High-Level Production of Human Parathyroid Hormone (hPTH) by Induced Expression inSaccharomyces cerevisiae

Saccharomyces cerevisiaewas used as host for high-level production of intact human parathyroid hormone (hPTH). The yield increased about 30-fold by changing from the constitutive MFα promoter to the inducibleCUP1promoter in the expression cassettes, use of another host strain, and optimization of growth conditions where especially the pH value was crucial. The secreted products consisted mainly of intact hormone, hPTH(1-84). In addition, two C-terminally truncated forms that lacked the four or five last amino acid residues, hPTH(1-80) and hPTH(1-79), were identified. These hPTH forms migrated aberrantly by SDS–PAGE as 14-kDa proteins, while the real masses measured by mass spectrometry on HPLC-purified products were about 9 kDa. Availability of such easily purified truncated forms will be valuable for studies of how the C-terminal residues affect the structure and function of the hormone. Combination of mutations and disruptions of the host genes encoding proteinase A, B, carboxypeptidase Y, and Kex1p or Mkc7p did not influence the C-terminal deletions. The secretion of hPTH could be enhanced by overexpression of the yeast syntaxin geneSSO2, but the total level of the hormone was not improved due to impaired growth.     

A micromodification of the electroimmunoassay

A micromodification of the Laurell electroimmunoassay is presented. The immunoelectrophoresis is run vertically in 50-μl capillary tubes keeping the immunoglobulin-containing agarose gel. The method has high accuracy, and for proteins the detection limit is 0.1 ng. The quantitative limit is 1.0 ng with a mean variation coefficient of 6.8%. Besides the sensitivity the method also has practical advantages as it is easy to handle and requires no expensive equipment. In addition, the consumption of immunoglobulins is extremely low, which renders the method rather economical. The antibody-containing gel tubes can be stored for long periods. This makes it possible to run several samples against the same antibody-gel batch, which increases the speed and simplicity of the method.     

P2X5 and P2X7 receptors in human warts and CIN-612 organotypic raft cultures of human papillomavirus infected keratinocytes

Purinergic receptors, which bind adenosine 5′-triphosphate (ATP), are expressed on human cutaneous keratinocytes and in squamous cell carcinomas. Studies on normal human epidermis and primary keratinocyte cultures have suggested that P2X₅ receptors are likely to be involved in keratinocyte differentiation and P2X₇ receptors are likely to be part of the machinery of end stage terminal differentiation/apoptosis of keratinocytes. P2X₇ receptor agonists can significantly reduce primary keratinocyte cell numbers in culture. Human papillomaviruses are increasingly recognised as important human carcinogens in the development of non-melanoma skin cancers. In our study, immunohistochemical analysis for P2X₅ and P2X₇ receptors was performed on paraffin sections of normal human skin, warts, raft cultures of normal human keratinocytes and raft cultures of CIN 612 cells, a model of keratinocytes infected with human papillomavirus type 31. In warts there was up-regulation of the expression of P2X₅ receptors. A similar pattern was seen in the CIN 612 raft cultures. Both P2X₅ and P2X₇ receptors were found in the nuclei of koilocytes, abnormal keratinocytes characteristic of human papillomavirus infection. P2X₅ and P2X₇ receptors may provide a new focus for therapeutic research into treatments for warts because these receptors can induce cell differentiation and cell death.An erratum to this article can be found at     

Programmed death ligand-1 over-expression correlates with malignancy and contributes to immune regulation in ovarian cancer

The programmed death-1 (PD-1) pathway is important in the maintenance of peripheral tolerance and homeostasis through suppression of T cell receptor signaling. As such, it is employed by many tumors as a means of immune escape. We have investigated the role of this pathway in human ovarian cancer (OC) to assess its potential role as a diagnostic and/or prognostic marker and therapeutic target, following recent clinical trial success of antibody therapy directed at this pathway. We show programmed death ligand-1 (PD-L1) expression on monocytes in the ascites and blood of patients with malignant OC is strikingly higher than those with benign/borderline disease, with no overlap in the values between these groups. We characterize the regulation of this molecule and show a role of IL-10 present in ascitic fluid. Flow cytometric analysis of T cells present in the ascites and blood showed a correlation of PD-1 expression with malignant tumors versus benign/borderline, in a similar manner to PD-L1 expression on monocytes. Finally, we demonstrate functional links between PD-L1 expression on monocytes and OC tumor cells with suppression of T cell responses. Overall, we present data based on samples obtained from women with ovarian cancer, suggesting the PD-1 pathway may be used as a reliable diagnostic marker in OC, as well as a viable target for use with PD-1/PD-L1-directed antibody immunotherapy.