GALT deficiency causes UDP-hexose deficit in human galactosemic cells.

Previously we reported that stable transfection of human UDP-glucose pyrophosphorylase (hUGP2) rescued galactose-1-phosphate uridyltransferase (GALT)-deficient yeast from "galactose toxicity." Here we test in human cell lines the hypothesis that galactose toxicity was caused by excess accumulation of galactose-1-phosphate (Gal-1-P), inhibition of hUGP2, and UDP-hexose deficiency. We found that SV40-transformed fibroblasts derived from a galactosemic patient accumulated Gal-1-P from 1.2+/-0.4 to 5.2+/-0.5 mM and stopped growing when transferred from 0.1% glucose to 0.1% galactose. Control fibroblasts accumulated little Gal-1-P and continued to grow. The GALT-deficient cells had 157+/-10 micromoles UDP-glucose/100 g protein and 25+/-5 micromoles UDP-galactose/100 g protein when grown in 0.1% glucose. The control cells had 236+/-25 micromoles UDP- glucose/100 g protein and 82+/-10 micromoles UDP-galactose/100 g protein when grown in identical medium. When we transfected the GALT-deficient cells with either the hUGP2 or GALT gene, their UDP-glucose content increased to 305+/-28 micromoles/100 g protein (hUGP2-transfected) and 210+/-13 micromoles/100 g protein (GALT-transfected), respectively. Similarly, UDP-galactose content increased to 75+/-12 micromoles/100 g protein (hUGP2-transfected) and 55+/-9 micromoles/100 g protein (GALT-transfected), respectively. Though the GALT-transfected cells grew in 0.1% galactose with little accumulation of Gal-1-P (0.2+/-0.02 mM), the hUGP2-transfected cells grew but accumulated some Gal-1-P (3.1+/-0.4 mM). We found that 2.5 mM Gal-1-P increased the apparent KM of purified hUGP2 for glucose-1-phosphate from 19.7 microM to 169 microM, without changes in apparent Vmax. The Ki of the reaction was 0.47 mM. Gal-1-P also inhibited UDP-N-acetylglucosamine pyrophosphorylase, which catalyzes the formation of UDP-N-acetylglucosamine. We conclude that intracellular concentrations of Gal-1-P found in classic galactosemia inhibit UDP-hexose pyrophosphorylases and reduce the intracellular concentrations of UDP-hexoses. Reduced Sambucus nigra agglutinin binding to glycoproteins isolated from cells with increased Gal-1-P is consistent with the resultant inhibition of glycoprotein glycosylation.

Vertebral collapse in childhood leukemia.

Leukemia is the most common form of cancer in children. At the time of initial presentation, 10% of children have normal peripheral blood counts. Appendicular skeletal involvement occurs in approximately 50% of cases. The literature does not clearly define the incidence of spinal involvement, with only a total of 31 cases located in a review of the literature. If case reports are excluded and series that reported the frequency of spinal involvement are summarized, only 16 of 615 cases had spinal involvement. In only one of 31 was it clear that the patient had a normal peripheral blood count at the time of diagnosis. This article adds to the literature two patients who had normal peripheral blood counts and spinal involvement with acute lymphocytic leukemia. Patients with leukemia may have significant symptoms and radiographic signs with normal peripheral cell blood counts at the time of initial presentation.

The Grice extra-articular subtalar arthrodesis in the treatment of spastic hindfoot valgus deformity.

The results of the Grice extra-articular subtalar arthrodesis were evaluated in 102 feet of 60 ambulatory patients with spasticity at an average of five years postoperatively. Results were satisfactory in 96 feet (94 per cent). Unsatisfactory results were attributed to recurrent deformity in four feet and over-correction in two. No other significant complications were identified. The Grice arthrodesis is recommended for hindfoot valgus deformity secondary to spasticity which is refractory to non-operative management.

Prostaglandin-induced hyperostosis. A case report.

The use of prostaglandin-E1 (PGE1) to maintain patency of the ductus arteriosus in infants with ductal-dependent congenital heart disease is now well established. A 2.5-month-old child with cyanotic heart disease who required long-term PGE1 infusions; developed widespread periosteal reactions during the course of therapy. Prostaglandin-induced subperiosteal hyperostosis should now be considered in the differential diagnosis of neonatal cortical proliferation.

Unrecognized pin penetration in slipped capital femoral epiphysis.

