High-performance liquid chromatographic determination of dihydroorotate dehydrogenase of Plasmodium falciparum and effects of antimalarials on enzyme activity

A reversed-phase high-performance liquid chromatographic technique for the determination of dihydroorotate dehydrogenase in Plasmodium falciparum was developed. The assay was applied to the evaluation of the effects of several antimalarial drugs on the enzyme. Treatment of both the asexual and gametocyte stages of P. falciparum in culture with menoctone, primaquine or the primaquine derivative WR 238605 led to depression of the enzyme activity, although the drugs did not appear to inhibit the enzyme directly.     

The increase in activity of acid hydrolases in muscles of rats after subcutaneous administration of dimethyl-para-phenylene diamine. A combined histochemical and biochemical investigation

Summary The increase in activity of acid hydrolases in skeletal muscles of rats after subcutaneous administration of dimethyl-para-phenylene diamine (DPPD) was studied with a combined histochemical and biochemical investigation. In part I the histochemical findings were presented. In this communication the biochemical findings are reported and compared with the histochemical findings.In homogenates of m. biceps femoris, m. gastrocnemius and m. rectus femoris of DPPD-treated rats, the activity of the lysosomal acid hydrolases, cathepsin D, acid maltase, acid phosphatase, and -glucuronidase was increased. This increase in activity was maximal after 7 to 9 days of DPPD treatment and ran parallel to the severity of the pathological changes. Statistical calculations clearly reveal that the increased activity of one acid hydrolase was significantly paralleled by an increased activity of a second acid hydrolase. Moreover these calculations reveal that the biochemical activity findings correlated with the histochemical activity findings. However it was remarkable that in the histochemical study, the estimated increase in acid phosphatase activity was much more than the increase in acid phosphatase activity found biochemically, whilst on the other hand the histochemically estimated increase in -glucuronidase activity corresponded with the biochemical observations. The results of gel filtration techniques have shown that this discrepancy of acid phosphatase activity was caused by different substrate specificity of the different isoenzymes of acid phosphatase and that as a result of the DPPD treatment the isoenzyme pattern had been altered. The elution patterns showed three distinct isoenzymes of acid phosphatase of normal and of DPPD treated rats. These isoenzymes, termed I, II and III, have molecular weights of: 200,000 or more, 83,500–104,500 and 14,500–18,100. Isoenzymes I and II split the substrates 4-methylumbelliferyl phosphate and naphthol AS-BI phosphate and the activity is strongly increased in the muscles of the DPPD treated rats. Isoenzyme III does not split naphthol AS-BI phosphate and the activity is not increased in the muscles of the DPPD treated rats. Considering the fact that it has been shown that the activity of isoenzyme III is high compared with that of the isoenzymes I and II, it is important to realise that by using naphthol AS-BI phosphate not all acid phosphatase can be demonstrated in sections of skeletal muscle.This study was partly supported by a grant from the Prinses Beatrix Fonds, 's Gravenhage, The Netherlands, and was mainly extracted from the Ph. D. thesis of D.E. Israël (1977).     

NEMO specifically recognizes K63‐linked poly‐ubiquitin chains through a new bipartite ubiquitin‐binding domain

An important property of NEMO, the core element of the IKK complex involved in NF‐κB activation, resides in its ability to specifically recognize poly‐ubiquitin chains. A small domain called NOA/UBAN has been suggested to be responsible for this property. We recently demonstrated that the C‐terminal Zinc Finger (ZF) of NEMO is also able to bind ubiquitin. We show here by ZF swapping and mutagenesis that this represents its only function. While neither NOA nor ZF shows any preference for K63‐linked chains, we demonstrate that together they form a bipartite high‐affinity K63‐specific ubiquitin‐binding domain. A similar domain can be found in two other proteins, Optineurin and ABIN2, and can be freely exchanged with that of NEMO without interfering with its activity. This suggests that the main function of the C‐terminal half of NEMO is to specifically bind K63‐linked poly‐ubiquitin chains. We also demonstrate that the recently described binding of NEMO to linear poly‐ubiquitin chains is dependent on the NOA alone and does not require the presence of the ZF.     

Preliminary Assessment of Gastrointestinal Parasites in Two Wild Groups of Endangered Moor Macaques (Macaca maura) from Sulawesi

International Journal of Primatology - Information on parasite biodiversity and abundance can improve our understanding of parasitic infections on endangered wildlife, as parasites can affect host...     

Atypical forms of incontinentia pigmenti in male individuals result from mutations of a cytosine tract in exon 10 of NEMO (IKK-gamma)

Familial incontinentia pigmenti (IP [MIM 308310]), or Bloch-Sulzberger syndrome, is an X-linked dominant and male-lethal disorder. We recently demonstrated that mutations in NEMO (IKK-gamma), which encodes a critical component of the NF-kappaB signaling pathway, were responsible for IP. Virtually all mutations eliminate the production of NEMO, causing the typical skewing of X inactivation in female individuals and lethality in male individuals, possibly through enhanced sensitivity to apoptosis. Most mutations also give rise to classic signs of IP, but, in this report, we describe two mutations in families with atypical phenotypes. Remarkably, each family included a male individual with unusual signs, including postnatal survival and either immune dysfunction or hematopoietic disturbance. We found two duplication mutations in these families, at a cytosine tract in exon 10 of NEMO, both of which remove the zinc (Zn) finger at the C-terminus of the protein. Two deletion mutations were also identified in the same tract in additional families. However, only the duplication mutations allowed male individuals to survive, and affected female individuals with duplication mutations demonstrated random or slight skewing of X inactivation. Similarly, NF-kappaB activation was diminished in the presence of duplication mutations and was completely absent in cells with deletion mutations. These results strongly indicate that male individuals can also suffer from IP caused by NEMO mutations, and we therefore urge a reevaluation of the diagnostic criteria.     

Altered expression of tyrosine kinases of the Src and Syk families in human T-cell leukemia virus type 1-infected T-cell lines

During the late phase of adult T-cell leukemia/lymphoma, a severe lymphoproliferative disorder caused by human T-cell leukemia virus type 1 (HTLV-1), leukemic cells no longer produce interleukin-2. Several studies have reported the lack of the Src-like protein tyrosine kinase Lck and overexpression of Lyn and Fyn in these cells. In this report we demonstrate that, in addition to the downregulation of TCR, CD45, and Lck (which are key components of T-cell activation), HTLV-1-infected cell lines demonstrate a large increase of FynB, a Fyn isoform usually poorly expressed in T cells. Furthermore, similar to anergic T cells, Fyn is hyperactive in one of these HTLV-1-infected T-cell lines, probably as a consequence of Csk downregulation. A second family of two proteins, Zap-70 and Syk, relay the signal of T-cell activation. We demonstrate that in contrast to uninfected T cells, Zap-70 is absent in HTLV-1-infected T cells, whereas Syk is overexpressed. In searching for the mechanism responsible for FynB overexpression and Zap-70 downregulation, we have investigated the ability of the Tax and Rex proteins to modulate Zap-70 expression and the alternative splicing mechanism which gives rise to either FynB or FynT. By using Jurkat T cells stably transfected with the tax and rex genes or inducibly expressing the tax gene, we found that the expression of Rex was necessary to increase fynB expression, suggesting that Rex controls fyn gene splicing. Conversely, with the same Jurkat clones, we found that the expression of Tax but not Rex could downregulate Zap-70 expression. These results suggest that the effect of Tax and Rex must cooperate to deregulate the pathway of T-cell activation in HTLV-1-infected T cells.