In situ PCR protocols.

This chapter provides detailed methods and material descriptions of in situ polymerase chain reaction protocols. It describes all the essential components of the technique, various protocols suitable for different kinds of tissues and cell preparations, and also gives various instructions for troubleshooting. We have declined to devote detailed discussions on in situ hybridization because there are several excellent articles and books available on this subject.

Latest developments in in situ PCR.

Since the first publication on the method of in situ polymerase chain reaction (PCR), several thousand research papers have appeared in peer-reviewed journals describing various findings based solely on the application of this method or combined with other more robust methods, including solution-based PCR, immunohistochemistry, Southern blot, etc. A few years after the advent of PCR, several investigators developed in situ PCR methods that differed considerably from each other with regard to tissue preparations, fixation, mounting of slides, reverse transcription technique, primer design, target selection, size, and amplicon size, and thermocycler designs and the use, among many other fine details. This chapter describes the detail procedures that are used in the author's laboratory. It also discusses the variations and modification that can be used for the specific needs of an investigator. This protocol should serve as a primer for the investigators, and each researcher must use his or her variation according to their needs.

Localization of human herpesvirus type 8 in human sperms by in situ PCR.

OBJECTIVES: Defining the mechanism of infection with human herpesvirus-8 (HHV-8) or Kaposi's sarcoma associated herpesvirus (KSHV) is an important clinical issue. HHV-8 has been linked to Kaposi's sarcoma (KS) development in HIV-1-infected individuals, and KS develops in 40% of those infected with both viruses. A series of epidemiological data suggest that sexual transmission is one of the routes of transmission for HHV-8. In our studies, we sought to assess the cellular reservoirs of HHV-8 DNA in the semen of HIV-1-infected men and the potential role of HHV-8 infected spermatozoa in horizontal transmission. DESIGN AND METHODS: A nested polymerase chain reaction (PCR), in situ PCR (ISPCR) and a sodium iodide (NaI) DNA isolation technique that extracts both nuclear and episomal DNA were utilized to amplify specific genes in vitro and within intact cells to evaluate the types of seminal cells infected with HHV-8 in HIV-1-infected and uninfected men. RESULTS: HHV-8 was present in the spermatozoa and mononuclear cells of the semen in 64 of 73 (88%) HIV-1 infected individuals. Both the sperms as well as the mononuclear cells of the semen specimens of HIV-1 infected men were found to be infected with HHV-8. Multiplex ISPCR revealed that a significantly higher percentage of semen cells were infected with HHV-8 than HIV-1 (p>0.001). Rare (less than one in a 100,000) sperm cells were co-infected with both viruses. A co-culture of HHV-8 infected sperm with uninfected 293 or Sup-T1 cell lines resulted in an abortive infection of these cells with HHV-8. DNA isolation by NaI yielded 73% of the positive sperm, whereas the standard phenol/chloroform method resulted in significantly lower positives (45%) from the same specimens. CONCLUSIONS: Design and methods: Our data strongly suggest a potential sexual/horizontal route of transmission of HHV-8, via the HHV-8 infected sperm and other semen cells, where a large percentage of HIV-1 infected men's sperm and other semen cells are infected with HHV-8. Co-culture studies have further supported the observations that HHV-8 in the sperm cells is infectious and capable of transmission of the virus to uninfected cells.

Role of zinc and zinc transporters in the molecular pathogenesis of diabetes mellitus.

Diabetes is one of the most common chronic diseases in the United States. An estimated 18.2 million people in the US (6.3%) have diabetes; among them 2.8 million are African Americans (AAs). On average, AAs are twice as likely to have diabetes as European Americans (EAs) of similar age. AAs disproportionately suffer from various diseases in the US. Many of these diseases include hypertension, cardiovascular disease (CVD), diabetes mellitus (DM-beta predominantly Type II), and cancers of the prostate and pancreas. A number of risk factors such as smoking, a high fat diet, little physical activity, stress, and meager access to health care have been the subject of numerous investigations. However, the factor of the interaction between genetics and the environment has received very little attention in the scientific community. Of note, the content of zinc in pancreatic beta gells is among the highest in the body; however, very little is known about the uptake and storage of zinc inside these cells. We hypothesize that one of the major reason AAs disproportionally suffer from DM (as well as some other illnesses like prostate cancer, CVD and hypertension) is due to their inherent inability to transport appropriate amount of zinc in the crucial cell types that require relatively higher amount of zinc than the other cell types. In this article, we will explore in detail the possible genetic and environmental link between human zinc transporters (hZIPs) and their differential expressions in the islet beta cells from AAs as compared to other racial groups, particularly EAs, in both normal healthy individuals and diabetic patients. We hypothesize that the hZIPs play an important role in the development of diabetes, and the main reason AAs disproportionately suffer from DM (as well as other illnesses like prostate and pancreatic cancers, hypertension, and CVD) as compared to EAs may be due the low degree of expressions of the critical zinc transporters in the beta cells. Understanding the molecular events in the pathogenesis of DM with regards to regulation of zinc uptake would be critical to the evaluation of the natural history of diabetes in humans and especially in various racial groups. If a direct link between zinc transport and diabetes can be established, then a special nutritional formula, medication or other intervention might be especially designed to test the ability to decrease the incidence of this disease in DM susceptible groups, particularly in AAs.