Correction: Ankyrin-G regulates forebrain connectivity and network synchronization via interaction with GABARAP.

In the original version of this article, affiliation 3 was given as: "Division of Life Sciences, State Key Laboratory of Molecular Neuroscience, Hong Kong, University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong, China". This has now been corrected to: "Division of Life Sciences, State Key Laboratory of Molecular Neuroscience, Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong, China".Additionally in the 'Data availability' section an incorrect accession code was given. The accession code has now been changed from 'PDB A9X (AnkG:GABARAPL)' to 'PDB 6A9X (AnkG:GABARAP)'.These errors have been corrected in both the PDF and HTML versions of the Article.

Ankyrin-G regulates forebrain connectivity and network synchronization via interaction with GABARAP.

GABAergic circuits are critical for the synchronization and higher order function of brain networks. Defects in this circuitry are linked to neuropsychiatric diseases, including bipolar disorder, schizophrenia, and autism. Work in cultured neurons has shown that ankyrin-G plays a key role in the regulation of GABAergic synapses on the axon initial segment and somatodendritic domain of pyramidal neurons, where it interacts directly with the GABAA receptor-associated protein (GABARAP) to stabilize cell surface GABAA receptors. Here, we generated a knock-in mouse model expressing a mutation that abolishes the ankyrin-G/GABARAP interaction (Ank3 W1989R) to understand how ankyrin-G and GABARAP regulate GABAergic circuitry in vivo. We found that Ank3 W1989R mice exhibit a striking reduction in forebrain GABAergic synapses resulting in pyramidal cell hyperexcitability and disruptions in network synchronization. In addition, we identified changes in pyramidal cell dendritic spines and axon initial segments consistent with compensation for hyperexcitability. Finally, we identified the ANK3 W1989R variant in a family with bipolar disorder, suggesting a potential role of this variant in disease. Our results highlight the importance of ankyrin-G in regulating forebrain circuitry and provide novel insights into how ANK3 loss-of-function variants may contribute to human disease.

Identifying areas of degrading and improving groundwater-quality conditions in the State of California, USA, 1974-2014.

Areas of improving and degrading groundwater-quality conditions in the State of California were assessed using spatial weighting of a new metric for scoring wells based on constituent concentrations and the direction and magnitude of a trend slope (Sen). Individual well scores were aggregated across 2135 equal-area grid cells covering the entire groundwater resource used for public supply in the state. Spatial weighting allows results to be aggregated locally (well or grid cell), regionally (groundwater basin), provincially, or statewide. Results differentiate degrading (increasing concentration trends) areas with low to moderate concentrations (unimpaired) from degrading areas with moderate to high concentrations (impaired). Results also differentiate improving areas (decreasing concentration trends) in the same manner. Multi-year to decadal groundwater-quality trends were computed from periodic, inorganic water-quality data for 38 constituents collected between 1974 and 2014 for compliance monitoring of nearly 13,000 public-supply wells (PSWs) in the State of California. Mann-Kendall (MK) rank correlations and Sen's slope estimator were used to detect statistically significant trends for the entire period of recorded data (long-term trend), for the period since 2000 (recent trend), for different pumping seasons (seasonal trend), and for reversals of trends. Statewide, the most frequently detected trends since 2000 were for nitrate (36%), gross alpha/uranium (10%), arsenic (14%), total dissolved solids (TDS) (23%), and the major ions that contribute to TDS (19-28%). The Transverse and Selected Peninsular Ranges (TSPR) and the San Joaquin Valley (SJV) hydrogeologic provinces had the largest percentage of areas with moderate to high nitrate concentrations and groundwater quality trends. Improving nitrate concentrations in parts of the TSPR is associated with long-term managed aquifer recharge that has replaced historical, agriculturally affected groundwater with low-nitrate recharge in parts of the TSPR. This example suggests that application of dilute, excess surface water to agricultural fields during the winter could improve groundwater-quality in the SJV over the long term.

Videolaryngoscopy versus direct laryngoscopy for emergency orotracheal intubation outside the operating room: a systematic review and meta-analysis.

