Forehead monitoring of heart rate in neonatal intensive care.

Heart rate is an extremely important physiological parameter to measure in critically unwell infants, as it is the main physiological marker that changes in response to a change in infant condition. Heart rate is routinely measured peripherally on a limb with a pulse oximeter. However, when infants are critically unwell, the blood supply to these peripheries is reduced in preference for central perfusion of vital organs such as the brain and heart. Measurement of heart rate with a reflection mode photoplethysmogram (PPG) sensor on the forehead could help minimise this problem and make it easier for other important medical equipment, such as cannulas, to be placed on the limbs. This study compares heart rates measured with a forehead-based PPG sensor against a wrist-based PPG sensor in 19 critically unwell infants in neonatal intensive care collecting 198 h of data. The two heart rates were compared using positive percentage agreement, Spearman's correlation coefficient and Bland-Altman analysis. The forehead PPG sensor showed good agreement with the wrist-based PPG sensor with limits of agreement of 8.44 bpm, bias of -0.22 bpm; positive percentage agreement of 98.87%; and Spearman's correlation coefficient of 0.9816. The analysis demonstrates that the forehead is a reliable alternative location for measuring vital signs using the PPG.

Maternity services' responses to the COVID-19 pandemic: how Public Health England guidance was implemented in practice.

INTRODUCTION: The rapidly evolving COVID-19 pandemic required systemic change in how healthcare was delivered to minimize virus transmission whilst maintaining safe service delivery. Deemed at 'moderate-high risk', maternity patients are an important patient group that require consideration. Public Health England (PHE) issued national guidance on how to adjust these services. AIM: To explore how maternity units in England implemented PHE guidance. METHODS: An online survey of 22 items was distributed to individuals that had worked on an England-based maternity unit during the COVID-19 pandemic. The questionnaire was designed and tested by the multidisciplinary research team. Data was collected from November 2020 to July 2021. FINDINGS: Forty-four participants across 33 maternity units responded. Ninety-three percent were able to test all women requiring an overnight stay for COVID-19. Only 27% reported birth partners were tested for COVID-19. Only 73% reported they were able to isolate all COVID-19-positive patients in single rooms. Eighty-four percent stated they were aware of current PHE guidance on personal protective equipment (PPE) and 82% felt 'confident' in donning/doffing of PPE. Priorities for the future include rapid testing and a focus on community service provision. CONCLUSIONS: PHE COVID-19 guidance was implemented differently in maternity units across England due to the varying resources available at each trust leading to variable ability to test and isolate patients as recommended. More specific, tailored guidance for infection control measures against COVID-19 is needed for maternity settings due to their unique position.

Implementation of Public Health England infection prevention and control guidance in maternity units in response to the COVID-19 pandemic.

BACKGROUND: This study aimed to explore the successes and barriers to the implementation of Public Health England (PHE) infection prevention and control guidance in English maternity units during the COVID-19 pandemic. METHODS: Qualitative semi-structured interviews with obstetricians, midwives and neonatologists who worked in a maternity unit in England, UK, between March 2020 and July 2021. A thematic analysis was performed. RESULTS: Successes to the implementation of PHE guidance were related to existing infrastructure, training satisfaction, and organisational culture where subthemes considered the importance of a multidisciplinary approach, COVID-19 dedicated roles and hospital-wide communication. Barriers to implementation related to the applicability of the guidance with subthemes highlighting contradictions between updates, specialties and hospitals, undesirable timings and frequency of guidance updates, reductions in staff compliance and delayed implementation. Finally, the layout of some units made it difficult to implement various aspects of the guidance (e.g., social distancing), and many detailed issues related to information technology compatibility, a lack of availability and accessibility to appropriate personal protective equipment (PPE), and variations in testing arrangements between units. CONCLUSIONS: This research provides information on the experiences of healthcare professionals working on maternity units during the COVID-19 pandemic. Findings illustrate the importance of effective hospital-wide communication and the need for consistent, easily understood guidance. These results will be used to inform the content of an expert panel consensus meeting.

