Search

Background and Objectives: Predicting the development of adjacent segment disease (ASD) after lumbar spine fusion would help guide preoperative and postoperative therapies to prevent reoperation. We sought to evaluate whether sarcopenia predicts the development of early ASD after transforaminal lumbar interbody fusion (TLIF)., Methods: Retrospective data were collected from 109 patients who underwent TLIF from 2013 to 2023. Patients older than 18 years who underwent elective posterior midline approach TLIF were included. Patients with prior lumbar instrumented fusions, cases of trauma, central nervous system infection, cancer, or long-construct thoracolumbar deformity corrections and those who lacked sufficient follow-up were excluded. The primary outcome was radiographic ASD development within 3 years of surgery. Psoas volumetric measurements were recorded from the most recent preoperative MRI. Odds ratios were calculated with logistic regression analyses., Results: In 109 patients undergoing elective TLIF, 22 (20.2%) developed ASD within 3 years. Gender, body mass index, and extent of surgery were not associated with ASD development. Multivariate analysis showed left/right psoas cross-sectional area, and psoas:vertebral body ratio (P:VBR) predicted early ASD (P < .0001). Sarcopenia was further categorized as having bilateral P:VBR ≥1 SD below gender mean (T-score -1). Of 18 sarcopenic patients, 15 developed early ASD (83.33%) vs 7 of 91 nonsarcopenic patients (7.69%; P < .0001). Postoperative mismatch between pelvic incidence and lumbar lordosis was predictive of ASD on univariate (P = .0480) but not multivariate analysis. Pelvic tilt and lumbar lordosis postoperatively were not associated with early ASD., Conclusion: Sarcopenia, measured by decreased psoas area and P:VBR, predicts ASD formation within 3 years of surgery. Morphometric analysis of psoas size is a simple tool to identify patients at risk of developing ASD. This information can potentially guide preoperative and postoperative therapies, affect surgical decision making, and effectively counsel patients on risks of reoperation., (Copyright © Congress of Neurological Surgeons 2024. All rights reserved.)

Background: C5 palsy (C5P) is a recognized potential postoperative complication of cervical spine surgery but has rarely been reported following an open esophageal diverticulectomy., Methods: A 61-year-old underwent an open esophageal diverticulectomy for symptomatic Zencker's diverticulum., Results: Postoperatively, she presented with right upper extremity weakness and sensory deficits consistent with a C5P that was later confirmed by electromyography., Conclusion: The potential for C5P after esophageal diverticulectomy for symptomatic Zencker's diverticulum is rare. Postoperative recognition and appropriate management are critical to recovery., Competing Interests: There are no conflicts of interest., (Copyright: © 2024 Surgical Neurology International.)

Background: Choroid plexus papillomas (CPPs) are rare neoplasms arising from choroid plexus epithelium representing <1% of all intracranial tumors. Symptoms vary based on location and regional mass effect; however, hydrocephalus is common due to cerebrospinal fluid flow obstruction and/or overproduction. Distant site metastasis or de novo formation in extraventricular sites is rare., Case Description: A 57-year-old female with a history of a 4 th ventricular CPP status post resection in 2004 and 2018 with subsequent gamma knife therapy in 2019 presented with increased thirst and urination. Since her initial surgery, she has experienced significant gait imbalance, diplopia, dysphagia, and right-sided hemiparesis and hemisensory loss. Magnetic resonance imaging revealed a new 1.5 × 1.8 cm suprasellar lesion. She underwent a left supraorbital craniotomy for tumor resection, with pathology revealing metastatic World Health Organization grade II CPP., Conclusion: Extraventricular manifestation of CPP is rare. De novo or metastatic involvement of the sella has seldom been reported. Treatment should target gross total surgical resection. Adjuvant chemotherapy and radiation may be useful in higher-grade lesions., Competing Interests: There are no conflicts of interest., (Copyright: © 2024 Surgical Neurology International.)

