In Vitro Immunological Cross-Reactivity of Thai Polyvalent and Monovalent Antivenoms with Asian Viper Venoms

The intravenous administration of polyclonal antibodies known as antivenom is the only effective treatment for snakebite envenomed victims, but because of inter-specific variation in the toxic components of snake venoms, these therapies have variable efficacies against different snake species and/or different populations of the same species. In this study, we sought to characterize the in vitro venom binding capability and in vitro cross-neutralizing activity of antivenom, specifically the Hemato Polyvalent antivenom (HPAV; The Queen Saovabha Memorial Institute (QSMI) of the Thai Red Cross Society, Thailand) and three monovalent antivenoms (QSMI) specific to Daboia siamensis, Calloselasma rhodostoma, and Trimeresurus albolabris venoms, against a variety of South Asian and Southeast Asian viper venoms (Calloselasma rhodostoma, Daboia russelii, Hypnale hypnale, Trimeresurus albolabris, Trimeresurus purpureomaculatus, Trimeresurus hageni, and Trimeresurus fucatus). Using ELISA and immunoblotting approaches, we find that the majority of protein components in the viper venoms were recognized and bound by the HPAV polyvalent antivenom, while the monospecific antivenom made against T.albolabris extensively recognized toxins present in the venom of related species, T. purpureomaculatus, T. hageni, and T. fucatus. In vitro coagulation assays using bovine plasma revealed similar findings, with HPAV antivenom significantly inhibiting the coagulopathic activities of all tested viper venoms and T. albolabris antivenom inhibiting the venoms from Malaysian arboreal pit vipers. We also show that the monovalent C. rhodostoma antivenom exhibits highly comparable levels of immunological binding and in vitro venom neutralization to venom from both Thailand and Malaysia, despite previous reports of considerable intraspecific venom variation. Our findings suggest that Thai antivenoms from QSMI may by useful therapeutics for managing snake envenomings caused by a number of Southeast Asian viper species and populations for which no specific antivenom currently exists and thus should be explored further to assess their clinical utility in treating snakebite victims.

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Examination of the Efficacy and Cross-Reactivity of a Novel Polyclonal Antibody Targeting the Disintegrin Domain in SVMPs to Neutralize Snake Venom

Toxins ◽

10.3390/toxins13040254 ◽

2021 ◽

Vol 13 (4) ◽

pp. 254

Author(s):

Shelby S. Szteiter ◽

Ilse N. Diego ◽

Jonathan Ortegon ◽

Eliana Salinas ◽

Abcde Cirilo ◽

...

Keyword(s):

Snake Venom ◽

Polyclonal Antibody ◽

Polyclonal Antibodies ◽

Cross Reactivity ◽

Snake Envenomation ◽

Disintegrin Domain ◽

The Common ◽

Neutralization Ability ◽

New Strategies

Snake envenomation can result in hemorrhage, local necrosis, swelling, and if not treated properly can lead to adverse systemic effects such as coagulopathy, nephrotoxicity, neurotoxicity, and cardiotoxicity, which can result in death. As such, snake venom metalloproteinases (SVMPs) and disintegrins are two toxic components that contribute to hemorrhage and interfere with the hemostatic system. Administration of a commercial antivenom is the common antidote to treat snake envenomation, but the high-cost, lack of efficacy, side effects, and limited availability, necessitates the development of new strategies and approaches for therapeutic treatments. Herein, we describe the neutralization ability of anti-disintegrin polyclonal antibody on the activities of isolated disintegrins, P-II/P-III SVMPs, and crude venoms. Our results show disintegrin activity on platelet aggregation in whole blood and the migration of the SK-Mel-28 cells that can be neutralized with anti-disintegrin polyclonal antibody. We characterized a SVMP and found that anti-disintegrin was also able to inhibit its activity in an in vitro proteolytic assay. Moreover, we found that anti-disintegrin could neutralize the proteolytic and hemorrhagic activities from crude Crotalus atrox venom. Our results suggest that anti-disintegrin polyclonal antibodies have the potential for a targeted approach to neutralize SVMPs in the treatment of snakebite envenomations.