Videolaryngoscopy (VL) may improve the success of orotracheal intubation compared with direct laryngoscopy (DL). We performed a systematic search of PubMed, Embase, and CENTRAL databases for studies comparing VL and DL for emergency orotracheal intubations outside the operating room. The primary outcome was rate of first-pass intubation, with subgroup analyses by location, device used, clinician experience, and clinical scenario. The secondary outcome was complication rates. Data are presented as [odds ratio (95% confidence intervals); P-values]. We identified 32 studies with 15 064 emergency intubations. There was no difference in first-pass intubation with VL compared with DL [OR=1.28, (0.99-1.65); P=0.06]. First-pass intubations were increased with VL compared with DL in the intensive care unit (ICU) [2.02 (1.43-2.85); P<0.001], and similar in the emergency department or pre-hospital setting. First-pass intubations were similar with GlideScope®, but improved with the CMAC® [1.32 (1.08-1.62); P=0.007] compared with DL. There was greater first-pass intubation with VL compared with DL amongst novice/trainee clinicians [OR=1.95 (1.45-2.64); P<0.001], but not amongst experienced clinicians or paramedics/nurses. There was no difference in first-pass intubation with VL compared with DL during cardiopulmonary resuscitation or trauma. VL compared with DL was associated with fewer oesophageal intubations [OR=0.32 (0.14-0.70); P=0.003], but more arterial hypotension [OR=1.49 (1.00-2.23); P=0.05]. In summary, VL compared with DL is associated with greater first-pass emergency intubation in the ICU and amongst less experienced clinicians, and reduces oesophageal intubations. However, VL is associated with greater incidence of arterial hypotension. Further trials investigating the utility of VL over DL in specific situations are required.

Paederus Dermatitis: Two Outbreaks of an Entomologic Disease in Austere Expeditionary Care.

Paederus dermatitis, also known as dermatitis linearis, is an acute cutaneous condition caused by contact with the potent vesicant toxin paederin, produced by endosymbiotic Pseudomonas-like bacteria within Paederus spp. beetles. Paederin is a protein synthesis inhibitor that halts cell division, leading to vesiculation and necrotic breakdown of the epidermis. The resultant damage facilitates secondary infection, especially in austere conditions. Between October 2022 and July 2023, several US special operations teams forward deployed to various countries in East Africa reported over 17 cases of painful, blistering skin eruptions consistent with Paederus dermatitis, 2 of which are summarized in this report. To confirm the best treatment approach, 1 team's medic reached back to a deployed infectious disease physician in the same Combatant Command as well as through the DoD ADVISOR line to a dermatologist in the United States. All cases were managed with irrigation, followed by a combination of topical steroid and antibacterial ointments, with resolution occurring after 3 to 7 days, whereas 38% of the cases in July 2023 developed secondary cellulitis by day 7, requiring oral antibiotic treatment. Strict preventative measures were implemented to limit exposure while carefully observing each case, as this was critical to optimize medical readiness of the team. Ultimately, we seek to highlight the identification of beetles specific to East Africa, seasonal prevalence, and the importance of preventative measures as they vary depending on specific beetle characteristics. Additionally, we reiterate classic presentation features that help avoid misdiagnosis for cases that occur outside of the typical epidemiologic criteria.

Optimization of patient positioning for improved healing after corneal transplantation.

Corneal transplantation is the only solution which avoids loss of vision, when endothelial cells are dramatically lost. The surgery involves injecting gas into the anterior chamber of the eye, to create a bubble that pushes onto the donor cornea (graft), achieving sutureless adherence to the host cornea. During the postoperative period, patient positioning affects the bubble. To improve healing, we study the shape of the gas-bubble interface throughout the postoperative period, by numerically solving the equations of fluid motion. Patient-specific anterior chambers (ACs) of variable anterior chamber depths (ACD) are considered, for either phakic (with natural lens) and pseudophakic (with artificial lens) eyes. For each AC, gas-graft coverage is computed for different gas fill and patient positioning. The results show that the influence of positioning is negligible, regardless of gas filling, as long as the ACD is small. However, when the ACD value increases, patient positioning becomes important, especially for pseudophakic ACs. The difference between best and worst patient positioning over time, for each AC, is negligible for small ACD but significant for larger ACD, especially for pseudophakic eyes, where guidelines for optimal positioning become essential. Finally, mapping of the bubble position highlights the importance of patient positioning for an even gas-graft coverage.

