Features of the immune proteasome expression in ascite Zajdela hepatoma after implantation into Brattleboro rats with the hereditary defect of arginine-vasopressin synthesis

The expression of the total proteasome pool, immune subunits LMP2 and LMP7, TAP1 and TAP2 transporters, and RT1A molecules of the major histocompatibility complex (MHC) class I in ascite Zajdela hepatoma cells was studied on the 10th day after implantation into Brattleboro rats with the hereditary defect in the synthesis of arginine-vasopressin (AVP) in the hypothalamus and WAG rats with normal AVP expression. Western-blot analysis revealed a threefold increase in the total number of proteasomes and immune subunit LMP2 and an eightfold increase in the immune subunits LMP7 in Zajdela hepatoma after its implantation in Brattleboro rats as compared with WAG rats. Differences in the expression of immune subunits LMP2 and LMP7 in Zajdela hepatoma in Brattleboro rats may contribute to different functions of these proteasomes, namely, the important role of the subunit LMP7 in antitumor immunity. Zajdela hepatoma growth in WAG rats was accompanied by a fall in both the total proteasome pool and immune proteasomes as compared with their content in Brattleboro rats, whose tumors regressed. The analysis of the content of peptide transporters TAP1 and TAP2 in Zajdela hepatoma implanted into Brattleboro and WAG rats showed their pronounced expression in tumor cells of both rat strains. In Zajdela hepatoma implanted into Brattleboro rats, a threefold increase in the basic molecule of MHC class I-RT1A was identified as compared with its expression in the tumor implanted to WAG rats. Furthermore, the content of CD8 and CD4 T-lymphocytes in the spleen of WAG and Brattleboro rats on the 10th day after implantation of Zajdela hepatoma was analyzed with flow cytometry. An increase in T-lymphocytes expressing the CD8 and CD4 antigens in the spleen of Brattleboro rats after implantation of the tumor as compared with WAG rats was shown. Increased numbers of both cytotoxic T lymphocytes and helper T-cells may facilitate tumor regression in Brattleboro rats. At the same time, a reduced number of subpopulations of T-lymphocytes in the spleen of WAG rats after implantation of hepatoma was accompanied by splenomegaly and growth of the tumor. Based on analysis of the data obtained it can be concluded that the deficiency of AVP in Brattleboro rats in Zajdela hepatoma leads to an increased expression of immune subunit LMP7 and basic molecules of MHC class I resulting in tumor immunogenicity and its elimination by the adaptive immune system.     

Mechanism of bFGF-binding Peptide Reversing Adriamycin Resistance in Human Gastric Cancer Cells

Development of drug resistance is a challenging problem in cancer chemotherapy. It has been shown that basic fibroblast growth factor (bFGF) plays an important role in an epigenetic mechanism of drug resistance. We have isolated a bFGF binding peptide P7 with inhibitory activity against bFGF-induced proliferation of human gastric cancer cells by screening a phage display library. In this study, we found that P7 peptide also has efficacy of reversing bFGF-induced resistance to Adriamycin (ADM) in human gastric cancer cells. Further investigations with SGC-7901 cells revealed that inhibition of Akt activation triggered by bFGF, and reversal of bFGF-induced up-regulation of Bcl-2 and XIAP and down-regulation of Bax, contribute to P7 peptide counteracting the anti-apoptotic effect of bFGF, and further reversing bFGF-induced resistance to ADM. The results suggested that the bFGF-binding peptide may have therapeutic potential of drug resistance in gastric cancer.     

Apparent Reduction of ADAM10 in Scrapie-Infected Cultured Cells and in the Brains of Scrapie-Infected Rodents

