Oil gland and oil pores in billfishes: in search of a function.

Billfishes are well known for their distinctive elongated rostra, i.e. bills. The functional significance of billfish rostra has been frequently discussed and the recent discovery of an oil gland (glandula oleofera) at the base of the rostrum in swordfish, Xiphias gladius, has added an interesting facet to this discussion regarding the potential co-evolution of gland and rostra. Here, we investigated the oil gland and oil pores (through which the oil is brought to the skin surface) of four billfish species - swordfish, Atlantic blue marlin (Makaira nigricans), Indo-Pacific sailfish (Istiophorus platypterus) and striped marlin (Kajikia audax) - and provide detailed evidence for the presence of an oil gland in the last three. All four species had a high density of oil pores on the forehead which is consistent with the hypothesis of hydrodynamic benefits of the oil. The extension of the pores onto the front half of the rostrum in sailfish and striped marlin, but not in swordfish or blue marlin, suggests that the oil may have additional functions. One such function could be linked to the antibacterial and anti-inflammatory properties of the oil. However, the available evidence on predatory rostrum use (and hence the likelihood of tissue damage) is only partly consistent with the extension of pores on rostra across species. We conclude that the oil gland probably serves multiple, non-mutually exclusive functions. More detailed information on rostrum use in blue marlin and swordfish is needed to better link behavioural and morphological data with the aim of accomplishing a full comparative analysis.

Alternative male mating tactics of the endemic Red Sea parrotfish Scarus ferrugineus.

The present study shows that small non-territorial terminal-phase males of the rusty parrotfish Scarus ferrugineus are reproductively active and are comparable with initial-phase males in behaviour, rates of participation during group-spawning and success in streaking into pair spawning. Large territorial terminal-phase males defend contiguous territories for several hours during the morning where they pair spawn with initial-phase females.

Seasonal and life-phase related differences in growth in Scarus ferrugineus on a southern Red Sea fringing reef.

Temporal trends in growth of the rusty parrotfish Scarus ferrugineus were studied on a southern Red Sea fringing reef that experiences seasonal changes in environmental conditions and benthic algal resources. Length increment data from tagging and recapture were compared among periods and sexes and modelled using GROTAG, a von Bertalanffy growth model. The growth pattern of S. ferrugineus was highly seasonal with a maximum occurring between April and June and a minimum between December and March. Body condition followed the seasonal variation in growth, increasing from April to June and decreasing from December to March. The season of maximum growth coincided with high irradiation, temperature increases and peak abundance of the primary food source, the epilithic algal community. There was a decline in growth rate during summer (July to October) associated with a combination of extreme temperatures and lowered food availability. There were strong sexual size dimorphism (SSD) and life-history traits. Terminal-phase (TP) males achieved larger asymptotic lengths than initial-phase individuals (IP) (L(∞) 34·55 v. 25·12 cm) with growth coefficients (K) of 0·26 and 0·38. The TPs were growing four times as fast as IPs of similar size. Three individuals changed from IP to TP while at liberty and grew eight times faster than IPs of similar size, suggesting that sex change in S. ferrugineus is accompanied by a surge in growth rate. The SSD in S. ferrugineus thus coincided with fast growth that started during sex change and continued into the TP. Faster growth during sex change suggests that the cost associated with sex change is limited.

An opinion paper: emphasis on white muscle development and growth to improve farmed fish flesh quality.

Due to rapid depletion of wild stocks, the necessity to cultivate fish is eminent. Current fish farming practices seek to improve flesh quality. The notion that white muscles are the main target of the fishing industry is emphasized. A novel approach is suggested based on the development of white muscles in wild fish from eggs to adults. A compilation of facts about white muscle structure, function and ontogeny is followed by an account of the changes in swimming behaviour and performance related to the use of white muscle during growth from larva to adult. Ecological data narrate early swimming performance with white muscle development and growth, unveiling some of the important natural selection factors eliminating weak swimmers and poor growers from the breeding stock. A comparison between fish culture practise and natural conditions reveals fundamental differences. New approaches following wild breeding processes promise several important advantages regarding the quality of white muscle.