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Development of Murine Monoclonal Antibodies for the Immunohistochemical Diagnosis of Systemic Bovine Aspergillosis

Journal of Veterinary Diagnostic Investigation ◽

10.1177/104063879600800111 ◽

1996 ◽

Vol 8 (1) ◽

pp. 68-75 ◽

Cited By ~ 31

Author(s):

H. E. Jensen ◽

B. Aalbaek ◽

P. Lind ◽

H. V. Krogh ◽

P. L. Frandsen

Keyword(s):

Monoclonal Antibodies ◽

Polyclonal Antibodies ◽

Fungal Species ◽

Cross Reactivity ◽

Water Soluble ◽

Enzyme Linked Immunosorbent Assay ◽

Immunohistochemical Techniques ◽

Immunohistochemical Diagnosis ◽

Murine Monoclonal Antibodies

Murine monoclonal antibodies (MAbs) against water-soluble somatic antigens (WSSA) and the wall fraction (WF) from Aspergillus fumigatus were produced by fusion of splenocytes from immunized BALB/c mice with mouse myeloma X63-Ag 8.653 cells. The supernatants of in vitro cultured hybridomas were initially screened for reactivity with the WSSA and the WF from A. fumigatus and WSSA of other fungi in an enzyme-linked immunosorbent assay (ELISA). Supernatants reacting only with A. fumigatus antigens were subsequently screened for homologous and heterologous reactivity with immunohistochemical techniques using formalin-fixed, paraffin-embedded tissues from experimentally infected mice. Because of a high immunohistochemical reactivity with homologous fungi, 4 MAbs raised against A. fumigatus WSSA and WF were selected for a further evaluation of cross-reactivity (diagnostic specificity) in immunohistochemical and immunoblotting assays. In immunohistochemical assays, all MAbs raised against WSSA cross-reacted heavily with a number of other fungal species. All 4 MAbs (MAb-WF-AF-1-4) raised against the WF reacted strongly with hyphae of Aspergillus spp.; hyphae of Scedosporium apiospermum were also strongly labeled by MAb-WF-AF-3 and-4. The 2 specifically reacting MAbs (MAb-WF-AF-1 and-2) were of the IgM biotype and were precipitating, and in immunoblotting experiments both bound to a 106-kD antigen of the WF, whereas they did not bind to WSSA of A. fumigatus. One of the 2 aspergillosis-specific MAbs (MAb-WF-AF-1) was used to screen 145 mycotic lesions of cattle. The diagnoses on bovine lesions obtained by MAb-WF-AF-1 were compared with results based on reactivity with heterologously absorbed polyclonal antibodies and, for some lesions, to culture results. In the vast majority of lesions ( n = 133), the MAb-WF-AF-1 and the polyclonal anti-Aspergillus antibodies reacted in a similar pattern, i.e., positively in 41 aspergillosis lesions and negatively in 92 zygomycotic lesions. Hyphae in 3 of 12 lesions that were not stained by the polyclonal antibodies reacted with the specific MAb-WF-AF-1; i.e., aspergillosis was diagnosed. The characteristics of the 2 MAbs (MAb-WF-AF-1 and-2) raised against the WF of A. fumigatus in ELISA and immunoblotting and immunohistochemical assays justify their application for the in situ diagnosis of systemic aspergillosis of cattle.

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Effects of chronic corticotrophin treatment on aldosterone metabolism in the rat

Journal of Endocrinology ◽

10.1677/joe.0.1370445 ◽

1993 ◽

Vol 137 (3) ◽

pp. 445-455 ◽

Cited By ~ 8

Author(s):

D. R. E. Abayasekara ◽

N. I. Onyezili ◽

B. J. Whitehouse ◽

S. M. Laird ◽

G. P. Vinson

Keyword(s):

