Public health impact of isoniazid-resistant Mycobacterium tuberculosis strains with a mutation at amino-acid position 315 of katG: a decade of experience in The Netherlands

A previous limited study demonstrated that Mycobacterium tuberculosis isolates with a mutation at amino-acid position 315 of katG (Delta315) exhibited high-level resistance to isoniazid and were more frequently resistant to streptomycin. In the present study, isoniazid-resistant M. tuberculosis isolates from 8,332 patients in The Netherlands (1993-2002) were screened for the Delta315 mutation. Isoniazid resistance was found in 592 (7%) isolates, of which 323 (55%) carried Delta315. IS6110 restriction fragment length polymorphism analysis showed that Delta315 isolates occurred in clusters, suggesting recent transmission, at the same frequency as isoniazid-susceptible isolates. In contrast, other isoniazid-resistant isolates clustered significantly less frequently. Delta315 isolates were high-level isoniazid-resistant, streptomycin-resistant and multidrug-resistant significantly more often, and may have a greater impact on public health, than other isoniazid-resistant isolates.     

Telomere structure, function and maintenance in Arabidopsis

The stability of eukaryotic genomes is provided in part by the integrity of telomeres, the nucleoprotein caps on the ends of chromosome. Recent studies reveal that proper telomere architecture is required for long-term proliferation capacity. Here we describe molecular mechanisms that protect and maintain chromosome ends and discuss why Arabidopsis is emerging as a powerful new model for elucidating fundamental aspects of telomere biology.     

Marfan syndrome: exclusion of genetic linkage to the COL1A2 gene

Marfan Syndrome is a genetic disorder of the connective tissue. Individuals from one large family with this disorder were genotyped for COL1A2 gene associated RFLPs. Our results demonstrated that the COL1A2 gene, encoding the proa2(I) collagen chain, segregated independently of the phenotype and it is therefore excluded as the mutant locus in this family.     

DNA cloned from the ayw subtype of hepatitis B virus

DNA from the ayw subtype of hepatitis B virus (HBV) was ligated into the EcorI site of DNA from plasmid pBR322 and propagated in E coli chi 776. A plasmid with a 3200 base pair insert (pHBV-1) was isolated and the cloned HBV DNA was mapped with restriction endonucleases. Differences were found in restriction endonuclease cleavage sites for DNAs from HBV of subtype ayw and adr.     

Restriction fragment differences between the genomes of the Oka varicella vaccine virus and American wild-type varicella-zoster virus

The Oka vaccine strains of varicella-zoster virus (VZV) have a significantly different BgII DNA restriction pattern from that of American wild-type isolates of VZV. This difference consists primarily of an additional BgII site, which lies within the BamHI "D" fragment. In conjunction with a study of the efficacy of an experimental Merck/Oka VZV vaccine, the area of the genome from which the most marked restriction pattern alteration arises was studied more closely to determine if there are other significant differences between the Oka strains and American wild-type strains. BamHI "D" fragments from the DNA of the Oka parent strain (the progenitor of the vaccine strain), the RIT/Oka vaccine strain (a derivative of the Oka parent strain), the Merck/Oka vaccine strain, and the EF strain (an American wild type), were submitted to extensive endonuclease digestion studies to ascertain if additional unique restriction sites are present in the Oka parent or vaccine strains. The extra BgII restriction site characteristic of the Merck/Oka vaccine strain is also present in the DNA of the parent virus as well as its derivatives and was therefore not produced by the "attenuation" process. No other novel sites were found in the Oka parent or Oka-derived strains in this section of the genome. The Merck/Oka vaccine strain of VZV, despite its Japanese origin, is therefore quite similar to circulating American varicella-zoster virus strains. Varicella-zoster virus DNA, at least in the area of the BamHI D fragment, also appears to be remarkably stable from strain to strain.     

Biochemical characterization of I-Ak proteins from mutant antigen-presenting B-cell--B-lymphoma hybridomas

Chemically induced mutants of an I-Ak,d expressing antigen-presenting B-cell--B-lymphoma hybridoma have recently been generated by immunoselection in vitro and were found to possess alterations in some of their serologically and functionally defined I-Ak region dependent functions. In order to identify at the structural level the origin of the differences in serological and functional properties of these mutants, I-Ak molecules from several of these mutant hybridomas were compared biochemically to wild-type I-Ak polypeptides by two-dimensional gel electrophoresis and high-pressure liquid chromatographic tryptic peptide analyses. Two-dimensional gel electrophoresis indicated that no major structural alterations, resulting in changes in mol. wt or charge, had occurred in the Ak alpha or Ak beta polypeptides from the mutant cells. Likewise, Ak alpha peptide maps of the mutants were indistinguishable from the normal Ak alpha peptide maps. However, two of the three mutants studied did exhibit one additional peptide in their Ak beta peptide maps. These results suggest that the major deficiencies in T-cell-activating functions of these mutants are a result of a limited alteration in the Ak beta polypeptide primary structure.     