An automated quasi-continuous capillary refill timing device.

Capillary refill time (CRT) is a simple means of cardiovascular assessment which is widely used in clinical care. Currently, CRT is measured through manual assessment of the time taken for skin tone to return to normal colour following blanching of the skin surface. There is evidence to suggest that manually assessed CRT is subject to bias from ambient light conditions, a lack of standardisation of both blanching time and manually applied pressure, subjectiveness of return to normal colour, and variability in the manual assessment of time. We present a novel automated system for CRT measurement, incorporating three components: a non-invasive adhesive sensor incorporating a pneumatic actuator, a diffuse multi-wavelength reflectance measurement device, and a temperature sensor; a battery operated datalogger unit containing a self contained pneumatic supply; and PC based data analysis software for the extraction of refill time, patient skin surface temperature, and sensor signal quality. Through standardisation of the test, it is hoped that some of the shortcomings of manual CRT can be overcome. In addition, an automated system will facilitate easier integration of CRT into electronic record keeping and clinical monitoring or scoring systems, as well as reducing demands on clinicians. Summary analysis of volunteer (n = 30) automated CRT datasets are presented, from 15 healthy adults and 15 healthy children (aged from 5 to 15 years), as their arms were cooled from ambient temperature to 5°C. A more detailed analysis of two typical datasets is also presented, demonstrating that the response of automated CRT to cooling matches that of previously published studies.

Forehead reflectance photoplethysmography to monitor heart rate: preliminary results from neonatal patients.

Around 5%-10% of newborn babies require some form of resuscitation at birth and heart rate (HR) is the best guide of efficacy. We report the development and first trial of a device that continuously monitors neonatal HR, with a view to deployment in the delivery room to guide newborn resuscitation. The device uses forehead reflectance photoplethysmography (PPG) with modulated light and lock-in detection. Forehead fixation has numerous advantages including ease of sensor placement, whilst perfusion at the forehead is better maintained in comparison to the extremities. Green light (525 nm) was used, in preference to the more usual red or infrared wavelengths, to optimize the amplitude of the pulsatile signal. Experimental results are presented showing simultaneous PPG and electrocardiogram (ECG) HRs from babies (n = 77), gestational age 26-42 weeks, on a neonatal intensive care unit. In babies ⩾32 weeks gestation, the median reliability was 97.7% at ±10 bpm and the limits of agreement (LOA) between PPG and ECG were +8.39 bpm and -8.39 bpm. In babies <32 weeks gestation, the median reliability was 94.8% at ±10 bpm and the LOA were +11.53 bpm and -12.01 bpm. Clinical evaluation during newborn deliveries is now underway.

Suboptimal maternal nutrition during early fetal kidney development specifically promotes renal lipid accumulation following juvenile obesity in the offspring.

Reduced maternal food intake between early-to-mid gestation results in tissue-specific adaptations in the offspring following juvenile-onset obesity that are indicative of insulin resistance. The aim of the present study was to establish the extent to which renal ectopic lipid accumulation, as opposed to other markers of renal stress, such as iron deposition and apoptosis, is enhanced in obese offspring born to mothers nutrient restricted (NR) throughout early fetal kidney development. Pregnant sheep were fed either 100% (control) or NR (i.e. fed 50% of their total metabolisable energy requirement from 30-80 days gestation and 100% at all other times). At weaning, offspring were made obese and, at approximately 1 year, kidneys were sampled. Triglyceride content, HIF-1α gene expression and the protein abundance of the outer-membrane transporter voltage-dependent anion-selective channel protein (VDAC)-I on the kidney cortex were increased in obese offspring born to NR mothers compared with those born to controls, which exhibited increased iron accumulation within the tubular epithelial cells and increased gene expression of the death receptor Fas. In conclusion, suboptimal maternal nutrition coincident with early fetal kidney development results in enhanced renal lipid deposition following juvenile obesity and could accelerate the onset of the adverse metabolic, rather than cardiovascular, symptoms accompanying the metabolic syndrome.

