Lactic acid bacteria isolated from canine faeces.

AIMS: Lactic acid bacteria (LAB) were isolated and sequenced from the faeces of healthy dogs. Five of these strains were selected and further characterized to clarify the potential of these strains as probiotics for canine. METHODS AND RESULTS: LAB were found in 67% (14/21) of the canine faeces samples when plated on Lactobacilli Selective Media without acetic acid. Out of 13 species identified with partial 16S rRNA gene sequencing, Lactobacillus fermentum LAB8, L. mucosae LAB12, L. rhamnosus LAB11, L. salivarius LAB9 and Weissella confusa LAB10 were selected as candidate probiotic strains based on their frequency, quantity in faeces, growth density, acid tolerance and antimicrobial activity. The minimal inhibitory concentration values of these isolates were determined for 14 antibiotics. L. salivarius LAB9, W. confusa LAB10 and L. mucosae LAB12 were viable in pH 2 for 4 h (mLBS), indicating tolerance to acidity and thus the potential to survive in gastrointestinal tract of the canine. The LAB8-LAB12 strains showed antimicrobial activity against Micrococcus luteus A1 NCIMB86166. CONCLUSIONS: Thirteen different LAB species were found from the faecal microbiota of the healthy canines. Five acid tolerant and antimicrobially active LAB strains with the capacity to grow to high densities both aerobically and anaerobically were chosen to serve as candidate probiotics. SIGNIFICANCE AND IMPACT OF THE STUDY: The selected LAB strains are among the first host-specific LAB with antimicrobial activity isolated from canines that could serve as potential probiotics for canine use.

Heat shock protein 90 and the nuclear transport of progesterone receptor.

Steroid receptors exist as large oligomeric complexes in hypotonic cell extracts. In the present work, we studied the nuclear transport of the 2 major components of the oligomeric complex, the receptor itself and the heat shock protein 90 (Hsp90), by using different in vitro transport systems: digitonin permeabilized cells and purified nuclei. We demonstrate that the stabilized oligomeric complex of progesterone receptor (PR) cannot be transported into the nucleus and that unliganded PR salt dissociated from Hsp90 is transported into the nucleus. When nonstabilized PR oligomer was introduced into the nuclear transport system, the complex dissociated and the PR but not the Hsp90 was transported into the nucleus. If PR exists as an oligomeric form after synthesis, as suggested by the experiments with reticulocyte lysate, the present results suggest that the complex is short-lived and is dissociated before or during nuclear transport. Thus, the role of Hsp90 in PR action is likely to reside in the Hsp90-assisted chaperoning process of PR preceding nuclear transport of the receptor.

The C-terminal half of Hsp90 is responsible for its cytoplasmic localization.

With some exceptions, research so far has shown heat shock protein (Hsp) 90 to be a cytoplasmic protein. Here, we studied the sequence determinants which dictate the subcellular localization of Hsp90. By constructing hybrid molecules between a nuclear protein, progesterone receptor (PR), and parts of Hsp90, we demonstrated that the C-terminal but not the N-terminal half of Hsp90 can prevent nuclear translocation of the PR. Studies with an antibody raised against a region which contains the major nuclear localization signal (NLS) of the PR suggest that the inhibition of nuclear localization is not due to steric hindrance of the NLS of the PR by Hsp90 sequences in hybrid molecules. In order to characterize further the cytoplasmic anchoring of Hsp90 we constructed four chimeric molecules between the C-terminal half of Hsp90 and estrogen receptor (ER) with different numbers of nuclear localization protosignals (proto-NLS). When the C-terminal half of Hsp90 was fused with ER containing no or one proto-NLS, the hybrid molecule was located exclusively in the cytoplasm. When the nuclear translocation signal was strengthened by adding two or three protosignals, the hybrid molecule was exclusively nuclear. These results suggest that the C-terminal half of Hsp90 contains a sequence which is responsible for the cytoplasmic localization of the protein. Further deletions of the molecule suggested that the cytoplasmic anchoring signal is located between amino acids 333 and 664.

Localization of glucocorticoid receptor interacting protein 1 in murine tissues using two novel polyclonal antibodies.

