A novel pathway of levodopa metabolism by commensal Bifidobacteria.

The gold-standard treatment for Parkinson's disease is levodopa (L-DOPA), which is taken orally and absorbed intestinally. L-DOPA must reach the brain intact to exert its clinical effect; peripheral metabolism by host and microbial enzymes is a clinical management issue. The gut microbiota is altered in PD, with one consistent and unexplained observation being an increase in Bifidobacterium abundance among patients. Recently, certain Bifidobacterium species were shown to have the ability to metabolize L-tyrosine, an L-DOPA structural analog. Using both clinical cohort data and in vitro experimentation, we investigated the potential for commensal Bifidobacteria to metabolize this drug. In PD patients, Bifidobacterium abundance was positively correlated with L-DOPA dose and negatively with serum tyrosine concentration. In vitro experiments revealed that certain species, including B. bifidum, B. breve, and B. longum, were able to metabolize this drug via deamination followed by reduction to the compound 3,4-dihydroxyphenyl lactic acid (DHPLA) using existing tyrosine-metabolising genes. DHPLA appears to be a waste product generated during regeneration of NAD +. This metabolism occurs at low levels in rich medium, but is significantly upregulated in nutrient-limited minimal medium. Discovery of this novel metabolism of L-DOPA to DHPLA by a common commensal may help inform medication management in PD.

Oncogenic mutations and dysregulated pathways in obesity-associated hepatocellular carcinoma.

Epidemiological studies showed that obesity and its related non-alcoholic fatty liver disease (NAFLD) promote hepatocellular carcinoma (HCC) development. We aimed to uncover the genetic alterations of NAFLD-HCC using whole-exome sequencing. We compared HCC development in genetically obese mice and dietary obese mice with wild-type lean mice fed a normal chow after treatment with diethylnitrosamine. HCC tumor and adjacent normal samples from obese and lean mice were then subjected to whole-exome sequencing. Functional and mechanistic importance of the identified mutations in Carboxyl ester lipase (Cel) gene and Harvey rat sarcoma virus oncogene 1 (Hras) was further elucidated. We demonstrated significantly higher incidences of HCC in both genetic and dietary obese mice with NAFLD development as compared with lean mice without NAFLD. The mutational signatures of NAFLD-HCC and lean HCC were distinct, with <3% overlapped. Eight metabolic or oncogenic pathways were found to be significantly enriched by mutated genes in NAFLD-HCC, but only two of these pathways were dysregulated by mutations in lean HCC. In particular, Cel was mutated significantly more frequently in NAFLD-HCC than in lean HCC. The multiple-site mutations in Cel are loss-of-function mutations, with effects similar to Cel knock-down. Mutant Cel caused accumulation of cholesteryl ester in liver cells, which led to induction of endoplasmic reticulum stress and consequently activated the IRE1α/c-Jun N-terminal kinase (JNK)/c-Jun/activating protein-1 (AP-1) signaling cascade to promote liver cell growth. In addition, single-site mutations in Hras at codon 61 were found in NAFLD-HCC but none in lean HCC. The gain-of-function mutations in Hras (Q61R and Q61K) significantly promoted liver cell growth through activating the mitogen-activated protein kinase (MAPK) and phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K)/3-phosphoinositide-dependent protein kinase-1 (PDK1)/Akt pathways. In conclusion, we have identified mutation signature and pathways in NAFLD-associated HCC. Mutations in Cel and Hras have important roles in NAFLD-associated hepatocellular carcinogenesis.

Selective 14-3-3γ induction quenches p-ß-catenin Ser37/Bax-enhanced cell death in cerebral cortical neurons during ischemia.

