Molecular cloning and characterization of FHL2, a novel LIM domain protein preferentially expressed in human heart

A full-length cDNA clone encoding a novel LIM-only protein was isolated and sequenced from a human fetal heart cDNA library. This full-length clone consists of 1416 base pairs and has a predicted open reading frame (ORF) encoding 279 amino acids. The ORF of this polypeptide codes for the human heart-specific

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our and a

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alf

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IM-only protein

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(FHL2). It possesses an extra zinc finger that is a half LIM domain and four repeats of LIM domain. When the human FHL2 cDNA probe was used to hybridize with poly-A RNA of various human tissues, a very strong signal could be seen in heart tissues, and only moderately low signals could be detected in placenta, skeletal muscle and ovary. Virtually no signal could be detected in brain, lung, liver, kidney, pancreas, spleen, thymus, prostate, testis, small intestine, colon or peripheral blood leukocyte. FHL2 was mapped to chromosome 2q12–q13 by fluorescent in-situ hybridization (FISH).     

Morphological development and allometric growth patterns in the juvenile seahorse Hippocampus kuda Bleeker

The morphological development and allometric growth patterns in the juvenile spotted seahorse Hippocampus kuda were studied under hatchery rearing conditions. Newborn spotted seahorses [mean ± s.d . standard length ( L _S) 9·33 ± 0·79 mm] were raised till the age of 124 days (119·35 ± 6·04 mm). Growth was characterized by three stages with two inflexion points occurring at day 21 and 76. The mean growth rates in the first, second and third stages were 0·68, 1·16 and 0·71 mm day⁻¹, respectively. The growth rate was most rapid in the second stage and was probably influenced by a behavioural shift from pelagic to benthic form. The mass ( M ) and L _S relationship was exponential ( M = 7·14 × 10⁻⁶ L _S^2·76), but the slope, b = 2·76, reflected negative allometric growth. Sexes could be distinguished at c. 110 days, and the sex ratio was unbiased. The L _S in males and females did not differ significantly. Morphological stageing series is proposed, which divides H. kuda juvenile development into eight stages based on the development of coronet, cheek and eye spines, keel and pigmentation. The morphometric ratios for all the body parts, except trunk length, showed considerable changes at a transition point occurring at c. 25 mm L _S. The high proportional growth in head length, head depth, pectoral fin base length, dorsal fin base length, snout length, snout depth and eye diameter at the initial stages, and the abrupt increase in tail length only after the first 2 weeks, possibly reflect development priorities during early development where important organs are being developed first for the enhancement of juvenile survival.     

In vitro rooting of sentang shoots (Azadirachta excelsa L.) and acclimatization of the plantlets

Summary In vitro proliferated sentang shoots were cultured onto half-strength Murashige and Skoog (MS) medium containing combinations of 1-naphthyleneacetic acid (NAA) and indole-3-butyric acid (IBA). Sentang shoots were unable to root in the absence of both auxins. A combination of 0.5 mg NAA per and 1 mg IBA per 1 induced the most shoots to form roots. With the addition of 2.5 g activated charcoal per 1 into half-strength MS medium containing 0.5 mg NAA per 1 and 1 mg IBA per 1, roots were more numerous and longer. Substances like gelrite and phloroglucinol and sugar content which would commonly influence in vitro rooting were inhibitory to adventitious root formation of sentang. Maximal rooting of 100% was achieved in “Culture Pack,” made of fluorocarbon polymer film containing charcoal-free medium with 0.5 mg NAA per 1 and 1 mg IBA per 1. Rooted shoots were acclimatized for 4 wk. Overall survival was 80%. These findings suggest the use of Culture Pack as the culture vessels, with 0.5 mg NAA per 1 and 1 mg IBA per 1 in half-stength MS media to effectively induce roots in sentang shoots.     

Protective immune responses induced by vaccination with an expression genomic library of Leishmania major.

To develop an effective vaccine against the intracellular protozoan parasite Leishmania spp., we investigated the feasibility of expression library immunization (ELI) in the mouse. Genomic expression libraries of L. major were constructed and used to immunize mice. One of the three libraries (L1, with 10(5) clones) induced a significant protective immune response and delayed the onset of lesion development in highly susceptible BALB/c mice after i.m. immunization, compared with control mice immunized with the empty vector (EV). L1 was then divided into five sublibraries of approximately 2 x 10(4) clones each. Mice immunized with one of the sublibraries (SL1A) developed an even stronger protective effect than that induced by L1. SL1A was further divided into 20 sublibraries (SL2) of approximately 10(3) clones each. One of the SL2 libraries (SL2G) induced a strong protective effect against L. major infection. In direct comparative studies, the protective effect of the sublibraries was in the order of SL2G > SL1A > L1. Lymphoid cells from mice vaccinated with SL2G produced more IFN-gamma and NO, compared with cells from control mice injected with EV. Serum from the vaccinated mice also contained more parasite-specific IgG2a Ab, compared with controls. Therefore, these data demonstrate that ELI is feasible against this complex intracellular parasitic infection, by preferentially inducing the development of Th1 responses. Furthermore, by sequential division of the libraries, this approach may be used to enrich and identify protective genes for effective gene vaccination against other parasitic infections.     

