Isolation and characterization of mutants as an approach to a transformation system in Kluyveromyces marxianus

 A method to obtain K. marxianus mutants has been developed. Different auxotrophic mutants were isolated by nystatin and snail-enzyme enrichment procedures using an incubation time of 2 h before adding the antibiotic or the enzyme respectively. All his mutants analyzed by complementation tests turned out to belong to the same complementation group. Some of them were transformed and complemented by the S. cerevisiae HIS3 gene. These non-reverting his3 mutants contain no heterologous sequence, which is essential to make them acceptable for application in the food industry. Received: 15 November/21 December 1995     

ATM germline mutations in Spanish early-onset breast cancer patients negative for BRCA1/BRCA2 mutations

Heterozygous carriers of ATM (ataxia telangiectasia mutated gene) mutations have increased risk of breast cancer (BC). We have estimated the prevalence of mutations in the ATM gene among Spanish patients with early-onset BC. Forty-three patients diagnosed with BC before the age of 46 years, and negative for BRCA1 and BRCA2 mutations, were analysed for the presence of ATM mutations. A total of 34 ATM sequence variants were detected: 1 deleterious mutation, 10 unclassified variants and 23 polymorphisms. One patient (2.3%) carried the ATM deleterious mutation (3802delG that causes ataxia telangiectasia in the homozygous state) and 13 patients carried the 10 ATM unclassified variants. The truncating mutation 3802delG and eight of the rare variants were not detected in a control group of 150 individuals. Different bioinformatic sequence analysis tools were used to evaluate the effects of the unclassified ATM changes on RNA splicing and function protein. This in silico analysis predicted that the missense variants 7653 T>C and 8156 G>A could alter the splicing by disrupting an exonic splicing enhancer motif and the 3763 T>G, 6314 G>C, and 8156 G>A variants would affect the ATM protein function. These are the initial results concerning the prevalence of germline mutations in the ATM gene among BC cases in a Spanish population, and they suggest that ATM mutations can confer increased susceptibility to early-onset BC.     

Radiographic assessment of knee–ankle alignment after total knee arthroplasty for varus and valgus knee osteoarthritis

Background

There are unanswered questions about knee–ankle alignment after total knee arthroplasty (TKA) for varus and valgus osteoarthritis (OA) of the knee. The aim of this retrospective study was to assess knee–ankle alignment after TKA.

Flow cytometric detection of intracellular myeloperoxidase, CD3 and CD79a. Interaction between monoclonal antibody clones, fluorochromes and sample preparation protocols

Detection of intracellular myeloperoxidase (MPO), CD79a and CD3 has become the most specific tool for the assignment of myeloid, B- and T-lymphoid lineages in acute leukemias. In order to establish the best combination of monoclonal antibody reagent and sample preparation technique for the intracellular detection of these three markers, we compared six different cell fixation-permeabilization kits (Cytofix/Cytoperm, Fix and Perm, Intraprep, Intrastain, Permeacyte and Permeafix) using 12 fluorochrome conjugates derived from seven monoclonal antibody (mAb) clones. A total of 21 samples corresponding to normal peripheral blood (n=4), normal bone marrow (n=3), acute myeloblastic leukemia (AML, n=6), precursor B-acute lymphoblastic leukemia (ALL, n=6) and T-ALL (n=2) cases, were analysed in two centers. All fixation/permeabilization methods resulted in decreased side scatter and mostly increased forward scatter as compared to erythrocyte-lyse-washed and 1% paraformaldehyde fixed samples. The autofluorescence levels of the leukocyte populations was only significantly increased with use of the Cytofix/Cytoperm kit and mildly with the other techniques. In addition, non-specific staining increased significantly for combinations of any anti-MPO mAb with the Cytofix/Cytoperm kit and for the CD3 clone S4.1 combined with any intracellular method. Anti-MPO antibodies gave a stronger fluorescence signal when conjugated to PE than when coupled to FITC. In conclusion, MPO-7-PE, UCHT-1-PE (CD3) and any HM57-PE conjugate (CD79a) in combination with Fix and Perm, Intraprep, Intrastain or Permeafix, provided specific staining of the respective markers in sufficient intensities. Thus, combined selection of fixation/permeabilization kits and monoclonal antibody reagents against CD3, CD79a and MPO is required for obtaining optimal cytoplasmic detection of these antigens.     

Dynamics of dominance of a dipeptide insertion in reverse transcriptase of HIV-1 from patients subjected to prolonged therapy

A small proportion (0.8%) of individuals of a cohort of HIV-1 infected patients subjected to prolonged therapy with nucleoside analogues included a recently recognised dipeptide insertion in their RT (Ser-Ser or Ser-Gly between RT codons 69 and 70). To study the dynamics of dominance of genomes with this genetic change, sequential HIV-1 isolates from two patients were analyzed with regard to consensus sequences and complexity of mutant spectra. The two patients displayed completely different, complex evolutionary patterns leading to temporary dominance of dipeptide insertions. In one patient, a virus very closely related to an ancestor virus from the same patient overtook the population at late times, displacing genomes encoding a Ser-Ser insertion. In another patient the sequential dominance of genomes with Ser-Ser insertion-->no insertion-->Ser-Gly insertion was observed. These three types of genomes coexisted in the mutant spectrum of one HIV-1 isolate. Complexity was also reflected in the shape of phylogenetic trees derived with genomes from the mutant spectrum at each time point. The results suggest that HIV-1 genomes encoding a dipeptide insertion between RT codons 69 and 70 do not show a clear selective advantage over other genomes lacking the insertion. Such an absence of a clear selective advantage will favor that such genomes encoding this RT insertion become dominant only in a transient fashion, and following disparate kinetics in different patients.     

