-like gene revealed an open reading frame of 1422 nucleotides with a coding capacity of 474 amino acids and a predicted molecular weight of 50,155. The deduced amino acid sequence of the htrA-like gene showed 42% and 36% identity with B. abortus and E. coli HtrAs respectively. Western blotting of E. coli lysate containing the htrA-like gene was not recognized by sera from B. abortus-infected cattle or mice. B. abortus htrA but not htrA-like relieved the temperature sensitive phenotype and permitted growth of an E. coli htrA mutant at 42 degrees C. B. abortus htrA and htrA-like mutants were constructed and their survival and growth in BALB/c mice was compared to the parental strain S2308.(ABSTRACT TRUNCATED AT 250 WORDS)     

Corticostriatal mechanisms of behavior

This article presents the results of three series of experiments on cats, dogs, and lower primates, performed to investigate the structural, neurophysiological, and mediator mechanisms of the corticostriatal systems involved in the organization of behavior. Morphological studies of corticostriatal connections showed that along with the diffuse distribution of afferent terminals within the striatum, there were also elements of topical organization defined by anteroposterior and mediolateral gradients. Neurophysiological experiments on dogs and lower primates were used to study the spike activity of the prefrontal region of the cortex and the head of the caudate nucleus during training to conditioned first- and second-order reflexes and during the solution of complex problems involving delayed spatial selection. Studies demonstrated that while in dogs, most of the neurons recorded showed a transition to responses to the conditioned signal at a particular stage of carrying out a conditioned response, in monkeys all cells recorded showed specific responses at different periods of solving the task at all stages of the study. Neuropharmacological experiments on dogs showed that agents blocking glutamine receptors in the caudate nucleus had more pronounced effects at the phase of developing conditioned movement reflexes. Administration of these agents during the reflex reinforcement phase affected only the differentiation of inhibition. These results lead to the conclusion that the prefrontal area of the cortex and, to some extent, the caudate nuclei, act on incoming information specifying the current dominant need and the states of the external and internal environments, to carry out programmed actions and assess the results of these actions.     

Quantitative correlation of breast tissue parameters using magnetic resonance and X-ray mammography

BACKGROUND: Disassembly of cytoplasmic microtubules by nocodazole in cultured mammalian cells leads to the disruption of the continuous ribbonlike Golgi apparatus and dispersal of the Golgi elements from their normal juxtanuclear location, close to the microtubule-organizing center (MTOC), toward the cell periphery. Clearing of the drug induces reassembly of the microtubules from the MTOC and reorganization of the Golgi elements into a continuous ribbonlike juxtanuclear structure. In the yeast Saccharomyces cerevisiae, the Golgi apparatus does not form a continuous structure as in mammalian cells but instead constitutes independent units dispersed throughout the cytoplasm. It is the purpose of this article to investigate the role of microtubules in the structure and distribution of the Golgi elements in S. cerevisiae by studying the ultrastructure of cell organelles either in mutant cells deficient in beta-tubulin or in wild-type cells treated with the microtubule-depolymerizing drug nocodazole. METHODS: Two S. cerevisiae yeast strains were used in this study: a control wild-type strain, CUY226 (ade2-101, his3-delta 200, leu2-delta 1, lys2-801, ura3-52 Mat alpha), and a mutant strain, CUY66 (tub2-401, ade2-101, ura3-52, Mat alpha). Nocodazole was added to the wild-type cells cultivated at 30 degrees C, and cells were fixed 5 min, 20 min, and 60 min, respectively, after adding the drug to the culture. Both strains were fixed and examined 5 min, 20 min, and 60 min after shifting the cultures from the permissive temperature of 30 degrees C to the restrictive temperature of 14 degrees C. Cells were fixed in 2% glutaraldehyde, treated for 15 min in 1% sodium metaperiodate, postfixed in reduced osmium, and embedded in Epon. To visualize the three-dimensional configuration of cell organelles, stereopairs were prepared from sections stained with lead citrate and tilted at +/- 15 degrees from the 0 degree position of the goniometric stage of the electron microscope. RESULTS: In mutant cells shifted to restrictive temperature and wild-type cells treated with nocodazole, the main ultrastructural modification was a fragmentation of networks of membranous tubules, which probably correspond to the yeast Golgi apparatus. Secretion granules were still present in growing buds, and they were dispersed in the cytoplasm, which contained in addition numerous small vesicles in the 30-60-nm diameter range. CONCLUSIONS: In normal cells, small vesicles may originate from the endoplasmic reticulum and fuse together to give rise to Golgi networks (Rambourg et al. 1994. Anat. Rec., 240:32-41). If this hypothesis is correct, the observations reported might indicate that intact microtubules orient the flow of small vesicles and favour their fusion into Golgi networks.     

