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We have purified the factor VIII from a CRM+ Hemophilia A plasma (90 U/dL VIII:Ag but 0 U/dL VIII:C) and analyzed the protein before and after thrombin activation by Western blotting with monoclonal antibodies (MoAbs). Normal or patient citrated plasma was ultracentrifuged, cryo-ethanol-precipitated and chromatographed on Sepharose 6B. The void volume fractions were reduced and subjected to ion exchange chromatography yielding material of specific activity approximately 1,000 U/mg protein (VIII:C or VIII:Ag). Factor VIII purified in this way from normal plasma is fully activatable by thrombin with proteolytic fragmentation as previously described by F. Rotblat et al (Biochemistry 24: 4294, 1985). Factor VIII 1,689-Cys has the normal distribution of factor VIII light and heavy chains prior to thrombin activation. After exposure to thrombin the heavy chain polypeptides were fully proteolysed but the light chain was totally resistant to cleavage. This is consistent with the demonstration in the patient's leucocyte DNA of a C to T transition in codon 1,689 converting Arg to Cys at the light chain thrombin cleavage site as previously described by J. Gitschier et al (Blood 72:1022, 1988). Uncleaved light chain of Factor VIII 1,689-Cys is not released from von Willebrand factor (vWF) by thrombin, but this is not the sole cause of the functional defect since the protein purified free of vWF has no coagulant activity. We conclude that the functional defect in factor VIII 1,689-Cys is a consequence of failure to release the acidic peptide from the light chain upon thrombin activation.

Tissue factor (TF) is a 263 amino acid membrane-bound procoagulant protein that serves as a cofactor for the serine protease factor VII (fVII). Recombinant human TF (rTF) produced in both human kidney 293 cells and Escherichia coli has been immunoaffinity purified by using a TF-specific monoclonal antibody. Recombinant TF produced in 293 cells is glycosylated and migrates on reducing SDS-PAGE with an apparent molecular weight (Mr) of 45K. Some interchain disulfide-bonded rTF dimers are observed under nonreducing conditions. The E. coli produced rTF has a molecular weight of 33K and 35K, with the 33K band missing nine amino acids at the carboxy terminus. Although the E. coli produced rTF does not contain any carbohydrate, it is fully functional in both a chromogenic assay and a one-stage prothrombin time assay. A variant has been constructed wherein the cytoplasmic cysteine (residue 245) has been mutagenized to a serine residue. The amount of disulfide-linked aggregates is dramatically reduced following immunoaffinity purification of this four-cysteine variant (C2455), which is active in the chromogenic and prothrombin time assays.

A murine monoclonal antibody has been produced (RFF-VIII:R/2) that binds specifically to human factor VIII-related antigen (VIII:RAg) in plasma and in vascular endothelial cells but has no reactivity with factor VIII procoagulant antigen (VIII:cAg). This antibody is a potent inhibitor of von Willebrand factor activity (VIII:vWF) in that it can totally neutralize ristocetin-induced aggregation of platelet rich plasma and inhibit platelet adhesion at high flow rates. RFF-VIII:R/2 can be used in a one-stage, fluid phase immunoradiometric assay that can detect VIII:RAg at concentrations of 0.001 u/ml. This method has been used to analyse plasma from patients with von Willebrand's disease (vWD). Results obtained in these patients showed a high degree of correlation between the monoclonally-defined epitope and VIII:vWF levels measured by ristocetin-induced aggregation of washed platelets. This correlation was maintained in those patients with the 'variant' types of vWD who exhibit highly disparate VIII:vWF and VIII:RAg levels when the latter is determined using polyclonal antisera. It appears that this monoclonal antibody recognizes a site on the VIII:RAg molecule which is associated with its interaction with the platelet membrane. Immunoradiometric assays using RFF-VIII:R/2 offer a simplified, reproducible means of detecting functionally-active VIII:RAg as an alternative or supplement to techniques involving platelet interactions.

