A Balanced Ratio of Proteins from Gene G and Frameshift-Extended Gene GT Is Required for Phage Lambda Tail Assembly

In bacteriophage λ, the overlapping open reading frames G and T are expressed by a programmed translational frameshift similar to that of the gag-pol genes of many retroviruses to produce the proteins gpG and gpGT. An analogous frameshift is widely conserved among other dsDNA tailed phages in their corresponding “G” and “GT” tail genes even in the absence of detectable sequence homology. The longer protein gpGT is known to be essential for tail assembly, but the requirement for the shorter gpG remained unclear because mutations in gene G affect both proteins. A plasmid system that can direct the efficient synthesis of tails was created and used to show that gpG and gpGT are both essential for correct tail assembly. Phage complementation assays under conditions where levels of plasmid-expressed gpG or gpGT could be altered independently revealed that the correct molar ratio of these two related proteins, normally determined by the efficiency of the frameshift, is also crucial for efficient assembly of functional tails. Finally, the physical connection between the G and T domains of gpGT, a consequence of the frameshift mechanism of protein expression, appears to be important for efficient tail assembly.     

Site on inhibition of the spino-bulbo-spinal reflex on stimulation of the anterior lobe of the cerebellum.

The effect of stimulation of the anterior lobe (AL) of the cerebellum on spinobulbo-spinal (SBS) reflex activity evoked by somatic and splanchnic afferentation was studied in chloralose-anaesthetized cats. Unit activity synchronous with the SBS component of the efferent discharge was observed at the level of motoneurons, descending axons of the dorsolateral and ventral funiculi and neurones of the reticular formation (RF). Conditioning stimulation of the AL inhibited this unit activity. Reticular formation units influenced from the AL had direct contact with segmental structures; these results showed that disappearance of the SBS reflex during AL conditioning is associated with the depressant effect of the cerebellar cortex on the reticular formation.     

Bacillus Calmette-Guérin plus interleukin-2 and/or granulocyte/macrophage-colony-stimulating factor enhances immunocompetent cell production of interferon-γ, which inhibits B16F10 melanoma cell growth in vitro

 Although immunotherapy with bacillus Calmette Guérin (BCG) is an established adjuvant treatment for malignant melanoma, the mechanism of its role in this process is unclear. To investigate the possible contribution of tumor-inhibitory cytokines induced by BCG, B16F10 melanoma cell growth in culture was assessed in response to purified cytokines and conditioned media of BCG-stimulated splenocytes. Interferon-γ (IFNγ) was the most potent single agent (IC₅₀≈50 pg/ml). Tumor necrosis factor α was substantially weaker (IC50>10 ng/ml) but provided synergy with IFNγ. None of the other cytokines such as interleukin-2 (IL-2), IL-4, IL-6, IL-10, IL-12, or granulocyte/macrophage-colony-stimulating factor had direct antitumor activity against B16F10 melanoma cells. However, when IL-2 and/or GM-CSF were combined with BCG either by exogenous addition or through endogenous production by novel cytokine-secreting recombinant BCG (rBCG), a substantial increase in INFγ production by splenocytes was observed. Antitumor activity of this conditioned medium directly correlated with IFNγ concentration and was completely blocked by neutralizing antibody to IFNγ. These results suggest that BCG may exert part of its antitumor action on melanoma through the induction of IFNγ, which can be greatly enhanced through the concomitant addition of IL-2 and/or GM-CSF. Furthermore, by utilizing rBCG that secrete these cytokines, it may be possible to potentiate the antitumor effect of BCG directly at the site of BCG inoculation. Received: 29 January 1996 / Accepted: 9 April 1996     

Explaining marriage patterns in a globally representative sample through socio-ecology and population history: A Bayesian phylogenetic analysis using a new supertree

Comparative analyses have sought to explain variation in human marriage patterns, often using predictions derived from sexual selection theory. However, most previous studies have not controlled for non-independence of populations due to shared ancestry. Here we leverage a phylogenetic supertree of human populations that includes all 186 populations in the Standard Cross-Cultural Sample (SCCS), a globally representative and widely-used sample of human populations. This represents the most comprehensive human phylogeny to date, and allows us not only to control for non-independence, but also to quantify the role of population history in explaining behavioral variation, in addition to current socio-ecological conditions. We use multiple imputation to overcome missing data problems and build a comprehensive Bayesian phylogenetic model of marriage patterns with two correlated response variables and eleven minimally collinear predictors capturing various socio-ecological conditions. We show that ignoring phylogeny could lead to both false positives and false negatives, and that the phylogeny explained about twice as much variance as all the predictors combined. Pathogen stress and assault frequency emerged as the predictors most strongly associated with polygyny, which had been considered evidence for female choice of good genes and male intra-sexual competition or male coercion, respectively. Mixed support was found for a polygyny threshold based on variance in male wealth, which is discussed in light of recent theory. Barring caveats, these findings refine our understanding of the evolution of human marriage systems, and highlight the value of combining population history and current socio-ecology in explaining human behavioral variation. Future studies using the SCCS should do so using the present supertree.     

