Bifidobacterium as an oral delivery carrier of oxyntomodulin for obesity therapy: inhibitory effects on food intake and body weight in overweight mice.

INTRODUCTION: Oxyntomodulin (OXM) is a gut hormone released from intestinal L cell. Synthetic OXM and its analog reduce food intake and body weight in both rodents and human beings by being administered intravenously. However, people find intravenous administration difficult because of its side effects and inconvenience. The aim of this study is to develop a novel oral delivery system for OXM and its analog using genetically engineered Bifidobacterium as the carrier. METHODS: An OXM gene expression vector pBBADs-OXM for the Bifidobacterium genus was constructed. Human OXM sequence was fused with extracellular exo-xylanase (XynF) signal peptide (Xs) from Bifidobacterium longum under the control of the pBAD promoter. B. longum NCC2705 was transformed with the recombinant plasmid pBBADs-OXM by electroporation, and the transformed B. longum was selected using MRS plates containing 60 microg ml(-1) ampicillin. The OXM expression in vitro was identified by western blot and enzyme-linked immunosorbent assay (ELISA) assay after L-arabinose induction. Overweight BALB/c mice were treated with B. longum transformed with OXM after 0.2% L-arabinose induction every day for 4 weeks to investigate the effects of OXM-transformed B. longum on food intake and body weight by oral administration. The B. longum transformed with the green fluorescent protein (GFP) gene was used as negative control; orlistat, a gastrointestinal lipase inhibitor, was used as positive control; Normal saline (NS, 0.9% saline) was used as blank control. The food intakes of each group were measured every day, and body weights were measured once a week. Normal BALB/c (2 months old) mice were treated with OXM-transformed B. longum after induction by intragastric administration every day for 6 days to reveal the mechanism of transformed B. longum, with OXM exerting its biological function by oral administration. Plasma OXM, plasma ghrelin and the OXM of intestinal contents were detected by the ELISA method. Plasma glucose and triglyceride levels were analyzed using the Automatic Biochemistry Analyzer. RESULTS: Transformed B. longum with OXM was selected and identified without biological and morphological alteration. An approximately 4-5 kDa OXM peptide was detected in both the supernatant and the cell pellet of transformed B. longum after L-arabinose induction in vitro. The food intake, body weight and blood triglyceride level of overweight mice treated with OXM-transformed B. longum were all significantly reduced compared with that of the GFP negative control group and NS control group (P<0.01). Interestingly, the plasma triglyceride level of the GFP group was significantly decreased compared with that of the NS control group (P<0.01). The OXM level in the intestinal contents of the OXM group was significantly increased compared with that of the GFP negative control group and the NS group (P<0.05). The plasma ghrelin level of the OXM group was significantly decreased compared with that of the GFP and NS groups (P<0.01). Unexpectedly, the ghrelin level of the GFP group was significantly increased compared with that of the NS control group (P<0.01). CONCLUSION: A novel oral delivery system of Bifidobacterium for human OXM has been successfully established. The expression of recombinant OXM can be detected in the supernatant and cell pellet of transformed B. longum. OXM-transformed B. longum reduces food intake, body weight and plasma lipid level in overweight mice by oral administration.

Phosphomannomutase activity in congenital disorders of glycosylation type Ia determined by direct analysis of the interconversion of mannose-1-phosphate to mannose-6-phosphate by high-pH anion-exchange chromatography with pulsed amperometric detection.

Congenital disorders of glycosylation (CDG) are a group of multisystemic disorders resulting from defects in the synthesis and processing of N-linked oligosaccharides. The most common form, CDG type Ia (CDG-Ia), results from a deficiency of the enzyme phosphomannomutase (PMM). PMM converts mannose 6-phosphate (man-6-P) to mannose-1-phosphate (man-1-P), which is required for the synthesis of GDP-mannose, a substrate for dolichol-linked oligosaccharide synthesis. The traditional assay for PMM, a coupled enzyme system based on the reduction of NADP(+) to NADPH using man-1-P as a substrate, has limitations in accuracy and reproducibility. Therefore, a more sensitive, direct test for PMM activity, based on the detection of the conversion of man-1-P to man-6-P by high-pH anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD), was developed. Using this assay, the activity of PMM was markedly deficient in fibroblasts and lymphoblasts from 23 patients with CDG-Ia (range 0-15.3% of control, average 4.9+/-4.7%) and also decreased in seven obligate heterozygotes (range 33.0-72.0% of control, average 52.2+/-14.7%). Unlike the spectrophotometric method, there was no overlap in PMM activity among patients, obligate heterozygotes, or controls. Thus, the PMM assay based on HPAEC-PAD has increased utility in the clinical setting, and can be used, together with transferrin isoelectric focusing, to diagnose patients with CDG-Ia and to identify heterozygotes when clinically indicated.

