A straightforward approach to isolate DNA sequences with potential linkage to the retinoblastoma locus

Summary From a human-Chinese hamster somatic cell hybrid a clone was derived containing chromosome 13 in duplicate as its only human material. This clone was used to construct a human chromosome 13-specific recombinant DNA-library. Overlapping Sau3AI DNA sequences (11.9–17.2 kb) from the cell hybrid were inserted into the lambda phage vector EMBL4. From eleven recombinants having a human insert thirteen putative unique DNA sequences were isolated and cloned into the plasmid vector pBR329. A human-mouse hybrid containing a human chromosome 13 with a deletion of 13q14 and lacking its undeleted homologue was constructed to be used in a selection procedure for DNA sequences belonging to band q14. Three probes originating from two different phages were assigned to 13q14 because they did not hybridise to DNA from this cell hybrid. One of these 13q14 probes detects a low frequency (2/44) Msp I restriction fragment length polymorphism. The probes are now being used for screening a cosmid library to find adjacent polymorphic sequences with a RFLP information content suitable for application in the diagnosis of hereditary retinoblastoma.Preliminary reports of these experiments were presented at the 8th International Workshop on Human Gene Mapping, Helsinki, August 4–10, 1985, and the 13th International Congress of Biochemistry, Amsterdam, August 25–30, 1985 (Scheffer et al. 1985a,b)     

Regional LCA in a global perspective. A basis for spatially differentiated environmental life cycle assessment

Background, aim and scope  

In the context of environmental life cycle assessment (LCA), life cycle impact assessment (LCIA) is one of the central issues with respect to modelling and methodological data collection. The thesis described in this paper focusses on the assessment of toxicity-related impacts, and on the collection of normalisation data. A view on the complementary roles of LCA toxicity assessment on the one hand and human and environmental risk assessment (HERA) on the other is presented, and the global, spatially differentiated LCA toxicity assessment model GLOBOX for the assessment of organics and metals is described. Normalisation factors for the year 2000 are calculated on a global as well as on a European level.     

Microbial engineering of dehydro-amino acids and lanthionines in non-lantibiotic peptides

This minireview focusses on the use of bacteria to introduce dehydroresidues and (methyl)lanthionines in (poly)peptides. It mainly describes the broad exploitation of bacteria containing lantibiotic enzymes for the engineering of these residues in a wide variety of peptides in particular in peptides unrelated to lantibiotics. Lantibiotic dehydratases dehydrate serines and threonines present in peptides preceded by a lantibiotic leader peptide thus forming dehydroalanine and dehydrobutyrine, respectively. These dehydroresidues can be coupled to cysteines thus forming (methyl)lanthionines. This coupling is catalysed by lantibiotic cyclases. The design, synthesis, and export of microbially engineered dehydroresidue and or lanthionine-containing peptides in non-lantibiotic peptides are reviewed, illustrated by some examples which demonstrate the high relevance of these special residues. This minireview is the first with special focus on the microbial engineering of nonlantibiotic peptides by exploiting lantibiotic enzymes.     

Disruption of sexual communication in the mirid bug Lygocoris pabulinus by hexyl butanoate

1 The metathoracic scent gland in Lygocoris pabulinus contains mostly hexyl butanoate. As secretions of this gland in Heteroptera may serve as an alarm pheromone, we determined whether hexyl butanoate is released by disturbed bugs, and whether this compound disrupts sexual attraction of L. pabulinus males towards females. 2 Undisturbed males and females, and disturbed males released less than 100 ng/h hexyl butanoate, whereas disturbed females released a highly variable amount, ranging from 25 ng/h to more than 1 μg/h. 3 In the field, traps with virgin females and rubber septa containing 20 mg hexyl butanoate, caught a total of one male in a month. In control traps without hexyl butanoate, 36 males were caught in the same period. 4 In Y‐track olfactometer tests, males were not attracted to virgin females when a dispenser with 20 mg hexyl butanoate was placed in the bottle with females. Males were attracted to females when the dispenser was placed downwind from the females, but upwind from the point of male release. 5 These results suggest that males are not repelled by hexyl butanoate, but that this compound inhibits sex pheromone release in females. Application possibilities for pest management are discussed.     

