Diagnosis of Carcinoma and Benign Cysts of the Breast: The Value of Needle Aspiration

Diagnosis of solid breast masses by needle aspiration with cytological examination of the aspirate has been practiced for some time in several centers in this country and abroad. It has been proposed as an alternative to the conventional excisional biopsy for the diagnosis of carcinoma of the breast.At the same time, simple needle aspiration of benign cysts as an office procedure has gained new favor as a means of proving the presence of benign disease at the first office visit and thus avoiding the loss of time, and the expense and worry of surgical excision in a hospital. From a review of the reliability and practical usefulness of both methods, it is concluded that aspiration biopsy for the diagnosis of carcinoma is less reliable than conventional excisional biopsy and offers very little practical advantage.Simple aspiration of cysts, on the other hand, appears to offer a true saving of time, expense and worry, and to be a reliable method, if used properly.     

The comparison of rat liver rough endoplasmic reticulum membrane proteins before and after in vitro removal of its bound ribosomes

The comparison of the proteins of rat liver rough membrane after stripping with EDTA or KCl-puromycin by two dimensional gel electrophoresis is described. By stripping the membrane with EDTA, most of the basic ribosomal proteins are still attached to the membrane; in contrast to the EDTA stripping method, treatment with KCl-puromycin removes most of the ribosomal proteins and does not remove any of the membranal proteins.     

Reconstitution of the vascular wall in vitro. A novel model to study interactions between endothelial and smooth muscle cells

To study the biology of the endothelium under conditions that mimic the architecture of the vessel wall, endothelial cells were grown on a collagen lattice containing a multilayer of smooth muscle cells. Light and electron microscopy of such cultures revealed a confluent monolayer of flattened endothelial cells. In co-culture, endothelial cells tend to elongate, whereas in the absence of smooth muscle cells, the endothelial cells show the polygonal morphology typical for cultures of endothelial cells grown on polystyrene substrates. As conditioned culture media of endothelial cells contain substances that may both promote or inhibit the growth of smooth muscle cells, the availability of this vessel wall model prompted us to examine to what extent endothelial cells regulate the proliferation of smooth muscle cells when these cells are maintained in co-culture. Here we show that endothelial cells suppress the proliferation of co-existing smooth muscle cells. This finding suggests that under physiological conditions the balance of the action of growth-promoting and growth-inhibiting substances produced by endothelial cells is in favour of the latter.     

Effect of apolipoproteins E and C-III on the interaction of chylomicrons with parenchymal and non-parenchymal cells from rat liver.

[3H]Triacylglycerol-labelled chylomicrons were isolated from intestinal lymph, obtained from rats made hypolipidaemic by treatment with pharmacological amounts of 17 alpha-ethynyloestradiol. Oestrogen treatment results in a large reduction in the content of apolipoproteins (apo) E and C of lymph chylomicrons. Upon incubation in vitro with freshly isolated parenchymal and non-parenchymal cells the apo E-, apo C-poor chylomicrons became readily cell-associated. With increasing chylomicron concentrations this cell-association was saturable and half-maximal cell-association was achieved at about 0.55 mg of triacylglycerol/ml. The cell-association was time- and temperature-dependent. A more than 90% inhibition of the cell-association of the [3H]triacylglycerol moiety was observed with both parenchymal and non-parenchymal cells when pure apo C-III (12.6 micrograms/mg of triacylglycerol) was incorporated into the chylomicrons. These data indicate that apo E-, apo C-poor chylomicrons are bound to both parenchymal and non-parenchymal liver cells at a high-affinity site of limited capacity and that binding to this site is strongly inhibited by apo C-III. With apo C-III-enriched chylomicrons simultaneous determination of the cell-association of the 125I-apo C-III and the [3H]triacylglycerol moiety indicated that more 125I-apo C-III becomes associated to the cells than expected on the basis of [3H]triacylglycerol radioactivity measurements. It is suggested that upon cell-association of apo C-III its binding to the chylomicron particles is lost. Consequently the occupation of the cellular recognition site by apo C-III prevents further chylomicron binding and thus leads to a decrease of the cell-association level of the [3H]triacylglycerol moiety. Apo E enrichment of the chylomicrons led to an increased cell-association rate with parenchymal cells and to a marked increase of the cell-association level with non-parenchymal cells. The cell-association of the apo E radioactivity followed closely the [3H]triacylglycerol radioactivity, indicating that the particle-apo E complex is bound as a unity. The apo E effects were opposed by apo C-III. With apo E-, apo C-III-enriched chylomicrons more 125I-apo E became associated with the cells than could be expected on the basis of the [3H]triacylglycerol measurements. It is concluded that apo C-III can weaken the interaction of apo E with the chylomicrons leading to the cell-association of free apo E. It appears that subtle changes in the apo E and/or apo C-III content of chylomicrons can influence the interaction with both parenchymal and non-parenchymal liver cells.(ABSTRACT TRUNCATED AT 400 WORDS)     

CRISPR-Cas9 mediated gene deletions in lager yeast <Emphasis Type="Italic">Saccharomyces pastorianus</Emphasis>

Background

The ease of use of CRISPR-Cas9 reprogramming, its high efficacy, and its multiplexing capabilities have brought this technology at the forefront of genome editing techniques. Saccharomyces pastorianus is an aneuploid interspecific hybrid of Saccharomyces cerevisiae and Saccharomyces eubayanus that has been domesticated for centuries and is used for the industrial fermentation of lager beer. For yet uncharacterised reasons, this hybrid yeast is far more resilient to genetic alteration than its ancestor S. cerevisiae.

