Localization of Apaf1 gene expression in the early development of the mouse by means of in situ reverse transcriptase-polymerase chain reaction.

Apoptosis is an essential ubiquitous process that controls the duration of the life span of cells, thus playing a crucial role in morphogenetic, histogenetic, and phylogenetic developmental processes. Apaf1 (apoptosis protease activating factor 1) is one of the central mediators of the intrinsic apoptotic pathway and a part of the apoptosome, which activates procaspase-3 and promotes cell death. Gene knockout of Apaf1 in mice leads to late embryonic lethality with malformations such as the persistence of interdigital webs and hyperplasia of brain and retina. Therefore, Apaf1 is generally believed to play a crucial role in developmental apoptosis and have a widespread expression. However, its pattern of expression in early development remains unknown. To specify whether Apaf1 indeed plays this key role, we investigated the pattern of gene expression for Apaf1 in mouse embryos on day 7, 9, and 12 of development. Our results show, that gene expression for Apaf1 first occurs within the embryo between day 7 and 9 of development, becoming more widespread toward day 12 and then includes structures, such as yolk sac, mesenchyme, cartilage, heart anlage, otic vesicle, peridermis, and anlagen of the spinal ganglia and vertebral bodies. Our results also show that gene expression for Apaf1 is not ubiquitous in early mouse development. This finding indicates that cell death processes are independent of or less dependent on Apaf1 during this time. Of interest, an active gene expression for Apaf1 is also present in organ anlagen such as heart or intestine, in which no obvious phenotype is seen after Apaf1 deletion. This finding suggests a possible role for Apaf1 in such anlagen as a putative alternative compensatory pathway, which could be switched on in the case of defects in the mediators that are normally involved in such organs.     

Detergent fractionation with subsequent subtractive suppression hybridization as a tool for identifying genes coding for plasma membrane proteins

Abstract:  The identification of tumor-specific proteins located at the plasma membrane is hampered by numerous methodological pitfalls many of which are associated with the post-translational modification of such proteins. Here, we present a new combination of detergent fractionation of cells and of subtractive suppression hybridization (SSH) to gain overexpressed genes coding for membrane-associated or secreted proteins. Fractionation of subcellular components by digitonin allowed sequestering mRNA of the rough Endoplasmatic reticulum and thereby increasing the percentage of sequences coding for membrane-bound proteins. Fractionated mRNAs from the cutaneous T-cell lymphoma (CTCL) cell line HuT78 and from normal peripheral blood monocytes were used for SSH leading to the enrichment of sequences overexpressed in the tumor cells. We identified some 21 overexpressed genes, among them are GPR137B, FAM62A, NOMO1, HSP90, SLIT1, IBP2, CLIF, IRAK and ARC. mRNA expression was tested for selected genes in CTCL cell lines, skin specimens and peripheral blood samples from CTCL patients and healthy donors. Several of the detected sequences are clearly related to cancer, but have not yet been associated with CTCL. qPCR confirmed an enrichment of these mRNAs in the rough endoplasmic reticulum fraction. RT-PCR confirmed the expression of these genes in skin specimens and peripheral blood of CTCL patients. Western blotting verified protein expression of HSP90 and IBP2 in HuT78. GPR137B could be detected by immunohistology in HuT78 and in keratinocytes of dysplastic epidermis, but also in sweat glands of healthy skin. In summary, we developed a new technique, which allows identifying overexpressed genes coding preferentially for membrane-associated proteins.     

Making the transition from information systems of the 1970s to medical information systems of the 1990s: The role of the Physician's Workstation

Many hospitals today have implemented widely disparate information systems on mainframe and mini-computer hardware. The advent of network technology in hospitals has made it possible to access information in these systems. Unfortunately, the user interfaces to applications on these systems are unique and difficult to learn, which makes them unsuitable for use by clinical services. In this paper we describe the development of a Physician's Workstation which integrates information from multiple existing information systems and discuss how the workstation makes it possible to move from the departmental systems of the present to the computer-based medical record system of the future.     

The influence of different levels of dietary fat on the incidence and growth of MNU-induced mammary carcinoma in rats

This study describes the influence of isocalorically fed diets (containing different amounts of fat) on tumor incidence and parameters of fat metabolism in female Sprague-Dawley rats. Comparisons are made between rats induced with methylnitrosourea (25 mg/kg body wt) and untreated controls (Group I). The animals received either control diets (3.9% fat by weight, Groups I and II) or fat-enriched diets (10.7%, Group III; 15.6%, Group IV; 21.4%, Group V) over a period of 180 days. At the termination of the experiment, intake of the diet containing 10.7% fat by weight (24% fat per total calories) was associated with the highest tumor incidence. Comparing the different diets, liver lipid concentrations in the individual groups increased with increasing dietary fat, whereas the total lipid in plasma decreased. During the feeding period, total lipid of the liver and plasma, and plasma cholesterol, increased in all groups, but triglycerides of plasma decreased. However, when plasma cholesterol and triglycerides were calculated as a relative amount of total lipid in plasma, cholesterol was found to be significantly decreased in Groups III and IV, and triglycerides were increased in Group III but decreased significantly in Groups I, II, and V at the end of the experiment.     

