Constitutive expression in gal7 mutants of Kluyveromyces lactis is due to internal production of galactose as an inducer of the Gal/Lac regulon

The induction process of the galactose regulon has been intensively studied, but until now the nature of the inducer has remained unknown. We have analyzed a delta gal7 mutant of the yeast Kluyveromyces lactis, which lacks the galactotransferase activity and is able to express the genes of the Gal/Lac regulon also in the absence of galactose. We found that this expression is semiconstitutive and undergoes a strong induction during the stationary phase. The gal1-209 mutant, which has a reduced kinase activity but retains its positive regulatory function, also shows a constitutive expression of beta-galactosidase, suggesting that galactose is the inducer. A gal10 deletion in delta gal7 or gal1-209 mutants reduces the expression to under wild-type levels. The presence of the inducer could be demonstrated in both delta gal7 crude extracts and culture medium by means of a bioassay using the induction in gal1-209 cells. A mutation in the transporter gene LAC12 decreases the level of induction in gal7 cells, indicating that galactose is partly released into the medium and then retransported into the cells. Nuclear magnetic resonance analysis of crude extracts from delta gal7 cells revealed the presence of 50 microM galactose. We conclude that galactose is the inducer of the Gal/Lac regulon and is produced via UDP-galactose through a yet-unknown pathway.     

Multiple peaks in high-performance liquid chromatography of proteins. beta-Lactoglobulins eluted in a hydrophobic interaction chromatography system

The hypothesis of Geisler (Brain Res. 212 (1981) 198-201), in which the different spontaneous-rate classes of primary auditory neurons were accounted for by the different sizes of uniquantal EPSPs relative to the gap between resting membrane and threshold potentials, was represented with an expanded model which included relative refractory effects. The spike rates generated by the expanded model, when plotted vs. estimated sound level, are qualitatively similar to those of experimentally obtained rate-level curves. The hypothesis is also consistent with recent ultrastructural data which suggest that average quantal-release rates for any particular primary auditory neuron are inversely related to its spontaneous rate. The model's recovery processes following spike generation (hazard functions) are also similar to those observed experimentally.     

Major internal nuclear matrix proteins are common to different human cell types

Cancer invasion and metastasis are associated with matrix degradation. We describe a novel in vivo model of invasion by squamous epithelial neoplastic cells derived from transgenic mice grown on acellular human dermis. Human dermis was subjected to multiple freeze-thaw cycles to render it acellular, maintaining the basement membrane of the former dermal-epidermal junction. Cells representing discrete stages of a multistep transgenic mouse model of epidermal carcinogenesis (neonatal transgenic keratinocytes, moderately/poorly differentiated squamous cell carcinoma, and lymph node metastasis) were seeded onto the basement membrane surface, grown in culture for 4 days, grafted in a subpannicular pocket of athymic mice, and harvested after 3 weeks. Histological analysis demonstrated that neonatal transgenic keratinocytes did not degrade the basement membrane or invade the underlying dermis. In contrast, malignant cells derived from both a moderately differentiated squamous carcinoma and a lymph node metastasis were highly invasive. Immunohistochemical analysis revealed collagenase only in nests of invading malignant cells in contact with the dermal matrix, but not in the tumor mass remaining above the basement membrane, suggesting that this proteinase may be required for stromal invasion. This novel model recapitulates the events seen in malignant invasion: transgenic keratinocytes are unable to penetrate the dermis while cells from a moderately differentiated carcinoma and from lymph node metastasis consistently invade.     

Late rectal sequelae following definitive radiation therapy for carcinoma of the uterine cervix: a dosimetric analysis

OBJECTIVE: The structure of the collagen scar during healing of a myocardial infarction is a determinant of the function of the remodeled tissue. We hypothesize that the passive deformations of both scar and normal tissue are related to the underlying collagen uncoiling as the tissue stretches, and that the unloaded tortuosity of the collagen may be a determinant of tissue stiffness at low ventricular pressure. Hence collagen uncoiling and tissue strain were measured during passive loading in normal tissue, and in healing infarct tissue. METHODS: Left ventricles of rats were infarcted by ligation of the left anterior descending artery for 2 weeks. Surface strains were measured during passive inflation in the scar region in one set of excised hearts, and other arrested hearts were fixed at different ventricular pressures, after which collagen tortuosity was measured in the infarcted and normal tissue. RESULTS: Passive loading strains were smaller in the scar in both the fiber and cross-fiber directions. Tortuosity decreased with load in normal and infarcted tissue, with fibrils tending to straighten more in the scar tissue at higher pressures (1.056 +/- 0.009 vs. 1.024 +/- 0.009 at P = 20 mmHg) with similar tortuosities at zero pressure (1.110 +/- 0.012 vs. 1.098 +/- 0.019). The decrease in tortuosity with strain was greater for the infarcted tissue. CONCLUSIONS: The greater stiffness of infarcted tissue at low pressure is not due to 'straightened' collagen fibers, and there may be a different three-dimensional structure of infarct vs. normal coiled collagen fibers which can affect the material properties of these tissues.     

