Influence of food restriction during gestation on craniofacial growth of the weanling rat

Holtzman rats were subjected to food restriction during gestation or lactation, or both periods (overall stress). At weaning, male pup skulls were measured and female brains and cranial masticatory muscles were weighed and a neuromuscular index was calculated. It was found that gestational protein-calorie malnutrition (PCM) without suckling restoration accounted for about 30% of the growth delay observed under overall stress. That effect disappeared after a normal suckling restoration. Under the same conditions of maternal food restriction in both periods, growth delay in the offspring was greater during lactation than gestation. As in lactation, craniofacial changes during gestational restriction were due to an adjustive response of bone growth to PCM. This response seemed to accrue from an altered relationship between the growth of the brain-less sensitive and highly restorable-and the growth of the masticatory muscles-more sensitive and less restorable. Some degree of delay in orthocephalization would be the skeletal outcome of such adjustive neuromuscular response.     

A novel robust heat-inducible promoter for heterologous gene expression in Tetrahymena thermophila

An increasing amount of data has revealed the importance of inducible promoters in ciliate research and in ciliate-related industries. However, knowledge about these promoters and related genes is relatively sparse. Here we report a novel inducible promoter from a Tetrahymena cytoplasmic Hsp70 gene member, HSP70-2. The reported promoter was able to induce the endogenous gene up to ～9000-fold after a short heat shock treatment and this remarkable feature has been retained when a relatively short region of the promoter was introduced into a reporter construct followed by transformation. During the recovery period following a short heat shock, both the mRNA and protein levels of the reporter gene were maintained high up to two hours. A constant heat shock treatment to the transformed cells led to a stabilization of the reporter mRNA up to at least six hours and the reporter protein continued to accumulate up to around three hours. The promoter strength appears to be similar to that of the cadmium-induced metallothionein gene (MTT1) promoter. Therefore, the HSP70-2 promoter represents an attractive alternative for the over-expression of proteins in Tetrahymena, and the promoter-reporter gene construct used in this study is an ideal tool to help in understanding the regulation mechanisms of heat shock genes in ciliates.     

Orthocephalization in the postweaning squirrel monkey

Twenty male squirrel monkeys (Saimiri sciureus boliviensis) raised in captivity were allotted to one of the following groups: weanling control (C6) sampled at 6 months of age; young control (C24) fed ad libitum on a control diet and killed at 24 months of age; and malnourished (M24) fed ad libitum on a low-protein diet and sampled at 24 months of age. Cranial points and the lateral semicircular canals were marked. On each skull, a strict lateral teleradiograph was taken, and the lengths of the midsagittal chords and their angles with respect to the vestibular line were measured. Age changed the lengths in about 70% of the chords and more than 50% of the angles. Malnutrition arrested about 50% of the lengths, but the angles were practically not affected. It is concluded that the postweaning Saimiri sciureus undergoes orthocephalization according to a general pattern already observed in rodents and suggested for pongids. Postweaning malnutrition affected growth in size but not shape changes related to the orthocephalization of the Saimiri skull. © 1996 Wiley-Liss, Inc.     

Repression of the RcsC-YojN-RcsB phosphorelay by the IgaA protein is a requisite for Salmonella virulence

Bacterial pathogenesis relies on regulators that activate virulence genes. Some of them act, in addition, as repressors of specific genes. Intracellular-growth-attenuator-A (IgaA) is a Salmonella enterica membrane protein that prevents overactivation of the RcsC-YojN-RcsB regulatory system. This negative control is critical for growth because disruption of the igaA gene is only possible in rcsC, yojN or rcsB strains. In this work, we examined the contribution of this regulatory circuit to virulence. Viable igaA point mutant alleles were isolated and characterized. These alleles encode IgaA variants leading to different levels of activation of the RcsC-YojN-RcsB system. IgaA-mediated repression of the RcsB-YojN-RcsC system occurred at the post-translational level, as shown by chromosomal epitope tagging of the rcsC, yojN and rcsB genes. The activity of the RcsC-YojN-RcsB system, monitored with the product of a tagged gmd-3xFLAG gene (positively regulated by RcsC-YojN-RcsB), was totally abolished by wild-type bacteria in mouse target organs. Such tight repression occurred only in vivo and was mediated by IgaA. Shutdown of the RcsC-YojN-RcsB system is a requisite for Salmonella virulence since all igaA point mutant strains were highly attenuated. The degree of attenuation correlated to that of the activation status of RcsC-YojN-RcsB. In some cases, the attenuation recorded was unprecedented, with competitive index (CI) values as low as 10(-6). Strikingly, IgaA is a protein absolutely dispensable for virulence in mutant strains having a non-functional RcsC-YojN-RcsB system. To our knowledge, IgaA exemplifies the first protein that contributes to virulence by exclusively acting as a negative regulator upon host colonization.     

