Glycolytic and non-glycolytic functions of Mycobacterium tuberculosis fructose-1,6-bisphosphate aldolase, an essential enzyme produced by replicating and non-replicating bacilli

The search for antituberculosis drugs active against persistent bacilli has led to our interest in metallodependent class II fructose-1,6-bisphosphate aldolase (FBA-tb), a key enzyme of gluconeogenesis absent from mammalian cells. Knock-out experiments at the fba-tb locus indicated that this gene is required for the growth of Mycobacterium tuberculosis on gluconeogenetic substrates and in glucose-containing medium. Surface labeling and enzymatic activity measurements revealed that this enzyme was exported to the cell surface of M. tuberculosis and produced under various axenic growth conditions including oxygen depletion and hence by non-replicating bacilli. Importantly, FBA-tb was also produced in vivo in the lungs of infected guinea pigs and mice. FBA-tb bound human plasmin(ogen) and protected FBA-tb-bound plasmin from regulation by α₂-antiplasmin, suggestive of an involvement of this enzyme in host/pathogen interactions. The crystal structures of FBA-tb in the native form and in complex with a hydroxamate substrate analog were determined to 2.35- and 1.9-Å resolution, respectively. Whereas inhibitor attachment had no effect on the plasminogen binding activity of FBA-tb, it competed with the natural substrate of the enzyme, fructose 1,6-bisphosphate, and substantiated a previously unknown reaction mechanism associated with metallodependent aldolases involving recruitment of the catalytic zinc ion by the substrate upon active site binding. Altogether, our results highlight the potential of FBA-tb as a novel therapeutic target against both replicating and non-replicating bacilli.     

Major morphological effects of a regulatory gene: Pgm1-t in rainbow trout

We have investigated the morphological effects of a genetic locus, Pgm1- t, that affects the expression of a phosphoglucomutase locus (Pgm1) in liver of rainbow trout (Salmo gairdneri). We have previously shown that embryos with liver Pgm1 expression hatch earlier than those without liver Pgm1 expression. We predicted that this difference in developmental rate should cause a reduction in meristic counts in the more rapidly developing fish with liver Pgm1 expression. Eight meristic (countable) characters in nine full-sib groups segregating for the presence or absence of liver Pgm1 expression are in agreement with this prediction. In eight of the nine families, there is a significant difference in the multivariate distribution of the eight meristic counts between full sibs with and without liver Pgm1 expression. This separation in multivariate space is based on a tendency for lower meristic counts in fish with liver Pgm1 expression. The magnitude of these morphological differences is similar to that between two subspecies of cutthroat trout (Salmo clarki) that show substantial genetic divergence at structural loci encoding enzymes (Nei's D = 0.34). These data support the view that small changes in the developmental process caused by genetic differences at regulatory genes can have large effects on morphology.      

Results of treatment of extensive volar wrist lacerations: the spaghetti wrist

A series of 38 volar wrist lacerations is reviewed with regard to epidemiologic aspects and results. In general, return of tendon function was quite good, and return of nerve function in this series was also satisfying. We attribute the generally good results to immediate repair of all structures, microscopic repair of significant arterial injuries, microscopic grouped fascicular nerve repair, early mobilization (dynamic splinting and intensive occupational therapy), and a generally youthful group of patients. Review of this series has strengthened our opinion that aggressive primary repair of all injured structures is appropriate for these extensive lacerations.     

Genetic analysis in families with van der Woude syndrome

We have brought together information on 864 affected individuals in 164 families (including three new pedigrees) reported in the 137 year period since 1845 when Demarquay first described a family with what was later called van der Woude syndrome (VWS). Both types of oral cleft, cleft palate (CP) and cleft lip with or without CP (CLP), segregate in these families together with lower lip pits or fistulae in an autosomal dominant mode with high penetrance estimated to be K = .89 and .99 by different methods. Cleft types (CLP and CP) occur in VWS in the same proportions as in the general non-VWS population, ie, about twice as many cleft-bearing individuals have CLP as have CP. On the other hand, we do not find the usually observed excess of females with CP and excess of males with CLP; in VWS the sex ratios are more nearly equal. Lip pits also are equally distributed between the sexes. Affected males and females are equally likely to transmit VWS. However, there is an excess of less severely affected individuals among transmitters and a deficiency of more severely affected, brought about by a proband bias and differential fecundity. The expression of VWS is significantly modified by the genetic background: More extreme phenotypes in parents tend to produce more extreme expression in their children. For a VWS gene carrier the relative risk of transmitting a cleft is 26.45%; that of transmitting lower lip pits is 23.55%. Three pedigrees of lip pits in the literature show no clefts among a significant number of affected individuals. Control of gene expression in VWS in the three target tissues appears to be independent and separately designated. Mutation rate of the VWS gene is calculated to be 1.8 X 10(-5).     

