| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Regular Articles |
From the Division of Bacteriology and Immunology,*
the
Haartman Institute, University of Helsinki, Helsinki, Finland; and the
Department of Internal Medicine,
the Division
of Nephrology, University of Michigan, Ann Arbor, Michigan
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Experimental models of glomerular disease are important tools for investigating the functional significance of the novel molecules by providing a means to effectively modulate the basic glomerular functions. Thus, even if transgenic and knockout mouse models are extremely useful in the targeted analysis of molecules, the well-established experimental models in the rat remain best characterized and most widely used for glomerular pathophysiology. In particular, the classic models of Heyman nephritis, which mimicks membranous nephropathy, and anti-GBM nephritis and puromycin aminonucleoside (PAN) nephrosis of the rat, which models minimal change nephropathy, are still widely used.10-13 Similar modeling is often impossible in the mouse.
Kestilä et al14 recently cloned a new gene, NPHS1, which is mutated in the congenital nephrotic syndrome of the Finnish type (CNF). The current treatment of CNF with early nephrectomy and final renal transplantation appears to cure all symptoms.15 After transplantation, no new symptoms are reported after several years of follow-up. The results of Kestilä et al14 showed that NPHS1 is only expressed in the kidney glomerulus. However, little is known thus far of the regulation of this gene of apparently great functional significance in various proteinuria-associated diseases.
Here we report the molecular cloning and characterization of rat nephrin cDNA.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Male Sprague-Dawley rats weighing 175–190 g, with free access to tapwater and standard rat chow throughout the study, were used.
To induce PAN nephrosis, puromycin aminonucleoside (20 mg/100 g; Sigma Chemical Company, St Louis, MO) in phosphate-buffered saline (PBS) was given by a single intraperitoneal injection to 10 rats, as described earlier.16 All injected animals developed significant albuminuria (>30 mg/24 hours) by day 5 after injection, as measured by nephelometry (Behring Nephelometer 100 Analyzer; Behringwerke, Marburg, Germany). Five animals were sacrificed at day 3 before the onset of albuminuria (<10 mg of urinary albumin/24 hours), and five at day 10 at the peak of albuminuria (>100 mg/24 hours). One kidney from each animal was used for immediate isolation of cortical RNA. For immunofluorescence studies the remaining kidney was frozen in isopentane and stored appropriately at -70°C until used.
Cloning of Rat Nephrin cDNA
For the cloning of the rat nephrin cDNA, a rat glomerular cDNA library was prepared and used as previously described.17 Briefly, the library was constructed using total RNA isolated from glomeruli of 1-month-old rats by the isothiocyanate/CsCl- method.18 Furthermore, poly(A+) mRNA was isolated using a PolyATrac mRNA isolation System III kit (Promega, Madison, WI). mRNA was used to construct a poly(T)-primed directional glomerular library, using a ZAP Express cDNA Synthesis kit and a ZAP Express cDNA Gigapack II Cloning kit (Stratagene, La Jolla, CA).
For the library screening 50 x 103
plaque-forming
units of the library per 13-cm plate was transferred to a nylon
membrane (Hybond N+; Amersham Life Sciences, Buckinghamshire, England),
according to the instructions of the manufacturer. The prehybridization
and further hybridization were performed as described
previously.17
Briefly, the filter was prehybridized at
42°C and further hybridized in the hybridization solution containing
the 32P probe (107
cpm; see below) labeled with
[
-32P]CTP (3000 Ci/mmol, 10 mCi/ml; Amersham), using a
Random priming kit (Boehringer Mannheim, Germany). After an overnight
hybridization the filter was washed twice with low-stringency buffer at
42°C, twice with high-stringency buffer at 50°C, and exposed to the
film (Fuji Photo Film, Japan) with intensifying screens at -70°C.
