Measurements of subcutaneous tissue PO2 reflect oxygen metabolism of the small intestinal mucosa during hemorrhage and resuscitation. An experimental study in pigs.

BACKGROUND: The first tissues to be deprived of perfusion and oxygenation in a hypovolemic situation and the last ones to be reperfused are the subcutaneous tissue and the gastrointestinal mucosa. We hypothesized that measurements of subcutaneous tissue gases and pH might reflect simultaneous changes in oxygenation of the intestinal mucosa. The aim of this study was to evaluate tissue gases and pH as measures of tissue oxygenation and tissue oxygen metabolism in subcutaneous and intestinal tissues simultaneously. MATERIAL AND METHODS: Five out of 17 domestic pigs (weight 21-25 kg) were used as controls without bleeding. Twelve animals were bled in 3 steps, 10% of their calculated blood volume at each step. The removed blood, crystalloid and colloid were thereafter infused, and animals were stabilized for 30 min. Measurements were made after each step of bleeding, retransfusion and stabilization. Before bleeding, all animals had a sensor (Paratrend 7) implanted subcutaneously in the left groin for measurements of tissue gases and pH (P(sc)O(2), P(sc)CO(2) and pH(sc)). Catheters were positioned in the jugular vein, portal vein, carotid artery, pulmonary artery and femoral artery for infusion, bleeding and oxygen monitoring. Via a midline laparotomy, 2 silicon tonometers (TRIP sigmoid catheters) were positioned in the ileum and sigmoid colon for measurements of PCO(2) and pH (P(iI)CO(2), P(si)CO(2), pH(iI) and pH(si)). Blood flow in the portal vein was measured by an ultrasound probe (H6SB) and a Clark electrode (Cardiff tissue oxymeter) was used for serosal PO(2) measurements of the ileum (P(iI)O(2)) and sigmoid colon (P(si)O(2)). RESULTS: After the first step of bleeding, P(sc)O(2) decreased from 64 +/- 17 to 56 +/- 22 mm Hg (SD; p < 0.05). P(sc)CO(2) and pH(sc) did not change. P(iI)CO(2) increased from 64 +/- 14 to 79 +/- 14 mm Hg (p < 0.05), P(si)CO(2) increased from 77 +/- 16 to 90 +/- 18 mm Hg (p < 0.05). pH(iI) decreased from 7.15 +/- 0.09 to 7.03 +/- 0.09 (p < 0.05). P(iI)O(2) and P(si)O(2) decreased, but not significantly until steps of further bleeding. After re-transfusion and stabilization, P(sc)O(2) and P(iI)CO(2) returned to baseline. CONCLUSION: Measurements of subcutaneous PO(2) are sensitive to bleeding and resuscitation and reflect oxygen metabolism in the small intestinal mucosa as measured by PCO(2) and pH.

Role of CD18-dependent neutrophil recruitment in skin and intestinal wound healing.

CD11/CD18 is an important adhesion molecule mediating recruitment of leukocytes, which, in turn, may cause postoperative injury in the skin and gastrointestinal tract. The objective of the present study was to investigate the effects of inhibiting the function of CD18 on surgery-induced dermal and intestinal infiltration of neutrophils and on the healing of surgical skin flaps and colonic anastomosis. A flap in the dorsal skin or an end-to-end colonic anastomosis were created in Sprague-Dawley rats. Skin necrosis and anastomotic breaking strength were analyzed 6 and 3 days after surgery, respectively. Tissue myeloperoxidase (MPO) was used as a marker of neutrophil recruitment. Administration of a monoclonal antibody directed against rat CD18 (WT.3, 2 mg/kg) significantly decreased dermal and anastomotic MPO activity by more than 80%. Passive immunization against CD18 significantly improved flap survival, i.e. the survival was 80% in the anti-CD18 antibody group as compared to 38% in the control group. In contrast, this passive immunization against CD18 had no effect on the reconstitution of the integrity of the colonic anastomosis, i.e. the anastomotic breaking strength was 1.3 +/- 0.1 and 1.3 +/- 0.3 N in the control and anti-CD18 antibody group, respectively. These findings suggest that specific inhibition of CD18 function and reduced neutrophil recruitment may improve the survival of experimental skin flaps and, thus, may represent a potential target for therapeutic intervention. In contrast, we also found that blocking CD18-dependent neutrophil infiltration in the intestine had no effect on breaking strength of colonic anastomosis. Thus, neutrophils may influence the wound-healing process differently in specific organs and this needs to be considered when applying an anti-inflammatory treatment regime in order to improve tissue healing.