Lipopolysaccharide structure modulates cationic biocide susceptibility and crystalline biofilm formation in Proteus mirabilis.

Chlorhexidine (CHD) is a cationic biocide used ubiquitously in healthcare settings. Proteus mirabilis, an important pathogen of the catheterized urinary tract, and isolates of this species are often described as "resistant" to CHD-containing products used for catheter infection control. To identify the mechanisms underlying reduced CHD susceptibility in P. mirabilis, we subjected the CHD tolerant clinical isolate RS47 to random transposon mutagenesis and screened for mutants with reduced CHD minimum inhibitory concentrations (MICs). One mutant recovered from these screens (designated RS47-2) exhibited ~ 8-fold reduction in CHD MIC. Complete genome sequencing of RS47-2 showed a single mini-Tn5 insert in the waaC gene involved in lipopolysaccharide (LPS) inner core biosynthesis. Phenotypic screening of RS47-2 revealed a significant increase in cell surface hydrophobicity and serum susceptibility compared to the wildtype, and confirmed defects in LPS production congruent with waaC inactivation. Disruption of waaC was also associated with increased susceptibility to a range of other cationic biocides but did not affect susceptibility to antibiotics tested. Complementation studies showed that repression of smvA efflux activity in RS47-2 further increased susceptibility to CHD and other cationic biocides, reducing CHD MICs to values comparable with the most CHD susceptible isolates characterized. The formation of crystalline biofilms and blockage of urethral catheters was also significantly attenuated in RS47-2. Taken together, these data show that aspects of LPS structure and upregulation of the smvA efflux system function in synergy to modulate susceptibility to CHD and other cationic biocides, and that LPS structure is also an important factor in P. mirabilis crystalline biofilm formation.

Spectrin: a structural mediator between diverse plasma membrane proteins and the cytoplasm.

The spectrin skeleton of non-erythroid cells is likely to interact with a variety of integral membrane proteins and participate both in stable linkages as well as dynamic structures capable of rapid disassembly and assembly. The basis for diversity of roles for spectrin includes multiple, functionally distinct isoforms of spectrin, ankyrin and other associated proteins, regulation of protein interactions through phosphorylation and calcium/calmodulin, as well as differential expression of accessory proteins that determine the organization and localization of spectrin in cells. Spectrin is highly conserved from Drosophila to man and is likely to be involved in fundamental aspects of membrane structure requiring long range order and organization. Spectrin is a candidate to interact with many integral membrane proteins in roles basic to metazoan cells which must associate into tissues. Organization of cells into tissues requires loss of cell motility, formation of specialized membrane domains and assembly of cell junctions, which are all activities potentially involving spectrin. Future challenges lie in devising direct experiments to evaluate the functions of spectrin in cells and tissues.

Ankyrins and cellular targeting of diverse membrane proteins to physiological sites.

Ankyrins are spectrin-binding proteins that associate via ANK repeats with a variety of ion channels/pumps, calcium release channels and cell adhesion molecules. Recent studies in mice indicate that ankyrins have a physiological role in restricting voltage-gated sodium channels and members of the L1 CAM family of cell adhesion molecules to excitable membranes in the central nervous system and in targeting calcium-release channels to the calcium homeostasis compartment of striated muscle.

Review of preventative behavioural interventions for dermal and respiratory hazards.

