The potential use of DNA probes to identify and type strains within the Mycobacterium tuberculosis complex

DNA was extracted and purified from 11 strains of Mycobacterium bovis isolated from cattle in Ireland. After digestion with restriction endonuclease PvuII and electrophoresis on an agarose gel, the separated DNA fragments were transferred to a nylon membrane and sequentially hybridized with three DNA probes derived from BCG.
None of the three probes detected restriction fragment length polymorphism (RFLP) within the 11 M. bovis strains, indicating a very close genetic relationship. One probe, pBCG12, detected RFLPs between the M. bovis strains and a reference PvuII digest of DNA from M. tuberculosis R37Rv, confirming that M. bovis and M. tuberculosis are closely related though genetically distinct.     

Dot-blot assays and their use as a direct antigen-binding method to screen monoclonal antibodies to 1,4-beta- and 1,3-beta-glucan synthases

A rapid method has been developed to assay beta-glucan synthases spotted on a nitrocellulose sheet. The sensitivity of this method allows screening of hybridoma-making monoclonal antibodies in a direct antigen-binding assay by measurement of the activity of the enzymes retained by the antibodies previously fixed on nitrocellulose.     

Complete complementary DNA of rat tyrosine aminotransferase messenger RNA. Deduction of the primary structure of the enzyme

The primary structure of rat tyrosine aminotransferase (L-tyrosine:2-oxoglutarate aminotransferase; EC 2.6.1.5), a liver-specific enzyme involved in gluconeogenesis, has been deduced from the nucleotide sequence of a cloned full-length cDNA. The mRNA is 2362 nucleotides long (excluding the poly(A) tail) and codes for a polypeptide of 454 amino acids with a molecular weight of 50634. Unambiguous identification was obtained by comparison of this sequence with the amino acid sequences of several peptides obtained from the purified enzyme.     

Mycobacterial disease—a challenge to biotechnology

Summary The two principal mycobacterial diseases — tuberculosis and leprosy — remain major causes of human suffering in many parts of the world. The struggle against these diseases has been hampered by the inadequacy of methods for their diagnosis, prevention and therapy. Improvements in diagnostic methods, especially for tuberculosis, are required as existing bacteriological techniques are time consuming and insensitive, but immunological tests suffer from a lack of specificity and a failure to distinguish active disease from past sensitization. Tests are required that will either detect the presence of mycobacterial antigens or the occurrence of immune reactions specific to active infection. Prophylaxis by BCG vaccine is never complete and in some regions appears virtually ineffective. In order to determine the reason for this variation, to produce better vaccines, and to use BCG more effectively, a deeper understanding of the immune reactions in mycobacterial disease is required. In particular, the bacterial determinants of virulence and protection and the mode of action of BCG require detailed study. The therapy of tuberculosis and, to a lesser extent, leprosy has undergone an extensive transformation since the introduction of rifampicin. Nevertheless the cost of modern short course regimens for tuberculosis limit their application. Even shorter regimens, made possible by more potent bactericidal drugs or by agents that stimulate the host's defences are thus required. Recent advances in biotechnology could therefore lead to significant advances in the control of these diseases but only if they are integrated into local, self-reliant public health initiatives.

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Enfermedades producidas por microbacterias. Un desafío a la biotecnologia

Resumen La dos principales enfermedades producidas por micobacterias — tuberculosis y lepra —persisten como las causas más importantes del sufrimiento humano en muchas partes del mundo. La lucha contra estas enfermedades se ha visto dificultada por lo inadecuado de los métodos para su diagnosis, prevención y terapia. Es necesaria una mejora de los métodos de diagnosis, especialmente en el caso de la tuberculosis, ya que las técnicas bacteriológicas existentes son poco sensibles y consumen mucho tiempo y los tests inmunológicos son inespecíficos y no permiten distinguir entre la enfermedad activa y la sensibilidad adquirida. Se necesitan tests capaces de detectar la presencia de antígenos microbacterianos o la existencia de reacciones de inmunización específicas de la infección activa. La profilaxis con la vacuna BCG no es nunca completa y en algunas regiones es virtualmente enefectiva. Con objeto de determinar las razones de esta variación, de producir mejores vacunas y de usar la BCG de forma más eficiente, se necesita entender con más profundidad las reacciones de inmunización en las enfermedades por micobacterias. En particular los determinantes bacterianos de la virulencia y de la protección y la forma de actuar de la BCG precisan de un estudio detallado. La terapia de la tuberculosis y en menor grado la de la lepra, han sufrido una extensa transformación desde la introducción de la rifampicina. Sin embargo, el costo de los modernos tratamientos a corto plazo de la tuberculosis limítan su aplicación. Se requieren tratamientos aún más cortos basados en el desarrollo de drogas bactericidas más potentes o de agentes que estimulen las defensas del enfermo. Los recientes avances en biotecnología podrían por tanto conducir a avances importantes en el control de estas enfermedades pero solamente si se integran en iniciativas médicas públicas consistentes.