How swifts control their glide performance with morphing wings.

Gliding birds continually change the shape and size of their wings, presumably to exploit the profound effect of wing morphology on aerodynamic performance. That birds should adjust wing sweep to suit glide speed has been predicted qualitatively by analytical glide models, which extrapolated the wing's performance envelope from aerodynamic theory. Here we describe the aerodynamic and structural performance of actual swift wings, as measured in a wind tunnel, and on this basis build a semi-empirical glide model. By measuring inside and outside swifts' behavioural envelope, we show that choosing the most suitable sweep can halve sink speed or triple turning rate. Extended wings are superior for slow glides and turns; swept wings are superior for fast glides and turns. This superiority is due to better aerodynamic performance-with the exception of fast turns. Swept wings are less effective at generating lift while turning at high speeds, but can bear the extreme loads. Finally, our glide model predicts that cost-effective gliding occurs at speeds of 8-10 m s(-1), whereas agility-related figures of merit peak at 15-25 m s(-1). In fact, swifts spend the night ('roost') in flight at 8-10 m s(-1) (ref. 11), thus our model can explain this choice for a resting behaviour. Morphing not only adjusts birds' wing performance to the task at hand, but could also control the flight of future aircraft.

Leading-edge vortex lifts swifts.

The current understanding of how birds fly must be revised, because birds use their hand-wings in an unconventional way to generate lift and drag. Physical models of a common swift wing in gliding posture with a 60 degrees sweep of the sharp hand-wing leading edge were tested in a water tunnel. Interactions with the flow were measured quantitatively with digital particle image velocimetry at Reynolds numbers realistic for the gliding flight of a swift between 3750 and 37,500. The results show that gliding swifts can generate stable leading-edge vortices at small (5 degrees to 10 degrees) angles of attack. We suggest that the flow around the arm-wings of most birds can remain conventionally attached, whereas the swept-back hand-wings generate lift with leading-edge vortices.

Interference of PR3-ANCA with the enzymatic activity of PR3: differences in patients during active disease or remission of Wegener's granulomatosis.

Anti-neutrophil cytoplasmic antibodies (ANCA) against proteinase 3 (PR3) are strongly associated with Wegener's granulomatosis (WG) and are thought to be involved in its pathogenesis. Levels of PR3-ANCA do not always correspond to clinical disease activity. To investigate the relationship between functional effects of PR3-ANCA and disease activity, we tested the effect of IgG samples from sera of 43 WG patients, taken during active disease or remission, for their capacity to interfere with the proteolytic activity of PR3. Furthermore, longitudinal sera of seven WG patients were included. The enzymatic activity of PR3 was determined (1) with casein or with a small synthetic substrate and (2) by complexation of PR3 with alpha1-antitrypsin (alpha1-AT). With a fixed concentration (100 microg/ml) of IgG, PR3-ANCA from patients during an active phase of WG had a higher inhibitory capacity towards the proteolytic activity of PR3 and complexation of PR3 with alpha1-AT than did PR3-ANCA from WG patients during remission. However, the number of PR3-ANCA units that gave 50% inhibition of the PR3 enzymatic activity and its complexation with alpha1-AT was lower for patients during remission than for patients during an active phase of WG, indicating a stronger inhibitory capacity at a molar base. In conclusion, PR3-ANCA from patients during remission had a relatively higher inhibitory capacity towards the enzymatic activity of PR3 than PR3-ANCA from patients during an active phase. This may indicate that during active disease the ANCA titre is increased, but the number of active ANCA molecules that recognize the enzyme-inhibiting epitopes is not increased.

How the body contributes to the wake in undulatory fish swimming: flow fields of a swimming eel (Anguilla anguilla).

