Rapid and simultaneous typing of hemoglobin S, hemoglobin C, and seven Mediterranean beta-thalassemia mutations by covalent reverse dot-blot analysis: application to prenatal diagnosis in Sicily

The molecular lesions causing beta-thalassemia in Sicily can be subdivided into two groups. One that occurs at a 71% frequency and consists of the beta 39, IVS 1,110 and IVS 1,6 mutations and the other group at a 20% frequency comprising the -87, beta s, IVS 1,1 and IVS 2,745 mutations. The identification of all these mutations by polymerase chain reaction (PCR) and conventional dot-blot hybridization has been time consuming and expensive. In this article, we describe the implementation of the reverse dot-blot (RDB) hybridization as a rapid nonradioactive method for the identification of the nine most frequent molecular lesions in the beta-globin gene (-87, beta s, beta c, IVS 1,1, IVS 1,6, IVS 1,110, beta 39, IVS 2,1, IVS 2,745) in Sicily. Sixty prenatal diagnoses were performed by this RDB assay, each of which was confirmed by dot-blot/ASO hybridization; thus demonstrating the accuracy of the RDB. The main advantage of this assay is the rapid typing of an individual's DNA for many mutations in a single working day. Because the mutations in this assay are representative for the Mediterranean region, this mutational panel can also be extended to the screening of beta-thalassemia from other Mediterranean regions.     

Elevation of serum cystathionine levels in patients with cobalamin and folate deficiency

Homocysteine can be methylated to form methionine by the cobalamin- (Cbl) and folate-dependent enzyme, methionine synthase; serum levels of total homocysteine are elevated in greater than 95% of patients with either Cbl or folate deficiency. Homocysteine can also condense with serine to form cystathionine in a pyridoxal phosphate-dependent reaction catalyzed by cystathionine beta-synthase. Cystathionine is subsequently cleaved to cysteine and alpha-ketobutyrate by the pyridoxal phosphate-dependent enzyme gamma-cystathionase. To assess levels of cystathionine in Cbl and folate deficiency, we developed a new capillary gas chromatographic-mass spectrometric assay and measured cystathionine in the serum of normal subjects and patients with clinically confirmed deficiencies of these vitamins. The normal range for serum cystathionine was 65 to 301 nmol/L (median = 126 nmol/L) for 50 normal blood donors. In 30 patients with clinically confirmed Cbl deficiency, values for cystathionine ranged from 208 nmol/L to 2,920 nmol/L (median = 816 nmol/L) and 26 (87%) had levels above the normal range. In 20 patients with clinically confirmed folate deficiency, cystathionine concentrations ranged from 138 nmol/L to 4,150 nmol/L (median = 1,560 nmol/L) and 19 (95%) had values above the normal range. Five homozygotes for cystathionine beta-synthase deficiency had high values for serum-total homocysteine and low or low-normal values for serum cystathionine that ranged from 30 nmol/L to 114 nmol/L even though they were on treatment with pyridoxine and had partially responded. One patient with a defect in the synthesis of 5-CH3- tetrahydrofolate and five patients with defects in the synthesis of CH3- Cbl had high values for serum-total homocysteine and high values for cystathionine that ranged from 311 nmol/L to 1,500 nmol/L even though they were on treatment with folic acid and Cbl, respectively, and had partially responded. We conclude that levels of cystathionine are evaluated in the serum of most patients with Cbl and folate deficiency and that they are useful in the differential diagnosis of an elevated serum-total homocysteine level.     

Acquired immunodeficiency syndrome-associated non-Hodgkin's lymphomas and other malignancies in patients with hemophilia

Non-Hodgkin's lymphoma (NHL) is the most common human immunodeficiency virus (HIV)-associated malignancy in hemophiliacs. We studied the incidence and clinicopathologic features of NHL in 3,041 hemophiliacs followed at 18 US Hemophilia Centers between 1978 and 1989. Of the 1,295 (56.6%) who were HIV(+), 253 (19.5%) developed acquired immunodeficiency syndrome (AIDS), of whom 14 (5.5%) developed NHL. Three NHL occurred in HIV(-) hemophiliacs, for a 36.5-fold greater risk in HIV(+) than HIV(-) hemophiliacs (P < .001). The NHL incidence rate was 29-fold greater than in the US population by Surveillance, Epidemiology, and End Results (SEER) estimates (P < .001). Between 0 and 4 lymphomas have been observed per year between 1978 and 1989. At presentation 13 (92.9%) of the HIV(+) NHL were extranodal. Ten were stage IV, 1 stage II, and 3 stage IE. Ten (71.4%) were high-grade, 3 (21.4%) intermediate-grade, and 1 (7.1%) was a low-grade B-cell lymphoma. Epstein-Barr virus (EBV) DNA was detected in 36% by in situ hybridization, including one central nervous system (CNS) lymphoma. The mean CD4 cell count at NHL diagnosis was 64/mm3, the mean latency from initial HIV infection was estimated to be 59 months, and the median survival was 7 months. The incidence of basal cell carcinoma in HIV(+) hemophiliacs was 18.3-fold greater than in HIV(-) hemophiliacs (P < .001) and 11.4-fold greater than in the US population (P < .001). In conclusion, incidence rates of NHL and basal cell carcinoma in HIV(+) hemophiliacs are significantly increased over rates in HIV(-) hemophiliacs and over rates in the US population. Clinicopathologic presentation of NHL in HIV(+) hemophiliacs is similar to that in HIV(+) homosexual men.     