Absence of Diabetes and Pancreatic Exocrine Dysfunction in a Transgenic Model of Carboxyl-Ester Lipase-MODY (Maturity-Onset Diabetes of the Young)

Background

CEL-MODY is a monogenic form of diabetes with exocrine pancreatic insufficiency caused by mutations in CARBOXYL-ESTER LIPASE (CEL). The pathogenic processes underlying CEL-MODY are poorly understood, and the global knockout mouse model of the CEL gene (CELKO) did not recapitulate the disease. We therefore aimed to create and phenotype a mouse model specifically over-expressing mutated CEL in the pancreas.

Methods

We established a monotransgenic floxed (flanking LOX sequences) mouse line carrying the human CEL mutation c.1686delT and crossed it with an elastase-Cre mouse to derive a bitransgenic mouse line with pancreas-specific over-expression of CEL carrying this disease-associated mutation (TgCEL). Following confirmation of murine pancreatic expression of the human transgene by real-time quantitative PCR, we phenotyped the mouse model fed a normal chow and compared it with mice fed a 60% high fat diet (HFD) as well as the effects of short-term and long-term cerulein exposure.

Results

Pancreatic exocrine function was normal in TgCEL mice on normal chow as assessed by serum lipid and lipid-soluble vitamin levels, fecal elastase and fecal fat absorption, and the normoglycemic mice exhibited normal pancreatic morphology. On 60% HFD, the mice gained weight to the same extent as controls, had normal pancreatic exocrine function and comparable glucose tolerance even after resuming normal diet and follow up up to 22 months of age. The cerulein-exposed TgCEL mice gained weight and remained glucose tolerant, and there were no detectable mutation-specific differences in serum amylase, islet hormones or the extent of pancreatic tissue inflammation.

Conclusions

In this murine model of human CEL-MODY diabetes, we did not detect mutation-specific endocrine or exocrine pancreatic phenotypes, in response to altered diets or exposure to cerulein.     

Characterization of Zn2+-binding nuclear proteins present in the myocardium

Nonhistone nuclear proteins were isolated from 3–5 day old neonatal as well as 3 month-old adult myocardium. The nuclear proteins were separated and analyzed by two-dimensional polyacrylamide gel electrophoresis. Using a blot transfer technique equilibrated with⁶⁵Zn²⁺, at least four polypeptides exhibited Zn²⁺-binding activity over the spectrum of nonhistone nuclear proteins. A protein with a molecular weight of 68kDa pI7.8, which has been characterized for its involvement in nucleosome structure, consistently binds Zn²⁺ in both the neonatal and adult myocardium. This nuclear protein has now been further characterized by partial amino acid microsequencing. It was found that this novel polypeptide is distinct from the pore-complex lamina proteins. Three other polypeptides with M90kDa, pI7.8, M68kDa, pI6.5 and M35 kDa, pI7.5 exhibited increased Zn²⁺-binding activity in neonatal myocardium as compared to adult myocardium. Together with results from our previous studies, this study provides the first evidence implicating Zn⁺⁺-binding nuclear proteins in the processes of growth and differentiation of myocardial development. (Mol Cell Biochem121: 175–179, 1993)     

Binding interaction of quercetin-3-beta-galactoside and its synthetic derivatives with SARS-CoV 3CL(pro): structure-activity relationship studies reveal salient pharmacophore features

The 3C-like protease (3CL^pro) of severe acute respiratory syndrome-associated coronavirus (SARS-CoV) is one of the most promising targets for discovery of drugs against SARS, because of its critical role in the viral life cycle. In this study, a natural compound called quercetin-3-β-galactoside was identified as an inhibitor of the protease by molecular docking, SPR/FRET-based bioassays, and mutagenesis studies. Both molecular modeling and Q189A mutation revealed that Gln189 plays a key role in the binding. Furthermore, experimental evidence showed that the secondary structure and enzymatic activity of SARS-CoV 3CL^pro were not affected by the Q189A mutation. With the help of molecular modeling, eight new derivatives of the natural product were designed and synthesized. Bioassay results reveal salient features of the structure–activity relationship of the new compounds: (1) removal of the 7-hydroxy group of the quercetin moiety decreases the bioactivity of the derivatives; (2) acetoxylation of the sugar moiety abolishes inhibitor action; (3) introduction of a large sugar substituent on 7-hydroxy of quercetin can be tolerated; (4) replacement of the galactose moiety with other sugars does not affect inhibitor potency. This study not only reveals a new class of compounds as potential drug leads against the SARS virus, but also provides a solid understanding of the mechanism of inhibition against the target enzyme.     