OBJECTIVE: Glucocorticoid receptor interacting protein 1 (GRIP1) is a coactivator that binds to the nuclear hormone receptors in a ligand-dependent manner and mediates transcriptional activation of the target genes. The aim of this study was to investigate GRIP1 expression in various murine tissues and whether the protein is nuclear, cytoplasmic, or both. DESIGN: Two novel polyclonal antibodies against amino acids 34-47 and 468-481 of GRIP1 were raised and characterized in order to study the GRIP1 expression with immunohistochemistry. RESULTS: Transient transfection studies with COS cells showed a clearly nuclear staining pattern and also immunohistochemical localization of GRIP1 was mainly nuclear, but cytoplasmic expression was seen as well. GRIP1 was expressed in epithelial cells of the submandibular gland, gastrointestinal tract, pancreas, kidney, uterus, mammary gland, testis, prostate, trachea, lungs and adrenal gland. GRIP1 was also detected in stromal cells of colon, rectum, urinary bladder, vagina, uterus, mammary gland and trachea, and to a lesser extent in esophagus, ureter, urethra, thymus and spleen. Smooth muscle cells of the gastrointestinal and urinary tract, uterus, epididymis, prostrate and bronchioles expressed GRIP1. Blood vessels of many organs, capsule of the kidney and prostate, mesovarium, adipocytes of the mammary gland, pericardium and cartilage of the trachea were also GRIP1-positive. Liver, thyroid gland and striated muscle did not express GRIP1. CONCLUSIONS: GRIP1 was expressed in a wide variety of murine organs, and expression varied between cell types and organs. In addition to mainly nuclear localization of endogenous GRIP1, cytoplasmic expression was seen as well.

Detection of progesterone receptor forms A and B by immunohistochemical analysis.

AIM: The measurement of progesterone receptors (PR) is recommended as part of the clinical management of breast and endometrial cancers, and immunohistochemistry on formalin fixed tissue is now the method of choice. PR is expressed as two isoforms, PRA and PRB, and although both these proteins are expressed in hormone dependent cancers, there is evidence that a large proportion of tumours express a predominance of one isoform. Therefore, it is essential to document the individual detection of PRA and PRB by the presently available anti-PR antibodies. The aim of this study is to investigate the detection of PR isoforms A and B in formalin fixed, paraffin wax embedded cell lines and tissue sections by immunohistochemistry, using a panel of commercial and in house antibodies to human PR. METHODS: PR negative cell lines stably transfected to express only PRA (MCF-7Mll/PRA) or PRB (MDA-MB-231/PRB), and tissue sections of human breast carcinoma and normal endometrium were stained using an immunoperoxidase method. A panel of primary PR specific antibodies was evaluated for ability to detect both PRA and PRB proteins, and for intensity and distribution of positive staining under optimal conditions. RESULTS: Of the 11 antibodies assessed, only four recognised PRA and PRB similarly. Six recognised PRA proteins but were unable to detect PRB expression in the cell lines expressing only PRA or PRB. In tissues expressing high amounts of PRA and PRB, all antibodies tested demonstrated positive PR staining. However, in tissues expressing a predominance of PRB, differential staining patterns were observed, with variations in staining intensity and in the proportion of cells positive for PR. CONCLUSIONS: Most PR specific antibodies tested failed to detect PRB in formalin fixed tissue by immunohistochemical techniques, despite their ability to do so by immunoblot analysis. These observations suggest that there are conformational differences between PRA and PRB that mask epitopes on the PRB protein recognised by most anti-PR antibodies. The selection of antibodies that recognise both PRB and PRA in formalin fixed tissue is essential for the accurate evaluation of PR positivity in clinical specimens.

Vitamin D and prostate cancer.

