Use of protoplasts in the improvement of filamentous microorganisms. I. Action of N-methyl-N′-nitro-N-nitrosoguanidine on protoplasts of Streptomycetes

Summary The effects of N-methyl-N–nitro-N-nitroso-guanidine (NTG) on protoplasts of Streptomycetes are markedly different from its action on spores, showing high mutagenic activity even at concentrations having no marked effect on protoplast survival. Strain improvement, eg in chlorotetracycline-producing strains of S. aureofaciens, was most effective when protoplasts were subjected to prolonged treatment (2 h) with low concentrations of NTG (50 /ug/ml).     

Diurnal rhythm of hypophyseal cells in mice. IV. Pars Distalis∗

Abstract

Using various staining techniques and methods, the following cells were differentiated from pars distalis of the pituitary gland in the mouse: corticotropic, somatotropic, lactotropic, thyrotropic and gonadotropic cells.

Diurnal activity of the differentiated cells in pars distalis of the pituitary gland was determined by the method of karyometry.

The karyometric analysis showed the existence of a distinct diurnal rhythm in nuclear volume in all five types of the differentiated cells in both sexes.     

Determination of CO₂ sensitivity of micro-organisms in shaken bioreactors. II. Novel online monitoring method

In the present study, a new online monitoring method for the determination of the CO? sensitivity of micro-organisms, based on the values of the respiration factors [OTR (oxygen transfer rate) and CTR (carbon dioxide transfer rate)], obtained by using the RAMOS (respiratory activity monitoring system) device considering a variety of aeration rates in the measuring flask, is investigated. Based on the data of the OTR, obtained by RAMOS under a variety of specific aeration rates, the proposed new method was developed as an online monitoring method for CO? sensitivity of micro-organisms in shaken bioreactors. A maximum accumulated CO? concentration of 12% was derived in applied methods, provided that the cultivation system is carried out under optimal conditions. Additionally, to predict these conditions, an unsteady-state gas transfer model in shaken bioreactors would be very advantageous. The data of OTR obtained using the RAMOS device were analysed and recalculated by a programme considering the calibration factor (Cf). The major advantage of the new method is the possibility to determine the metabolic activity, regardless of manual sampling.     

Neural stimulation of mitotic activity in the crypts of lieberkühn in rat jejunum.

The influence of nerve stimulation and sham-stimulation on the mitotic rate in epithelial cells lining the crypts of Lieberkühn in the jejunum of anaesthetized rats was studied. Administration of the anaesthetic and opening the abdominal cavity was without significant effect on the crypt cell mitotic rate. However, externalizing a loop of jejunum and applying sham-stimuli to its mesenteric nerves resulted in a significant decrease in the crypt cell mitotic rate in that loop. Application of electrical stimuli to the mesenteric nerves of another externalized jejunal loop resulted in a significant increase in the mitotic rate in the crypt cells of that segment. Similar acceleration of crypt cell proliferation by electrical stimuli applied to mesenteric nerves was also seen in chemically sympathectomized rats.     

Distribution of Legionella spp. in hydrothermal areas in continental Portugal and the island of S?o Miguel, Azores.

Nineteen aquatic environment sites from three hydrothermal areas on continental Portugal and one area on the island of S?o Miguel, Azores, were examined for the recovery of Legionella spp. Physicochemical and bacteriological parameters were also determined for each site. Water temperatures varied between 22 and 67.5 degrees C, although the majority had temperatures above 40 degrees C; the pH varied between 5.5 and 9.2. The number of Legionella spp. recovered varied between 5.0 x 10(2) and 2.3 x 10(6) CFU/liter. A total of 288 isolates from 14 sites were identified by indirect immunofluorescence assay. The majority of the isolates belonged to Legionella pneumophila (74.3%), of which most belong to serogroup 1, but the relative proportion of L. pneumophila serogroups varied considerably. L. pneumophila serogroup 1 constituted 96.2% of the isolates in area 2 from central Portugal, but no isolates of this serogroup were recovered from S?o Miguel, where serogroup 6 strains were the predominant isolates. Ninety-six percent of the L. pneumophila serogroup 1 isolates belonged to monoclonal antibody subgroups OLDA and Bellingham. Other species identified were L. bozemanii serogroup 2, L. dumoffii, L. micdadei, L. moravica, L. oakridgensis, L. sainticrucis, and L. sainthelensi. Two undescribed species, which react by indirect immunofluorescence assay to antisera to "L. londoniensis" and "L. nautarum" and a group of isolates with strong cross-reaction to L. cincinnatiensis/L. sainticrucis/L. longbeachae by indirect immunofluorescence assay were also recovered. The latter were the only isolates recovered from area 3, in east central Portugal, over a period of 1 year.     

