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The major sperm proteins (MSPs) are a family of closely related, small, basic proteins comprising 15% of the protein in Caenorhabditis elegans sperm. They are encoded by a multigene family of more than 50 genes, including many pseudogenes. MSP gene transcription occurs only in late primary spermatocytes. In order to study the genomic organization of transcribed MSP genes, probes specific for the 3' untranslated regions of sequenced cDNA clones were used to isolate transcribed genes from genomic libraries. These and other clones of MSP genes were located in overlapping cosmid clones by DNA fingerprinting. These cosmids were aligned with the genetic map by overlap with known genes or in-situ hybridization to chromosomes. Of 40 MSP genes identified, 37, including all those known to be transcribed, are organized into six clusters composed of 3 to 13 genes each. Within each cluster, MSP genes are not in tandem but are separated by at least several thousand bases of DNA. Pseudogenes are interspersed among functional genes. Genes with similar 3' untranslated sequences are in the same cluster. The six MSP clusters are confined to only three chromosomal loci; one on the left arm of chromosome II and two near the middle of chromosome IV. Additional sperm-specific genes are located in one cluster of MSP genes on chromosome IV. The multiplicity of MSP genes appears to be a mechanism for enhancing MSP synthesis in spermatocytes, and the loose clustering of genes could be a result of the mechanism of gene duplication or could play a role in regulation.

Experimental spinal cord injury was produced in rats by dropping a 10 g weight from 30 cm upon dura-invested exposed spinal cord. Proteolytic activities at neutral (pH 7.6) and acid (pH 5.5 and 3.6) pH were determined in whole homogenate and the cytosolic fraction of the lesion (lumbar) and cervical control segments. The enzyme activity was monitored by SDS-PAGE analysis of the extent of substrate myelin basic protein (MBP) degradation. Activities (neutral and cathepsin B-like) in the sham-operated spinal cord were lower than those of cervical autologous control at 24 h after injury. The increase in neutral proteinase activity was progressive and greater in the lesion than the autologous control. A 61.5% +/- 3.5 loss of MBP was observed at 2 h following injury and increased at 24 h (78.2% +/- 3.4). The loss of MBP coincided with the appearance of several low molecular weight peptides. The cathepsin B-like and cathepsin D activities were also increased in the lesion but to a lesser extent than the neutral proteinase. The neutral proteinase and cathepsin B-like activity were inhibited by leupeptin and not by pepstatin while the converse obtained for cathepsin D activity. The release of neutral proteolytic activity which is nonlysosomal in origin suggests a novel hypothesis for the mechanism of traumatic axon-myelin injury.

The acylation of myelin proteolipid protein (PLP) and intermediate protein (IP) was investigated in an in vitro system of tissue slices prepared from actively myelinating rat brainstem. The incorporation of [(3)H]palmitate into the proteins in nine subcellular fractions including myelin and other cellular membranes which are actively involved in the synthesis and intracellular transport of the proteins was measured. More than 80% of [(3)H]palmitate-labeled proteins were recovered in myelin. The incorporation was highest in the heavy myelin and lowest in the light myelin subfraction. Appreciable acylation was also detected in the myelin-like fraction. On the other hand, the remaining fractions comprising a variety of endo- and ectomembranes, which harbored over 90% of newly synthesized PLP and IP as seen from [(3)H]leucine labeling showed practically no [(3)H]palmitate incorporation. The results indicate that the acylation of PLP and IP is a late event in their posttranslational processing and occurs only at their entry into the myelin sheath.

The incorporation of radioactive label into the myelin basic protein isolated from whole brain and from purified myelin of Quaking mice and normal littermates was compared. four Quaking mice (32 days) and 4 littermate controls were injected intracranially with 150 muCi [2-3H]glycine and 25 muCi of [2-14C]glycine, respectively. One hour later, the 8 mice were sacrificed and their brains combined for common homogenization. The 3H/14C ratios of the small and large basic protiens in whole brain were 3.44 and 2.48 respectively, while the 3H/14C ratios for these proteins in myelin were 0.79 and 1.00, respectively. In the same experiment, the microsomal fraction had a 3H/14C ratio of 2.98 which is the expected ratio for normal incorporation. The results indicate that the synthesis of basic protein in whole brain of Quaking mouse proceeds at a normal rate, but specifically, the incorporation of basic protein into myelin is depressed suggesting a defect at the step of assembly of myelin components into a final membrane product.