Our recent epidemiological study (Ahonen et al., Cancer Causes Control 11(2000) (847-852)) suggests that vitamin D deficiency may increase the risk of initiation and progression of prostate cancer. The nested case-control study was based on a 13-year follow-up of about 19000 middle-aged men free of clinically verified prostate cancer. More than one-half of the serum samples had 25OH-vitamin D (25-VD) levels below 50 nmol/l, suggesting VD deficiency. Prostate cancer risk was highest among the group of younger men (40-51 years) with low serum 25-VD, whereas low serum 25-VD appeared not to increase the risk of prostate cancer in older men (>51 years). This suggests that VD has a protective role against prostate cancer only before the andropause, when serum androgen concentrations are higher. The lowest 25-VD concentrations in the younger men were associated with more aggressive prostate cancer. Furthermore, the high 25-VD levels delayed the appearance of clinically verified prostate cancer by 1.8 years. Since these results suggest that vitamin D has a protective role against prostate cancer, we tried to determine whether full spectrum lighting (FSL) during working hours could increase serum 25-VD concentrations. After 1-month exposure, there was no significant increase in the serum 25-VD level, although there was a bias towards slightly increasing values in the test group as opposed to decreasing values in controls. There was no significant change in the skin urocanic acid production. The possibility to use FSL in cancer prevention is discussed. In order to clarify the mechanism of VD action on cell proliferation and differentiation, we performed studies with the rat and human prostates as well prostate cancer cell lines. It is possible that 25-VD may have a direct role in the host anticancer defence activity, but the metabolism of vitamin D in the prostate may also play an important role in its action. We raised antibodies against human 1alpha-hydroxylase and 24-hydroxylase. Our preliminary results suggest that vitamin D is actively metabolised in the prostate. Vitamin D appears to upregulate androgen receptor expression, whereas androgens seem to upregulate vitamin D receptor (VDR). This may at least partially explain the androgen dependence of VD action. VD alone or administered with androgen causes a suppression of epithelial cell proliferation. VD can activate mitogen-activated kinases, erk-1 and erk-2, within minutes and p38 within hours. Also, auto/paracrine regulation might be involved, since keratinocyte growth factor (mRNA and protein) was clearly induced by VD. Based on these studies, a putative model for VD action on cell proliferation and differentiation is presented.

A comparison of the hemodynamic effects of paracervical block and epidural anesthesia for labor analgesia.

BACKGROUND: Both paracervical block (PCB) and epidural analgesia are sometimes associated with hemodynamic effects potentially harmful to the well-being of the fetus. Our study was designed to test the hypothesis that PCB would have a more profound effect on maternal and fetal blood flow than epidural analgesia. METHODS: Forty-four healthy primiparous parturients were randomized to receive either PCB (n=21) or epidural analgesia (n= 23) with 25 or 30 mg of bupivacaine, respectively, for labor analgesia. Maternal blood pressure and fetal heart rate were recorded. Blood flow was measured using a color Doppler device. The blood flow measurements consisted of assessment of the pulsatility indices (PI) of the right maternal femoral artery and the main branch of the uterine artery (placental side), the umbilical artery and the fetal middle cerebral artery. The measurements were performed before administration of analgesia and approximately 15-20 min later after the onset of analgesia. RESULTS: Both methods provided in general good analgesia, but rescue medication was required more often after PCB. Epidural analgesia decreased maternal blood pressure more than PCB and the PI of maternal femoral artery decreased after onset of epidural analgesia, indicating epidural-induced vasodilation. The PI of the uterine artery increased after the onset of PCB, indicating vasoconstriction of this artery. No significant adverse effects or differences in the well-being of the newborn were observed, as indicated by similar Apgar scores and pH-status. CONCLUSION: There were small differences in the effects of PCB and epidural analgesia on uteroplacental circulation as well as on maternal hemodynamics. PCB may have a vasoconstrictive effect on the uterine artery. This and the fact that the parturients required rescue analgesia more frequently after PCB than after epidural block speaks for the feasibility of the latter in obstetrics.

Distribution of progesterone receptor in female mouse tissues.

Two novel antibodies against the mammalian progesterone receptor (PR) were raised and characterized to study the distribution of PR and the effect of estrogen on PR expression in various female murine tissues by immunohistochemistry. There were estrogen-independent constitutive PR expressions in the smooth muscle cells of uterus, uterine blood vessels, urinary bladder, duodenum, and jejunum of ovariectomized mice. Uterine stromal cells, capsular cells of kidney and adrenal gland, and the epithelial cells of submandibular gland expressed PR constitutively. PR expression was detected in some thymic cells and the number of PR-positive thymic cells increased markedly after estrogen treatment. Estrogen induced PR expression in the epithelial cells of uterus, vagina, urethra, and skin and the stromal cells of vagina, urethra, and pancreatic ducts, as well as the smooth muscle cells of some blood vessels. These results suggest cell-specific progesterone actions in the urinary tract, skin, and gastrointestinal organs, on the immune functions, and on the regulation of local blood flow.

Mice with an aspartylglucosaminuria mutation similar to humans replicate the pathophysiology in patients.