Testosterone is essential for cocaine sensitization in male rats

Most studies agree that males and females respond differently to drugs of abuse. In females, estradiol enhances the behavioral response to cocaine. However, studies on the role of testosterone and the locomotor response to psychostimulants in the male rat are inconclusive. Our study was designed to determine the behavioral effects of testosterone on the development and persistence of cocaine sensitization in male rats. We tested different doses of cocaine (10, 15 and 30 mg/kg) to determine which dose induced locomotor sensitization in intact (INT) and gonadectomized (GDX) animals. We also investigated if GDX males with testosterone replacement (GDX-T) showed a similar locomotor response to cocaine as INT males.Our data showed that gonadectomy enhanced the locomotor response to a single cocaine injection. This effect was not observed in gonadectomized rats that received testosterone replacement. However, GDX rats did not show a progressive increase in their locomotor response to repeated cocaine administration (15 and 30 mg/kg) (sensitization) as did INT and GDX-T animals. It is possible that in GDX males, the initial high locomotor response to cocaine creates a ceiling effect that limits further increase in cocaine-induced hyperactivity. These findings indicate that testosterone not only modulates the behavioral response to a single and to repeated cocaine injections, but is also essential for male rats to become sensitized to cocaine.     

Extraperitoneal endometriosis: a diagnosis to be considered

Summary BACKGROUND: Endometriosis is a relatively common pathology in women of childbearing age and of low parity but rarely shows extraperitoneal involvement. The main aim of this paper is to raise the attention of specialists to the necessity of carrying out penetrating diagnosis of nonspecific extraperitoneal masses occurring in women of reproductive age. METHODS: We performed a retrospective review of six patients diagnosed with extraperitoneal endometriosis who were treated at the Vega Baja University Hospital (Spain) during the last 5 years. RESULTS: Surgical treatment had positive results in five patients. The preoperative diagnosis was correctly made in only two patients. The accurate aetiology of endometriosis remains unknown and diagnosis is based on clinical and cytopathological findings. CONCLUSIONS: Surgical treatment of extraperitoneal endometriosis is recommended. However, postoperative follow-up is obligatory and hormonal suppressive therapy may be necessary. Medical treatment with gestagens, Danazol, or agonists of the gonadotropin-releasing hormone are ineffective in endometriomas which are bigger than 2 cm.      

某些免疫增强剂对照射小鼠的抗辐射效果

本文结果表明,免疫增强剂对受照射小鼠有抗辐射作用。照射8Gy的小鼠,照后30min注入I-RNA、MDP+CP-CWS、BCG-CWS+CP-CWS,它们的30天活存率分别为78.4%、73.3%、53.3%、57.1%和50.0%,而对照组只为5.7%。照射8.5Gy的小鼠注入MDP+CP-CWS、TP+CP-CWS,活存率分别为34.8%和57.1%,对照组只为2.08%。照射9Gy的小鼠注入TP+CP-CWS、TP其活存率分别为52.7%、37.5%,对照组全部死亡。     

H亚型血管与骨质疏松症

目前,骨质疏松症的传统治疗方法主要集中于抑制骨破坏和促进骨形成方面,但此单一的治疗方法很难彻底解决多重因素致病的骨质疏松症。近来,许多文献报道血管形成与骨形成之间存在偶联关系,其中骨骼中新发现的H亚型血管成为骨质疏松治疗的新焦点。根据骨骼血管内皮细胞位置和功能的区别,可分为H亚型血管和L亚型血管,而H亚型血管可促进其周围骨祖细胞的分化与增殖。H亚型血管主要分布于干骺端和骨膜下,表现为血小板-内皮细胞粘附分子抗体(CD31)和内皮粘蛋白抗体(endomucin,EMCN)强阳性;而L亚型血管表现为两种内皮细胞抗体(CD31或EMCN)弱阳性,分布于骨干区。实验表明,H亚型内皮细胞与周围的骨祖细胞数量会随着小鼠的老龄化呈正比例下降,而且小鼠体内缺氧诱导型转录因子1α缺失后会出现H亚型内皮细胞和骨祖细胞数量明显降低,这些均提示H亚型血管与骨形成存在偶联关系。笔者着重对H亚型内皮细与骨形成之间存在的偶联关系及其机制进行综述,探讨H亚型内皮细胞与骨质疏松症的关系,以进一步加深对骨质疏松症发生、发展的认识,为骨质疏松症治疗提供新的思路与方法。     

机器学习在放射治疗中的临床应用

放射治疗是癌症的主要治疗手段之一,以机器学习为代表的人工智能飞速发展,可应用于放射治疗临床实践的各个环节,包括临床决策支持、自动勾画靶区、预测疗效和副反应等,提高准确性与效率.尽管面临着结构化数据缺乏、模型可解释性差等挑战,机器学习在放射治疗中的应用将日趋深刻而广泛.本文从机器学习简介、在放射治疗中的临床应用研究进展和...