Results

This study reports a new CRISPR-Cas9 method for accurate gene deletion in S. pastorianus. This method combined the Streptococcus pyogenes cas9 gene expressed from either a chromosomal locus or from a mobile genetic element in combination with a plasmid-borne gRNA expression cassette. While the well-established gRNA expression system using the RNA polymerase III dependent SNR52 promoter failed, expression of a gRNA flanked with Hammerhead and Hepatitis Delta Virus ribozymes using the RNA polymerase II dependent TDH3 promoter successfully led to accurate deletion of all four alleles of the SeILV6 gene in strain CBS1483. Furthermore the expression of two ribozyme-flanked gRNAs separated by a 10-bp linker in a polycistronic array successfully led to the simultaneous deletion of SeATF1 and SeATF2, genes located on two separate chromosomes. The expression of this array resulted in the precise deletion of all five and four alleles mediated by homologous recombination in the strains CBS1483 and Weihenstephan 34/70 respectively, demonstrating the multiplexing abilities of this gRNA expression design.

Conclusions

These results firmly established that CRISPR-Cas9 significantly facilitates and accelerates genome editing in S. pastorianus.

     

Butanol recovery from fermentations by liquid-liquid extraction and membrane solvent extraction

Extraction can successfully be used for in-situ alcohol recovery in butanol fermentations to increase the substrate conversion. An advantage of extraction over other recovery methods may be the high capacity of the solvent and the high selectivity of the alcohol/water separation. Extraction, however, is a comprehensive operation, and the design of an extraction apparatus can be complex. The aim of this study is to assess the practical applicability of liquid-liquid extraction and membrane solvent extraction in butanol fermentations. In this view various aspects of extraction processes were investigated.Thirty-six chemicals were tested for the distribution coefficient for butanol, the selectivity of alcohol/water separation and the toxicity towards Clostridia. Convenient extractants were found in the group of esters with high molar mass.Liquid-liquid extraction was carried out in a stirred fermenter and a spray column. The formation of emulsions and the fouling of the solvent in a fermentation broth causes problems with the operation of this type of equipment. With membrane solvent extraction, in which the solvent is separated from the broth by a membrane, a dispersion-free extraction is possible, leading to an easy operation of the equipment. In this case the mass transfer in the membrane becomes important.With membrane solvent extraction the development of a process is emphasized in which the extraction characteristics of the solvent are combined with the property of silicone rubber membranes to separate butanol from water. In the case of apolar solvents with a high molar mass, the characteristics of the membrane process are determined completely by the solvent. In the case of polar solvents (e.g. ethylene glycol), the permselectivity of the membrane can profitably be used. This concept leads to a novel type of extraction process in which alcohol is extracted with a water-soluble solvent via a hydrophobic semipermeable membrane. This extraction process has been investigated for the recovery of butanol and ethanol from water. A major drawback in all processes with membrane solvent extraction was the permeation of part of the solvent to the aqueous phase.The extraction processes were coupled to batch, fed batch and continuous butanol fermentations to affirm the applicability of the recovery techniques in the actual process. In the batch and fed batch fermentations a three-fold increase in the substrate consumption could be achieved, in the continuous fermentation about 30% increase.     

Isolation of DNA markers informative in purebred dog families by genomic representational difference analysis (gRDA)

Genomic Representational Difference Analysis (gRDA) is a subtractive DNA method to clone the differences between two related genomes, called tester and driver. We have evaluated this method to obtain polymorphic DNA markers for pedigree dogs. Amplified size-selected genomic restriction fragments (amplicons) of two dog littermates were repeatedly hybridized to each other in order to remove (subtract) those restriction fragments common to both sibs. Already after two rounds of subtractive hybridization, a clear enrichment of presumably tester-specific restriction fragments was observed, which was even more pronounced after the third round of subtraction. A plasmid library of 3000 recombinant clones was constructed of the second round and of the third round difference product. DNA sequence determination of randomly chosen clones of each difference product showed that approximately 1000 unique clones were obtained in the second-round difference product and approximately 500 in the third-round difference product. About half of the clones identified in the second-round difference product were also present in the third-round difference product. Of the second-round difference product, 39 different gRDA fragments could be identified, of which 21 were tester specific. In the third-round difference product, 22 different gRDA fragments were identified, of which 18 were tester specific. There were 13 fragments in common, resulting in a total of 48 different fragments. In order to establish the localization of these markers, we performed mapping using the dog radiation hybrid panel RHDF5000. Of 39 mapped clones, 29 were mapped to 20 existing RH groups, and 10 remained unlinked. It is concluded that gRDA is suitable to generate DNA markers to track disease genes within lines of pedigree dogs. Received: 26 April 2000 / Accepted: 11 May 2000