Functional modulation of caecal fermentation and microbiota in rat by feeding bean husk as a dietary fibre supplement.

A feeding study using rats was conducted to evaluate the utility of lablab bean husk and soya bean husk as sources of potential prebiotic fibre. Twenty 5-week-old Sprague Dawley rats were divided into 4 groups and fed one of the following diets for 3 weeks: purified diet (AIN93 G) containing 5% cellulose (CEL), or the same diet in which cellulose was replaced by corn starch (STA), lablab bean husk (LBH), or soya bean husk (SBH). Rats were sacrificed at 8 weeks of age and caecal digesta were collected. Feed intake, body weight, anatomical parameters, and caecal ammonia level did not differ significantly among diets. Rats on LBH and SBH showed higher concentrations of caecal short-chain fatty acid and lactate than those on CEL. Rats on CEL, SBH, and LBH exhibited lower caecal indole and skatole levels. LBH yielded increased caecal abundance of Akkermansia muciniphila and Oscillibacter relatives, as demonstrated by either qPCR, MiSeq, or clone library analysis. SBH favoured the growth of lactobacilli as assessed by both qPCR and MiSeq, and favoured the growth of bifidobacteria as assessed by MiSeq. In comparison with STA, LBH and SBH yielded lower caecal abundance of bacteria related to Dorea massiliensis, as demonstrated by qPCR, MiSeq, and clone library analysis. Both types of bean husk were found to contain oligosaccharides that might selectively stimulate the growth of beneficial bacteria. Based on these results, the two species of bean husk tested are considered potentially functional for promoting the gut health of monogastric animals.

Binding to the naturally occurring double p53 binding site of the Mdm2 promoter alleviates the requirement for p53 C-terminal activation.

Genotoxic stress activation of the tumor suppressor transcription factor p53 involves post-translational C-terminal modifications that increase both protein stability and DNA binding activity. We compared the requirement for p53 protein activation of p53 target sequences in two major p53-regulated genes, p21/WAF1 (encoding a cell cycle inhibitory protein) and Mdm2 (encoding a ubiquitin ligase that targets p53 for proteolytic degradation). The p53 binding site in the proximal p21/WAF1 promoter contains a single p53 binding consensus sequence, while the p53 binding site in the Mdm2 promoter contains two consensus sequences linked by a 17 bp spacer. Binding of recombinant p53 protein to the p21/WAF1 binding site required monoclonal antibody PAb421, which can mimic activating phosphorylation and/or acetylation events at the C-terminus. In contrast, recombinant p53 bound strongly to the Mdm2 binding site in the absence of PAb421 antibody. Separate binding to each consensus sequence of the Mdm2 binding site still required PAb421, indicating that p53 binding was not simply due to greater affinity to the Mdm2 consensus sequences. Linking two p21/WAF1 binding sites with the 17 bp spacer region from the Mdm2 gene eliminated the PAb421 requirement for p53 binding to the p21/WAF1 site. These results suggest a mechanism for regulation of Mdm2 gene transcription that differs from that other p53-induced genes by its lack of a requirement for C-terminal activation of p53 protein. A steady induction of Mdm2 protein would maintain p53 protein at low levels until post-translational modifications following DNA damage increased p53 activity towards other genes, mediating p53 growth inhibitory and apoptotic activities.

Real time fixation point monitoring system for photocoagulation of juxtafoveal neovascularisation.

BACKGROUND/AIMS: A new real time monitoring system has been developed to locate the fixation point during juxtafoveal laser photocoagulation. METHOD: The red diode laser beam is combined coaxially with the illumination beam to image a cross in the focal plane of the slit lamp, which allows projection of a red cross onto the patient's fundus. 27 patients with juxtafoveal choroidal neovascularisation were treated by photocoagulation using this system. RESULTS: 13 (48%) patients whose visual acuity ranged from 20/200 to 20/40 answered that it was easier to keep the focus on the cross target image than on the aiming beam. The patient maintained stable fixation throughout the treatment. The laser treatment was completed without foveal damage near the fixation point in all patients. CONCLUSION: The real time fixation monitoring system should allow surgeons to treat juxtafoveal lesions with laser photocoagulation more safely and accurately.

Effect of heat stress on tomato fruit protein expression.

We delayed the ripening of tomato fruit for several days (average 5 days) by a 1-day heat treatment at 37-42 degrees C. We analyzed the tomato fruit pericarp proteins, which were altered by the heat stress, using two-dimensional electrophoresis. Heat stress caused about 23.7% of the proteins in the pericarp to disappear and about 1.1% of new proteins to appear. We determined their apparent molecular mass, isoelectric point, and N-terminal amino acid sequence. Identified proteins included antioxidant enzymes, heat shock proteins, cell-wall-related proteins, etc.