Background: Malignant peripheral nerve sheath tumors (MPNSTs) are rare malignant soft-tissue sarcomas arising from peripheral nerves. Little data exist regarding MPNST originating intracranially. Here, we present a 7 th /8 th nerve complex MPNST, discuss the treatment strategy and patient outcome, and provide a comprehensive review of existing literature., Methods: Using Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines, PubMed and crossed references were queried, yielding 37 publications from 1952 to the present. Fifty-three cases of primary intracranial and extra-axial MPNST were identified., Results: We additionally report a 40-year-old female presented with acute onset dizziness and subsequent hearing loss with associated right-sided facial numbness. Magnetic resonance imaging revealed a 0.5 cm × 1.7 cm enhancing lesion within the right internal auditory canal extending into the cerebellopontine angle. The patient was initially treated with retro sigmoid craniotomy for tumor resection followed by a trans labyrinth approach for residual tumor resection. She completed adjuvant fractionated radiation therapy and underwent facial nerve transfer to restore complete hemifacial paralysis. The most common cranial nerves involved were V and VIII (43.4% each), with 66% of patients male and 34% female. The average age was 43.4 ± 17.4 years. The mean survival time for reported non-survivors after tissue diagnosis was 15 ± 4 months. Two-year survival for patients receiving gross total resection was 33.3% versus 22.8% with subtotal resection., Conclusion: MPNSTs comprise a group of highly aggressive neoplasms that rarely arise intracranially. Gross total surgical resection should be pursued when feasible., Competing Interests: There are no conflicts of interest., (Copyright: © 2024 Surgical Neurology International.)

Background: Myelopathy and nerve root dysfunction resulting from the imperceptible growth of intraspinal schwannomas have been well documented.[1] Thoracic spine schwannomas, in particular, have exceptional growth potential due to the presence of the posterior mediastinum and retropleural spaces accommodating insidious and often subclinical tumor expansion.[5] Extraspinal extension of these lesions, however, poses a distinct challenge for surgeons.[3,4]., Case Description: Here, we provide a two-dimensional intraoperative video demonstrating the technical nuances concerning maximally safe resection of a partially cystic thoracic dumbbell schwannoma having extraspinal extension with associated bony remodeling of the T10 vertebral body and neural foramen in a middle-aged male. A posterolateral approach with T8-T12 fusion, retropleural thoracotomy, facetectomies, and pediculectomies allowed for gross total resection. No intraoperative or postoperative complications were observed, and the parietal pleura was kept intact throughout the surgery. In addition, the patient continued to have improved symptoms and was ambulatory at 6-month follow-up., Conclusion: Gross total resection of a partially cystic thoracic dumbbell schwannoma was achieved without complications. Our use of a preoperative three-dimensional reconstruction for surgical planning,[2] intraoperative ultrasound,[6] and a durable instrumentation construct were essential for a successful outcome. Moreover, great care was taken to avoid violating the tumor-parietal pleura plane, which would have resulted in postoperative respiratory complications., Competing Interests: There are no conflicts of interest., (Copyright: © 2024 Surgical Neurology International.)

Background: Calcium pyrophosphate deposition disease (CPPD), also known as "pseudogout," is a crystal deposition arthropathy involving the synovial and periarticular tissues. Pseudogout rarely presents in the axial spine. Here, we present the case of an 80-year-old female patient admitted after a mechanical fall, initially misdiagnosed on computed tomography (CT)/magnetic resonance studies with cervical osteodiscitis/ventral epidural abscess that proved to be pseudogout., Case Description: An 80-year-old female was admitted after a mechanical fall. The initial cervical CT scan showed multilevel degenerative changes with an acute C6 anterior wedge compression fracture, focal kyphosis, C5-6 disc space collapse, and endplate destruction. The magnetic resonance imaging showed marked contrast enhancement of the C5-6 vertebral bodies and disc space. An interventional radiology-guided biopsy of the C5-6 vertebral bodies and disc space was consistent with calcium pyrophosphate deposits, was diagnostic for pseudogout, and was negative for infection. She was managed conservatively with a rigid collar and seven days of oral prednisone., Conclusion: CPPD involvement in the axial spine is rare. Prompt pathologic diagnosis should be pursued to rule out an infectious process., Competing Interests: There are no conflicts of interest., (Copyright: © 2024 Surgical Neurology International.)

Background: Thymomas rarely metastasize to the spine. Here, we present a 69-year-old female diagnosed with stage IV thymoma, which subsequently developed a symptomatic epidural thoracic spinal lesion causing thoracic myelopathy., Case Description: The patient initially presented with paraspinal rib pain, lower extremity weakness, and gait imbalance. The magnetic resonance revealed a T10 vertebral body lesion with epidural extension causing severe spinal cord compression. A T9-T10 hemilaminotomy for tumor resection was performed; this was followed by adjuvant chemotherapy and radiation. Gross total resection was achieved, and the final pathology was metastatic thymoma. Postoperatively, the patient significantly improved., Conclusion: Metastatic thymomas to the thoracic spine are rare. For those presenting with epidural lesions causing myelopathy, surgical resection is beneficial and may be accompanied by adjunctive radiation and chemotherapy., Competing Interests: There are no conflicts of interest., (Copyright: © 2023 Surgical Neurology International.)