DNA polymorphism among isolates from multiple sites of a patient with chronic herpes simplex virus, type 1 infection

DNA polymorphisms among independent isolates of herpes simplex virus (HSV) type 1 were studied from a 7-year-old male patient with recurrent infections of the skin and internal organs. In the patient's serum, HSV antibodies could not be detected by complement fixation, enzyme-linked immunosorbent assay (ELISA), or neutralization tests. ELISA tests for the presence of antibodies to human immunodeficiency virus were also negative. One HSV isolate was obtained from mesenteric nodes biopsied in 1983; one from skin in 1984; and three (postmortem) from brain, lungs, and liver in 1985. Restriction enzymes Eco RI, Bgl II, Hind III, Kpn I, and Bam H1 digestion patterns of the five isolates were similar. However, Sal I digests of isolates from skin, mesenteric nodes, lungs, and liver showed variations that were distinct from that of the brain isolate. Although Sal I digests of skin, mesenteric nodes, lungs, and liver isolates share a common variation in lacking F and G, the liver isolate can be further differentiated because of the gain of a restriction site on the H fragment. Thus, the three distinct variants observed were the isolates from brain (variant 1); from skin, mesenteric nodes, and lungs (variant 2); and from liver (variant 3). The fragments involved in variations among these isolates (presence or absence of Sal, G and H) are from the unique short and long regions (invariable regions) of the genome and therefore do not show heterogeneity in size. The extent of variation among these isolates is less than that seen among epidemiologically unrelated strains, suggesting that they originated from a single infecting strain, probably the brain isolate.     

Membrane attack complex of complement and 20 kDa homologous restriction factor (CD59) in myocardial infarction

In order to investigate the mechanism of deposition of the complement membrane attack complex (MAC) in cardiomyocytes in areas of human myocardial infarction, the 20 kDA homologous restriction factor of complement (HRF20; CD59) and complement components (C1q, C3d and MAC) were analysed immunohistochemically using specific antibodies. Myocardial tissues obtained at autopsy from nine patients who died of acute myocardial infarction were fixed in acetone and embedded in paraffin. The ages of the infarcts ranged from about 3.5 h to 12 days. In cases of myocardial infarction of 20 h or less, MAC deposition was shown in the infarcted cardiomyocytes without loss of HRF20. Where the duration was 4 days or more, the cardiomyocytes with MAC deposition in the infarcted areas also showed complete loss of HRF20. Outside the infarcts, HRF20 in the cardiomyocytes was well preserved without MAC deposition. The present study suggests that the initial MAC deposition in dead cardiomyocytes can occur as a result of degradation of plasma-membrane by a mechanism independent of complement-mediated injury to the membrane. Loss of HRF20 from dead cardiomyocytes may not be the initial cause of MAC deposition, but may accelerate the deposition process of MAC in later stages of infarction.     

The effect of exhaustive exercise on the antioxidant enzyme system in skeletal muscle from calcium-deficient rats

 The aim of the current study was to elucidate the synergism of dietary calcium restriction and exhaustive exercise in the antioxidant enzyme system of rat soleus muscle, and to investigate the involvement of neutrophils in exercise-induced muscle damage. Forty-eight male Wistar rats were assigned to the following groups: control (C) or calcium-restricted [1 month (1 M) or 3 months (3 M)]. Each group was subdivided into acutely exercised or non-exercised groups. Soleus muscle from each rat was analysed to determine the levels of antioxidant enzymes [Mn-superoxide dismutase (SOD), Cu,Zn-SOD, glutathione peroxidase (GPX), and catalase (CAT)]. Dietary calcium restriction resulted in calcium deficiency and upregulated the antioxidant enzymes examined except GPX. Conversely, exhaustive exercise significantly decreased GPX and CAT, but not SODs activities in the calcium-restricted (1 M and/or 3 M) rats. Contents of immunoreactive Mn-SOD and Cu,Zn-SOD were only increased in the 3 M rats. During calcium restriction, the mRNA expression of both forms of SOD showed initial upregulation, followed by downregulation. Exhaustive exercise significantly increased the mRNA expressions only in the 3 M rats. Moreover, exhaustive exercise markedly increased myeloperoxidase activity in soleus muscles from the 1 M and 3 M rats compared with the C rats, and significantly enhanced the ability of neutrophils to generate superoxide in the 3 M rats. The results demonstrate that dietary calcium restriction upregulates certain antioxidant enzyme activities in rat soleus muscle, indicating an enhanced resistance to potential increases in intracellular reactive oxygen species. The results also suggest that exhaustive exercise may cause oxidative damage in soleus muscle of calcium-deficient rats through the activation of neutrophils. Received: 4 August 1997 / Received after revision: 29 September 1997 / Accepted: 26 November 1997