It has been described that A disintegrin and metalloproteinase (ADAM10) may involve in the physiopathology of prion diseases, but the direct molecular basis still remains unsolved. In this study, we confirmed that ADAM10 was able to cleave recombinant human prion protein in vitro. Using immunoprecipitation tests (IP) and immunofluorescent assays (IFA), reliable molecular interaction between the native cellular form of PrP (PrP^C) and ADAM10 was observed not only in various cultured neuronal cell lines but also in brain homogenates of healthy hamsters and mice. Only mature ADAM10 (after removal of its prodomain) molecules showed the binding activity with the native PrP^C. Remarkably more prion protein (PrP)-ADAM10 complexes were detected in the membrane fraction of cultured cells. In the scrapie-infected SMB cell model, the endogenous ADAM10 levels, especially the mature ADAM10, were significantly decreased in the fraction of cell membrane. IP and IFA tests of prion-infected SMB-S15 cells confirmed no detectable PrP-ADAM10 complex in the cellular lysates and PrP-ADAM10 co-localization on the cell surface. Furthermore, we demonstrated that the levels of ADAM10 in the brain homogenates of scrapie agent 263K-infected hamsters and agent ME7-infected mice were also almost diminished at the terminal stage, showing time-dependent decreases during the incubation period. Our data here provide the solid molecular basis for the endoproteolysis of ADAM10 on PrP molecules and interaction between ADAM10 and PrP^C. Obvious loss of ADAM10 during prion infection in vitro and in vivo highlights that ADAM10 may play essential pathophysiological roles in prion replication and accumulation.     

Heme Oxygenase-2 Suppress TNF-α and IL6 Expression via TLR4/MyD88-Dependent Signaling Pathway in Mouse Cerebral Vascular Endothelial Cells

Heme oxygenase (HO) represents an intrinsic antiinflammatory system based on its ability to inhibit expression of proinflammatory cytokines. The constitutive isoform heme oxygenase-2 (HO-2) has high expression and activity in cerebral microvascular endothelial cells (CMVEC). This study was undertaken to evaluate the role of HO-2 in regulation of TLR4/MyD88-dependent signaling and to study the effect of HO-2 on the expression and secretion of the proinflammatory cytokines tumor necrosis factor α (TNF-α) and Interleukin-6 (IL6) in CMVEC. HO-2 short hairpin RNA (shRNA) and HO-2 overexpression plasmids were used to observe the effect of HO-2 on proinflammatory cytokines in CMVEC in vitro, and the results showed that the messenger RNA (mRNA) and protein levels of TNF-α and IL6 were increased and decreased, respectively, compared with control groups. LPS-stimulated TNF-α and IL6 mRNA and protein were also reduced in CMVEC treated with an inhibitor of TLR4 signaling, CLI-095, or HO-2 overexpression. CLI-095 and HO-2 overexpression both reduced TLR4 expression in CMVEC, and HO-2 shRNA blocked these effects of CLI-095. CLI-095 and HO-2 overexpression potently suppressed TLR4/MyD88-dependent proinflammatory cytokine expression in CMVEC. These results suggest that HO-2 plays an important role in protecting CMVEC against cytokine-mediated inflammation.     

Expression of TNF-α, IFN-γ, TGF-β, and IL-4 in the Spinal Tuberculous Focus and Its Impact on the Disease

To investigate the expression of TNF-α, IFN-γ, TGF-β, and IL-4 in the spinal tuberculous focus and its relationship with the lesions type, severity, and bone destruction. The pathological samples of patients with spinal tuberculosis (TB) were divided into hyperplasia group and necrosis group according to their intra-operative and post-operative pathological findings. Normal bone tissues were taken as the control group. Pathology and expression of TNF-α, IFN-γ, TGF-β, and IL-4 in different tissues were compared among these three groups using immunohistochemical staining, quantitative image analysis, and measurement of bone tissue. 286 granulomas observed in the 14 samples in the hyperplasia group, which included 84 necrotizing and 202 non-necrotizing granulomas. As for the 20 samples in the necrosis group, there were 356 necrotizing and 186 non-necrotizing granulomas among all the 542 granulomas. The proportion of necrotizing granulomas in the necrosis group was significantly higher than that of the hyperplasia group. By inter-group comparison, expression of TNF-α, IFN-γ of granulomas in the hyperplasia group was significantly higher than that of the necrosis group, while the expression of TGF-β, IL-4 of granulomas in the necrosis group was significantly higher than that of the hyperplasia group. Also, expression of IFN-γ of non-necrotizing granulomas was significantly higher than that of necrotizing granulomas in the hyperplasia group, and expression of TGF-β in necrotizing granulomas was significantly higher than that of non-necrotizing granulomas in the necrosis group. The lesions were mainly bone resorption in the hyperplasia group, whereas mostly necrotic bones accompanied by local fibrosis in the necrosis group. Expression levels of TNF-α, IFN-γ in the hyperplasia group have a positive correlation to bone loss, whereas expression levels of TGF-β, IL-4 in the necrosis group have a positive correlation to the bone formation. The high expressions of TNF-α, IFN-γ in the spinal tuberculous focus were associated with protective immune cells. TGF-β and IL-4 were related to allergic lesions, fibrosis and osteogenesis. Expression imbalance of TNF-α, IFN-γ, TGF-β, and IL-4 might aggravate the allergy of TB.     