The majority of spinal nerve sheath tumors are within the intradural/extramedullary compartment. A subset of these tumors develop extraforaminal components that gradually expand into potential spaces. Herein, the authors provide a 2D video demonstrating the technical nuances concerning resection of cervical dumbbell schwannomas with extraspinal extension. Although nerve sheath tumors with large extraforaminal extension are often associated with complications and pose unique challenges to surgeons, circumferential exposure with intradural exploration allows for gross-total resection and nerve root preservation, without need for adjuvant treatments. The use of intraoperative ultrasound, neurophysiological monitoring, Doppler imaging, and meticulous surgical techniques aided to circumvent complications., Competing Interests: Disclosures The authors report no conflict of interest concerning the materials or methods used in this study or the findings specified in this publication.The authors report no conflict of interest concerning the materials or methods used in this study or the findings specified in this publication., (© 2023, The Authors.)

Background: Retro-odontoid pseudotumors are rare inflammatory complications of atlantoaxial instability often associated with cervical degenerative disease and rheumatoid arthritis. While propagation of these lesions has been shown to cause spinal cord compression and cervical myelopathy, intradural extension has rarely been reported., Methods: In this manuscript and 2-dimensional illustrative intraoperative video, we demonstrate cervical decompression, removal of the intradural component, and stabilization with C1-2 instrumentation using a posterior approach. A 71-year-old patient presented with progressive cervical myelopathy. Preoperative imaging demonstrated a large retro-odontoid pannus causing severe spinal cord compression and an associated contrast-enhancing intradural lesion, in the absence of obvious C1-2 instability or fractures on computed tomography scan. C1-2 posterior decompression and fusion were performed with maximally safe intradural pannus resection and ventral dural reconstruction., Results: Postoperatively, the patient experienced significant improvement in myelopathic symptoms. Imaging demonstrated good spinal cord decompression with complete intradural pannus resection and debulking of the extradural component., Conclusions: Our outcome in this rare complication suggests a posterior approach may be effective in treating similar patients., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Background: The coronavirus (COVID-19) pandemic has caused unprecedented suspensions of neurosurgical elective surgeries, a large proportion of which involve spine procedures. The goal of this study is to report granular data on the impact of early COVID-19 pandemic operating room restrictions upon neurosurgical case volume in academic institutions, with attention to its secondary impact upon neurosurgery resident training. This is the first multicenter quantitative study examining these early effects upon neurosurgery residents caseloads., Methods: A retrospective review of neurosurgical caseloads among seven residency programs between March 2019 and April 2020 was conducted. Cases were grouped by ACGME Neurosurgery Case Categories, subspecialty, and urgency (elective vs. emergent). Residents caseloads were stratified into junior (PGY1-3) and senior (PGY4-7) levels. Descriptive statistics are reported for individual programs and pooled across institutions., Results: When pooling across programs, the 2019 monthly mean (SD) case volume was 214 (123) cases compared to 217 (129) in January 2020, 210 (115) in February 2020, 157 (81), in March 2020 and 82 (39) cases April 2020. There was a 60% reduction in caseload between April 2019 (207 [101]) and April 2020 (82 [39]). Adult spine cases were impacted the most in the pooled analysis, with a 66% decrease in the mean number of cases between March 2020 and April 2020. Both junior and senior residents experienced a similar steady decrease in caseloads, with the largest decreases occurring between March and April 2020 (48% downtrend)., Conclusions: Results from our multicenter study reveal considerable decreases in caseloads in the neurosurgical specialty with elective adult spine cases experiencing the most severe decline. Both junior and senior neurosurgical residents experienced dramatic decreases in case volumes during this period. With the steep decline in elective spine cases, it is possible that fellowship directors may see a disproportionate increase in spine fellowships in the coming years. In the face of the emerging Delta and Omicron variants, programs should pay attention toward identifying institution-specific deficiencies and developing plans to mitigate the negative educational effects secondary to such caseloads reduction., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2022 The Author(s).)

Background: Novel methods in predicting survival in patients with spinal metastases may help guide clinical decision-making and stratify treatments regarding surgery vs palliative care., Objective: To evaluate whether the frailty/sarcopenia paradigm is predictive of survival and morbidity in patients undergoing surgery for spinal metastasis., Methods: A total of 271 patients from 4 tertiary care centers who had undergone surgery for spinal metastasis were identified. Frailty/sarcopenia was defined by psoas muscle size. Survival hazard ratios were calculated using multivariate analysis, with variables from demographic, functional, oncological, and surgical factors. Secondary outcomes included improvement of neurological function and postoperative morbidity., Results: Patients in the smallest psoas tertile had shorter overall survival compared to the middle and largest tertile. Psoas size (PS) predicted overall mortality more strongly than Tokuhashi score, Tomita score, and Karnofsky Performance Status (KPS). PS predicted 90-d mortality more strongly than Tokuhashi score, Tomita score, and KPS. Patients with a larger PS were more likely to have an improvement in deficit compared to the middle tertile. PS was not predictive of 30-d morbidity., Conclusion: In patients undergoing surgery for spine metastases, PS as a surrogate for frailty/sarcopenia predicts 90-d and overall mortality, independent of demographic, functional, oncological, and surgical characteristics. The frailty/sarcopenia paradigm is a stronger predictor of survival at these time points than other standards. PS can be used in clinical decision-making to select which patients with metastatic spine tumors are appropriate surgical candidates., (Copyright © 2020 by the Congress of Neurological Surgeons.)