Application of in vitro pollination of opened ovaries to obtain Brassica oleracea L. × B. rapa L. hybrids

This study presents the results of experiments concerning: (1) interspecific hybridization of Brassica oleracea × Brassica rapa via application of in vitro placental pollination and (2) embryological analysis of the process of resynthesis of Brassica napus. In order to overcome certain stigma/style barriers, B. rapa pollen was placed in vitro on an opened B. oleracea ovary (with style removed). Pollinated ovaries were cultured on Murashige and Skoog (MS) medium. After 24-d culture, the developing embryos were isolated from immature seeds and transferred onto MS medium supplemented with 0.47 μM kinetin, 0.49 μM 1-naphthaleneacetic acid, and 10% (v/v) coconut water. When the embryos had turned green, they were immediately placed onto MS medium with 100 μM kinetin. After development of the seedling, plantlets were transferred to soil. Chromosome doubling was achieved after another week. Cytometric analysis of nuclear DNA confirmed the hybrid nature of the plants. Resynthesis of B. napus can be performed through interspecific hybridization of B. oleracea × B. rapa followed by embryo rescue and genome doubling.     

The utility of trnK intron 5′ region in phylogenetic analysis of Ulmaceae s. l.

The sequences of chloroplast trn K intron 5' region were determined for seven genera of Ulmaceae s.l. ( 12 species), two genera of Cannabaceae, two of Moraceae and one genus from each of the three families, i.e. , Urticaceae, Eucommiaceae and Malvaceae. The aligned sequences used in PAUP analyses were 805 bp, and the maximum parsimony analysis resulted in a single most parsimonious tree with tree length = 665, CI = 0.7714 and RI = 0.7965. The topology of the tree is relatively strongly supported by the bootstrap test and congruent with those generated from rbc L sequence analysis and cpDNA restriction site analysis. The phylogenetic tree indicated that Ulmaceae s. l. is polyphyletic, and that the monophyletic Ulmaceae s. str. is sister to the other Urticales taxa. Cannabaceae, represented by Humulus and Cannabis, is nested within Celtidaceae, revealing that Celtidaceae is a paraphyletic group. Gironniera and Aphananthe are both clades of Celtidaceae. 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Misunderstanding in the classification of diabetes mellitus. What's in a name?

To assess whether physicians, residents, medical students, hospital diagnosis coders, and patients properly use the designations insulin-dependent diabetes mellitus (IDDM) and non-insulin-dependent diabetes mellitus (NIDDM) that were established by criteria of the National Diabetes Data Group, we reviewed clinic and hospital records and administered questionnaires. Although essentially all cases of true IDDM were identified as such and most cases of NIDDM not requiring insulin therapy were correctly identified by all groups, patients with NIDDM on insulin therapy were misidentified as having IDDM by 38% of residents in internal medicine clinics and 68% of primary care and surgical subspecialty residents. On a survey, of 22 patients with NIDDM on insulin therapy, 17 (77%) considered themselves to have IDDM. Thus, patients who have NIDDM by the established criteria who are on insulin therapy are commonly mislabeled as having IDDM. We present an approach for dealing with this problem by adapting nomenclature focusing on insulin deficiency and resistance. It would probably also be helpful to separately identify the subset of patients with "insulin-deficient diabetes" who are ketosis-prone. It is important to use immunologic profiling (islet cell antibody testing) and insulin sensitivity or deficiency testing (C-peptide levels).     

Clinicians’ Involvement of Patients in Decision Making. A Video Based Comparison of Their Behavior in Public vs. Private Practice

Background

Little is known about the extent to which Peruvian physicians seek to involve patients in shared decision making, or about the variation in these efforts across different settings.

Objective

To measure the extent to which Peruvian clinicians involve their patients in decision making and to explore the differences between clinicians’ behavior in private vs. public practice.

