Numerical chromosomal aberrations of chromosome 1 and 7 in dysplastic cervical smears

Cervical smears with Papanicolaou's staining (PAP) reveal only morphological characteristics of epithelial cells of the cervix uteri. Since chromosomal aberrations are known to play a role in malignant transition, we analyzed cervical smears for numerical changes of the chromosomes 1 and 7 with fluorescence in-situ hybridization to probe for a diagnostic value of these chromosomes in the characterization of cervical dysplasia. Cervical smears were collected from 21 patients with suspect histology of curettage or biopsy specimen, 14 of them having been subsequently graded as cervical intraepithelial neoplasia (CIN) III and 5 as CIN II. Nineteen normal cervical smears (PAP I-II) served as controls. Smears were hybridized with chromosomal enumeration probes for chromosome 1 and 7. Disomic cells (2 copies of chromosome 1 and 7) were decreased in the CIN II (63%) and CIN III group (57%) with respect to the control group (77%). Cells with 3 signals for chromosome 7 were significantly more frequent in the CIN III and the CIN II group than in the control group (6.7, 6.4 and 0.7%, respectively). Only the CIN II group (10%), but not CIN II (6%), showed a significant trisomy for chromosome 1 as compared with the controls (3.8%). A close correlation between the incidence of trisomy 1 or 7 and PAP grading was observed. PAP III-IIID smears with high trisomy 1 counts corresponded to CIN III histology, while all CIN II patients were PAP III-IIID with low incidence of trisomy 1. We conclude that trisomy of chromosome 7 is a feature of cervical dysplasia and seems to be an early event in dysplastic transition. In contrast, trisomy of chromosome 1 is observed only in high grade dysplasia and may be a marker for pre-malignant lesions.     

Cell-microcarrier adhesion to gas-liquid interfaces and foam

The interaction of microcarriers, both with and without cells attached, with gas bubbles was studied. These studies consisted of qualitative microscopic observations of microcarriers with bubbles, quantitative measurements of microcarrier entrapment in foam, and quantitative measurements of the effect of bubble rupture at gas-medium interfaces. Ten different "protective additives" were evaluated for their ability to change the dynamic surface tension of the culture media and to prevent microcarrier adhesion to air bubbles during gas sparging and to prevent entrapment in the foam layer. These studies indicate that microcarriers, with and without cells, readily attach to gas-medium interfaces; yet unlike suspended cells, cells attached to microcarriers are not damaged by bubble ruptures at gas-medium interfaces. Only one surfactant was found to substantially prevent microcarrier entrapment in the foam layer; however, this surfactant was toxic to cells. No correlation was observed between surface tension and the prevention of microcarrier adhesion to gas-liquid interfaces. It is suggested that cell damage as a result of sparging in microcarrier cultures is the result of cells, attached to microcarriers, attaching to rising bubbles and then detaching from the microcarrier as this combination rises through the medium. It is further suggested that the hydrodynamic drag force of the rising microcarrier is sufficiently high to remove the bubble-attached cell from the microcarrier.     

Neural Induction of the Blood–Brain Barrier: Still an Enigma

1. The study of the blood–brain barrier and its various realms offers a myriad of opportunities for scientific exploration. This review focuses on two of these areas in particular: the induction of the blood–brain barrier and the molecular mechanisms underlying this developmental process.2. The creation of the blood–brain barrier is considered a specific step in the differentiation of cerebral capillary endothelial cells, resulting in a number of biochemical and functional alterations. Although the specific endothelial properties which maintain the homeostasis in the central nervous system necessary for neuronal function have been well described, the inductive mechanisms which trigger blood–brain barrier establishment in capillary endothelial cells are unknown.3. The timetable of blood–brain barrier formation is still a matter of debate, caused largely by the use of varying experimental systems and by the general difficulty of quantitatively measuring the degree of blood–brain barrier tightness. However, there is a general consensus that a gradual formation of the blood–brain barrier starts shortly after intraneural neovascularization and that the neural microenvironment (neurons and/or astrocytes) plays a key role in inducing blood–brain barrier function in capillary endothelial cells. This view stems from numerous in vitro experiments using mostly cocultures of capillary endothelial cells and astrocytes and assays for easily measurable blood–brain barrier markers. In vivo, there are great difficulties in proving the inductive influence of the neuronal environment. Also dealt with in this article are brain tumors, the least understood in vivo systems, and the induction or noninduction of barrier function in the newly established tumor vascularization.4. Finally, this review tries to elucidate the question concerning the nature of the inductive signal eliciting blood–brain barrier formation in the cerebral microvasculature.     

The transmembrane domain 10 of the yeast Pdr5p ABC antifungal efflux pump determines both substrate specificity and inhibitor susceptibility

We have previously shown that a S1360F mutation in transmembrane domain 10 (TMD10) of the Pdr5p ABC transporter modulates substrate specificity and simultaneously leads to a loss of FK506 inhibition. In this study, we have constructed and characterized the S1360F/A/T and T1364F/A/S mutations located in the hydrophilic face of the amphipatic Pdr5p TMD10. A T1364F mutation leads to a reduction in Pdr5p-mediated azole and rhodamine 6G resistance. Like S1360F, the T1364F and T1364A mutants were nearly non-responsive to FK506 inhibition. Most remarkably, however, the S1360A mutation increases FK506 inhibitor susceptibility, because Pdr5p-S1360A is hypersensitive to FK506 inhibition when compared with either wild-type Pdr5p or the non-responsive S1360F variant. Hence, the Pdr5p TMD10 determines both azole substrate specificity and susceptibility to reversal agents. This is the first demonstration of a eukaryotic ABC transporter where a single residue change causes either a loss or a gain in inhibitor susceptibility, depending on the nature of the mutational change. These results have important implications for the design of efficient reversal agents that could be used to overcome multidrug resistance mediated by ABC transporter overexpression.     