The study of heat stress in tomato fruits by NMR microimaging.

The ripening of the tomato fruit was delayed for several days (average 5 days) by a 1-day heat treatment at 42 degrees C. Ethylene production increased during the first 3 h, but, after 6 h inhibition was almost total in tomato fruit incubated at 42 degrees C. However, recovery of ethylene production was rapid if fruits were returned to a temperature of 25 degrees C after heating. In NMR microimaging, three imaging pulse sequences with different repetition and echo times at 42 degrees C were used to obtain the proton density (TR = 6000 ms, TE = 15 ms), the T1 weighted image (TR = 1000 ms, TE = 15 ms) and the T2-weighted image (TR = 6000 ms, TE = 120 ms). After 12 h heating, the water in locular tissues began to show shorter T1 and T2 values. Though the tomatos were returned to 25 degrees C and preserved one more day, the water having a shorter T2 value in locular tissues, did not change. These results show that tomato fruit do not fully recover from heating even after one day, although ethylene production is recovered almost immediately. For this reason, we suggest that some denaturation event inside the tomato, which goes on after the end of heating, is the cause of the delay in tomato ripening.

Two short sequences have positive effects on the human p27Kip1 gene transcription.

The cyclin-dependent kinase (Cdk) inhibitor p27Kip1 plays an important role in the progression from G1 to S phase in the cell cycle. To study the activities of its promoter and other regulatory elements, we have cloned and characterized the 5'-flanking region of the human p27Kip1 gene. This region, about 3kb in length, is GC-rich and shares homology with that of the mouse p27Kip1 gene. Transcription start points (tsp) determined by the oligo-capping method are mapped in two regions, the cluster I (-479 to -403) and cluster II (-280 to -273). The cluster I was the primary functional site in transcription initiation. The luciferase activities of serial deletion mutants indicated that two short sequences (-581 to -557 and -556 to -526) had positive effects on transcription. The gel shift assay showed that factors in HeLa nuclear extract bound to these sequences. Sp1 was the major binding factor to the sequence of -556 to -526, wheres yet unidentified positive factors bound to the sequence of -581 to -557.

A core promoter and a frequent single-nucleotide polymorphism of the mismatch repair gene hMLH1.

The hMLH1 gene encodes a protein that is involved in the DNA mismatch repair system. The coding region of the hMLH1 gene has been known to be mutated in a subset of patients with hereditary nonpolyposis colorectal cancer (HNPCC). Our current research characterized the promoter region of the hMLH1 gene and searched for mutations correlating to HNPCC. Utilizing the oligo-capping method, major transcription start sites of the hMLH1 gene were mapped at two locations. The core promoter region of about 180 bp was determined by the luciferase assay of serial deletion mutants. Although we did not find any pathogenic mutation in the hMLH1 promoter region by PCR-SSCP, we found a single-nucleotide polymorphism at position -93 nt from the adenine residue of the start codon. By PCR-RFLP analysis with Pvu II for this polymorphism, we detected LOH in four tumors from three patients. An easy detection of this polymorphism with PCR-RFLP and high incidence ( approximately 50%) of informative cases make this polymorphism a suitable marker for the detection of hMLH1 allelic losses.

Up-regulation of thioredoxin expression in motor neurons after nerve injury.

A substantial up-regulation of thioredoxin, a dithiol/disulfide oxido-reductase, in adult rat motoneurons following hypoglossal nerve axotomy, was demonstrated by using both in situ hybridization and immunohistochemistry. Although thioredoxin is normally accumulated more in the nucleus of a motoneuron rather than in the cytoplasm, a dramatic increase of thioredoxin in the cytoplasmic region after nerve injury was observed. The up-regulation of mRNA lasted more than 9 weeks, whereas, the detectable up-regulation of protein was observed for more than 5 weeks.

Promoter analysis of the human mismatch repair gene hMSH2.

The human DNA mismatch repair gene homologue hMSH2 is involved in hereditary nonpolyposis colorectal cancer. We isolated and characterized the 5' upstream region, about 4.4kbp, of the hMSH2 gene. This region contains CpG islands and a number of elements involved in constitutive expression, but there is no TATA-box nearby the transcription start points. This is the typical structure for many promoters of housekeeping genes. Alu sequences and mononucleotide repeats are clustered in this region and there are two transcription start points. Deletion analysis revealed that less than 300bp was sufficient to initiate transcription. Although no mutation that influences promoter activity of this region was found, a polymorphism was detected by PCR-RFLP analysis. Because informative cases (C/T heterozygous) were relatively high ( approximately 30%), this polymorphism is suitable for a marker to examine allelic losses.

