Isolation of a 30 kDa immunoglobulin binding protein from Pseudomonas maltophilia

We have demonstrated that Pseudomonas maltophilia (ATCC No. 13637) possesses an exposed, immunologically accessible protein which binds to the Fc region of several species of immunoglobulins. Whole bacteria suspensions were incubated for 18 h with purified 125I-labelled antibodies with and without added non-labelled immunoglobulins. The suspensions were centrifuged for 30 min and the pellet containing bacteria was assessed for radioactivity. Using this crude assay, the whole organism bound 125I-labelled rabbit and mouse immunoglobulins and the purified Fc portion of human IgG. All of these labelled preparations were competitively displaced by unlabelled rabbit and mouse immunoglobulins, and Fc of human IgG, as well as human immunoglobulin subclasses. The organism was sonicated to solubilize this immunoglobulin binding protein. Using this sonicated preparation, it was shown that unlabelled Fc of IgG, unlabelled mouse and rabbit immunoglobulins, all competitively displaced 125I-labelled human Fc of IgG in a dose-response manner. A partially purified protein was prepared by Sephacryl S-300 followed by Sephadex G-100 column chromatography. This preparation was incubated with 125I-Fc gamma and with the following purified unlabelled preparations: F(ab')2 of IgG, Fc of IgG, murine monoclonal IgA, IgG1, IgG2, IgG3, and IgG4. All except F(ab')2 of IgG produced dose response competitive displacement. The molecular weight, as estimated by SDS-PAGE and Western blot, was 30,000 daltons. In Western blots, Fc gamma, murine monoclonal IgA, and human immunoglobulin subclasses, all showed affinity for the immobilized protein. Human F(ab')2 fragments did not show affinity for the protein. Radioiodinated pseudomonal Ig-binding protein showed affinity for human IgG coupled to Sepharose, and was displaced by unlabelled pseudomonal Ig-binding protein. Scatchard analysis of binding showed two binding affinities: two distinct types of Ig-binding proteins were obtained, a high affinity with Kd = 1.54 x 10(-10) and a lower affinity with Kd = 2.36 x 10(-8). This immunoglobulin binding protein may be useful in immunoglobulin purification or identification.     

REM sleep deprivation inhibits LTP in vivo in area CA1 of rat hippocampus

Rapid eye movement (REM) sleep deprivation has previously been shown to interfere with normal learning and memory and to inhibit long-term potentiation (LTP) in vitro. Previous studies on REM sleep deprivation and LTP have relied on in vitro analysis in isolated brain slices taken from animals following several days of sleep deprivation. LTP in the hippocampus in situ may differ from LTP in vitro due to modulatory inputs from other brain regions, which are altered after REM sleep deprivation. Here, we examined LTP in unanesthetized, behaving animals on the first and second recovery days following REM sleep deprivation to determine if similar effects are seen in vivo as previously reported in vitro. We found that LTP was significantly impaired in REM sleep-deprived animals on the second recovery day but not the first recovery day. Our results extend previous findings by showing that REM sleep deprivation continues to affect hippocampal function for more than 24h following the end of deprivation. Our results also suggest the presence of a modulatory process not present in vitro. Our findings are not explained by stress during REM sleep deprivation because equivalent circulating corticosterone levels (an index of stress) were found during both REM sleep deprivation and control treatment.     

Lung vascular smooth muscle as a determinant of pulmonary hypertension at high altitude.

The pulmonary hypertensive response to chronic hypoxia varies markedly among mammalian species. An explanation for this variability was sought by exposing seven species to hypobaric hypoxia (PB equal to 435 mmHg) for 19-48 days. Control animals were studied at 1,600 m (PB equal to 630 mmHg). The pulmonary hypertension that developed varied in the following order of decreasing severity: calf and pig (severe); rat and rabbit (moderate); sheep, guinea pig, and dog (mild). Right ventricular hypertrophy developed in proportion to the elevation in right ventricular systolic pressure. These interspecies variations in response were not correlated with the degree of arterial hypoxemia, degree of polycythemia, elevation in heart rate, or postnatal age. However, the medial thickness of the small pulmonary arteries in control animals was highly correlated with the development of pulmonary hypertension and right ventricular hypertrophy in hypoxic animals. Thus, the amount of lung vascular smooth muscle inherent within each species is a major determinant of the pulmonary hypertensive response to high altitude and contributes to the interspecies variability in this response.     

Genotoxic evaluation of welders occupationally exposed to chromium and nickel using the Comet and micronucleus assays

Chromium (Cr) and nickel (Ni) are widely used industrial chemicals. Welders in India are inclined to possible occupational Cr and Ni exposure. The carcinogenic potential of metals is a major issue in defining human health risk from exposure. Hence, in the present investigation, 102 welders and an equal number of control subjects were monitored for DNA damage in blood leucocytes utilizing the Comet assay. The two groups had similar mean ages and smoking prevalences. A few subjects were randomly selected for estimation of Cr and Ni content in whole blood by inductively coupled plasma mass spectrometry. The Comet assay was carried out to quantify basal DNA damage. The mean comet tail length was used to measure DNA damage. Welders had higher Cr and Ni content when compared with controls (Cr, 151.65 versus 17.86 micro g/l; Ni 132.39 versus 16.91 micro g/l; P < 0.001). The results indicated that the welders had a larger mean comet tail length than that of the controls (mean +/- SD, 23.05 +/- 3.86 versus 8.94 +/- 3.16; P < 0.001). In addition, the micronucleus test on buccal epithelial cells was carried out in a few randomly selected subjects. Welders showed a significant increase in micronucleated cells compared with controls (1.30 versus 0.32; P < 0.001). Analysis of variance revealed that occupational exposure (P < 0.05) had a significant effect on DNA mean tail length, whereas smoking and age had no significant effect on DNA damage. The current study suggested that chronic occupational exposure to Cr and Ni during welding could lead to increased levels of DNA damage.     