Ischemia-induced cell death is a major cause of disability or death after stroke. Identifying the key intrinsic protective mechanisms induced by ischemia is critical for the development of effective stroke treatment. Here, we reported that 14-3-3γ was a selective ischemia-inducible survival factor in cerebral cortical neurons reducing cell death by downregulating Bax depend direct 14-3-3γ/p-ß-catenin Ser37 interactions in the nucleus. 14-3-3γ, but not other 14-3-3 isoforms, was upregulated in primary cerebral cortical neurons upon oxygen-glucose deprivation (OGD) as measured by quantitative PCR, western blot and fluorescent immunostaining. The selective induction of 14-3-3γ in cortical neurons by OGD was verified by the in vivo ischemic stroke model. Knocking down 14-3-3γ alone or inhibiting 14-3-3/client interactions was sufficient to induce cell death in normal cultured neurons and exacerbate OGD-induced neuronal death. Ectopic overexpression of 14-3-3γ significantly reduced OGD-induced cell death in cultured neurons. Co-immunoprecipitation and fluorescence resonance energy transfer demonstrated that endogenous 14-3-3γ bound directly to more p-ß-catenin Ser37 but not p-Bad, p-Ask-1, p-p53 and Bax. During OGD, p-ß-catenin Ser37 but not p-ß-catenin Ser45 was increased prominently, which correlated with Bax elevation in cortical neurons. OGD promoted the entry of 14-3-3γ into the nuclei, in correlation with the increase of nuclear p-ß-catenin Ser37 in neurons. Overexpression of 14-3-3γ significantly reduced Bax expression, whereas knockdown of 14-3-3γ increased Bax in cortical neurons. Abolishing ß-catenin phosphorylation at Ser37 (S37A) significantly reduced Bax and cell death in neurons upon OGD. Finally, 14-3-3γ overexpression completely suppressed ß-catenin-enhanced Bax and cell death in neurons upon OGD. Based on these data, we propose that the 14-3-3γ/p-ß-catenin Ser37/Bax axis determines cell survival or death of neurons during ischemia, providing novel therapeutic targets for ischemic stroke as well as other related neurological diseases.

Detection of animal viruses using nucleic acid sequence-based amplification (NASBA).

As seen in recent avian influenza outbreaks in Asia, prevention is the key to fighting infectious disease successfully. Efficient disease surveillance systems on the basis of molecular diagnostics will help monitor the emergence of viruses in the early stage and thus prompt containment measures can be in place to minimize disease spread. Here we describe and review molecular diagnostics focusing on nucleic acid sequence-based amplification (NASBA) technology in detecting viruses causing animal diseases, such as avian influenza, foot-and-mouth disease, and Newcastle disease. NASBA offers high sensitivity, specificity, accuracy, and speed of availability of results, and NASBA would be the most applicable molecular diagnostics for disease surveillance and control.

A NASBA method to detect high- and low-pathogenicity H5 avian influenza viruses.

Nucleic acid sequence-based amplification (NASBA) allows the rapid amplification of specific regions of nucleic acid obtained from a diverse range of sources. It is especially suitable for amplifying RNA sequences. A NASBA technique was developed that allows the detection of avian influenza A subtype H5 from allantoic fluid harvested from inoculated chick embryos. The amplified viral RNA is detected by electrochemiluminescence. The described NASBA technique is a specific, rapid, and sensitive method of detection of influenza A subtype H5 viruses. More importantly, it can be used to distinguish high- and low-pathogenicity strains of the H5 subtype.

Cloning and identification of differentially expressed transcripts in primary culture of GABAergic neurons.

A RNA based arbitrarily primed polymerase chain reaction (RAP-PCR) was used to identify differentially expressed transcripts in primary cultures of cerebral cortical neurons prepared from E16 mouse cerebral cortex. The majority of neurons found in this culture preparation are known to be GABAergic. Different primer combinations were used, and the PCR products were separated on PAGE. Visualization by silver staining revealed a high resolution RNA fingerprint pattern with a total of about 200 transcripts. Six differentially expressed cDNA fragments were recovered, cloned and sequenced. The results of a NCBI database search showed that 6 clones were highly homologous to known genes and expressed sequence tags (ESTs), and that they were either up-regulated or down-regulated during development. Among these clones, Clone 3.1.7 shared 99% sequence homology to mouse Reelin, a neuronal migration and positioning related protein. Clone 4.6.2 shared 91% homology to Rat prepro bone morphogenetic protein-3 mRNA. Clone 6.10.2 had 90% homology to a novel orphan gene of calcium-independent alpha-latrotoxin receptor, which stimulates presynaptic neurotransmitter release. Northern blot analysis confirmed the up-regulated expression profile of Clone 6.10.2 in neuron from Day 2 to 7 during stages of differentiation and development.

Bcl-2, Bcl-x, and Bax expression by immunohistochemistry in inclusion body myositis: a study of 27 cases.