Polyclonal Antibodies ◽

Cross Reactivity ◽

Plasma Aldosterone ◽

Adrenal Vein ◽

Metabolic Clearance ◽

Sprague Dawley ◽

Peripheral Plasma ◽

Acth Treatment

ABSTRACT Chronic treatment with high doses of ACTH leads to marked reduction in aldosterone biosynthesis and secretion both in vivo and in vitro. In contrast, it has been reported that peripheral plasma aldosterone levels may be elevated following prolonged ACTH treatment. The present study attempts to determine the reason(s) for this apparently paradoxical finding. ACTH treatment (40 μg/100 g body weight) of male Sprague–Dawley rats for 7 days caused a decrease of more than 90% in aldosterone secretion into the adrenal vein in vivo and aldosterone production by intact adrenal capsules incubated in vitro. In contrast, peripheral plasma aldosterone levels appeared to be increased when measured by radioimmunoassay using two different polyclonal antibodies (antibody 1 (AB1) raised against aldosterone-3-carboxymethyloxime–bovine serum albumin (BSA) and antibody 2 (AB2) raised against aldosterone-21-hemisuccinate–BSA). However, when a highly specific monoclonal antibody (raised against aldosterone-3-carboxymethyloxime–BSA and showing low cross-reactivity to aldosterone metabolites) was used, peripheral plasma aldosterone levels appeared to be reduced in ACTH-treated rats. Following chromatographic fractionation of peripheral plasma, significantly more material with aldosterone-like immunoreactivity, but which was less polar than authentic aldosterone in chromatographic mobility, was detected in the fractions using antibodies AB1 and AB2. The absence of this material from fractions of adrenal vein plasma leads us to infer that this material is generated in the peripheral circulation, probably as a result of hepatic metabolism. In addition, the overall metabolic clearance rate (MCR) of [3H] aldosterone was found to be significantly decreased following prolonged ACTH treatment. We conclude that the seemingly discrepant findings with regard to the effects of chronic ACTH treatment on peripheral plasma aldosterone levels and the secretion of aldosterone in vivo can be reconciled by (1) the changes in the overall MCR of aldosterone and (2) the generation of increased quantities of aldosterone metabolites such as 5α-dihydroaldosterone and 3α,5β-tetrahydroaldosterone which show significant cross-reactivity with some aldosterone antibodies. Journal of Endocrinology (1993) 137, 445–455

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Generation of High Affinity Anti-Peptide Polyclonal Antibodies Recognizing Goat αs1-Casein

Molecules ◽

10.3390/molecules25112622 ◽

2020 ◽

Vol 25 (11) ◽

pp. 2622

Author(s):

Aliah Zannierah Mohsin ◽

Rashidah Sukor ◽

Jinap Selamat ◽

Anis Shobirin Meor Hussin ◽

Intan Hakimah Ismail ◽

...

Keyword(s):

Binding Affinity ◽

Analytical Methods ◽

Polyclonal Antibodies ◽

Sequence Similarity ◽

High Specificity ◽

Cross Reactivity ◽

Enzyme Linked Immunosorbent Assay ◽

High Sequence Similarity ◽

High Affinity

The chemical, technological and allergy properties of goat’s milk are significantly affected by the level of αs1-casein. Detection and quantification of αs1-casein requires high-specificity methods to overcome high-sequence similarity between this protein and others in the casein family. Unavailability of antibodies with high affinity and specificity towards goat αs1-casein hinders the development of immuno-based analytical methods such as enzyme-linked immunosorbent assay (ELISA) and biosensors. Here, we report the generation of polyclonal antibodies (or immunoglobulins, IgGs) raised towards goat αs1-casein N- (Nter) and C-terminal (Cter) peptide sequences. The Nter and Cter peptides of goat αs1-casein were immunized in rabbits for the generation of antisera, which were purified using protein G affinity chromatography. The binding affinity of the antisera and purified IgGs were tested and compared using indirect ELISA, where peptide-BSA conjugates and goat αs1-casein were used as the coating antigens. The Nter antiserum displayed higher titer than Cter antiserum, at 1/64,000 and 1/32,000 dilutions, respectively. The purification step further yielded 0.5 mg/mL of purified IgGs from 3 mL of antisera. The purified Nter IgG showed a significantly (p < 0.05) higher binding affinity towards peptide-BSA and goat αs1-casein, with lower Kd value at 5.063 × 10−3 μM compared to 9.046 × 10−3 μM for the Cter IgG. A cross-reactivity test showed that there was no binding in neither Nter nor Cter IgGs towards protein extracts from the milk of cow, buffalo, horse and camel. High-quality antibodies generated will allow further development of immuno-based analytical methods and future in vitro studies to be conducted on goat αs1-casein.

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Comparative Study of Two Insulinlike Proteases in Cryptosporidium parvum

Microorganisms ◽

10.3390/microorganisms9040861 ◽

2021 ◽

Vol 9 (4) ◽

pp. 861

Author(s):

Wei He ◽

Cong Lai ◽

Fuxian Yang ◽

Yu Li ◽

Na Li ◽

...