Adipose tissue and fetal programming.

Adipose tissue function changes with development. In the newborn, brown adipose tissue (BAT) is essential for ensuring effective adaptation to the extrauterine environment, and its growth during gestation is largely dependent on glucose supply from the mother to the fetus. The amount, location and type of adipose tissue deposited can also determine fetal glucose homeostasis. Adipose tissue first appears at around mid-gestation. Total adipose mass then increases through late gestation, when it comprises a mixture of white and brown adipocytes. BAT possesses a unique uncoupling protein, UCP1, which is responsible for the rapid generation of large amounts of heat at birth. Then, during postnatal life some, but not all, depots are replaced by white fat. This process can be utilised to investigate the physiological conversion of brown to white fat, and how it is re-programmed by nutritional changes in pre- and postnatal environments. A reduction in early BAT deposition may perpetuate through the life cycle, thereby suppressing energy expenditure and ultimately promoting obesity. Normal fat development profiles in the offspring are modified by changes in maternal diet at defined stages of pregnancy, ultimately leading to adverse long-term outcomes. For example, excess macrophage accumulation and the onset of insulin resistance occur in an adipose tissue depot-specific manner in offspring born to mothers fed a suboptimal diet from early to mid-gestation. In conclusion, the growth of the different fetal adipose tissue depots varies according to maternal diet and, if challenged in later life, this can contribute to insulin resistance and impaired glucose homeostasis.

Influence of prenatal nutrition and obesity on tissue specific fat mass and obesity-associated (FTO) gene expression.

The recent discovery of an association between body composition, energy intake and the fat mass and obesity-associated (FTO) gene represents a promising new therapeutic target in obesity prevention. In a well, pre-established large animal model, we investigated the regulation of FTO gene expression under conditions either leading to obesity or increased risk of obesity related disorders: i) a sedentary 'Western' lifestyle and ii) prenatal exposure to nutrient restriction. Pregnant sheep were either fed to fully meet their nutritional requirements throughout gestation or 50% of this amount from early-to-mid gestation. Following weaning, offspring were either made obese through exposure to a sedentary obesogenic environment or remained lean. A significant positive relationship between placental FTO gene expression and fetal weight was found at 110 days gestation. In both the newborn and adult offspring, the hypothalamus was the major site of FTO gene expression. Hypothalamic FTO gene expression was upregulated by obesity and was further increased by prenatal nutrient restriction. Importantly, we found a strong negative relationship between the hypothalamic FTO gene expression and food intake in lean animals only that may imply FTO as a novel controller of energy intake. In contrast, FTO gene expression in the heart was downregulated in obese offspring born to nutrient restricted mothers. In addition, FTO gene expression was unaffected by obesity or prenatal diet in insulin-dependent tissues, where it changed with age possibly reflecting adaptations in cellular energetic activity. These findings extend information gained from human epidemiology and provide new insights into the regulation of in vivo energy metabolism to prevent obesity.

Session on 'Obesity'. Adipose tissue development, nutrition in early life and its impact on later obesity.

It is now apparent that one key factor determining the current obesity epidemic within the developed world is the extent to which adipose tissue growth and function can be reset in early life. Adipose tissue can be either brown or white, with brown fat being characterised as possessing a unique uncoupling protein (uncoupling protein 1) that enables the rapid generation of heat by non-shivering thermogenesis. In large mammals this function is recruited at approximately the time of birth, after which brown fat is lost, not normally reappearing again throughout the life cycle. The origin and developmental regulation of brown fat in large mammals is therefore very different from that of small mammals in which brown fat is retained throughout the life cycle and may have the same origin as muscle cells. In contrast, white adipose tissue increases in mass after birth, paralleled by a rise in glucocorticoid action and macrophage accumulation. This process can be reset by changes in the maternal nutritional environment, with the magnitude of response being further determined by the timing at which such a challenge is imposed. Importantly, the long-term response within white adipocytes can occur in the absence of any change in total fat mass. The present review therefore emphasises the need to further understand the developmental regulation of the function of fat through the life cycle in order to optimise appropriate and sustainable intervention strategies necessary not only to prevent obesity in the first place but also to reverse excess fat mass in obese individuals.

