The Src/Csk regulatory circuit arose early in metazoan evolution.

We have identified a gene encoding a member of the Csk family of non-receptor protein-tyrosine kinases (PTKs) in the early-diverging metazoan Hydra. In situ hybridization analysis of the distribution of RNA from the Hydra Csk gene indicates that it is expressed in most of the epithelial cells of the adult polyp and in gametogenic cells. Comparison of the expression pattern of Hydra Csk with that of STK, the Hydra Src gene orthologue, reveals that the two genes are largely co-expressed. Such co-expression is consistent with a role for Hydra Csk in regulation of STK activity. This possibility was tested directly by coexpressing Hydra Csk with STK in yeast. Co-expression suppressed the growth inhibition seen when STK alone is expressed in yeast. Suppression was dependent on the presence of the putative regulatory tyrosine in the carboxyl-terminal tail of STK. Phosphotyrosine immunoblot analysis confirmed that expression of Csk resulted in suppression of STK kinase activity. Taken together these data indicate that the regulatory circuit involving Src and Csk PTKs was established prior to the divergence of the phylum Cnidaria from the rest of the metazoans.

Response to insulin and the expression pattern of a gene encoding an insulin receptor homologue suggest a role for an insulin-like molecule in regulating growth and patterning in Hydra.

A gene encoding a receptor protein-tyrosine kinase closely related to the vertebrate insulin receptor has been identified in the Cnidarian Hydra vulgaris. The gene is expressed in both epithelial layers of the adult polyp. A particularly high level of expression is seen in the ectoderm of the proximal portions of the tentacles and in a ring of ectodermal cells at the border between the foot basal disk and body column. The expression pattern of the gene in asexual buds is dynamic; expression is high throughout the newly emerging bud but the area of high expression becomes restricted to the apex as the bud lengthens. When the bud begins hypostome and tentacle formation, a high level of expression appears at the bases of the emerging tentacles. Finally, a ring of high expression appears just above the foot of the bud, completing the pattern seen in the adult polyp. The presence of this receptor and its pattern of expression suggested that an endogenous molecule related to insulin plays a role in regulating cell division in the body column and in differentiation of the tentacle and foot cells in Hydra, with the switch between the two being determined by the level of the receptor. Treatment of Hydra polyps with mammalian insulin caused an increase in the number of ectodermal and endodermal cells undergoing DNA synthesis.

Determination of the efficiency of K99-F41 fimbrial antigen vaccine in newborn calves.

Semipurified K99 and F41 fimbrial antigens were used to prepare an oil-emulsified vaccine against bovine enterotoxigenic colibacillosis. Nine Nelore cows about 7 months pregnant were divided into 3 groups (A, B and C) of 3 animals each, which received different doses of vaccine (1,500 HU, 750 HU and 380 HU, respectively) 8 and 2 weeks before delivery, in the neck by the subcutaneous route. As a control (group D), 3 pregnant cows of the same breed were not vaccinated for later challange of their calves. Vaccine efficiency was measured by the serological tests double diffusion and ELISA. Challenge of calves from the vaccinated and from the three control unvaccinated cows was carried out with the virulent Escherichia coli B41 strain (0101, STa+, K99+, F41+). Two of the 3 calves from the unvaccinated cows died within 48 h with acute diarrhea. E. coli B41 was recovered as pure culture from their stools. In contrast, none of the calves born from vaccinated cows presented diarrhea. These data suggest that the antibody transfer to calves through colostrum gave full protection against the challenge. This semipurified fimbrial vaccine against K99-F41-harboring strains is the first oil-emulsified immunogen prepared in Brazil, which was not only efficient, but also had no adverse effects on vaccinated pregnant cows.

Determination of the efficiency of K99-F41 fimbrial antigen vaccine in newborn calves

Semipurified K99 and F41 fimbrial antigens were used to prepare an oil-emulsified vaccine against bovine enterotoxigenic colibacillosis. Nine Nelore cows about 7 months pregnant were divided into 3 groups (A, B and C) of 3 animals each, which received different doses of vaccine (1,500 HU, 750 HU and 380 HU, respectively) 8 and 2 weeks before delivery, in the neck by the subcutaneous route. As a control (group D), 3 pregnant cows of the same breed were not vaccinated for later challange of their calves. Vaccine efficiency was measured by the serological tests double diffusion and ELISA. Challenge of calves from the vaccinated and from the three control unvaccinated cows was carried out with the virulent Escherichia coli B41 strain (0101), STa+, K99+, F41+). Two of the 3 calves from unvaccinated cows died within 48 h with acute diarrhea. E. coli B4 was recovered as pure culture from their stools. In contrast, none of the calves born from vaccinated cows presented diarrhea. These data suggest that the antibody transfer to calves through colostrum gave full protection aginst the challenge. This semipurified fimbrial vaccine against K99-F41-harboring strains is the first oil-emulsified immunogen prepared in Brazil, which was not only efficient, but also had no adverse effects on vaccinated pregnant cows