Evaluation of splanchnic perfusion and oxygenation during positive end-expiratory pressure ventilation in relation to subcutaneous tissue gases and pH. An experimental study in pigs with oleic acid-induced lung injury.

Evaluation of splanchnic perfusion and oxygenation was performed by measurements of serosal tissue oxygen tension (PserO2) and intramucosal pH (pHi) in relation to subcutaneous oxygen tension (PscO2), subcutaneous carbon dioxide tension (PscCO2) and subcutaneous pH (pHsc) in pigs subjected to oleic acid-induced lung injury during ventilation with increasing levels of positive end-expiratory pressure (PEEP). Lung injury resulted in a general hypoxia and redistribution of perfusion away from the subcutaneous and splanchnic tissues, illustrated by a decrease in PaO2 from 93 to 37 mm Hg (p < 0.01), PscO2 from 45 to 17 mm Hg (p < 0.01), PserO2 from 80 to 30 mm Hg (p < 0.01) and pHi from 6.84 to 6.74 (p < 0.05) and a decrease of porta flow from 0.77 to 0.57 l/min. Application of PEEP up to 10-15 cm H2O resulted in an increase of portal vein oxygen tension (PportaO2) from 21 to 34 mm Hg (p < 0.01), PscO2 from 17 to 26 mm Hg (p < 0.05) and PserO2 from 30 to 55 mm Hg (p < 0.05). At PEEP 20 cm H2O PserO2 decreased to 47 mm Hg (p < 0.05). Porta flow decreased continuously with increasing levels of PEEP. PserO2 correlated with PportaO2 (r = 0.7, p < 0.001). pHi correlated poorly with PportaO2 (r = 0.2) and porta flow (r = 0.4). PscO2 and PserO2 correlated well (r = 0.8, p < 0.001). In summary, splanchnic perfusion and oxygenation was better reflected by serosal oxygen tension than pHi in the colon. Changes in serosal oxygenation of the colon paralleled changes in subcutaneous tissue oxygenation.

Diverting colostomy increases anastomotic leakage in the rat colon.

In the present study, we examined the effect of a diverting colostomy on the intestinal healing of colonic anastomosis in the rat. For this purpose, we created a colonic stenosis 2 days prior to the formation of a distal one-layer end-to-end anastomosis with or without a proximal double-barreled deviation colostomy in the rats. Radiological examination of anastomotic leakage was performed daily for 4 days and on day 7 after the operation. We found that anastomotic leakage was markedly increased in rats with a diverting colostomy compared to control animals; i.e. the leakage index (percentage of days with leakage during the experimental period) in colostomy rats was 29%, whereas in animals with no colostomy, the leakage index was only 7%. Interestingly, it was observed that anastomosis formation was associated with a higher mortality rate in rats with colostomy diversion (36%) compared to control animals (7%). However, there was no difference in suture holding capacity on day 7. Moreover, body weight decreased significantly in the colostomy group compared to rats without surgical defunctioning when followed for up to 7 days after surgery. Taken together, our novel findings suggest that a diverting colostomy may increase intestinal leakage after anastomosis formation in the rat colon. Thus, the role of proximal colostomy in the protection of colorectal anastomosis needs to be reevaluated and further investigations are required to resolve the influence of surgical defunctioning on intestinal healing.

Conventional running suture and continuous double loop closure: an experimental study of wound strength.

OBJECTIVE: To study the strength of laparotomy wounds closed by a continuous double loop technique or a conventional running suture, taking into account the ratio of suture length: wound length. DESIGN: Experimental study. ANIMALS: 60 Sprague-Dawley rats. INTERVENTIONS: Midline laparotomy incisions were closed with either a conventional running suture or a continuous double loop. Wounds were allocated to closure with a suture length: wound length ratio of 3, 4 and 7. MAIN OUTCOME MEASURES: Bursting pressure, bursting volume and the way the suture cut through the tissues. RESULTS: With a suture length: wound length ratio of 3 or 4 bursting pressure and bursting volume were lower with a continuous double loop closure. A conventional running suture and a continuous double loop produced similar bursting pressure and bursting volume only if closure was with a ratio of 7. CONCLUSIONS: Wound bursting strength is higher with a conventional running suture than with a continuous double loop closure when the effect of the suture length: wound length ratio is accounted for.