Background: Resection of intradural spinal tumors typically utilizes a posterior approach and often contributes to significant biomechanical instability and sagittal deformity., Methods: We searched PubMed for studies regarding pre- and postoperative spine biomechanics/alignment in patients with intradural tumors undergoing posterior decompressions., Results: Three patients underwent posterior decompressions with instrumented fusions to preserve good sagittal alignment postoperatively. Variables analyzed in this study included the extent of preoperative and postoperative deformity, the number of surgical levels decompressed and fused, the different frequencies of instability following the resection of cervical versus thoracic versus lumbar lesions, and whether pediatric patients were most likely to develop instability., Conclusion: Simultaneously performing instrumented fusions following posterior spinal decompressions for tumor removal proved optimal in preventing postoperative spinal deformity. Further, "open" surgical procedures offered more optimal/definitive tumor removal versus minimally invasive approaches, and the greater operative exposure and resultant increased risk for instability were remediated by performing simultaneous fusion., Competing Interests: There are no conflicts of interest., (Copyright: © 2020 Surgical Neurology International.)

Background: Subdural hygromas are excess fluid accumulations in the subdural compartment, likely occurring via tears in the arachnoid membrane causing cerebrospinal fluid (CSF) leakage into the subdural space. Treatment recommendations for spinal subdural hygromas are lacking., Case Description: We report a case of a 30-year-old man who developed delayed-onset cauda equina syndrome after a motor vehicle accident. Magnetic resonance imaging of the thoracic and lumbar spine revealed a CSF intensity collection involving most of the thoracic spine and extending toward the distal end of the thecal sac with ventral displacement of the spinal cord and nerve roots. The patient was successfully treated using interventional radiology-guided lumbar puncture., Conclusions: Posttraumatic spinal subdural hygromas are rare complications, as evidenced by the lack of literature and treatment guidelines. Using lumbar puncture, we demonstrate clinical and radiographic resolution of a traumatic subdural hygroma. This outcome suggests lumbar puncture may be an effective treatment modality for similar patients, and can potentially be used to avoid a more invasive surgical decompression., (Copyright © 2019 Elsevier Inc. All rights reserved.)

The mechanisms underlying zebrafish pancreatic islet vascularization have not been well characterized. We sought to determine the angiogenic factors responsible for islet vascularization and assess whether an absence of endothelial cells affects beta-cell and alpha-cell formation. We used a double transgenic zebrafish Tg(fli1:EGFP; insa:tagRFP) to label endothelial cells and beta-cells, respectively. Beta-cells developed adjacent to endothelial cells and by 72 hours post fertilization (hpf) the zebrafish pancreatic islet was highly vascularized. Zebrafish beta-cells express vascular endothelial growth factors (vegf), vegfaa and vegfab. Double knockdown of vegfaa and vegfab or the primary Vegfa receptors (Vegfr2), kdr and kdrl, resulted in vessel deficient islets. While beta-cell and alpha-cell numbers remained unchanged in vessel deficient islets, insulina expression was downregulated relative to controls. Vegfaa/Vegfab-Vegfr2 signaling is necessary for proper islet vessel development, but not for the initial formation of beta-cells and alpha-cells.

Co-translational protein targeting to the endoplasmic reticulum (ER), represents an evolutionary-conserved mechanism to target proteins into the secretory pathway. In this targeting pathway proteins possessing signal sequences are recognised at the ribosome by the signal recognition particle while they are still undergoing synthesis. This triggers their delivery to the ER protein translocation channel, where they are directly translocated into the ER. Here we review the current understanding of this translocation pathway and how molecular details obtained in the related bacterial system have provided insight into the mechanism of targeting and translocation. This article is part of a Special Issue entitled: Functional and structural diversity of endoplasmic reticulum., (Copyright © 2013. Published by Elsevier B.V.)