The positive plaques were picked and used for successive rounds of
screening. After single-clone excision protocol (ZAP Express cDNA
Gigapack II Cloning kit; Stratagene), the DNA of the pBK-CMV phagemid
clone was isolated (Quiagen plasmid Mini Kit; Quiagen, Hilden, Germany)
and sequenced from three clones along both strands (ABIPrism 310;
Perkin-Elmer Applied Biosystems, Foster City, CA), and the sequences
were screened for homology with database sequences, using the BLAST
search algorithm at the National Center for Biotechnology (Washington,
DC) via the Internet as described earlier.19
Probes
For screening of the rat glomerular cDNA library the probe (HN3') was constructed by polymerase chain reaction (PCR) amplification of normal human kidney cortex cDNA, using primer pair (numbering according to NPHS1; Gene Bank accession AF035835) NPHS-2606U (5'-cca aca tcg ttt tca ctt gg) and NPHS-3515L (5'-ggg aag gcc ata tcc tca t), producing a 909-bp-long PCR product, which was gel-purified after cloning to pGEM-T vector (Promega). The clone was digested by restriction enzymes ApaI and SacI, yielding a 777-bp-long fragment that was further used as a probe.
For screening the long transcripts from the library, a probe for the 5' end of rat nephrin (RN5') was constructed by PCR amplification of rat kidney cortex cDNA. The primers were designed according to the sequence of mouse nephrin (Holzman et al., manuscript in preparation) for the regions conserved in human and mouse. PCR reaction with the primers RN-S1 (5'-cca cct cag cac ctc gag) and RN-AS1 (5'-gag aca cga gct cgg gac c) produced a PCR product of 293 bp, which was cloned to pGEM-T vector as above. The isolated insert was further used as a probe.
Screening of the Splicing Variants
Screening of the splicing variants of rat nephrin gene by PCR was performed by amplification of normal rat kidney cortex cDNA, using sense primer RN-S4 (5'-ctgctgcctgtgctctttgc) and antisense primer RN-AS2 (5'-ggccataggctctctccact), corresponding to nucleotides 3172–3541 (all numberings according to rat nephrin gene; Gen Bank accession number AF125521).
PCR screening was also done by nested PCR, using primers (sense primer: RN-S3 5'-agcctcttgaccatcgctaa; antisense primer: RN-AS2) flanking the transmembrane region corresponding to nucleotides 2692–3541. The nested sense primer was RN-S2 (5'-tccaggtctccgtcactacc), and the antisense primer was RN-AS2, as in the first reaction.
All PCR reactions were performed as before,19 using AmpliTaq DNA polymerase (Perkin Elmer) and the following amplification program: after the initial denaturation at 94°C for 5 minutes, DNA was amplified using 30 cycles (94°C 1 minute, 55°C 1 minute, and 72°C 1 minute), followed by final elongation at 72°C for 10 minutes. In the nested PCR reactions, denaturation, annealing, and extension times were reduced to 30 seconds with 20 cycles.
Tissue Distribution of Nephrin by Northern Blotting and RT-PCR
Twenty-five micrograms of total RNAs from rat tissues (kidney cortex, liver, heart, muscle, testis, brain, lung, and spleen) were used for Northern blottings as previously described.20 To reveal the transcript levels of rat nephrin, the respective cDNA probes as described above were used. To control the total RNA content and lack of degradation in the preparations, blots were hybridized with a ß-actin probe.19 For autoradiography of Northern blots the filters were exposed on Fuji Bas IIIS Imaging Plates, and the expression was recorded using a Fuji phosphoimager and accompanying MacBAS software (Fuji Photo Film Co, Tokyo, Japan).
Because Northern blotting was not sensitive enough for detection of nephrin mRNA in renal cortex and other tissues mentioned above, we determined its transcript levels by reverse transcriptase-polymerase chain reaction (RT-PCR) with controls as previously used.19
Semiquantitative RT-PCR
RNA-samples from isolated kidney cortex of the PAN rats were used as a starting material in this analysis.19,20 These analyses were done in triplicate. Sequence-specific primers RN-S3 (see above) and RN-AS3 (5'-ccc agt cag cgt gaa ggt ag) yielded a 302-bp product. The semiquantitation of nephrin expression was done as previously described,19 using serial dilutions of sample cDNAs in the linear range of amplification and normalization to the amount of ß-actin amplification product.
