The antihypertensive effect of fermented milk in individuals with prehypertension or borderline hypertension.

Fermented milk (FM) with putative antihypertensive effect in humans could be an easy applicable lifestyle intervention against hypertension. The mode of action is supposed to be through active milk peptides, shown to possess in vitro ACE-inhibitory effect. Blood pressure (BP) reductions upto 23 mm Hg have been reported in spontaneously hypertensive rats fed FM. Results from human studies of the antihypertensive effect are inconsistent. However, many studies suffer from methodological weaknesses, as insufficient blinding and the use of office BP measurements. We conducted a randomised, double-blind placebo-controlled study of the antihypertensive effect of Lactobacillus helveticus FM in 94 prehypertensive and borderline hypertensive subjects. The participants were randomised into three treatment groups with a daily intake of 150 ml of FM, 300 ml of FM or placebo (chemically acidified milk). The primary outcome was repeated 24-h ambulatory BP measurements. There were no statistically significant differences in the outcome between the groups (systolic BP (SBP), P=0.9; diastolic BP (DBP), P=0.2). However, the group receiving 300 ml FM had reduced BP across the 8-week period in several readings, which could be compatible with a minor antihypertensive effect. Heart rate and lipids remained unchanged between groups. Hence, our study does not support earlier studies measuring office BP-measurements, reporting antihypertensive effect of FM. Based on straight performed 24-h ambulatory BP measurements, milk fermented with Lactobacillus helveticus does not posses significant antihypertensive effect.

Photoreceptor localization of the KIF3A and KIF3B subunits of the heterotrimeric microtubule motor kinesin II in vertebrate retina.

The heterotrimeric microtubule motor kinesin II has been shown to be required for morphogenesis and maintenance of both motile flagella and immotile sensory cilia. Recently, we showed that the KIF3A subunit of kinesin II is concentrated in the inner segment and connecting cilium of fish photoreceptors. Here we report the gene structure of human KIF3A (HsKIF3A) and describe its localization in human and monkey retina. We also describe the localization of both KIF3A and KIF3B kinesin II subunits in Xenopus retina. Using a portion of HsKIF3A we had amplified from adult human retinal cDNA, we found by a GenBank database search that an identical sequence had already been obtained by the Human Genome Center at Lawrence Berkeley National Laboratories in a direct sequencing analysis of 680 kb of human chromosome 5q31. By comparing the genomic sequence of HsKIF3A to the open reading frame (ORF) of the highly homologous mouse Kif3A, we determined that the HsKIF3A gene has 17 exons and an ORF of approximately 2.1 kb, predicting a protein of 80.3 kDa. Antibodies against sea urchin KRP85, a KIF3A homologue, bound to a single band of approximately 85 kDa in immunoblots of total retina protein from human, monkey and Xenopus. In these same samples, a single band of approximately 95 kDa is recognized by antibodies against Xklp3, a Xenopus KIF3B homologue. In sections of Xenopus retina, both antibodies strongly labelled photoreceptor inner segments and the outer limiting membrane. Both antibodies also labelled photoreceptor axonemes. The axonemal localization of kinesin II subunits suggests that kinesin II may play a role in transport of materials from the photoreceptor cell body to the outer segment.

Characterization of a novel C-kinesin (KIFC3) abundantly expressed in vertebrate retina and RPE.

Many forms of intracellular transport are mediated by microtubule-dependent motors of the kinesin superfamily (KIFs). To identify kinesins expressed in human retina and RPE, we used degenerate primer RT-PCR to amplify a approximately 440 bp kinesin motor domain fragment from human retinal and RPE messenger RNAs. Four distinct kinesins were detected: one C-kinesin (HsKIFC3); one kinesin from the unc104/KIF1 family [HsKIF1A]; and the ubiquitous and neuronal forms of conventional kinesin heavy chain [HsuKHC and HsnKHC]. The C-kinesin HsKIFC3 comprised 33.3% of the retinal clones and was 60% identical to FKIF2, the most abundant kinesin detected in a previous screen of fish retina and 95% identical to a fragment of MmKifC3 recently amplified from mouse brain. Elsewhere we have reported the sequence of HsKIFC3 and shown that it maps to the same locus on chromosome 16q13-q21 as Bardet-Biedl syndrome Type II, a hereditary retinal degeneration. We describe here the kinesin PCR screen of human retina and RPE and examine the tissue and subcellular distribution of KIFC3 in both fish and human retina using an antibody raised against a peptide conserved between FKIF2 and HsKIFC3. This peptide antibody identified a single approximately 80 kDa band in Western blots of fish and human retina and RPE. In both fish and human retina this antibody strongly labeled photoreceptor terminals in the outer plexiform layer, suggesting that FKIF2/KIFC3 may play some role in the photoreceptor synapse.