Aspartyglucosaminuria (AGU) is a lysosomal storage disease with autosomal recessive inheritance that is caused by deficient activity of aspartylglucosaminidase (AGA), a lysosomal enzyme belonging to the newly described enzyme family of N-terminal hydrolases. An AGU mouse model was generated by targeted disruption of the AGA gene designed to mimic closely one human disease mutation. These homozygous mutant mice have no detectable AGA activity and excrete aspartylglucosamine in their urine. Analogously to the human disease, the affected homozygous animals showed storage in lysosomes in all analyzed tissues, including the brain, liver, kidney and skin, and lysosomal storage was already detected in fetuses at 19 days gestation. Electron microscopic studies of brain tissue samples demonstrated lysosomal storage vacuoles in the neurons and glia of the neocortical and cortical regions. Magnetic resonance images (MRI) facilitating monitoring of the brains of living animals indicated cerebral atrophy and hypointensity of the deep gray matter structures of brain-findings similar to those observed in human patients. AGU mice are fertile, and up to 11 months of age their movement and behavior do not differ from their age-matched littermates. However, in the Morris water maze test, a slow worsening of performance could be seen with age. The phenotype mimics well AGU in humans, the patients characteristically showing only slowly progressive mental retardation and relatively mild skeletal abnormalities.

Molecular cloning, chromosomal assignment, and expression of the mouse aspartylglucosaminidase gene.

Aspartylglucosaminidase (AGA) is a lysosomal enzyme, the deficiency of which leads to human lysosomal storage disease aspartylglucosaminuria. Here, we describe isolation, chromosomal location, genomic structure, and tissue-specific expression of the mouse Aga gene as well as the intracellular processing of the mouse Aga polypeptide and compare these characteristics to human AGA. The mouse Aga gene was localized to the central area of the B region of chromosome 8, which represents the synteny group in the human chromosome 4q telomeric region where the human AGA gene is located. The mouse gene spans an 11-kb genomic region and contains nine exons and eight introns, which is analogous to the human gene. Furthermore, the exon-intron boundaries of the mouse and human genes are identically positioned. The nucleotide sequence identity of the cDNA and deduced amino acid sequence identity of the protein are 84.4 and 82.4%, respectively. However, the mouse Aga cDNA contains untranslated regions that are shorter than those in the human cDNA, and only one 1.2-kb mRNA transcript is produced in mouse versus two transcripts in human. Expression of the mouse Aga cDNA in COS-1 cells showed that the mouse Aga polypeptide was processed similarly to the human counterpart.

Deletion of the C-terminal end of aspartylglucosaminidase resulting in a lysosomal accumulation disease: evidence for a unique genomic rearrangement.

Aspartylglucosaminuria (AGU) is an inborn error of glycoprotein catabolism and represents the only known human deficiency of an amidase, aspartylglucosaminidase (AGA, EC 3.5.1.26). We report here a detailed characterization of a unique 2 kb deletion of the AGA gene in a North American AGU patient. To facilitate the characterization of the deletion, genomic lamda clones spanning the 3' flanking region of human AGA were isolated and sequenced. The breakpoint of the deletion was determined from the patient's DNA by sequencing the genomic region containing the novel junction. The rearrangement involved a nonhomologous recombination with only 2 bp of homology at the deletion breakpoint. The deletion's 5' breakpoint was located in the last intron of AGA, thus abolishing the normal C-terminal exon. This is in contrast to our previous findings indicating that the deletion in the AGA gene would contain only the complete 3' untranslated region and leave the coding region intact (1). The unique feature of this deletion is a triplication of 19 thymidine nucleotides of an inverted Alu repeat, which is located at the deletion 3' breakpoint. The analysis of the patient's AGA cDNA revealed an open reading frame containing a novel C-terminal exon, coding for a 64 amino acid sequence, which has no homology to the normal exon 9 of AGA. This new exon has a functional splice acceptor site at its 5' end, a stop codon, and a polyadenylation signal at the 3' end. Expression of the mutant AGA cDNA in COS cells showed that mutant mRNA is synthesized in equal amounts compared with normal.(ABSTRACT TRUNCATED AT 250 WORDS)

Quantitative determination of rare mRNA species by PCR and solid-phase minisequencing.

We present a new method for quantification of mRNA, in which the limitations of the current quantitative PCR methods can be overcome. A known amount of a synthetic RNA standard differing from the mRNA to be quantified by a single nucleotide is reverse-transcribed and amplified together with the mRNA template using a biotinylated primer. The biotinylated PCR product is immobilized on a streptavidin-coated solid support and denatured. The ratio between the two amplified sequences is determined by separate "mini-sequencing" reactions, in which a detection step primer annealing immediately adjacent to the site of the variable nucleotide is elongated by a single labeled dNTP complementary to the nucleotide at the variable site. The ratio between the incorporated labels accurately determines the ratio between the two sequences in the original RNA sample. We applied this method to quantify the mRNA of human aspartylglucosaminidase (AGA) in tissues and cultured cells. AGA is a lysosomal enzyme participating in the degradation of glycoproteins. A mutation in the AGA gene abolishes the enzyme activity and leads to aspartylglucosaminuria (AGU), a recessively inherited metabolic disorder. The mRNA quantification revealed that the normal and mutant genes are expressed at similar levels in kidney, liver, and cultured fibroblast, whereas the amount of AGA mRNA in normal placenta and brain is significantly higher than that found in the corresponding samples from AGU patients. The method presented here is generally applicable for PCR-based quantification of rare mRNAs and DNA as well.