CONTEXT: Bcl-2, Bcl-x, and Bax are among the variety of proteins that have been described as being involved in the regulation of apoptotic cell death. Bcl-2 and Bcl-x(L) inhibit apoptosis, and Bax is proapoptotic. OBJECTIVE: To evaluate the expression of Bcl-2, Bcl-x, and Bax in inclusion body myositis (IBM).Design.-We examined muscle specimens from 27 patients (17 men, 10 women) with IBM to evaluate Bcl-2, Bcl-x, and Bax expression by immunohistochemistry. RESULTS: Patient ages ranged from 29 to 80 years (mean 62.2 years). All biopsies were marked by endomysial chronic inflammation, muscle fiber necrosis, and regeneration. Rimmed (autophagic) vacuoles were present in all cases. Ragged red fibers were noted in 4 biopsies (15%), and cytochrome oxidase-deficient fibers were found in 10 biopsies (37%). Ultrastructural evidence of intranuclear or cytoplasmic tubulofilamentous inclusions, confirming the diagnosis of IBM, were noted in all cases. Paracrystalline mitochondrial inclusions were seen in 5 biopsies (18.5%). Inflammatory cells stained positively with Bcl-2 in all biopsies, Bax in 26 biopsies (96%), and Bcl-x in 8 biopsies (30%). Degenerating muscle fibers were highlighted with Bax in 24 biopsies (89%), Bcl-2 in 2 biopsies (7%), and Bcl-x in 3 biopsies (11%). Regenerative muscle fibers were noted to stain with Bax in 24 muscles (89%), Bcl-2 in 21 muscles (78%), and Bcl-x in 4 muscles (15%). Rimmed vacuoles were highlighted by Bax in 24 biopsies (89%) and only rarely by Bcl-2 (n = 2, 7%) and Bcl-x (n = 3, 11%). A subsarcolemmal staining pattern was observed in 21 biopsies (78%) with Bax, 6 biopsies (22%) with Bcl-2, and only 1 biopsy (4%) with Bcl-x. CONCLUSIONS: (1) Bax (proapoptotic) immunostaining highlighted most autophagic vacuoles; (2) subsarcolemmal Bax and Bcl-2 immunoreactivity may be associated with mitochondrial defects that are commonly noted in IBM; (3) Bcl-2 and Bax immunoreactivity were not confined to degenerating muscle fibers and in fact appeared to be expressed more commonly in regenerating fibers, suggesting that their expression may be independent of apoptosis in the setting of IBM.

Ischemia-induced apoptosis in primary cultures of astrocytes.

Astrocytes participate in a wide variety of important physiological processes and pathological insults, including ischemia. Information on the mechanism of astroglial injury and death during ischemic insult, however, is scarce. In this study, we investigated the mode of astrocytic cell death using an in vitro ischemic model. Cultured astrocytes exhibited several distinct morphological and biochemical features of apoptosis under ischemia. At 4 h of ischemia, Annexin V staining demonstrated an early commitment of some astrocytes to apoptosis. Condensed nuclei became visible from 4 h and the number increased with ischemic incubation time. Electron microscopy showed compacted and segregated chromatin along the edges of nuclear membranes. The number of TUNEL-positive nuclei and the degree of DNA laddering increased with ischemic incubation. Caspase-3, but not caspase-1, activity was increased in ischemia-injured astrocytes. Swollen mitochondria and vacuoles found in some cells with chromatin condensation indicated that these apoptotic-like cells might die of necrosis. The results imply that astrocytes are capable of undergoing apoptosis without the presence of other cell types, such as neurons. Ischemia can induce apoptosis in astrocytes contributing to the pathogenesis of ischemic injury in the CNS.

Astrocytes produce and release interleukin-1, interleukin-6, tumor necrosis factor alpha and interferon-gamma following traumatic and metabolic injury.

The brain is no longer considered immune-privileged due to its capability of producing cytokines in response to neurotrauma; however, the cellular sources of cytokines have not been defined. This study focused on the production of four inflammatory cytokines, interleukin-1 (IL-1alpha), interleukin-6 (IL-6), tumor necrosis factor alpha (TNFalpha), and interferon gamma (IFN-gamma) in primary culture of astrocytes under two different injury models which simulated in vivo mechanical trauma (scratch injury) and ischemia. Results demonstrated that astrocytes after scratch injury were positively immunostained with IL-1alpha, IL-6, and TNFalpha. A slot-blot study of culture media showed that the release of IL-1alpha, IL-6, TNFalpha, and IFN-gamma by astrocytes subsequent to scratch and ischemic injury reached approximately twice the control values. The temporal expression of these cytokines was different for the two models. All four cytokines began to increase 1 h postscratch and remained at high levels throughout the experiment. In the ischemic model, however, the increase of cytokine expression was delayed until 4-8 h of ischemia, when sharp increases were seen in all four cytokines. In this culture system, the exogenous influence of blood-borne factors and leukocytes, which occur with in vivo trauma and ischemia, was eliminated. Accordingly, the cytokines detected in the culture media were derived from astrocytes. This study provides the first evidence that astrocytes, without the influence from other cell types, can produce and release cytokines following mechanical and ischemic injury.