TLR-3 is Present in Human Adipocytes,but Its Signalling is Not Required for Obesity-Induced Inflammation in Adipose Tissue In Vivo

Innate immunity plays a pivotal role in obesity-induced low-grade inflammation originating from adipose tissue. Key receptors of the innate immune system including Toll-like receptors-2 and -4 (TLRs) are triggered by nutrient excess to promote inflammation. The role of other TLRs in this process is largely unknown. In addition to double-stranded viral mRNA, TLR-3 can also recognize mRNA from dying endogenous cells, a process that is frequently observed within obese adipose tissue. Here, we identified profound expression of TLR-3 in adipocytes and investigated its role during diet-induced obesity. Human adipose tissue biopsies (n=80) and an adipocyte cell-line were used to study TLR-3 expression and function. TLR-3-/- and WT animals were exposed to a high-fat diet (HFD) for 16 weeks to induce obesity. Expression of TLR-3 was significantly higher in human adipocytes compared to the non-adipocyte cells part of the adipose tissue. In vitro, TLR-3 expression was induced during differentiation of adipocytes and stimulation of the receptor led to elevated expression of pro-inflammatory cytokines. In vivo, TLR-3 deficiency did not significantly influence HFD-induced obesity, insulin sensitivity or inflammation. In humans, TLR-3 expression in adipose tissue did not correlate with BMI or insulin sensitivity (HOMA-IR). Together, our results demonstrate that TLR-3 is highly expressed in adipocytes and functionally active. However, TLR-3 appears to play a redundant role in obesity-induced inflammation and insulin resistance.     

Identification of Regions Critical for the Integrity of the TSC1-TSC2-TBC1D7 Complex

The TSC1-TSC2-TBC1D7 complex is an important negative regulator of the mechanistic target of rapamycin complex 1 that controls cell growth in response to environmental cues. Inactivating TSC1 and TSC2 mutations cause tuberous sclerosis complex (TSC), an autosomal dominant disorder characterised by the occurrence of benign tumours in various organs and tissues, notably the brain, skin and kidneys. TBC1D7 mutations have not been reported in TSC patients but homozygous inactivation of TBC1D7 causes megaencephaly and intellectual disability. Here, using an exon-specific deletion strategy, we demonstrate that some regions of TSC1 are not necessary for the core function of the TSC1-TSC2 complex. Furthermore, we show that the TBC1D7 binding site is encoded by TSC1 exon 22 and identify amino acid residues involved in the TSC1-TBC1D7 interaction.     

Endocytes and breakdown of ribonuclease oligomers by sinusoidal rat liver cells in vivo: II. Effect of charge

Experiments presented in this paper suggest that sinusoidal rat liver cells recognize basic on proteins and that this recognition results in endocytosis of the proteins. Evidence for involvement of basic groups was obtained in two ways.Firstly, we changedd the opsitively charged amino groups of the cross-linked ribonuclease molecules to neutral or negative by acetylation or succinylation, respectively. The modified proteins did not contain easily reducible disulfide bonds and they were not very sensitive to endoproteases, suggesting that they were not denatured by the acetylation procedures. Acetylation and succinylation reduced uptake of the injected cross-linked ribonuclease derivatives by liver and spleen and abolsihed their rapid clearance from plasma. In nephrectomized rats about 75% of the polymer, 36% of the acetylated polumer and 32% of the succinylated polumer were endocytosed by liver after 6 h. For the dimer fraction these values were 59%, 23% and 27%, respectively. Autoradiography and subcellular fractionation of liver 30 min post-injection localized the acetylated polumer in the lysosomal/microsomal fraction of sinusoidal liver cessls, probably endothelial cells.Secondly, a positive correlation was found between binding of a number of ribonuclease derivatives of the cation exchanges SP-Sephadex G-25 and the rate of endocytosis by sinusoidal liver cells.     

A three-dimensional analysis of tooth-root morphology in living bears and implications for feeding behaviour in the extinct cave bear

ABSTRACT

The morphology of both crowns and tooth-roots reflects dietary specialisation in mammalian carnivores. In this article, we analyse the tooth-root morphology of maxillary teeth from CT scans of living bears (Ursus arctos, Ursus americanus, Ursus maritimus, Ursus thibetanus, Melursus ursinus, Helarctos malayanus, Tremarctos ornatus and Ailuropoda melanoleuca) in order to make inferences about the diet and feeding behaviour of the extinct cave bear (Ursus spelaeus sensu lato). Specifically, we investigate two major mitochondrial clades of extinct cave bears recognized by previous authors: Ursus ingressus and Ursus spelaeus (U. spelaeus spelaeus, U. spelaeus ladinicus, U. spelaeus eremus). Our results indicate a close association between tooth-root surface area and feeding behaviour in all living bear species. Tooth-root surface area values of cave bears suggest that they relied more on vegetative matter than living brown bears (Ursus arctos) but subtle differences between these species/subspecies could also indicate different feeding strategies among the members of cave bear complex.