The previous literature has reported that when children are asked to judge the truth or falsity of universally quantified conditional sentences of the form If a thing is P then it is Q they typically give responses, e.g., responding "true" whenever there is a case of P and Q even if there are also cases of P and not-Q. Three experiments are reported that address possible sources of this error. Experiment 1 shows that the error survives on sentences that refer to particular things as well as to things of a particular kind, and further shows that articulating the necessity of the consequent (... then it has to be Q) eliminates the error for adults and reduces it for fifth graders, although it does not affect second grade performance. Experiment 2 shows that for second and fifth graders the error survives to problems that are not universally quantified and for second graders to problems that are not conditionals although are otherwise structurally similar. Experiment 3 compares various verbal formulations of such universally quantified conditionals: Second and fifth graders do not make the error when the quantification is expressed with the surface structure that makes its universality most explicit (all things ...); the error tendency is greatest when the indefinite article is used (if a thing ...); and formulations using any fall in between. We argue that such erroneous evaluations of universally quantified conditionals have more to do with the quantificational aspect than the conditional aspect of the problems; children interpret the indefinite article as existential, although they resist the error when the cue to universal quantification is completely clear. The error appears to result more from the surface-structure form of the stimuli than from an inability of children to appreciate the logic of universally quantified conditionals.

Human factor VIII:C has been purified over 300 000-fold from cryoprecipitate by polyelectrolyte purification followed by affinity chromatography on Sepharose linked to antibody to factor VIIIR:Ag (monoclonal or polyclonal) and Sepharose linked to monoclonal antibody to factor VIII:C. The purified material has been analyzed by polyacrylamide gel electrophoresis (PAGE) and Western blotting using monoclonal antibodies. PAGE shows predominant bands at 360K (unreduced), 210K, and 90K and an 80K/79K doublet; Western blotting showed all the monoclonal antibodies used bound the 360K form. In a small-scale purification, plasma from blood taken directly into thrombin inhibitor Kabi S-2581 was applied directly to the monoclonal anti-factor VIII:C column. Western blot analysis of this material showed the 360K band on reduction. The purified factor VIII:C could be activated 13-fold by human thrombin. Gel analysis of the activated material showed intensification followed by fading of the band at 90K and generation of bands at 70K/69K, 55K, and 40K. Western blotting shows that the 70K/69K doublet derives from the 80K/79K moiety and the 40K peptide derives from the 90K and is presumed to contain the active site. From these studies an epitope map of the factor VIII:C molecule has been constructed.

A series of renal tumors associated with renal cysts is presented. Prior to surgical exploration appropriate diagnostic studies failed to indicate evidence of the presence of neoplastic lesions. The importance of surgical exploration of avascular "benign" renal masses is emphasized.

Progressive dysmetria was observed at four months and six months of age in two female Labrador retriever littermates. Neurological examinations indicated a cerebellar disorder. Laboratory analyses were normal. The dogs were studied clinically until they were killed for necropsy at nine months and eleven months of age. Both dogs had normal brain size, shape, and calvarial symmetry; the older dog had body weight loss, reduced brain weight, and muscle atrophy. Microscopically, there were vacuoles, hypertrophied fibrous astrocytes, myelin loss, and prominent blood vessels, distributed symmetrically in the subcortical and deep white matter of all lobes of the cerebrum; in the folial and deep white matter of the cerebellum; in the tracts of some cranial nerves; in the thalamic area, midbrain and brainstem; and in the white matter of the spinal cord. There was no significant myelinolysis, inflammation, or axonal degeneration. Ultrastructurally, there were intramyelinic vacuoles with separation of lamellae at intraperiod lines and larger spaces formed by coalescence of ruptured vacuoles. Hypertrophied fibrous astrocytes had abundant glial filaments, edematous cytosol, membrane-bound crystalline inclusions, dilated cytocavitary systems, and abnormal mitochondria. The clinical, histological, and ultrastructural findings resembled those reported for the juvenile form of Canavan's disease (van Bogaert and Bertrand type) in children.

