Detergent-shock response in enteric bacteria

Our work on bacterial detergent resistance started with the realization that bacteria growing in a sink full of soap must be resistant to the detergents in that soap. We chose sodium dodecyl sulphate (SDS) as a model detergent and decided to see how much SDS the bacterium growing in the sink could tolerate. The research program thus initiated has shown that bacteria such as Enterobacter cloacae can grow in up to 25% SDS and that SDS-shock proteins constitute c. 8% of the proteins synthesized by SDS-grown Escherichia coli. It has also provided explanations why enteric bacteria are oxidase negative, and how pyrroloquinoline quinone (PQQ) enters the periplasmic space. Finally, for E. coli, it has provided evidence for an alternate, phosphate-limited, aquatic life style which places greater emphasis on the Entner-Doudoroff pathway. Detergent resistance is important both medically and ecologically, e.g. entry of pathogens via bile-salt-containing intestinal tracts and biodegradation of detergent-like pollutants such as those resulting from oil spills. Our current research is focused on SDS-induced modifications of the cytoplasmic membrane and the presence of SDS in the periplasm.     

Nucleotide Sequence of a 28-kb Mouse Genomic Region Comprising the Imprinted Igf2 Gene

The mouse insulin-like growth factor II gene (Igf2) is physicallylinked to the insulin II gene (Ins2) and both are subject totissue-specific genomic imprinting. The paternal-specific expressionof Igf2 has been associated with hypermethylation of some CpGsites in the 5' flanking region and in the body of the gene.As a first step in analyzing the structural features of thisimprinted locus, we here report the complete nucleotide sequenceof Igf2, including all introns and the intergenic region adjacentto Ins2. This 28-kb segment of mouse chromosome 7 exhibits 80%overall identity with the corresponding rat sequence and hasa high GC content of 52%. In addition to the known CpG islandwithin the second Igf2 promoter, another island was identifiedapproximately 2 kb 5' to the first exon. Other features of thislocus include a 35-fold tandem repeat of an 11-bp sequence thatoverlaps Igf2 pseudo-exon 2, and a B2 repeat element in theintergenic region between Ins2 and Igf2. The GC-richness andthe presence of CpG islands associated with tandem repeats arecommon features of imprinted genes and thus may play a rolein the imprinting mechanism.     

Mosquito trypsin: immunocytochemical localization in the midgut of blood-fed Aedes aegypti (L.)

Summary A polyclonal antibody was raised against trypsin purified from the midgut of blood-fed Aedes aegypti. Using this antibody and our modification of the peroxidase-antiperoxidase immunocytochemical reaction, strong activity was found in the lumen of the midgut at the light-microscopical level. The activity was localized mainly in the posterior part of the distensible, abdominal midgut, along the periphery of the blood bolus and within the peritrophic membrane. Immunoreactivity appeared 8 h after the blood meal and was most prominent around 24 h, coinciding with our previous spectrophotometric determinations of trypsin.At the electron-microscopical level, secretory granules, immunocytochemically labelled with anti-trypsin antibody and protein A-colloidal gold, were first detected about 12 h after the blood meal. At 18 h, the secretory pathway could be followed immunocytochemically from the formation of granules in the Golgi complex until their release by exocytosis in the midgut lumen. By 24 h, there was a reduction in secretory granules, and large lysosomes appeared.The process of secretion described for this mosquito is comparable to similar events in vertebrate secretory systems and the presence of an intracellular trypsinogen is suggested.     

17 Beta-hydroxysteroid dehydrogenase in human breast cancer: analysis of kinetic and clinical parameters

In this study, we assessed the rate of estradiol degradation via the 17 beta-hydroxysteroid dehydrogenase (HSD) enzyme in breast tumors from postmenopausal women. We initially studied the effects of time, level of enzyme activity, amount of tissue assayed, and substrate concentration on the linearity of conversion of estradiol to estrone in breast tumor homogenates. The reaction was demonstrated to be linear when less than 15% conversion of estradiol to estrone occurred over 30 min with homogenates produced from 2.5 mg of tissue. Detailed kinetic experiments demonstrated the presence of two classes of enzyme activity, one with high affinity and the other with low affinity. In 83% of the tumors examined, the high affinity form was present and had a median Km of 0.62 microM and Vmax of 82 nmol/g protein/h. In 29 tumors, HSD activity could be precisely quantified and correlated with clinical parameters. No statistically significant correlation of enzyme activity with estrogen receptor (r2 = 0.06) or progesterone receptor (r2 = 0.006) or with patient age could be detected (r2 = 0.001). In 12 additional tumors, activity exceeded 15% conversion of estradiol to estrone at 30 min and precise quantitation was not possible. The average content of progesterone receptor was similar for these 12 tumors as for the 19 with lower HSD activity. However, estrogen receptor content and patient age were lower in the group with high HSD activity. The finding of a high affinity form of HSD in this study provides support for the biological importance of this enzyme in breast cancer tissues.