Keyword(s):

Life Cycle ◽

Polyclonal Antibodies ◽

Parasite Load ◽

Cross Reactivity ◽

Binding Motif ◽

Apical Region ◽

Biological Functions ◽

Native Proteins ◽

Genes Encoding

Cryptosporidiumparvum is a common protozoan pathogen responsible for moderate-to-severe diarrhea in humans and animals. The small genome of C. parvum has 22 genes encoding insulinlike proteases (INS) with diverse sequences, suggesting that members of the protein family may have different biological functions in the life cycle. In this study, two members of the INS family, CpINS-4 and CpINS-6 with the Zn2+-binding motif “HXXEH” but different numbers of function domains, were expressed in Escherichia coli and used in the generation of polyclonal antibodies. In both recombinant and native proteins, CpINS-4 and CpINS-6 were spliced into multiple fragments. The antibodies generated recognized their respective recombinant and native proteins and the spliced products, but had minimum cross-reactivity with each other. Anti-CpINS-4 antibodies reacted with the middle region of sporozoites and merozoites, while CpINS-6 had the highest reactivity to the apical region. Polyclonal anti-CpINS-4 antibodies produced 36% reduction in parasite load in HCT-8 cultures at 24 h, while those against CpINS-6, which has one of the function domains missing, failed in doing so. The genes encoding both CpINS-4 and CpINS-6 had the highest expression in the invasion phase of in vitro C. parvum culture. These data suggest that CpINS-4 and CpINS-6 might be expressed in different organelles and play different biological functions in the life cycle of C. parvum.

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Immunohistochemical Diagnosis of Systemic Bovine Zygomycosis by Murine Monoclonal Antibodies

Veterinary Pathology ◽

10.1177/030098589603300207 ◽

1996 ◽

Vol 33 (2) ◽

pp. 176-183 ◽

Cited By ~ 24

Author(s):

H. E. Jensen ◽

B. Aalbæk ◽

P. Lind ◽

H. V. Krogh

Keyword(s):

Monoclonal Antibodies ◽

Lymph Nodes ◽

Rhizopus Oryzae ◽

Polyclonal Antibodies ◽

Cross Reactivity ◽

Water Soluble ◽

Enzyme Linked Immunosorbent Assay ◽

The Family ◽

Murine Monoclonal Antibodies

Murine monoclonal antibodies (Mabs) against water-soluble somatic antigens (WSSA) and the wall fraction (WF) from Rhizopus arrhizus (Rhizopus oryzae) were produced in vitro by fusion of splenocytes from immunized BALB/c mice with mouse myeloma X63-Ag 8.653 cells. Supernatants reacting only with homologous antigens in an enzyme-linked immunosorbent assay were subsequently screened for reactivity with homologous fungi in immunohistochemical techniques. All four Mabs raised against the WF of A. arrhizus failed to react on tissues. However, four of the Mabs raised against the WSSA of R. arrhizus (Mab-WSSA-RA-1 through Mab-WSSA-RA-4) revealed a high homologous reactivity on tissues and the cross-reactivity of these were subsequently evaluated on tissues containing other members of the family Mucoraceae and other unrelated fungi. On tissues and on immunoblots all four Mabs reacted identically and specifically with members of the family Mucoraceae, i.e., Absidia corymbifera, R. arrhizus, and Rhizomucor pusillus. The Mabs were all isotyped as IgM antibodies, were nonprecipitating, and reacted with homologous antigens with molecular masses from 14 to 110 kDa. With WSSA from A. corymbifera and R. pusillus the four Mabs were bound to antigens from 14 to 52 kDa and from 20 to 28 kDa, respectively. The diagnosis of 145 bovine lesions obtained by one of the specific Mabs (Mab-WSSA-RA-1) were compared to results obtained by heterologously absorbed polyclonal antibodies. In most lesions ( n = 140 [∼ 97%]) the Mab and the polyclonal antibodies reacted in a similar pattern, i.e., positively for zygomycosis in 89 lesions, negatively in 41 aspergillosis lesions, and negatively in 10 undiagnosed lesions. Hyphae within two of four lesions in lymph nodes, which were not stained by the polyclonal antibodies, reacted with the specific Mab. However, in another three lesions of lymph nodes stained by the polyclonal antibodies no reactivity was seen with the Mab-WSSA-RA-1. The immunoreactivity of the Mabs (Mab-WSSA-RA-1 through Mab-WSSA-RA-4) raised against WSSA of R. arrhizus justify their application for the in situ diagnosis of systemic bovine zygomycosis.

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184 Two types of anti-TIGIT antibodies with distinct binding epitope and functional activities

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2020-sitc2020.0184 ◽

2020 ◽

Vol 8 (Suppl 3) ◽

pp. A198-A198

Author(s):

Tingting Zhong ◽

Xinghua Pang ◽

Zhaoliang Huang ◽

Na Chen ◽

Xiaoping Jin ◽

...