Critical role of P-selectin-dependent rolling in tumor necrosis factor-alpha-induced leukocyte adhesion and extravascular recruitment in vivo.

Leukocyte-endothelium interactions are dependent on a coordinated expression and function of specific adhesion molecules. The objective of the present study was to examine the role of selectin function and leukocyte rolling in tumor necrosis factor-alpha (TNF-alpha)-induced leukocyte adhesion and extravasation in venules in vivo. For this purpose, we used intravital microscopy in the mouse cremaster muscle stimulated for 2-3 h with TNF-alpha intrascrotally. Pretreatment with fucoidan, which inhibits P- and L-selectin, and a P-selectin monoclonal antibody (RB40.34) abolished TNF-alpha-stimulated leukocyte rolling. This great reduction in rolling caused a marked attenuation of firm adhesion and extravascular accumulation of leukocytes. When fucoidan and RB40.34 were administrated after stimulation with TNF-alpha, it was found that leukocyte rolling was greatly reduced whereas the number of firmly adherent leukocytes was completely unchanged, suggesting that the inhibitory effect of blocking P-selectin function on firm leukocyte adhesion and recruitment was due to the reduction in leukocyte rolling along the endothelium. Moreover, pretreatment with a monoclonal antibody against intercellular adhesion molecule-1 (ICAM-1) and a platelet-activating factor (PAF)-receptor antagonist had no effect of TNF-alpha-induced leukocyte rolling and adhesion, indicating that molecules other than ICAM-1 and PAF mediate firm adhesion and recruitment of leukocytes in TNF-alpha-activated tissues. Taken together, our data demonstrate that P-selectin function plays an important role in TNF-alpha-induced inflammatory cell recruitment by mediating leukocyte rolling as a precondition for cytokine-provoked firm adhesion and transmigration in vivo. These findings, thus, suggest that inhibition of P-selectin may be a central target for pharmacological intervention in inflammatory diseases.

(2S,4R)-4-methylglutamic acid (SYM 2081): a selective, high-affinity ligand for kainate receptors.

Glutamic acid activates ionotropic glutamate receptors that mediate excitatory transmission in the central nervous system. The introduction of a methyl group at position 4 of glutamic acid imparts selectivity for kainate receptors, relative to other (N-methyl-D-aspartate and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid) ionotropic glutamate receptors. Among the stereoisomers of 4-methylglutamic acid, the potency of the (2S,4R)-isomer (SYM 2081) to inhibit [3H]kainic acid binding to both wild-type (rat forebrain) and recombinant (GluR6) kainate receptors (IC50 values of approximately 32 and 19 nM, respectively) was comparable to that of kainic acid (IC50 values of approximately 13 and 28 nM, respectively). SYM 2081 was approximately 800- and 200-fold less potent as an inhibitor of radioligand binding to wild-type (rat forebrain) alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid and N-methyl-D-aspartate receptors, respectively. Preexposure of human embryonic kidney 293 cells stably expressing GluR6 receptors to low concentrations of SYM 2081 (30-300 nM) resulted in a reversible blockade of the rapidly desensitizing currents produced by kainate application. At higher concentrations, SYM 2081 (EC50 of approximately 1 microM) elicited kainate-like, rapidly desensitizing, inward currents. Pretreatment of recombinant GluR6 receptors with concanavalin A both abolished the effect of SYM 2081 to block kainate-induced currents and revealed nondesensitizing currents induced by SYM 2081 alone. The latter observations provide strong support for the hypothesis that SYM 2081 blocks kainate-induced currents through a process of agonist-induced desensitization. SYM 2081 also activated alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor currents in primary cultures of cerebral cortex and, consistent with data obtained by radioligand binding, was approximately 5-fold less potent than kainate (EC50 values of 325 and 70 microM, respectively) in this measure. SYM 2081 is a high-affinity, selective, kainate agonist that may prove useful both as a probe to examine the physiological functions of kainate receptors and as the prototype of a novel class of therapeutic agents.

Cytotoxic effects of kainate ligands on HEK cell lines expressing recombinant kainate receptors.