Mechanism of inhibition of wt-dihydrofolate reductase from E. coli by tea epigallocatechin-gallate

Dihydrofolate reductase (DHFR) is a ubiquitous enzyme involved in major biological process, including DNA synthesis and cancer inhibition, and its modulation is the object of extensive structural, kinetic, and pharmacological studies. In particular, earlier studies showed that green tea catechins are powerful inhibitors of bovine liver and chicken liver DHFR. In this article, we report the results of inhibition kinetics for the enzyme from another source (DHFR from E. coli) exerted by (-)-epigallocatechingallate (EGCG). Using different analytical techniques, we reported that EGCG acts as a bisubstrate inhibitor on the bacterial DHFR. Moreover, the combined approach of biosensor, kinetic, and molecular modelling analysis disclosed the ability of EGCG to bind to the enzyme both on substrate (DHF) and cofactor (NADPH) site. Collectively, our data have confirmed the selectivity of antifolate compounds with respect to the different source of enzyme (bacterial or mammalian DHFR) and the possible role of tea catechins as chemopreventive agents.     

Genetic heterogeneity of variable number tandem repeats in thymidylate synthase gene in colorectal cancer patients

PURPOSE: To analyze the genetic variability in a variable number of tandem repeats (VNTR) in the thymidylate synthase (TS) enhancer promoter region and assess the influence of functional alterations in mismatch repair genes by analyzing constitutional and tumoral DNA from patients with colorectal adenocarcinoma with a high microsatellite instability (MSI-H) or microsatellite stability (MSS) status. PATIENTS AND METHODS: Patients who underwent surgery for colorectal adenocarcinoma were selected from the colorectal database of our institute and, on the basis of MSI status, assigned to a study group and a control group: group A, MSI-H; group B, MSS. Microsatellite status was investigated using the Bethesda recommended panel (BAT-26, BAT-25, D2S123, D5S346, D17S250). In MSI-H patients an additional analysis was made of the microsatellite loci D18S61 and D18S58, both mapping in the region containing the TS gene (18p11.2-11.32). Based on the number of altered microsatellites (> or = 2, 1, or 0), tumors were considered as having high (MSI-H) or low (MSI-L) instability or microsatellite stability (MSS), respectively. Genotyping for thymidylate synthase promoter polymorphism was carried out on constitutional and tumor DNA of each patient by PCR amplification of the polymorphic region. RESULTS: MSI-H was found in 55 patients (group A) and MSS in 50 patients (group B). In none of the MSI-H patients was microsatellite instability found in the additional D18S61 and D18S58 loci. In five group A and ten group B cases the analysis was not performed because constitutional DNA and/or tumoral DNA were not amplifiable. Homozygotes for the triple repeat variant (3R/3R) displayed only the large PCR product, homozygotes for the double repeat variant (2R/2R) displayed only the smaller PCR product, while heterozygotes (2R/3R) displayed both the larger and smaller PCR products. In 3/50 (6%) group A patients and 5/40 (12%) group B patients repeat variations were found in tumoral DNA. CONCLUSION: Our findings demonstrate that there is genetic homogeneity between constitutional and tumoral DNA but do not support the hypothesis that mismatch repair genes are involved in VNTR recombinant events in TS gene variability.