Construction of alpha-alpha domains structure in recombinant monkey metallothionein-1

The differences in metal-thiolate coordination and reactivity of mammalian metallothionein (MT) domains are closely related to their distinct, highly conservative cysteine number and position. Monkey metallothionein-1, containing a beta-domain with Cd(3)S(9) cluster and an alpha-domain with Cd(4)S(11) cluster, was used to evaluate the role of cysteine residues in the formation of MT's metal-thiolate clusters. The possible influence of cysteine residues on the binding and stability of MT domains has been examined with the metallothionein mutants: N4C, T27C and N4C/T27C, which possess ten or eleven cysteine residues in the re-constructed beta-domain, respectively. Assisted by study of UV, CD and electrospray ionization mass spectroscopy (ESI-MS) and their reactivity with DTNB (5,5'-dithiobis (2-nitrobenzoic acid)), we found that besides the original alpha-domain, some kinds of new domain containing 4-cadmium-thiolate clusters were formed in the N4C and N4C/T27C mutants of mkMT1. These new domains displayed metal binding and kinetic reactivity with DTNB similar to the alpha-domain. However, the thermal stability of the mutants was less stable than that of WT mkMT1. This might result from the disturbance of the inter-domains hydrogen bonds and of the non-cysteine amino acid residue arrangement.     

Astrocyte-specific inactivation of the neurofibromatosis 1 gene (NF1) is insufficient for astrocytoma formation

Individuals with the neurofibromatosis 1 (NF1) inherited tumor syndrome develop low-grade gliomas (astrocytomas) at an increased frequency, suggesting that the NF1 gene is a critical growth regulator for astrocytes. In an effort to determine the contribution of the NF1 gene product, neurofibromin, to astrocyte growth regulation and NF1-associated astrocytoma formation, we generated astrocyte-specific Nf1 conditional knockout mice (Nf1(GFAP)CKO) by using Cre/LoxP technology. Transgenic mice were developed in which Cre recombinase was specifically expressed in astrocytes by embryonic day 14.5. Successive intercrossing with mice bearing a conditional Nf1 allele (Nf1flox) resulted in GFAP-Cre Nf1flox/flox (Nf1(GFAP)CKO) animals. No astrocytoma formation or neurological impairment was observed in Nf1(GFAP)CKO mice after 20 months, but increased numbers of proliferating astrocytes were observed in several brain regions. To determine the consequence of Nf1 inactivation at different developmental times, the growth properties of embryonic day 12.5 and postnatal day 2 Nf1 null astrocytes were analyzed. Nf1 null astrocytes exhibited increased proliferation but lacked tumorigenic properties in vitro and did not form tumors when injected into immunocompromised mouse brains in vivo. Collectively, our results suggest that loss of neurofibromin is not sufficient for astrocytoma formation in mice and that other genetic or environmental factors might influence NF1-associated glioma tumorigenesis.     

Comparison of Some Photosynthetic Characters Between Two Hybrid Rice Combinations Differing in Yield Potential

Photosynthetic characteristics of two hybrid rice combinations, Peiai 64S/E32 and Shanyou 63, were compared at the panicle differentiation stage. As compared with Shanyou 63, the new combination Peiai 64S/E32 showed a significantly higher net photosynthetic rate (P _N), apparent quantum yield of carbon assimilation (_c), carboxylation efficiency (CE), and photorespiratory rate (R _P) as well as leaf chlorophyll content, but a significantly lower dark respiration rate (R _D) and compensation irradiance (I _c). It also showed a slightly higher photochemical efficiency (F_v/F_m and F/F_m) of photosystem 2, a lower non-photochemical quenching (q_N), and a similar CO₂ compensation concentration () as compared to Shanyou 63.     

The zinc ion in the HNH motif of the endonuclease domain of colicin E7 is not required for DNA binding but is essential for DNA hydrolysis

The HNH motif was originally identified in the subfamily of HNH homing endonucleases, which initiate the process of the insertion of mobile genetic elements into specific sites. Several bacteria toxins, including colicin E7 (ColE7), also contain the 30 amino acid HNH motif in their nuclease domains. In this work, we found that the nuclease domain of ColE7 (nuclease-ColE7) purified from Escherichia coli contains a one-to-one stoichiometry of zinc ion and that this zinc-containing enzyme hydrolyzes DNA without externally added divalent metal ions. The apo-enzyme, in which the indigenous zinc ion was removed from nuclease-ColE7, had no DNase activity. Several divalent metal ions, including Ni²⁺, Mg²⁺, Co²⁺, Mn²⁺, Ca²⁺, Sr²⁺, Cu²⁺ and Zn²⁺, re-activated the DNase activity of the apo-enzyme to various degrees, however higher concentrations of zinc ion inhibited this DNase activity. Two charged residues located at positions close to the zinc-binding site were mutated to alanine. The single-site mutants, R538A and E542A, showed reduced DNase activity, whereas the double-point mutant, R538A + E542A, had no observable DNase activity. A gel retardation assay further demonstrated that the nuclease-ColE7 hydrolyzed DNA in the presence of zinc ions, but only bound to DNA in the absence of zinc ions. These results demonstrate that the zinc ion in the HNH motif of nuclease-ColE7 is not required for DNA binding, but is essential for DNA hydrolysis, suggesting that the zinc ion not only stabilizes the folding of the enzyme, but is also likely to be involved in DNA hydrolysis.     