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Les maladies à mycobactéries — Un enjeu pour les biotechnologies

Résumé Les deux principales maladies à mycobactéries — la tuberculose et la lèpre — sont toujours des causes majeures de souffrance humaine dans de nombreuses parties du monde. La lutte contre ces maladies est entravée par l'inadéquation des méthodes diagnostiques, préventives et thérapeutiques. Spéciallement en ce qui concerne la tuberculose, l'amélioration des moyens de diagnostic est rendue nécessaire par le fait que les techniques bactériologiques existantes sont longues et peu sensibles, et que les tests immunologiques, n'étant pas spécifiques, ne permettent pas de distinguer entre une maladie en évolution et une sensibilisation ancienne. On a besoin de tests permettant de déceler soit la présence d'antigènes mycobactériens, soit l'apparition de réactions immunitaires spécifiques au cours d'une infection évolutive. La prophylaxie par le BCG n'est jamais complète et parait être pratiquement inefficace dans certaines régions. Pour expliquer cette variation géographique, produire de meilleurs vaccins, et utiliser le BCG efficacement, il convient de mieux connaître les réactions immunitaires au cours des maladies à mycobactéries. En particulier, les déterminants bactériens de la virulence et de la protection, ainsi que le mode d'action du BCG sont à approfondir. La rifampicine a considérablement transformé la thérapeutique de la tuberculose et, à un moindre degré, de la lèpre. Néanmoins, le coût des nouveaux traitements intensifs de la tuberculose limite leur application. Il est nécessaire que des médicaments bactéricides plus puissants ou des agents stimulant les défenses de l'hôte puissent raccourcir encore davantage la durée des traitements. Le progrès biotechnologique peut donc améliorer de façon significative le contrôle des maladies considérées, à la seule condition que ce but soit volontairement intégré dans les initiatives locales en matière de santé publique.

     

Simultaneous potentiation and fatigue in quadriceps after a 60-second maximal voluntary isometric contraction

Potential mechanisms of fatigue (metabolic factors) and potentiation (phosphate incorporation by myosin phosphorylatable light chains) were investigated during recovery from a 60-s maximal voluntary isometric contraction (MVC) in the quadriceps muscle of 12 subjects. On separate days before and for 2 h after the 60-s MVC, either a 1-s MVC or electrically stimulated contractions were used as indexes to test muscle performance. Torque at the end of the 60-s MVC was 57% of the initial level, whereas torques from a 1-s MVC and 50-Hz stimulation were most depressed in the immediate recovery period. At this time, muscle biopsy analyses revealed significant decreases in ATP and phosphocreatine and a 19-fold increase in muscle lactate. Conversely, isometric twitch torque and torque from a 10-Hz stimulus were the least depressed of six contractile indexes and demonstrated potentiation of 25 and 34%, respectively, by 4 min of recovery (P less than 0.05). At this time, muscle lactate concentration was still 16 times greater than at rest. An increased phosphate content of the myosin phosphorylatable light chains (P less than 0.05) was also evident both immediately and 4 min after the 60-s MVC. We conclude that the 60-s MVC produced marked force decreases likely due to metabolic displacement, while the limited decline in the twitch and 10-Hz torques and their significant potentiation suggested that myosin phosphorylation may provide a mechanism to enhance contractile force under conditions of submaximal activation during fatigue.     

Technical considerations for assessing alterations in skeletal muscle sarcoplasmic reticulum Ca++-sequestration functionin vitro

