To screen specific DNA probes by double hybridization from the constructed DNA li- brary of serotype A Cryptococcus neoformans.On the basis of the nucleotide sequence of vector pUC18, a pair of primers was synthesized.The insert fragments were amplified from the library on a PCR pro- cessor.The PCR products were spotted onto hybond-N~+ membranes.All inserts amplified from the ge- nomic library by PCR were screened by dot blot with 26 ~(32)P-labelled DNA from infectious agents for dif- ferentiation and from healthy persons.Twenty-eight candidate clones were obtained.The twenty-eight clone inserts got from low melting point agar were further characterized by dot blot with above 27 kinds of DNA for differentiation.Three specific DNA probes from the library of serotype A Cryptococcus neo- formans were obtained.Colony pCNIIA6 was serotype A-specific,which gave signals only with sertype A strain and did not cross hybridize with other DNAs.Colony pCNIIB5 was species-specific which gave signals only with DNA from Cryptococcus neoformans.Colony pCNIIIG1 was specific for var.neofor- mans,which gave signals only with serotype A and D strains.We can make a rapid diagnosis of Crypto- coccus neoformans infection at an early stage and distinguish the variants of C.neoformans and C.gattii using above specific probes.

Polymerase chain reaction(PCR) was used for the first time in China for the diagnosis of cryptococcal meningitis by amplification of specific sequence of the rDNA genes of C.neoformans.All 11 strains of C.neoformans yielded a specific 136 bp fragment but 21 non-C,neoformans strains did not. The sensitivity of the amplification was about 10 cfu.With the aid of the rDNA-PCR,23 of the 23 cere- brospinal fluids (CSF) specimens which had been confirmed C.neoforrnans positive by smear and/or cul- ture were PCR-positive(100%),and 13 of the 14 CSF specimens which had been confirmed C.neofor- mans negative by smear and culture were PCR-negative (93%).The results of the present study suggest that rDNA-PCR is a sensitive and specific method for rapid diagnosis of cryptococcal meningitis.

Objective: To study the meal qanity of the patient with chronic renal failure(CRF). Methods: The nutritional state of 140 CRF patients was researched,basing on the diet history. Results: With the progression of course of CRF ,the nutritional states of patients with CRF were getting worse . Conclusions: The nutritional state of CRF patients should be paid attention to.

Southern blotting with a labeled and linearized pUC 19 DNA containing a specific fragment of 0. 24 kb DNA of Plasmodium vivax asexual blood stages (kindly offered by Dr. C. Kidson )was used for further identification of blood samples showing positive reaction by dot-blot hybridization. The results showed that those with positive reaction from patients with P. vivax, with P. falciparum or with fever but with negative microscopic findings were also positive by Southern blotting. It was confirmed that some of those positive with P. falciparum were likely to infect P. vivax at the same time. So did a part of those with fever but negative in the blood films (Figs. 1,2).

AIM: To investigate the protective effects and mechanisms of iptakalim hydrochloride(Ipt)on H_2O_2 induced neurotoxity. METHODS: Neurotoxity injury was induced by H_2O_2 in PC12 cells. The cell viability was tested by MTT assay. The glutamate released from PC12 cells was measured by HPLC combined with fluorescent detector analysis. Changes in the intracellular free Ca 2+ concentration ([Ca 2+ ]_i) were determined in fluo-3 AM loaded PC12 cells. RESULTS: Ipt (1, 10 and 100 ?mol?L -1 ) markedly mitigated H_2O_2-induced neurotoxity, 10 ?mol?L -1 Ipt inhibited the release of glutamate and the increase of [Ca 2+ ]_i induced by H_2O_2 .The protective effects was incompletely blocked by 5-HD which is a mitochondrial K_ ATP channels antagnist. CONCLUSION: Ipt provides neuroprotective effects on H_2O_2 induced cytoxixity in cultured PC12 cells and the protective effects may be partially related with mitochondrial KATP channels.

Objective To investigate the effect of kangaroo mother care on children′s comfort with intramuscular injection. Methods One hundred children with an intramuscular injection were grouped according to the injection date. Those treated with injection on odd-numbers days were assigned as the intervention group , and those with injection on even-numbered days as the control group, with 50 cases in each group. The control group received routine care and the intervention group with kangaroo mother care. The two groups were compared in terms of comfort level. Result The comfort level of the observation group was higher than that of the control group with statistical significance (P<0.01). Conclusion Kangaroo mother care can improve the comfort level of children patients with intramuscular injection. It is a simple, safe and effective in non-pharmaceutical pain therapy.