The differential effects of the timing of maternal nutrient restriction in the ovine placenta on glucocorticoid sensitivity, uncoupling protein 2, peroxisome proliferator-activated receptor-gamma and cell proliferation.

Nutrient restriction (NR) during critical windows of pregnancy has differential effects on placento-fetal growth and development. Our study, therefore, investigated developmental and metabolic adaptations within the ovine placenta following NR at different critical windows during the first 110 days of gestation (term=147 days). Thus, the effects of NR on cell proliferation, glucocorticoid sensitivity, IGF1 and 2 receptor, peroxisome proliferator-activated receptor gamma (PPARG), and uncoupling protein (UCP)2 gene expression in the placenta were examined. Singleton bearing sheep (n=4-8 per group) were fed either 100% of their total metabolizable energy requirements throughout the study or 50% of this amount between 0-30, 31-65, 66-110, and 0-110 days gestation. A significant reduction in cell proliferation and increased gene expression for the glucocorticoid and IGF2 receptors, PPARG, and UCP2 were detected in placentae sampled from mothers who were nutrient restricted between days 66 and 110 of gestation, only, relative to controls. This window of gestation coincides with the maximum placental growth and the start of exponential growth of the fetus when there are substantially increased metabolic demands on the placenta compared with earlier in gestation. Consequently, increased glucocorticoid sensitivity and suppressed IGF2 action could contribute to a switch in the placenta from proliferation to differentiation, thereby improving its nutrient transfer capacity. Upregulation of PPARG and UCP2 would promote placental fatty acid metabolism thereby limiting glucose utilization. These compensatory placental responses may serve to maintain fetal growth but could result in adverse adaptations such as the early onset of the metabolic syndrome in later life.

The role of semen in induction of maternal immune tolerance to pregnancy.

Successful pregnancy requires a state of maternal immune 'tolerance' to accommodate antigens expressed by the conceptus. Implantation failure and placental pathologies largely reflect insufficiencies in maternal immune adaptation, but progress in devising therapeutic strategies to treat these conditions is stalled because the mechanisms underlying the induction and maintenance of maternal tolerance are unknown. Increasingly, clinical and experimental data support the proposal that insemination has consequences for the reproductive process beyond delivery of male gametes. An emerging hypothesis, based mainly on clinical observations and experiments in mice, is that insemination is causally linked to the activation and expansion of populations of lymphocytes mediating forms of 'active' immune tolerance in the implantation site. This review examines existing evidence for a role for semen in the immunology of pregnancy, highlighting the limitations of our existing knowledge and the prospects for future research and its clinical application.

Phylogeographic differentiation in the mitochondrial control region in the koala, Phascolarctos cinereus (Goldfuss 1817).

The koala, Phascolarctos cinereus, is a geographically widespread species endemic to Australia, with three currently recognized subspecies: P.c. adustus, P.c. cinereus, and P.c. victor. Intraspecific variation in the mitochondrial DNA (mtDNA) control region was examined in over 200 animals from 16 representative populations throughout the species' range. Eighteen different haplotypes were defined in the approximately 860 bp mtDNA control region, as determined by heteroduplex analysis/temperature gradient gel electrophoresis (HDA/TGGE). Any single population typically possessed only one or two haplotypes yielding an average within-population haplotypic diversity of 0.180 +/- 0.003, and nucleotide diversity of 0.16%. Overall, mtDNA control region sequence diversity between populations averaged 0.67%, and ranged from 0% to 1.56%. Nucleotide divergence between populations averaged 0.51%, and ranged from 0% to 1.53%. Neighbour-joining methods revealed limited phylogenetic distinction between geographically distant populations of koalas, and tentative support for a single evolutionarily significant unit (ESU). This is consistent with previous suggestions that the morphological differences formalized by subspecific taxonomy may be interpreted as clinal variation. Significant differentiation in mtDNA-haplotype frequencies between localities suggested that little gene flow currently exists among populations. When combined with microsatellite analysis, which has revealed substantial differentiation among koala populations, we conclude that the appropriate short-term management unit (MU) for koalas is the local population.

