Three Japanese patients demonstrated plasma prekallikrein (PK) deficiency (PKD) after an examination of the proband family line named 'PKD Seki'. A molecular genetic analysis of these PK genes showed homozygous amino acid substitutions Gly104Arg and Asn124Ser in exon 5, which encodes part of the apple domain 2 (A2) of the heavy chain. This is the first case involving substitutions in the heavy chain of the PK gene which affected blood coagulation. Because the apple domains of PK bind to the C-terminal domain (D6(H)) of high-molecular weight kininogen (HMWK), the two substitutions in A2 may therefore be the main cause of PKD Seki. We subsequently investigated the effects of amino acid substitutions in A2 to elucidate the binding activity of PK to HMWK using mutant A2 proteins produced in Escherichia coli. We clearly demonstrated that the Gly104Arg-substitution with the Asn124Ser-substitution in A2 reduce the binding activity of A2 to HMWK. PKD Seki is the first significant case to show the amino acid substitutions in the A2 affecting the binding capacity of PK with HMWK. Our findings therefore suggest that the binding of PK to HMWK may play a crucial role in the first step of blood coagulation.

Background: Andrographolide (Andro) is a diterpenoid component of the plant Andrographis paniculata that is known for its anti-tumor activity against a variety of cancer cells.   Methods: We studied the effects of Andro on the viability of the human leukemia monocytic cell line THP-1 and the human multiple myeloma cell line H929. Andro was compared with cytosine arabinoside (Ara-C) and vincristine (VCR), which are well-established therapeutics against hematopoietic tumors. The importance of reactive oxygen species (ROS) production for the toxicity of each agent was investigated by using an inhibitor of ROS production, N-acetyl-L-cysteine (NAC).    Results:  Andro reduced the viability of THP-1 and H929 in a dose-dependent manner. H929 viability was highly susceptible to Andro, although only slightly susceptible to Ara-C. The agents Andro, Ara-C, and VCR each induced apoptosis, as shown by cellular shrinkage, DNA fragmentation, and increases in annexin V-binding, caspase-3/7 activity, ROS production, and mitochondrial membrane depolarization. Whereas Ara-C and VCR increased the percentages of cells in the G0/G1 and G2/M phases, respectively, Andro showed little or no detectable effect on cell cycle progression. The apoptotic activities of Andro were largely suppressed by NAC, an inhibitor of ROS production, whereas NAC hardly affected the apoptotic activities of Ara-C and VCR.  Conclusions: Andro induces ROS-dependent apoptosis in monocytic leukemia THP-1 and multiple myeloma H929 cells, underlining its potential as a therapeutic agent for treating hematopoietic tumors. The high toxicity for (thus forming: The high toxicity for H929 cells, by a mechanism that is different from that of Ara-C and VCR, is encouraging for further studies on the use of Andro against multiple myeloma.) H929 cells, by a mechanism that is different from that of Ara-C and VCR, is encouraging for further studies on the use of Andro against multiple myeloma.

Background: Andrographolide (Andro) is a diterpenoid component of the plant Andrographis paniculata that is known for its anti-tumor activity against a variety of cancer cells.   Methods: We studied the effects of Andro on the viability of the human leukemia monocytic cell line THP-1 and the human multiple myeloma cell line H929. Andro was compared with cytosine arabinoside (Ara-C) and vincristine (VCR), which are well-established therapeutics against hematopoietic tumors. The importance of reactive oxygen species (ROS) production for the toxicity of each agent was investigated by using an inhibitor of ROS production, N-acetyl-L-cysteine (NAC).    Results:  Andro reduced the viability of THP-1 and H929 in a concentration-dependent manner. H929 viability was highly susceptible to Andro, although only slightly susceptible to Ara-C. The agents Andro, Ara-C, and VCR each induced apoptosis, as shown by cellular shrinkage, DNA fragmentation, and increases in annexin V-binding, caspase-3/7 activity, ROS production, and mitochondrial membrane depolarization. Whereas Ara-C and VCR increased the percentages of cells in the G0/G1 and G2/M phases, respectively, Andro showed little or no detectable effect on cell cycle progression. The apoptotic activities of Andro were largely suppressed by NAC, an inhibitor of ROS production, whereas NAC hardly affected the apoptotic activities of Ara-C and VCR.  Conclusions: Andro induces ROS-dependent apoptosis in monocytic leukemia THP-1 and multiple myeloma H929 cells, underlining its potential as a therapeutic agent for treating hematopoietic tumors. The high toxicity for H929 cells, by a mechanism that is different from that of Ara-C and VCR, is encouraging for further studies on the use of Andro against multiple myeloma.

Background: Andrographolide (Andro) is a diterpenoid component of the plant Andrographis paniculata that is known for its anti-tumor activity against a variety of cancer cells. Methods: We studied the effects of Andro on the viability of the human leukemia monocytic cell line THP-1 and the human multiple myeloma cell line H929. Andro was compared with cytosine arabinoside (Ara-C) and vincristine (VCR), which are well-established therapeutics against hematopoietic tumors. Results: Andro reduced the viability of THP-1 and H929 in a dose-dependent manner. H929 viability was highly susceptible to Andro, although only slightly susceptible to Ara-C. The agents Andro, Ara-C, and VCR each induced apoptosis, as shown by cellular shrinkage, DNA fragmentation, and increases in annexin V-binding, caspase-3/7 activity, reactive oxygen species (ROS) production, and mitochondrial membrane depolarization. The apoptotic activities of Andro were largely suppressed by N-acetyl-L-cysteine (NAC), an inhibitor of ROS production, whereas NAC hardly affected the apoptotic activities of Ara-C and VCR. Furthermore, whereas Ara-C and VCR increased the percentages of cells in the G0/G1 and G2/M phases, respectively, Andro showed little or no detectable effect on cell cycle progression. Conclusions: Andro induces ROS-dependent apoptosis in monocytic leukemia THP-1 and multiple myeloma H929 cells, underlining its potential as a therapeutic agent for treating hematopoietic tumors. Notably, the high sensitivity of H929 cells is encouraging for further studies on the use of Andro against multiple myeloma.

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