Two glucosamine-containing gangliosides, a ceramide pentasaccharide and ceramide heptasaccharide, have been purified from the gangliosides of chicken skeletal muscle. The lipids were extracted with a mixture of tetrahydrofuran and aqueous KCl and purified by anion-exchange and silicic acid column chromatography. Their structures were determined by specific enzymatic hydrolysis and methylation analysis. The proposed structures are sialosyl derivates of: (a) paragloboside, and (b) paragloboside with an additional lactosamine disaccharide: (a) NeuAc alpha 2 leads to 3Gal beta 1 leads to 4GlcNAc beta 1 leads to 3Gal beta 1 leads to 4GlcCer, (b) NeuAc alpha 2 leads to 3Gal beta 1 leads to 4GlcNAc beta 1 leads to 3Gal beta 1 leads to 4GlcNAc beta 1 leads to 3Gal beta 1 leads to 4GlcCer. The long base was predominantly sphingosine and the acyl groups were mainly palmitate, stearate and oleate.

The distribution of neutral, or asialosyl-, glycosphingolipids and gangliosides in a rhabdomyosarcoma of alveolar type have been studied. Histologically, this muscle tumor is composed primarily of two cell types: one with oval or round hyperchromatic nuclei and very little cytoplasm, and one a giant cell, with multiple, peripherally placed nuclei and weakly staining eosinophillic cytoplasm. In comparing glycolipids of the rhabdomyosarcoma with normal muscle from the same leg, the striking alteration in the tumor was a virtual disappearance of ganglioside GM2. There was also a slight increase in GM3 and a decrease in GD1a. The asialosyl derivative of GM2 (GalNac-Gal-Glc-Cer) was markedly increased in the tumor. A loss of glucosylceramide was also observed. The results are discussed in terms of glycolipid metabolic changes in muscle oncogenesis and their implications.

The activity of calcium-activated neutral proteinase (mM CANP) was determined in homogenate, myelin and supernatant of bovine brain corpus callosum. The enzyme activity in homogenate and myelin was increased eleven and thirteen-fold respectively by Triton X-100. Myelin prepared by the method of Norton and Poduslo as well as by a modified method, was shown to contain most (more than 50%) of homogenate mM CANP activity. The specific activity was highest in myelin, and increased almost three times more than the homogenate. Supernatant only contained 17% of enzyme activity. It is concluded from these studies that mM CANP is tightly bound to the membrane and predominantly associated with the myelin sheath.

A technique is described for direct detection by lectins conjugated to horseradish peroxidase of the oligosaccharides of glycosphingolipids on thin-layer chromatograms. Final visualization is accomplished using a substrate medium consisting of hydrogen peroxide and 3,3'-diaminobenzidine. The procedure is rapid, specific, sensitive and highly reproducible, and staining patterns are stable for years. In addition to providing preliminary structural information, this technique can be employed in combination with other methods, such as autoradiographic detection of tritium-labeled glycosphingolipids in the same chromatogram.

Experimental spinal cord trauma was produced in rats by dropping a 10-g weight from a height of 30 cm upon exposed spinal cord. The histological lesion consisted of edema, necrosis, and hemorrhage. The fine structure of the early traumatic lesion (4 to 12 hours) included granular dissolution of axons and a characteristic vesiculation of myelin. The predominant ultrastructural features of older lesions (12 to 72 hours) were intra-axonal calcification and lipid-laden macrophages. The yield of myelin and the activity of adenosine 2',3'-cyclic nucleotide 3'-phosphohydrolase (CNP) were reduced by approximately 15% at 4 hours and by 60% at 72 hours. Losses in all myelin proteins were observed, but were most severe and occurred earliest in the basic proteins. The ultrastructural and biochemical alterations observed in this study indicate that proteinase activity is increased and may be partially responsible for the traumatic myelinolysis in experimental spinal cord trauma.

A new quantitative procedure for the high-performance liquid chromatographic (HPLC) resolution of human brain gangliosides employing reversed-phase chromatography is described. To provide a derivative which can be determined by UV absorption techniques, p-nitrobenzyloxyamine was coupled to the gangliosides. Derivatization involves ozonation and cleavage of the ceramide double bond followed by oxime formation to the nascent aldehyde. Individual gangliosides, as they were resolved by HPLC, were collected. These fractions were then identified by thin-layer chromatography (TLC) and by gas chromatography of their monosaccharides. Quantitative results were obtained along with a marked increase in sensitivity over conventional resorcinol-hydrochloric acid quantitation of TLC-resolved gangliosides.