Crystal structure of Taq DNA polymerase in complex with an inhibitory Fab: the Fab is directed against an intermediate in the helix-coil dynamics of the enzyme.

We report the crystal structure of Thermus aquaticus DNA polymerase I in complex with an inhibitory Fab, TP7, directed against the native enzyme. Some of the residues present in a helical conformation in the native enzyme have adopted a gamma turn conformation in the complex. Taken together, structural information that describes alteration of helical structure and solution studies that demonstrate the ability of TP7 to inhibit 100% of the polymerase activity of the enzyme suggest that the change in conformation is probably caused by trapping of an intermediate in the helix-coil dynamics of this helix by the Fab. Antibodies directed against modified helices in proteins have long been anticipated. The present structure provides direct crystallographic evidence. The Fab binds within the DNA binding cleft of the polymerase domain, interacting with several residues that are used by the enzyme in binding the primer:template complex. This result unequivocally corroborates inferences drawn from binding experiments and modeling calculations that the inhibitory activity of this Fab is directly attributable to its interference with DNA binding by the polymerase domain of the enzyme. The combination of interactions made by the Fab residues in both the polymerase and the vestigial editing nuclease domain of the enzyme reveal the structural basis of its preference for binding to DNA polymerases of the Thermus species. The orientation of the structure-specific nuclease domain with respect to the polymerase domain is significantly different from that seen in other structures of this polymerase. This reorientation does not appear to be antibody-induced and implies remarkably high relative mobility between these two domains.

Structural studies on an inhibitory antibody against Thermus aquaticus DNA polymerase suggest mode of inhibition.

TP7, an antibody against Thermus aquaticus DNA polymerase I (TaqP), is used as a thermolabile switch in 'hot start' variations of PCR to minimize non-specific amplification events. Earlier studies have established that TP7 binds to the polymerase domain of TaqP, competes with primer template complex for binding and is a potent inhibitor of the polymerase activity of TaqP. We report crystallographic determination of the structure of an Fab fragment of TP7 and computational docking of the structure with the known three-dimensional structure of the enzyme. Our observations strongly suggest that the origin of inhibitory ability of TP7 is its binding to enzyme residues involved in DNA binding and polymerization mechanism. Although criteria unbiased by extant biochemical data have been used in identification of a putative solution, the resulting complex offers an eminently plausible structural explanation of biochemical observations. The results presented are of general significance for interpretation of docking experiments and in design of small molecular inhibitors of TaqP, that are not structurally similar to substrates, for use in PCR. Structural and functional similarities noted among DNA polymerases, and the fact that several DNA polymerases are pharmacological targets, make discovery of non-substrate based inhibitors of additional importance.

Isolation of a 90-kD Microtubule-Associated Protein from Tobacco Membranes.