Design

Videographic analysis.

Participants and Setting

Seven academic physicians who provided care to patients in a public and a private setting participate in this study. All the encounters in both settings were filmed on one random day of February 2012. Approach: Two raters, working independently and in duplicate used the 12-item OPTION scale to quantify the extent of physician effort to involve patients in shared decision making (with 0 indicating no effort and 100 maximum possible effort) in 58 video recordings of usual clinical encounters in private and public practice.

Results

The mean OPTION score was 14.3 (SD 7.0). Although the OPTION score in the private setting (mean 16.5, SD 7.3) was higher than in the public setting (mean 12.3 SD 6.1) this difference was not statistically significant (p = .09).

Conclusion

Peruvian academic physicians in this convenience sample barely sought to involve their patients in shared decision making. Additional studies are required to confirm these results which suggest that patient-centered care remains an unfulfilled promise and a source of inequity within and across the private and the public sectors in Peru.     

Loss of heterozygosity in somatic cells of the mouse. An important step in cancer initiation?

Loss of heterozygosity (LOH) of tumour suppressor genes is a crucial step in the development of sporadic and hereditary cancer. Recently, we and others have developed mouse models in which the frequency and nature of LOH events at an autosomal locus can be elucidated in genetically stable normal somatic cells. In this paper, an overview is presented of recent studies in LOH-detecting mouse models. Molecular mechanisms that lead to LOH and the effects of genetic and environmental variables are discussed. The general finding that LOH of a marker gene occurs frequently in somatic cells of the mouse without deleterious effects on cell viability, suggests that also tumour suppressor genes are lost in similar frequencies. LOH of tumour suppressor genes may thus be an initiating event in cancer development.     

Optimization of conditions for expression of recombinant interferon-γ in E.coli

Interferon gamma (IFN-γ) is an important immunoregulatory cytokine that has a central role against viral and bacterial infections. In this study, the cDNA encoding 141 amino acids of mature IFN-γ from mice splenocytes was cloned in a prokaryotic expression vector pQE 30. Optimization of expression conditions resulted in high IFN-γ protein. Western blot showed that recombinant IFN-γ was specifically recognized by its counterpart anti-mouse IFN-γ antibodies. In vitro dose-dependent studies, with A549 and HeLa cell lines, showed that cloned IFN-γ was safe and had no effect on cell proliferation. The protein prediction and analysis using SOPMA program, revealed that IFN-γ had 80 α-helices, 8 β-turns jointed by 9 extended strands and 44 random coils. A total of four major clusters were observed with murine IFN-γ sharing 39 % homology with human IFN-γ. Pair-wise alignment studies with human revealed 26 % identity and 43.3 % similarity. The recovery of bioactive proteins from inclusion bodies (IBs) is a complex process and various protocols have been developed. We report here a simple, robust and inexpensive purification approach for obtaining recombinant IFN-γ protein expressed as IBs in E.coli.     

Functional significance of amylase polymorphism in Drosophila melanogaster. III. Ontogeny of amylase and some α-glucosidases

Changes in amylase (E.C. 3.2.1.1), maltase (E.C. 3.2.1.20), sucrase, and PNPGase activities in relation to changes in wet weight and protein content were studied during the development of larvae and adult flies from two strains of Drosophila melanogaster, homozygous for different amylase alleles. All -glucosidase activities increase exponentially during a large part of larval development, parallel to the increase in weight, and drop at the end of the third instar. Amylase activity of the Amy ¹ strain follows the same pattern. In contrast, amylase activity of the Amy ^4,6 strain continues its exponential increase longer. In the third larval instar amylase activity in the Amy ^4,6 strain becomes much higher than in the Amy ¹ strain. During the first hours of adult life amylase activity of the two strains does not differ. Then Amy ^4,6 activity starts to rise and becomes much higher (4–5 times) than Amy ¹ amylase activity, which remains approximately constant. All adult enzyme activities are much higher than in larvae. Comparison of enzyme activity of amylase and -glucosidases in larvae and adults confirms that differences in amylase activities can become important only when starch is a limiting factor in the food.The investigations were supported by the Foundation for Fundamental Biological Research (BION), which is subsidized by the Netherlands Organization for the Advancement of Pure Research (Z.W.O.).