Making better transgenic models

Over the last decade transgenic mouse models have become a common experimental tool for unraveling gene function. During this time there has been a growing expectation that transgenes resemble the in vivo state as much as possible. To this end, a preference away from heterologous promoters has emerged, and transgene constructs often utilize the endogenous promoter and gene sequences in BAC, PAC and YAC form without the addition of selectable markers, or at least their subsequent removal. There has been a trend toward controlled integration by homologous recombination, either at a characterized chromosomal localization or in some cases within the allele of interest. Markers such as green fluorescent protein (GFP), beta-galactosidase (LacZ), and alkaline phosphatase (AP) continue to be useful to trace transgenic cells, or transgene expression. The development of technologies such as RNA interference (RNAi), are introducing new ways of using transgenic models. Future developments in RNAi technology may revolutionize tissue specific inactivation of gene function, without the requirement of generating conditionally targeted mice and tissue specific recombinase mice. Transgenic models are biological tools that aid discovery. Overall, the main consideration in the generation of transgenic models is that they are bona fide biological models that best impart the disease model or biological function of the gene that they represent. The main consideration is to make the best model for the biological question at heart and this review aims to simplify that task somewhat. Here we take a historical perspective on the development of transgenic models, with many of the important considerations to be made in design and development along the way.     

Novel role of Zn(II)-curcumin in enhancing cell proliferation and adjusting proinflammatory cytokine-mediated oxidative damage of ethanol-induced acute gastric ulcers

Alcohol consumption can induce gastric ulcers and zinc deficiency. Zinc complexes were reported to have anti-ulcer activity as it acts as an anti-inflammatory and antioxidant. Zn(II)-curcumin complex and its solid dispersions (SDs) were synthesized and evaluated for its gastroprotective activity and mechanism against ethanol-induced ulcer. The Swiss murine fibroblast cell line (3T3) was used as an alternative in vitro model to evaluate the effects of Zn(II)-curcumin on cell proliferation. Zn(II)-curcumin were administered orally for seven consecutive days prior to induction of ulcers using ethanol. Gross and microscopic lesions, immunological and biochemical parameters were taken into consideration. The results showed that solid dispersions (SDs) of Zn(II)-curcumin (2.5-20 μM) enhanced the proliferation of 3T3 cells more significantly than curcumin at the same concentrations (P<0.01). Oral administration of Zn(II)-curcumin (12, 24 and 48 mg/kg) SDs dose-dependently prevented formation of ulcer lesions induced by ethanol. The levels of proinflammatory cytokines tumor necrosis factor-α (TNF-α), interleukin 6 (IL-6), and oxidative stress superoxide dismutase (SOD), glutathione peroxidase (GPX-Px), malonaldehyde (MDA) and H(+)-K(+)-ATPase were in the rats exposed to ethanol in ulceration have been altered. Zn(II)-curcumin prevented formation of ulcer lesions, significantly inhibited TNF-α and IL-6 mRNA expression, increased the activity of SOD and GSH-Px, reduced MDA levels and H(+)-K(+)-ATPase in mucosa of rats compared to controls (P<0.05). These findings suggest that the gastroprotective activity of Zn(II)-curcumin complex might contribute in stimulating cell proliferation and adjusting the proinflammatory cytokine-mediated oxidative damage to the gastric mucosa.     

HIV and STI Prevalence among Female Sex Workers in C?te d'Ivoire: Why Targeted Prevention Programs Should Be Continued and Strengthened

Objective

To assess condom use and prevalence of STIs and HIV among female sex workers (FSWs), as part of a comprehensive monitoring and evaluation plan of a nationwide sex worker prevention project in Côte d''Ivoire.

Design and Methods

Cross sectional surveys were conducted among FSWs attending five project clinics in Abidjan and San Pedro (2007), and in Yamoussoukro and Gagnoa (2009). A standardized questionnaire was administered in a face-to-face interview, which included questions on socio-demographic characteristics, sexual behaviour and condom use. After the interview, the participants were asked to provide samples for STI and HIV testing.