The organization and function of microtubules in plant cells are important in key developmental events, including the regulation of directional cellulose deposition. Bridges connecting microtubules to each other and to membranes and other organelles have been documented by electron microscopy; however, the biochemical and molecular nature of these linkages is not known. We have partitioned proteins from a suspension culture of tobacco into cytosolic and membrane fractions, solubilized the membrane fraction with a zwitterionic detergent, and then used affinity chromatography and salt elution to isolate tubulin binding proteins. Dark-field microscopy of in vitro-assembled microtubules showed that the eluted proteins from both fractions induce microtubule bundling and, in the presence of purified tubulin, promote microtubule elongation. Gel electrophoresis of the eluted proteins revealed two distinct sets of polypeptides. Those in the membrane eluate included unique bands with apparent molecular masses of 98, 90, and 75 kD in addition to bands present in both eluates. The cytosolic eluate, in contrast, typically included relatively smaller proteins. The eluted proteins also bound to taxol-stabilized microtubules. Initial immunological characterization using monoclonal antibodies raised against the 90-kD polypeptide showed that it is colocalized in situ with cortical microtubules in tobacco protoplast ghosts.

Characterization of crystals of the thermostable DNA polymerase I from Thermus aquaticus.

Thermus aquaticus DNA polymerase I is an enzyme that is of both physiological and technological interest. It carries out template-directed polymerization of DNA at elevated temperatures and is widely used in polymerase chain reaction (PCR). We have obtained crystals of the enzyme that diffracts X-rays to at least 3.0 A resolution in a cubic space group. Determination of the three-dimensional structure of the native enzyme along with those of relevant complexes will greatly enhance our knowledge of molecular events involved in DNA replication, will permit improvements in PCR, and will add to our knowledge of the structural bases of thermostability in proteins.

Topographical characterization of the DNA polymerase from Thermus aquaticus. Defining groups of inhibitor mAbs by epitope mapping and functional analysis using surface plasmon resonance.

Among 24 unique monoclonal antibodies (mAb) generated against Taq polymerase (TaqPol) 13 are potent inhibitors of polymerase activity. These antibodies have been sorted into groups defined by their topographical or functional properties using surface plasmon resonance-based methods to examine three different types of interactions. An epitope map of all the pairwise interactions among all 24 antiTaqPol antibodies revealed the surface of TaqPol as a complex space populated by isolated antigenic domains with no evident relationship to each other. 11 discrete epitopes or epitope clusters were defined and potent inhibitors bound to sites within seven of them. The second method examined the ability of antiTaqPol mAbs to bind to recombinant forms of the constituent functional domains of TaqPol, the N-terminal 5'-nuclease domain and the C-terminal polymerase domain. Most of the antibodies demonstrated a clear specificity for one domain or the other. The third method measured the ability of each mAb to block the interaction of TaqPol with a preformed, immobilized primer:template complex (PTC). Some antibodies had no effect on this interaction while others effectively blocked it. Together these latter two methods resolved many of the antibodies into five distinct groups. In addition, antibodies that bound to overlapping epitopes in the pairwise interaction analysis were members of the same group by their interaction with the polymerase fragment and PTC. Three groups of polymerase inhibitors were clearly resolved by these analyses: (1) those that recognize an epitope on the 5'-nuclease domain and have no effect on the interaction of TaqPol with PTC; (2) those that recognize an epitope on the polymerase domain and block the interaction of TaqPol and PTC; and (3) those that recognize an epitope on the polymerase domain, but have no effect on the interaction of TaqPol with PTC. The surface of TaqPol bears at least three antigenic regions that are topographically and functionally distinct and may correspond to sites for inhibition of different steps in the enzymatic activity of TaqPol.

Monoclonal antibodies prepared against the DNA polymerase from Thermus aquaticus are potent inhibitors of enzyme activity.

Recent interest in the unique properties of the DNA polymerase from Thermus aquaticus (TaqPol) has stemmed from its use in many laboratories for the polymerase chain reaction. We have produced a panel of nine distinct monoclonal antibodies to a recombinant form of TaqPol that have the following properties: (1) each binds TaqPol with high affinity (Kd < 10 nM); (2) eight of the nine arbitrarily selected monoclonal antibodies inhibit TaqPol activity completely; (3) the weak inhibitor is specific for TaqPol only while all eight strong inhibitors cross-react with the DNA polymerase from at least one other Thermus species as detected by either competitive ELISA, Western blotting, inhibition of enzyme activity or determination of binding by surface plasmon resonance; (4) these antibodies can be distinguished from each other by heavy chain class, cross-reactivity patterns, isoelectric points, and epitope mapping; and (5) these antibodies define seven non-overlapping epitopes. In addition, we show data from a preliminary experiment that demonstrates that at least one of these antibodies inhibits TaqPol by preventing DNA binding.