Evaluation of an oil emulsified vaccine against bovine colibacillosis using semi-purified K99-F41 adhesins

Os antígenos K99-F41 foram extraídos da amostra de Escherichia coli B41 por aquecimento e semi-purificados pela precipitaçäo com sulfato de amônio e tratamento com desoxicolato de sódio (DOC). Os antígenos semi-purificados foram utilizados na produçäo de uma vacina oleosa contra a colibacilose bovina. Foram preparadas vacinas contendo em cada dose 1.500 HU (Unidades Hemaglutinantes), 750 HU e 380 HU. A eficiência da vacina foi avaliada através do ensaio de imunodifusäo dupla, ensaio imunoenzimático (ELISA) e por um desafio, em que a amostra de Escherichia coli virulenta foi inoculada nos bezerros nascidos de vacas vacinadas e näo vacinadas. Observamos que a vacina contendo 750 HU foi a que melhor induziu a produçäo de anticorpos nas vacas vacinadas, e que estes mostratam-se protetores, uma vez que os bezerros nascidos de vacas vacinadas e que mamaram o colostro, nada sofreram no desafio. Näo se verificou nenhum efeito colateral nas vacas vacinadas

A molecular snapshot of the metazoan 'Eve'.

A description of the molecular make-up of the ancestral multicellular animal is emerging from the growing availability of molecular biological and biochemical data gleaned from the study of modern members of ancient groups of animals. We use the distributions of classes of transcription factors, signal transduction systems and other molecular innovations among metazoan phyla to infer some of the characteristics of the first animals.

Expression of Cnox-2, a HOM/HOX gene, is suppressed during head formation in hydra.

Using PCR, six genes of the HOM/HOX class have been identified in hydra. One of them, Cnox-2, has been sequenced and resembles the Deformed gene of Drosophila. As shown previously, the expression pattern of Cnox-2 suggested that it may be involved in axial patterning as it was strongly expressed in the epithelial cells of the body column and foot, but only weakly expressed in the head. In addition, expression of Cnox-2 decreased sharply in body column tissue as it was converted into head tissue, thereby providing further support for a role in hydra pattern formation. To examine this possibility further, additional manipulations were carried out which convert body column tissue into either head or foot tissue. When body column tissue was converted into head tissue by regeneration, transplantation, or during budding, Cnox-2 expression was sharply reduced, indicating that expression of this gene is suppressed during head formation. When body column tissue was converted into foot tissue by regeneration or Li+ treatment, the changes in Cnox-2 expression patterns indicated that Cnox-2 had no direct role in foot formation. Surprisingly, however, the observed changes in Cnox-2 expression patterns indicate that following bisection, both head and foot regeneration processes are initiated at the injured edge of the bisected animal.

Expression of Cnox-2, a HOM/HOX homeobox gene in hydra, is correlated with axial pattern formation.

Cnox-2 is a HOM/HOX homeobox gene that we have identified in the simple metazoan Hydra vulgaris (Cnidaria: Hydrozoa). Cnox-2 is most closely related to anterior members of the Antennapedia gene complex from Drosophila, with the greatest similarity to Deformed. The Cnox-2 protein is expressed in the epithelial cells of adult hydra polyps in a region-specific pattern along the body axis, at a low level in the head and at a high level in the body column and the foot. The expression pattern of Cnox-2 is consistent with a role in axial pattern formation. Alteration of hydra axial patterning by treatment with diacylglycerol (DAG) results in an increase of head activation down the body column and in a coordinate reduction of Cnox-2 expression in epithelial cells in 'head-like' regions. These results suggest that Cnox-2 expression is negatively regulated by a signaling pathway acting through protein kinase C (PKC), and that the varying levels of expression of Cnox-2 along the body axis have the potential to result in differential gene expression which is important for hydra pattern formation.