Distinctly different gene structure of KLK4/KLK-L1/prostase/ARM1 compared with other members of the kallikrein family: intracellular localization, alternative cDNA forms, and Regulation by multiple hormones

The tissue kallikreins (KLKs) form a family of serine proteases that are involved in processing of polypeptide precursors and have important roles in a variety of physiologic and pathological processes. Common features of all tissue kallikrein genes identified to date in various species include a similar genomic organization of five exons, a conserved triad of amino acids for serine protease catalytic activity, and a signal peptide sequence encoded in the first exon. Here, we show that KLK4/KLK-L1/prostase/ARM1 (hereafter called KLK4) is the first significantly divergent member of the kallikrein family. The exon predicted to code for a signal peptide is absent in KLK4, which is likely to affect the function of the encoded protein. Green fluorescent protein (GFP)-tagged KLK4 has a distinct perinuclear localization, suggesting that its primary function is inside the cell, in contrast to the other tissue kallikreins characterized so far that have major extracellular functions. There are at least two differentially spliced, truncated variants of KLK4 that are either exclusively or predominantly localized to the nucleus when labeled with GFP. Furthermore, KLK4 expression is regulated by multiple hormones in prostate cancer cells and is deregulated in the androgen-independent phase of prostate cancer. These findings demonstrate that KLK4 is a unique member of the kallikrein family that may have a role in the progression of prostate cancer.     

Type II estrogen binding site agonist: synthesis and biological evaluation of the enantiomers of methyl-para-hydroxyphenyllactate (MeHPLA)

A high-affinity ligand for the type II estrogen binding site (EBS) was identified. Methyl para-hydroxyphenyllactate (MeHPLA) was observed to suppress the cellular proliferation of estrogen-sensitive MCF-7 breast cancer cells in vitro and to suppress the growth of rat uteri in vivo. The high affinity of MeHPLA for the type II EBS suggests that this interaction is responsible for the observed suppression of cell growth. In this study, the enantiomers of MeHPLA were synthesized and separated by three methods and evaluated for biological activity. When the methods were compared, it was found that the method using an optically pure amine to form the diastereomers of the enantiomers gave the superior yield. Binding studies for the enantiomers to the type II EBS showed that the S-MeHPLA had a higher affinity for the binding site. However, higher binding affinity did not translate into superior cell growth suppression. Both enantiomers suppressed cell growth equally.     

<Emphasis Type="Italic">Burkholderia</Emphasis> Hep_Hag autotransporter (BuHA) proteins elicit a strong antibody response during experimental glanders but not human melioidosis

Background  

The bacterial biothreat agents Burkholderia mallei and Burkholderia pseudomallei are the cause of glanders and melioidosis, respectively. Genomic and epidemiological studies have shown that B. mallei is a recently emerged, host restricted clone of B. pseudomallei.     

Bone formation rather than inflammation reflects Ankylosing Spondylitis activity on PET-CT: a pilot study

Introduction

Positron Emission Tomography - Computer Tomography (PET-CT) is an interesting imaging technique to visualize Ankylosing Spondylitis (AS) activity using specific PET tracers. Previous studies have shown that the PET tracers [¹⁸F]FDG and [¹¹C](R)PK11195 can target inflammation (synovitis) in rheumatoid arthritis (RA) and may therefore be useful in AS. Another interesting tracer for AS is [¹⁸F]Fluoride, which targets bone formation. In a pilot setting, the potential of PET-CT in imaging AS activity was tested using different tracers, with Magnetic Resonance Imaging (MRI) and conventional radiographs as reference.

Methods

In a stepwise approach different PET tracers were investigated. First, whole body [¹⁸F]FDG and [¹¹C](R)PK11195 PET-CT scans were obtained of ten AS patients fulfilling the modified New York criteria. According to the BASDAI five of these patients had low and five had high disease activity. Secondly, an extra PET-CT scan using [¹⁸F]Fluoride was made of two additional AS patients with high disease activity. MRI scans of the total spine and sacroiliac joints were performed, and conventional radiographs of the total spine and sacroiliac joints were available for all patients. Scans and radiographs were visually scored by two observers blinded for clinical data.