Undulatory swimmers generate thrust by passing a transverse wave down their body. Thrust is generated not just at the tail, but also to a varying degree by the body, depending on the fish's morphology and swimming movements. To examine the mechanisms by which the body in particular contributes to thrust production, we chose eels, which have no pronounced tail fin and hence are thought to generate all their thrust with their body. We investigated the interaction between body movements and the flow around swimming eels using two-dimensional particle image velocimetry. Maximum flow velocities adjacent to the eel's body increase almost linearly from head to tail, suggesting that eels generate thrust continuously along their body. The wake behind eels swimming at 1.5 Ls(-1), where L is body length, consisted of a double row of double vortices with little backward momentum. The eel sheds two vortices per half tail-beat, which can be identified by their shedding dynamics as a start-stop vortex of the tail and a vortex shed when the body-generated flows reach the 'trailing edge' and cause separation. Two consecutively shed ipsilateral body and tail vortices combine to form a vortex pair that moves away from the mean path of motion. This wake shape resembles flow patterns described previously for a propulsive mode in which neither swimming efficiency nor thrust is maximised but sideways forces are high. This swimming mode is suited to high manoeuvrability. Earlier recordings show that eels also generate a wake reflective of maximum swimming efficiency. The combined findings suggest that eels can modify their body wave to generate wakes that reflect their propulsive mode.

Photic entrainment of circadian activity patterns in the tropical labrid fish Halichoeres chrysus.

Yellow wrasses (Halichoeres chrysus) show clear daily activity patterns. The fish hide in the substrate at (subjective) night, during the distinct rest phase. Initial entrainment in a 12h:12h light-dark (12:12 LD) cycle (mean period 24.02h, SD 0.27h, n = 16) was followed by a free run (mean period 24.42h, SD 1.33h) after transition into constant dim light conditions. Light pulses of a comparable intensity as used in the light part of the LD cycles did not result in significant phase shifts of the free-running rhythm in constant darkness. Application of much brighter 3h light pulses resulted in a phase-response curve (PRC) for a fish species, with pronounced phase advances during late subjective night. The PRCs differed from those mainly obtained in other vertebrate taxa by the absence of significant phase delays in the early subjective night. At that circadian phase, significant tonic effects of the light pulses caused a shortening of the circadian period length. Entrainment to skeleton photoperiods of 1:11 LD was observed in five of six wrasses exposed, also after a 3h phase advance of this LD cycle. Subsequently, a 1:11.25 LD cycle resulted in entrainment in four of the six fish. It is suggested that the expression of the circadian system in fish can be interpreted as a functional response to a weak natural zeitgeber, as present in the marine environment. This response allows photic entrainment as described here in the yellow wrasse.

Hydrodynamics of unsteady fish swimming and the effects of body size: comparing the flow fields of fish larvae and adults.

Zebra danios (Brachydanio rerio) swim in a burst-and-coast mode. Most swimming bouts consist of a single tail flick and a coasting phase, during which the fish keeps its body straight. When visualising the flow in a horizontal section through the wake, the effects of the flow regime become apparent in the structure of the wake. In a two-dimensional, medio-frontal view of the flow, larvae and adults shed two vortices at the tail during the burst phase. These vortices resemble a cross section through a large-core vortex ring: two vortex cores packed close together with the central flow directed away from the fish. This flow pattern can be observed in larvae (body length approximately 4 mm) at Reynolds numbers below 100 as well as in adult fish (body length approximately 35 mm) at Reynolds numbers above 1000. Larval vortices differ from those of adult zebra danios mainly in their relatively wider vortex cores (higher ratio of core radius to ring radius) and their lower vortex circulation. Both effects result from the increased importance of viscosity on larval flows. During the coasting phase, larval and adult flows again differ because of the changing importance of viscosity. The high viscosity of the water causes large vortical flows adjacent to the larva's body. These regions of high vorticity represent the huge body of water dragged along by the larva, and they cause the larva to stop almost immediately after thrust generation ceases. No such areas of high vorticity are visible adjacent to adult zebra danios performing a comparable swimming manoeuvre. The rapid decrease in vortex circulation and the severe reduction in the coasting distance due to viscous drag contribute to the high cost that larvae - unlike adult fish - face when using a burst-and-coast swimming style.