BACKGROUND: No previous systematic review of the evidence base has been undertaken to help occupational health professionals understand how to reliably lower the instance of occupational ill-health through reducing risk-taking behaviour. AIMS: To evaluate the effectiveness and processes of occupational-based behavioural interventions for workers exposed to dermal and respiratory hazards. METHODS: A systematic review was conducted. Sixteen electronic databases were searched using key words. Bibliography, health and safety websites and hand searches of key journals were also undertaken. Articles were included if they evaluated an intervention targeting workers' behavioural compliance, addressed dermal or respiratory hazards, used before and after measures with a control group comparison and used behaviour-related exposure indicators such as airborne exposure, health effects, behaviour observations and self-reported work practices. Data were extracted according to potential sources of bias, impact and behavioural change processes used. RESULTS: Ten of 550 articles identified as potentially relevant were included. A predominance of small effect sizes, particularly for larger samples, demonstrated limited but positive impact upon exposure. Studies contained too much heterogeneity for reliable meta-analysis. None of the studies covered the full range of behaviour change components necessary for reducing exposure risk. CONCLUSIONS: We conclude that future interventions could enhance their effectiveness through improving design quality, reporting and basing their content upon evidence-based behavioural change approaches. Using a comprehensive range of evidence-informed behaviour change ingredients should improve occupational health professional's ability to reliably reduce occupational ill-health where exposure cannot totally be designed out of the workplace.

Drifting frozen waves.

We describe a dynamical state observed shortly above onset of the frozen wave instability. The transition to drifting waves, which are repeatedly created and destroyed, is a marked departure from the usual behavior of frozen waves, which are generally understood to remain motionless (on average) in the reference frame of the vibrating container. The spatial inhomogeneity of the underlying base flow, due both to the presence of the lateral walls and to the associated vibroequilibria effect, provides the driving mechanism. Energy arguments are used to understand the initial outward drift and the existence of a critical threshold which is estimated from the dependence of the drift velocity on the applied forcing. The dependence on container aspect ratio Γ is investigated, and drifting is seen to occur only when 1.5≲Γ≲3.5.

The ANK repeat: a ubiquitous motif involved in macromolecular recognition.

Many proteins rely on stable, noncovalent interactions with other macromolecules to perform their function. The identification of a repeated sequence motif, the ANK repeat, in diverse proteins whose common function involves binding to other proteins indicates one way nature may achieve a wide range of protein-protein interactions. In this article, we describe evidence that these ANK repeats are involved in the specific recognition of proteins and possibly DNA, and present a model for the folding of the motif.

Clathrin-coated vesicle assembly polypeptides: physical properties and reconstitution studies with brain membranes.

The assembly polypeptides are an integral component of coated vesicles and may mediate the linkage of clathrin to the vesicle membrane. We have purified assembly polypeptides in milligram quantities from bovine brain by an improved procedure. Hydrodynamic and chemical crosslinking studies indicate that the protein is an asymmetric heterotetramer with a molecular weight of 252,000, containing two subunits of Mr 98,000-115,000, one subunit of 52,000, and one subunit of 16,000. Two-dimensional peptide maps of the subunits show that the 16- and 52-kD polypeptides are not derived from the higher molecular weight species, and that the group of bands at 98-115 kD are related. Electron microscopic visualization shows an essentially globular protein with one or two knob-like tails. We demonstrate a specific membrane protein binding site for 125I-labeled assembly polypeptides in 0.1 N sodium hydroxide-extracted bovine brain membranes based on the following criteria: (a) binding is displaceable by unlabeled ligand, (b) the binding site is destroyed by protease treatment of the membranes, and (c) the distribution of binding between vesicle-depleted membranes and coated vesicle membranes parallels the in vivo localization of assembly polypeptides and clathrin. This binding site is likely to be an integral membrane protein because (a) it is enriched in the sodium hydroxide-extracted membranes stripped of most of their peripheral membrane proteins, and (b) the binding site is partially extracted by 0.5% Triton X-100. A similar binding site appears to be present in coated vesicles. Clathrin binds to the hydroxide-stripped membranes in an assembly polypeptides dependent manner, and this binding is diminished by Triton extraction of the membranes. This assay may aid in identification of the membrane receptor for the assembly polypeptides.

Ankyrin-G coordinates assembly of the spectrin-based membrane skeleton, voltage-gated sodium channels, and L1 CAMs at Purkinje neuron initial segments.