Multiple kinesin family members expressed in teleost retina and RPE include a novel C-terminal kinesin.

Kinesins comprise a large superfamily of microtubule-based motor proteins, individual members of which mediate specific types of motile processes. To identify kinesin family members (KIFs) that are critical to retinal function and thus to vision, a reverse transcriptase polymerase chain reaction (RT-PCR) cloning strategy was used to isolate putative KIFs expressed in the neural retina and retinal pigmented epithelium (RPE) of the striped bass, Morone saxatilus. Eleven fish KIFs (FKIFs) were isolated from neural retina and six of the same FKIFs were also isolated from RPE. One of the KIFs identified in this screen, FKIF2, was the most prevalent clone detected both in the retina (41% of clones) and RPE (72% of clones). Based on predicted amino acid sequence homology within the motor domain, seven of the FKIFs have been tentatively assigned to known kinesin families: the kinesin heavy chain family (FKIF1, 5 and 9), the unc104/KIF1 family (FKIF3 and 8), the KIF2 family (FKIF4), and the cKIF family (FKIF2). Northern blot analysis revealed that each detectable FKIF exhibited a unique tissue-specific expression pattern. Since FKIF2 was more highly expressed in retina than in any other tissue tested, including brain, and was the most abundant KIF message expressed in both retina and RPE, it was examined in more detail and the complete approximately 2.3 kb open reading frame for FKIF2 was cloned and sequenced. The predicted amino acid sequence indicates that FKIF2 has a C-terminal motor domain, and thus is a member of the cKIF family. FKIF2 is only 36.5% identical at the amino acid level to the most closely related cKIF in the database, suggesting that FKIF2 may be a novel member of this family. Antibodies raised against a unique peptide specific to FKIF2 recognize an approximately 80 kd protein in homogenates of retina, RPE, brain and kidney. The pronounced expression of FKIF2 in retina and RPE suggests that FKIF2 may play an important role in microtubule-dependent motile events in these two tissues.

Expression of kinesin heavy chain isoforms in retinal pigment epithelial cells.

To examine the possible role of kinesin in pigment granule migration in the retinal pigment epithelium (RPE) of teleosts, we investigated the expression and distribution of kinesin heavy chain (KHC) in RPE. Blots of fish RPE lysates probed with two well-characterized antibodies to KHC (H2 and HD) displayed a prominent band at 120 kD. A third KHC antibody (SUK4) recognized a band at 118 kD. The 118 kD band was also occasionally present in blots probed with H2, suggesting the presence of two KHC isoforms in teleost RPE. Reverse transcriptase-polymerase chain reaction (RT-PCR) of mRNA from RPE using primers homologous to conserved regions of the KHC motor domain resulted in the identification of two putative KHC genes (FKIF1 and FKIF5) based on partial amino acid sequences. Previous studies had demonstrated a requirement for microtubules in pigment granule aggregation in RPE. In addition, the reported microtubule polarity orientation in RPE apical projections is consistent with a role for kinesin in pigment granule aggregation. Immunofluorescent localization of KHC in isolated RPE cells using H2 revealed a mottled distribution over the entire cell body, with no detectable selective association with pigment granules, even in cells fixed while aggregating pigment granules. Microinjected KHC antibodies had no effect on pigment granule aggregation or dispersion, although each of the three antibodies has been shown to block kinesin function in other systems. Thus we found no evidence for KHC function in RPE pigment granule aggregation. However, the two KHC isoforms may participate in other microtubule-dependent processes in RPE.