Aquatic vertebrate locomotion: wakes from body waves.

Vertebrates swimming with undulations of the body and tail have inflection points where the curvature of the body changes from concave to convex or vice versa. These inflection points travel down the body at the speed of the running wave of bending. In movements with increasing amplitudes, the body rotates around the inflection points, inducing semicircular flows in the adjacent water on both sides of the body that together form proto-vortices. Like the inflection points, the proto-vortices travel towards the end of the tail. In the experiments described here, the phase relationship between the tailbeat cycle and the inflection point cycle can be used as a first approximation of the phase between the proto-vortex and the tailbeat cycle. Proto-vortices are shed at the tail as body vortices at roughly the same time as the inflection points reach the tail tip. Thus, the phase between proto-vortex shedding and tailbeat cycle determines the interaction between body and tail vortices, which are shed when the tail changes direction. The shape of the body wave is under the control of the fish and determines the position of vortex shedding relative to the mean path of motion. This, in turn, determines whether and how the body vortex interacts with the tail vortex. The shape of the wake and the contribution of the body to thrust depend on this interaction between body vortex and tail vortex. So far, we have been able to describe two types of wake. One has two vortices per tailbeat where each vortex consists of a tail vortex enhanced by a body vortex. A second, more variable, type of wake has four vortices per tailbeat: two tail vortices and two body vortices shed from the tail tip while it is moving from one extreme position to the next. The function of the second type is still enigmatic.

Burrow ventilation in the tube-dwelling shrimp Callianassa subterranea (Decapoda: thalassinidea). I. Morphology and motion of the pleopods, uropods and telson.

The morphology of the pleopods, uropods and telson of the tube-dwelling shrimp Callianassa subterranea have been studied using dissection microscopy and scanning electron microscopy. The kinematics of these appendages were examined by motion analysis of macro-video recordings of ventilating shrimps in transparent artificial burrows. The pleopods show the usual crustacean biramous anatomy, but all segments are rostro-caudally flattened. The protopodite bears a triangular medially oriented endopodite and a scoop-shaped exopodite. The contralateral endopodites are linked by the appendix interna, ensuring a perfect phase relationship between contralateral pleopods. The outer rims of the exopodites are fringed with long fern-leaf-like plumose setae bearing flattened setules. These setae have very mobile joints and can be turned caudally. The slits between contralateral endopodites have rims of leaf-like setae as well. Setae of the same leaf-like type fringe the uropods, but these are non-motile. The telson has a narrow fringe of leaf-like setae, locally interrupted by long mechanoreceptory setae. A shrimp, wandering through the burrow or resting, holds its pleopods against the abdomen with the exopodites and their setal fringes retracted medially. The uropods are folded medially as well, probably to reduce the shrimp's drag. During ventilation, the uropods are extended against the tube wall, leaving only a small opening for flow ventral from the telson, and the pleopods beat at a frequency of approximately 1 Hz (0.9+/-0.2 Hz). Fourier analysis of pleopod kinematics showed that the motion pattern of the first flow-generating pleopod pair (PP1) consisted mainly of the first harmonic (75 %) and to a lesser extent of the third harmonic (20 %), resulting in almost perfect sinusoidal motion. The motion patterns of PP2 and PP3 could be modelled by assigning pure sinusoids with a 120 degrees phase shift and a rostro-caudal ranking to the three pairs of pleopods.

Burrow ventilation in the tube-dwelling shrimp callianassa subterranea (Decapoda: thalassinidea). II. The flow in the vicinity of the shrimp and the energetic advantages of a laminar non-pulsating ventilation current.