Key enzymes of degradation and angiogenesis as factors of tumor progression for squamous-cell cervical carcinoma

Destruction of the connective tissue matrix (CTM) and angiogenesis are the two processes playing a key role in tumor progression. Matrix metalloproteinases (MMPs) play a leading role in processes of tissue destruction. Tissue collagenases MMP-1 and MT1-MMP hydrolyze fibrillar collagens constituting the base of CTM and enable tumor invasion. Gelatinases A and B (MMP-2 and MMP-9) hydrolyze type IV collagen which is the main component of basal membranes and contribute to the development of metastases. Endogenous activators and inhibitors are involved in the regulation of expression and activity of these enzymes. MMP-9 was shown to release vascular endothelial growth factor (VEGF), the principal inductor of angiogenesis, bound to CTM. Angiotensin-converting enzyme (ACE) is involved in the induction of VEGF synthesis and stimulation of endothelial cell proliferation mediated by angiotensin II (AII) and its type 1 receptor (AT1R). Experiments reported in the present article addressed the distinctive features of expression of key degradation and angiogenesis enzymes in squamous cell carcinoma (SCC) of the cervix. MMP-1, MT1-MMP, MMP-2, and MMP-9 and their endogenous regulators TIMP-1 and TIMP-2, as well as ACE, were the objects of research. Experiments were performed with clinical specimens including tumor tissue samples, for which presence or absence of metastasis to regional lymph nodes was taken into account, and morphologically normal tissue samples. Increased expression of MMP-1, MT1-MMP, and MMP-9 and decreased expression of TIMP-1 and TIMP-2 were shown to make the principal contribution to the destructive (invasive) potential of cervical carcinomas; the effect of changes in MMP-2 expression was less pronounced. Dramatically increased expression of MMP-1 and MMP-9 was evident in metastasizing tumors. ACE activity in tumor samples was generally higher than activity in normal tissue. Substantial expression of MMP-1, MMP-2, MMP-9, and ACE was detected in morphologically normal tissue adjacent to the tumor; this can contribute to increased destructive potential of a tumor. The data reported are important for understanding the mechanisms of tumor progression, have prognostic value, and may affect the choice of individual therapeutic strategy for the patients.     

CpG-mediated modulation of MDSC contributes to the efficacy of Ad5-TRAIL therapy against renal cell carcinoma

Tumor progression occurs through the modulation of a number of physiological parameters, including the development of immunosuppressive mechanisms to prevent immune detection and response. Among these immune evasion mechanisms, the mobilization of myeloid-derived suppressor cells (MDSC) is a major contributor to the suppression of antitumor T-cell immunity. Patients with renal cell carcinoma (RCC) show increased MDSC, and methods are being explored clinically to reduce the prevalence of MDSC and/or inhibit their function. In the present study, we investigated the relationship between MDSC and the therapeutic potential of a TRAIL-encoding recombinant adenovirus (Ad5-TRAIL) in combination with CpG-containing oligodeoxynucleotides (Ad5-TRAIL/CpG) in an orthotopic mouse model of RCC. This immunotherapy effectively clears renal (Renca) tumors and enhances survival, despite the presence of a high frequency of MDSC in the spleens and primary tumor-bearing kidneys at the time of treatment. Subsequent analyses revealed that the CpG component of the immunotherapy was responsible for decreasing the frequency of MDSC in Renca-bearing mice; further, treatment with CpG modulated the phenotype and function of MDSC that remained after immunotherapy and correlated with an increased T-cell response. Interestingly, the CpG-dependent alterations in MDSC frequency and function did not occur in tumor-bearing mice complicated with diet-induced obesity. Collectively, these data suggest that in addition to its adjuvant properties, CpG also enhances antitumor responses by altering the number and function of MDSC.     