Design of Synthetic Peptides
Sequence-specific polypeptides against intracellular and
extracellular nephrin domains were selected over the human nephrin
sequence, using the MacDNAsis and DNAStar programs as described. The
sequence from amino acid 1101 to amino acid 1126 for the intracellular
domain (Figure 1)
was selected. This
sequence was identical with the human sequence, whereas no homology to
other known protein sequences could be found as screened by the
PredictProtein program (European Molecular Biology Laboratory,
Heidelberg, Germany). The peptides, coupled to a high-density multiple
antigenic peptide-polylysine matrix,21
were synthesized and
purified at a local peptide synthesis unit (Haartman Institute,
University of Helsinki).
|
The polyclonal antipeptide antibodies were produced in the rabbit. Briefly, the synthetic peptides in Freund's complete adjuvant were injected into two rabbits. After three booster immunizations 4 weeks apart, peptide-specific IgG fractions were further immunoaffinity purified on CNBr-Sepharose coupled to the corresponding peptides. The specificity of the antisera was tested by immunofluorescence (IF) on kidney sections and by Western blotting of rat glomerular extracts.22
Immunofluorescence Microscopy
To study the presence and tissue distribution of nephrin, frozen sections of the rat kidneys were cut at 5 µm, fixed in acetone at -20°C for 5 minutes, and washed in PBS.9 Thereafter, the rabbit antibodies were flooded over sections for 1 hour. After washing the tissue sections were further incubated with rat anti-rabbit IgG (Boehringer Mannheim, Mannheim, Germany) coupled with fluorescein isothiocyanate (FITC). An Olympus OX50 microscope equipped with an epiilluminator and a filter system for FITC fluorescence was used for microscopy.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Molecular cloning of the rat nephrin cDNA was undertaken to generate reagents necessary for studying nephrin in rat model systems. A human nephrin cDNA probe was amplified by PCR and was used to screen the rat glomerular cDNA library. Additional library screening was necessary to obtain the entire open reading frame of the rat nephrin sequence. Using the mouse nephrin cDNA sequence (Holzman, manuscript in preparation), we designed primers that allowed the amplification of 5' rat nephrin cDNA probe. This cDNA fragment showed 93% sequence identity with mouse nephrin and was successfully employed to assemble the full-length cDNA sequence.
The rat nephrin cDNA coding region of 1234 nucleotides revealed 82%
sequence identity with the open reading frame of the human nephrin cDNA
sequence. The predicted amino acid sequence, with a calculated
molecular mass of 134 kd was 89% identical to the human sequence (see
Figure 1
) and the extracellular, transmembrane, and intracellular
domains showed 90%, 99%, and 83% sequence identity with human
nephrin. A highly conserved general structure of the extracellular
region with the conserved Ig-like and fibronectin type III-like modules
and signal sequence were found as in human nephrin. Putative
N-glycosylation sites were also identically located in human
and rat nephrin. Cysteine residues were also similarly conserved,
showing similarity in location, and two additional cysteine residues in
rat (positions 192 and 356) could be found. In addition to the whole
coding sequence, two splicing variants (rat nephrin- and
nephrin-ß; see below) could be detected with library screening (see
Figure 2
).
|
Our previous data suggested the presence of splice isoforms of
human nephrin. Evidence of alternatively spliced rat nephrin cDNA
clones prompted further examination for alternatively spliced forms.
Primers RN-S2, RN-AS2, RN-S3, and RN-S4 (see Figure 2
) were
particularly designed to reveal possible additional splicing of the
transmembrane coding sequence in the rat.
PCR with primers RN-S4 and RN-AS2 amplified the expected full-size
product (370 bp) as well as a product that was about 100 bp smaller.
After isolation and direct sequencing, the smaller PCR product was
identified as a splicing variant rat nephrin-
(Figure 2)
.
