Rapid susceptibility testing for slowly growing nontuberculous mycobacteria using a colorimetric microbial viability assay based on the reduction of water-soluble tetrazolium WST-1.

Rapid susceptibility testing for slowly growing nontuberculous mycobacteria (NTM) using a colorimetric microbial viability assay based on the reduction of the water-soluble tetrazolium salt {2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt (WST-1)} using 2,3,5,6-tetramethyl-1,4-benzoquinone as an electron mediator was developed. Using the Clinical and Laboratory Standards Institute (CLSI) method, a long-term incubation time (7-14 days) was required to determine the minimum inhibitory concentrations (MICs) of the slowly growing NTM. The MICs for a variety of different antibiotics against the slowly growing NTM were determined by the WST-1 colorimetric method and compared with those obtained using the broth microdilution methods approved by the CLSI. Good agreement was found between the MICs determined after 3-4 days using the WST-1 colorimetric method and those obtained after 10-14 days using the broth microdilution method. The results suggest that the WST-1 colorimetric assay is a useful method for the rapid determination of the MICs for the slowly growing NTM.

Are animal models useful for understanding the pathophysiology of lysosomal storage disease?

UNLABELLED: Spontaneously occurring genetic lysosomal storage diseases are as rare in other mammalian species as in man. However, the advent of gene targeting technology has revolutionized the state of animal models of genetic diseases. Nearly all lysosomal storage diseases known in man have been duplicated in the mouse. The technology now allows, not only complete inactivation of endogenous genes, but also the introduction of essentially any type of mutation. These animal models can overcome many of the limitations inherent in studies of human patients--rarity of the disease, extremely complex genetic background and logistical and ethical constraints in the design and execution of experiments with human subjects. For example, genetic manipulations of germ cells or cross-breeding experiments between two mutants are readily feasible with animal models. Two major areas of the utility of animal models are the clarification of the pathophysiology/pathogenetic mechanism of disease and the exploration of therapeutic approaches. Examples of experiments using animal models of lysosomal storage disease are presented, primarily from studies undertaken in our own laboratory. CONCLUSION: Animal models have proved invaluable in extending our knowledge of the lysosomal storage diseases and exploring potential therapies.

Paradoxical influence of acid beta-galactosidase gene dosage on phenotype of the twitcher mouse (genetic galactosylceramidase deficiency).

We have cross-bred twitcher mice (galactosylceramidase deficiency) and acid beta-galactosidase knockout mice (G(M1) gangliosidosis) and found that the acid beta-galactosidase gene dosage exerts an unexpected and paradoxical influence on the twitcher phenotype. Twitcher mice with an additional complete deficiency of acid beta-galactosidase have the mildest phenotype with the longest lifespan and nearly rescued CNS pathology. In contrast, twitcher mice with a single functional acid beta-galactosidase gene have the most severe disease with the shortest lifespan, despite the fact that G(M1) gangliosidosis carrier mice with an otherwise normal genetic background are phenotypically normal. A significant proportion of these galc(-/-), bgal(+/-) mice clinically develop additional extreme hyper-reactivity and generalized seizures not seen in any other genotypes. Consistent with the clinical seizures, widespread neuronal degeneration is present in the galc(-/-), bgal(+/-) mice, most prominently in the CA3 region of the hippocampus. The double knockout mice show a massive accumulation of lactosylceramide in all tissues. The brain inexplicably contains only a half-normal amount of galactosylceramide, which may account for the mild clinical and pathological phenotype. On the other hand, brain psychosine level is increased in all twitcher mice, but galc(-/-), bgal(+/-) mice show a significantly higher level than other genotypes. The reduced galactosylceramide in the brain of the double knockout mice and the significantly higher psychosine in the brain of the galc(-/-), bgal(+/-) mice cannot readily be explained from the genotypes of these mice. These observations are contrary to the expected outcome of Mendelian autosomal recessive single gene disorders and may also be interpreted as that the acid beta-galactosidase gene functions as a modifier gene for the phenotypic expression of genetic galactosylceramidase deficiency.

Biochemistry and neuropathology of mice doubly deficient in synthesis and degradation of galactosylceramide.