High Fungal Diversity and Abundance Recovered in the Deep-Sea Sediments of the Pacific Ocean

Knowledge about the presence and ecological significance of bacteria and archaea in the deep-sea environments has been well recognized, but the eukaryotic microorganisms, such as fungi, have rarely been reported. The present study investigated the composition and abundance of fungal community in the deep-sea sediments of the Pacific Ocean. In this study, a total of 1,947 internal transcribed spacer (ITS) regions of fungal rRNA gene clones were recovered from five sediment samples at the Pacific Ocean (water depths ranging from 5,017 to 6,986 m) using three different PCR primer sets. There were 16, 17, and 15 different operational taxonomic units (OTUs) identified from fungal-universal, Ascomycota-, and Basidiomycota-specific clone libraries, respectively. Majority of the recovered sequences belonged to diverse phylotypes of Ascomycota (25 phylotypes) and Basidiomycota (18 phylotypes). The multiple primer approach totally recovered 27 phylotypes which showed low similarities (≤97 %) with available fungal sequences in the GenBank, suggesting possible new fungal taxa occurring in the deep-sea environments or belonging to taxa not represented in the GenBank. Our results also recovered high fungal LSU rRNA gene copy numbers (3.52?×?10⁶ to 5.23?×?10⁷copies/g wet sediment) from the Pacific Ocean sediment samples, suggesting that the fungi might be involved in important ecological functions in the deep-sea environments.     

A Systems Biology Approach to Study the Biology Characteristics of Esophageal Squamous Cell Carcinoma by Integrating microRNA and Messenger RNA Expression Profiling

Esophageal squamous cell carcinoma (ESCC) is one of the most malignant tumors. The aim of this study was to investigate the biology characteristics of ESCC by analyzing microRNA and mRNA expression profile. We used BRB-array tools to analyze the deregulated microRNA and mRNA between esophageal squamous cell carcinomas and paired normal adjacent tissues. We used miRTrail and protein–protein interaction methods to explore the related pathways and networks of deregulated microRNA and mRNA. By combining the results of pathways and networks, we found that the deregulated microRNA and their deregulated target mRNA are enriched in the following pathways: DNA replication, cell cycle, ECM-receptor interaction, focal adhesion, mismatch repair, and pathways in cancer. The results showed that many deregulated microRNAs and mRNAs may play a vital role in the pathogenesis of ESCC, and the systems biology approach is very helpful to explore molecular mechanism of ESCC.     

Cathepsin B is involved in the heat shock induced cardiomyocytes apoptosis as well as the anti-apoptosis effect of HSP-70

Cathepsin B is one of the major lysosomal cysteine proteases that plays an important role in apoptosis. Herein, we investigated whether Cathepsin B is involved in cardiomyocyte apoptosis caused by hyperthermic injury (HI) and heat shock protein (HSP)-70 protects these cells from HI-induced apoptosis mediated by Cathepsin. HI was produced in H9C2 cells by putting them in a circulating 43 °C water bath for 120 min, whereas preinduction of HSP-70 was produced in H9C2 cells by mild heat preconditioning (or putting them in 42 °C water bath for 30 min) 8 h before the start of HI. It was found that HI caused both cardiomyocyte apoptosis and increased Cathepsin B activity in H9C2 cells. E-64-c, in addition to reducing Cathepsin B activity, significantly attenuated HI-induced cardiomyocyte apoptosis (evidenced by increased apoptotic cell numbers, increased tuncated Bid (t-Bid), increased cytochrome C, increased caspase-9/-3, and decreased Bcl-2/Bax) in H9C2 cells. In addition, preinduction of HSP-70 by mild heat preconditioning or inhibition of HSP-70 by Tripolide significantly attenuated or exacerbated respectively both the cardiomyocyte apoptosis and increased Cathepsin B activity in H9C2 cells. Furthermore, the beneficial effects of pre-induction of HSP-70 by mild heat production in reducing both cardiomyocyte apoptosis and increased Cathepsin B activity caused by HI can be significantly reduced by Triptolide preconditioning. These results indicate that Cathepsin B is involved in HI-induced cardiomyocyte apoptosis in H9C2 cells and HSP-70 protects these cells from HI-induced cardiomyocyte apoptosis through Cathepsin B pathways.     