The ventilation flow in the vicinity of the pleopod-pumping thalassinid shrimp Callianassa subterranea in an artificial transparent burrow has been mapped using particle image velocimetry. The flow in the tube in front of the shrimp was unidirectional, laminar and steady, with a parabolic cross-sectional velocity profile. The mean flow velocity was 2.0+/-0.1 mm s-1. The flow passed the thorax of the shrimp along the lateral and ventral sides. Ventral to the abdomen, the flow was dominated by the metachronally oscillating pleopods. The water around a pleopod is accelerated caudally and ventrally during the power stroke, and decelerated to a much lesser extent during the recovery stroke owing to a reduction in pleopod area. On average, the flow ventral to the abdomen converged towards the small opening underneath the telson, simultaneously increasing in velocity. A jet with a core velocity of 18-20 mm s-1 entered the area behind the shrimp from underneath the telson. This caused a separation zone with backflow caudal to the telson. Owing to the high rates of shear, the jet diverged and re-adjusted to a parabolic cross-sectional profile within 1-2 body lengths behind the shrimp, showing no traces of pulsation. The metachronal pleopod movements in combination with the increase in flow velocity at the constriction in the tube caused by the uropods and the telson probably prevented pulsation. The energetic consequences of pulsating and steady flows in combination with several tube configurations were evaluated. The results suggested that, by constricting the tube and keeping the flow steady, C. subterranea saves on ventilation costs by a factor of up to six compared with oscillatory flow in a tube without the tail-fan constriction.

Burrow ventilation in the tube-dwelling shimp Callianassa subterranea (Decapoda: thalassinidea). III. Hydrodynamic modelling and the energetics of pleopod pumping.

The process of flow generation with metachronally beating pleopods in a tubiform burrow was studied by designing a hydrodynamic model based on a thrust-drag force balance. The drag of the tube (including the shrimp) comprises components for accelerating the water into the tube entrance, for adjusting a parabolic velocity profile, for accelerating the flow into a constriction due to the shrimp's body and another constriction due to the extended tail-fan, for shear due to separation and for the viscous resistance of all tube parts. The thrust produced by the beating pleopods comprises components for the drag-based thrust and for the added-mass-based thrust. The beating pleopods are approximated by oscillating flat plates with a different area and camber during the power stroke and the recovery stroke and with a phase shift between adjacent pleopod pairs. The added mass is shed during the second half of the power stroke and is minimized during the recovery stroke. A force balance between the pleopod thrust and the tube drag is effected by calculating the mean thrust during one beat cycle at a certain flow velocity in the tube and comparing it with the drag of the tube at that flow velocity. The energetics of the tube and the pump are derived from the forces, and the mechanical efficiency of the system is the ratio of these two. Adjusted to standard Callianassa subterranea values, the model predicts a mean flow velocity in the tube of 1.8 mm s-1. The mean thrust force, equalling the drag, is 36. 8 microN, the work done by the pleopod pump per beat cycle is 0.91 microJ and the energy dissipated by the tube system is 0.066 microJ per cycle. The mechanical efficiency is therefore 7.3 %. Pump characteristics that may be varied by the shrimp are the beat frequency, the phase shift, the amplitude and the difference in pleopod area between the power and recovery strokes. These parameters are varied in the model to evaluate their effects. Furthermore, the moment of added mass shedding, the distance between adjacent pleopods, the number of pleopods and the total tube drag were also varied to evaluate their effects.

Fish foot prints: morphology and energetics of the wake behind a continuously swimming mullet (Chelon labrosus Risso).

The structure of the wake behind a continuously swimming mullet was analysed qualitatively and quantitatively by applying two-dimensional particle image velocimetry. A detailed analysis of the flow pattern and of the swimming movements of the fish allowed us to derive a kinematic explanation of the flow pattern as well as an estimate of the relative contributions of the body and the tail to thrust production. During active propulsion, the undulatory swimming fish shed a wake consisting in the medio-frontal plane of a rearward, zigzagging jet flow between alternating vortices. The fish shed one vortex per half tailbeat when the tail reached its most lateral position. Part of the circulation shed in the vortices had been generated previously on the body by the transverse body wave travelling down the body. This undulatory pump mechanism accounted for less than half of the energy shed in the wake. The remainder was generated by the tail. The vortex spacing matched the tailbeat amplitude and the stride length.