Exposure of neurons either for prolonged periods of time or to high concentrations of excitatory amino acids (EAA), such as glutamate, results in neuronal death. Kainate also causes cell toxicity through the glutamate receptors. However, it is unclear whether the kainate receptor itself mediates any of the toxic responses. In the present study, HEK cells expressing the GluR6 +/- KA2 receptor subunit(s) were studied for their susceptibility to toxicity through the kainate receptor by kainate ligands. The natural ligand, glutamate, did not result in toxicity to the recombinant cell lines over that observed with the untransfected HEK cells, whereas kainate produced a 2-3-fold increase in LDH in both the HEK/GluR6 (ANOVA, P = 0.0001) and HEK/GluR6 + KA2 (ANOVA, P = 0.0002) cell lines following treatment with various dosages, but did not affect the HEK cells. Similar 2-3-fold increases in LDH activity were detected in both recombinant cell lines following treatment with 100 nM of SYM2081 ((2S,4R)-4-methylglutamic acid), a dose at which agonistic activity is elicited. The rank order potencies for eliciting toxicity are consistent with the previously reported EC50 values (SYM2081 > kainate > > > glutamate). Surprisingly, the kainate antagonist, NBQX, was the most toxic of the compounds tested although it had an affinity for the kainate receptor similar to glutamate. Treatment with as little as 10 nM elicited a dramatic increase in toxicity (6-10-fold) in the recombinant cell lines. At 1 microM, NBQX was significantly more toxic (Fisher PLSD, P < 0.05) than any of the other compounds tested. Thus, it appears that cell toxicity can be mediated via kainate receptor through two independent mechanisms: activation and blockage of the kainate receptor.

Effect of cysteine substitutions on the mitogenic activity and stability of recombinant human keratinocyte growth factor.

Human Keratinocyte growth factor (hKGF), a member of the FGF family of growth factors, contains five cysteines at amino acid positions 1, 15, 40, 102, and 106. We expressed five cysteine mutants of hKGF in which the cysteines were cumulatively replaced with alanine or serine, starting with cysteine-1. Recombinant hKGF has an inherently higher mitogenic activity and stability to heat and acid than reported for glycosylated hKGF. Mitogenic activity is increased an additional 2.6 fold by substitution of cysteine-1 with alanine. Mutants with the conserved cysteine substituted at position 40 were more susceptible to heat inactivation than rhKGF, but showed no significant difference in acid inactivation. Cysteine-free rhKGF is mitogenic, demonstrating that neither cysteines nor disulfide bonds are required for mitogenic activity. However, cysteine-free rhKGF does not bind Heparin-Sepharose and is unstable to heat and acid compared to rhKGF, suggesting that the cysteines have a role in maintaining KGF's structure. This information will useful in the development of a more stable and more potent wound healing agent from hKGF.

Characterization of the hamster DDT-1 cell aFGF/HGBF-I gene and cDNA and its modulation by steroids.

Syrian hamster DDT-1 cells are derived from smooth muscle of the ductus deferens. DDT-1 cell growth is increased by the addition of testosterone (T). Acidic fibroblast growth factor (aFGF) or basic fibroblast growth factor (bFGF) also known as heparin binding growth factor I and II (HBGF-I and HBGF-II) can replace T in the stimulation of growth in these cells. This phenomenon is correlated with testosterone's ability to elevate aFGF/HBGF-I mRNA. The increase steady-state levels of aFGF/HBGF-I mRNA were documented by northern blots and by in situ hybridization. Using a 520 bp human aFGF/HBGF-I cDNA probe, a genomic clone with a 38 kb DNA insert was isolated from a cosmid library. By restriction enzyme analysis and southern hybridization, it was determined that there are three coding exons. DNA sequence analysis showed all of the coding region and 3' noncoding sequences were on this clone. A 5' noncoding exon not in the 38 kb insert is indicated, based on the cDNA sequences and genomic sequences of aFGF/HBGF-I's from hamster DDT-1 cells and several other species. The cDNA for hamster aFGF/HBGF-I was isolated from a DDT-1 lambda gt11 library and sequenced. Comparison of the coding region of aFGF/HBGF-I from four species shows a greater than 90% conservation of amino acid sequence.