We have generated mice doubly deficient in both synthesis and degradation of galactosylceramide by cross-breeding twitcher mice and galactosylceramide synthase (UDP-galactose:ceramide galactosyltransferase, CGT) knockout mice. The prediction that the phenotype of the doubly deficient mice should be the same as the cgt -/- mice, since the degrading enzyme should not be necessary if the substrate is not synthesized, proved to be only partially correct. In early stages of the disease, the doubly deficient mice (galc -/-, cgt -/-) were essentially indistinguishable from the cgt -/- mice. However, the doubly deficient mice had a much shorter life span than cgt -/- mice. Both galactosylceramide and galactosylsphingosine (psychosine), were undetectable in the brain of the cgt -/- and the doubly deficient mice. The characteristic twitcher pathology was never seen in the galc -/-, cgt -/- mice. However, after 43 days, neuronal pathology was observed in the brainstem and spinal cord. This late neuronal pathology has not been seen in the CGT knockout mice but has been described in some long surviving bone marrow-transplanted twitcher mice. Furthermore, the motor segment of the trigeminal nerve of the galc -/-, cgt -/- mice showed severe degeneration not seen in either twitcher or CGT knockout mice. Thus, the galc -/-, cgt -/- mice, while primarily showing the cgt -/- phenotype as predicted, develop late pathology that is seen only in twitcher mouse and also a unique pathology in the trigeminal nerve. These observations indicate that the functional relationship between galactosylceramidase and galactosylceramide synthase is complex.

Twitcher mice with only a single active galactosylceramide synthase gene exhibit clearly detectable but therapeutically minor phenotypic improvements.

Cross-breeding of mouse mutants, each defective in either synthesis (CGT knockout) or degradation (twitcher) of galactosylceramide, generates hybrids with a genotype of galc -/-, cgt +/-, in addition to doubly deficient mice. They are ideally suited to test the potential usefulness of limiting synthesis of the substrate as a treatment of genetic disorders due to degradative enzyme defects. The rate of accretion of galactosylceramide in the brain of CGT knockout carrier mice (cgt +/-) is approximately two-thirds of the normal, suggesting a gene-level compensation for the reduced gene dosage. Phenotype of twitcher mice with a single dose of normal cgt gene was indeed milder with statistical significance, albeit only slightly. Compared among 10 paired littermates, the difference in the life span was 7+/-3.9 days (S.D.) and the difference in the maximum attained body weight was 1.9+/-1.2 g (S.D.). Neuropathologists were able to distinguish blindly galc -/-, cgt +/- mice from galc -/-, cgt +/+ mice. The brain psychosine level in galc -/-, cgt +/- mice was also approximately two-thirds of the galc -/-, cgt +/+ mice. These observations indicate that reduction of galactosylceramide synthesis to two-thirds of the normal level results in minor but clearly detectable phenotypic improvements. Because of the detrimental consequences of drastic reduction in galactosylceramide synthesis that may be required for pragmatically meaningful improvements, this approach by itself is unlikely to be useful as the sole treatment but may be helpful as a supplement to other therapies.

Ceramide accumulation is associated with increased apoptotic cell death in cultured fibroblasts of sphingolipid activator protein-deficient mouse but not in fibroblasts of patients with Farber disease.

Ceramide is recognized as an intracellular mediator of cell growth, differentiation and apoptosis. Tumour necrosis factor, anti-fas antibody, radiation and anticancer drugs such as actinomycin D are known to induce apoptosis in several cell types through generation of ceramide by activation of the sphingomyelinase pathway or ceramide synthetase. In this study, we examined the occurrence of apoptosis in fibroblasts from patients with Farber disease and from sphingolipid activator protein-deficient (sap -/-) mouse. These cells accumulate ceramide as the result of genetic deficiency of acid ceramidase and the ceramidase activator (sap-D), respectively. Amounts of ceramide in fibroblasts from Farber patients and in fibroblasts from sap -/- mouse were increased 2.9-fold and 2.8-fold, respectively, over the level of controls. Despite the similar degree of ceramide accumulation, cells exhibiting apoptotic features were increased only in fibroblasts from the sap -/- mouse but not those from the Farber patients. Thymidine uptake of Farber fibroblasts was normal while that of sap -/- mouse fibroblasts was twice normal, consistent with the apparently normal growth and the different rates of apoptotic cell death in these two cell lines. These data suggest that intralysosomal accumulation of ceramide due to defective acid ceramidase or its activator may not play an important role as a mediator of apoptosis. The increased apoptosis in the cultured fibroblasts from the sap -/- mouse may be caused by mechanisms other than the ceramide accumulation. Although more frequent than normal, significant apoptotic cell death was not observed in sap -/- mouse brain in vivo.