Use of the polymerase chain reaction in the diagnosis of leprosy

A case is described in which a pericardial branch of a nongrafted left internal mammary artery communicated directly with the distal left anterior descending artery, following saphenous vein bypass grafting. This type of collateralization following coronary artery bypass surgery seems to be very rare, and perhaps could protect the myocardium from severe ischemia.     

A T42A Ran mutation: differential interactions with effectors and regulators, and defect in nuclear protein import

Ran, the small, predominantly nuclear GTPase, has been implicated in the regulation of a variety of cellular processes including cell cycle progression, nuclear-cytoplasmic trafficking of RNA and protein, nuclear structure, and DNA synthesis. It is not known whether Ran functions directly in each process or whether many of its roles may be secondary to a direct role in only one, for example, nuclear protein import. To identify biochemical links between Ran and its functional target(s), we have generated and examined the properties of a putative Ran effector mutation, T42A-Ran. T42A-Ran binds guanine nucleotides as well as wild-type Ran and responds as well as wild-type Ran to GTP or GDP exchange stimulated by the Ran-specific guanine nucleotide exchange factor, RCC1. T42A-Ran.GDP also retains the ability to bind p10/NTF2, a component of the nuclear import pathway. In contrast to wild-type Ran, T42A-Ran.GTP binds very weakly or not detectably to three proposed Ran effectors, Ran-binding protein 1 (RanBP1), Ran-binding protein 2 (RanBP2, a nucleoporin), and karyopherin beta (a component of the nuclear protein import pathway), and is not stimulated to hydrolyze bound GTP by Ran GTPase-activating protein, RanGAP1. Also in contrast to wild-type Ran, T42A-Ran does not stimulate nuclear protein import in a digitonin permeabilized cell assay and also inhibits wild-type Ran function in this system. However, the T42A mutation does not block the docking of karyophilic substrates at the nuclear pore. These properties of T42A-Ran are consistent with its classification as an effector mutant and define the exposed region of Ran containing the mutation as a probable effector loop.     

HoxA is a transcriptional regulator for expression of the hup structural genes in free-living Bradyrhizobium japonicum

A chromosomally integrated Bradyrhizobium japonicum hoxA mutant is unable to oxidize hydrogen in free-living conditions. Derepressing conditions that induce hydrogenase activity in free-living, wild-type B. japonicum cells cannot induce expression of the hydrogenase structural genes in the hoxA mutant. The DNA-binding capacity of HoxA at the hup promoter region was studied by means of gel retardation. Both heterotrophically growing cells and cells induced to express hydrogenase activity contain a protein that specifically binds to the hup promoter region. Crude protein extracts isolated from a B. japonicum hoxA mutant do not contain this binding compound. The HoxA protein was overexpressed in E. coli and isolated in the form of a maltose-binding protein (MBP)-HoxA fusion. The MBP-HoxA hybrid protein specifically bound to a 50 bp region of the hupSL promoter known to be important for regulation of hupSL expression.     

Isolation of a genetic locus associated with metronidazole resistance in Helicobacter pylori

We examined the molecular mechanism of metronidazole resistance by constructing a lambda-Zap II phagemid expression library with genomic DNA from a metronidazole-resistance strain of Helicobacter pylori. Twenty-two clones were found to have elevated MTZ resistances in XLOLR strain of E. coli. Phagemids belonging to the twenty two clones were extracted and then retransformed into the XLOLR strain of E. coli. After MTZ selection, five clones could confer metronidazole resistance consistently. According to Southern hybridization and DNA sequencing, the five clones contained a same locus, recA. In addition, transforming the five clones into BL21 strain of E. coli produced a higher resistance to MTZ. Interestingly, electroporation of one of the five phagemid clones into two MTZ sensitive H. pylori yielded MTZ resistant strains. Comparing amino acid sequence in MTZ resistant with sensitive isolates revealed two point mutations at this locus. Above results suggest that mutation in recA may be associated with metronidazole resistance of H. pylori.