Results

A total of 1110 FSWs participated in the surveys. There were large differences in socio-demographic and behavioural characteristics between FSW coming for the first time as compared to FSW coming on a routine visit. The prevalence of N. gonorrhoeae or C.trachomatis was 9.1%, 11.8% among first vs. 6.9% routine attendees (p = 0.004). The overall HIV prevalence was 26.6%, it was lower among first time attendees (17.5% as compared to 33.9% for routine attendees, p<0.001). The HIV prevalence among first attendees was also lower than the proportion of HIV positive tests from routine testing and counselling services in the same clinics.

Conclusions

The results show a relatively high STI and HIV prevalence among FSWs in different cities in Côte d''Ivoire. In the light of these results, prevention efforts should continue to focus on FSWs in the country.     

Targeting antibody responses to the membrane proximal external region of the envelope glycoprotein of human immunodeficiency virus

Although human immunodeficiency type 1 (HIV-1) infection induces strong antibody responses to the viral envelope glycoprotein (Env) only a few of these antibodies possess the capacity to neutralize a broad range of strains. The induction of such antibodies represents an important goal in the development of a preventive vaccine against the infection. Among the broadly neutralizing monoclonal antibodies discovered so far, three (2F5, Z13 and 4E10) target the short and hidden membrane proximal external region (MPER) of the gp41 transmembrane protein. Antibody responses to MPER are rarely observed in HIV-infected individuals or after immunization with Env immunogens. To initiate antibody responses to MPER in its membrane-embedded native conformation, we generated expression plasmids encoding the membrane-anchored ectodomain of gp41 with N-terminal deletions of various sizes. Following transfection of these plasmids, the MPER domains are displayed on the cell surface and incorporated into HIV virus like particles (VLP). Transfected cells displaying MPER mutants bound as efficiently to both 2F5 and 4E10 as cells transfected with a plasmid encoding full-length Env. Mice immunized with VLPs containing the MPER mutants produced MPER-specific antibodies, the levels of which could be increased by the trimerization of the displayed proteins as well as by a DNA prime-VLP boost immunization strategy. Although 2F5 competed for binding to MPER with antibodies in sera of some of the immunized mice, neutralizing activity could not be detected. Whether this is due to inefficient binding of the induced antibodies to MPER in the context of wild type Env or whether the overall MPER-specific antibody response induced by the MPER display mutants is too low to reveal neutralizing activity, remains to be determined.     

Regulation of the mutually exclusive exons 8a and 8 in the CaV1.2 calcium channel transcript by polypyrimidine tract-binding protein

CaV1.2 calcium channels play roles in diverse cellular processes such as gene regulation, muscle contraction, and membrane excitation and are diversified in their activity through extensive alternative splicing of the CaV1.2 mRNA. The mutually exclusive exons 8a and 8 encode alternate forms of transmembrane segment 6 (IS6) in channel domain 1. The human genetic disorder Timothy syndrome is caused by mutations in either of these two CaV1.2 exons, resulting in disrupted Ca(2+) homeostasis and severe pleiotropic disease phenotypes. The tissue-specific pattern of exon 8/8a splicing leads to differences in symptoms between patients with exon 8 or 8a mutations. Elucidating the mechanisms controlling the exon 8/8a splicing choice will be important in understanding the spectrum of defects associated with the disease. We found that the polypyrimidine tract-binding protein (PTB) mediates a switch from exon 8 to 8a splicing. PTB and its neuronal homolog, nPTB, are widely studied splicing regulators controlling large sets of alternative exons. During neuronal development, PTB expression is down-regulated with a concurrent increase in nPTB expression. Exon 8a is largely repressed in embryonic mouse brain but is progressively induced during neuronal differentiation as PTB is depleted. This splicing repression is mediated by the direct binding of PTB to sequence elements upstream of exon 8a. The nPTB protein is a weaker repressor of exon 8a, resulting in a shift in exon choice when nPTB replaces PTB in cells. These results provide mechanistic understanding of how these two exons, important for human disease, are controlled.     