Objective:To study the feasibility of 3-D reconstruction model in the observation of tetrachlorodibenzo-p-dioxin(TCDD) effected palatal organ development of fetal mouse.Methods:Kunming mice treated 40 ug/kg TCDD by lavage on day 12.5 of pregnancy were used as in the experimental group,isodose corn oil treated in the control group.On day 13.5,14.5 and 15.5 of pregnancy heads of the fetal mice were taken out and fixed.Conventional paraffin serial sections of palatal organ were preparated and dyed by hematoxylin-eosin,images of the palatal organs were collected and photoshop treated,3-D reconstruction of the palatal organ was performed by 3D-DOCTOR software.Results:3-D reconstruction images showed that palatal organs moved from on both sides above the tongue and gradually closed and merged in the control group.In the experimental group,the palatal organs moved from on both sides above the tongue was later than control group,gradually closed,but not merged,formed cleft palate.Conclusion:3D-DOCTOR software reconstruction can be used for the study of the development process effected by TCDD in the pregnant mouse.

Objective To study the gene homology of intestinal colonized and infectious bacteria in ICU patients to provide the epidemiological and molecular biological basis for formulating the control measures of multi resistant bacterial hospital infection. Methods The multi-drug resistant colonized bacteria isolated from the anal swabs and the same multi-drug resistant bacteria isola-ted from the clinical samples in the same patients were matched.The Diversilab automatic repetitive extragenic palindromic(REP)-PCR typing system was adopted to analyze the gene homology of multi-drug resistant colonized bacteria and infectious bacteria in the intestinal tract.Results 4 pairs of multi-drug resistant colonized bacteria and the same multi-drug resistant bacteria isolated from the clinical samples on admission in the same patients were selected and performed the homology detection,2 pairs had the ho-mology and 2 pairs had no homology;4 pairs of multi-drug resistant colonized bacteria and the same multi-drug resistant bacteria isolated from the clinical samples in the patients with hospital infection were performed the homology detection,4 pairs all showed the homology.Conclusion The multi-drug resistant colonized bacteria and the infectious bacteria in ICU patients have the homolo-gy.The multi-drug resistant colonized bacteria can cause the occurrence of hospital infection,so their management should be strengthened in clinic.

OBJECTIVETo detect mutations of the retinitis pigmentosa GTPase regulator (RPGR) gene in two Chinese X-linked retinitis pigmentosa families.

METHODSFragments of exons 1-19 of the RPGR gene were amplified with intronic primers, using genomic DNA as template. The polymerase chain reaction (PCR) products were analysed by single-strand conformation polymorphism (SSCP) and direct sequencing. Mutations were identified by comparing DNA sequences of the patients with those of the normal controls.

RESULTSTwo novel mutations, c1536delC and E332X, were identified in exons 12 and 9 of the RPGR gene in both families. Each mutation was the first mutation found in their respective exons. Both mutations were predicted to cause premature termination, which resulted in truncated proteins without normal functions of the RPGR products.

CONCLUSIONSBoth mutations are the genetic basis of the pathogenesis in the respective families. Our data might be helpful in analysing the function of the RPGR protein.

Carrier Proteins ; genetics ; Eye Proteins ; Female ; Genetic Linkage ; Humans ; Male ; Mutation ; Polymerase Chain Reaction ; Polymorphism, Single-Stranded Conformational ; Retinitis Pigmentosa ; genetics ; Sequence Analysis, DNA ; X Chromosome

OBJECTIVE: To determine the mass scores of naringin and neohesperidin in Fructus aurantii and Citrus chang-shan-huyou with their processed products and evaluate the quality of Fructus aurantii and Citrus changshan-huyou with their pro-cessed products. METHODS:According to the requirements of Chinese Pharmacopoeia(2010 edition)and Zhejiang Province Tradi-tional Chinese Medicine Preparation Standards (2005 edition),the moisture and ash of F. aurantii and C. changshan-huyou with their processed products were detected. And the contents of naringin and neohesperidin were determined. The ZORBAX SB-C18 column was used with the mobile phase of acetonitrile-water(20∶80,V/V)at the flow rate of 1.0 ml/min. The detection wave-length was set at 283 nm,and the column temperature was 40℃.The samples size was 10μl. RESULTS:The moisture of F. au-rantii and C. changshan-huyou was decreased after processing with no obvious change for ash. The contents of naringin and neohes-peridin were decreased,significantly for F. aurantii,and all consistent with the requirements of Chinese Pharmacopoeia(2010 edi-tion)except F. aurantii. The linear range was 0.028 45-0.284 5μg(r=0.999 7)for naringin and 0.085 9-0.858 6μg(r=0.999 6)for neohesperidin;the RSDs of precision,stability and reproducibility tests were no more than 1.36% and the average recovery was re-spectively 96.45%-100.43%(RSD=1.45%,n=6) and 98.36%-102.00%(RSD=1.26%,n=6). CONCLUSIONS: There was no significant difference in the inspection and determination re-sults in F. aurantii and C. changshan-huyou. It is suggested to adjust the limitation of content determination in the Chinese Pharmacopoeia(2010 edition)and processed standards.