The axon initial segment is an excitable membrane highly enriched in voltage-gated sodium channels that integrates neuronal inputs and initiates action potentials. This study identifies Nav1.6 as the voltage-gated sodium channel isoform at mature Purkinje neuron initial segments and reports an essential role for ankyrin-G in coordinating the physiological assembly of Nav1.6, betaIV spectrin, and the L1 cell adhesion molecules (L1 CAMs) neurofascin and NrCAM at initial segments of cerebellar Purkinje neurons. Ankyrin-G and betaIV spectrin appear at axon initial segments by postnatal day 2, whereas L1 CAMs and Nav1.6 are not fully assembled at continuous high density along axon initial segments until postnatal day 9. L1 CAMs and Nav1.6 therefore do not initiate protein assembly at initial segments. betaIV spectrin, Nav1.6, and L1 CAMs are not clustered in adult Purkinje neuron initial segments of mice lacking cerebellar ankyrin-G. These results support the conclusion that ankyrin-G coordinates the physiological assembly of a protein complex containing transmembrane adhesion molecules, voltage-gated sodium channels, and the spectrin membrane skeleton at axon initial segments.

Distinct ankyrin isoforms at neuron cell bodies and nodes of Ranvier resolved using erythrocyte ankyrin-deficient mice.

Isoforms of ankyrin (ankyrinsR) immunologically related to erythrocyte ankyrin (ankyrinRo) are associated with distinct neuronal plasma membrane domains of functional importance, such as cell bodies and dendrites, axonal hillock and initial segments, and nodes of Ranvier. AnkyrinRo is expressed in brain, and accounts for at least one of the ankyrinR isoforms. Another ankyrin isoform of brain, ankyrinB, is encoded by a distinct gene and is immunologically distinct from ankyrinsR. Mutant mice with normoblastosis (nb/nb) constitute the first described genetic model of ankyrin deficiency: they display a severe hemolytic anemia due to a significantly reduced expression of the ankyrinRo gene in reticulocytes as well as brain (Peters L. L., C. S. Birkenmeier, R. T. Bronson, R. A. White, S. E. Lux, E. Otto, V. Bennett, A. Higgins, and J. E. Barker. 1991. J. Cell Biol. 114:1233-1241). In the present report, we distinguish between ankyrinRo and other ankyrinR isoforms using immunoblot analysis and immunofluorescence localization of ankyrinsR throughout the nervous system (forebrain, cerebellum, brain stem, spinal cord, and sciatic nerve) of nb/nb and normal mice. This is the first immunocytochemical characterization of the neurological component of the nb mutation and shows the following. (a) The isoform of ankyrin at the nodes of Ranvier and initial axonal segments is present in the nb/nb mice and does not cross-react with an ankyrinRo-specific antibody; this isoform, therefore, is distinct from both ankyrin isoforms identified in brain, ankyrinRo and ankyrinB, and is probably the product of a distinct gene and a unique component of the specialized membrane skeleton associated with nodes of Ranvier. (b) AnkyrinRo missing from nb/nb mice is selectively associated with neuronal cell bodies and dendrites, excluded from myelinated axons, and displays a selective pattern of expression in the nervous system whereby expression is almost ubiquitous in neurons of the cerebellum (Purkinje and granule cells) and spinal cord, and restricted to a very minor subset of neurons in hippocampus and neocortex of forebrain.

Restriction of 480/270-kD ankyrin G to axon proximal segments requires multiple ankyrin G-specific domains.

AnkyrinG (-/-) neurons fail to concentrate voltage-sensitive sodium channels and neurofascin at their axon proximal segments, suggesting that ankyrinG is a key component of a structural pathway involved in assembly of specialized membrane domains at axon proximal segments and possibly nodes of Ranvier (Zhou, D., S. Lambert, D.L. Malen, S. Carpenter, L. Boland, and V. Bennett, manuscript submitted for publication). This paper addresses the mechanism for restriction of 270-kD ankyrinG to axon proximal segments by evaluation of localization of GFP-tagged ankyrinG constructs transfected into cultured dorsal root ganglion neurons, as well as measurements of fluorescence recovery after photobleaching of neurofascin- GFP-tagged ankyrinG complexes in nonneuronal cells. A conclusion is that multiple ankyrinG-specific domains, in addition to the conserved membrane-binding domain, contribute to restriction of ankyrinG to the axonal plasma membrane in dorsal root ganglion neurons. The ankyrinG-specific spectrin-binding and tail domains are capable of binding directly to sites on the plasma membrane of neuronal cell bodies and axon proximal segments, and presumably have yet to be identified docking sites. The serine-rich domain, which is present only in 480- and 270-kD ankyrinG polypeptides, contributes to restriction of ankyrinG to axon proximal segments as well as limiting lateral diffusion of ankyrinG-neurofascin complexes. The membrane-binding, spectrin-binding, and tail domains of ankyrinG also contribute to limiting the lateral mobility of ankyrinG-neurofascin complexes. AnkyrinG thus functions as an integrated mechanism involving cooperation among multiple domains heretofore regarded as modular units. This complex behavior explains ability of ankyrinB and ankyrinG to sort to distinct sites in neurons and the fact that these ankyrins do not compensate for each other in ankyrin gene knockouts in mice.