Results

No increased [¹⁸F]FDG and [¹¹C](R)PK11195 uptake was noticed on PET-CT scans of the first 10 patients. In contrast, MRI demonstrated a total of five bone edema lesions in three out of 10 patients. In the two additional AS patients scanned with [¹⁸F]Fluoride PET-CT, [¹⁸F]Fluoride depicted 17 regions with increased uptake in both vertebral column and sacroiliac joints. In contrast, [¹⁸F]FDG depicted only three lesions, with an uptake of five times lower compared to [¹⁸F]Fluoride, and again no [¹¹C](R)PK11195 positive lesions were found. In these two patients, MRI detected nine lesions and six out of nine matched with the anatomical position of [¹⁸F]Fluoride uptake. Conventional radiographs showed structural bony changes in 11 out of 17 [¹⁸F]Fluoride PET positive lesions.

Conclusions

Our PET-CT data suggest that AS activity is reflected by bone activity (formation) rather than inflammation. The results also show the potential value of PET-CT for imaging AS activity using the bone tracer [¹⁸F]Fluoride. In contrast to active RA, inflammation tracers [¹⁸F]FDG and [¹¹C](R)PK11195 appeared to be less useful for AS imaging.     

Enzymatic histochemistry of mouse kidney in plastic

Two-micrometer sections of methacrylate-embedded kidney were used to investigate the enzymatic activities of mouse kidney where the proximal tubule and Bowman's capsule from the same corpuscle were viewed in the same section. Alkaline phosphatase, acid phosphatase, 5'-nucleotidase, gamma-glutamyl transpeptidase, N-acetyl-beta-glucosaminidase, leucine aminopeptidase, alpha-naphthyl butyrate esterase, and adenosine triphosphatase activities were observed in the proximal tubule, but only 5'-nucleotidase, alpha-naphthyl butyrate esterase, and alkaline phosphatase were observed in the squamous portion of the parietal epithelium of Bowman's capsule. The use of methacrylate-embedded tissue allowed more precise localization of enzymatic activity than is possible with most frozen sections. This may provide interesting applications not only for characterization of kidney diseases but also for characterization of other normal and abnormal tissues.     

Mutant KRAS in aberrant crypt foci (ACF): initiation of colorectal cancer?

Since aberrant crypt foci (ACF) were first described in 1987, they have been the subjects of hundreds of papers; however, the debate continues about their role in colorectal tumorigenesis. This review focuses on the many phenotypic, genetic and epigenetic alterations in ACF that support the hypothesis that ACF are putative precursors of colorectal cancer in both humans and experimental animals. Human ACF, both with and without dysplasia, are monoclonal and display evidence of chromosomal instability. Both of these characteristics are shared by colorectal cancers. While most ACF do not have APC mutations, a large proportion has KRAS mutations and methylated SFRP1 and SFRP2 genes. This epigenetic inactivation gives rise to constitutive Wnt signaling in these putative precursors of colorectal cancer.     

Separation of epithelial cells from suspensions of cells from the hamster parotid gland in an isokinetic density gradient of Ficoll in tissue culture medium.

Epithelial cells were separated from suspensions of hamster parotid cells by velocity sedimentation in an isokinetic gradient and by isopycnic sedimentation. Epithelial cells were 48.1 ± 18.0% of the cells in the starting sample suspensions of cells from the disaggregated hamster parotid glands. The purest gradient fractions following velocity sedimentation in a previously described isokinetic gradient contained 98.8 ± 1.8% epithelial cells. The purest fractions obtained from isopycnic sedimentation contained 99.9 ± 0.2% epithelial cells. Purification of parotid epithelial cells by velocity sedimentation in the isokinetic gradient seems preferable to purification using isopycnic centrifugation because a larger proportion of the epithelial cells are obtained in the zone of the gradient which contains highly purified epithelial cells and because velocity sedimentation requires lower centrifugal forces for a shorter period of time.