Protein translocation across the endoplasmic reticulum membrane occurs at the Sec61 translocon. This has two essential subunits, the channel-forming multispanning membrane protein Sec61p/Sec61α and the tail-anchored Sss1p/Sec61γ, which has been proposed to "clamp" the channel. We have analyzed the function of Sss1p using a series of domain mutants and found that both the cytosolic and transmembrane clamp domains of Sss1p are essential for protein translocation. Our data reveal that the cytosolic domain is required for Sec61p interaction but that the transmembrane clamp domain is required to complete activation of the translocon after precursor targeting to Sec61p.

An accurate and precisely annotated genome assembly is a fundamental requirement for functional genomic analysis. Here, the complete DNA sequence and gene annotation of mouse Chromosome 11 was used to test the efficacy of large-scale sequencing for mutation identification. We re-sequenced the 14,000 annotated exons and boundaries from over 900 genes in 41 recessive mutant mouse lines that were isolated in an N-ethyl-N-nitrosourea (ENU) mutation screen targeted to mouse Chromosome 11. Fifty-nine sequence variants were identified in 55 genes from 31 mutant lines. 39% of the lesions lie in coding sequences and create primarily missense mutations. The other 61% lie in noncoding regions, many of them in highly conserved sequences. A lesion in the perinatal lethal line l11Jus13 alters a consensus splice site of nucleoredoxin (Nxn), inserting 10 amino acids into the resulting protein. We conclude that point mutations can be accurately and sensitively recovered by large-scale sequencing, and that conserved noncoding regions should be included for disease mutation identification. Only seven of the candidate genes we report have been previously targeted by mutation in mice or rats, showing that despite ongoing efforts to functionally annotate genes in the mammalian genome, an enormous gap remains between phenotype and function. Our data show that the classical positional mapping approach of disease mutation identification can be extended to large target regions using high-throughput sequencing., Competing Interests: The authors have declared that no competing interests exist.

Glucosidase II (GluII) is a glycan-trimming enzyme active on nascent glycoproteins in the endoplasmic reticulum (ER). It trims the middle and innermost glucose residues (Glc2 and Glc1) from N-linked glycans. The monoglucosylated glycan produced by the first GluII trimming reaction is recognized by calnexin/calreticulin and serves as the signal for entry into this folding pathway. GluII is a heterodimer of alpha and beta subunits corresponding to yeast Gls2p and Gtb1p, respectively. While Gls2p contains the glucosyl hydrolase active site, the Gtb1p subunit has previously been shown to be essential for the Glc1 trimming event. Here we demonstrate that Gtb1p also determines the rate of Glc2 trimming. In order to further dissect these activities we mutagenized a number of conserved residues across the protein. Our data demonstrate that both the MRH and G2B domains of Gtb1p contribute to the Glc2 trimming event but that the MRH domain is essential for Glc1 trimming.

Spermatogenesis can be divided into three stages: spermatogonial mitosis, meiosis of spermatocytes, and spermiogenesis. During spermiogenesis, spermatids undergo dramatic morphological changes including formation of a flagellum and chromosomal packaging and condensation of the nucleus into the sperm head. The genes regulating the latter processes are largely unknown. We previously discovered that a bi-functional gene, Spag16, is essential for spermatogenesis. SPAG16S, the 35 kDa, testis-specific isoform derived from the Spag16 gene, was found to bind to meiosis expressed gene 1 product (MEIG1), a protein originally thought to play a role in meiosis. We inactivated the Meig1 gene and, unexpectedly, found that Meig1 mutant male mice had no obvious defect in meiosis, but were sterile as a result of impaired spermatogenesis at the stage of elongation and condensation. Transmission electron microscopy revealed that the manchette, a microtubular organelle essential for sperm head and flagellar formation was disrupted in spermatids of MEIG1-deficient mice. We also found that MEIG1 associates with the Parkin co-regulated gene (PACRG) protein, and that testicular PACRG protein is reduced in MEIG1-deficient mice. PACRG is thought to play a key role in assembly of the axonemes/flagella and the reproductive phenotype of Pacrg-deficient mice mirrors that of the Meig1 mutant mice. Our findings reveal a critical role for the MEIG1/PARCG partnership in manchette structure and function and the control of spermiogenesis.

Background: ENU-mutagenesis is a powerful technique to identify genes regulating mammalian development. To functionally annotate the distal region of mouse chromosome 4, we performed an ENU-mutagenesis screen using a balancer chromosome targeted to this region of the genome., Results: We isolated 11 lethal lines that map to the region of chromosome 4 between D4Mit117 and D4Mit281. These lines form 10 complementation groups. The majority of lines die during embryonic development between E5.5 and E12.5 and display defects in gastrulation, cardiac development, and craniofacial development. One line displayed postnatal lethality and neurological defects, including ataxia and seizures., Conclusion: These eleven mutants allow us to query gene function within the distal region of mouse chromosome 4 and demonstrate that new mouse models of mammalian developmental defects can easily and quickly be generated and mapped with the use of ENU-mutagenesis in combination with balancer chromosomes. The low number of mutations isolated in this screen compared with other balancer chromosome screens indicates that the functions of genes in different regions of the genome vary widely.