Keyword(s):

T Cells ◽

Mixed Culture ◽

Cell Activity ◽

Cross Reactivity ◽

Binding Activity ◽

Jurkat Cells ◽

List Type ◽

Dependent Manner ◽

Vitro Assay

BackgroundTIGIT is an inhibitory receptor mainly expressed on natural killer (NK) cells, CD8+ T cells, CD4+ T cells and Treg cells. TIGIT competes with CD226 for binding with CD155. In cancers, CD155 has been reported to up-regulate on tumor cells, and TIGIT was found to increase on TILs.1 Activation of TIGIT/CD155 pathway would mediate immunosuppression in tumor; while blockade of TIGIT promotes anti-tumor immune response.MethodsAK126 and AK113 are two humanized anti-human TIGIT monoclonal antibodies developed by Akesobio. Binding activity of AK126 and AK113 to human TIGIT, and competitive binding activity with CD155 and CD112, were performed by using ELISA, Fortebio, and FACS assays. Cross-reactivity with cynomolgus monkey TIGIT and epitope binning were also tested by ELISA assay. In-vitro assay to investigate the activity to promote IL-2 secretion was performed in mixed-culture of Jurkat-TIGIT cells and THP-1 cells.ResultsAK126 and AK113 could specifically bind to human TIGIT with comparative affinity and effectively blocked the binding of human CD155 and CD112 to human TIGIT. X-ray crystal structure of TIGIT and PVR revealed the C’-C’’ loop and FG loop regions of TIGIT are the main PVR interaction regions.2 The only amino acid residue differences in these regions between human and monkey TIGIT are 70C and 73D. AK126 binds to both human and monkey TIGIT, AK113 binds only to monkey TIGIT. This suggests that these residues are required for AK113 binding to human TIGIT, but not required for AK126. Interestingly, results from cell-based assays indicated that AK126 and AK113 showed significantly different activity to induce IL-2 secretion in mixed-culture of Jurkat-TIGIT cells and THP-1 cells (figure 1A and B), in which AK126 had a comparable capacity of activity to 22G2, a leading TIGIT mAb developed by another company, to induce IL-2 secretion, while, AK113 showed a significantly higher capacity than 22G2 and AK126.Abstract 184 Figure 1Anti-TIGIT Antibodies Rescues IL-2 Production in Vitro T-Cell Activity Assay in a dose dependent manner. Jurkat-TIGIT cells (Jurkat cells engineered to over-express human TIGIT) were co-cultured with THP-1 cells, and stimulated with plate-bound anti-CD3 mAb in the presence of TIGIT ligand CD155 (A) or CD112 (B) with anti-TIGIT antibodies. After incubated for 48h at 37° C and 5.0% CO2, IL-2 levels were assessed in culture supernatants by ELISA. Data shown as mean with SEM for n = 2.ConclusionsWe discovered two distinct types of TIGIT antibodies with differences in both epitope binding and functional activity. The mechanism of action and clinical significance of these antibodies require further investigation.ReferencesSolomon BL, Garrido-Laguna I. TIGIT: a novel immunotherapy target moving from bench to bedside. Cancer Immunol Immunother 2018;67:1659–1667.Stengel KF, Harden-Bowles K, Yu X, et al. Structure of TIGIT immunoreceptor bound to poliovirus receptor reveals a cell-cell adhesion and signaling mechanism that requires cis-trans receptor clustering. Proc Natl Acad Sci USA 2012;109:5399–5404.

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Previous Humoral Immunity to the Endemic Seasonal Alphacoronaviruses NL63 and 229E Is Associated with Worse Clinical Outcome in COVID-19 and Suggests Original Antigenic Sin

Life ◽

10.3390/life11040298 ◽

2021 ◽

Vol 11 (4) ◽

pp. 298

Author(s):

Daniele Focosi ◽

Angelo Genoni ◽

Ersilia Lucenteforte ◽

Silvia Tillati ◽

Antonio Tamborini ◽

...

Keyword(s):

Humoral Immunity ◽

Cross Reactivity ◽

Clinical Severity ◽

World Health ◽

Laboratory Findings ◽

Cellular And Humoral Immunity ◽

Rate Ratios ◽

Original Antigenic Sin

Antibody-dependent enhancement (ADE) of severe acute respiratory syndrome coronavirus-2 (SARS CoV-2) infection has been hypothesized. However, to date, there has been no in vitro or in vivo evidence supporting this. Cross-reactivity exists between SARS CoV-2 and other Coronaviridae for both cellular and humoral immunity. We show here that IgG against nucleocapsid protein of alphacoronavirus NL63 and 229E correlate with the World Health Organization’s (WHO) clinical severity score ≥ 5 (incidence rate ratios was 1.87 and 1.80, respectively, and 1.94 for the combination). These laboratory findings suggest possible ADE of SARS CoV-2 infection by previous alphacoronavirus immunity.