Aesthetic aspects in reconstructive microsurgery

The reconstruction of a part of the body that has lost or inhibited function has been the most important aspect of reconstructive surgery in the past. Because of the existence of better techniques and the patient's wish for social reintegration, the plastic surgeon today is forced to consider the aesthetic results of his work more than in the past. Sometimes microsurgical procedures in reconstructive surgery are the only chance for the patient to be healed or palliated. Experience with more than 350 free tissue transfers gave us the opportunity to estimate the value of a donor flap with respect to the requirements of the receiving site. To satisfy the high aesthetic demands in these cases, it was necessary for the right place and shape the flap to fit as closely as possible, even if further corrections were necessary to achieve a satisfactory result. The transfer of latissimus dorsi and radial forearm flaps to the face and lower leg is discussed in cases of tumorus diseases and severe injuries of young and old patients.Presented to the IXth Congress of the International Society of Aesthetic Plastic Surgery, New York, October 13, 1987     

Tropical spastic paraparesis-like illness occurring in a patient dually infected with HIV-1 and HTLV-II

We describe a 34-year-old man from southern Florida with a history of intravenous drug use, dually infected with human immunodeficiency virus type 1 (HIV-1) and human T-lymphotropic virus type II (HTLV-II), who developed a myelopathy clinically indistinguishable from HTLV-I-associated myelopathy/tropical spastic paraparesis (HAM/TSP). This myelopathy was characterized by spastic lower extremity weakness, distal paresthesias, sensory loss with a discrete thoracic level to pinprick, back pain, impotence, and sphincter disturbances. Nerve conduction studies revealed an associated mixed axonal and demyelinative neuropathy. Despite a lack of response to 10 months of zidovudine therapy, the myeloneuropathy improved dramatically 2 years after its onset in the absence of any therapeutic intervention.     

Vaccination against hepatitis B in liver transplant recipients: pilot analysis of cellular immune response shows evidence of HBsAg-specific regulatory T cells.

After liver transplantation for hepatitis-B-related diseases, patients currently receive lifelong treatment with hepatitis B immunoglobulin to prevent endogenous reinfection with hepatitis B virus (HBV). Active immunization with hepatitis B vaccine would be a preferable alternative; however, most attempts to immunize these patients with standard vaccine have failed. A recent study with a new adjuvanted hepatitis B vaccine was exceptionally successful, leading to a high-titered long-lasting antibody response in 80% of all vaccinees. To identify the immunological mechanisms behind these unexpected results, the successfully vaccinated participants were tested for hepatitis B surface antigen (HBsAg)-specific T and B cells, and their cellular responses to revaccination with conventional vaccine were studied. HBsAg-specific CD4(+) T lymphocytes could be detected in 13 of 16 patients after immunization with the new vaccine. Unexpectedly, these T cells produced almost exclusively interleukin (IL)-10 and had a CD4(+)/CD25(+) phenotype. They were functionally active, suppressing cytokine secretion in HBsAg-specific (Th1) cells, thus representing antigen-specific regulatory T cells (T(Reg)). Following a booster dose with conventional vaccine 22-31 months after completion of the initial vaccination series, the T-cell pattern in the revaccinated individuals changed substantially: 7 days after revaccination 9 of 11 individuals showed a switch to a Th1-type immune response with HBsAg-specific T cells secreting IL-2, interferon gamma and tumor necrosis factor alpha as observed in healthy controls. Four weeks after the booster, 4 patients still showed a Th1-type cytokine pattern, whereas in 5 patients only IL-10-secreting cells were detectable. After 1 year, in 3 of 4 revaccinated individuals only IL-10-secreting cells could be found, whereas the specific T cells of the fourth patient still showed a Th1-type of response. HBsAg-specific T(Reg) cells could be demonstrated in HBV-positive liver transplant recipients successfully immunized with a new adjuvanted vaccine. Revaccination led to immediate disappearance of the these cells and the appearance of HBsAg-specific T cells with a Th1-type cytokine profile, which in most cases were replaced by the IL-10-secreting regulatory cells during the following months. The specific induction of T(Reg) cells could contribute to the poor response of liver transplant recipients to conventional vaccine. In conclusion,, for successful vaccination of these patients, a vaccine with a strong inhibitory effect on T(Reg) cells would be desirable.     

Generation of cell diversity and segmental pattern in the embryonic central nervous system of Drosophila.

Development of the central nervous system (CNS) involves the transformation of a two-dimensional epithelial sheet of uniform ectodermal cells, the neuroectoderm, into a highly complex three-dimensional structure consisting of a huge variety of different neural cell types. Characteristic numbers of each cell type become arranged in reproducible spatial patterns, which is a prerequisite for the establishment of specific functional contacts. The fruitfly Drosophila is a suitable model to approach the mechanisms controlling the generation of cell diversity and pattern in the developing CNS, as it allows linking of gene function to individually identifiable cells. This review addresses aspects of the formation and specification of neural stem cells (neuroblasts) in Drosophila in the light of recent studies on their segmental diversification.