Protein translocation into the endoplasmic reticulum occurs at pore-forming structures known as translocons. In yeast, two different targeting pathways converge at a translocation pore formed by the Sec61 complex. The signal recognition particle-dependent pathway targets nascent precursors co-translationally, whereas the Sec62p-dependent pathway targets polypeptides post-translationally. In addition to the Sec61 complex, both pathways also require Sec63p, an integral membrane protein of the Hsp40 family, and Kar2p, a soluble Hsp70 located in the ER lumen. Using a series of mutant alleles, we demonstrate that a conserved Brl (Brr2-like) domain in the COOH-terminal cytosolic region of Sec63p is essential for function both in vivo and in vitro. We further demonstrate that this domain is required for assembly of two oligomeric complexes of 350 and 380 kDa, respectively. The larger of these corresponds to the heptameric "SEC complex" required for post-translational translocation. However, the 350-kDa complex represents a newly defined hexameric SEC' complex comprising Sec61p, Sss1p, Sbh1p, Sec63p, Sec71p, and Sec72p. Our data indicate that the SEC' complex is required for co-translational protein translocation across the yeast ER membrane.

Glucosidase II is essential for sequential removal of two glucose residues from N-linked glycans during glycoprotein biogenesis in the endoplasmic reticulum. The enzyme is a heterodimer whose alpha-subunit contains the glycosyl hydrolase active site. The function of the beta-subunit has yet to be defined, but mutations in the human gene have been linked to an autosomal dominant form of polycystic liver disease. Here we report the identification and characterization of a Saccharomyces cerevisiae gene, GTB1, encoding a polypeptide with 21% sequence similarity to the beta-subunit of human glucosidase II. The Gtb1 protein was shown to be a soluble glycoprotein (96-102 kDa) localized to the endoplasmic reticulum lumen where it was present in a complex together with the yeast alpha-subunit homologue Gls2p. Surprisingly, we found that Deltagtb1 mutant cells were specifically defective in the processing of monoglucosylated glycans. Thus, although Gls2p is sufficient for cleavage of the penultimate glucose residue, Gtb1p is essential for cleavage of the final glucose. Our data demonstrate that Gtb1p is required for normal glycoprotein biogenesis and reveal that the final two glucose-trimming steps in N-glycan processing are mechanistically distinct.

Posttranslational translocation of prepro-alpha-factor (ppalphaF) across the yeast endoplasmic reticulum membrane begins with the binding of the signal sequence to the Sec complex, a membrane component consisting of the trimeric Sec61p complex and the tetrameric Sec62p/63p complex. We show by photo-cross-linking that the signal sequence is bound directly to a site where it contacts simultaneously Sec61p and Sec62p, suggesting that there is a single signal sequence recognition step. We found no evidence for the simultaneous contact of the signal sequence with two Sec61p molecules. To identify transmembrane segments of Sec61p that line the actual translocation pore, a late translocation intermediate of ppalphaF was generated with photoreactive probes incorporated into the mature portion of the polypeptide. Cross-linking to multiple regions of Sec61p was observed. In contrast to the signal sequence, neighboring positions of the mature portion of ppalphaF had similar interactions with Sec61p. These data suggest that the channel pore is lined by several transmembrane segments, which have no significant affinity for the translocating polypeptide chain.

Sec61p is required both for protein translocation and dislocation across the membrane of the endoplasmic reticulum (ER). However, the cellular role of the Sec61p homolog Ssh1p has not been clearly defined. We show that deltassh1 mutant cells have strong defects in both SRP-dependent and -independent translocation. Moreover, these cells were also found to be induced for the unfolded protein response and to be defective in dislocation of a misfolded ER protein. In addition, deltassh1 mutant cells rapidly became respiratory deficient. The other defects discussed above were suppressed in the respiratory-deficient state or under conditions where the rate of polypeptide translation was artificially reduced. These data identify Ssh1p as a component of a second, functionally distinct translocon in the yeast ER membrane.

In the context of the EUROFAN programme, we report the deletion and functional analysis of six open reading frames (ORFs) on the right arm of chromosome XII of Saccharomyces cerevisiae. Using a PCR-based gene replacement strategy, we have systematically deleted individual ORFs and subjected the heterozygous diploids and haploid knockout strains to basic genetic and phenotypic characterization. Two ORFs, YLR127c and YLR129w, are essential for viability, whereas no growth phenotype could be detected following deletion of YLR124w, YLR125w, YLR126c or YLR128w. For each of the individual ORFs, a kanMX4 replacement cassette and the corresponding cognate wild-type gene were cloned into appropriate plasmids., (Copyright 2000 John Wiley & Sons, Ltd.)