Catecholamine-depletion-induced catalepsy isolates and leaves intact an aggregate of allied reflexes (e.g., righting, standing still, bracing, and clinging) which involve all the body and limb segments in defending stable static equilibrium. Because other movement subsystems (locomotion, orienting, scanning, directed use of mouth or forepaws) are depressed, such animals cling in a vertical position for an abnormally long period of time. As a consequence, grasping reflexes may be studied independently of other responses. Haloperidol, a dopamine antagonist, abolishes visually elicited reaching and grasping, but leaves intact tactile and proprioceptive control of grasping. The grasping of haloperidol-treated rats can be further simplified by the pharmacological removal of the remaining sensory controls. The addition of morphine to haloperidol abolishes tactile grasping, while the addition of diazepam to haloperidol abolishes both tactile and proprioceptive (traction-elicited) grasping. Although visual, tactile, and proprioceptive grasping are abolished by haloperidol-plus-diazepam, some vestibular input to clinging remains: such rats, in response to being held vertically upright in the air, flex their digits with sufficient strength to allow them to cling vertically. The strength of forepaw digit flexion is severely diminished by labyrinthectomy, but the digits of the hindpaws appear to be unaffected. This residual non-labyrinthine digit gripping appears to be induced by proprioceptive inputs from the head, neck and torso in response to the vertical body position. Wrapping an elastic bandage snugly around the head and neck of a labyrinthectomized rat given haloperidol-plus-diazepam further diminishes the strength of forepaw digit flexion, and to a lesser degree hindpaw digit flexion.(ABSTRACT TRUNCATED AT 250 WORDS)

Spongy degeneration of the CNS was diagnosed in 2 female Labrador Retriever littermates. The dogs had histories of progressive cerebellar ataxia and episodes of extreme extensor rigidity beginning at 4 and 6 months of age. One had a history of hearing difficulties. When examined at 9 months of age, both dogs had moderate ataxia and dysmetria of the head, trunk, and limbs, hyperreflexia with clonus, and extensor rigidity with episodes of exaggerated rigidity and opisthotonos. One dog was euthanatized and the other was monitored for 2 months, during which time her signs progressed to severe dysmetria, weakness, muscle atrophy, and rigidity with marked hyperreflexia. Necropsy of both dogs revealed astrocytic abnormalities and spongy degeneration of the white matter of the CNS. The clinical signs and postmortem findings were similar to the juvenile form of spongy degeneration in man.

Cross reactive antigens to factor VIII have been measured in a range of vertebrate phyla. VIII coagulant antigen (VIII:CAg) measured using a human antibody was in general, much lower than reported values of VIII coagulant activity (VIII:C) except in simians and the guinea pig. The dose response curve for the assay was parallel in all cases. Factor VIII-related antigen (VIIIR:Ag) measured using rabbit antibody to human VIIIR:Ag was low or absent in most species except simians and the dose response curve was non-parallel to the human standard except with goat plasma. Avians lacked cross-reactive material.

We reviewed 4 patients with urothelial bladder cancer and hypercalcemia but without evidence of bony metastasis. Of the patients 2 presented with a leukemoid reaction (1 also had thrombocytosis). None of the patients had evidence of bone metastases or other causes of hypercalcemia, such as hyperparathyroidism, sarcoidosis or vitamin D intoxication. All 4 patients received aggressive therapy for the tumors. In each instance the serum calcium returned to normal following radiation therapy or tumor removal, lending support to the theory of humoral hypercalcemia of cancer. A diagnostic and therapeutic approach to such patients is outlined.

Papillary cystadenoma of the epididymis is a rare benign tumor, accounting for only 4 per cent of all epididymal tumors. Histologically, it can be confused with metastatic renal cell carcinoma. We report a case of bilateral involvement of the epididymides and infertility. Its diagnostic significance is its association with the von Hippel-Lindau syndrome, necessitating close surveillance.

The deduced amino acid sequence of human factor VIII, obtained from the DNA sequence, predicts a mature polypeptide of 2,332 amino acids containing a triplicated domain structure. The polypeptide has 35% sequence homology with the copper-binding plasma protein, ceruloplasmin. Determination of the thrombin cleavage sites in plasma-derived factor VIII polypeptides allows prediction of the domains involved in the associated activation and inactivation of the protein.

In a comparative study of the diagnostic value of epinephrine renal venography (ERV) and angiography in eight patients with severe inflammatory disease of the kidneys, the spectrum of abnormalities exhibited by ERV was more striking than the angiographic findings. Complete disorganization of the renal venous architecture with linear streaking of contrast agent is a sign of renal inflammatory disease. ERV is a useful adjunctive diagnostic procedure in cases of a nonfunctioning kidney.