Endothelin-1 protects astrocytes from hypoxic/ischemic injury.

Under pathological conditions such as ischemia (I), subarachnoid hemorrhage, and Alzheimer's disease, astrocytes show a large increase in endothelin (ET) -like immunoreactivity. However, it is not clear whether ET is protective or destructive to these cells during brain injury. Using astrocytes from ET-1-deficient mice, we determined the effect of ET-1 on these cells under normal, hypoxic (H), and hypoxic/ischemic (H/I) conditions. Under normal culture conditions, astrocytes from wild-type and ET-1-deficient mice showed no difference in their morphology and cell proliferation rates. ET-3 and ETA receptor mRNAs were up-regulated whereas ETB receptor mRNA was down-regulated in ET-1-deficient astrocytes, suggesting that ET-1 and ET-3 may complement each other's functions and that the expressions of these endothelins and their receptors are regulated by a complex feedback mechanism. Under H and H/I conditions, ET-1 peptide and mRNA were up-regulated in wild-type astrocytes, and the astrocytes without ET-1 died faster than the wild-type astrocytes, as indicated by greater efflux of lactate dehydrogenase. The present study suggests that astrocytes without ET-1 are more vulnerable to H and H/I injuries and that the up-regulation of astrocytic ET-1 is essential for the survival of astrocytes.

Quercetin inhibits c-fos, heat shock protein, and glial fibrillary acidic protein expression in injured astrocytes.

Quercetin, a bioflavonoid, is found widely in many kinds of fruits and vegetables. It is known to engage in many bioactivities, such as interfering with of the progress of stress responses to injury. In the present study, we investigated the effects of quercetin on some injury responses in primary cultures of astrocytes. These injury responses included the elevation of c-fos protein, heat shock protein (HSP70), and glial fibrillary acidic protein (GFAP). After heat shock insult, the levels of c-fos protein and HSP70 in astrocytes increased. With quercetin treatment, these proteins were significantly reduced. The inhibition of these injury responses by quercetin in astrocytes indicated a dose dependency, with the highest effect at 100 microM. We have previously established a scratch injury model in a primary culture of astrocytes. In that model, astrocytes responded to the scratch injury by an elevation in their GFAP level and formation of hypertrophic cell processes, which extend into the scratch areas. Quercetin treatment reduced the number of hypertrophic cell processes being extended into the scratch areas. With 100 microM of quercetin, there was a complete inhibition of the formation of the hypertrophic cell process. Western blot analysis for GFAP indicated that quercetin significantly reduced the induction of GFAP in the scratch model. At 100 microM, the total GFAP content in the injured cultures was reduced to a level lower than that of the control. This implied that quercetin might possess an antigliotic property.

Multi-lipofection efficiently transfected genes into astrocytes in primary culture.

This study demonstrated that liposome-mediated transfection - lipofection - is suitable for delivering genes into astrocytes. By repeatedly lipofecting the same astrocyte cultures, a process we call multi-lipofection, the transfection efficiency of the beta-galactosidase (beta-gal) gene was improved from 2.6+/-0.6 to 17. 4+/-1.1%. This is the highest efficiency ever reported in gene-transfer with Lipofectin(R) in a primary culture of mouse cerebral cortical astrocytes. Furthermore, multi-lipofection did not cause observable disturbance to astrocytes as indicated by insignificant changes in the glial fibrillary acidic protein content in the cultures. In order to demonstrate that the transfected gene achieved a physiologically relevant expression level, a plasmid containing the pEF-hsp70 protein gene was lipofected into astrocytes. This produced colonies of astrocytes showing an increased resistance to heat-induced cell death. A similar experiment was performed with the glial-derived neurotrophic factor (GDNF) gene. Control astrocytes had no detectable GDNF. In the transfected astrocytes, the GDNF protein could be identified intracellularly by immunocytochemistry. Western blot analysis revealed, as compared to astrocytes with one lipofection, a 2.9-fold increase of GDNF with four lipofections. GDNF remained detectable in astrocytes 2 weeks after four lipofections. Thus, multi-lipofection provides a mild and efficient means of delivering foreign genes into astrocytes in a primary culture, making astrocytes good candidate vehicle cells for gene/cell therapy in the CNS.