A sunflower oleosin was expressed in yeast to study the in vivo insertion of the protein into the endoplasmic reticulum (ER) and subsequent transfer to lipid bodies. The oleosin cDNA was expressed in a range of yeast secretory (sec) mutants to determine the precise targeting pathway of the oleosin to the ER. Subcellular fractionation experiments indicated that the signal recognition particle (SRP) is required for oleosin targeting to the ER and hence subsequent deposition on the lipid bodies in vivo. The expression of oleosin in a range of sec61 mutant alleles confirmed the role of the SEC61 translocon in insertion of oleosin into the ER membrane, as well as indicating an unusual substrate/translocon interaction for one particular allele (sec61-3). Mistargeting of the oleosin due to impaired SRP function resulted in enhanced proteolysis of the plant protein in the transformed yeast, as determined by pulse-chase analysis. These data therefore provide the first in vivo evidence for the SRP-dependent targeting of the oleosin to the ER, and the subsequent requirement for a functional SEC61 translocon to mediate the correct insertion of the protein into the membrane.

The translocation of secretory polypeptides into and across the membrane of the endoplasmic reticulum (ER) occurs at the translocon, a pore-forming structure that orchestrates the transport and maturation of polypeptides at the ER membrane. Recent data also suggest that misfolded or unassembled polypeptides exit the ER via the translocon for degradation by the cytosolic ubiquitin/proteasome pathway. Sec61p is a highly conserved multispanning membrane protein that constitutes a core component of the translocon. We have found that the essential function of the Saccharomyces cerevisiae Sec61p is retained upon deletion of either of two internal regions that include transmembrane domains 2 and 3, respectively. However, a deletion mutation encompassing both of these domains was found to be nonfunctional. Characterization of yeast mutants expressing the viable deletion alleles of Sec61p has revealed defects in post-translational translocation. In addition, the transmembrane domain 3 deletion mutant is induced for the unfolded protein response and is defective in the dislocation of a misfolded ER protein. These data demonstrate that the various activities of Sec61p can be functionally dissected. In particular, the transmembrane domain 2 region plays a role in post-translational translocation that is required neither for cotranslational translocation nor for protein dislocation.

We have analyzed how the signal sequence of prepro-alpha-factor is recognized during the first step of posttranslational protein transport into the yeast endoplasmic reticulum. Cross-linking studies indicate that the signal sequence interacts in a Kar2p- and ATP-independent reaction with Sec61p, the multispanning membrane component of the protein-conducting channel, by intercalation into transmembrane domains 2 and 7. While bound to Sec61p, the signal sequence forms a helix that is contacted on one side by Sec62p and Sec71p. The binding site is located at the interface of the protein channel and the lipid bilayer. Signal sequence recognition in cotranslational translocation in mammals appears to occur similarly. These results suggest a general mechanism by which the signal sequence could open the channel for polypeptide transport.

The Sec61 protein is required for protein translocation across the ER membrane in both yeast and mammals and is found in close association with polypeptides during their membrane transit. In Saccharomyces cerevisiae Sec61p is essential for viability and the extent of sequence similarity between the yeast and mammalian proteins (55% sequence identity) suggests that the role of Sec61p in the translocation mechanism is likely to be conserved. In order to further our understanding of the structure and function of Sec61p we have cloned homologues from both Schizosaccharomyces pombe and Yarrowia lipolytica. The S. pombe gene comprises six exons encoding a 479 residue protein which we have immunolocalised to the endoplasmic reticulum. Sequence comparisons reveal that S. pombe Sec61p is 58.6% identical to that of S. cerevisiae. The deduced amino acid sequence of the Y. lipolytica protein shares 68.8% sequence identity with S. cerevisiae Sec61p. Gene disruption studies have shown that the SEC61 is required for viability in both S. pombe and Y. lipolytica demonstrating that the essential nature of this protein is not unique to S. cerevisiae. Moreover, heterologous complementation studies indicate that the Y. lipolytica SEC61 gene can complement a null mutation in S. cerevisiae. Sequence comparisons between the various eukaryotic Sec61p homologues reveal a number of highly conserved domains, including several transmembrane sequences and the majority of cytosolic loops. These comparisons will provide an important framework for the detailed analysis of interactions between Sec61p and other components of the translocation machinery and between Sec61p and translocating polypeptide chains.