Quantitative flow analysis around aquatic animals using laser sheet particle image velocimetry

Two alternative particle image velocimetry (PIV) methods have been developed, applying laser light sheet illumination of particle-seeded flows around marine organisms. Successive video images, recorded perpendicular to a light sheet parallel to the main stream, were digitized and processed to map the flow velocity in two-dimensional planes. In particle tracking velocimetry (PTV), displacements of single particles in two subsequent images were determined semi-automatically, resulting in flow diagrams consisting of non-uniformly distributed velocity vectors. Application of grid-cell averaging resulted in flow field diagrams with uniform vector distribution. In sub-image correlation PIV (SCPIV), repetitive convolution filtering of small sub-areas of two subsequent images resulted in automatic determination of cross-correlation peaks, yielding flow field diagrams with regularly spaced velocity vectors. In both PTV and SCPIV, missing values, caused by incomplete particle displacement information in some areas of the images or due to rejection of some erroneous vectors by the vector validation procedure, were interpolated using a two-dimensional spline interpolation technique. The resultant vector flow fields were used to study the spatial distribution of velocity, spatial acceleration, vorticity, strain and shear. These flow fields could also be used to test for flow in the third dimension by studying the divergence, and to detect the presence and location of vortices. The results offer detailed quantitative descriptions of the flow morphology and can be used to assess dissipated energy. The versatile character of the technique makes it applicable to a wide range of fluid mechanical subjects within biological research. So far it has been successfully applied to map the flow around swimming copepods, fish larvae and juvenile fish and the ventilation current of a tube-living shrimp.

Tuning in to fish swimming waves: body form, swimming mode and muscle function

Most fish species swim with lateral body undulations running from head to tail. These waves run more slowly than the waves of muscle activation causing them, reflecting the effect of the interaction between the fish's body and the reactive forces from the water. The coupling between both waves depends on the lateral body shape and on the mechanical properties of the tail. During steady swimming, the length of each myotomal muscle fibre varies cyclically. The phase relationship between the strain (muscle length change) cycle and the active period (when force is generated) determines the work output of the muscle. The muscle power is converted to thrust either directly by the bending body or almost exclusively by the tail, depending upon the body shape of the species and the swimming kinematics. We have compared the kinematics and muscle activity patterns from seven species of fish with different body forms and swimming modes and propose a model which yields a consistent pattern, with at least three extremes. Subtle tuning of the phase relationship between muscle strain and activation cycles can lead to major changes in the way muscles function in different swimming modes.

Body surface adaptations to boundary-layer dynamics.

Evolutionary processes have adapted nektonic animals to interact efficiently with the water that surrounds them. Not all these adaptations serve the same purpose. This paper concentrates on reduction of drag due to friction in the boundary layer close to the body surface. Mucus, compliant skins, scales, riblets and roughness may influence the flow velocity gradient, the type of flow and the thickness of the boundary layer around animals, and may seriously affect their drag in a positive or negative way. The long-chain polymers found in mucus decrease the pressure gradient and considerably reduced drag due to friction. The effect is probably due to channelling of the flow particles in the direction of the main flow, resulting in a reduction of turbulence. Compliant surfaces could probably reduce drag by equalising and distributing pressure pulses. However, the existing evidence that drag reduction actually occurs is not convincing. There is no indication that instantaneous heating, reducing the viscosity in the boundary layer, is used by animals as a drag-reducing technique. Small longitudinal ridges on rows of scales on fish can reduce shear stress in the boundary by a maximum of 10% compared with the shear stress of a smooth surface. The mechanism is based on the impedance of cross flow under well-defined conditions. The effect has been visualized with the use of particle image velocimetry techniques. The function of the swords and spears of several fast, pelagic, predatory fish species is still enigmatic. The surface structure of the sword of a swordfish is shown to be both rough and porous. The height of the roughness elements on the tip of the sword is close to the critical value for the induction of a laminar-to-turbulent flow transition at moderate cruising speeds. A flow tank is described that is designed to visualize the effects of surface imperfections on flow in the boundary layer in direct comparison with a smooth flat wall. The flow in a 1 m long, 10 cm high and 1 cm wide channel is visualized by illuminating the particles in a thin laser light sheet. The first results show that a rough surface increases the shear stress in the boundary layer and makes it thinner. The function of the roughness on the sword of a swordfish is probably to reduce the total drag by generating premature turbulence and by boundary layer thinning, despite an increased friction over the surface of the sword. The function of the porous surface structures on the sword, and of the porous skins of sharks and of the castor oil fish, will probably be discovered soon using new particle image velocimetry techniques applied under strong magnification to visualize the local behaviour of the flow.