The modulation of excitatory synaptic transmission by adenosine in area CA1 of the rat hippocampus is temperature dependent

We tested the possibility that extracellular adenosine concentration varies with tissue temperature by measuring the tonic adenosinergic inhibition of excitatory synaptic transmission at different temperatures in the in vitro rat hippocampus. Application of the A1 receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) enhanced population excitatory postsynaptic potentials (EPSPs) by antagonizing tonic adenosinergic inhibition; this effect was greatest at 25 degrees C, and was progressively reduced at 35 and 37.5 degrees C. These results demonstrate that tonic adenosinergic inhibition is inversely related to temperature. In a second experiment, an exogenous A1 agonist, N6-cyclohexyladenosine (CHA), was applied to slices to inhibit evoked EPSPs. CHA inhibition of EPSPs was greater at 35 than at 25 degrees C, demonstrating that the reduced adenosinergic inhibition at higher temperatures is not a result of reduced A1 receptor function.     

Role of the left interventricular sulcus in formation of the interventricular septum and crista supraventricularis in normal human cardiogenesis

Serial sections of normal human embryos were studied and three-dimensional images reconstructed to determine the early development of the interventricular septum. The position of the interventricular septum is determined in stage 9 of normal development by the formation of the left interventricular sulcus. As a result of unknown properties of the cells of the myocardial layer, the left interventricular sulcus persists while the right disappears, producing the initial lateral asymmetry of the primary heart tube. By stage 14, the left interventricular sulcus forms a spiral which is continuous with the developing interventricular septum. The dorsal limb of the spiral passes to the right between the atrioventricular canal and the origin of the outflow tract, and is lost in the wall of the trabeculated right ventricle. It appears that this dorsal limb of the spiral is the precursor of part of the cirsta supraventricularis. The midportion of the sulcus, the bulboventricular groove, becomes the socalled fibrous continuity between the aortic and mitral valves. The ventral limb of the spiral passes caudally in the anterior interventricular groove and then dorsally and cranially toward the dorsal cushion of the atrioventricular canal. The ventral limb of the spiral is continuous with the crest of the muscular interventricular septum, which develops by apposition of tissue from the expanding right and left ventricles. From stage 14 to stage 19, the muscular interventricular septum, the atrioventricular endocardial cushions, and the ventricular end of the spiral ridges of the outflow tract appose and fuse. Subsequent formation of the membranous interventricular septum completes the physical separation of the right and left ventricles.     

Superantigen-induced T cell responses in acute rheumatic fever and chronic rheumatic heart disease patients

CD4+ and CD8+ T cells from healthy donors, acute rheumatic fever (ARF) and chronic rheumatic heart disease (CRHD) patients responded variably to a superantigen from Streptococcus pyogenes--Streptococcal pyrogenic erythrogenic toxin A (SPE-A). In vitro culture of CD4+ T cells from ARF patients (CD4-ARF) with SPE-A exhibited a Th1 type of response as they produced high levels of IL-2, while CD4+ T cells from CRHD patients (CD4-RHD) secreted IL-4 and IL-10 in large amounts, i.e. Th2 type of cytokine profile. The skewing of human CD4+ T cells (in response to SPE-A stimulation) to Th1 or Th2 type reflects the role of the two subsets in a disorder with differing intensities at the two extremes of the spectrum. Moreover, the anergy induction experiments revealed that CD8-ARF and CD8-RHD undergo anergy (to different extents), whereas CD4+ T cells do not, in response to re-stimulation by SPE-A. These results initially demonstrate that both CD4+ and CD8+ T cells respond differentially to SPE-A, and hence it is an important observation with respect to the pathogenesis of ARF/CRHD. Anergy in CD8+ T cells in the presence of SPE-A in vitro goes a step further to show the clinical relevance of these cells and their possible role in suppression of the disease.     

Amyotrophic cerebellar hypoplasia

Summary Lower motor neuron degeneration, cerebellar hypoplasia, atrophy of pons, olives, and cerebellum, sclerosis of thalamus and pallidum, and deficient myelination were found in a 2-months-old baby with laryngeal paralysis, mental retardation, progressive amyotrophy, and slow nerve conduction velocity. Such changes seem characteristic of an unusual syndrome previously referred to as cerebellar hypoplasia in Werdnig-Hoffmann disease, or anterior horn cell disease with pontocerebellar hypoplasia. Although the pathologic changes in lower motor neurons are indistinguishable from those in other cases of infantile spinal muscular atrophy, the consistent reproducibility of a complex pathologic pattern suggests that this is probably a manifestation of a separate disease process. The term amyotrophic cerebellar hypoplasia (ACH) is a convenient designation for the syndrome.Supported in part by National Institutes of Health grant no. RR75