Antibodies as thermolabile switches: high temperature triggering for the polymerase chain reaction.

We demonstrate the utility of antibodies to the DNA polymerase from Thermus aquaticus (TaqPol) as thermolabile inhibitors of TaqPol activity. One of the limitations of the polymerase chain reaction (PCR) is the co-amplification of non-specific products caused by TaqPol activity on low stringency templates present in the initial cycle of PCR. We have used anti-TaqPol antibodies as thermolabile switches that inhibit TaqPol activity at low temperatures (20-40 degrees C) and release fully active TaqPol when they are inactivated by elevated temperatures in the PCR thermal cycling (70-98 degrees C). Several in a set of high affinity anti-TaqPol monoclonal antibodies fully inhibited TaqPol activity at 37 degrees C. The capacity for inhibition was ablated by incubation at temperatures high enough to denature antibodies but not sufficiently high to significantly reduce TaqPol activity. In a PCR model system, preincubation of TaqPol with these antibodies yielded PCR product consisting entirely of the intended product and the absence or significant reduction of non-specific products and primer dimers. In evaluation of clinical samples such antibody triggering yielded defined PCR product and higher sensitivity because of the absence of non-specific products.

Developmental regulation of asparagine-linked oligosaccharide synthesis in Dictyostelium discoideum.

In the preceding report we demonstrated that the expression of two developmentally regulated alpha-mannosidase activities is induced in Dictyostelium discoideum during its differentiation from single-cell amoebae to multicellular organism (Sharkey, D. J., and Kornfeld, R. (1991) J. Biol. Chem. 266, 18477-18484). These activities, designated membrane alpha-mannosidase I (MI) and membrane alpha-mannosidase II (MII), were shown to have several properties in common with rat liver Golgi alpha-mannosidases I and II, respectively, suggesting that MI and MII may play a role in the processing of asparagine-linked oligosaccharides in developing D. discoideum. In this study we analyzed the structures of the asparagine-linked oligosaccharides synthesized by D. discoideum at various stages of development to determine the timing and extent of asparagine-linked oligosaccharide processing. Cells were labeled with [2-3H] mannose, and then total cellular glycoproteins were digested with Pronase to generate glycopeptides that were fractionated on concanavalin A-Sepharose. Glycopeptides from each fraction were digested with endoglycosidase H, both before and after desulfation by solvolysis, and the released, neutral oligosaccharides were sized by high pressure liquid chromatography. At early stages of development, D. discoideum contain predominantly large high mannose-type oligosaccharides (Man9GlcNAc and Man8GlcNAc). Some of these are modified by GlcNAc residues attached beta 1-4 to the mannose-linked alpha 1-6 to the beta-linked core mannose (the "intersecting" position), as well as by fucose, sulfate, and phosphate. In contrast, the oligosaccharides found at late stages of development (18-24 h) have an array of sizes from Man9GlcNAc to Man3GlcNAc. These are still modified by GlcNAc, fucose, sulfate, and phosphate, but the percent of larger high mannose oligosaccharides that are modified with GlcNAc in the intersecting position decreases after 6 h of development, in parallel with the decrease in the intersecting GlcNAc transferase activity. Similarly, the changes in the size of asparagine-linked oligosaccharides synthesized during development correlate well with the appearance of MI and MII activities and suggest that these developmentally regulated alpha-mannosidase activities function in the processing of these oligosaccharides. This is supported further by the observation that oligosaccharide processing was inhibited in late stage cells labeled in the presence of either deoxymannojirimycin, an inhibitor of MI, or swainsonine, an inhibitor of MII.