Nested amplification using primers RN-S2 and RN-AS2 (first PCR reaction
with primers RN-S3 and RN-AS2) showed five reaction products. In
addition to the expected full-size product (460 bp) and the already
identified variants nephrin-, -ß and -, a fourth splicing
variant giving a 230-bp product (nephrin-
) was seen and verified by
direct sequencing of the PCR product. The sequences of all splicing
variants translated the COOH-terminal rat nephrin in the correct
reading frame.
Tissue Distribution of Rat Nephrin Specific mRNA
The tissue distribution of rat nephrin mRNA was further studied
using Northern blotting and RT-PCR of the following tissues: liver,
skeletal muscle, cardiac muscle, spleen, kidney cortex, and brain.
Whereas the sensitivity of Northern blotting was found to be too weak
for clear detection of the nephrin specific RNA (data not shown), the
RT-PCR analysis could verify expression in kidney cortex and in the
spleen (Figure 3)
.
|
In semiquantitative RT-PCR analysis of rat cortical kidney in the
PAN nephrosis model, a distinct down-regulation of the rat nephrin
homolog was constantly seen (see Figure 4
). Interestingly, this down-regulation
up to 60% was observed already at 3 days after the single injection of
puromycin amino nucleoside and further decreased down to 20% of the
level of controls at day 10 in this model.
|
Immunohistochemical staining of cortical kidney with antipeptide
antibodies to the intracellular nephrin domain showed distinct
reactivity exclusively with glomeruli (Figure 5)
. Notably, finely granular dotted lines
of reactivity resembling a podocyte-like reactivity at the elements
facing the urinary space could be seen (Figure 5)
. In the kidneys of
PAN rats a systematic decrease (from intense +++ to faint/moderate
+/++) in glomerular nephrin reactivity could be seen (data not shown).
|
.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
The characteristic massive proteinuria of the congenital nephrotic syndrome of the Finnish type has long been considered a unique model disease of the perturbed glomerular filtration function.15,19,20,23 Thus, the recent identification of the NPHS1 gene14 responsible for this disease phenotype has suggested that this gene and the respective protein product nephrin play some key roles in the maintenance of the glomerular filtration barrier. Kestilä et al14 showed that NPHS1 encodes a putative transmembrane protein with sequence similarity to that of cell adhesion molecules of the immunoglobulin superfamily and with expression in the kidney but not in other tissues. Here we show that in addition to the kidney, nephrin can be found in the spleen of the rat. The significance of this additional expression remains to be studied in detail.
The cDNA sequence cloned from the rat glomerular cDNA library shows 82% identity with the respective human cDNA sequence. No other related sequences were obtained in similarity searches. Furthermore, the rat nephrin cDNA bears high sequence similarity to human14 and mouse cDNA (Holzman et al, manuscript in preparation) nephrin sequences. Similarly, the rat nephrin cDNA sequence translates into a closely related amino acid sequence of 89% homology to human nephrin. Indeed, the translated open reading frame includes conserved extra-, intra-, and transmembrane domains, identically located cysteines, signal sequence, and glycosylation sites. Together this suggests that our rat nephrin cDNA is a true homolog of the respective human nephrin cDNA.
Our results have indicated (Holthöfer et al, manuscript submitted
for publication) that NPHS1-specific mRNA is spliced in the
human kidney glomeruli with a variant lacking exon 24 covering the
whole transmembrane domain. Interestingly, the splicing at this
apparently important functional domain appears to be more complex in
the rat with all of the splicing variants identified here. The
unspliced form appears as the dominant one, whereas the less dominant
forms lacking human exons 24–27 are found in the order of
nephrin- > ß > > . Kendall and Thomas
have shown suppression of endothelial cell growth factor activity by a
secreted splice variant of vascular endothelial cell growth factor
receptor lacking the transmembrane domain.24
In addition,
similar transmembrane-negative modifications generated by alternative
splicing have been reported, e.g., for the T-cell
receptor,25
angiotensin-converting enzyme,26
and interleukin-6 receptor.27
Furthermore, the distinct
functional role of such splicing variants was recently shown for the
soluble receptor of advanced glycosylation,28
which appears
to be an important physiological mechanism regulating atherogenesis in
hyperglycemia.28
What is the significance of the splicing
variants of nephrin for glomerular functions and whether their mutual
ratios are changing in experimental glomerular diseases is currently
being studied in detail.