pol mutations conferring zidovudine and didanosine resistance with different effects in vitro yield multiply resistant human immunodeficiency virus type 1 isolates in vivo.

Specific mutations in the human immunodeficiency virus type 1 (HIV-1) pol gene that cause zidovudine (3'-azido-2',3'-dideoxythymidine; AZT) and didanosine (2',3'-dideoxyinosine; ddI) resistance were studied. The 50% inhibitory concentrations (IC50s) of nucleosides for cloned viruses containing these mutations were compared with the IC50s of the corresponding triphosphate analogs for mutant recombinant-expressed reverse transcriptases (RTs). Changes in ddATP inhibition of RNA-dependent DNA polymerase activity fully accounted for the ddI resistance of the virus caused by a Leu-74-->Val substitution in RT, including an augmentation by the AZT-selected substitutions Thr-215-->Tyr and Lys-219-->Gln in RT. In contrast, the AZT-selected substitutions studied did not cause as great a change in the IC50 of AZT-triphosphate (AZT-TP) for polymerase as they did in the IC50 of AZT for mutant virus. In addition, the mutation at codon 74 suppressed AZT resistance in the virus caused by the mutations at codons 215 and 219 but did not suppress the AZT-TP resistance of enzyme containing these same mutations in RT. The mutation at codon 74 was found in clinical isolates whether or not the patient had received AZT prior to starting ddI therapy. AZT resistance coexisted with ddI resistance following acquisition of Leu-74-->Val in three clinical isolates, indicating that the suppressive effect of Val-74 on the AZT resistance of the virus does not occur in all genetic contexts. When this suppression of AZT resistance was seen in the virus, Val-74 did not appear to cause mutually exclusive changes in AZT-TP and ddATP binding to RT in vitro. The results of the in vitro experiments and characterization of clinical isolates suggest that there are differences in the functional effects of these AZT and ddI resistance mutations.

Two-drug combinations of zidovudine, didanosine, and recombinant interferon-alpha A inhibit replication of zidovudine-resistant human immunodeficiency virus type 1 synergistically in vitro.

Optimal management of human immunodeficiency virus type 1 (HIV-1) infections may require combinations of anti-HIV-1 agents. Zidovudine (AZT, 3'-azido-3'-deoxythymidine), didanosine (ddI, 2',3'-dideoxyinosine), and recombinant interferon-alpha A (rIFN-alpha A) were evaluated in two-drug regimens against replication of AZT-resistant HIV-1 in vitro. AZT-sensitive and AZT-resistant isolate pairs derived from two individuals before and after extended AZT monotherapy were studied. Drug interactions using peripheral blood mononuclear cells infected with HIV-1 were evaluated mathematically. Synergistic interactions were seen among AZT, ddI, and rIFN-alpha A in two-drug regimens against AZT-resistant HIV-1 in vitro, even when AZT was included in the treatment regimen. Mixtures of wild-type and mutant reverse transcriptase genes were found in one of the late-AZT therapy isolates, suggesting that the mechanism of synergy of AZT-containing regimens may involve inhibition of AZT-sensitive viruses in the viral pool. These studies suggest that AZT may be useful in drug combination regimens, even when AZT-resistant viruses are isolated in vitro.