Heparin-binding (fibroblast) growth factors type one and two genes are co-expressed in proliferating normal human vascular endothelial and smooth muscle cells in culture.

Complimentary ribonucleic acid (cRNA) probes were used to detect expression of the genes for heparin binding (fibroblast) growth factor type one (HBGF-1) and two (HBGF-2) in cultured endothelial and smooth muscle cells from normal human blood vessels. Hybridization in situ revealed that transcripts for both HBGF-1 and HBGF-2 are expressed in endothelial cells from both umbilical vein and aorta. Relative intensity of radioactive grains suggest that HBGF-1 gene expression may exceed HBGF-2 expression in aortic smooth muscle cells. Collective expression of both HBGF-1 and HBGF-2 in smooth muscle cells may exceed that in endothelial cells.

Androgens and glucocorticoids modulate heparin-binding growth factor I mRNA accumulation in DDT1 cells as analyzed by in situ hybridization.

The ductus deferens smooth muscle tumor cell line (DDT1-MF-2) is very sensitive to steroids. Treatment with 10 nM testosterone accelerates the growth of DDT1 cells in the absence of serum. Glucocorticoids in the presence or absence of androgens inhibits growth. Stimulation of growth of DDT1 cells by testosterone can be replaced by the addition of heparin-binding growth factor I and II (HBGF). Addition of testosterone plus HBGF growth factors results in a further increase in cell number. By in situ hybridization, accumulation of HBGF-I mRNA is significantly increased by testosterone treatment of low density cultures. Testosterone treatment of high density cultures results in no stimulation of HBGF-I mRNA accumulation. Glucocorticoids alone, which block growth of DDT1 cells, have no effect of HBGF-I mRNA accumulation. However, the simultaneous addition of glucocorticoid and androgens to DDT1 cells results in a rapid accumulation of HBGF-I mRNA by 12 h, although growth is inhibited by the presence of both steroid analogs.

Heparin-binding growth factor type 1 (acidic fibroblast growth factor): a potential biphasic autocrine and paracrine regulator of hepatocyte regeneration.

Heparin-binding growth factor type 1 (HBGF-1; sometimes termed acidic fibroblast growth factor) is potentially an important factor in liver regeneration. HBGF-1 alone (half-maximal effect at 60 pM) stimulated hepatocyte DNA synthesis and bound to a high-affinity receptor (Kd = 62 pM; 5000 per cell). Epidermal growth factor (EGF) neutralized or masked the mitogenic effect of HBGF-1 concurrent with appearance of low-affinity HBGF-1 binding sites. HBGF-1 reduced the inhibitory effect of transforming growth factor type beta (TGF-beta) on the EGF stimulus. Nanomolar levels of HBGF-1 decreased the EGF stimulus. An increase in hepatic HBGF-1 gene expression after partial hepatectomy precedes increases in expression of the EGF homolog, TGF-alpha, and nonparenchymal-cell-derived TGF-beta in the regenerating liver. Expression of HBGF-1 mRNA occurs in both hepatocytes and nonparenchymal cells and persists for 7 days in liver tissue after partial hepatectomy. HBGF-1 acting through a high-affinity receptor is a candidate for the early autocrine stimulus that drives hepatocyte DNA synthesis prior to or concurrent with the EGF/TGF-alpha stimulus. It may allow hepatocyte proliferation to proceed in the presence of low levels of TGF-beta. An EGF/TGF-alpha-dependent change in HBGF-1 receptor phenotype and increasing levels of nonparenchymal-cell-derived HBGF-1 and TGF-beta may serve to limit hepatocyte proliferation.

Heparin-binding growth factor gene expression and receptor characteristics in normal rat prostate and two transplantable rat prostate tumors.

Heparin-binding polypeptide growth factors (HBGF) are essential mitogens for isolated prostate cells. HBGF type one (HBGF-1) mRNA was expressed specifically in the epithelial cells of prostates from normal 6- to 8-week-old rats. Expression declined significantly at 14 weeks and was undetectable in 35-week-old animals. Slow-growing, androgen-responsive, nonmetastatic Dunning R3327PAP tumors, which are composed of a well-defined epithelium and stroma, expressed HBGF-1 mRNA constitutively in specifically the mesenchymal cells. A rapid-growing, androgen-independent, metastatic variant (Dunning R3327AT-3), which was composed of a single clonogenic cell type, expressed both HBGF-1 and HBGF type two (HBGF-2) mRNA. HBGF activity in the extracts of normal and tumor tissues correlated with mRNA levels. Epithelial cells from the R3327PAP tumor and the single cell type that composed the R3327AT-3 tumor exhibited alterations in HBGF receptor characteristics that correlated with increased sensitivity to mitogenic effects of HBGF. The results suggest that alterations in HBGF gene expression in both prostate epithelial and mesenchymal cells and in properties of the receptor in specifically epithelial cells may contribute to differential growth rates and malignancy of different prostatic tumors.