Trypanocidal and cytotoxic evaluation of synthesized thiosemicarbazones as potential drug leads against sleeping sickness

Thiosemicarbazones have become one of the promising compounds as new clinical candidates due to their wide spectrum of pharmaceutical activities. The wide range of their biological activities depends generally on their related aldehyde or ketone groups. Here, we report the pharmacological activities of some thiosemicarbazones synthesized in this work. Benzophenone and derivatives were used with N(4)-phenyl-3-thiosemicarbazide to synthesize corresponding five thiosemicarbazones (1–5). Their structures were characterized by spectrometrical methods analysis IR, NMR ¹H & ¹³C and MS. The compounds were then screened in vitro for their antiparasitic activity and toxicity on Trypanosoma brucei brucei and Artemia salina Leach respectively. The selectivity index of each compound was also determined. Four thiosemicarbazones such as 4, 2, 3 and 1 reveal interesting trypanocidal activities with their half inhibitory concentration (IC₅₀) equal to 2.76, 2.83, 3.86 and 8.48 μM respectively, while compound 5 (IC₅₀ = 12.16 μM) showed a moderate anti-trypanosomal activity on parasite. In toxicity test, except compound 1, which showed a half lethal concentration LC₅₀ >281 μM, the others exerted toxic effect on larvae with LC₅₀ of 5.56, 13.62, 14.55 and 42.50 μM respectively for thiosemicarbazones 4, 5, 3 and 2. In agreement to their selectivity index, which is greater than 1 (SI >1), these compounds clearly displayed significant selective pharmaceutical activities on the parasite tested. The thiosemicarbazones 2–5 that displayed significant anti-trypanosomal and cytoxicity activities are suggested to have anti-neoplastic and anti-cancer activities.     

Confirmation of putative HIV-1 group P in Cameroon

We report the second human immunodeficiency virus (HIV) belonging to the new HIV type 1 (HIV-1) group P lineage that is closely related to the simian immunodeficiency virus found in gorillas. This virus was identified in an HIV-seropositive male hospital patient in Cameroon, confirming that the group P virus is circulating in humans. Results from screening 1,736 HIV-seropositive specimens collected in Cameroon indicate that HIV-1 group P infections are rare, accounting for only 0.06% of HIV infections. Despite its rarity, group P shows evidence of adaptation to humans.     

Cysteine-rich secretory protein 2 binds to mitogen-activated protein kinase kinase kinase 11 in mouse sperm

Cysteine-rich secretory protein (CRISP) 2 (previously TPX1) is a testis-enriched member of the CRISP family, and has been localized to both the sperm acrosome and tail. Like all members of the mammalian CRISP family, its expression pattern is strongly suggestive of a role in male fertility, but functional support for this hypothesis remains limited. In order to determine the biochemical pathways within which CRISP2 is a component, the putative mature form of CRISP2 was used as bait in a yeast two-hybrid screen of a mouse testis expression library. One of the most frequently identified interacting partners was mitogen-activated protein kinase kinase kinase 11 (MAP3K11). Sequencing and deletion experiments showed that the carboxyl-most 20 amino acids of MAP3K11 interacted with the CRISP domain of CRISP2. This interaction was confirmed using pull-down experiments and the cellular context was supported by the localization of CRISP2 and MAP3K11 to the acrosome of the developing spermatids and epididymal spermatozoa. Interestingly, mouse epididymal sperm contained an approximately 60-kDa variant of MAP3K11, which may have been a result of proteolytic cleavage of the longer 93-kDa form seen in many tissues. These data raise the possibility that CRISP2 is a MAP3K11-modifying protein or, alternatively, that MAP3K11 acts to phosphorylate CRISP2 during acrosome development.