Selective release of glutamate from cerebellar granule cells differentiating in culture

The aim of the present study was to assess whether endogenous and newly synthesized glutamate can be released from differentiating cultured cerebellar granule cells in a way compatible with a neurotransmitter role. Granule cells from 8-day-old rat cerebella were grown in basal Eagle's medium with 10% fetal calf serum for 2-12 days in vitro (DIV), then washed with Krebs-Ringer medium, and labeled for 45 min with tracer amounts of radioactive glutamine. Subsequently, the release of endogenous glutamate and of newly formed radioactive glutamate was measured in basal conditions and upon depolarization with elevated K⁺ concentration or veratridine. At 2 DIV, the release of endogenous and newly synthesized glutamate evoked by high K⁺ concentration was small and Ca²⁺ independent, but it progressively and steadily increased (up to 8- to 10-fold) and became Ca²⁺ dependent (up to 80-85%) at later stages (4, 8, and 12 DIV). Veratridine was almost ineffective with cells at 2 DIV but greatly increased glutamate release (endogenous and neosynthesized) at 8 DIV, and its action was totally antagonized by tetrodotoxin. The level and synthesis of glutamate remained fairly constant in cells from 2 to 12 DIV. γ-Aminobutyric acid synthesis from radioactive glutamine was about 3% of that of glutamate, and γ-aminobutyric acid release (endogenous and neosynthesized) was not measurable. Aspartate synthesis was about 10% of that of glutamate, and the high K⁺ concentration-evoked release of this amino acid was modest and scarcely affected by Ca²⁺. Neither high K⁺ concentration nor veratridine was able to induce glutamate release from confluent cerebellar astrocyte cultures at 14 DIV, although the level and synthesis of the amino acid were comparable to those in granule cells. In conclusion, the data show that a stimulus-coupled release of endogenous and neosynthesized glutamate is progressively expressed by cerebellar granule cells differentiating in culture, and this strongly supports the concept that glutamate is the neurotransmitter of these cells.     

Hemostatic plug formation in normal and von Willebrand pigs: the effect of the administration of cryoprecipitate and a monoclonal antibody to Willebrand factor

Hemostatic plug (HP) formation was investigated in the ear bleeding time incision in normal and von Willebrand pigs. HP volume was calculated by integrating the areas of serial sections. In normal pigs (n = 11), platelets immediately formed a layer on the surface of the cut channel. Platelet aggregates formed at the ends of transected vessels and gradually enlarged. Finally, all transected vessels were occluded by HP and bleeding stopped. In contrast, large HPs were formed in the incision in von Willebrand's disease (vWD) pigs (n = 4); these HPs did not cover the ends of the transected vessels, which continued to bleed, allowing the formation of large hemostatically ineffective platelet aggregates in the incision. Canals traversed these HPs, and bleeding from the open vessels may have continued through them. After infusion of cryoprecipitate into a vWD pig, the bleeding time shortened, and the morphological findings of the HPs were similar to those of normal pigs. In normal pigs (n = 3) infused with an anti- Willebrand factor monoclonal antibody, which prolonged the bleeding time, a large HP formed in the incision, similar to that observed in the vWD pig. The volume of the normal and vWD HPs increased with time. These in vivo findings suggest that Willebrand factor is involved in the localization of the HP to the damaged vessel and may also play a role in platelet-platelet interaction. A computerized morphometric technique was used for measuring the volume of the hemostatic plugs and the distance of sequential points on the perimeter of the HP from the center of selected bleeding vessels.     

Human herpesvirus 7 is a T-lymphotropic virus and is related to, but significantly different from, human herpesvirus 6 and human cytomegalovirus.

An independent strain (JI) of human herpesvirus 7 (HHV-7) was isolated from a patient with chronic fatigue syndrome (CFS). No significant association could be established by seroepidemiology between HHV-7 and CFS. HHV-7 is a T-lymphotropic virus, infecting CD4+ and CD8+ primary lymphocytes. HHV-7 can also infect SUP-T1, an immature T-cell line, with variable success. Southern blot analysis with DNA probes scanning 58.8% of the human herpesvirus 6 (HHV-6) genome and hybridizing to all HHV-6 strains tested so far revealed homology to HHV-7 with only 37.4% of the total probe length. HHV-7 contains the GGGTTA repetitive sequence, as do HHV-6 and Marek's disease chicken herpesvirus. DNA sequencing of a 186-base-pair fragment of HHV-7(JI) revealed an identity with HHV-6 and human cytomegalovirus of 57.5% and 36%, respectively. Oligonucleotide primers derived from this sequence (HV7/HV8, HV10/HV11) amplified HHV-7 DNA only and did not amplify DNA from other human herpesviruses, including 12 different HHV-6 strains. Southern blot analysis with the p43L3 probe containing the 186-base-pair HHV-7 DNA fragment hybridized to HHV-7 DNA only. The molecular divergence between human cytomegalovirus, on the one hand, and HHV-6 and HHV-7, on the other, is greater than between HHV-6 and HHV-7, which, in turn, is greater than the difference between HHV-6 strains. This study supports the classification of HHV-7 as an additional member of the human beta-herpesviruses.