The distribution and kinetics of nuclei in rat osteoclasts

Osteoclast development and growth were studied by determining the number of labelled nuclei in osteoclasts of different sizes (based on the number of nuclei per osteoclast, N/O) and the number of osteoclasts with labelled nuclei at various intervals after tritiated thymidine [( 3H]TdR) injection in young rats. The osteoclast smears were made from the cellular periosteum of the proximal tibia. The frequency distribution of the N/O osteoclasts types in the smears had profiles similar to that of in situ osteoclasts in whole mounts of proximal tibia, which indicates that the osteoclast population of the smears was representative of that on the bone surface. A vast majority of the osteoclasts had a 1-6 N/O, and a number of the cells had as many as 26 or more nuclei. Furthermore, profiles of N/O frequency distributions were similar over the course of the study. Nuclei with [3H]TdR label were initially observed in osteoclasts between 4 and 12 hr after isotope injection. However, fusion of labelled nuclei to osteoclasts continued for at least 150 hr. In general, the labelled osteoclasts exhibited a significantly larger number of nuclei than the unlabelled osteoclasts. The probability of an osteoclast incorporating one or more labelled nuclei increased with time after injection and with an increase in N/O. Labelling intensity decreased with time post injection and with an increase in N/O. The data suggest that turnover of nuclei is more rapid in osteoclasts with high N/O values.     

Evidence for a type-IV-related collagen in Drosophila melanogaster. Evolutionary constancy of the carboxyl-terminal noncollagenous domain

Type IV collagen, a major structural component of basement membrane, has been characterized only in vertebrates. It is unique among the collagenous proteins in that it forms specific lattice networks by end-to-end interactions. In particular, in mammals the C-terminal noncollagenous domain (NCl) of collagen IV was shown to be one of the major cross-linking sites in the network assembly. Here, we give the first direct evidence of type-IV-related collagen in invertebrates by sequence analysis of cDNA and genomic DNA clones for the 3'-end of a previously characterized Drosophila collagen gene. The data describe the C-terminal 190 amino acid residues of the triple helix and the entire noncollagenous domain (231 amino acids) of the chain encoded for by this gene. Comparison with data reported for human and mouse alpha 1(IV) chains reveals that triple-helix regions are quite different, while NC1 structures are very similar. This suggests different constraints on triple-helix and NC1 domains during evolution. Present data support the assumption that the NC1 structure originated from duplication of an ancestral sequence; the extent of both interspecies and intramolecular homologies suggests the maintenance in vertebrates and invertebrates of an ancestral specific function.     

Patterns of spontaneous unit preoptic neurosecretory cell discharges in the rainbow trout, Salmo gairdneri

Extracellular antidromic potentials recorded from the neurosecretory cell body were characterized by the following criteria: constant latency, the ability to follow a high frequency rate of stimulation and the collision test. The latency of the antidromic potentials ranged from 12 to 24 ms (17.46 +/- 3.10 SD) which gave a mean conduction velocity of 0.19 m/s, typical of unmyelinated nerve fibers. Two components could be clearly distinguished in the antidromic potential. A small "A" spike which showed constant latency and a large "B" spike with a variable latency and amplitude. A delay of 6.5 ms between the two spikes could occur and sometimes the "B" spike was blocked leaving only the "A" spike. Four patterns of spontaneous activity seem to emerge: Type I (26% of units, M +/- SD = 0.77 +/- 0.32 sp/s) corresponds to a slow and irregular pattern of activity; Type II (28% of units, M = 1.58 +/- 0.47 sp/s) is hard to classify and may be related to an irregular bursting pattern of activity; Type III (28% of units, M = 2.59 +/- 1.19 sp/s) corresponds to a continuous pattern of activity; Type IV (18% of units) represents a rhythmic pattern of activity with an active phase of about 3 min (M = 2.42 +/- 0.90 min), a silent phase of about 4 min (M = 3.89 +/- 3.02 min) and a maximal frequency of unit discharge in the range 2-18 sp/s. No statistical differences exist for the mean dorsal aortic pressure (DAP) between the four types of neurosecretory cell activity.     

Intraventricular somatostatin-14, arginine vasopressin, and oxytocin: analgesic effect in a patient with intractable cancer pain

The analgesic effect of intraventricular somatostatin-14 (SOM-14), arginine vasopressin (AVP), and oxytocin (OT) were tested in one terminally ill cancer patient with a diffuse mesothelioma suffering intractable continuous and incapacitating thoracic pain. SOM-14 reduced pain by 90% for 48 min; AVP reduced pain by 95% for 75 min, and OT reduced pain by 88% for 77 min. The only notable side effects were seen after the administration of AVP, which induced anesthesia and flaccid paralysis of the lower limbs, from which the patient fully recovered after 20 h.     