Detection of N-(deoxyguanosin-8-yl)-2-fluorenamine in DNA of peritoneal serosa and liver after intraperitoneal exposure of rats to N-hydroxy-N-2-fluorenylbenzamide or N-hydroxy-N-2-fluorenylacetamide

DNA adduct formation was examined in rat peritoneal serosa, a tumor target for i.p. administered aqueous suspensions of N-hydroxy-N-2-fluorenylbenzamide (N-OH-2-FBA) and N-hydroxy-N-2-fluorenylacetamide (N-OH-2-FAA), and compared to that in the liver, which is a tumor target for N-OH-2-FAA in the male rat. 32P-Postlabeling analyses showed the presence of a single adduct, N-(deoxyguanosin-8-yl)-2-fluorenamine (dG-C8-FA), from activation of both hydroxamic acids by the serosa and liver in vitro and in vivo. The relatively low levels of dG-C8-FA (60-80 fmol/micrograms DNA) from N-OH-2-FBA in vitro were increased 2.7- and 35-fold upon the addition of acetyl coenzyme A (AcCoA) to the serosal cytosol and hepatic cytosol or microsomes respectively. By contrast, addition of AcCoA led to a decrease (approximately 34%) in the high level of dG-C8-FA (4330 fmol/micrograms DNA) from activation of N-OH-2-FAA by hepatic cytosol and did not alter the levels from activation by hepatic microsomes and serosal cytosols (530 and 78.3 fmol/micrograms DNA respectively). These data and the previously reported hydroxamic acid activation enzyme activities in the serosa and liver indicated that the precursor of dG-C8-FA, N-acetoxy-N-2-fluorenamine, was formed from N-OH-2-FAA chiefly via an intramolecular N,O-acetyltransfer and from N-OH-2-FBA via a two-step sequence of N-debenzoylation and AcCoA-dependent O-acetylation. The levels of dG-C8-FA were approximately 2- to 3-fold higher in the serosal DNA (up to 515 and 1012 fmol/micrograms DNA) after one (30 mumol/rat) and ten or eleven (cumulative dose of approximately 275 mumol/rat) injections of N-OH-2-FBA or N-OH-2-FAA than in the hepatic DNA. This correlated with the carcinogenicities of the hydroxamic acids, but was inversely proportional to the rates and extents of their activation in vitro. Multiple injections affected hepatic enzyme activities related to the activation of the hydroxamic acids in that the cytosolic N-debenzoylation of N-OH-2-FBA increased (approximately 1.7-fold) whereas N-OH-2-FAA acetyltransferase and sulfotransferase activities decreased. The effect of treatment with N-OH-2-FBA was greater than that with N-OH-2-FAA and was greater on the sulfotransferase activity (approximately 88% decrease). The latter suggested that N-OH-2-FBA, although a poor acceptor for an enzymatic sulfate transfer, may be carcinogenic for the rat liver.(ABSTRACT TRUNCATED AT 400 WORDS)     

11Beta-HSD and 17beta-HSD as biological markers of depression: sex differences and correlation with symptom severity

There is growing interest in the hydroxysteroid dehydrogenases (HSDs) as local modulators of hormone action. We have studied urinary steroid profiles from nine men and nine women with Major Depression to determine whether 11beta-HSD and 17beta-HSD activity are altered. Urinary steroid profiles were determined by high resolution gas chromatography. The ratio of 11-oxo over 11beta-hydroxy metabolites of cortisol was used as the index of 11beta-HSD activity and the ratio of dehydroepiandrosterone (DHA) over androstenediol-17beta was used as the index of 17beta-HSD activity. Symptom severity was assessed using the Hamilton Depression Rating Scale (HDRS). None of the measures of cortisol production, 9 AM plasma cortisol, 24 hour urinary free cortisol or 24 hour total urinary cortisol metabolites, correlated with HDRS in either men or women. 11Beta-HSD activity was altered and correlated with symptom severity in depressed women (r=0.688, p<0.05) but not men (r=0.132). In both depressed men and women, 17beta-HSD activity was altered and correlated with HDRS scores (r=-0.796, p<0.05 and r=0.688, p<0.05 respectively). However, although the ratio was changed in the same direction in depressed men and women. the correlation with symptom severity was positive in women but negative in men. We conclude that markers of subtle change such as these may prove more useful and informative than gross changes in plasma cortisol in depression.     