Convenient and quantitative determination of the frequency of a mutant allele using solid-phase minisequencing: application to aspartylglucosaminuria in Finland.

Aspartylglucosaminuria (AGU) is a recessively inherited lysosomal disease caused by inadequate aspartylglucosaminidase (AGA) activity. The disease is prevalent in the genetically isolated Finnish population. We have used a new method, solid-phase minisequencing, to determine the frequency of two missense mutations in the AGA gene in this population. In samples from 70% of the Finnish AGU families, we found that the two nucleotide changes were always associated, and they were identified in 98% of the AGU alleles analyzed. Thus, the high prevalence of AGU in the Finnish population is the consequence of a founder effect of one ancient mutation. The identification of asymptomatic carriers by the minisequencing test proved to be unequivocal. The method also allowed quantification of a mutated nucleotide sequence present in less than 1% of a sample. The frequency of AGU carriers in this population was 1/36 when estimated by quantifying the mutated AGU allele in a pooled leukocyte sample from 1350 normal Finnish individuals.

Spectrum of mutations in aspartylglucosaminuria.

Aspartylglucosaminuria (AGU) is an inherited lysosomal storage disorder caused by the deficiency of aspartylglucosaminidase. We have earlier reported a single missense mutation (Cys163----Ser) to be responsible for 98% of the AGU alleles in the isolated Finnish population, which contains about 90% of the reported AGU patients. Here we describe the spectrum of 10 AGU mutations found in unrelated patients of non-Finnish origin. Since 11 out of 12 AGU patients were homozygotes, consanguinity has to be a common denominator in most AGU families. The mutations were distributed over the entire coding region of the aspartylglucosaminidase cDNA, except in the carboxyl-terminal 17-kDa subunit in which they were clustered within a 46-amino acid region. Based on the character of the mutations, most of them are prone to affect the folding and stability and not to directly affect the active site of the aspartylglucosaminidase enzyme.

Infantile form of neuronal ceroid lipofuscinosis (CLN1) maps to the short arm of chromosome 1.

The neuronal ceroid lipofuscinoses (CLNs) are one of the most common progressive encephalopathies of childhood in Western countries. They are divided into three main types: infantile, late infantile, and juvenile. The inheritance of all forms is autosomal recessive, and the biochemical background is totally unknown. The infantile type (CLN1) demonstrates the earliest onset of symptoms and the most severe clinical course. CLN1 is enriched in the Finnish population with incidence of 1:20,000, and only about 50 cases have been reported from other parts of the world. We have collected 15 Finnish CLN1 families with one or two diseased children for a linkage analysis with polymorphic probes randomly localized on human chromosomes. After studying 42 polymorphic protein and DNA markers, we found definitive proof of linkage with three different probes on the short arm of chromosome 1, with maximum lod scores of 3.38 at theta = 0.00 (0.00-0.08) for D1S57 (pYNZ2), 3.56 at theta = 0.00 (0.00-0.09) for D1S7 (lambda MS1), and 3.56 at theta = 0.00 (0.00-0.11) for D1S79 (pCMM8). With the assignment of the CLN1 gene, our study demonstrates the power of multiallelic VNTR probes in the search for linkage of a rare recessive disorder using limited family material.

Infantile neuronal ceroid-lipofuscinosis is not an allelic form of Batten disease: exclusion of chromosome 16 region with linkage analyses.

Infantile neuronal ceroid-lipofuscinosis (CLN1) is the form of neuronal ceroid-lipofuscinoses (NCL) with the earliest onset of symptoms. The locus of the most common form of these disorders, juvenile NCL (CLN3), has been mapped to chromosome 16. We report here linkage data of the same region in Finnish CLN1 families. Our results indicate that CLN1 is not allelic with CLN3 but represents a different locus, which is not located within about 70 cM in chromosome 16.