Nucleofection of human embryonic stem cells.

The ability to realize the full potential of human pluripotent stem cells (hPSCs) as tools for -understanding human development and advancing the field of regenerative medicine is dependent on efficient methods to genetically manipulate these cells. There are several methods for introducing foreign DNA into cells such as electroporation, lipid-based transfection technology, and viral transduction. We describe here a method to transfect human embryonic stem cells (hESCs) using nucleofection technology. This unique method uses the Nucleofector II Device that combines the use of a cell type-specific Nucleofector Solution and preprogrammed electrical parameters to efficiently deliver DNA into the cell nucleus. The use of this technology allows high-efficiency transfer of nucleic acids into hESCs enabling both transient and stable manipulation of gene expression in these cells.

Regulation of IL-13 production by histamine in cloned murine T helper type 2 cells.

Histamine affects the balance of T helper type 1 (Th1) and T helper type 2 (Th2) cytokines by shifting cytokine production from a Th1 to a Th2 pattern. Interleukin-13 (IL-13) is an important autacoid mediator that has been implicated in the development of allergic disease. This study was designed to investigate the mechanisms of regulation of IL-13 by histamine in Th2 cells. D10.G4.1 cells, a murine Th2 cell line, were treated with histamine (10(-8)-10(-4) M) and then activated with PMA (phorbol 12 myristate 13-acetate) plus ionomycin or alphaCD3. Levels of IL-13 production were then measured by enzyme-linked immunosorbent assay (ELISA) and semiquantitative reverse transcription-polymerase chain reaction (RT-PCR). Cells were pretreated with histamine receptor antagonists pyrilamine, ranitidine, cimetidine and thioperamide to determine the involvement of histamine receptors. Cells were also pretreated with protein kinase A (PKA) inhibitors N-[2-(methylaminoethyl)]-5-isoquinoline-sulfonamide (H-8) and Rp-diastereomer of adenosine cyclic 3'5'-phosphorothionate (Rp-cAMPS), and Janus kinase-signal transducer and activator of transcription (Jak-STAT) inhibitor tyrphostin AG490 prior to the addition of histamine. H-8 is an inhibitor of the catalytic subunit of PKA while Rp-cAMPS is an inhibitor of the regulatory subunit of PKA. Tyrphostin is an inhibitor of Jak2, Jak3, STATI, STAT3 and STAT5. Finally, cells were pretreated with IL-12, a monokine known to repress STAT6 DNA binding. We found that histamine dose-dependently enhanced IL-13 secretion and mRNA levels in Th2 cells via H1 and H2 receptors. Pretreatment of cells with H-8, Rp-cAMPS and tyrphostin prevented histamine-induced secretion and transcription of IL-13. Likewise, pretreatment of Th2 cells with IL-12 also reversed histamine's effects on IL-13 secretion from stimulatory to inhibitory. These observations suggest a role for PKA and the Jak-STAT pathway in histamine-mediated elevation of IL-13 secretion and transcription.

Promoter regions of the extA extensin gene from Brassica napus control activation in response to wounding and tensile stress.

To identify controlling cis acting promoter regions in the B. napus extA extensin gene, expression in transgenic tobacco of 5' - 159, -433, -664, -789 and -940 bp promoter truncations linked to the uidA (B-glucuronidase) reporter coding sequence were analysed. The - 159 and -433 bp truncations directed non specific expression in all cell types within the plant. An activator region which increased expression levels 10 fold in all cell types was located between - 159 to -433 bp. A repressor region was found between -664 to -789 bp; removal of this region resulted in a 15 fold increase in expression. Histochemical analysis showed that transgenics containing the -664, -789 and -940 bp truncations directed expression of the fusion gene only in the phloem. A negative regulatory region located between -433 to -664 bp repressed expression in non-phloem cell types. In areas of the plant subject to tensile stress, the repression exerted by the negative regulatory region was overcome, allowing expression in all cell types. The quantitative repressor and activator regions which controlled absolute expression levels in all cell types were separate from the negative regulatory region which controlled cell type specific expression in response to tensile stress. A wound responsive region was found to be located between -940 to -3500 bp. Thus, the extA gene is under complex control, being regulated by 4 sets of positively and negatively acting cis regions, which control wound inducibility, activation in response to tensile stress, and quantitative expression levels.

Managing patients with vulvovaginal candidiasis.

Epidemiologic studies have demonstrated a continuing increase in the prevalence of vulvovaginal candidiasis. Although in the past most of these infections were caused by Candida albicans, an increasing percentage are caused by non-albicans Candida species that are less sensitive to the most frequently used antifungal agents. An accurate diagnosis of these infections and the subsequent choice of the most appropriate therapy can only be made after a thorough evaluation of the patient. Successful treatment of vulvovaginal candidiasis is dependent on compliance with therapy; thus, the treatment regimen chosen should fit the patient's daily lifestyle. Newer single-dose regimens offer the option of completing therapy with a single treatment for most patients with uncomplicated vaginal candidiasis. Use of topical agents avoids the potential systemic adverse effects and drug interactions that have been noted with oral antifungals. Patient education and support can also enhance satisfaction with the treatment plan and promote compliance.

Demonstration of gamma-glutamyl transferase, alkaline phosphatase, CEA and HCG in human lung cancer.

In contrast to its absence in normal and hyperplastic bronchial mucosa, gamma glutamyl transferase (GGT) has been demonstrated on the luminal surface of adenocarcinoma cells of bronchogenic cancer in three cases studied. On the other hand, little or no GGT was demonstrable in four epidermoid carcinomas and one oat cell cancer. Metaplastic alveolar cells appearing as cuboidal epithelial cells were uniformly positive for GGT and L-homoarginine-sensitive alkaline phosphatase. Whereas interest in GGT has centered in the past on experimental hepatocarcinogenesis, the current results demonstrate an ectopic expression of GGT in metaplastic alveolar cells and in adenocarcinoma of the lung. These findings merit further exploration to document the extent of the expression of GGT in bronchogenic cancer and to attempt to explain its presence in alveolar cell metaplasia.

The torture or stretch arthritis syndrome (a modern counterpart of the medieval 'manacles' and 'rack').

A characteristic symmetrical bilateral polyarthritis syndrome is described, affecting the compartements of the wrist joints in a characteristic sequence. The pisiform-triquetral joint gives first, exposing the inferior radioulnar joint so that pisiform pain and tenderness are followed by painful supination. The proximal radiocarpal joint slackens next, followed by the mid-carpal joints. The lunates subluxate as their posterior attachments stretch. Synovial oedema may produce additional median and ulnar carpal tunnel syndromes. The best radiologic sign of this distraction is anterior subluxation of the lunates. Nocturnal arthralgia becomes severe; grip and the ability to write are lost and stretching now shows as a traumatic arthritis in the clavicular joints. The patient is incapacitated and therfore progression halts. A history of excessive straining and lifting is obtained eg with a wheel-barrow, iron pots or strenuous rowing. All serologic tests for rheumatoid disease are negative. Serum uric acid levels and blood sedimentation rates remain normal. There are clear analogies with old descriptions of the effects of torture by stretching from manacles or gauntlets or by the rack.