Spinal cord injury was produced in rats by dropping a 10 g weight from 30 cm upon dura-invested exposed spinal cord. Examination of the fine structure of the traumatic lesion (15 min to 30 min) revealed granular degeneration of axons and occasional loosening of myelin lamellae. Older lesions (4 to 72 hours) showed degeneration of axons and vesiculation of myelin. At 15 minutes there is more loss of neurofilament proteins than of myelin proteins. Substantial decreases in the neurofilament and myelin proteins were observed at 30 minutes and the losses were even greater 2-72 hours after injury. This indicates that degeneration of axons may precede degradation of the myelin sheath and also that increased proteinase(s) activity, possibly activated by calcium, mediates the traumatic axonolysis and myelinolysis in experimental spinal cord trauma.

The serum concentration and composition of gangliosides were examined in 80 humans including 10 normal subjects. A significant increase was found in the total gangliosides of serum in 7 patients with cerebral astrocytomas. There was also an increased percentage of serum gangliosides with simpler structure, particularly GM3. The serum of patients with other intracranial tumors, including pituitary adenomas, ependymoma, teratoma, and metastases, did not show an increase in total ganglioside; however the pattern of simplification was found in these and in a few patients with extracranial tumors as well. The findings suggest that astrocytoma tumors shed sialoglycolipids into the circulation, and their assay may be useful in monitoring oncological therapy.

Lead encephalopathy was induced in developing Long-Evans rats by adding lead carbonate (4% w/w) to the diet of nursing mother immediately after delivery. The morphological and biochemical features of cerebral ontogenesis were studied in 30-day-old rats. By the 30th postnatal day, the overall effect of lead intoxication was retardation of brain growth. The mass of both the cerebral gray and white matter was appreciably reduced in the lead rats without any reduction in cell populations. While the neuronal population was preserved, the growth of neurons was reduced and their maturation retarded. The retarded neuronal growth was characterized by the limited proliferation of processes in the neuropil and by the reduction in the number of synapses per neuron. However, synaptogenesis was neither delayed nor perturbed but reduced by the limited development of neuronal dendritic fields. The myelination was altered and its cerebral content significantly reduced. The effect of lead on myelination was one of hypomyelination. The hypomyelination appears to be primarily related to retarded growth and maturation of the neuron and is not a reflection of a defect in the myelinating glia or a delay in the initiation of myelination.

The synthesis and acylation of proteolipid proteins where investigated in tissue slices prepared from 19-day-old quaking and normal littermate mouse brain. The mutant CNS had a normal rate of total protein synthesis but synthesis of the myelin-specific proteins, proteolipid protein (PLP) and intermediate protein (IP), was impaired to approximately 50% of control. The acylation of myelin proteins with labeled palmitate was reduced to only about 20% of control. The acylation of two nonmyelin proteolipid proteins was also significantly reduced. The incorporation of palmitate into phospholipids was identical in control and mutant. The results indicate impaired synthesis of PLP and IP and a general deficit in protein acylation in quaking brain.

Individual ganglioside species (possessing the gangliotetrose oligosaccharide) were purified from bovine brain gray matter and applied in varying concentrations to the culture medium of mouse neuroblastoma cells (N2A) in vitro. After 48 hr of incubation, the cells were stained, and the neuritogenic response quantitated with a video analysis system, employing a program to measure three parameters of neuroblastoma differentiation: neurites per cell (sprouting), neurite length (extension), and degree of neurite branching (arborization). All the individual gangliosides tested promoted neurite extension in a dose-dependent fashion. Asialogangliosides ("neutral" glycosphingolipids) were without effect, which suggests that sialic acid (N-acetylneuraminic acid) is necessary to elicit this cellular response. With increasing concentrations of GM1 (5 to 500 micrograms/ml), the average cellular neurite length increased significantly, whereas the number of neurites per cell decreased. With the trisialoganglioside GT1b, neurite length did not increase to the extent seen with GM1, but an increase in the number of neurites per cell (sprouting) and branch points per neurite (arborization) was observed. These results suggest that the in vitro neuronal response to exogenous gangliosides may combine specific responses to individual species making up the total.

An increased accumulation of tissue thromboxane A2 occurred shortly after spinal cord injury. Prostacyclin formation was not affected. The magnitude of the increase in thromboxane and the extent of post-traumatic vascular damage as determined by extravasation of 125I-labeled human serum albumin were both dependent on the degree of injury. These findings raise the possibility that activation of arachidonic acid metabolism with a preponderance in thromboxane formation may contribute to microvascular injury in experimental spinal cord contusion.