Childhood-onset schizophrenia/autistic disorder and t(1;7) reciprocal translocation: identification of a BAC contig spanning the translocation breakpoint at 7q21.

Childhood-onset schizophrenia (COS) is defined by the development of first psychotic symptoms by age 12. While recruiting patients with COS refractory to conventional treatments for a trial of atypical antipsychotic drugs, we discovered a unique case who has a familial t(1;7)(p22;q21) reciprocal translocation and onset of psychosis at age 9. The patient also has symptoms of autistic disorder, which are usually transient before the first psychotic episode among 40-50% of the childhood schizophrenics but has persisted in him even after the remission of psychosis. Cosegregating with the translocation, among the carriers in the family available for the study, are other significant psychopathologies, including alcohol/drug abuse, severe impulsivity, and paranoid personality and language delay. This case may provide a model for understanding the genetic basis of schizophrenia or autism. Here we report the progress toward characterization of genomic organization across the translocation breakpoint at 7q21. The polymorphic markers, D7S630/D7S492 and D7S2410/D7S646, immediately flanking the breakpoint, may be useful for further confirming the genetic linkage for schizophrenia or autism in this region. Am. J. Med. Genet. (Neuropsychiatr. Genet.) 96:749-753, 2000. Published 2000 Wiley-Liss, Inc.

A genome-wide search for chromosomal loci linked to mental health wellness in relatives at high risk for bipolar affective disorder among the Old Order Amish.

Bipolar affective disorder (BPAD; manic-depressive illness) is characterized by episodes of mania and/or hypomania interspersed with periods of depression. Compelling evidence supports a significant genetic component in the susceptibility to develop BPAD. To date, however, linkage studies have attempted only to identify chromosomal loci that cause or increase the risk of developing BPAD. To determine whether there could be protective alleles that prevent or reduce the risk of developing BPAD, similar to what is observed in other genetic disorders, we used mental health wellness (absence of any psychiatric disorder) as the phenotype in our genome-wide linkage scan of several large multigeneration Old Order Amish pedigrees exhibiting an extremely high incidence of BPAD. We have found strong evidence for a locus on chromosome 4p at D4S2949 (maximum GENEHUNTER-PLUS nonparametric linkage score = 4.05, P = 5. 22 x 10(-4); SIBPAL Pempirical value <3 x 10(-5)) and suggestive evidence for a locus on chromosome 4q at D4S397 (maximum GENEHUNTER-PLUS nonparametric linkage score = 3.29, P = 2.57 x 10(-3); SIBPAL Pempirical value <1 x 10(-3)) that are linked to mental health wellness. These findings are consistent with the hypothesis that certain alleles could prevent or modify the clinical manifestations of BPAD and perhaps other related affective disorders.

The characterization and sequence analysis of thirty CTG-repeat containing genomic cosmid clones.

We have systematically isolated and characterized DNA containing large CTG (n > 7) repeats from a human cosmid genomic DNA library. Using a CTG10 probe, more than 100 cosmid clones were identified, and 30 of these have been extensively characterized. The sequenced cosmids contain repeats that are between three and 19 perfect units (average 10 perfect repeats). The cosmids map to at least 12 different chromosomes. Sequence analysis of flanking regions suggests that more than one third of the repeats occur in exons, and many share strong sequence identity with databank sequences, including the gene involved in dentatorubral pallidoluysian atrophy (DRPLA). Genotyping of human DNA samples demonstrates that more than half of the repeats are polymorphic. This and similar collections of clones containing trinucleotide repeats should aid in the identification of genes that may contain expansions of trinucleotide repeats involved in human disease.

Large CAG/CTG repeats are associated with childhood-onset schizophrenia.

Recent studies have shown an association between trinucleotide repeat expansions (TREs) and adult-onset schizophrenia (AOS). Childhood-onset schizophrenia (COS) is a severe variant of schizophrenia with onset of symptoms before age 12 years. We have used the repeat expansion detection (RED) method to investigate the occurrence of repeat expansions in a group of well-characterized COS patients as well as a set of clinically related childhood-onset psychosis cases labeled 'multidimensionally impaired' (MDI). The difference observed in the CAG/CTG RED product distribution between normal (n = 44) and COS (n = 36) samples was only marginally significant (P = 0.036). However, male COS samples (n = 20) had a significantly different RED product distribution compared to male controls (n = 25, P = 0.002) with longer RED products in COS. No such difference was seen in females (ncont = 19; ncos = 16; P = 0.236). The difference remained significant between male COS (n = 12) and male controls (n = 24) when only Caucasian samples were used (P = 0.003). Similarly, the RED product distribution in male MDI samples (n = 18) was significantly different compared to male controls (P = 0.018). Some of the detected TREs in all three populations (COS, MDI and control) correlated with expanded alleles found at the CTG18.1 locus on chromosome 18. In conclusion, we have found an association between TREs and COS. This association is specifically significant in the male population. Thus, the occurrence of an expanded trinucleotide repeat may contribute to the genetic risk of COS, possibly in combination with other factors.

