Clinical importance of inducible beta-lactamases in gram-negative bacteria

The clinical problems caused by inducible beta-lactamases in certain gram-negative bacteria are being recognized with increasing frequency. These problems include the rapid emergence of multiple beta-lactam resistance during therapy with many of the newer beta-lactam antibiotics. Such multiply resistant organisms are now spreading within the hospital and have become important nosocomial pathogens. This has been a particularly difficult problem for intensive care units, cystic fibrosis centers and burn units where there are clusters of patients who are highly susceptible to infections with organisms likeEnterobacter spp.,Serratia spp. andPseudomonas aeruginosa, which possess inducible beta-lactamases. Only through an awareness of these problems, their cause, and restriction of the use of certain newer betalactam antibiotics can these problems be controlled.     

Acid phosphatase in the lysosomal and microsomal fractions of human prostatic epithelium

Polyacrylamide gel electrophoresis of acid phosphatase from prostatic tissue reveals one more band than seminal plasma. It was attempted to ascertain which subcellular fraction was responsible for that intracellularly localized enzyme. Prostatic epithelium from patients with prostatic hyperplasia was homogenized, and a lysosomal and microsomal fraction were prepared by differential centrifugation. These two fractions were further centrifuged on an isopycnic Percoll gradient. The intracellularly localized form of acid phosphatase was associated with the lysosomal as well as with the microsomal fraction. In a fused rocket electrophoresis experiment these acid phosphatases cross-reacted with antiserum from seminal plasma. After neuraminidase treatment of the acid phosphatase of lysosomal and microsomal origin, only one activity band was found in polyacrylamide gels. It is concluded that only one acid phosphatase protein exists in prostatic epithelium; differences in electrophoretic mobility are caused mainly by different amounts of sialic acid residues, coupled to the same protein backbone.     

Impact of clinical history on fracture detection with radiography

The effect of knowledge of localizing symptoms and signs in the detection of fractures was studied. Forty radiographs of the extremities were examined twice by seven radiologists; the sessions were separated by 4 months. In 26 cases, a subtle fracture was present; 14 cases were normal. In half of the cases at each session, the precise location of pain, tenderness, or swelling was provided. The observer was asked to determine if the case was normal or abnormal (provide the exact location of the fracture) and to indicate the degree of confidence in the diagnosis. Responses were converted to a numeric scale for analysis. Analysis of receiver operator characteristic parameters indicates that clues regarding location of trauma facilitate detection of fractures. The improvement is based largely on an increased true-positive rate without an increased false-positive rate, regardless of the decision criteria of the radiologist (overall willingness to "overread" or "underread"). This has direct clinical applicability and reinforces the plea of radiologists for precise clinical information.     

Infectious salmon anemia virus (ISAV) genomic segment 3 encodes the viral nucleoprotein (NP), an RNA-binding protein with two monopartite nuclear localization signals (NLS)

Infectious salmon anemia virus (ISAV) is the type species of the genus Isavirus belonging to the Orthomyxoviridae, and causes serious disease in Atlantic salmon (Salmo salar). This study presents the expression and functional analysis of the ISAV genome segment 3, and provides further evidence that it encodes the viral nucleoprotein (NP). The encoded protein was expressed in a baculovirus system, and Western blot analysis showed that it corresponds to the 66-71 kDa structural protein previously found in purified ISAV preparations. RNA-binding activity was established by the interaction of viral and recombinant NP with single-stranded RNA transcribed in vitro. Immunofluorescence studies of infected cells showed the ISAV NP to be an early protein. It locates to the nucleus of infected cells before it is transported to the cytoplasm prior to virus assembly. A similar localization pattern was observed in cells transfected with the NP gene, confirming that the encoded protein has an intrinsic ability to be imported into the nucleus. Two monopartite nuclear localization signals (NLS) at amino acids (230)RPKR(233) and (473)KPKK(476) were identified by computer analysis, and validated by site-directed mutagenesis. In contrast to other orthomyxovirus-NPs, that have several NLSs that function independent of each other, both NLSs had to be present for the ISAV NP protein to be transported into the nucleus, indicating that these motifs cooperate to target the protein to the nucleus.     