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Influences of advanced glycosylation end products on the inner blood–retinal barrier in a co-culture cell model in vitro

Open Life Sciences ◽

10.1515/biol-2020-0067 ◽

2020 ◽

Vol 15 (1) ◽

pp. 619-628

Author(s):

Chen Yuan ◽

Ya Mo ◽

Jie Yang ◽

Mei Zhang ◽

Xuejun Xie

Keyword(s):

Electrical Resistance ◽

Cell Model ◽

Polyclonal Antibodies ◽

Time Dependent ◽

Dependent Manner ◽

Blood Retinal Barrier ◽

Advanced Glycosylation End Products ◽

End Products ◽

Advanced Glycosylation

AbstractAdvanced glycosylation end products (AGEs) are harmful factors that can damage the inner blood–retinal barrier (iBRB). Rat retinal microvascular endothelial cells (RMECs) were isolated and cultured, and identified by anti-CD31 and von Willebrand factor polyclonal antibodies. Similarly, rat retinal Müller glial cells (RMGCs) were identified by H&E staining and with antibodies of glial fibrillary acidic protein and glutamine synthetase. The transepithelial electrical resistance (TEER) value was measured with a Millicell electrical resistance system to observe the leakage of the barrier. Transwell cell plates for co-culturing RMECs with RMGCs were used to construct an iBRB model, which was then tested with the addition of AGEs at final concentrations of 50 and 100 mg/L for 24, 48, and 72 h. AGEs in the in vitro iBRB model constructed by RMEC and RMGC co-culture led to the imbalance of the vascular endothelial growth factor (VEGF) and pigment epithelial derivative factor (PEDF), and the permeability of the RMEC layer increased because the TEER decreased in a dose- and time-dependent manner. AGEs increased VEGF but lowered PEDF in a dose- and time-dependent manner. The intervention with AGEs led to the change of the transendothelial resistance of the RMEC layer likely caused by the increased ratio of VEGF/PEDF.

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The centrin-based cytoskeleton of Chlamydomonas reinhardtii: distribution in interphase and mitotic cells.

The Journal of Cell Biology ◽

10.1083/jcb.107.2.635 ◽

1988 ◽

Vol 107 (2) ◽

pp. 635-641 ◽

Cited By ~ 151

Author(s):

J L Salisbury ◽

A T Baron ◽

M A Sanders

Keyword(s):

Nuclear Envelope ◽

Polyclonal Antibodies ◽

Flagellar Apparatus ◽

Immunogold Labeling ◽

Basal Bodies ◽

Flagellar Roots ◽

Cell Biol ◽

Immunofluorescence Studies ◽

Descending Fibers

Monoclonal and polyclonal antibodies raised against algal centrin, a protein of algal striated flagellar roots, were used to characterize the occurrence and distribution of this protein in interphase and mitotic Chlamydomonas cells. Chlamydomonas centrin, as identified by Western immunoblot procedures, is a low molecular (20,000-Mr) acidic protein. Immunofluorescence and immunogold labeling demonstrates that centrin is a component of the distal fiber. In addition, centrin-based flagellar roots link the flagellar apparatus to the nucleus. Two major descending fibers extend from the basal bodies toward the nucleus; each descending fiber branches several times giving rise to 8-16 fimbria which surround and embrace the nucleus. Immunogold labeling indicates that these fimbria are juxtaposed to the outer nuclear envelope. Earlier studies have demonstrated that the centrin-based linkage between the flagellar apparatus and the nucleus is contractile, both in vitro and in living Chlamydomonas cells (Wright, R. L., J. Salisbury, and J. Jarvik. 1985. J. Cell Biol. 101:1903-1912; Salisbury, J. L., M. A. Sanders, and L. Harpst. 1987. J. Cell Biol. 105:1799-1805). Immunofluorescence studies show dramatic changes in distribution of the centrin-based system during mitosis that include a transient contraction at preprophase; division, separation, and re-extension during prophase; and a second transient contraction at the metaphase/anaphase boundary. These observations suggest a fundamental role for centrin in motile events during mitosis.

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