VEGF-targeted cancer therapy strategies: current progress, hurdles and future prospects

Despite setbacks, the clinical development of antiangiogenic agents has accelerated remarkably over the past 3-4 years. Consequently, there are currently three direct inhibitors of the VEGF pathway approved for use in cancer therapy. Other agents that block the VEGF pathway are in advanced stages of clinical development and have shown promising results. With these exciting developments come crucial questions regarding the use of these new molecular-targeted agents, alone or in combination with standard cytotoxic or targeted agents. Importantly, the mechanisms of action of anti-VEGF therapy remain unknown. Here, we discuss several potential mechanisms of action such as tumor vascular normalization, bone marrow-derived cell recruitment blockade and cytostatic effects of anti-VEGF therapy. We review the current progress, the major stumbling blocks and the future directions for anti-cancer therapy using anti-VEGF agents, emphasizing clarification of the underlying molecular mechanisms of action and biomarker identification and validation.     

Rab11-FIP3 is critical for the structural integrity of the endosomal recycling compartment

Rab11-FIP3 is an endosomal recycling compartment (ERC) protein that is implicated in the process of membrane delivery from the ERC to sites of membrane insertion during cell division. Here we report that Rab11-FIP3 is critical for the structural integrity of the ERC during interphase. We demonstrate that knockdown of Rab11-FIP3 and expression of a mutant of Rab11-FIP3 that is Rab11-binding deficient cause loss of all ERC-marker protein staining from the pericentrosomal region of A431 cells. Furthermore, we find that fluorophore-labelled transferrin cannot access the pericentrosomal region of cells in which Rab11-FIP3 function has been perturbed. We find that this Rab11-FIP3 function appears to be specific because expression of the equivalent Rab11-binding deficient mutant of Rab-coupling protein does not perturb ERC morphology. In addition, we find that other organelles such as sorting and late endosomes are unaffected by loss of Rab11-FIP3 function. Finally, we demonstrate the presence of an extensive coiled-coil region between residues 463 and 692 of Rab11-FIP3, which exists as a dimer in solution and is critical to support its function on the ERC. Together, these data indicate that Rab11-FIP3 is necessary for the structural integrity of the pericentrosomal ERC.     

Effects of Kupffer cell blockade on the hepatic expression of metallothionein and heme oxygenase genes in endotoxemic rats with obstructive jaundice

AimsHeme oxygenase (HO) and metallothionein (MT) genes are rapidly upregulated in the liver by pro-inflammatory cytokines and/or endotoxin as protection against cellular stress and inflammation. Gadolinium chloride (GdCl₃)-induced Kupffer cell blockade has beneficial consequences in endotoxemia following bile duct ligation. Herein we further characterized the effects of Kupffer cell inhibition on the activation of the antioxidant defense system (HO and MT gene expressions, and antioxidant enzyme activities) in response to endotoxemia and obstructive jaundice.Main methodsThe isoform-specific expression of MT and HO genes was assessed (RT-PCR) in rat livers following 3-day bile duct ligation, 2-h lipopolysaccharide treatment (1 mg/kg) or their combination, with or without GdCl₃ pretreatment (10 mg/kg, 24 h before endotoxin). Lipid peroxidation, DNA damage and hepatic antioxidant enzyme activities were also assessed.Key findingsAll these challenges induced similar extents of DNA damage, whereas the lipid peroxidation increased only when endotoxemia was combined with biliary obstruction. The MT and HO mRNA levels displayed isoform-specific changes: those of MT-1 and HO-2 did not change appreciably, whereas those of MT-2 and HO-1 increased significantly in 2-h endotoxemia, with or without obstructive jaundice. Among the enzymes reflecting the endogenous protective mechanisms, the catalase and copper/zinc-superoxide dismutase levels decreased, while that of Mn-SOD slightly increased. Interestingly, GdCl₃ alone induced lipid peroxidation, DNA damage and MT-2 expression. In response to GdCl₃, HO-1 induction was significantly lower in each model.SignificanceDespite its moderate hepatocellular toxicity, the ameliorated stress-induced hepatic reactions provided by GdCl₃ may contribute to its protective effects.