There are at least nine different techniques recommended to lower the incidence of femoral head penetration by fixation pins in the pinning of slipped capital femoral epiphyses. The routine use of fluoroscopy and placement of the fixation pins no closer than 8 mm (or one-third the radius of the femoral head) from the subchondral bone will leave a safe, workable margin. Confirmation of subchondral location after the procedure can be done with fluoroscopy. With the leg in maximal internal rotation, the fluoroscopic unit is rotated from anteroposterior (AP) to lateral under continuous monitoring to detect anterolateral and posteromedial quadrant penetrations. With the fluoroscopic unit in the lateral position, the foot is externally rotated 90 degrees, again under continuous monitoring, to visualize the anteromedial and posterolateral quadrants. If the femoral head cannot be well-visualized in the lateral view, use of a lateral x-ray machine and AP fluoroscopy can be helpful. Another useful technique when the head cannot be well-visualized is the injection of contrast medium through a cannulated screw.

Irreducible fracture of the calcaneus in a child.

Displaced fractures of the calcaneus are rare in children, and most do not require open reduction as a treatment modality. The case of a 4-year 8-month-old boy who sustained a fracture of the anterior process of the calcaneus requiring open reduction is presented; no similar fracture in childhood has been previously reported. Treatment guidelines for calcaneal fractures in children are reviewed.

Congenital scoliosis and urinary tract abnormalities: are intravenous pyelograms necessary?

One hundred patients with a diagnosis of congenital scoliosis were reviewed with regard to their urologic history, chemistries, and excretory urography. A 40% incidence of urologic abnormality was found in these asymptomatic patients. Twenty-five additional patients were evaluated by excretory urography and ultrasonography. Of six patients identified by ultrasonography as having abnormalities five had results confirmed by excretory urography. Urologic evaluation of all patients with congenital scoliosis is recommended; however, diagnostic ultrasonographic evaluations of the urinary tract have proven to be an acceptable alternative as an initial screening modality.

Preparation of partially protected des-alanineB30-insulin-B-chain-disulphide and its use in semisynthesis.

The preparation of N alpha 1 N epsilon 29-bis-methylsulphonylethyloxycarbonyl-des-alanineB30-B-chain-disulphide-O gamma 13 O gamma 21-dimethyl ester is described. Starting with the di-S-sulphonate-B-chain it was prepared by i) removal of the C-terminal amino acid by digestion with carboxypeptidase A, ii) reduction and oxidation to form the cyclic disulphide, iii) esterification of the three carboxyl groups (alpha COOH = LysB29, gamma COOH - GluB13 and GluB21) followed by selective tryptic hydrolysis of the alpha-carboxyl ester, and iv) the protection of both amino groups (alpha NH2 = PheB1, epsilon NH2 = LysB29). This B-chain derivative was activated selectively at the C-terminal carboxyl group by formation of a mixed anhydride, and condensed with the dipeptide Thr-Arg. All protecting groups were removed by treatment with alkali. The human-B-chain C-terminal elongated with Arg was obtained in a yield of 30% based on the protected des-AlaB30-B-chain derivative.

[Improved preparation of des-alanylB30-insulin (author's transl)]. / Verbesserte Darstellung von Des-alanylB30-insulin.

Des-AlaB30-insulin was prepared by incubation of insulin with carboxypeptidase A. The C-terminal alanineB30 of the B-chain was quantitatively liberated without liberating asparagineA21 of the A-chain in ammonium hydrogencarbonate buffer.

[A21-Asparaginimide] insulin. Saponification of insulin hexamethyl ester, I.

[Asn A21]Insulin is formed as the main product during alkaline saponification of insulin hexamethyl ester. Purification was achieved by gel chromatography followed by ion-exchange chromatography on carboxymethyl cellulose at pH 4 or by preparative isoelectric focusing in a granulated gel over a narrow pH range. Two main products could be isolated. One of them showed the electrophoretic behaviour of insulin (A), whilst the other corresponded to insulin with a blocked carboxyl function (B). Incubation of this product B with carboxypeptidase A liberated only the C-terminal alanine of the B-chain, but not the asparagine of the C-terminus of the A-chain. Chymotryptic digestion of the isolated S-sulfonate A-chain derivative (C) followed by high-voltage electrophoresis confirmed that the carboxyl function of asparagine A21 was blocked. In order to determine the free carboxyl functions of the A-chain derivative C, it was coupled with glycine methyl ester yielding D. Amino acid analysis of the chymotryptic peptides of D showed that the carboxyl functions of glutamic acid A4 and A17 had been free prior to coupling. The amino acid analysis of the enzymatic hydrolysate (subtilisin, aminopeptidase M) of the A-chain derivative C showed an additional peak with an elution position identical to the model compound aminosuccinimide. The biological activity of the [Asm A21[insulin was found to be about 40% in the fat cell test and 13.2 units/mg measured by the mouse convulsion method.