Impregnation of the Bacterial Cellulose Membrane with Biologically Produced Silver Nanoparticles

Different wound dressings with antibacterial property have been surveyed and one among them is bacterial cellulose (BC). Since the BC does not have antibacterial property, the biologically produced silver nanoparticles (SNPs) were impregnated into the BC. For the BC production, Hestrin–Schramm broth was used. Formation of the BC was proven by enzymatic hydrolysis. For SNPs production, the bacterial supernatant was treated with AgNO₃ and formation of SNPs was monitored through spectrophotometer, TEM and XRD. For impregnation of SNPs into the BC, the cleaned membrane was placed in the bacterial supernatant that contained 1 mmol of AgNO₃. The antibacterial assay was done for the BC/SNPs. Enzymatic hydrolysis proved the presence of the BC. Spectrophotometer and XRD results showed the formation of SNPs. TEM analysis revealed the presence of SNPs with sizes around 5–100 nm. SEM micrographs showed the impregnation of SNPs into the BC. Antibacterial test exhibited the antibacterial activity of the BC/SNPs.     

AE37 peptide vaccination in prostate cancer: identification of biomarkers in the context of prognosis and prediction

A fundamental challenge in administering immunotherapies for cancer is the establishment of biomarkers that can predict patients’ responsiveness to treatment. In this study, our aim was to predict the immunologic and clinical responses of vaccination therapy with an Ii-key-modified HER-2/neu peptide (Ii-key/HER-2(776–790) or AE37), applied in our recent phase I study in patients with prostate cancer. To this end, we retrospectively analyzed our data derived from immunologic determinations before, during and after primary series of vaccinations with AE37, as well as after one AE37 booster injection. Using the obtained data, we then observed the relationship between the immunologic parameters and clinical outcome of patients. We found that preexisting levels of transforming growth factor beta (TGF-β) had an inverse correlation with in vivo and in vitro immunologic responses to the AE37 vaccine which were measured as delayed-type hypersensitivity (DTH) and interferon gamma (IFN-γ) production in response to the native HER-2(776–790) (or AE36) peptide, respectively. Patients with preexistent IFN-γ immunity to AE36 developed positive DTH reactions after primary vaccinations and booster. Moreover, we could detect a direct correlation between IFN-γ production and DTH reactions in response to AE36 challenge in our vaccinated patients. DTH reactions were a stronger indicator for patients’ overall survival (OS) than preexistent or vaccine-induced IFN-γ immunity. In contrast, we found that preexisting TGF-β levels were correlated with shorter patients’ OS. These retrospective analyses suggest that the above biomarkers at the time-points measured offer promise for evaluating immunologic and clinical responses to AE37-based vaccinations.     

GRP78 promotes the invasion of pancreatic cancer cells by FAK and JNK

The major characteristics of pancreatic cancer are its excessive local invasion and early systemic dissemination. The glucose-regulated protein is over-expressed in many human cancers including pancreatic cancer and correlated with invasion and metastasis in many cancers. To investigate the effect of Grp78 on the invasion of pancreatic cancer, we used western blot and Transwell assay. We found Grp78 is expressed at lower levels in capan-2 and higher expressed in MiaPaCa-2 cells, and Grp78 expression levels were correlated with the invasion potentials of tumor cells. Then,we increased the expression of Grp78 in capan-2 cells and decreased the expression of Grp78 in MiaPaCa-2 cells. We found that over-expression of Grp78 caused significant increase in the expression of TIMP-1, TIMP-2, MMP-14, MMP-2, and MMP-9 in Capan-2 cells. Consistently, knockdown of Grp78 decreased the expression of them in MiaPaCa-2 cells. Gelatin zymography showed Grp78 over-expression stimulated the activities of MMP-2 and MMP-9, while GRP78 knockdown reduced the activities of MMP-2 and MMP-9. Cytoskeleton staining showed that knockdown of Grp78 caused a marked increase in cytoskeleton F-actin stress fibers in MiaPaCa-2 cells. Consistently, GRP78 knockdown hyperactivated RhoA and inhibited significantly Rac activity. Grp78 over-expression decreases the RhoA and stimulated Rac activity. We also found that Grp78 modulated FAK and JNK signaling pathways. Over-expression of GRP78 in Capan-2 activated FAK and JNK. Finally, we demonstrated that knockdown of FAK by shRNA in combination with blockade of JNK signaling pathway with SP600125 completely inhibited GRP78-induced cancer cell invasion. GRP78 is involved in the regulation of pancreatic cancer invasion. FAK and JNK are the key downstream effectors of GRP78.     