HLA antigens in childhood onset schizophrenia.

Evidence of immune system abnormalities in adult schizophrenia has prompted examination of the human leukocyte antigen (HLA) system. Childhood onset schizophrenia offers a unique opportunity to test neurodevelopmental hypotheses of schizophrenia, including those which implicate components of the immune system. In the present study, class I and II HLA antigens were typed using sequence-specific primers and the polymerase chain reaction in 28 childhood onset schizophrenics and 51 ethnically matched healthy subjects. Groups were compared for frequencies of HLA antigens reported to be associated with schizophrenia and/or autoimmune disorders. We hypothesized that antigen frequencies would differ between schizophrenic and healthy children, suggesting that some dimension of the neurodevelopmental disturbance experienced by these children may be mediated by subtle abnormalities of immune function. There were no significant differences between schizophrenic and healthy subjects in the frequency of any antigen tested. These findings do not support HLA-associated pathology in childhood onset schizophrenia.

Chromosome 22q11.2 interstitial deletions among childhood-onset schizophrenics and "multidimensionally impaired".

Since its first description almost a century ago schizophrenia with childhood onset, a rare yet devastating disorder, has been diagnosed in children as young as age 5. Recently, the velocardiofacial syndrome, whose underlying cause is interstitial deletions of 22q11.2, was found in 2 of 100 cases of schizophrenics with adult onset [Karayiorgou et al., Proc Natl Acad Sci USA 92: 7612-7616, 1995]. No study has documented the prevalence of velocardiofacial syndrome and the 22q11.2 deletion in a population of schizophrenics with childhood onset. Here we describe the result of such a study in a sample originally selected for a trial of atypical antipsychotic drugs. A separate group of patients was also included in the study; they can best be accounted for as a variant of childhood-onset schizophrenia (COS) and had been provisionally termed "multidimensionally impaired." Fluorescent in situ hybridization screening of 32 COS and 21 multidimensionally impaired patients revealed 1 COS patient with an interstitial deletion spanning at least 2.5 megabases.

Multiple endocrine neoplasia syndrome, type III: review and case report.

Successful treatment of the multiple endocrine neoplasia type III (MEN III) syndrome requires early diagnosis. It is highly possible that the patient's dentist may be the first practitioner with the opportunity to diagnose this potentially fatal syndrome. Additionally, patients with this syndrome having a pheochromocytoma and needing invasive dental treatment pose a life-threatening dental management risk. This article presents a review of the MEN II syndrome and a case report on the surgical management of a MEN III patient with a pheochromocytoma.

N-methyl-D-aspartate induces a rapid, reversible, and calcium-dependent intracellular acidosis in cultured fetal rat hippocampal neurons.

The ability of NMDA to alter intracellular pH (pHi) was studied in fetal rat hippocampal neurons and glia using the pH-sensitive fluorescent indicator 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF). Brief exposure (60 sec) of hippocampal neurons to NMDA (2.5-250 microM) results in a rapid, and in most cells reversible, reduction in pHi, with full recovery to baseline pHi values taking several minutes following removal of NMDA. In contrast, little or no change in pHi was observed in glial cells exposed to these same concentrations of NMDA. The NMDA-induced acidification of neurons was concentration and time dependent, with an EC50 of 39 microM and Emax (delta pH) of -0.53. More prolonged exposure to NMDA (> or = 10 min) resulted in a more prolonged reduction in pHi values over the ensuing 20 min observation period. The intracellular acidification resulting from NMDA exposure of hippocampal neurons was blocked by the NMDA receptor antagonist 3-((+/-)-2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid (CPP). Moreover, removal of extracellular Ca2+ eliminated both the selective NMDA-induced elevation in [Ca2+]i and the reduction in pHi, indicating that Ca2+ influx may be required for the decrease in pHi induced by NMDA receptor activation. Finally, the NMDA-induced reduction in pHi was not significantly attenuated when extracellular [H+] was decreased by increasing extracellular pH to 8.0. The latter suggests that an intracellular source of H+ is responsible for the NMDA-induced reduction in neuronal pHi. The reduction in neuronal pHi induced by NMDA receptor activation may mediate some of the physiological and (or) pathophysiological actions of glutamate.

Cathepsin D as a prognostic indicator for node-negative breast cancer patients using both immunoassays and enzymatic assays.