The expression of Cdk5, p35, p39, and Cdk5 kinase activity in developing, adult, and aged rat brains.

The expression of cyclin-dependent kinase 5 (Cdk5) and its regulatory subunits, p35 and p39, was investigated in rat brain from embryonic day 12 (E12) to postnatal 18 months (18M). The Cdk5 protein levels increased from E12 to postnatal day 7 (P7) and remained at this level until 18M. The Cdk5 kinase activity and the levels of both p35 mRNA and protein were low at E12, became prominent at E18-P14 but then decreased in the adult and aged rat brains of 3M to 18M. In comparison, the expression pattern of p39 appeared to have an inverse relationship to that of Cdk5 and p35. In regional distribution studies, p35 protein levels and Cdk5 kinase activity were significantly higher in the cerebral cortex and hippocampus, but lower in the cerebellum and striatum. These results suggested that Cdk5, p35 and p39 might have region-specific and developmental stage-specific functions in rat brain.

Dexmedetomidine-induced stimulation of glutamine oxidation in astrocytes: a possible mechanism for its neuroprotective activity.

Dexmedetomidine is a highly specific alpha2-adrenergic agonist, which is used clinically as an anesthetic adjuvant and in animal experiments has a neuroprotective effect during ischemia. The current study showed that dexmedetomidine enhances glutamine disposal by oxidative metabolism in astrocytes. This effect occurs at pharmacologically relevant concentrations. It is exerted on alpha2-adrenergic receptors and not on imidazoline-preferring sites, and it is large enough to reduce the availability of glutamine as a precursor of neurotoxic glutamate.

Protection against ischemic injury in primary cultured mouse astrocytes by bis(7)-tacrine, a novel acetylcholinesterase inhibitor [corrected].

The effects of bis(7)-tacrine, a novel acetylcholinesterase inhibitor, on ischemia-induced cell death and apoptosis were investigated in primary cerebral cortical astrocytes of mice. Following a 6 h in vitro ischemic incubation of the cultures, a marked decrease in the percentage of viable cells was observed by lactate dehydrogenase (LDH) release assay. Furthermore, using bisbenzimide staining, we determined that approximately 65% of the cells underwent apoptosis. Treatment with bis(7)-tacrine (1-10 nM) during ischemic incubation effectively inhibited the ischemia-induced apoptosis, as demonstrated by morphological and biochemical tests. Our results demonstrated that bis(7)-tacrine could protect astrocytes against ischemia-induced cell injury, indicating that the drug might be beneficial for the treatment of vascular dementia, in addition to Alzheimer's disease.

Neuronal-astrocytic and cytosolic-mitochondrial metabolite trafficking during brain activation, hyperammonemia and energy deprivation.

A novel concept is described, according to which both neurons and astrocytes are capable of metabolizing glucose all the way to CO(2) and water, but in addition interact metabolically in a process generating glutamate from glucose, and subsequently, metabolizing excess glutamate to CO(2) and water Hertz, L., Dringen, R., Schousboe, A., Robinson, S.R., 1999. Astrocytes: Glutamate producers for neurons (Journal of Neuroscience Research 57, 417-428). The proposed metabolic degradation of glucose via glutamate serves the purpose of adjusting transmitter pools of glutamate to the demands for glutamatergic transmission, and it must account for a major fraction of glucose utilization. Evidence in favor of this concept is presented and a multitude of in vivo data are interpreted in the context of metabolic trafficking between neurons and astrocytes. In addition, intracellular trafficking occurs between cytosol and mitochondria during synthesis of transmitter glutamate, partly explaining a robust quantitative correlation between glutamine synthesis, as a measure of release of transmitter glutamate, and glucose utilization, reported by several authors. Both intracellular and intercellular metabolic trafficking may be affected during pathological conditions, as evidenced by effects of hyperammonemia (mimicking hepatic encephalopathy) and energy deprivation (mimicking stroke). It is suggested that neuronal-astrocytic interactions may also be impaired during degenerative dementing diseases.

Expression of interleukin-1 alpha, tumor necrosis factor alpha and interleukin-6 genes in astrocytes under ischemic injury.