Human erythrocyte myosin: identification and purification.

Human erythrocytes contain an Mr 200,000 polypeptide that cross-reacts specifically with affinity-purified antibodies to the Mr 200,000 heavy chain of human platelet myosin. Immunofluorescence staining of formaldehyde-fixed erythrocytes demonstrated that the immunoreactive myosin polypeptide is present in all cells and is localized in a punctate pattern throughout the cell. Between 20-40% of the immunoreactive myosin polypeptide remained associated with the membranes after hemolysis and preparation of ghosts, suggesting that it may be bound to the membrane cytoskeleton as well as being present in the cytosol. The immunoreactive myosin polypeptide was purified from the hemolysate to approximately 85% purity by DEAE-cellulose chromatography followed by gel filtration on Sephacryl S-400. The purified protein is an authentic vertebrate myosin with two globular heads at the end of a rod-like tail approximately 150-nm long, as visualized by rotary shadowing of individual molecules, and with two light chains (Mr 25,000 and 19,500) in association with the Mr 200,000 heavy chain. Peptide maps of the Mr 200,000 heavy chains of erythrocyte and platelet myosin were seen to be nearly identical, but the proteins are distinct since the platelet myosin light chains migrate differently on SDS gels (Mr 20,000 and 17,000). The erythrocyte myosin formed bipolar filaments 0.3-0.4-micron long at physiological salt concentrations and exhibited a characteristic pattern of myosin ATPase activities with EDTA, Ca++, and Mg++-ATPase activities in 0.5 M KCl of 0.38, 0.48, and less than 0.01 mumol/min per mg. The Mg++-ATPase activity of erythrocyte myosin in 0.06 M KCl (less than 0.01 mumol/min per mg) was not stimulated by the addition of rabbit muscle F-actin. The erythrocyte myosin was present in about 6,000 copies per cell, in a ratio of 80 actin monomers for every myosin molecule, which is an amount comparable to actin/myosin ratios in other nonmuscle cells. The erythrocyte myosin could function together with tropomyosin on the erythrocyte membrane (Fowler, V.M., and V. Bennett, 1984, J. Biol. Chem., 259:5978-5989) in an actomyosin contractile apparatus responsible for ATP-dependent changes in erythrocyte shape.

LAD-1, the Caenorhabditis elegans L1CAM homologue, participates in embryonic and gonadal morphogenesis and is a substrate for fibroblast growth factor receptor pathway-dependent phosphotyrosine-based signaling.

This study shows that L1-like adhesion (LAD-1), the sole Caenorhabditis elegans homologue of the L1 family of neuronal adhesion molecules, is required for proper development of the germline and the early embryo and embryonic and gonadal morphogenesis. In addition, the ubiquitously expressed LAD-1, which binds to ankyrin-G, colocalizes with the C. elegans ankyrin, UNC-44, in multiple tissues at sites of cell-cell contact. Finally, we show that LAD-1 is phosphorylated in a fibroblast growth factor receptor (FGFR) pathway-dependent manner on a tyrosine residue in the highly conserved ankyrin-binding motif, FIGQY, which was shown previously to abolish the L1 family of cell adhesion molecule (L1CAM) binding to ankyrin in cultured cells. Immunofluorescence studies revealed that FIGQY-tyrosine-phosphorylated LAD-1 does not colocalize with nonphosphorylated LAD-1 or UNC-44 ankyrin but instead is localized to sites that undergo mechanical stress in polarized epithelia and axon-body wall muscle junctions. These findings suggest a novel ankyrin-independent role for LAD-1 related to FGFR signaling. Taken together, these results indicate that L1CAMs constitute a family of ubiquitous adhesion molecules, which participate in tissue morphogenesis and maintaining tissue integrity in metazoans.

