Development of a semi-nested PCR using degenerate primers for the generic detection of small ruminant lentivirus proviral DNA.

A PCR assay was developed for the reliable detection of small ruminant lentivirus (SRLV) proviral DNA. The method involved the use of degenerate deoxyinosine-substituted primers and a second semi-nested PCR step that increased the polyvalency and sensitivity of the detection, respectively. Primers were designed from the pol gene conserved motifs of 85 SRLV isolates and were evaluated using different SRLV isolates together with Maedi-Visna virus (MVV) and caprine arthritis-encephalitis virus (CAEV) reference strains.

Antigenic profile of human recombinant PrP: generation and characterization of a versatile polyclonal antiserum.

We describe the quality of a rabbit polyclonal antiserum (Sal1) that was raised against mature human recombinant prion protein (rhuPrP). Epitope mapping demonstrated that the Sal1 antiserum recognized six to eight linear antigenic sites, depending on the animal species. The versatility of the antiserum was evident from the range of animal species and immunochemical techniques where it could be applied successfully.

A short purification process for quantitative isolation of PrPSc from naturally occurring and experimental transmissible spongiform encephalopathies.

Background: Transmissible spongiform encephalopathies (TSEs) are neurodegenerative diseases affecting both humans and animals. They are associated with post-translational conversion of the normal cellular prion protein (PrPC) into a heat- and protease-resistant abnormal isoform (PrPSc). Detection of PrPSc in individuals is widely utilized for the diagnosis of prion diseases.

Increased incidence of sporadic Creutzfeldt-Jakob disease on the island of Crete associated with a high rate of PRNP 129-methionine homozygosity in the local population.

Since the spring of 1997, when the Neurology Department of the University Hospital of Crete admitted its first patient, nine cases (eight neuropathologically confirmed and one probable) of sporadic Creutzfeldt-Jakob disease (sCJD) have been recorded. This represents an annual incidence five-fold higher than expected based on the island's population (0.54 million).

Marked increase of neuronal prion protein immunoreactivity in Alzheimer's disease and human prion diseases.

In neurodegenerative disorders including Alzheimer's disease (AD), free radical damage to lipids, carbohydrates, proteins and DNA has been demonstrated to play a key pathogenetic role. In vitro studies have suggested a function of the cellular prion protein (PrPc) in the defense against oxidative stress. Therefore, we investigated the distribution of PrPc immunoreactivity in hippocampus (sectors CA4-CA1), subiculum (Sub), entorhinal (EC), and temporal cortex (TC) in sections from AD, human transmissible spongiform encephalopathy (TSE) and control brains.

Characterization of the murine BSE infectious agent.

Bovine spongiform encephalopathy (BSE) is a prion-associated disease where the infectious agent is thought to be a host-encoded protein with a protease-resistant conformation (PrP(Sc)). Here, data are presented on the solubilization of purified murine BSE material, using guanidine-HCl as a denaturing agent. This treatment led to loss of infectivity, which was partially recovered on renaturation after dialysis to remove the chaotropic agent.

Glycosaminoglycan analysis in brain stems from animals infected with the bovine spongiform encephalopathy agent.

Increasing evidence suggests that the pathological alterations observed in brains affected by neurodegenerative disorders such as Creutzfeldt-Jakob disease and Alzheimer's disease also involve changes in glycosaminoglycans (GAGs). In the present study, we have isolated, purified, and characterized total GAGs from brain stems of healthy cows or those infected with the bovine spongiform encephalopathy (BSE) agent and we report on the differences between the two groups. Purification of the GAGs was achieved by gel filtration after homogenization, delipidation, and sequential treatment with pronase, DNase, and alkali borohydride.

Expression of the prion protein in the rat forebrain--an immunohistochemical study.

The cellular prion protein (PrP(C)) is crucial for the development of transmissible spongiform encephalopathies (TSEs), where the pathogenic scrapie isoform (PrP(Sc)) of the same protein, is considered to be the principal or sole infectious agent. Here, we report findings on PrP(C) expression in the rat forebrain, using immunohistochemical techniques on free floating sections of 60 microm thickness. Along with neurons and astrocytes in the gray matter, PrP(c) was detected for the first time in glial cells of the white matter and in cells of circumventricular organs.

Preliminary findings on the expression of thymosin beta-10 in human breast cancer.

Paraffin sections from 30 human breast tissue specimens were stained with a specific antibody for thymosin beta-10, ATB10(38-43). The results showed that thymosin beta-10 was detected mainly in the malignant tissue, particularly in the cancerous cells, whereas the normal cell population around the lesions showed very weak staining. Also, the intensity of staining in the cancerous cells was proportionally increased with the increasing grade of the lesions.