Relationship between cAMP and Ca2+ fluxes in human platelet membranes

The effect of cAMP (which involved a 23 kDa protein phosphorylation) has been studied on the Ca2+ uptake and Ca2+ release from a human platelet membrane vesicle fraction. It was tested in the presence of the catalytic subunit of the cAMP-dependent protein kinase (C Sub). The addition of C Sub increased the steady state level of the Ca2+ uptake into the membrane vesicles. The effect was enhanced when tested in the absence of Ca2+ precipitating agent. The response was proportional to the dose of C Sub. Moreover, the effect varied with the Ca2+ concentration. The effect of C Sub has been tested on the inositol 1,4,5-trisphosphate (IP3)-induced Ca2+ release. A phosphorylated state of the 23 kDa protein appeared to be necessary. Indeed, a phosphorylation inhibition prevented the IP3 effect and the addition of C Sub increased the percentage of released Ca2+ (without modification of the time course). However, the C Sub dose-dependent response was not linear. The effect of cAMP on the two functions (Ca2+ uptake and Ca2+ release) appears to be different. Therefore, these results led us to suggest a more complex role of cAMP in the regulation of platelet Ca2+ concentration.     

Leishmaniasis in Bolivia. IV. The dog in the cycles of leishmaniasis in Bolivia

In Bolivia, the dog is involved in the cycle of visceral leishmaniasis (Leishmania (Le.) chagasi) in the Yungas (alt. 1,000-2,000 m), and also in the cycle of cutaneous leishmaniasis (Le. (V.) braziliensis) in the Alto Beni (alt. 400-600 m). But it plays a different role in the two cycles. In the Yungas focus, it is the main reservoir of Le. (Le.) chagasi and the source of contamination for man. In the Alto Beni focus, it is only a "victim-host", like man, of Le. (V.) braziliensis; the reservoir of which is unknown. Wild mammals are very likely to be involved.     

Molecular cloning and expression in Escherichia coli of a cellodextrinase gene from Bacteroides succinogenes S85

A DNA fragment coding for a cellodextrinase of Bacteroides succinogenes S85 was isolated by screening of a pBR322 gene library in Escherichia coli HB101. Of 100,000 colonies screened on a complex medium with methylumbelliferyl-beta-D-cellobioside as the indicator substrate, two cellodextrinase-positive clones (CB1 and CB2) were isolated. The DNA inserts from the two recombinant plasmids were 7.7 kilobase pairs in size and had similar restriction maps. After subcloning from pCB2, a 2.5-kilobase-pair insert which coded for cellodextrinase activity was isolated. The enzyme was located in the cytoplasm of the E. coli host. It exhibited no activity on carboxymethyl cellulose, Avicel microcrystalline cellulose, acid-swollen cellulose, or cellobiose but hydrolyzed p-nitrophenyl-beta-D-cellobioside and p-nitrophenyl-beta-D-lactoside. The Km (0.1 mM) for the hydrolysis of p-nitrophenyl-cellobioside by the enzyme expressed in E. coli was similar to that reported for the purified enzyme from B. succinogenes. Expression of the cellodextrinase gene was subjected to catabolite repression by glucose and was not induced by cellobiose. The origin of the DNA insert from B. succinogenes was confirmed by Southern blot analysis. Western blotting (immunoblotting) using antibodies raised against the purified B. succinogenes cellodextrinase revealed a protein with a molecular weight of approximately 50,000 in E. coli clones which comigrated with the native enzyme isolated from B. succinogenes. These data indicate that the cellodextrinase gene expressed in E. coli is fully functional and codes for an enzyme with properties similar to those of the native enzyme.     

Sequence divergence and open regions of RNA secondary structures in the envelope regions of the 17 human immunodeficiency virus isolates.

Genetic variation during the course of infection of an individual is a remarkable feature of the acquired immune deficiency syndrome (AIDS) disease. This variation has been studied for the envelope protein encoding regions of seventeen different sequences from various isolates of human immunodeficiency virus (HIV) using multiple sequence comparison and calculation of variability. The open regions with little intramolecular base pairing in these envelope sequences are predicted by a recently developed statistical method. The minimum length L for a run of hypervariable sites, conserved sites, or open regions that gives significance at the 1% (or 0.1%) level is then determined by a scan statistical method. The results show that significant clusters of open regions predicted at the RNA levels correlate with significant clusters of hypervariable sites in the HIV envelope gene. Those significant genomic variations in HIVs seem to be manifested mainly in the extracellular portion of the envelope protein. Twelve potential antigenic determinants are predicted using an antigenic index method. Interestingly, the majority of the significant hypervariable regions in the exterior envelope protein (gp120) were predicted potential epitopes.     

Two types of K+ channels in the apical membrane of rabbit proximal tubule in primary culture

The patch-clamp technique was used to investigate ionic channels in the apical membrane of rabbit proximal tubule cells in primary culture. Cell-attached recordings revealed the presence of a highly selective K+ channel with a conductance of 130 pS. The channel activity was increased with membrane depolarization. Experiments performed on excised patches showed that the channel activity depended on the free Ca2+ concentration on the cytoplasmic face of the membrane and that decreasing the cytoplasmic pH from 7.2 to 6.0 also decreased the channel activity. In symmetrical 140 mM KCl solutions the channel conductance was 200 pS. The channel was blocked by barium, tetraethylammonium and Leiurus quinquestriatus scorpion venom (from which charybdotoxin is extracted) when applied to the extracellular face of the channel. Barium and quinidine also blocked the channel when applied to the cytoplasmic face of the membrane. Another K+ channel with a conductance of 42 pS in symmetrical KCl solutions was also observed in excised patches. The channel was blocked by barium and apamin, but not by tetraethylammonium applied to the extracellular face of the membrane. Using the whole-cell recording configuration we determined a K+ conductance of 4.96 nS per cell that was blocked by 65% when 10 mM tetraethylammonium was applied to the bathing medium.