Characterization of the gene encoding a histidine and aspartic acid-rich protein from Pneumocystis carinii

A cDNA clone derived from Pneumocystis carinii contained an unusual sequence (GTGATG)2(ATGGTG)4(ATG)4 and many GAT repeats. It was found to encode a histidine and aspartic acid-rich protein (HARP). The complete cDNA contained an 888-bp open reading frame encoding a putative protein of 32.6 kDa. The deduced HARP protein contained 39 aspartic acid and 22 histidine residues. The genomic copy of the HARP gene (1203 bp in length) was found to contain 3 small introns of 46, 44, and 38 bp, respectively. HARP was predicted by computer programs to be a plasma membrane protein with nickel-binding activity.     

Protein translocon at the Arabidopsis outer chloroplast membrane.

Chloroplasts are organelles essential for the photoautotrophic growth of plants. Their biogenesis from undifferentiated proplastids is triggered by light and requires the import of hundreds of different precursor proteins from the cytoplasm. Cleavable N-terminal transit sequences target the precursors to the chloroplast where translocon complexes at the outer (Toc complex) and inner (Tic complex) envelope membranes enable their import. In pea, the Toc complex is trimeric consisting of two surface-exposed GTP-binding proteins (Toc159 and Toc34) involved in precursor recognition and Toc75 forming an aequeous protein-conducting channel. Completion of the Arabidopsis genome has revealed an unexpected complexity of predicted components of the Toc complex in this plant model organism: four genes encode homologs of Toc159, two encode homologs of Toc34, but only one encodes a likely functional homolog of Toc75. The availability of the genomic sequence data and powerful molecular genetic techniques in Arabidopsis set the stage to unravel the mechanisms of chloroplast protein import in unprecedented depth.     

Cloning and expression of the malolactic gene of Pediococcus damnosus NCFB1832 in Saccharomyces cerevisiae

Wine production is characterized by a primary alcoholic fermentation, conducted by Saccharomyces cerevisiae, followed by a secondary malolactic fermentation (MLF). Although most lactic acid bacteria (LAB) have the ability to metabolize L-malate, only a few species survive the high ethanol and SO2 levels in wine. Wines produced in colder viticultural regions have a lower pH than wines produced in warmer regions. The decarboxylation of L-malate in these wines leads to an increase in pH, more organoleptic complexity and microbiological stability. MLF is, however, difficult to control and problems often occur during filtering of such wines. Pediococcus spp. are known to occur in high pH wines and have strong malolactic activity. However, some pediococci synthesize exocellular polysaccharides, which may lead to abnormal viscosity in wine. In this study, the malolactic gene from Pediococcus damnosus NCFB1832 (mleD) was cloned into S. cerevisiae and co-expressed with the malate permease gene (mae1) of Schizosaccharomyces pombe. Expression of the mleD gene was compared to the expression of two other malolactic genes, mleS from Lactococcus lactis MG1363 and mleA from Oenococcus oeni Lal1. The genetically modified strain of S. cerevisiae decreased the level of L-malate in grape must to less than 0.3 gl(-1) within 3 days. This is the first expression of a malolactic gene from Pediococcus in S. cerevisiae.     

Growth factor regulation of autophagy and cell survival in the absence of apoptosis

In animals, cells are dependent on extracellular signals to prevent apoptosis. However, using growth factor-dependent cells from Bax/Bak-deficient mice, we demonstrate that apoptosis is not essential to limit cell autonomous survival. Following growth factor withdrawal, Bax-/-Bak-/- cells activate autophagy, undergo progressive atrophy, and ultimately succumb to death. These effects result from loss of the ability to take up sufficient nutrients to maintain cellular bioenergetics. Despite abundant extracellular nutrients, growth factor-deprived cells maintain ATP production from catabolism of intracellular substrates through autophagy. Autophagy is essential for maintaining cell survival following growth factor withdrawal and can sustain viability for several weeks. During this time, cells respond to growth factor readdition by rapid restoration of the ability to take up and metabolize glucose and by subsequent recovery of their original size and proliferative potential. Thus, growth factor signal transduction is required to direct the utilization of sufficient exogenous nutrients to maintain cell viability.     

Biocatalytic production of perillyl alcohol from limonene by using a novel Mycobacterium sp. cytochrome P450 alkane hydroxylase expressed in Pseudomonas putida

A number of oxygenated monoterpenes present at low concentrations in plant oils have anticarcinogenic properties. One of the most promising compounds in this respect is (-)-perillyl alcohol. Since this natural product is present only at low levels in a few plant oils, an alternative, synthetic source is desirable. Screening of 1,800 bacterial strains showed that many alkane degraders were able to specifically hydroxylate l-limonene in the 7 position to produce enantiopure (-)-perillyl alcohol. The oxygenase responsible for this was purified from the best-performing wild-type strain, Mycobacterium sp. strain HXN-1500. By using N-terminal sequence information, a 6.2-kb ApaI fragment was cloned, which encoded a cytochrome P450, a ferredoxin, and a ferredoxin reductase. The three genes were successfully coexpressed in Pseudomonas putida by using the broad-host-range vector pCom8, and the recombinant converted limonene to perillyl alcohol with a specific activity of 3 U/g (dry weight) of cells. The construct was subsequently used in a 2-liter bioreactor to produce perillyl alcohol on a scale of several grams.