Serum interferon as a biological marker in malignant tumors

A total of 745 serum interferon (IFN) determinations were carried out on the serum of 332 cancer patients with a variety of tumors. Disease activity was defined as either 'active' or 'nonactive' as based on clinical examination, the results of laboratory tests and radiological criteria. IFN levels were estimated for both disease activity and mode of treatment. A statistically significant higher level of IFN in the serum of active, as compared to that of nonactive disease, was established. Furthermore, a slight increase in the IFN level was observed in patients before initiating chemotherapy as compared to untreated patients, followed by a decrease in those patients undergoing chemotherapy. These differences between groups were not found to be statistically significant. However, in the cases of rectum, prostate and female genital cancers, a significant elevated level of serum IFN was observed at a late stage of the disease, followed by a decrease after chemotherapy treatment. In addition, a significant lower level of serum IFN was detected in breast cancer patients shortly after radiotherapy as compared to those before radiotherapy. We conclude that IFN, known to play a role in the control mechanisms of developing malignant processes, can be determined in the serum and used to monitor disease activity in individual patients. The inhibition of IFN release depends in part on therapy and can be used for monitoring treatment schedules along the course of disease.     

Protective effect of cholesterol on Friend leukemic cells against photosensitization by hematoporphyrin derivative

The cytotoxic effects of hematoporphyrin derivative (HPD) on Friend erythroleukemic cells were studied. Upon binding of the porphyrin to the cells, the fluorescence spectra was shifted from 613 to 633 nm regarding the main band and from 676 to 667 nm concerning the secondary band. The kinetics of HPD binding was then determined. Maximum binding already occurred at 60 s after exposure of the cells to HPD. It could be demonstrated that the effect of the photoactivated HPD on cell viability was drug, dose, and light fluorescence dependent. Cellular protein synthesis and Friend virus complex release from the cells were equally inhibited by the photodynamic sensitization of the drug, indicating no specific effect on virus maturation. Since cholesterol affects the fluidity of cell membranes, it was important to study the effect of cholesterol enrichment on the photodynamic sensitization by HPD. It was found that, while a 50% reduction in protein synthesis was monitored following treatment with 20 micrograms of HPD per ml and illumination by a 6-milliwatt white light for 60 s, no inhibition was observed following preenrichment of the cells with 0.5, 1, or 2% of cholesterol hemisuccinate. The same trend of cholesterol protection was demonstrated with longer illumination periods up to 10 min. The protective effect of cholesterol hemisuccinate was also seen using scanning electron microscopy. It is thus concluded that the cholesterol hemisuccinate content of Friend erythroleukemic cell membranes is an important factor in regulating the cytotoxicity of photoactivated HPD.     

Specific antigrowth effect of interferon on mouse cells transformed by murine sarcoma virus

The growth rate of NIH/3T3 mouse fibroblasts transformed by the Moloney strain of murine sarcoma virus was investigated following interferon (IFN) treatment. These cells were found to be sensitive to the antigrowth effect of IFN as indicated by a slower growth rate in its presence. The effect was most efficiently expressed when cells were grown at low serum concentrations, e.g., 2.5%. Likewise, IFN treatment caused a reduction in the rate of DNA synthesis as measured by [3H]thymidine incorporation. In contrast, these effects were observed only to a very minor extent with uninfected NIH/3T3 cells or with NIH/3T3 cells chronically infected with murine leukemia virus. In addition, IFN treatment significantly decreased the cloning efficiency of murine sarcoma virus-transformed cells in semi-solid agar. Furthermore, an even stronger effect on the cloning efficiency was observed in liquid medium supplied with 2.5% serum, indicating a direct inhibitory effect on the growth of these cells. Under these conditions, NIH/3T3 or NIH/3T3(MLV) cells remained unaffected by IFN treatment, nor was this effect evident when the transformed cells were grown in the presence of 10% serum. A 4-fold increase in the level of (2'-5')oligoadenylate synthetase following IFN treatment was observed in murine sarcoma virus-transformed cells as compared to either NIH/3T3 or NIH/3T3(MLV) cells.     

Computational Discovery of Internal Micro-Exons

Very short exons, also known as micro-exons, occur in large numbers in some eukaryotic genomes. Existing annotation tools have a limited ability to recognize these short sequences, which range in length up to 25 bp. Here, we describe a computational method for the identification of micro-exons using near-perfect alignments between cDNA and genomic DNA sequences. Using this method, we detected 319 micro-exons in 4 complete genomes, of which 224 were previously unknown, human (170), the nematode Caenorhabditis elegans (4), the fruit fly Drosophila melanogaster (14), and the mustard plant Arabidopsis thaliana (36). Comparison of our computational method with popular cDNA alignment programs shows that the new algorithm is both efficient and accurate. The algorithm also aids in the discovery of micro-exon-skipping events and cross-species micro-exon conservation.     

合欢皮中新皂甙的结构鉴定

从合欢皮(Albizziae cortex)的95%乙醇提取物的正丁醇萃取部分中分离得到3个新的三萜皂甙,用化学方法及¹H-和¹³C-NMR,DEPT,COSY,TOCSY,HMQC-COSY,HMQC-TOCSY,HMBC,NOESY等波谱方法鉴定其结构为:I(1个三萜,9个糖,2个单萜),命名为合欢皂甙(julibroside)J₁;II(1个三萜,8个糖,2个单萜),命名为合欢皂甙J_{2;II(1个三萜,9个糖,2个单萜),命名为合欢皂甙J3。}