A new 440-kD isoform is the major ankyrin in neonatal rat brain.

This report describes initial characterization of a 440-kD isoform of brain ankyrin (ankyrinB) representing an alternatively spliced mRNA product of the gene encoding the major isoform of ankyrin in adult human brain (Otto, E., M. Kunimoto, T. McLaughlin, V. Bennett, J. Cell Biology. 114:241-253). Northern and immunoblot analyses indicate that 440-kD ankyrinB includes the spectrin and membrane-binding domains as well as a regulatory domain of the major 220-kD isoform. 440-kD ankyrinB contains, in addition, a sequence of a predicted size of 220 kD which is inserted between the regulatory domain and spectrin/membrane-binding domains. 440-kD ankyrinB has properties expected of a peripherally associated membrane-skeletal protein: it is exclusively present in the particulate fraction of brain homogenates, is extracted with NaOH, and remains associated with Triton-X-100-resistant structures. Expression of 440-kD ankyrinB in rat brain began at birth before other ankyrins could be detected, peaked 10 d after birth, and then decreased progressively to 30% of the maximum in adults. Expression of the 220-kD ankyrinB and ankyrinR (erythroid ankyrin) began approximately 10 d after the 440-kD isoform, increased rapidly between 10 and 15 d after birth, and finally achieved their maximal levels in adults. 440-kD ankyrinB is present in approximately equivalent amounts in all regions of neonatal brain while in adult brain it is present in highest levels in cerebellum and lowest in brain stem. 440-kD ankyrinB was localized by immunofluorescence in regions of neonatal and adult brain containing primarily dendrites and unmyelinated axons. 440-kD ankyrinB thus may play a specialized role in neuronal processes.

Isolation and characterization of cDNAs encoding human brain ankyrins reveal a family of alternatively spliced genes.

Ankyrins are a family of membrane-associated proteins that can be divided into two immunologically distinct groups: (a) erythrocyte-related isoforms (ankyrinR) that have polarized distributions in particular cell types; and (b) brain-related isoforms (ankyrinB) that display a broader distribution. In this paper, we report the isolation and sequences of cDNAs related to two ankyrinB isoforms, human brain ankyrin 1 and 2, and show that these isoforms are produced from alternatively spliced mRNAs of a single gene. Human brain ankyrin 1 and 2 share a common NH2-terminus that is similar to human erythrocyte ankyrins, with the most striking conservation occurring between areas composed of a repeated 33-amino acid motif and between areas corresponding to the central portion of the spectrin-binding domain. In contrast, COOH-terminal sequences of brain ankyrin 1 and 2 are distinct from one another and from human erythrocyte ankyrins, and thus are candidates to mediate protein interactions that distinguish these isoforms. The brain ankyrin 2 cDNA sequence includes a stop codon and encodes a polypeptide with a predicted molecular mass of 202 kD, which is similar to the Mr of the major form of ankyrin in adult bovine brain membranes. Moreover, an antibody raised against the conserved NH2-terminal domain of brain ankyrin cross-reacts with a single Mr = 220 kD polypeptide in adult human brain. These results strongly suggest that the amino acid sequence of brain ankyrin 2 determined in this report represents the complete coding sequence of the major form of ankyrin in adult human brain. In contrast, the brain ankyrin 1 cDNAs encode only part of a larger isoform. An immunoreactive polypeptide of Mr = 440 kD, which is evident in brain tissue of young rats, is a candidate to be encoded by brain ankyrin 1 mRNA. The COOH-terminal portion of brain ankyrin 1 includes 15 contiguous copies of a novel 12-amino acid repeat. Analysis of DNA from a panel of human/rodent cell hybrids linked this human brain ankyrin gene to chromosome 4. This result, coupled with previous reports assigning the human erythrocyte ankyrin gene to chromosome 8, demonstrates that human brain and erythrocyte ankyrins are encoded by distinct members of a multigene family.