A multiple measurement system for assessing sarcoplasmic reticulum (SR) Ca⁺⁺-ATPase activity and Ca⁺⁺-uptake was used to examine the effects of SR fractionation and quick freezing on rat white (WG) and red (RG) gastrocnemius muscle.In vitro measurements were performed on whole muscle homogenates (HOM) and crude microsomal fractions (CM) enriched in SR vesicles before and after quick freezing in liquid nitrogen. Isolation of the CM fraction resulted in protein yields of 0.96±0.1 and 0.99±0.1 mg/g in WG and RG, respectively. The percent Ca⁺⁺-ATPase recovery for CM compared to HOM was 14.5% (WG) and 10.1% (RG). SR Ca⁺⁺-activated Ca⁺⁺-ATPase activity was not affected by quick freezing of HOM or CM, but basal ATPase was reduced (P<0.05) in frozen HOM (5.12±0.18–3.98±0.20 mole/g tissue/min in WG and from 5.39±0.20–4.48±0.24 mole/g tissue/min in RG). Ca⁺⁺-uptake was measured at a range of physiological free [Ca⁺⁺] using the Ca⁺⁺ fluorescent dye Indo-1. Maximum Ca⁺⁺-uptake rates when corrected for initial [Ca⁺⁺]_f were not altered in HOM or CM by quick freezing but uptake between 300 and 400nM free Ca⁺⁺ was reduced (P<0.05) in quick frozen HOM (1.30±0.1–0.66±0.1 mole/g tissue/min in WG and 1.04±0.2–0.60±0.1 mole/g tissue/min in RG). Linear correlations between Ca⁺⁺-uptake and Ca⁺⁺-ATPase activity measured in the presence of the Ca⁺⁺ ionophore A23187 were r=+0.25, (P<0.05) and r=+0.74 (P<0.05) in HOM and CM preparations, respectively, and were not altered by freezing. The linear relationships between HOM and CM maximum Ca⁺⁺-uptake (r=+0.44, P<0.05) and between HOM and CM Ca⁺⁺-ATPase activity (r=+0.34, P<0.05) were also not altered by tissue freezing. These data suggest that alterations in maximal SR Ca⁺⁺-uptake function and maximal Ca⁺⁺-ATPase activity may be measured in both HOM and CM fractions following freezing and short term storage. (Mol Cell Biochem139, 41–52, 1994)     

Specific Activity of Brain Palmitoyl-CoA Pool Provides Rates of Incorporation of Palmitate in Brain Phospholipids in Awake Rats

Abstract: In vivo rates of palmitate incorporation into brain phospholipids were measured in awake rats following programmed intravenous infusion of unesterified [9,10-³H]palmitate to maintain constant plasma specific activity. Animals were killed after 2–10 min of infusion by microwave irradiation and analyzed for tracer distribution in brain phospholipid and phospholipid precursor, i.e., brain unesterified palmitate and palmitoyl-CoA, pools. [9,10-³H]Palmitate incorporation into brain phospholipids was linear with time and rapid, with >50% of brain tracer in choline-containing glycerophospholipids at 2 min of infusion. However, tracer specific activity in brain phospholipid precursor pools was low and averaged only 1.6–1.8% of plasma unesterified palmitate specific activity. Correction for brain palmitoyl-CoA specific activity increased the calculated rate of palmitate incorporation into brain phospholipids (0.52 nmol/s/g) by ∼60-fold. The results suggest that palmitate incorporation and turnover in brain phospholipids are far more rapid than generally assumed and that this rapid turnover dilutes tracer specific activity in brain palmitoyl-CoA pool owing to release and recycling of unlabeled fatty acid from phospholipid breakdown.     

Failure of short term stimulation to reduce sarcoplasmic reticulum Ca2+-ATPase function in homogenates of rat gastrocnemius

To examine the effect of short term intense activity on sarcoplasmic reticulum (SR) Ca²⁺ sequestering function, the gastrocnemius (G) muscles of 11 anaesthetized male rats (weight, 411±8 g,X±SE) were activated using supramaximal, intermittent stimulation (one train of 0.2 msec impulses per sec of 100 msec at 100 Hz). Homogenates were obtained from stimulated white (WG-S) and red (RG-S) tissues, assayed for Ca²⁺ uptake and maximal Ca²⁺ ATPase activity and compared to contralateral controls (WG-C, RG-C). Calcium uptake (nmoles/mg protein/min) determined using Indo-l and at [Ca²⁺]_f concentrations between 300–400 nM was unaffected (p>0.05) by activity in both WG (6.14+0.43 vs 5.37+0.43) and RG (3.21+0.18 vs 3.07+0.20). Similarly, no effect (p>0.05) of contractile activity was found for maximal Ca²⁺ ATPase activity (mole/mg protein/min) determined spectrophotometrically in RG (0.276+0.03 vs 0.278+0.02). In WG, Ca²⁺ ATPase activity was 15% higher in WG-S compared to WG-C (0.412+0.03 vs 0.385+0.04). Repetitive stimulation resulted in a reduction in tetanic tension of 74% (p<0.05) by 2 min in the G muscle. By the end of the stimulation period, ATP concentration was reduced (p<0.05) by 57% in the WG and by 47% in the RG. These results indicate that the repeated generation of maximal tetanic force, at least for short term periods, need not adversely affectin vitro homogenate determination of Ca²⁺ sequestering function in spite of severe alterations in energy potential and that some other mechanism must be involved to explain the depression in Ca²⁺ uptake and Ca²⁺ ATPase activity previously noted with short term intense exercise.