Ischemic myocardial injury in cultured heart cells: Leakage of cytoplasmic enzymes from injured cells

Summary An in vitro model of myocardial ischemia has been established with primary monolayer cultures of postnatal rat myocardial cells. Ischemic conditions were simulated in vitro by subjecting the myocardial cell cultures to various levels of oxygen and glucose deprivation. The experimental protocol consisted of treatment with 20% or 0% O₂ and 1000, 500 or 0 mg glucose per 1 of medium for 4 or 24 hr. Control cultures were treated with 20% O₂ and 1000 mg glucose. After the ischemic treatments, cultures of beating muscle (M) cells were evaluated for signs of injury, i.e. leakage of cytoplasmic enzymes into the culture medium. Differences were found in leakage of lactate dehydrogenase (LDH) and creatine phosphokinase (CPK) from the cultures that were exposed to partial ischemia of glucose deprivation and from those cultures that were exposed to total ischemia of oxygen and glucose deprivation. Glucose deprivation alone resulted in a slight-to-moderate loss of LDH and CPK from the cells, whereas total ischemia resulted in a significant release of the two cytoplasmic enzymes. When the cultures were allowed to recover after ischemic treatment in complete medium (1000 mg glucose) and a normal atmosphere of 20% O₂, they had levels of LDH leakage comparable to those of control cultures. Cell viability and total protein content of the ischemic cultures did not differ significantly from controls. This study was supported by Research Grant HL 18647 from the National Heart, Lung, and Blood Institute.     

Successful treatment of acute vascular insufficiency in a hand by intra-arterial fibrinolysin, heparin, and reserpine. Case report.

A patient is presented who presumably had multiple drugs injected intra-arterially in the right hand. Clinical signs and angiographic evidence of severe vascular insufficiency were well demonstrated. Therapy with fibrinolysin, reserpine, and heparin was followed by a return of normal circulation.     

Is reversed venous flow safe in free-flap transfer? A dilemma with the radial forearm flap.

The distally based radial artery forearm flap has become our workhorse flap for hand and finger coverage, relying on reversed or retrograde venous outflow through the venae comitantes. Free-flap transfer, however, has been used by us only with antegrade venous anastomoses. This study was intended to determine if a single retrograde venous anastomosis would be adequate for flap viability. Six groups of saphenous flaps were developed in New Zealand White rabbits. In situ flaps compared antegrade with retrograde venous outflow in groups 1 and 2. Microvascular venous anastomoses with antegrade or retrograde outflow were compared in groups 3 and 4. Free-flap transfer with antegrade or retrograde venous outflow was compared in groups 5 and 6. No significant differences in survival was found between groups 1 and 2. A significant difference in survival (p = 0.025) was found between groups 3 and 4, but technical differences make these groups incomparable. Significantly better survival (p = 0.014, chi-squared test) was found in group 5 with antegrade outflow versus group 6 with retrograde outflow.     

Impedance plethysmography: a new method for continuous muscle perfusion monitoring

Vigilant postoperative monitoring of the buried muscle flap is critical after free transfer because early diagnosis of vascular insufficiency is essential to allow prompt correction. We have identified a monitoring method utilizing needle electrodes and impedance plethysmography that gives a beat-to-beat representation of muscular perfusion. In 25 New Zealand White rabbits the gastrocnemius muscle was isolated on its vascular pedicle, and two intramuscular needle electrodes were placed. The instantaneous impedance changes of the muscle (corresponding to the pulsatile volume changes of perfusion) were measured and recorded. Using this representation of perfusion, an independent judge was able to correctly diagnose muscular ischemia 100 percent of the time (n = 25). Further, the judge was able to correctly distinguish the ischemia as arterial (n = 10) or venous (n = 10) in origin 100 percent of the time. Additionally, we monitored muscle perfusion transcutaneously in five free muscle flaps and demonstrated a reliable impedance signal that correlated with perfusion.     

Phorid flies, Pseudacteon spp. (Diptera: Phoridae), affect forager size ratios of red imported fire ants Solenopsis invicta (Hymenoptera: Formicidae) in Texas

Multiple species of Pseudacteon phorid flies (Diptera: Phoridae) are currently being released throughout the southern United States to aid biological control of red imported fire ants, Solenopsis invicta Buren (Hymenoptera: Formicidae). It is anticipated that these flies will interfere with S. invicta foraging, allowing native ant assemblages to outcompete S. invicta for available resources. Numerous studies have shown a decrease in S. invicta foraging intensity when exposed to phorids. This study documents a behavioral change in phorid-exposed S. invicta colonies at a phorid release site in central Texas. Significant differences in forager size ratios were detected between phorid-exposed and phorid-absent colonies. A similar phenomenon was recently documented in the native range of these insects in South America as well. Experimental manipulation of ratios of S. invicta worker sizes has been shown to have important effects on colony success. This newly documented phorid-mediated S. invicta colony-level effect represents a significant shift in S. invicta foraging dynamics and may provide an additional mechanism by which phorids can influence S. invicta populations in their United States range.