The heterotrimeric Sec61p complex is a key component of the protein translocation apparatus of the endoplasmic reticulum membrane. The complex characterized from yeast includes Sec61p, a 10-transmembrane-domain membrane protein which has a direct interaction with Sss1p, a small C-terminal anchor protein. In order to gain some insight into the architecture of this complex we have functionally expressed Sec61p as complementary N- and C-terminal fragments. Chemical crosslinking of Sss1p to specific Sec61p fragments in these functional combinations and suppression of sec61 mutants by over-expression of Sss1p have led to identification of the region which includes transmembrane domains TM6, TM7 and TM8 (amino acid residues L232-R406) of Sec61p as a major site of interaction with Sss1p.

Eukaryotic cells are characterized by the existence of membrane-bound subcellular compartments which perform a variety of specialized functions. Proteins destined for these compartments begin their synthesis in the cytosol and must be subsequently targeted to their functional compartment by specific signal sequences present in the newly synthesized polypeptide chain. The translocation of preproteins across biological membranes is a fundamental process of intracellular trafficking and organelle biogenesis. Entry into the secretory pathway occurs by translocation of proteins into or across the membrane of the endoplasmic reticulum (ER). This process involves two distinct steps which are dependent on the orchestrated action of several proteins. The initial step of targeting involves recognition of the signal sequence and delivery of the protein precursor to the ER in a translocation competent conformation. The subsequent translocation event is characterized by interaction of the preprotein with the translocation channel followed by unidirectional movement across the lipid bilayer of the ER membrane into the lumenal space. The study of the mechanism of the translocation process is one of the most intriguing and rapidly advancing areas in cell biology. Here we review recent findings in both the yeast Saccharomyces cerevisiae and mammals concerning the mechanisms of the translocation step and discuss the roles of the proteins implicated in this process.

Sec61p is a highly conserved integral membrane protein that plays a role in the formation of a protein-conducting channel required for the translocation of polypeptides into, and across, the membrane of the endoplasmic reticulum. As a major step toward elucidating the structure of the endoplasmic reticulum translocation apparatus, we have determined the transmembrane topology of Sec61p using a combination of C-terminal reporter-domain fusions and the in situ digestion of specifically inserted factor Xa protease cleavage sites. Our data indicate the presence of 10 transmembrane domains, including several with surprisingly limited hydrophobicity. Furthermore, we provide evidence for complex intramolecular interactions in which these weakly hydrophobic domains require C-terminal sequences for their correct topogenesis. The incorporation of sequences with limited hydrophobicity into the bilayer may play a vital role in the formation of an aqueous membrane channel required for the translocation of hydrophilic polypeptide chains.

A small-scale functional analysis screen has revealed several new phenotypes associated with a large deletion of GDH3, one of two Saccharomyces cerevisiae genes known to encode NADP-linked glutamate dehydrogenase. Diploids heterozygous for the deletion are able to sporulate in rich media, while haploid deletants produce dark, wrinkled colonies containing pseudohyphal cells. The haploid cells rapidly lose viability upon starvation.

The possible role of Candida species in carcinogenesis at the uterine cervix was investigated in 226 females attending a colposcopy clinic. Approximately 34% of the 226 subjects harbored Candida species in cervical/vaginal secretions, but there was no association with any particular histologic abnormality. Two independent analytical procedures were used for strain discrimination of the isolates of C. albicans, but again no relationship was found between individual strains and histologic diagnoses. Only three C. glabrata strains were isolated, but they were all in association with cervical intraepithelial neoplasia (CIN) II or III. A total of 18 strains of C. albicans, one C. glabrata and one C. parapsilosis all inhibited the formation of the nitrosamine nitrosodimethylamine (NDMA) from precursors. Furthermore, C. albicans strains did not convert NDMA to carcinogenic metabolites. The results of this study do not suggest that C. albicans has a role in cervical carcinogenesis.

The repetitive DNA sequence poly[d(GT).d(CA)],(polyGT) can be used to generate DNA fingerprints that distinguish different yeast genera. In this study we demonstrate that the probe can also be used to distinguish individual strains of clinical isolates of Candida albicans. Isolates were fingerprinted by probing Southern blots of restriction enzyme-cleaved DNA samples with radioactively labelled polyGT. The discrimination between strains was clearer than can be achieved by direct visualization of ethidium bromide stained gels and was comparable to that achieved with previous DNA probes. However, the advantage of this probe is that it is not limited to Candida species since polyGT sequences appear to be ubiquitous in eukaryotes. Fingerprints were also generated from Southern blots using a commercial (AMBIS) radioanalytical imaging computer system. A proprietary software package (MICRO PM) was effective in discriminating between the C. albicans strains. These preliminary results indicate the potential value of this probe for discrimination between Candida isolates in epidemiological studies of candidosis.