Isolation and characterization of the 5' region of the human mismatch repair gene hPMS1.

The hPMS1 gene encodes a mutL homolog that is implicated in DNA mismatch repair and was found to be mutated in the germline of a patient with hereditary nonpolyposis colorectal cancer (HNPCC). To understand transcriptional regulation and to perform mutational analysis in the promoter region, we cloned and characterized the genomic sequence of the 5' region of the gene. hPMS1 has an intron upstream of the initiation codon. There were several transcripts with alternative splicing sites and multiple transcriptional start sites. The cloned 1.4-kbp fragment of the 5' region contains a CpG island but no TATA-boxes, typical for promoters of housekeeping genes. The promoter activity of the fragment was almost equal to that of the SV40 early promoter. Deletion analysis showed that about a 300-bp region was sufficient to initiate transcription. Although we searched for mutations in the hPMS1 promoter region in HNPCC kindreds, neither germline nor somatic mutations were detected. However, we found a highly informative polymorphism in the first exon that is useful for searching allelic losses because no polymorphic changes in hPMS1 have been reported previously.

Identification of a novel transmembrane semaphorin expressed on lymphocytes.

Semaphorin (also known as collapsin) members are thought to be involved in axon guidance during neural network formation. Here, we report the isolation of a novel member, mouse semaphorin G (M-sema G), which encodes a semaphorin domain followed by a single putative immunoglobulin-like domain, a transmembrane domain, and a cytoplasmic domain. M-sema G is most closely related to M-sema F, which we previously reported, and semB and semC. These four members appear to constitute a transmembrane type subfamily in mouse semaphorins. In contrast to the predominant expression of M-sema F mRNAs in the nervous tissues, M-sema G mRNAs are strongly expressed in lymphoid tissues, especially in the thymus, as well as in the nervous tissues. The mRNAs are also detected in various cell lines from hematopoietic cells. By generating specific antibodies, we confirmed the strong expression of M-Sema G proteins on the surface of lymphocytes. These results provide the first evidence that semaphorin is expressed on lymphocytes and suggest that semaphorins may play an important role in the immune system, as well as in the nervous system.

Differential distribution of mRNA encoding cAMP-specific phosphodiesterase isoforms in the rat brain.

We studied the distributions of four different cyclic AMP-specific phosphodiesterase isoform mRNAs (APDE1-4) and compared them with that of 63 kDa calmodulin-stimulated phosphodiesterase (CPDE) in the rat brain by in situ hybridization histochemistry using specific radiolabeled oligonucleotides. The distribution patterns were unique for all the APDE isoforms examined here. Although no significant signals for APDE1 could be detected anywhere in the rat brain, all other isoforms were expressed ubiquitously but unevenly and showed overlapping distribution patterns. Among all the APDE isoforms studied here, APDE3 showed the strongest and the most extensive expression. Its distribution pattern implies that it may modulate different cellular processes associated with learning and memory. Compared to APDE3, the levels of expression of APDE2 and APDE4 were weaker, the latter showing the weakest expression. Our study suggests that different isoforms of APDE are expressed together in the same class of neurons implying complex interactions among different signaling pathways, thereby mediating distinct and specific functions.

Localization of mRNAs for Rlim-1, the rat Xlim-1 homolog, in the developing rat brain.

We studied the localization of Rlim-1 mRNAs, the rat Xlim-1 homolog, in the developing rat brain using in situ hybridization histochemistry. On embryonic day 13 (E13), strong signals were observed in the most superficial layer of the telencephalon, the zonalimitans intrathalamica, the ventral thalamus, some nuclei of the hypothalamus, the tectum, the cerebellum, the lower brainstem and the spinal cord. In the above-mentioned regions except the cerebellum, the distribution pattern remained almost the same from embryonic stage to adulthood but the intensity of expression gradually decreased after birth. In the cerebellum, the distribution pattern changed. during development; all the primordium of cerebellum in E13, the external granular and the Purkinje cell layers in postnatal day 7 (P7), and only the Purkinje cell layer in the adult expressed positive signals. These results suggest that Rlim-1 may be involved in region specification.

Identification of a member of mouse semaphorin family.