Fecal incontinence was created by bilateral pudendal neurectomy in 12 dogs. In six dogs, the fecal incontinence was treated by implantation of a polyester-impregnated silicone elastomer sling to form an artificial external anal sphincter. Six dogs underwent the same operative procedure without the implant to serve as controls. Five dogs regained total continence and one dog regained partial continence after implantation of the sling. Four control dogs remained incontinent and two control dogs regained partial continence. The difference between the dogs with slings and the controls was statistically significant (P less than 0.01). The only complication was infection in one dog, which was treated successfully. Histologic reaction to the implant was confined to a zone of fibrosis 1 to 3 mm surrounding the implant. The sling was well tolerated clinically and appeared to offer a successful method for correction of fecal incontinence in dogs.

The first reported case of traumatic rupture of a horseshoe kidney with partial ureteral duplication and an associated supernumerary kidney is presented. A review of the incidence, hypothesis of embryogenesis, associated anomalous development, and acute surgical management of this unique renal anomaly is provided.

In this article, we show that feline predation involves a continuous gradient of activation between defense and attack and that predatory "play" results from an interaction of the two. Benzodiazepines (oxazepam, diazepam) escalated attack toward killing, so that cats that had avoided mice prior to the drug now played with them, cats that had originally played now killed, and cats that killed mice now did so with less preliminary contact. In such shifts, no sharp demarcation between play and predation was evident. Lateral hypothalamic lesions disrupted the escalation of attack. During recovery, attack was escalated once again along the gradient toward killing, but in the absence of both defense and play. A similar result was obtained in intact killers and nonkillers by the application of mild tail pinch. These results suggest that play with prey is a misnomer for predatory behavior that fails to escalate along the gradient between defense and attack. Movement notation analysis revealed that playful movements are adaptive in that they protect the cat from injury.

Our ten-year experience with 141 kidney transplants is reported. Fifty per cent of the grafts are presently functioning, and 22 patients have been followed more than five years. A urologic complication rate of 13 per cent occurred, but 84 per cent of those patients retained their grafts. Only 1.3 per cent of patients with urologic complications died. Improved results can be achieved by good operative technique and aggressive management of complications.

In an attempt to prevent ejaculatory dysfunction associated with retroperitoneal lymphadenectomy, the dissection has been modified to stay above the inferior mesenteric artery. Using this approach, there has been no adverse impact on survival (at least 18 months follow-up) in 18 patients with nonseminomatous germ cell tumors.

Mild tail-pinch induces eating in animals which are aphagic following lateral hypothalamic lesions. This study compares the specificity of the behavior produced by tail-pinch to that produced by vestibular stimulation in cats which are akinetic and aphagic following lateral hypothalamic lesions. In these cats, tail-pinch elicited licking and biting at food while vestibular stimulation preferentially elicited forward locomotion. These results suggest that such stimuli activate specific movement subsystems rather than producing general, nonspecific arousal.

We present a case of complete scrotal destruction secondary to acute necrotizing fasciitis. Full scrotal reconstruction utilizing bilateral gracilis musculocutaneous flaps was achieved with excellent results. This technique is described.

We reviewed 63 consecutive cases of gunshot wounds of the ureter seen during a 15-year period. The importance of prompt recognition and aggressive surgical management is emphasized, with particular attention being placed on adequate debridement and spatulated splinted watertight ureteroureterostomy. Our philosophy of management for individual injuries is discussed along with our complications and results. This series is the largest reported to date of ureteral gunshot wounds.

Condylomata acuminata and verrucous carcinoma are 2 lesions rarely seen in the bladder. Condylomata of the bladder can be treated successfully by local therapy but underlying carcinoma must be considered if the lesion is extensive or progressive in nature. Vigorous biopsies of the base of the lesions are necessary to eliminate the possibility of malignancy. If verrucous carcinoma is found the patient should be treated surgically. A case of condylomata acuminata of the bladder undergoing malignant change to verrucous carcinoma is reported.