Proteasome Inhibition-Induced Downregulation of Akt/GSK-3β Pathway Contributes to Abnormality of Tau in Hippocampal Slice

Proteasome inhibition can induce abnormal accumulation and phosphorylation of microtubule-associated protein tau. The major function of tau protein is to promote microtubules assembly and stabilization, and abnormal tau protein would disturb its microtubule-binding function. In this study, proteasome inhibitor MG132 was used to treat hippocampal slices to explore the role and mechanism of Akt/glycogen synthase kinase-3β (GSK-3β) in proteasome inhibition-induced tau abnormality. During the culture period, we measure the lactate dehydrogenase (LDH) content to assay the viability of hippocampal slices. Following 2.5 and 5 μM MG132 treatment for 6 h, we detected the expression, phosphorylation modification, and microtubule-binding function of tau protein of slices. We also analyzed the changed activities of glycogen synthase kinase-3β (GSK-3β) and protein kinase B (PKB/Akt) and the level of heat shock protein 90 (Hsp90) in the process. In addition, co-immunoprecipitation was used to investigate the interaction between Akt and Hsp90, Akt and protein phosphatase-2A (PP2A) in the MG132-treated organotypic hippocampal slices. Our results indicated that proteasome inhibition led to degradation obstacles and abnormal phosphorylation of tau protein. The downregulated Akt/GSK-3β signaling pathway might be responsible for the abnormal phosphorylation of tau protein at multiple sites which further reduced the microtubule-binding function of tau protein. Furthermore, proteasome inhibition decreased the binding capacity of Akt-Hsp90 while increased the Akt-PP2A binding ability which mediated Akt inactivity. This current study establishes a hippocampal slice model targeting Akt/GSK-3β signaling pathway to explore the pivotal role of proteasome inhibition in tau pathology.     

Structural and functional analysis of the anti-malarial drug target prolyl-tRNA synthetase

Aminoacyl-tRNA synthetases (aaRSs) drive protein translation in cells and hence these are essential enzymes across life. Inhibition of these enzymes can halt growth of an organism by stalling protein translation. Therefore, small molecule targeting of aaRS active sites is an attractive avenue from the perspective of developing anti-infectives. Febrifugine and its derivatives like halofuginone (HF) are known to inhibit prolyl-tRNA synthetase of malaria parasite Plasmodium falciparum. Here, we present functional and crystallographic data on P. falciparum prolyl-tRNA synthetase (PfPRS). Using immunofluorescence data, we show that PfPRS is exclusively resident in the parasite cytoplasm within asexual blood stage parasites. The inhibitor HF interacts strongly with PfPRS in a non-competitive binding mode in presence or absence of ATP analog. Intriguingly, the two monomers that constitute dimeric PfPRS display significantly different conformations in their active site regions. The structural analyses presented here provide a framework for development of febrifugine derivatives that can seed development of new anti-malarials.     

The biogeochemical cycle of the adsorbed template

Experimental results are presented for the verification of the first adsorption step of the ‘adsorbed template’ biogeochemical cycle, a simple model for a primitive prebiotic replication system. The adsorption of Poly-C, Poly-U, Poly-A, Poly-G, and 5′-AMP, 5′-GMP, 5′-CMP and 5′-UMP onto gypsum was studied. It was found that under the conditions of the experiment, the polymers have a very high affinity for the mineral surface, while the monomers adsorb much less efficiently.     

G6PD Punjab,a dialysis sensitive variant of human glucose-6-phosphate dehydrogenase

Erythrocyte samples from 101 individuals, originally from Punjab and living at the time of investigation in England, were screened for glucose-6-phosphate dehydrogenase (G6PD) variants by Beutler’s fluorescent spot test and standard cellulose acetate gel (Cellogel) electrophoresis. All but 2 of the 40 males in the study were found to be indistinguishable from normal G6PD B. One of the variants had 2% of the normal activity and resembled G6PD Mediterranean in electrophoretic behaviour. The other variant showed 52% of the normal activity and migrated slower than G6PD B in Cellogel with about half of the normal band intensity. A set of physicochemical characteristics of the variant determined by conventional methods distinguished it from the variants reported so far. It was designated as G6PD Punjab, and the corresponding allele asG6PD PUN. The most striking feature of G6PD Punjab is a remarkable alteration in its electrophoretic behaviour after dialysis.