This is a retrospective study on 162 node-negative patients, with both biochemical and clinical factors being measured for determination of prognostic markers. Steroid receptors were measured on all tumors, while tumor size, histological grade, ploidy status, and cell cycle kinetics indicators could not be found or measured on 25 or less of the patient group. The primary focus of this study was the measurement of cathepsin D, analyzed by two different procedures, and 161 of the 162 patients had at least one value. The antigenic assay was performed using the US-CIS kit, and it was sensitive and reproducible. A biochemical assay using the enzymatic activity of cathepsin D was developed, and it gave proportional values, compared to the antigenic assay values (r2 = 0.79). Our results indicated that the mean antigenic levels were 20% higher than the biochemical assay levels (P = 0.001). High levels of cathepsin D by the antigenic assay predicted poor relapse-free (P = 0.0001) and overall (P = 0.0004) survival. High levels of cathepsin D by the biochemical assay also predicted poor relapse-free (P = 0.031) and overall (P = 0.0013) survival. The cathepsin D values were still useful as predictors of outcome after multivariate analysis. Several other factors, such as grade and S phase, were useful as additional prognostic indicators. In conclusion, cathepsin D is the most useful marker in node-negative patients, and the analysis can be performed by both a biochemical and an antigenic assay.

Re-evaluation of the linkage relationship between chromosome 11p loci and the gene for bipolar affective disorder in the Old Order Amish.

Reanalysis of an Old Order Amish pedigree, to include several new individuals and two changes in clinical status, markedly reduces the probability of linkage between bipolar affective disorder and the Harvey-ras-1 oncogene and insulin loci on chromosome 11. This linkage can be excluded using a large lateral extension of the original Amish pedigree.

Implantation metastasis as a cause of local recurrence of colorectal carcinoma.

Local recurrence of colorectal carcinoma postoperatively is due to locally unresected tumor, lymphatic permeation by tumor, or intraoperative implantation of viable shed cancer cells. One hundred eighty-five patients with colorectal carcinoma underwent resection for cure. Of these patients, 40 received diluted formalin intraluminally for prevention of local recurrence, and the remainder received no cancericidal agent. Distribution by tumor size and stage was similar in both groups. Local recurrence occurred in 2.6 percent of formalin-treated patients and 14.3 percent of untreated patients. The difference in recurrence rates was significant (p less than 0.05). No significant difference between the two groups was present in the actuarial survival curves. The 5-year survival rate was 66.6 percent in the formalin-treated group and 50.5 percent in the control group. No suture line recurrences were observed in the treated group. Our results indicate that intraluminal fixation of cancer cells before opening the bowel is an effective method of reducing local recurrence after resection of colorectal cancer.

Localization of immunoreactive thyrotropin releasing hormone in the lower brainstem of the rat.

The distribution of thyrotropin releasing hormone (TRH) in individual nuclei of the rat brainstem was examined by radioimmunoassay. TRH was detectable in 36 of 40 brainstem nuclei investigated and the localization of TRH in the brainstem was unlike other known brainstem neuropeptides. By far the highest concentration of TRH in the brainstem (1.2 ng/mg protein) is present in the nucleus of the solitary tract. The concentration of TRH was relatively high in the motor nuclei of the IIIrd, Vth, VIIth, Xth and XIIth cranial nerves, and less high in the area postrema, nucleus gracilis, locus coeruleus, lateral reticular nucleus (A1-catecholamine cell group), dorsal raphe and central gray matter. Cerebellum and pontine nuclei contained very low levels (less than 0.03 ng/mg protein) of TRH.

Substance P activity in the bullfrog retina: localization and identification in several vertebrate species.

Immunoreactive substance P is present in the bullfrog retina, possibly in two types of stratified amacrine cells, with their somas in the inner nuclear layer and their neuronal processes entering the inner plexiform layer and ramifying in sublayers 3 or 4 (or both). Occasionally, polygonal somas positive for substance P were found in the ganglion cell layer. Approximately 75 percent of the cell bodies positive for substance P and 65 percent of the radioimmunoassayable substance P were found in the superior half of the frog retina. On the basis of high-performance liquid chromatography, the immunoreactive substance P in the neural retina of the rat, monkey, or chick is similar to synthetic substance P, whereas this is not true of the immunoreactive substance P in the bullfrog or carp retina.

alpha-Melanocyte-stimulating hormone: reduction in adult rat brain after monosodium glutamate treatment of neonates.

Intraperitoneal injection of monosodium glutamate in neonatal rates resulted in a 90 percent loss of alpha-melanocyte-stimulating hormone in hypothalamic and extrahypothalamic areas of the brain, whereas the amount of hormone in the pituitary gland did not change. The dramatic reduction of alpha-melanocyte-stimulating hormone in the brain suggests that is primary source there is the neuronal perikarya of the arcuate nucleus.