Application of an automatic molecular-replacement procedure to crystal structure analysis of cytochrome c2 from Rhodopseudomonas viridis.

An automatic molecular-replacement procedure has been applied to solve the crystal structure of cytochrome c(2) from Rhodopseudomonas viridis. The structure was solved on the basis of the structure of tuna cytochrome c as a search model using an automatic processing program system, AUTOMR. The refinements by molecular dynamics and restrained least-squares methods result in a current crystallographic R factor of 0.219 for diffraction data at 3 A resolution.

Structural similarity of cytochrome c2 from Rhodopseudomonas viridis to mitochondrial cytochromes c revealed by its crystal structure at 2.7 A resolution.

The crystal structure of cytochrome c2 from Rhodopseudomonas viridis has been refined using molecular dynamics and restrained least-squares methods to a crystallographic R-factor of 0.216 at 2.7 A resolution. A structural comparison between Rps. viridis cytochrome c2 and the other bacterial cytochromes c2 or mitochondrial cytochromes c indicates that the overall protein foldings are very similar to each other with the exception of the surface loop and terminal region of the polypeptide chain. However, the position and hydrogen-bond pattern of the evolutionarily conserved water molecule buried within the heme binding pocket in Rps. viridis cytochrome c2 are common to those in the mitochondrial cytochromes c. This fact indicates that Rps. viridis cytochrome c2 is structurally more similar to mitochondrial cytochromes c than to the other bacterial cytochromes c2.

Determination of free N-acetylneuraminic acid in human body fluids by high-performance liquid chromatography with fluorimetric detection.

Determinations of both the free and bound form of N-acetyl-neuraminic acid (NANA) in several human body fluids, such as serum, cerebrospinal fluid (CSF), saliva, urine, amniotic fluid, and milk were carried out by HPLC with fluorimetric detection. The method utilized 1,2-diamino-4,5-methylenedioxybenzene dihydrochloride (DMB) as a fluorimetric derivatizing reagent. Free-form NANA was obtained from the body fluids after ultrafiltration with Microcon 10 (YM-10 cellulose membrane, filtration limit M(r) = 10,000, Amicon). The DMB derivative of NANA was separated isocratically by a Nucleosil 5C18 column with a mixture of 0.1 M sodium phosphate buffer (pH 2.0)-methanol (75:25, v/v). A gradient elution system was used for urine analysis. Analysis times were 10-30 min. Recoveries of free NANA by ultrafiltration were satisfactory: 95.66 +/- 1.80% for serum and 97.27 +/- 1.55% for CSF, respectively. The high sensitivity and specificity render this method applicable to all the body fluids tested. Although a physiological role for free NANA has not yet been elucidated, the method presented promises to contribute to the basic understanding of the NANA metabolism.

A case of Canavan disease: the first biochemically proven case in a Japanese girl.

Canavan disease (CD) has only been diagnosed on autopsy or brain biopsy, however, specific biochemical markers, such as N-acetylaspartic acid (NAA) and aspartoacylase activity, have recently been described in CD. We report a case of CD having the above biochemical markers. High levels of NAA were found in her urine, serum and CSF. Fibroblasts did not exhibit aspartoacylase activity. Clinically, she presented progressive psychomotor retardation, cerebellar signs, pyramidal signs and relative megalencephaly. CT and MRI showed findings of leukodystrophy. The evoked potentials showed widespread involvement in the brainstem. Magnetic resonance spectra showed a high level of NAA in the white matter. In Japan, this case is the first of CD determined on the basis of biochemical markers.

Crystallization and preliminary X-ray diffraction studies of C-phycocyanin from a red alga, Porphyra tenera.

C-Phycocyanin from a red alga, Porphyra tenera, has been crystallized by the vapor-diffusion procedure. Both orthorhombic and hexagonal forms were obtained from ammonium sulfate solutions, whereas only the orthohombic form was selectively grown from sodium citrate solutions. The orthorhombic crystals are more suitable for further crystallographic work; their space group is P2(1)2(1)2(1), with unit-cell dimensions of a = 105, b = 121, and c = 184 A. The asymmetric unit comprises two (alpha beta)3 trimer molecules of C-phycocyanin. These crystals diffract X-rays up to about 3 A resolution.