Identification of novel mutations in MLC1 responsible for megalencephalic leukoencephalopathy with subcortical cysts

Megalencephalic leukoencephalopathy with subcortical cysts (MLC) is an inherited neurologic disorder with macrocephaly before the age of one and slowly progressive deterioration of motor functions. Magnetic resonance imaging shows diffusely abnormal and swollen white matter of the cerebral hemispheres and the presence of subcortical cysts in the anterior-temporal region and often also in the frontoparietal region. Mutations in the MLC1 gene, encoding a putative membrane protein, have been recently identified as a cause for MLC. Here, we describe 14 new mutations in 18 patients. Two identified polymorphisms lead to alterations of amino acid residues. The role, suggested by others, of a mutation in the MLC1gene in catatonic schizophrenia and the possible function of the MLC1 protein as a cation channel are discussed.     

Probing the binding states of GDP to Cdc42 using urea interaction

The inactive state of the small G protein Cdc42, the Cdc42.GDP.Mg(2+) ternary complex, was investigated using fluorescence, Mn(2+) substituted electron paramagnetic resonance, and (31)P nuclear magnetic resonance spectroscopy at various urea concentrations. The urea interaction with the protein was used to probe the binding state of GDP.Mg(2+) to Cdc42. Two binding states of the Cdc42.GDP.Mg(2+) ternary complex with different binding stability were observed. The two binding states were characterized by two sets of (31)P resonance of GDP phosphate groups, namely P(alpha) and P(beta), P('alpha), and P('beta). The high populated binding state I (P(alpha) and P(beta)) was more stable and less sensitive to the urea interaction. Yet the population of binding state II (P('alpha) and P('beta)) was lower, and the binding of GDP.Mg(2+) to Cdc42 in this state was more sensitive to the urea interaction. The release of GDP.Mg(2+) from the ternary complex in binding state II was faster than in state I.     

The size of lipid rafts: an atomic force microscopy study of ganglioside GM1 domains in sphingomyelin/DOPC/cholesterol membranes

Atomic force microscopy has been used to study the distribution of ganglioside GM1 in model membranes composed of ternary lipid mixtures that mimic the composition of lipid rafts. The results demonstrate that addition of 1% GM1 to 1:1:1 sphingomyelin/dioleoylphosphatidylcholine/cholesterol monolayers leads to the formation of small ganglioside-rich microdomains (40-100 nm in size) that are localized preferentially in the more ordered sphingomyelin/cholesterol-rich phase. With 5% GM1 some GM1 microdomains are also detected in the dioleoylphosphatidylcholine-rich phase. A similar preferential localization of GM1 in the ordered phase is observed for bilayers with the same ternary lipid mixture in the upper leaflet. The small GM1-rich domains observed in these experiments are similar to the sizes for lipid rafts in natural membranes but considerably smaller than the ordered bilayer domains that have been shown to be enriched in GM1 in recent fluorescence microscopy studies of lipid bilayers. The combined data from a number of studies of model membranes indicate that lateral organization occurs on a variety of length scales and mimics many of the properties of natural membranes.     

Isolation of differentially expressed genes in human heart tissues

We applied RNA arbitrarily primed-PCR (RAP-PCR) to screen the genes differentially expressed between common congenital heart defects (CHD) [atrial septal defect, ventricular septal defect, Tetrology of Fallot (TOF)] and normal human heart samples. Three of these differentially amplified fragments matched cDNA sequences coding for proteins of unknown function in humans: hCALO (human homologue of calossin), NP79 (coding for a nuclear protein of 79KD) and SUN2 (Sad-1 unc-84 domain protein 2). The other four fragments were from known human genes: apolipoprotein J, titin, dystrophin and protein kinase C-delta. Northern blot analysis confirmed that all of these genes are expressed in the human heart. The results of RAP-PCR were reconfirmed by quantitative RT-PCR in TOF and control heart samples. Both techniques showed the levels of expression of hCALO, NP79 and SUN2 to be comparable in TOF and control samples and the level of expression of dystrophin and titin, both coding for cytoskeletal proteins, to be significantly upregulated in TOF samples. In summary, we have shown that the RAP-PCR technique is useful in the identification of differentially expressed gene from biopsy samples of human CHD tissues. In this manner, we have identified three novel genes implicated in the normal function of the human heart and two known genes upregulated in TOF samples.     

Structure-function relationship of three neurotoxins from the venom of Naja kaouthia: a comparison between the NMR-derived structure of NT2 with its homologues, NT1 and NT3

Three homologous short-chain neurotoxins, named NT1, NT2 and NT3, were purified from the venom of Naja kaouthia. NT1 has an identical amino acid sequence to cobrotoxin from Naja naja atra [Biochemistry 32 (1993) 2131]. NT3 shares the same sequence with cobrotoxin b [J. Biochem. (Tokyo) 122 (1997) 1252], whereas NT2 is a novel 61-residue neurotoxin. Tests of their physiological functions indicate that NT1 shows a greater inhibition of muscle contraction induced by electrical stimulation of the nerve than do NT2 and NT3. Homonuclear proton two-dimensional NMR methods were utilized to study the solution tertiary structure of NT2. A homology model-building method was employed to predict the structure of NT3. Comparison of the structures of these three toxins shows that the surface conformation of NT1 facilitates the substituted base residues, Arg28, Arg30, and Arg36, to occupy the favorable spatial location in the central region of loop II, and the cation groups of all three arginines face out of the molecular surface of NT1. This may contribute greatly to the higher binding of NT1 with AchR compared to NT2 and NT3.