Elongation of very long-chain fatty acids is enhanced in X-linked adrenoleukodystrophy

X-linked adrenoleukodystrophy (X-ALD) is a progressive neurodegenerative disorder characterized by the accumulation of saturated and mono-unsaturated very long-chain fatty acids (VLCFA) and reduced peroxisomal VLCFA beta-oxidation activity. In this study, we investigated the role of VLCFA biosynthesis in X-ALD fibroblasts. Our data demonstrate that elongation of both saturated and mono-unsaturated VLCFAs is enhanced in fibroblasts from patients with peroxisomal beta-oxidation defects including X-ALD, and peroxisome biogenesis disorders. These data indicate that enhanced VLCFA elongation is a general phenomenon associated with an impairment in peroxisomal beta-oxidation, and not specific for X-ALD alone. Analysis of plasma samples from patients with X-ALD and different peroxisomal beta-oxidation deficiencies revealed increased concentrations of VLCFAs up to 32 carbons. We infer that enhanced elongation does not result from impaired peroxisomal beta-oxidation alone, but is due to the additional effect of unchecked chain elongation. We demonstrate that elongated VLCFAs are incorporated into complex lipids. The role of chain elongation was also studied retrospectively in samples from patients with X-ALD previously treated with "Lorenzo's oil." We found that the decrease in plasma C26:0 previously found is offset by the increase of mono-unsaturated VLCFAs, not measured previously during the trial. We conclude that evaluation of treatment protocols for disorders of peroxisomal beta-oxidation making use of plasma samples should include the measurement of saturated and unsaturated VLCFAs of chain lengths above 26 carbon atoms. We also conclude that chain elongation offers an interesting target to be studied as a possible mode of treatment for X-ALD and other peroxisomal beta-oxidation disorders.     

Thermal gradients in microtitration plates. Effects on enzyme-linked immunoassay

Temperature studies of microtitration plates demonstrate that the use of a common bacteriology incubator for heating the plates can cause a phase lag of over 30 min for the fluid in the wells to reach 37°C from ambient temperature, and that a temperature gradient of as much as 1.6°C can exist between the peripheral and center wells. This gradient is a cause of the ‘rim’ or edge effect noted in enzyme immunoassay using microtitration plates. The problem is corrected by the use of a specially designed forced air microtitration plate incubator.     

Determinants of CD4 Counts Among HIV-Negative Ethiopians: Role of Body Mass Index,Gender, Cigarette Smoking,Khat (<Emphasis Type="Italic">Catha Edulis</Emphasis>) Chewing,and Possibly Altitude?

To study the determinants of CD4% and CD4 counts among HIV-negative Ethiopians, and to identify factors susceptible to explain the low CD4 counts observed among Ethiopian subjects. Cohort studies among factory workers in Akaki and Wonji, Ethiopia. Clinical and laboratory examinations, including determination of HIV serological status and T-cell subsets, were performed during follow-up visits every six months. In addition, micronutrients (retinol, carotenoids, tocopherol, transferrin receptor, and selenium) plasma concentrations were determined in a subset of 38 HIV-positive and 121 HIV-negative participants. HIV-negative participants with at least one CD4 count measurement were 157 females in Akaki, 203 males in Akaki, and 712 males in Wonji. CD4 counts were independently and positively associated with body mass index (through an increase in lymphocyte counts), female gender (through an increase in CD4%), cigarette smoking (through an increase in CD4%), khat chewing (through an increase in both lymphocyte counts and CD4%), and Akaki study site (through a large increase in lymphocyte counts compensating a decrease in CD4%). Intestinal parasitic infections were not associated with CD4% or CD4 counts. Retinol, carotenoids, and -tocopherol plasma concentrations decreased with HIV infection and advancing immunosuppression, but were not associated with CD4 counts among HIV-negative subjects. Low body mass index among Ethiopians may have contributed to their overall low CD4 counts. Other factors remain to be elucidated.     