Structure of the androgen dependent SVS VI protein as derived from cDNA.

The 11S poly(A+)RNA from rat seminal vesicle was cloned. By hybrid selection of clones reacting to the low molecular weight region of the 11S peak, a clone containing a new seminal vesicle cDNA sequence called SVS VI was isolated. The DNA sequence of two overlapping cDNAs (pSV24 and pSV33) is presented. The sequence of SVS VI was compared to the previously isolated SVS IV and SVS V cDNAS. Dot hybridization showed that SVS VI is androgen responsive after giving testosterone to castrated rats. The hydrophilicity was analyzed using standard Bionet procedures. All three proteins are extremely variable, rich in alpha-helix and very water soluble. The computer predicted hydrophilicity is compared for SVS VI, V and IV. A small region in the 3'-non-coding area of SVS VI has high similarity to a region in SVS IV mRNA.

High and low affinity binding of heparin-binding growth factor to a 130-kDa receptor correlates with stimulation and inhibition of growth of a differentiated human hepatoma cell.

The differentiated human hepatoblastoma-derived cell line, HepG2, displayed two classes of specific membrane receptors for heparin-binding growth factor type 1 (HBGF-1). Specific membrane receptors were distinguished from nonreceptor heparin-like binding sites. Receptors with an apparent Kd of 9.2 +/- 0.9 pM and present at 15,000 +/- 900/cell correlated with HBGF-1 stimulation of HepG2 growth. Receptors with an apparent Kd of 2 +/- 0.4 nM and present at 180,000 +/- 18,000/cell correlated with inhibition of growth and changes in secretory products. Other hepatoma cell lines exhibited a simple positive mitogenic response to HBGF-1 and a single class of high affinity binding sites. HBGF-1 covalently cross-linked to hepatoma cell surface polypeptides of apparent mean molecular mass of 130 kilodaltons. At 37 degrees C, receptor-bound HBGF-1 was internalized (t 1/2 = 45 min) but not degraded for up to 6 h. The display of receptors decreased with increased cell density and expression of HBGF-1 mRNA and HBGF-1-like activity in the culture medium. Proliferating normal human hepatocytes also exhibited two classes of binding sites with affinities for HBGF-1 and apparent molecular weight similar to HepG2 cells. These results implicate HBGF-1 or homologues in human hepatoma cell growth and normal liver cell regeneration.

Seminal vesicle secretion IV gene: allelic difference due to a series of 20-base-pair direct tandem repeats within an intron.

The rat seminal vesicle secretion IV (SVS IV) gene was isolated from a lambda Charon 4A library. The SVS IV gene transcription unit was found to be on one 3.3-kilobase (kb) EcoRI fragment. Restriction mapping and DNA sequence analysis demonstrated that the entire length of the SVS IV transcription unit is 1,930 base pairs (bp) and contains two introns. The 3.3-kb EcoRI fragment contains 144 bp of 5'-flanking region. At -113 bp from the presumed transcription initiation site an interesting structure with perfect dyad symmetry is noted. In another lambda clone, a 3.5-kb EcoRI fragment was isolated that contains the SVS IV gene and was shown to be identical to the 3.3-kb EcoRI fragment except for 180 bp of DNA in the second intron. The extra DNA consists of several (8-10) 20-bp tandem repeats flanked on each side by seven or eight copies of this same 20-bp repeat. Fisher X Sprague-Dawley hybrid rats, which contain both the EcoRI 3.5-kb form and the 3.3-kb form of the SVS IV gene, were crossed with each other. Analysis of the F1 generation demonstrated that the presence or absence of the 180-bp intronic insertion in the SVS IV gene defines an allelic difference. This report also presents the DNA sequence of the transcription unit and flanking regions of the SVS IV gene.