Application of rDNA-PCR amplification and DGGE fingerprinting for detection of microbial diversity in a Malaysian crude oil

Two culture-independent methods, namely ribosomal DNA libraries and denaturing gradient gel electrophoresis (DGGE), were adopted to examine the microbial community of a Malaysian light crude oil. In this study, both 16S and 18S rDNAs were PCR-amplified from bulk DNA of crude oil samples, cloned, and sequenced. Analyses of restriction fragment length polymorphism (RFLP) and phylogenetics clustered the 16S and 18S rDNA sequences into seven and six groups, respectively. The ribosomal DNA sequences obtained showed sequence similarity between 90 to 100% to those available in the GenBank database. The closest relatives documented for the 16S rDNAs include member species of Thermoincola and Rhodopseudomonas, whereas the closest fungal relatives include Acremonium, Ceriporiopsis, Xeromyces, Lecythophora, and Candida. Others were affiliated to uncultured bacteria and uncultured ascomycete. The 16S rDNA library demonstrated predomination by a single uncultured bacterial type by >80% relative abundance. The predomination was confirmed by DGGE analysis.     

Molecular phylogenetic and morphological analyses of Oidium heveae, a powdery mildew of rubber tree

Powdery mildew of rubber tree caused by Oidium heveae is an important disease of rubber plantations worldwide. Identification and classification of this fungus is still uncertain because there is no authoritative report of its morphology and no record of its teleomorphic stage. In this study, we compared five specimens of the rubber powdery mildew fungus collected in Malaysia, Thailand, and Brazil based on morphological and molecular characteristics. Morphological results showed that the fungus on rubber tree belongs to Oidium subgen. Pseudoidium. Nucleotide sequence analysis of the ribosomal DNA internal transcribed spacer (ITS) region and the large subunit rRNA gene (28S rDNA) were conducted to determine the relationships of the rubber powdery mildew fungus and to link this anamorphic fungus with its allied teleomorph. The results showed that the rDNA sequences of the two specimens from Malaysia were identical to a specimen from Thailand, whereas they differed by three bases from the two Brazilian isolates: one nucleotide position in the ITS2 and two positions in the 28S sequences. The ITS sequences of the two Brazilian isolates were identical to sequences of Erysiphe sp. on Quercus phillyraeoides collected in Japan, although the 28S sequences differed at one base from sequences of this fungus. Phylogenetic trees of both rDNA regions constructed by the distance and parsimony methods showed that the rubber powdery mildew fungus grouped with Erysiphe sp. on Q. phillyraeoides with 100% bootstrap support. Comparisons of the anamorph of two isolates of Erysiphe sp. from Q. phillyraeoides with the rubber mildew did not reveal any obvious differences between the two powdery mildew taxa, which suggests that O. heveae may be an anamorph of Erysiphe sp. on Q. phillyraeoides. Cross-inoculation tests are required to substantiate this conclusion.     

Altered patterns of differentiation in karyotypically abnormal human embryonic stem cells

Upon prolonged culture, human embryonic stem (hES) cells undergo adaptation, exhibiting decreased population doubling times and increased cloning efficiencies, often associated with karyotypic changes. To test whether culture adaptation influences the patterns of differentiation of hES cells, we compared the expression of genes indicative of distinct embryonic lineages in the embryoid bodies produced from two early passage, karyotypically normal hES cell lines, and two late passage, karyotypically abnormal hES cell lines. One of the abnormal lines was a subline of one of the normal early passage lines. The embryoid bodies from each of the lines showed evidence of extensive differentiation. However, there were differences in the expression of several genes, indicating that the culture adapted hES cells show altered patterns of differentiation compared to karyotypically normal hES cells. The loss of induction of alphafetoprotein in the culture-adapted cells was especially marked, suggesting that they had a reduced capacity to produce extra-embryonic endoderm. These changes may contribute to the growth advantages of genetically variant cells, not only by reflecting an increased tendency to self renewal rather than to differentiate, but also by reducing spontaneous differentiation to derivatives that themselves may produce factors that could induce further differentiation of undifferentiated stem cells.