Analysis of mammographic density and breast cancer risk from digitized mammograms

To evaluate the association between mammographic density and breast cancer risk, a simple, observer-assisted technique called interactive thresholding was developed that allows reliable quantitative assessment of mammographic density with use of a computer workstation. Use of this technique helps confirm that mammographic density is one of the strongest risk factors for breast cancer and is present in a large proportion of breast cancer cases. The strong relationship between mammographic density and breast cancer risk suggests that the causes of breast cancer may be better understood by identifying the factors associated with mammographically dense tissue and determining how such tissue changes as these factors vary. Furthermore, because it can be modified, mammographic density may also be a good vehicle for the development and monitoring of potential preventive strategies. Areas of ongoing investigation include evaluating a potential genetic component of mammographic density by comparing density measurements in twins and understanding changes in density relative to age, menopausal status, exogenous hormone use, and exposure to environmental carcinogens. In addition, work is ongoing to establish measurements from imaging modalities other than mammography and to relate these measurements directly to breast cancer risk.     

External carotid artery ligation for life-threatening hemorrhage in exsanguinating orbital facial congenital hemangiopericytoma

A 7-week-old infant with a locally invasive, orbital, congenital hemangiopericytoma underwent emergency external carotid artery (ECA) ligation for exsanguinating hemorrhage from an intraoral biopsy site. ECA ligation was successful in controlling the life-threatening hemorrhage and in reducing tumor size and vascularity. The location and extensive nature of the tumor prevented primary excision. Preoperative adjuvant chemotherapy was unsuccessful in controlling tumor growth. After ECA ligation, with reduction in tumor bulk and blood supply, the tumor was radically excised. This technique has important implications in the management of patients with extensive hemangiopericytomas or sino-facial tumors previously regarded as unresectable and which present with life-threatening hemorrhage.     

The exposed ocular surface and its relationship to spontaneous eyeblink rate in elderly caucasians

1. Mechanical forces associated with blood flow play important roles in the acute control of vascular tone, the regulation of arterial structure and remodelling and the localization of atherosclerotic plaque. Uraemia is a proatherogenic process and is expected to be associated with impaired vascular reactivity.2. To study this, 12 male Wistar rats were rendered uraemic by five-sixths nephrectomy and 12 control rats were sham operated simultaneously. After 8 weeks a tail-cuff systolic blood pressure was recorded, blood samples were taken and the animals killed. Isolated femoral arteries were dissected and mounted on a pressure myograph and myogenic tone was assessed over a range of intravascular pressures from 40 to 160 mmHg. Histologically the arteries were comparatively examined for gross morphology, calcification and deposition of collagen.3.Biochemically the serum urea and creatinine were greater in the uraemic compared with the control rats (urea: 23.5+/-6 mmol/l and 6.8+/-01 mmol/l respectively, P not significant; creatinine: 130.7+/-13 mmol/l and 70.3+/-5 mmol/l respectively, P<0.01) but systolic blood pressure was the same in both groups (control, 97+/-1 mmHg; uraemic, 98+/-2 mmHg), compatible with mild uraemia.4. Myogenic tone was significantly greater in uraemic vessels (7.3+/-1.8% versus 2.3+/-0. 4% in control, P=0.01). The actual vessel lumen diameter was also smaller in pressurized uraemic vessels compared with control vessels (471+/-30 microm versus 604+/-33 microm, P<0.01) after equilibration in physiological salt solution. However, when incubated in calcium-free physiological salt solution, the passive internal diameter was similar in uraemic vessels (538+/-25 microm compared with 595+/-31 microm in control). Histologically, there were no differences between the two groups.5. We conclude that some aspects of vascular reactivity are altered in mild experimental uraemia as shown by a reduced internal lumen diameter and increased myogenic tone. Furthermore, these changes are apparent in the absence of hypertension and precede structural changes.