Multiple levels of analysis of an IGHG4 gene deletion

Summary Human immunoglobulin heavy chain constant region (IGHC) genes constitute a typical multigene family, usually comprising eleven genes on the telomere of chromosome 14 (14q32). In this region, deleted and duplicated haplotypes have been reported to exist with considerable frequency. Their origin is the result of either unequal crossing-over or looping out excision. In this paper, we report the characterization of a new type of deletion, involving the IGHG4 gene, in a subject who also carries a larger deletion of a previously described type on the second chromosome. Employment of several methods (polymerase chain reaction, standard Southern blot, pulsed field gel electrophoresis, serological techniques) to analyze these deleted haplotypes has resulted in a level of accuracy in their characterization that has not been achieved in previous cases. The site of recombination responsible for the IGHG4 deletion was restricted to a 2.5-kb region 3 of the G4 gene; this rules out any possible involvement of the S regions in the recombination process. The usefulness of the various techniques in the characterization of the deletions is also discussed, together with possible future applications in the field.     

Flavonols and fertilization in Petunia hybrida: localization and mode of action during pollen tube growth

Flavonols form an important class of flavonoids which serve an essential function during plant reproduction. Flavonoid biosynthesis is initiated by the enzyme chalcone synthase (CHS). A high abundance of flavonols and chs mRNA was demonstrated in male and female reproductive organs of Petunia hybrida. Detailed analyses revealed precise spatial and temporal regulation of the chs promoter and flavonol synthesis in the stigma, style and ovules. Transgenic plants were generated with a complete block of flavonol biosynthesis as the result of anti-sense inhibition of chs gene activity. The absence of flavonols by this dominant mutation rendered these plants self-sterile. Pollination experiments with wild-type and mutant plants revealed that the production of flavonols in either the anthers or the pistils was required for pollen tube growth and seed set. Mutant pollen without flavonols in their exine germinated normally. However, after a short period of in vitro pollen tube growth the tips of these tubes disrupted and the protoplasm was disloaded leading to the death of the pollen grain. Addition of flavonol aglycones but not other flavonoids complemented this phenotype. Confocal laser scanning microscopy revealed the localization of high levels of flavonols throughout the wild-type pollen tube. These compounds were not detected in the exine or cell wall of growing tubes. In addition, it was observed that the flavone apigenin could completely inhibit pollen tube growth. Taken together, it is shown that flavonols play an important role in the growth of the pollen tube and their mode of action is discussed.     

Envisioning the reward

The primary visual cortex (area V1) is for vision. At least, that is what most researchers believe. However, in a recent issue of Science, Shuler and Bear demonstrate a correlate of reward timing in area V1. This surprising result indicates that brain circuits for reward processing are more extensive than expected and that area V1 has more functionality than previously thought.     

Modeling conversion and transport phenomena in solid-state fermentation: a review and perspectives

Solid-state fermentation (SSF) is accompanied inevitably by development of concentration and temperature gradients within the substrate particles and microbial biofilms. These gradients are needed for driving the transport of substrates and products. In addition, concentration gradients have been suggested to be crucial for obtaining the characteristics that define the products of SSF; nevertheless, gradients are also known to result in reduced productivity and unwanted side reactions. Solid-state fermentations are generally batch processes and this further complicates their understanding as conditions change with time. Mathematical models are therefore needed for improving the understanding of SSF processes and allowing their manipulation to achieve the desired outcomes. Existing models of SSF processes describe coupled substrate conversion and diffusion and the consequent microbial growth. Existing models disregard many of the significant phenomena that are known to influence SSF. As a result, available models cannot explain the generation of the numerous products that form during any SSF process and the outcome of the process in terms of the characteristics of the final product. This review critically evaluates the proposed models and their experimental validation. In addition, important issues that need to be resolved for improved modeling of SSF are discussed.     

Methacrylamide polymers with hydrolysis-sensitive cationic side groups as degradable gene carriers

Water-soluble polymers with hydrolyzable cationic side groups (structure of the monomers are shown in Figure 1) were synthesized and evaluated as DNA delivery systems. The polymers, except for pHPMA-NHEM, were able to condense plasmid DNA into positively charged nanosized particles. The rate of hydrolysis at 37 degrees C and pH 7.4 of the side groups differed widely; the fastest rate of hydrolysis was observed for HPMA-DEAE (half-life of 2 h), while HPMA-DMAPr had the lowest rate of hydrolysis (half-life of 70 h). In line with this, pHPMA-DEAE-based polyplexes showed the fastest destabilization of the polyplexes at 37 degrees C and pH 7.4. Polyplexes based on pHPMA-DEAE, pHPMA-DMAE, and pHPMA-MPPM showed release of intact DNA within 24, 48, and 48 h, respectively, after incubation at 37 degrees C and pH 7.4. PHPMA-DEAE and pHPMA-MPPM based polyplexes showed the highest transfection activity (almost twice as active as pEI). Importantly, the pHPMA-DEAE, pHPMA-MPPM, and pHPMA-BDMPAP polyplexes preserved their transfection activity in the presence of serum proteins. All polymers investigated showed a substantial lower in vitro cytotoxicity than pEI. In conclusion, pHPMA-based polyplexes are an attractive class of biodegradable vectors for nonviral gene delivery.     

Preadipocyte Number in Omental and Subcutaneous Adipose Tissue of Obese Individuals

Objective: To determine the variation in preadipocyte isolation procedure and to assess the number and function of preadipocytes from subcutaneous and omental adipose tissue of obese individuals. Research Methods and Procedures: The preadipocyte number per gram of adipose tissue in the abdominal‐subcutaneous and abdominal‐omental adipose stores of 27 obese subjects with a BMI of 44 ± 10 kg/m² and an age of 40 ± 9 years was determined. Results: The assessment of the preadipocyte number was found to be labor intensive and error prone. Our data indicated that the number of stromal vascular cells (SVCs), isolated from the adipose tissue by collagenase digestion, was dependent on the duration of collagenase treatment and the size and the origin of the biopsy. In addition, the fat accumulation and leptin production by differentiated SVCs were dependent on the number of adherent SVCs (aSVCs) in the culture plate and the presence of proteins derived from serum and peroxisome proliferator‐activated receptor ligands. Discussion: Using our standardized isolation and differentiation protocol, we found that the number of SVCs, aSVCs, leptin production, and fat accumulation still varied considerably among individuals. Interestingly, within individuals, the number of SVCs, aSVCs, and the leptin production by differentiating aSVCs from both the subcutaneous and the omental fat depots were associated, whereas fat accumulation was not. In obese to severely obese subjects, differences in BMI and age could not explain differences in SVCs, aSVCs, leptin production, and fat accumulation.