Astrocytes form an integral part of the blood brain barrier and are the first cell type in the central nervous system to encounter insult if there is an ischemic attack. The immunologic reaction of astrocytes to an ischemic insult would be affective to the subsequent responses of other nerve cells. We previously showed that ischemia caused an increase in the levels of interleukin 1alpha (IL-1alpha), tumor necrosis factor alpha (TNF alpha), and interleukin 6 (IL-6) in the culture medium of mouse cerebral cortical astrocyte. We did not have evidence on the source of these cytokines. This study aimed to investigate the expressions of these cytokine mRNAs in the astrocytes under ischemia. Results demonstrated that ischemia could induce necrosis and apoptosis in astrocytes. By using the RT-PCR method, we demonstrated for the first time that the mRNA levels of IL-1alpha, TNF alpha and IL-6 in normal astrocyte was very low, but their expressions could be induced quickly under ischemia. These cytokines might be interactive as indicated by the difference in time course of their expressions, with IL-1alpha being the earliest and IL-6 being the latest. The result provided some understanding of the induction and progression of these immunologic responses in astrocytes under ischemia. It also supported our previous findings that astrocytes contributed to the cytokines released under ischemia.

A locus for brachydactyly type A-1 maps to chromosome 2q35-q36.

Brachydactyly type A-1 (BDA1) was, in 1903, the first recorded example of a human anomaly with Mendelian autosomal dominant inheritance. Two large families, the affected members of which were radiographed, were recruited in the study we describe here. Two-point linkage analysis for pedigree 1 (maximum LOD score [Zmax] 6.59 at recombination fraction [theta] 0.00) and for pedigree 2 (Zmax=5.53 at straight theta=0.00) mapped the locus for BDA1 in the two families to chromosome 2q. Haplotype analysis of pedigree 1 confined the locus for family 1 within an interval of <8.1 cM flanked by markers D2S2248 and D2S360, which was mapped to chromosome 2q35-q36 on the cytogenetic map. Haplotype analysis of pedigree 2 confined the locus for family 2 within an interval of <28. 8 cM flanked by markers GATA30E06 and D2S427, which was localized to chromosome 2q35-q37. The two families had no identical haplotype within the defined region, which suggests that the two families were not related.

Epidemiology of diabetes mellitus in children in Hong Kong: the Hong Kong childhood diabetes register.

OBJECTIVES: To establish a registry for Chinese children with onset of type 1 (insulin dependent) diabetes mellitus before 15 years of age and to determine the incidence of childhood onset type 1 diabetes mellitus in Chinese children in Hong Kong. RESEARCH DESIGN AND METHODS: A registry was established in 1997 to collect childhood diabetes cases retrospectively from all districts in Hong Kong. The study included all newly diagnosed cases of diabetes with onset < 15 yr of age from 1st January 1984 to 31 December 1996. Primary ascertainment was based on review of medical records at all regional public hospitals in Hong Kong and survey of all the registered practitioners in Hong Kong. The secondary source of validation was made impractical, if not impossible, because of the recent implementation of the Personal Data Privacy Ordinance in Hong Kong. RESULTS: A total of 255 diabetic cases were identified, 227 type 1 diabetes mellitus (218 were Chinese), 18 type 2 diabetes mellitus and 11 secondary diabetes. 246 patients were Chinese and 9 non-Chinese. The age-standardized incidence of type 1 and type 2 diabetes mellitus in southern Chinese children in Hong Kong was 1.4/100,000/yr and 0.1/100,000/yr respectively for children < 15 yr of age during the study period. The incidence rates for type 1 diabetes were 0.9, 1.5 and 1.7 per 100,000/yr for 0-4 years, 5 to 9 years and 10 to 14 years age-groups respectively. The incidence for males was 1.2/100,000/yr and for females 1.7/100,000/yr. A significant increase in the incidence was demonstrated during the study period by simple linear regression (slope 0.14/100,000/year, r2 = 0.73, p = 0.0002) CONCLUSIONS: A diabetic registry is established in Hong Kong. This study documents a very low incidence rate of childhood type 1 diabetes mellitus in southern Chinese children in Hong Kong and we have seen an increasing incidence of the disease in the past 13 years.

[A brief introduction to the methods for novel gene cloning].

There are a lot of methods for novel gene cloning, but how to clone candidate gene(s) quickly and correctly? This is a brief introduction to methods of novel gene cloning, these methods includes: differential display reverse transcriptase polymerase chain reaction(DD RT-PCR), suppression subtractive hybridization(SSH), RNA arbitrarily primed PCR(RAP-PCR), representational difference analysis(RDA), yeast two-hybrid system, cDNA capturation, et al. We not only introduced these methods, but also discussed the advantages and disadvantages of them. However, no single method is omnipotent, one should pick up the method most suitable for a special purpose.