PAN nephrosis in rats is a model for minimal change nephropathy in humans. Interestingly, our initial results on PAN nephrosis show distinct down-regulation of rat nephrin gene-specific mRNA to 60% of the level of controls at day 3 before peak proteinuria, and an additional drop to only 20% at day 10 of the controls at the highest proteinuria. This was supported by findings from immunofluorescence microscopy in which weaker fluorescence was observed in the PAN rat kidney glomeruli, especially at day 10, although immunofluorescence can be considered semiquantitative at best. Currently nothing is known of the metabolic rates of nephrin, but our results suggest a relatively slow degradation. Further studies are in progress to accurately study the decrease in the specific mRNA and the respective nephrin protein in various experimental models of glomerular disease in the rat.
A double band in immunoblotting at 200 kd, the molecular mass also found for human nephrin (Holthöfer et al, manuscript submitted for publication), suggests that two protein transcripts of nephrin are actively produced in the normal rat kidney. The calculated molecular mass of nephrin is 134 kd; the difference from the observed molecular mass is most likely explained by glycosylation at the multiple glycosylation sites of the nephrin sequence.
Taken together, the data presented here show the close identity of rat nephrin cDNA with the respective human cDNA. The observed characteristic alternative splicing suggests that the splicing variants may be important for nephrin functions. The widely used experimental models of glomerular damage in the rat may now be fully exploited to study the functions of nephrin. (Review started on January 22, 1999).
|
Acknowledgements |
|---|
|
Footnotes |
|---|
Supported by the Academy of Finland, a research grant from Helsinki University Hospital, the Finnish Foundation of Heart Disease, and the Sigrid Juselius Foundation.
Accepted for publication May 22, 1999.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
This article has been cited by other articles:
 |
|
 |
|
  P. Aaltonen and H. Holthofer The nephrin-based slit diaphragm: new insight into the signalling platform identifies targets for therapy Nephrol. Dial. Transplant., December 1, 2007; 22(12): 3408 - 3410. [Full Text] [PDF]
|
|
|
|
 |
|
 K. Suzuki, G. D. Han, N. Miyauchi, T. Hashimoto, T. Nakatsue, Y. Fujioka, H. Koike, F. Shimizu, and H. Kawachi Angiotensin II Type 1 and Type 2 Receptors Play Opposite Roles in Regulating the Barrier Function of Kidney Glomerular Capillary Wall Am. J. Pathol., June 1, 2007; 170(6): 1841 - 1853. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 N. Miyauchi, A. Saito, T. Karasawa, Y. Harita, K. Suzuki, H. Koike, G. D. Han, F. Shimizu, and H. Kawachi Synaptic Vesicle Protein 2B Is Expressed in Podocyte, and Its Expression Is Altered in Proteinuric Glomeruli J. Am. Soc. Nephrol., October 1, 2006; 17(10): 2748 - 2759. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. D. Cohen, P. P. Doran, S. M. Blattner, M. Merkle, G. Q. Wang, H. Schmid, P. W. Mathieson, M. A. Saleem, A. Henger, M. P. Rastaldi, et al. Sam68-Like Mammalian Protein 2, Identified by Digital Differential Display as Expressed by Podocytes, Is Induced in Proteinuria and Involved in Splice Site Selection of Vascular Endothelial Growth Factor J. Am. Soc. Nephrol., July 1, 2005; 16(7): 1958 - 1965. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Lehtonen, E. Lehtonen, K. Kudlicka, H. Holthofer, and M. G. Farquhar Nephrin Forms a Complex with Adherens Junction Proteins and CASK in Podocytes and in Madin-Darby Canine Kidney Cells Expressing Nephrin Am. J. Pathol., September 1, 2004; 165(3): 923 - 936. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Patari, C. Forsblom, M. Havana, H. Taipale, P.-H. Groop, and H. Holthofer Nephrinuria in Diabetic Nephropathy of Type 1 Diabetes Diabetes, December 1, 2003; 52(12): 2969 - 2974. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Ahola, E. Heikkila, E. Astrom, M. Inagaki, I. Izawa, H. Pavenstadt, D. Kerjaschki, and H. Holthofer A Novel Protein, Densin, Expressed by Glomerular Podocytes J. Am. Soc. Nephrol., July 1, 2003; 14(7): 1731 - 1737. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. J. Kelly, P. Aaltonen, A. J. Cox, J. R. Rumble, R. Langham, S. Panagiotopoulos, G. Jerums, H. Holthofer, and R. E. Gilbert Expression of the slit-diaphragm protein, nephrin, in experimental diabetic nephropathy: differing effects of anti-proteinuric therapies Nephrol. Dial. Transplant., July 1, 2002; 17(7): 1327 - 1332. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Rantanen, T. Palmen, A. Patari, H. Ahola, S. Lehtonen, E. Astrom, T. Floss, F. Vauti, W. Wurst, P. Ruiz, et al. Nephrin TRAP Mice Lack Slit Diaphragms and Show Fibrotic Glomeruli and Cystic Tubular Lesions J. Am. Soc. Nephrol., June 1, 2002; 13(6): 1586 - 1594. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Yuan, E. Takeuchi, G. A. Taylor, M. McLaughlin, D. Brown, and D. J. Salant Nephrin Dissociates from Actin, and Its Expression Is Reduced in Early Experimental Membranous Nephropathy J. Am. Soc. Nephrol., April 1, 2002; 13(4): 946 - 956. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. A. Dworak, M. A. Charles, L. B. Pellerano, and H. Sink Characterization of Drosophila hibris, a gene related to human nephrin Development, November 1, 2001; 128(21): 4265 - 4276. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Simons, K. Schwarz, W. Kriz, A. Miettinen, J. Reiser, P. Mundel, and H. Holthofer Involvement of Lipid Rafts in Nephrin Phosphorylation and Organization of the Glomerular Slit Diaphragm Am. J. Pathol., September 1, 2001; 159(3): 1069 - 1077. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. BENIGNI, S. TOMASONI, E. GAGLIARDINI, C. ZOJA, J. A. GRUNKEMEYER, R. KALLURI, and G. REMUZZI Blocking Angiotensin II Synthesis/Activity Preserves Glomerular Nephrin in Rats with Severe Nephrosis J. Am. Soc. Nephrol., May 1, 2001; 12(5): 941 - 948. [Abstract] [Full Text]
|
|
|
|
|
|
J. PATRAKKA, V. RUOTSALAINEN, I. KETOLA, C. HOLMBERG, M. HEIKINHEIMO, K. TRYGGVASON, and H. JALANKO Expression of Nephrin in Pediatric Kidney Diseases J. Am. Soc. Nephrol., February 1, 2001; 12(2): 289 - 296. [Abstract] [Full Text]
|
|
|
|
|
|
H. PUTAALA, K. SAINIO, H. SARIOLA, and K. TRYGGVASON Primary Structure of Mouse and Rat Nephrin cDNA and Structure and Expression of the Mouse Gene J. Am. Soc. Nephrol., June 1, 2000; 11(6): 991 - 1001. [Abstract] [Full Text]
|
|
|
|
|
|
H. Holthofer, H. Ahola, M.-L. Solin, S. Wang, T. Palmen, P. Luimula, A. Miettinen, and D. Kerjaschki Nephrin Localizes at the Podocyte Filtration Slit Area and Is Characteristically Spliced in the Human Kidney Am. J. Pathol., November 1, 1999; 155(5): 1681 - 1687. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