Expression of TGFβ1/β3 during early chick embryo development

We have used an antibody against a TGFβ peptide fragment to localize this growth factor in the early chick embryo from laying to the ten-somite stage of development. Western blotting showed that the antibody reacted with both mammalian TGFβ1 and chicken TGFβ3. By immunocytochemistry we find that at the earliest developmental stage (stage X of Eyal-Giladi and Kochav) immunoreactivity to this antibody is primarily located in the cells of the area opaca and marginal zone, as well as in the most peripheral edge cells of the blastoderm. The yolk is non-reactive, except in a highly localized region subjacent to the edge cells. This pattern persists at stage XII, and at both stages individual isolated cells in the epiblast and hypoblast are also reactive. By the time to gastrulation, reactivity in the epiblast is polarized to the ventral extremity of the cells, and again some isolated cells in this layer are intensely immunoreactive. At this stage also, the endoderm cells, particularly those underlying the primitive streak, are positive, as are the mesoderm cells lateral to the streak. At somite stages, the neuroepithelium is not reactive but the ectoderm lateral to it is strongly positive. At the caudal primitive streak levels of early somite embryos, the ectoderm and endoderm are immunoreactive while the mesoderm loses the reactivity it showed at the early gastrulation stages. The neuroepithelial cells later show reactivity at their apical poles, and, as at the earlier stages, individual cells show intense labelling. These results indicate that TGFβ1 and/or TGFβ3 immunoreactivity is developmentally regulated from very early stages of morphogenesis in the chick, and together with data from earlier functional studies, suggest that this factor has roles in embryonic axis formation and in blastoderm expansion. © 1994 Wiley-Liss, Inc.     

Repeat sizes at CAG/CTG loci CTG18.1, ERDA1 and TGC13-7a in schizophrenia

A number of studies using the repeat expansion detection (RED) technique have suggested an association between unknown large CAG/CTG repeats and schizophrenia. The polymorphic CAG/CTG repeat loci CTG18.1 and ERDA1 have been reported to account for a high proportion (approximately 90%) of the large repeats detected by RED and may therefore be responsible for the cited association. The recently described locus TGC13-7a contains a highly polymorphic CTA/TAG and CAG/CTG composite repeat, and is thus another authentic candidate. In the present investigation, each locus was analysed for association with schizophrenia in a sample of 206 patients and 219 group-matched controls. No evidence for association of CTG18.1, ERDA1 and/or TGC13-7a with schizophrenia was found. The combined data accounted for only 54% of the CAG/CTG arrays of > 40 repeats found in our previous RED analysis.     

Reversibility of Effects of Very Hypotonic Fluids on in Vitro Frog Gastric Mucosa: A Functional and Morphological Study

Functional and morphological properties of the in vitro frog gastric mucosa were studied during and after exposure to very hypotonic (? 25 mOsM) solutions. Within 20 min the acid secretory rate decreased to zero, but it returned to normal levels after isotonic fluids had been restored. The potential difference (PD) dropped within the first minutes after the exposure to hypotonic solutions, and became inverted. Following the return of isotonic conditions the PD increased to levels higher than in the controls. The electrical resistance increased about 10–fold during the hypotonic period, but decreased to near normal values when isotonic conditions were restored. By light and electron microscopy the cells of the hypotonic mucosae appeared greatly swollen, and the alterations were assessed by morphometric methods. The gland lumina were almost obliterated, and the lamina propria was reduced to about 60% of its former volume. After the return to isotonic conditions normal morphology was restored. It is conceivable that the great increase in resistance during the hypotonic period was caused by the occlusion of the gland lumina. Quantitative analyses of the Na, K, and C1 tissue concentrations indicated a large loss of these ions during the hypotonic state. Presumably the epithelial cells in the hypotonic mucosae avoid bursting by rapidly letting large numbers of ions exit, which results in a cellular osmolarity close to that of the bathing fluids.