Grasshopper semaphorin I (Sema I) and its related proteins, chick collapsin and mouse Sema III contribute to the axon guidance by their repellent actions [5,9,12]. We have identified a member of semaphorin gene family from the mouse brain and named it M-Sema F. The N-terminal encodes a semaphorin domain that is similar between Sema I-III [6] followed by a single putative immunoglobulin-like domain, a transmembrane domain, and a proline-rich intracellular domain. M-Sema F mRNA is expressed widely in the nervous tissues during development. These suggest that M-Sema F is a transmembrane member of the semaphorin family of the vertebrate which may function in the developing neuronal network.

Localization of 63-kDa calmodulin-stimulated phosphodiesterase mRNA in the rat brain by in situ hybridization histochemistry.

We studied the localization of 63-kDa Ca2+/calmodulin stimulated phosphodiesterase mRNA in the rat brain by in situ hybridization histochemistry using an oligonucleotides probe specific to this enzyme. The signals were especially concentrated in several brain regions such as the olfactory tubercle, accumbens nucleus, caudate putamen, fundus striati, dentate gyrus of the hippocampus, pontine nuclei and dorsal tegmental nucleus. These results suggest that in the neuronal groups containing the strong signals this enzyme is involved in calcium-dependent signal transduction system coupled to cyclic nucleotides messenger systems.

Distribution of Rlim, an LIM homeodomain gene, in the rat brain.

We examined the distribution of Rlim, a homologue to Xlim-1, in the rat brain. Rlim, a LIM class homeodomain gene, was isolated from rat brain, and localized in the adult brain by in situ hybridization histochemistry. The expression of Rlim was found in discrete regions, such as the Purkinje cell layer of the cerebellum and several nuclei of the hypothalamus, midbrain and pons. This suggests that Rlim is related to regulation of genes that are specific to some neurons such as Purkinje cells in the adult.

Distinct regulation of sodium channel types I, II and III following nerve transection.

We investigated mRNA expression of type I, II and III voltage-sensitive sodium channels in motoneurons following facial nerve transection by means of in situ hybridization histochemistry. Type I mRNA expression decreased markedly after nerve transection, while that of type III increased. Type II expression underwent no detectable change following nerve transection. These results suggest that type III sodium channels may be involved in regeneration and plasticity.

Production of anti-tumor-promoting iridoid glucosides in Genipa americana and its cell cultures.

The Genipa americana plant contains geniposide [3] and geniposidic acid [2] in the fruits and geniposidic acid [2] in the leaves. On callus induction, the plant produces tarennoside [1], geniposidic acid [2], and gardenoside [4] in high levels. The leaves of Ge. americana plants redifferentiated from the callus tissues produce 2.

Orientation and reactivity of NADH kinase in proteoliposomes.

NADH kinase was reconstituted in liposomes by employing phosphatidylcholine and phosphatidylethanolamine with n-octyl-beta-D-thioglucoside as a detergent. An analogous molecular organization of the NADH kinase to that in the mitochondrial inner membrane was ascertained to exist in the liposomal membrane. Michaelis constants for NADH and ATP were determined as 27 and 133 microM, respectively. Both values were lower than that of the solubilized enzyme. The catalytic center of NADH kinase was exposed on the outer surface of the reconstituted liposomes. The NADH kinase reconstituted with ADP/ATP carrier protein catalyzed the phosphorylation of exogenously supplied NADH by the use of ATP entrapped in the liposomal matrix.

Localization of the NADH kinase in the inner membrane of yeast mitochondria.

The bulk of NADH kinase of Saccharomyces cerevisiae was recovered in the mitochondrial fraction prepared from spheroplasts. Most of the NADH kinase was localized in the inner membrane fraction, which was separated from other mitochondrial components by the combined swelling, shrinking, and sonication procedure. Treatment of mitoplasts with antiserum against the NADH kinase caused inactivation of the enzyme. On the contrary, no influence was observed upon the same treatment of intact mitochondria. p-Chloromercuribenzoate and eosin-5-maleimide inactivated the enzyme without affecting the matrix ATPase. The NADH kinase was enzymatically iodinated in mitoplasts, but not in the intact mitochondria. These results support the conclusion that NADH kinase is localized and functions at the intermembrane space side of the mitochondrial inner membrane. It is evident that the NADH kinase is encoded by nuclear gene(s) because it is synthesized in the presence of chloramphenicol or acriflavine, and a significant amount of the enzyme was detected in mitochondrial DNA-deficient mutants.